1. health and fitness
2. drugs

Indian Journal of Pharmaceutical Education and Research
The Official Journal of Association of
Pharmaceutical Teachers of India
(Registered under Registration of Societies Act XXI of 1860
No. 122 of 1966-1967, Lucknow)
Abstracted in International Pharmaceutical Abstract
Vol. 40 (6)*
Editor
B.G. Nagavi, Ph.D.
Mysore.
1997 - till date
Founder Editor
B.M. Mithal, Ph.D.
Pilani.
1967 - 1974
Editorial Advisory Board
Dr. P. Gundurao
Head, R&D Division,
Divis Laboratories,
Hyderabad
Dr. B. Suresh
President,
Pharmacy Council of
India, New Delhi
Prof. P. Ramarao
Director,
NIPER,
Chandigarh
Mr. David Cosh
Consultant Pharmacist
Adelaide, Australia
Mr. Frank May
University of Kentucky
USA
Mrs. Claire Anderson
University of Nottingham,
UK
Prof. V.I. Hukkeri
President, APTI
Prof. B.G. Shivananda
Secretary, APTI
Number 02
April - June 2006
70
Editorial
71
Invited Editorial
Third Phase of Development of Pharmacy Training - P. Velayudha Panicker
73
77
84
93
Invited Article
Pharmacist Conducted Medication Review in Australia: is there Relevance to
Practice in India? - David Cosh
Review Articles
New Generation Antipsychotics: A Review
- Devender Pathak, Rajiv Dahiya, Kamla Pathak and Sunita Dahiya
Pharmacogenomics: The Search for the Individualized Therapy
- C.S.Magdum, V.S.Velingkar and Meenu K.Gupta
Harmonization of Excipients: The Indian Perspective
- S.S.Poddar, Chivate Amit and Prajapati Paresh
Research Articles
100 Stability-indicating HPTLC Determination of Donepezil HCl in Pharmaceutical
Dosage Form - Vinay Saxena, Zahid Zaheer and Mazhar Farooqui
106 Chitosan-Based Nanoparticles for Delivery of Proteins and Peptides
- V.J.Mohanraj, Y.Chen and B.Suresh
Article
116 Rhabdomyolysis - Milind Parle, Mamta Farswan and Arvind Semwal
Short Communications
122 Protease Inhibitors – Spanking Agents for Respiratory Disorder
- S. Ponnusankar, R. Senthil, Sandip Kumar Bhatt and B. Suresh
126 Pharmacist: Against Drug Abuse - P. G. Yeole
129 Bibliometric Analysis for Identifying the Spectrum of Cardiovascular
Pharmacological Research and Leading Nations
- P.M.K. Reddy, M. Jayanthi, K.M. Ravindra, R. Kesavan and S.A. Dkhar
132 TQM of Pharmacy Teachers: Need and Challenges - Neelam Mahajan
Ph.D. Thesis
135 Chemotaxonomical Significance of Anthraquinone Glycosides in Local Cassia
Species - Mohib Khan
136 Formulation and Evaluation of Mucoadhesive Drug Delivery Systems of Some
Antiasthmatic Drugs - T.M. Pramod Kumar
136 Studies on Karnataka state pharmacists’ attitudes and behaviours towards patient
counselling and use of patient information leaflets. - Adepu Ramesh
137 Preparation and evaluation of waxes/ fat microspheres loaded with hydrophilic and
lipophilic drugs for controlled release - D.Vishakante Gowda
139 Book Reviews
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
69
* Number in bracket indicates the new number after registration of IJPE with registrar of news papers for India , New Delhi.
Editorial
Patient Counseling – Heart of Professional Practice
One day Alice came to a fork in the road and saw a Cheshire cat in a tree.
“ Which road do I take?” she asked.
His response was a question: “ Where do you want to go?”
I do not know” Alice answered.
“Then” said the cat, “ It doesn’t matter”
- Lewis Carroll - Alice in wonderland.
Patient counseling is the most important component
their professional responsibilities. But in reality patient
in the delivery of pharmaceutical care services. Many
counseling was rarely done in the pharmacies due to
scientific studies have proven the patient counseling
inadequate knowledge to offer counseling, lack of proper
influence on improved patient medication adherence and
infrastructure, peak work load and business attitude of
health related quality of life. Internationally, pharmacists
the practicing pharmacists, non-legalisation of patient
proved themselves as professionals by virtue of patient
counseling and lack of remuneration. Doctor dispensing
counseling. In some countries it is mandatory and also a
is also another important threat to offer patient counseling
remunerative service.
especially in urban areas. Majority patient information
In India, inspite of considerable efforts by the
leaflets (95%) produced by the pharmacists and
various professional associations, in restructuring the
manufacturers did not meet the readability and lay out
curriculum, introducing subjects focusing on professional
requirements.
practices, patient counseling remained a dream and
The research is a first ever Ph. D level work
pharmacists still behave as traders than professionals.
conducted in India and the candidate was awarded Ph. D
Thus it has become necessary to ascertain pharmacists’
in Pharmacy (Pharmacy Practice) with focus in community
attitudes responsible for the trader behaviour.
pharmacy by Rajiv Gandhi University of Health Sciences,
A detailed study was conducted in the state of
Karnataka. in February 2006.
Karnataka, to assess the attitudes and behaviors of
The authors of the research work Dr. Adepu Ramesh
practicing community pharmacists towards patient
and Dr. B. G. Nagavi have put forth the following
counseling and use of patient information leaflets. As a
recommendations to improve the present pharmacy
part of the study public and prescribers perception
practice situation in India.
towards pharmacists’ services were also taken in to
• Minimum qualification for registration of Pharmacists
account. The responded public opined community
to be raised from the present diploma to degree
pharmacists as traders and expected sincere and honest
qualification to achieve patient counseling.
professional services from them. The prescribers opined
• Schedule K of Drugs & Cosmetics act (1940) to be
that, pharmacists are the right persons to take the
amended to delete the doctor dispensing in urban
responsibility of patient counseling and providing product
areas.
information to ease their professional burden. But in
• Prepare and implement the professional standards for
order to provide these services, prescribers desire that,
practicing pharmacists by PCI and state pharmacy
pharmacists should have B.Pharm or M.Pharm
councils.
qualification.
• The regulatory authorities shall encourage
Out of 258 respondent pharmacists more than 50 %
pharmacists opening new pharmacies.
mentioned that, they were not aware about the
• Legalize patient counseling and allow the pharmacists
professional responsibilities of community pharmacists
to charge nominal professional fee and insist on
recommended by World Health Organisation (WHO) or
patient counseling cubicles in community pharmacy
International Pharmaceutical Federation (F.I.P). Results
and hospital.
of critical survey of community pharmacies revealed that,
• Conduct continuous professional development (CPD)
4% of the pharmacies did not meet the schedule N
programs for the practicing pharmacists through state
requirement, 9% of the pharmacies did not meet the
pharmacy councils, with the help of local pharmacy
schedule C & C1 and only 7% of them had computers,
colleges and the regulatory authorities.
which were used only for business operations. About
• All the pharmacy colleges should have an attached
19% of pharmacies were having patient counseling
community pharmacy (Drug Store) in community set
cubicle but did not use for the counseling purpose.
up offering professional dispensing and patient
Majority of the respondents (78%) agreed that, the
counseling to the public.
patient counseling and providing information leaflets are
IJPER profusely thank All India Council for Technical Education (AICTE) New Delhi, for part financial assistance for
printing/publishing of Indian Journal of Pharmaceutical Education and Research during financial year 2005-06.
70
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Invited Editorial
Third Phase of Development of Pharmacy Training
P.Velayudha Panicker
Visiting Professor, Al-Shifa College of Pharmacy, Perinthalmanna, Kerala-679 325
The awareness that alternative systems of therapy
using herbal and other natural products as well as some
ethnical technique is slowly catching the attention of a
cross- section of professional and policy making
organizations. The query that is posing the Pharmacists
and other medical professionals is how best the
alternative traditional systems could be attuned without
losing their basic idiom to the modern therapeutic
modulation in the present scenario of globalization.
The strong conviction I cherish is the role of a
HERBAL PHARMACIST distinctive from Herbal chemist,
who is to link between the two divergent systems in a
hospital environment practice. The solution of ‘AlloAyurveda’ or ‘Allo-Traditional’ as envisaged by a section
of professionals, is not a practical solution in my opinion
because two divergent systems should stay alive as
parallel systems under the same roof keeping the idiom
of respective system intact. This context has prompted
me to suggest a third phase of development in Pharmacy
curriculum and training to incorporate a concept of a
HERBAL PHARMACIST. Since surgical therapy and
technique are advancing in India to cope up with any
advanced country, it is out of context to link this area
with tradition. But there is a dire need for linking
YESTERDAYS (Wealth & resources) and TODAY
(Utilization) with TOMORROW (Target fixed for 2020 AD)
in an ongoing drug therapy. I would try to endorse in
this article three phases of profession, one that of past,
the second that of present and the third of future.
First Phase
The modern pharmaceutical education was started
in the mid 1930’s under the valiant and perceptive
leadership of Prof M.L.Schroff. This was a period when
all out emphasis of profession was concentrated on
Pharmaceutical technology available that time and more
significance was given to dispensing pharmacy. The only
Indian pharmaceutical manufacturing unit worth
mentioning was Bengal chemicals which was an archetype.
The dawn of independence witnessed a host of
multinational technology coming to the country. When
the first IP was born in 1955 as a twin brother to BP,
many professors used to advise us to refer either; but
preferably BP to evade spelling mistakes. Later editions
of IP even though followed the pattern of BP, the latest
IP deviated the pattern by deleting many natural products.
The mid 1970’s witnessed a rapid pace of growth of Indian
pharmaceutical companies even to a competitive level to
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
multinationals by 1990’s. This created on one side a
decline in Dispensing practice, and on the other side an
explosiveness of market products and combinations. The
impact of this has reflected in irrational prescription
practice and drug interaction with drugs and food and
lead to a second phase of development in pharmacy.
Second Phase
The Pharmacy curricula of today give equal
importance to Industrial and Clinical Pharmacy. Clinical
pharmacy program facilitate focus work groups to
establish safe and appropriate use of drugs and
disseminate timely information of new drugs and services
to physicians and also to provide consultant services to
hospital professionals in formulary management.
The college of Pharmaceutical Sciences at
Trivandrum Medical College as early as mid 1980’s
implemented interaction with the referral hospital in the
following service
·
Antigenic preparation of local allergens for
department of chest and respiratory diseases
·
Toxicological analysis of poison cases of patients
and reporting the toxin and toxic level in body fluids
to decide on course of treatment
·
Manufacture of morphine in its various forms for
the registered terminal patients of cancer for RCC
·
Instrumental in making a hospital formulary for the
state
Third Phase
Having established and gaining momentum in the
two phases discussed, yet another area for pharmacy is
wide open which I have been protecting through IJPE
column [Ref: IJPE Vol.30, Dec. 1996;Vol 36(4) Oct-Dec.
2002 and Vol39 (2) April-June 2005] over a period. It paves
foundation for the concept of a HERBAL PHARMACIST
distinctive from Herbal Chemist engaged in molecular
level of research and study. The present undergraduate
level syllabus approved by AICTE also substantiate this
concept by including various traditional formulations in
the curricula at least on a preliminary basis. It becomes
an easier task for modern Pharmacognosist to cope up
with different traditional forms like Ayurveda, Sidha,
Unani, Naturopathy, and Tribal medicines rather than
starting suitable B.Pharm courses in the respective
branches. Such a HERBAL PHARMACIST has to act as
a link between the two divergent systems. I could
personally convince at least two modern speciality
71
hospitals the need to implement holistic system side by
side in certain areas of ailment and the HERBAL
PHARMACIST would assist the traditional practitioner
on patients referred from modern hospitals in recording
clinical status, standardize raw materials, and formulate
the flexible formulation depending on the need of
particular patient, recording the raw materials for further
reference and finally the clinical status of the treated
patient to be referred back to modern physicians.
The two systems can never meet and can only coexist and the bridging link could be a HERBAL
PHARMACIST. During the pre-medieval period, all
systems were based on holistic principle, until the French
apothecary Derosne in 1803 isolated the first molecule
and alkaloid named then as narcotine and Schiff in 1870
established structure of coniine and Ladenburg in 1889
synthesised coniine and so on paving a new vista to
develop new molecules and indexing them on special
therapeutic basis. Thus the branch was accepted
worldwide scientifically and the research and development
adopted a pattern of going to theory, then practical
associated with action based observation on animals and
further through different phases of clinical trials before
being introduced to market.
72
While, looking at the holistic systems, it generally
start from observation and experience on a particular
patient and then goes to hypothesis and translate to
dispensing, If we look at the different holistic systems,
we find Greek system has completely collapsed while
Indian, Chinese or Mesopotamian and Egyptian systems
are still alive. In India, the Adharva veda contributed to
a branch Ayurveda. The pre-islamic holistic culture in
Mesopotamia and Egypt during post-islamic period came
to India in the form of Unani and Sidha which are very
much alive along with a host of diverse tribal medicines.
For instance, the holistic approach of Ayurveda seeks
the cause of an illness and restores balance, using the
insight of the elemental creation of the universe. Similarly,
each traditional system possesses its own philosophy in
its implementation.
These different systems scattered around cannot
be integrated unless these systems co-exist with modern
hospitals giving priority to the regional need and
availability and HERBAL PHARMACIST can play an
important role in linking the systems. It would also
contribute HERBAL CHEMIST to take up traditional
drugs for research in molecular level to preserve our
tradition intact in the present global scenario.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Invited Article
Pharmacist Conducted Medication Review in Australia: is there
Relevance to Practice in India?
David Cosh
Clinical Pharmacist, Adelaide, South Australia
Mysore Seminar on Home Medication Review
th
On November 30 2005 in Mysore, India, a group of
Indian and Australian pharmacists conducted a one-day
seminar on home medication review (HMR) under the
auspices of Dr B. G. Nagavi and the JSS College of
Pharmacy, Mysore. The stimulus to run such a workshop
came from a belief that a new model of professional
practice that has evolved in Australia over the last 5
years may have relevance to practice in India.
In Australia, as is the case in India, doctors
prescribe and pharmacists dispense - processes that are
separated both intellectually and physically. The doctor
rarely seeks the advice of the pharmacist in choosing a
drug for his patient, and the pharmacist dispenses and
interacts with the patient in isolation from the doctor.
Without knowing what the other has told the patient,
both the prescriber and pharmacist hope that the patient
will abide by the instructions that they have been given.
Hopefully the advice of one will not contradict that of
the other, but of course this can and does happen, and if
it does, can we assume that the patient will be able to
decide on which advice they should follow?
HMR Programme in Australia
In 2000, the Australian government believing the
medication use in the elderly could be better managed,
offered to pay doctors and pharmacists to liaise together
and conduct medication reviews for patients living at
home and for those being cared for in aged care facilities
(hostels and nursing homes). The service was primarily
targeted at the elderly because as a group they take more
drugs than their younger counterparts and are known to
be risk of the adverse effects of many of the drugs they
consume. A study that was published over 20 years ago
summarises as well as any paper published since, the
problems faced by both prescriber and elderly patient
when using drugs to treat illness.1
The Australian government had been encouraged
to make funds available for pharmacist conducted
medication review by the results of study by South
Australian pharmacists and doctors that took place over
12 months beginning in March 1999 when 1,000 patients
in both city and rural areas had medication reviews
conducted by pharmacists with the approval of their
doctors. Sixty-three pharmacists and 129 doctors
participated in the study. A large number (2764) of
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
problems were identified and 37% related to medicine
selection, whilst in 17.5% a further laboratory or related
test was assessed as being needed. Twenty percent of
problems related to patient knowledge. Nine hundred and
seventy eight problems were acted upon and in 81% of
these the doctor reported that the problem had been
resolved, well managed or improving.
HMR – a case study
To illustrate how a home medication review can
assist a patient’s management, I describe a lady I saw in
July 2005 when I was asked by her doctor to conduct a
home medication review. At that time, Mrs LF was 73
years old and suffered from deteriorating memory, poor
eyesight, ischaemic heart disease, depression and
osteoporosis. She had undergone recent surgery for
cancer of the bowel and at the time of my visit, took
seven regular medications with two drugs on a ‘when
required’ basis (table 1). She lived at home with her
husband who had retired from work because of ill health.
Her doctor requested a medication review on the basis
of Mrs LF’s declining memory and his concerns about
her compliance with her medications.
Table 1: Mrs LF’s Medication Review
Aspirin
100mg morning
Calcium carbonate
1200mg night
Dothiepin
75mg night
Atorvastatin
Metoprolol
80mg night
25mg twice a day
Indapamide
2.5mg morning
Enalapril
20mg morning
Temazepam
10mg night when required
Paracetamol
1,000mg 4 times a day when
required
In my discussions with Mrs LF I found her to be an
anxious lady who felt very tired and described dizziness
on standing. The diagnosis of bowel cancer had left her
concerned of a reoccurrence of her disease. I asked her
to bring all her medications to the kitchen table and soon
discovered that she was, by mistake, taking 5mg of the
diuretic indapamide (she had two packets of different
brands of the drug each of 2.5mg strength).
As pharmacists we know that many people achieve
adequate control of their blood pressure with doses of
73
indapamide lower than 2.5mg and whilst some do need
this dose for adequate control, few if any should receive
5mg a day as the risk of dangerous electrolyte and
metabolic disturbances and postural drops in blood
pressure is unacceptably high. I immediately asked her
to stop taking the drug, and delivered a note to her
doctor’s office within the hour. I also visited her
community pharmacist and explained the situation.
In the comfortable and familiar environment of Mrs
LF’s home I was able to provide reassurance that her
overall treatment was appropriate and that she was no
different to many other Australians in terms of her
illnesses and the medicines that she needed to take. I
was able to ascertain that she was not taking her
medicines as both her doctor and community pharmacist
had advised. Unfortunately she had become confused
over two different brands of the same drug and was taking
a potentially dangerous dose of a drug that may have
explained her dizziness and lethargy.
This lady’s doctor is a very conscientious
practitioner and I have found, in conducting medication
reviews for many of his patients, that most are well
informed about their medical conditions and medications.
Likewise the town’s community pharmacist is also a very
competent pharmacist who spends a lot of time talking
with his patients. However even with such good people
caring for Mrs LF she was at risk of avoidable illness
because, despite the best efforts of all concerned, she
had become confused about her medicines. It took a home
visit focussed specifically on her medication use to
resolve a misunderstanding that had the potential to
cause serious morbidity.
The benefit of pharmacist conducted medication
reviews has been well stated by Dr Joe Neary, Chair of
Clinical Network, Royal College of General Practitioners
in the UK when he said in a published letter in response
to a paper by Zermansky “The challenge of adequately
reviewing complex drug regimens cannot be
accommodated in the 7-10 minute intervals that define
general practitioners’ clinical practice in the United
Kingdom. The role of the pharmacist is also degraded,
being reduced to that of a passive dispenser, who might
occasionally issue warnings in the case of overlooked
interactions. Zermansky et al show how different things
could be with a structured, shared approach, the
respective talents of doctor and pharmacist could be better
harnessed, to the benefit of both patient care and the
professional satisfaction of both parties. It would be
interesting to see the results of longer term follow up of
these cohorts of patients to see if differences in outcome
emerged. The true long term impact on the workload and
74
job satisfaction of doctors and pharmacists could also
be assessed.” 3,4
So far I have restricted my comments to pharmacist
conducted medication review for patients living in their
own homes. At 30 June 2004 Australia had available
156,580 places for elderly people who could no longer
manage to live at home. Most of these places are located
in nursing homes and hostels where, depending on the
level of disability, care ranges from a low level of support
for those with a degree of independence to high-level 24
hours a day care for those with major disabilities/illnesses.
The Australian government funds a similar system of
pharmacist conducted medication review for people who
live in these nursing homes.
The government has demanded that, for an
Australian pharmacist to be paid for conducting a
medication review, he or she must be accredited to
conduct medication reviews. Accreditation can be
obtained from two organisations: The Australian
Association of Consultant Pharmacy (AACP) and the
Society of Hospital Pharmacists of Australia (SHPA). As
at 20 June 2005 there were 1617 pharmacists accredited
by the AACP to conduct and be paid for medication
review. The SHPA program is not well supported by the
profession and only a very small number of pharmacists
have sought accreditation via their program.
Not all has happened, as it should have in Australia
in establishing medication review services. The initial
decision not to pay doctors to be involved in medication
review in aged care homes compared with reviews
conducted for patients in their own homes where the
doctor is paid to participate was a mistake that has made
it difficult for many pharmacists to obtain medical support
for aged care home review services.1 Some pharmacists
and pharmacy representative organisations have failed
to appreciate the level of clinical knowledge that is
required by a pharmacist to be successful in conducting
medication reviews that are useful for both doctor and
patient. Australian academic and former hospital
pharmacist Jeff Hughes writing in the SHPA’s Journal of
Pharmacy Practice and Research made a case for hospitals
to provide training places for pharmacists wanting to
undertake medication review work. Hughes said,
“teaching hospitals provide pharmacists with interaction
with patients, carers (e.g., relatives), doctors, nurses and
other health professionals. These hospitals provide an
environment where pharmacists can hone their skills in
the establishment of patient profiles; identification of
1
This changed in 2005 with further changes that will
make it easier for pharmacists and doctors to work
together in residential care planned for 2006
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
drug-related problems and the development of patient
care plans. They learn to prioritise, develop a sound
understanding of the clinical decision-making process,
and have the opportunity to provide input into that
process through one-on-one interaction with clinicians
and through participation in ward rounds or meetings.
They develop an appreciation that health professionals
looking at the same clinical problem may arrive at different
therapeutic decisions and that as a patient advocate, if
an intervention is warranted, they develop the confidence
and capacity to advance an argument to support their
point of view. Importantly, the ability to articulate the
problem, the relevant clinical findings and their evidencebased recommendations in the manner routinely used by
clinicians become part of their professional repertoire. A
fundamental aspect of the training and experience
required to function as a consultant pharmacist is the
development of an understanding that even the best
therapeutic decisions may not achieve the desired
therapeutic outcome.”5
Australian pharmacy undergraduate courses cannot
be expected to train graduates to a level of competence
required to conduct effective medication reviews and it
is my view that the accreditation process adopted by the
Australian Association of Consultant Pharmacy lacks the
necessary rigour to ensure that those accredited by the
AACP can perform the task of medication review
competently. However, as is the case in any new initiative,
things will never go as well as one might hope and
importantly those involved in Australia are seeking to
make the processes stronger so one can be reasonably
optimistic that this new professional service, that is
cognitive in nature rather than supply orientated, as is
the case with traditional dispensing, will grow in
importance as an important service offered by Australian
pharmacists working more closely with doctors.
Why run a medication review seminar in Mysore?
The answer is that, in my opinion, the model has relevance
to practice in India because hospital based clinical
pharmacy practice is established in India and the skills
developed by hospital practitioners can be taken out into
the community either directly by pharmacists who have
trained in hospitals and then transfer to community
practice or by the efforts of hospital trained pharmacists
who provide postgraduate education opportunities for
their community colleagues.
There are 2 intellectual levels of activity involved in
a medication review and I shall focus on the home setting
because it is here that I think the relevance to India lies.
When a pharmacist has the opportunity to visit a patient
in the familiar surroundings of the latter’s home many
questions that hitherto no one has been able to
confidently answer can be answered and the first and
most important is “is this person taking the medications
Table 2: Medication Review - practical questions
Is this person taking the medications his or
her doctor wants them to be taking?
The question the doctor most often wants answered
Are other medications that neither pharmacist
nor doctor knew about being taken?
Especially relevant in India where there are competing forms of
medicine e.g., Ayurveda, Siddha, Unani
Is the patient taking medicines that have
expired or are otherwise unsuitable for
administration?
Always ask patients to have their medicines ready for inspection
when arranging a visit to their home
Does the patient know how to use devices
such as inhalers, blood glucose monitors,
eye drops etc
The pharmacist can ask for a demonstration and assist in
training “on the spot”
What is the patient’s attitude towards their
medications?
Something that the patient may not be prepared to tell their
doctor but they may tell the pharmacist in the comfort of
their own home. Extremely useful information
What family supports are in place?
Spouse and children? – and can they be encouraged to
offer more support if necessary?
Are there risks associated with the structure
of the houses and furnishings etc?
Poor lighting, stairs, obstacles e.g., furnishings etc
that may increase the risk of falling? Many may not be
amenable to change but knowing that they exist is useful
information.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
75
his or her doctor wants them to be taking”? Table 2 lists
this question and a number of other very practical
questions that a competent and well-conducted
medication review can answer:
The list in Table 2 can be expanded but those
questions that I have listed are of a practical nature and
answers that might result in changes of environment,
attitudes and level of support on offer can be very helpful.
The second level of questioning is more a series
questions that the pharmacist might ask himself and it
relates to the clinical appropriateness of current drug
therapy. It is at this level of intellectual reasoning where
the pharmacist’s clinical acumen is very important. It is
easy to cause offence to the doctor by being seen to
question his prescribing decision-making and the
pharmacist needs to be very sure of his knowledge and
equally importantly, know how to convey that knowledge
without causing offence. I divide this second level of
expertise into two, 1) being able to form a clinically sound
opinion, and 2), being able to effectively convey that
opinion to the person who has the primary responsibility
for decision making on behalf of the patient i.e., the
doctor, in a way that will maximise the chance of that
doctor taking your advice seriously. As a medication
review pharmacist you may be correct in your opinion
that a dose of a drug or indeed the drug itself is not
suited to a patient, but if you are unable to convey that
opinion in an effective, polite and non-judgmental manner
then it is unlikely to be acted upon.
HMR – what can be done in India?
One can easily adapt the model to local
circumstances. For example in India, one could target
patients on medications for tuberculosis and simply ask
the questions 1) do they know what medications they
are meant to be taking and 2) are they taking them as
they should? Depending on the answers assistance may
be possible. Insufficient money to buy the drugs may
not easily be addressed but at least knowing that this is
the issue and knowing what the issues are for a large
number of patients who are not compliant is a very useful
database for planning initiatives that seek to help
patients.
Medication review takes the pharmacist out of the
shop into the community. How can it be funded? I suspect
that it will require Indian pharmacists with vision to
demonstrate, with pilot studies, that medication review
programs can work. A combination of pharmacist initiative
76
and political astuteness to source funding support may
see medication review become, as has been the case in
Australia, an exciting opportunity for Indian pharmacists
to contribute further to the health-care of their
communities.
Acknowledgements
Without the support of the following we would not
have been able to make a start in introducing the concept
of home medication review in India.
His Holiness Sri Sri Deshikendra Mahaswamiji for
his steadfast support for pharmacy practice within the
organisations of the JSS Mahavidyapeetha.
JSS Mahavidyapeetha Executive Secretary Sri
B.N.Betkerur for honouring the Mysore Home Medication
Review seminar by his presence and opening address on
November 30th 2005.
Professor B.G.Nagavi, Dr G Parthasarathi, Dr Adepu
Ramesh, and Mr Madhan Ramesh and their staff and
students at JSS College of Pharmacy Mysore.
My Australian pharmacist colleagues Mr Grant
Kardachi, Mrs Jody Kardachi and Professor Frank May,
who participated in the seminar
Professor B Suresh and his staff and students at
JSS College of Pharmacy, Ootacamund who continue to
support all matters associated with pharmacy practice.
The attendance of many of the Ooty students at the
seminar was appreciated.
References
1.
Medication for the Elderly. A report of the Royal
College of Physicians. J Royal College of Physicians.
1984;18:7-17.
2.
Gilbert AL, Roughead EE, Beilby J, Mott K, Barratt
JD. Collaborative medication management services:
improving patient care. Med J Aust 2002; 177:189192.
3.
Zermansky AG, Petty DR, Raynor D, Freemantle N,
Vail A, Lowe CJ. Randomised controlled trial of
medication review by a pharmacist of elderly patients
receiving repeat prescriptions in general practice.
BMJ 2001; 323:1340-3.
4.
Neary J. Clinical medication review by pharmacists
would improve care. BMJ 2002; 324:548.
5.
Hughes J. Hospital pharmacy–training ground of
consultant pharmacists for the future. J Pharm Pract
Res 2005; 3:175-6.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Review Article
Pharmacology
New Generation Antipsychotics:
A Review
Devender Pathak, Rajiv Dahiya, Kamla Pathak
and Sunita Dahiya
D. Pathak
Rajiv Dahiya
Received on 23.07.04
Rajiv Academy for Pharmacy, Mathura-281001, UP
Modified on 21.03.05
Kamla Pathak
Sunita Dahiya
Accepted on 01.09.05
ABSTRACT
Advances in understanding the pathology underlying psychotic disorders and the way in which antipsychotic
medication treat these disorders have altered clinicians’ pharmacologic approach to patients. This guided scientists to
develop new generation antipsychotic agents (NGA) exhibiting productive clinical outcomes. Extensive efforts in this
area provided several potent ‘atypical’ antipsychotic agents with greater efficacy against negative symptoms and almost
total avoidance of extrapyramidal side effects (EPS) which seemed to be major cause of non-compliance with traditional
agents. This review highlights benefits and risk factors associated with novel antipsychotic agents and outlines their
future scope.
INTRODUCTION
Among psychotic disorders, schizophrenia 1,2 i s a
chronic idiopathic psychiatric condition which causes
major public health problem. It has been estimated that
approximately 0.025% to 0.05% of the total world
population is treated for schizophrenia per year. Clinical
features of this mental illness can be divided into four
major types of symptoms
· Positive symptoms – hallucination, delusion,
agitation and disorganized
thinking
· Negative symptoms – introversion, apathy, personal neglect and catatonia
· Cognitive symptoms – poor memory, attention deficit, executive dysfunction
· Affective symptoms – depression, elation and
suicidal ideation
Most vital targets for the antipsychotic drugs are
dopamine and serotonin receptors,3,4 the blockade of
which contribute to their clinical efficacy. Dopamine (D1
– D7) receptors are associated with sleep, anxiety,
thermoregulation, neuronal excitation, locomotion and
CSF secretion whereas serotonin (5HT1 – 5HT7) receptors
are concerned with emotion, stereotypic behaviour, mood,
motor, endocrine and autonomic control. Akathisia,
dystonia, parkinsonism and tardive dyskinesia produced
by the traditional drugs are sources of great discomfort
to patients and lead them to refuse treatment. This results
in development of several new generation agents 5 viz.
clozapine, olanzapine, quetiapine, zotepine, risperidone,
ziprasidone, amisulpride, sertindole, iloperidone and
aripiprazole with improved side effect profiles. Besides
schizophrenia, these atypical agents are also
recommended for management of aggression, mania and
depression.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
NEW GENERATION ANTIPSYCHOTICS VS
TRADITIONAL AGENTS
Traditional agents are effective in only about 70 %
of schizophrenic patients whereas the remaining 30%
which are classed as ‘treatment-resistant’, represent a
major therapeutic problem. New Generation
Antipsychotics (NGA) are better choice for their treatment
and are considered atypical6-8 in four respects
· Decreased incidence of EPS
· Diminised hyperprolactinemia
· Demonstrable benefit in relieving negative symptoms
· Superior efficacy in refractory schizophrenia
In comparison to traditional agents which mainly
relieve positive symptoms, new generation antipsychotics
target both positive and negative symptoms and provided
significant benefits over them. Recently developed NGA
have chemical structures and pharmacologic profiles
dissimilar to those of older agents. They differ from each
other in higher affinity for serotonin receptors than for
dopamine receptors.9,10
MECHANISM OF ACTION AND ADVERSE EFFECTS
Like traditional antipsychotics, NGA alleviate
positive symptoms of schizophrenia by blocking
dopamine (DA) mainly at D2 receptors in the brain.
Additionally, new generation agents appear to have
blocking action on serotonin (5HT2A) receptors which is
concerned with relief of negative symptoms. NGA have
a improved side effect profile in comparison to older
agents. In particular, the incidence of EPS which is major
cause of intolerability and non-compliance with older
agents, is reduced to much lower level. Adverse effects 11,12
such as sedation, weight gain, somnolence and orthostatic
hypotension which are commonly associated with NGA
arise from the blockade of histaminic (H1 , H2 ) and
adrenergic (a 1, a 2 ) receptors. Besides antipsychotic
77
action, the ability of binding to D2 receptors is also
concerned with production of EPS. NGA are loosely
bound to D2 receptors in cell membrane and thereby,
concerned with negligible EPS whereas older agents are
tightly bound to D2 receptors and are associated with
increased incidence of EPS. Major adverse effects
concerned with NGA are concluded in Table I along with
their ability for different receptors in brain.
Table I: Side effect profile of new generation antipsychotic agents
Drug
Clozapine
Olanzapine
Quetiapine
Zotepine
Risperidone
Ziprasidone
Aripiprazole
Amisulpride
Sertindole
78
Affinity for receptors
High affinity for
serotonin 5HT2A, D1,D4
(in comparison to D2)
and muscarinic
receptors
Antagonism of
5HT2A/2C , D1-4,
muscarinic M1-4,
histaminic H1, aadrenergic receptors
Weak affinity for
GABAA and ßadrenergic receptors
Antagonism of serotonin
5HT1A, 5HT2, dopamine
D1 ,D2, Histamine H1,
adrenergic a 1, a 2receptors
High affinity for D1,
D2 – like receptors,
serotonin 5HT2A/2C,
5HT6/7 receptors,
inhibition of
reuptake of
noradrenaline
High affinity for D2,
5HT2, a1/a 2 receptors,
moderate affinity for
5HT1C/1D/1A receptors,
weak affinity for D1
receptors
Antagonism of D2/D3,
5HT2A/1D receptors,
agonism of serotonin
5HT1A receptors,
inhibition of reuptake of
noradrenaline
Partial agonism of
5HT1A and D2 receptors,
antagonism of 5HT2
receptors
High affinity for
dopamine D2 and D3
receptors
Antagonism of D2, 5HT2
and adrenergic a 1receptors
Common side effects
Sedation,
agranulocytosis, weight
gain and sialorrhoea
Other features
Associated with low risk
of relapse than traditional
agents
Drowsiness and
excessive appetite
with weight gain
Associated with low risk
of tardive dyskinesia than
clozapine
Greater tolerability and
less sexual dysfunction
Greater
tolerability and
than risperidone
Somnolence, orthostatic
hypotension
less sexual dysfunction
than risperidone
Significantly lower
incidence of substantial
EPS in comparison to
risperidone and
equivalent in efficacy
Weight gain,
somnolence, dizziness,
drowsiness and
headache
Less expensive than any
other atypical
antipsychotics
Headache, sedation,
anxiety and nausea
At least as effective as
haloperidol against
positive symptoms and
more effective in
relieving negative
symptoms
Less likely to cause
weight gain than
clozapine and
olanzapine
Somnolence, nausea
and orthostatic
hypotension
Headache, anxiety,
insomnia
Endocrine effects,
insomnia, anxiety and
agitation
Rhinitis, reduced
ejaculatory volume,
nasal congestion
Most likely to minimize
occurrence of tardive
dyskinesia than other
atypical agents
A dose dependent effect
on EPS like olanzapine
Essentially no EPS at any
dose but may cause
prolongation of QTc
interval
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
CHEMICAL CLASSIFICATION OF NEW GENERATION
ANTIPSYCHOTICS
New generation antipsychotic agents can be
classified as follows
1)
Benzodiazepine-type compounds
a. Dibenzodiazepine13-17 e.g. Clozapine
b. Thienobenzodiazepine18-21 e.g. Olanzapine
c. Dibenzothiazepine22-26 e.g. Quetiapine
d. Dibenzethiepine27-29 e.g. Zotepine
H
N
H
N
3) Quinolinone (Dihydrocarbostyril)37-41
Aripiprazole
CH3
S
Cl
Risperidone bears the structural feature of a hybrid
molecule between a butyrophenone antipsychotic and a
trazolone-like antidepressant and is especially effective
in individuals experiencing first episode of schizophrenia
where negative symptoms predominate. Ziprasidone has
11-fold affinity for the 5-HT2A receptor than for D2 receptor
but the drug’s efficacy in schizophrenia and
schizophrenia affective disorders is mediated through a
combined antagonistic effect. Like olanzapine, risperidone
and ziprasidone are associated with EPS in a dosedependent manner.
e.g.
N
N
N
N
Cl
N
H
CH3
Olanzapine
CH3
Clozapine
O
Cl
N
N
N
O
N
S
S
Aripiprazole
Cl
CH3
N
N
N
O
O
CH3
N
OH
Zotepine
Quetiapine
Clozapine is the prototype of new generation
antipsychotics which is effective against treatmentresistant schizophrenia. Both olanzapine and quetiapine
resemble clozapine in possessing tricyclic systems with
greater electron densities and are indicated for mood
disorders in addition to illnesses such as psychosis.
Olanzapine is especially effective in the treatment of
schizophrenia, schizoaffective and schizophreniform
disorders. Moreover, broad spectrum antipsychotic
‘zotepine’ shows efficacy for depressive symptoms of
refractory schizophrenia by blocking nor-epinephrine
uptake as well as by serotonin-dopamine antagonism.
2)
Benzisoazole-type compounds
a. Benzisoxazoles 30-33 e.g. Risperidone
b. Benzisothiazole34-36 e.g. Ziprasidone
O
N
4) Phenylindole derivative 42-44 e.g. Sertindole
F
N
Cl
N
N
Sertindole
H
N
O
CH 3
Risperidone
H
N
NH
Sertindole acts by blocking dopamine and serotonin
receptors in brain and is well accepted for paranoid,
hebephrenic and residual schizophrenia.
F
N
O
O
N
5) Benzamides 45-47 e.g. Amisulpride
O
N
Aripiprazole is a novel antipsychotic with a unique
receptor binding profile which differs from other atypical
antipsychotics in having ability to stimulate D2 receptors
rather than to block them. The agonism at dopamine D2,
serotonin 5HT 1A receptors and antagonism at 5HT 2 A
receptor in combination is responsible for its efficacy in
acute schizophrenic relapse and mania.
N
OCH3
CH3
Cl
H 3C
N
Ziprasidone
S
O
N
N
S
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
O
NH2
Amisulpride
79
Atypical profile of amisulpride depends on blockade
of the mesolimbic dopaminergic tracts rather than
nigrostriatal dopaminergic transmission and on a
preferential blockade of dopamine D3 receptors in the
limbic system. It is indicated for treatment of both acute
and chronic schizophrenic disorders.
DATA ON COMMERCIALLY AVAILABLE NEW
GENERATION ANTIPSYCHOTIC AGENTS
Information about dose strengths, dosage forms,
brand name and manufacture of novel antipsychotic
agents is summarized in Table II.
Table II: Marketed New Generation Antipsychotics
Generic Name
Clozapine
Brand Name
Clozaril
Dosage form and strength
Tablets
25 mg, 100 mg
Manufacturer
Novartis
Olanzapine
Olanzapine
Zyprexa
Lilly
Zyprexa
IntraMuscular
Tablets
2.5 mg, 5.0 mg, 7.5 mg 10 mg,
15 mg
Disintegrating Tablets
5 mg, 10 mg
IM
IM Injection
Injection
10
10 mg
mg
Quetiapine
Seroquel
(Quetiapine
fumarate)
Tablets
25 mg, 100 mg
200 mg, 300 mg
AstraZeneca
Zotepine
Zoleptil 
Tablets
25 mg, 50 mg, 100 mg
Orion
Pharmaceuticals
Risperidone
Risperdal
Tablets
0.25 mg, 0.5 mg, 1 mg
2 mg, 3 mg, 4 mg
Oral Solution
1 mg/ml
Disintegrating
Tablets
0.5 mg, 1 mg, 2 mg
Janssen
Zyprexa Zydis
Risperdal
M-Tab
80
Ziprasidone
Geodon
(Ziprasidone HCl)
Capsules
Capsules
20
mg,40
40mg
mg
20 mg,
60 mg, 80 mg Intramuscular
60 mg, 80 mg
Geodon for Injection Injection 20 mg/ml
GeodonÒ for Injection Intramuscular Injection
(Ziprasidone
(Ziprasidone mesylate) 20 mg/ml
mesylate)
Pfizer
Aripiprazole
Abilify
Bristol-Myers Squibb
Company
Amisulpride
Solian
Tablets
10 mg, 15 mg
20 mg, 30 mg
Tablets
100 mg, 200 mg, 400mg
Liquid 2.5 mg/ml
Sertindole
Serlect
Tablets
4 mg, 8 mg, 12 mg
16 mg, 20 mg, 24 mg
Abbott Laboratories
Lorex Synthelabo
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
ANTIPSYCHOTICS IN DEVELOPMENT PHASE
Besides targeting dopaminergic system and serotonin
signaling pathway, other neurotransmitters that may be
involved in the pathogenesis of schizophrenia include
neurokinin (NK) receptors, muscarinic receptors, glutamate
receptors, cannabinoid, kainate and sigma receptors. Two
novel antipsychotics 48-52 currently in development phase
are iloperidone (ILO-522) and bifeprunox (DU 127090).
Iloperidone which is a benzisoxazole derivative, acts by
equivalent blockade of both dopamine D2 a n d D3
receptors as well as serotonin 5HT2A and 5HT6 receptors
whereas bifeprunox is a partial dopamine D2 and
serotonin 5HT1A receptor agonist. Both of these novel
antipsychotics are presently in phase III clinical trials.
Other vital antipsychotics currently in clinical trials 53-56
are summarized in Table III.
Table III: Novel antipsychotics in development phase
Compound
DTA 201A
ORG 5222
(Asenapine)
Osanetant
(SR-142801)
Talnetant (SB223412)
Company
Knoll (BASF)
Pfizer/Organan
Method of Action
D 3 antagonist
5HT2/D2 antagonist
FDA trials phase
Phase III
Phase III
Sanofi-Aventis
Synthelabo
GlaxoSmithKline
Phase II
Palindore (DAB-452)
Wyeth
LY 354740
Eli-Lilly
SB-271046
GlaxoSmithKline
Lamictal
–
Mem 3454
Memory
Pharmaceuticals
SR141716
(Rimonabant)
Sanofi-Synthelabo
NK3 receptor
antagonist
NK3 receptor
antagonist
D 2/D 3 partial receptor
agonist
Metabotropic
glutamate agonist
5HT6 receptor
antagonist
Sodium channel
inhibitor
Partial agonist of
neuronal nicotinic a 7
receptor
Cannabinoid receptor
(CB 1) antagonist
EMR-62218
Merck
Pharmaceuticals
5HT2A receptor
antagonist with no
dopamine blockade
Phase I
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Phase II
Phase II
Phase II
Phase I
Phase III
Phase I
Phase III
81
CONCLUSION
There have been tremendous advances in the
development of the atypical antipsychotics since last
decade. New generation antipsychotic drugs have shown
clear improvement and protection against negative,
affective and cognitive symptoms. However, some of
these agents cause adverse effects such as weight gain
and sexual dysfunction, yet they have a more favorable
side effect profile when compared with traditional drugs.
Due to more of the benefits associated with NGA, they
are globally accepted and occupying a prominent position
in antipsychotic prescriptions now a days. Moreover,
NK3 antagonists are emerging as an entirely new class of
antipsychotic agents which would have a future role in
the treatment of schizophrenia and related disorders.
Nevertheless, the development of novel agents has
created hope for patients with schizophrenia and their
families helping them to obtain a more positive outlook
on life and future.
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83
Review Article
Pharmacology
Pharmacogenomics: The Search for the Individualized Therapy
C.S.Magduma , V.S.Velingkarb and Meenu K.Guptaa
a
Appasaheb Birnale College of Pharmacy, Sangli
Prin.K.M. Kundnani College of Pharmacy, Mumbai
b
Received on 05.08.2004
Modified on 29.04.2005
Accepted on 15.10.2005
ABSTRACT
Pharmacogenomics leads to a better understanding of interaction of drugs and organisms. In other words it is a
science that examines the inherited variations in genes that dictate drug response and explores the ways these variations
can be used to predict whether a patient will have a good, bad or no response at all. Pharmacogenomics hold the promise
that drug might one day be tailor made for individuals. The whole purpose of the study of genetic variations in drug
responses is to identify patients who may benefit from a particular medicine and avoid wasteful (or dangerous) prescription
to others.
INTRODUCTION
“Pharmacogenomics is the study of how an
individual’s genetic inheritance effects the body response
to drugs”. Pharmacogenetics describes the interaction
between drug and individual’s characteristics (which may
be related to inborn traits to a larger or lesser extent).
Thus, although both pharmacogenetics and
pharmacogenomics refer to the evaluation of drug effects
using nucleic acid technology, the directionalities of their
approaches are distinctly different: pharmacogenetics
represent the study of differences among a number of
individuals with regard to clinical response to a particular
drug, whereas pharmacogenomics represent the study of
differences among a number of compounds with regard
to gene expression response in a single (normative)
genome/ expressome. Accordingly, the fields of intended
use are distinct: the former will help in the clinical setting
to find the best medicine for a patient, the later in the
setting of pharmaceutical research and development to
find the best drug candidate from a given series of
compounds under evaluation.
PROMISES OF PHARMACOGENOMICS
Ø
Pharmacogenomics hold the promise that drug might
one day be tailor made for individuals 1 that is to say
as the tailor stitches the dress of an individual by
taking the size in the same way the dose of drug will
be decided by the individual makeup.
Ø The promise of Pharmacogenomics is that both the
choice of the drug and its dose will be determined
by the individual genetic make up leading to the
personalized, more efficacious and less harmful drug
therapy.
NECESSITY OF PHARMACOGENOMICS
Genetic variation holds the key of individuality.
Therefore, if the variation among individuals can be
established and documented, answer to differential drug
response of individuals can be worked out. Inter84
individual variability in drug response and ADRs
(Adverse Drug Reactions) are major public health
problems. ADRs are the fourth leading cause of death
after heart disease, cancer and stroke. 1998 reports of
USA suggest that 6.7% of the hospitalization was due to
under dosing, overdosing and prescription of unwanted
drugs. Among this 0.32% was fatal which led to 1,00,000
deaths in that year in USA. Just imagine what will be
the figure in whole world in that year and other years?
Are these deaths affordable by us? And 1998 report
reflects that United States had earned US $30 billion to
US $150 million a year2 because of ADRs. What a big
figure? The ADRs are the major cause on non-compliance
and failure of treatment, particularly for chronic
pathologies. For instance, the daily doses required to
treat patients vary by 20-fold for the warfarin, by 40-fold
for the antihypertensive drug propranolol and by 60-fold
for L-dopa for Parkinson’s disease.3 Other drugs have
clinical utility in a subset of patients with given
pathology, e.g., antipsychotic that are ineffective in 30%
of schizophrenics,4 suggesting the fact that such drugs
are only effective in patients with specific disease
etiologies. Many of these deaths could be avoided if the
physician had prior knowledge of patient’s genetic profile
of drug metabolizing enzymes and receptors, which
determine drug responses. In the near future, the present
prescription model of “one dose for all” will be replaced
by “individualized prescription-the right drug in right
dose for right person”. The whole purpose of the study
of genetic variations in drug responses is to identify
patients who may benefit from a particular medicine and
avoid wasteful (or dangerous) prescription to others.5
MOLECULAR DIAGNOSTICS AS A PREDICTIVE
TOOL: GENETICS OF DRUG EFFICACY AND
TOXICITY
Effects such as:
Genetics of drug response
Medications are given to patients to achieve a
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
defined therapeutic effect, yet it is recognized that few
drugs are universally effective. A recent review in the
journal suggested that efficacy rate for drug therapy of
most diseases is in the 25-80% range.6 Thus, even our
most effective therapies fail to work in 20% or more of
patients treated. At present, this lack of efficacy is
discovered by trial and error for most medications. More
troubling are those situations where the desired
therapeutic benefit is difficult or impossible to measure
in an individual (e.g. survival prolongation), or where
assessment of efficacy is made well into therapy, at a
point when treatment failure reduces the likelihood of
other therapies being successful (e.g. cancer
chemotherapy). Thus, the ability to predict an efficacious
response based on a genetic test, performed before the
initiation of therapy, has the potential to be of great
clinical value. Additionally, pharmacogenomics is likely
to significantly enhance our understanding of racial or
ethnic differences in drug response. Table 1 summarizes
several published examples of drug responses that are
associated with genetic variants, with additional
information on selected cases highlighted below.
Schizophrenia is a disease treated largely by
empirical approaches, and thus is one that could benefit
from genetically guided drug therapy. This approach was
taken in a recent study of schizophrenics treated with
clozapine, in which 19 polymorphisms in ten genes were
investigated for their relation to treatment outcome.8 A
combination of six polymorphisms provided a sensitivity
of 96% and 76% positive predictive value for treatment
success with clozapine. Additional studies are needed to
validate this model and extend the panel of gene
polymorphisms that can be used to guide clozapine
therapy. Clearly, the development of a diagnostic “chip”
that screens for polymorphisms that accurately predicted
response and (or) toxicity with neuroleptics would be of
great clinical value in the selection of treatment for this
complex disease.
Table 1: Examples of drug response associated with genetic variability
Drug response
Blood pressure lowering
Bronchodilation
Lipid lowering / decreased
cardiovascular events
Antiplatelet effects
Antipsychotic effects
Analgesia
Clinical improvement:
parkinson’s
Clinical improvement:
Alzheimer’s
Drug(s)/drug class(es)
Gene
ACE inhibitors, β-blockers,
thiazide diuretics, angiotensin
receptor blockers
β 2agonists,leukotriene modifiers
statins, fibrates
ACE, ADRB1, GNAS1, GNB3,
AGTR1, ADD1
Aspirin, glycoprotein IIb/IIIa
inhibitors
Typical and atypical
antipsychotics
Codeine, oxycodone,
hydrocodone, tramadol
Levodopa
ITGB3
Tacrine
APOE
Genetics of acquired drug resistance
There are some examples of acquired genetic
variation or genetic variants of pathogens that are used
clinically to determine appropriate drug therapy. The first
example is the anti-HER2 monoclonal antibody,
trastuzumab (Herceptin), a drug used in the treatment of
metastatic breast cancer. Early clinical trials indicated that
the drug was only effective in women overexpressing
the HER2 protein.9 Thus, subsequent premarketing studies
included only those women who had overexpression of
HER2 protein and current labeling of trastuzumab
stipulates that patients must be tested for HER2
overexpression before receiving the drug. Thus, although
this is not a direct genetic test for efficacy, it is presumed
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
ADRB2, ALOX5
APOE, CETP, TCF20
ADRD2, ADRD3, ADRD4,
5HT2A, 5HT6, 5HTT and others
CYP2D6
ADRD5, Parkin
that the variation in HER2 expression across breast cancer
patients is largely based on genetic differences in the
breast tumor. Genetic screening of HIV for prediction of
resistance is recommended by experts, and is being
increasingly used in the clinical setting.10
Genetics of adverse drug outcome
Essentially, all drugs that produce an efficacious
response also have the potential to produce adverse
effects. In some cases, the adverse effects are minor, yet
common, whereas other adverse effects can be serious
or even life threatening, but tend to be rare.
One of the best-studied examples in
pharmacogenetics is the genetic polymorphism of
85
thiopurine methyltransferase (TMPT). TMPT catalyses
the S-methylation of the thiopurines azathioprine,
mercaptopurine and thioguanine. 11 These agents are
commonly used in the treatment of leukemia, rheumatic
diseases, inflammatory bowel diseases, and solid organ
transplantation. Thiopurines are inactive prodrugs that
require metabolic conversion to thioguanine nucleotides
(TGN) to exert their effects, or they are inactivated via
either xanthine oxidase or TPMT. In hematopoietic
tissues, the level of xanthine oxidase is negligible, leaving
TPMT as the principal inactivation pathway, and patients
who inherit TPMT deficiency accumulate excessive TGN
concentrations with standard doses of these medications.
TPMT activity exhibits genetic polymorphism in all large
populations studied to date: ~90% of individuals inherit
high enzyme activity, 10% have immediate activity
because of heterozygosity, and 0.3% has low or no
detectable enzyme activity because they inherit two nonfunctional TPMT alleles. Numerous studies have shown
that TPMT-deficient patients are at a high risk for severe,
and sometimes fatal, hematological toxicity and patients
who are TPMT heterozygotes have an intermediate risk
of hematological toxicity. Patients who inherit two mutant
alleles should be started on 6-10% of the standard dose
of thiopurine. Treatment of heterozygous patients can
usually be initiated with full doses, but there is a
significantly higher probability these patients will require
a dose reduction to avoid toxicity. TPMT deficiency has
also been associated with a higher risk of irradiation
induced brain tumors in patients treated concomitantly
with thiopurines and radiation therapy. The molecular
basis for polymorphic TPMT activity has now been
defined, with three mutant alleles (TPMT*2, TPMT*3A,
or TPMT*3C) accounting for TPMT deficiency in >95%
of patients, and TPMT genotyping is now available as a
Clinical Laboratory improvement Act (CLIA)-certified
molecular diagnostic from reference laboratories (e.g.
Prometheus, San Diego, CA). A simple molecular method
for determining the molecular haplotype of the human
TPMT gene has been recently developed, providing even
greater accuracy of this molecular diagnostic.
Abacavir is a potent reverse-transcriptase inhibitor
used in the treatment of HIV infection and AIDS.
Approximately 5% of patients experience a
hypersensitivity reaction to Abacavir that can be severe
and rarely fatal. A recent study of 200 patients exposed
to Abacavir evaluated the association between major
histocompatibilty complex (MHC) alleles and abacavir
hypersensitivity. The presence of HLA-B*5701, HLA-DR7
and HLA-DQ3 had a 100% positive predictive value for
abacavir hypersensitivity and a negative predictive value
of 97%.12
86
SNP MAPPING-A TOOL FOR PERSONALIZED
GENETIC PROFILING
Following dogma: gene
protein
biochemical process
disease state became the model
for examining human disease. Following this scheme,
sickle cell anemia was the first trait to reveal that single
point mutation can change protein structure and lead to
a disease phenotype. Let us know what makes genes so
important factor.
Allele
One of the two or more different forms of genes
containing specific inheritable characteristic that occupies
corresponding position [loci] on paired chromosomes. In
case of pair of allele, one allele comes from father and
one from mother. A capital letter for a dominant and a
lower case letter for the recessive always indicate a pair
of allele. Identical allele, either dominant or recessive is
said to be homozygous for this gene. The union of
dominant gene and its recessive allele produces
heterozygous individual for that characteristic. More than
one inheritable characteristic may be present on some
pair of genes [alleles]. One form of allele is distinguished
from another form of allele by its particular nucleotide.
For e.g. if only one base pair is exchanged with other
base pair then only two different form of alleles exist in a
population for such a gene locus. If suppose two base
pairs are exchanged then four different alleles exist for
that gene locus. However, several different alleles may
exist in the population if the locus is defined by more
than one nucleotide as in case of micro satellites,
haplotypes.14
Single nucleotide polymorphism [SNP]
Pronounced as snips. SNPs are the hot spots on
the chromosome. SNP are biallelic i.e. one base pair is
exchanged with just one other base pair. Then only two
different forms of alleles exist. These SNPs will play a
major role in associating sequence variations with
heritable variation in drug response. Turning SNPs into
useful markers of drugs response for the following
reasons:
v SNPs are most common and most technically
accessible class of genetic variants 15.
v SNPs are much more frequent than microsatellite
repeats and are spread through out the genome.16
v SNPs are less prone to germline mutation, which
means that their inheritance is more stable.
v SNPs can occur within coding or regulatory region
of genes, which means that they can be directly
responsible for the traits studied.
v SNPs are biallelic, which make population frequency
estimation easier.17, 18
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Turning these SNPs into useful marker of drug
response is the goal of researcher in the field of
pharmacogenomics. Among the challenges of
pharmacogenomics is sorting through the vast number
of SNPs available and deciding how many and which
ones to analyze based on location, frequency, and type?
Once a large number of these SNPs and their frequencies
in different population are known, they can be used to
correlate an individual genetic “fingerprint” i.e. genetic
makeup with the probable individual drug response. Highdensity maps of SNPs in the human genome may allow
using these SNPs as markers of drug response even if
drug target remains unknown, providing a “drug response
profile” associated with contributions from multiple genes
to a drug response phenotype.19,20 Researchers are hoping
to use SNPs as genetic sign post for the gene
combinations that cause diseases. They could compare,
for example, SNPs in people with and without the diabetes,
to see which combinations of DNA correlate with the
disease. Doctors could then scan a patient’s genome to
see whether he or she carries the expected pattern of
SNPs associated with a disease risk. Scanning map of
SNPs will provide some sort of tools to unravel the
complex etiology of common genetic diseases.
Genome
Genome means all the genetic material present in
one cell of organism. Genomics is the study of genes
and their functions. Our genomic sequence provides
unique record of who we are and how we evolve as a
species, including the fundamental unity of all human
beings. 22, 23 Such an understanding will make it possible
to study how our genomic DNA varies among cohorts
of patients and especially the role of such variation in
the causation of important illnesses and responses to
pharmaceuticals.24, 25
(b) The largest known human gene being dystrophin
which have 2.4 million bases
(c) Chromosome 1 has highest genes that are 2968
genes
(d) Chromosome Y has fewest genes that are
231genes
4] The order of almost all [99.9%] nucleotide bases are
exactly the same in all people. That is to say only
0.1% variation is there in sequence of base pair.
These much 0.1% only leads to diversity in whole
of the human population in the world
5] (a) 3.7 million mapped human SNPs [single nucleotide
polymorphism]
(b) There is an average of 1 SNP per 1,250 base pair
or 1,331 base pair 26, 27. Thus we can say 2 cSNP exist in one gene26 or sometime it is said
there exist 4 c-SNP per gene.28, 29
(c) These SNPs may occur in exon, intron, intergenic
DNA or in regulatory region too
(d) Less than 1% of all known SNPs encode a direct
amino acid changes and thus a protein ultimately.
Thus, there are only thousands [not millions] of
genetic variations that directly contribute to the
structural protein diversity of human being.25
(e) For any given drug response, 10% of the
estimated 30,000 genes in the human genome are
involved, then 12,000 candidate c-SNPs should
exist
30,000 genes x 10% =3,000 genes
3,000 genes x 4 SNPs = 12,000 SNPs
(f) 40% of these are estimated to change amino acid,
in addition to yet unknown regulatory and noncoding SNPs may be the most promising SNPs to
examine. Therefore it is nearly to 4,800 SNPs
40% x 12,000 SNPs = 4,800 SNPs
What has been learned from analysis of the working
draft sequence of the human genome?
Thus, just only 4,800 SNPs are responsible for
variation in drug response.
1] There are 3.2 billion of nucleotide bases [A, T, G, and
C] 25
HUMAN GENOME PROJECT
2] (a) The total number of genes estimated are 30,000 to
35,000
(b) 99% of gene containing part of human sequence
finished to 99.99% accuracy [gene sequence]
(c) 15,000 full-length human c-DNA have been done
[gene identification]
3] (a) The average gene consists of 3,000 bases [but
sizes vary greatly]
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Human Genome Project is a massive, government
subsidized effort designed to map and sequence entire
DNA of a human. The Human Genome Project (HGP) is
an international 13-years effort formally started in October
1990.30 The U.S. Human Genome Project (HGP) had several
goals including mapping, sequencing, and identifying
genes; storing and analyzing data; and addressing the
ethical, legal, and social issues (ELSI) that may arise from
availability of personal genetic information. The goals
and their achievement have been shown in Table 2.
87
Table 2: Human Genome Project Goals and Completion Dates 30
Area
Goal
Achieved
1-cM resolution map
(3,000 markers)
52,000 STSs
Physical Map
99% of gene-containing
DNA Sequence
part of human sequence
finished to 99.99%
accuracy
Capacity and Cost Sequence 500 Mb/year at < Sequence >1,400
$0.25 per finished base
Mb/year at <$0.09 per
of Finished
finished base
Sequence
3.7 million mapped human
Human Sequence 100,000 mapped human
SNPs
SNPs
Variation
Gene
Full-length human cDNAs 15,000 full-length human
cDNAs
Identification
Finished genome
Model Organisms Complete
Complete
genome
genome
sequences of E. coli,
sequences of
S. cerevisiae , C. elegans,
E. coli, S. cerevisiae,
D.
melanogaster,
C. elegans, D. melanogaster
plus whole-genome drafts
of several others,
including C. briggsae,
D. pseudoobscura, mouse
and rat
Develop genomic -scale
High-throughput
Functional
technologies
oligonucleotide synthesis
Analysis
DNA microarrays
Eukaryotic, whole genome knockouts (yeast)
Scale-up of two-hybrid
system for protein-protein
interaction
Genetic Map
2- to 5-cM resolution map
(600 ---1,500 markers)
30,000 STSs
95% of gene-containing part
of human sequence finished
to 99.99% accuracy
ETHICAL CONSIDERATIONS IN
PHARMACOGENOMICS RESEARCH
CLINICAL
It is important to anticipate and consider the social,
legal and ethical consequences of this technology and
develop appropriate guidelines and policies for its use.
Genetic information is more vulnerable to violation of
privacy because it contains an individual’s probabilistic
“future diary”. However, although genetic information
is, by its nature, inherently personal, it is at the same
time familial and also communal. Therefore, there are
serious potential risks for discrimination and loss of
privacy, which need to be addressed to formulate a policy
to prevent such possible harm. 31 Thus, despite the
obvious scientific value of using families in
pharmacogenomic trials, such studies raise serious ethical
88
Year of
achievement
September
1994
October 1998
April 2003
November
2002
February
2003
March 2003
April 2003
1994
1996
1999
2002
concerns32 that ensue from the dynamic and social
significance of the family. Traditionally, the hallmark of
this relationship has been one of privacy, confidentiality
and beneficence.33 Genotyping and storage of DNA also
creates the potential for discrimination by employers and
the insurance industry.34, 35 An insurance company could
use predictive genetics to identify in advance and then
reject workers or policy applicants who are predisposed
to develop chronic diseases. “Without adequate
safeguards, the genetic revaluation could mean one step
forward for science and two steps backward for civil
rights”. Therefore, the introduction of “genetic ethics”,
before applying the genetic techniques through which
we can terminate the patient’s fear is mandatory. It is
also to introduce legislation that would prevent insurance
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
companies from requiring genetic testing and ban the
use of genetic information to deny coverage or to set
rates.
ANTICIPATED BENEFITS OF PHARMACOGENOMICS
1] More Powerful Medicines
Pharmaceutical companies will be able to create
drugs based on the proteins, enzymes, and RNA
molecules associated with genes and diseases. This will
facilitate drug discovery and allow drug makers to produce
a therapy more targeted to specific diseases. This
accuracy not only will maximize therapeutic effects but
also decrease damage to nearby healthy cells.
2] Better, Safer Drugs for the First Time
Instead of the standard trial-and-error method of
matching patients with the right drugs, doctors will be
able to analyze a patient’s genetic profile and prescribe
the best available drug therapy from the beginning. Not
only will this take the guesswork out of finding the right
drug, it will speed recovery time and increase safety as
the likelihood of adverse reactions is eliminated.
3] More Accurate Methods of Determining Appropriate
Drug Dosages
Current methods of basing dosages on weight and
age will be replaced with dosages based on a person’s
genetics—how well the body processes the medicine and
the time it takes to metabolize it. This will maximize the
value of therapy and decrease the likelihood of overdose.
4] Advanced Screening for Disease
Knowing one’s genetic code will allow a person to
make adequate lifestyle and environmental changes at
an early age so as to avoid or lessen the severity of a
genetic disease. Likewise, advance knowledge of
particular disease susceptibility will allow careful
monitoring, and treatments can be introduced at the most
appropriate stage to maximize their therapy.
5] Better Vaccines
Vaccines made of genetic material, either DNA or
RNA; promise all the benefits of existing vaccines without
all the risks. They will activate the immune system but
will be unable to cause infections. They will be
inexpensive, stable, easy to store, and capable of being
engineered to carry several strains of a pathogen at once.
6] Improvements in the Drug Discovery and Approval
Process
Pharmaceutical companies will be able to discover
potential therapies more easily using genome targets.
Previously failed drug candidates may be revived as they
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
are matched with the niche population they serve. The
drug approval process should be facilitated as trials are
targeted for specific genetic population groups —
providing greater degrees of success. The cost and risk
of clinical trials will be reduced by targeting only those
persons capable of responding to a drug.
7] Decrease in the Overall Cost of Health Care
Decreases in the number of adverse drug reactions,
the number of failed drug trials, the time it takes to get a
drug approved, the length of time that the patients are
on medication, the number of medications patients must
take to find an effective therapy, the effects of a disease
on the body (through early detection), and an increase
in the range of possible drug targets will promote a net
decrease in the cost of health care.
8] Organ transplantation
Several proteins are expressed as major
histocompatibility complexes (MHC) on the immune cells
depending upon the individual’s genetic makeup, which
would determine the development of immune reaction after
organ transplantation. Thus, the pharmacogenomics help
in establishing an exact match of recipient and donor
MHC’s, so that the organ rejection can be prevented.
IMPACT ON PHARMACY PROFESSION
Presently the doctors diagnose a disease [which is
based on the symptoms] and prescribe a drug on the
trial and error basis, which suits for the average patient.
Pharmacist currently advises about side effects and drugdrug interaction. But a day will come when you may go
for doctor visit and have your blood drawn for a genotype
to be done which would indicate what genes you have
for drug transporters, drug targets or drug elimination
enzymes. This is to say you will take a gene report instead
of blood reports. Thus after you are diagnosed by a
doctor, pharmacist would interpret the panels of genetic
results and advise you which drug would be best for
your particular gene so that you have fast recovery.
From this pharmacist will learn more about the
patient’s genetic information, issue of privacy will arise
which should be maintained by pharmacist. Protective
measures and standards will be required to control the
misuse of personal data and malpractice in testing
procedures. Thus it will lead to teamwork between genetic
counselor, doctor and a pharmacist.
DRUG DISCOVERY— GENETIC PERSPECTIVE
Pharmacogenomics will have a major influence on
drug discovery, through drug design, as well as clinical
practice.
89
GENOME
VALIDATED PROTEIN TARGETS
STRUCTURE DETERMINATION
LIGAND DISCOVERY
3D CRYSTAL STRUCTURES
HOMOLOGY MODELLING
MOLECULAR SIMILARITY
PROTEIN SITE ANALYSIS
LIGAND SUPER SURFACE
STRUCTURE-BASED LIGAND
NEW DRUGS LEADS
Fig 1 : Scheme for drug designs utilizing genomic data. The left track illustrates the process of drug design based on
structural determination of binding sites. The right track outlines design from known ligands.
FUTURE DIRECTIONS
New developments in pharmacogenomics will
impact on drug design at three main levels
Ø The interaction of the drug with its receptor
binding site,
Ø The absorption and distribution of the drug,
Ø The elimination of the drug from the body.
If we are ever to achieve personalized drug therapies,
the above issues will have to be addressed. SNPs in
terms of mutated drug binding sites and the resulting
changes that occur in response to medicines
Crystal structures or homology models of these
enzymes can be used to screen compounds designed by
de novo before synthesis has begun. This can be done
at two levels: The virtual compounds can be input into
metabolism prediction programs, such as Metabolexpert 33
or Meteor,34 to identify principal pathways of expected
drug metabolism.
BARRIERS TO PHARMACOGENOMICS PROGRESS
Pharmacogenomics is a developing research field
that is still in its infancy. Several of the following barriers
will have to be overcome before many Pharmacogenomics
benefits can be realized.
Ø Complexity of finding gene variations that affect
drug response : 1 SNPs occur every 1,250-1,331
90
base pair along the 3.2 billion base human genome,
therefore millions of SNPs must be identified and
analyzed to determine their involvement (if any)
in drug response. Further complicating the process
is our limited knowledge of which genes are
involved with each drug response. Since many
genes are likely to influence responses, obtaining
the big picture on the impact of gene variations is
highly time-consuming and complicated.
Ø Limited drug alternatives: Only one or two
approved drugs may be available for the treatment
of a particular condition. If patients have gene
variations that prevent them using these drugs,
they may be left without any alternatives for
treatment.
Ø Disincentives for drug companies to make
multiple pharmacogenomic products : M o s t
pharmaceutical companies have been successful
with their “one size fits all” approach to drug
development. Since it costs hundreds of millions
of dollars to bring a drug to market, will these
companies be willing to develop alternative drugs
that serve only a small portion of the population.
Ø The limitations of Pharmacogenomics are the
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
complexities of gene regulation, of proteomics, of
gene environment interactions and also of the
psychological complexities of interactions between
physicians and patients.
CONCLUSION
Ultimately pharmacogenomics encompasses a
genetic profile for each individual, containing sufficient
information to select which drugs are almost likely to be
safe and effective in that person. The opportunity for
pharmaceutical industry to embrace pharmacogenomics
cannot be ignored, integrated into clinical development
of new compounds. Pharmacogenomics studies have the
potential to demonstrate both whether a compound is
clinically effective or for whom it will be most effective.
The role of genes in determining disease
susceptibility, progression, complications and response
to treatment could all be potentially mapped.
Pharmacogenomics will yield drugs targeted to act at or
near the cause of a disease. Genetically defining patient
population will help to improve outcomes and genetic
prognostics will revolutionize treatment and improve costeffectiveness. Pharmacogenomics is already making an
impact in a wide array of disease states and drug therapy;
it will eventually become part of standard patient
management in selecting and monitoring drug therapy.
Pharmacogenomics will definitely help us to sharpen our
medical and pharmaceutical tools. Drugs will become more
precise and efficient and the risk of toxic side effects will
be reduced. But at the same time increasing amounts of
information will be collected, which may be put to a
variety of uses.
Over all the “racial problem” is the basic problem of
India and the same faced by whole world. We have
come to know from human genome project that only 0.1%
leads to diversity in whole of the human population in
world. Then the question arises why to fight? You know
we are fighting with our own nearly similar genetic makeup
person. We can say with our own brotherhood.
Acknowledgement
We are extremely thankful to Dr. A.Giri & Dr. D.
Sarkar, N.C.L,Pune for their valuable suggestions and
constant help. We cannot conclude without expressing
our gratitude to Mr. and Mrs.Gupta (Meenu’s papamummy) and her brother Dr. Yogesh Kumar Gupta and
her friend Rahul Jadhav.
3.
4.
5.
6.
7.
8.
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11.
12.
13.
14.
15.
16.
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Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Review Article
Pharmaceutics
Harmonization of Excipients: The Indian Perspective
S.S.Poddar, Chivate Amit and Prajapati Paresh
Prin K.M.Kundnani College of Pharmacy, Mumbai 400018
Received on 25.09.2004
Modified on 28.06.2005
Accepted on 01.09.2005
ABSTRACT
Excipients are critical in the formulations to ensure an acceptable and safe drug delivery to patients. The article
attempts to explain the importance of harmonization of its components. It also tries to bring about differences in limits,
test procedures, etc. among various pharmacopoeias regarding excipients. The article also highlights the role and
participation of Indian Pharmacopoeia in the direction of harmonization.
Although excipients do not have the therapeutic benefit they remain essential constituents of virtually every
pharmaceutical product. Excipients fulfill an extensive and diverse range of functions. They have critical role in the
formulation to ensure that drug is acceptable to patients. They also control the type and rate of drug release as well as
release mechanisms. The requirement for pharmaceutical manufacturers to test raw materials prior to their use in drug
products is clearly established by current Good Manufacturing Practices (cGMP). The United States Code of Federal
Regulations (CFR), Title 21, Part 211.84(d) (2) states, “Each component shall be tested for conformity with all appropriate
written specifications for purity, strength, and quality.” A component is defined in Part 210.3(b) (3) as “any ingredient
intended for use in the manufacture of a drug product.” This definition includes excipients, viz ingredients other than the
active pharmaceutical ingredient incorporated in the finished drug product.
In testing raw materials for pharmaceutical use, the ultimate goal is to ensure the safety of patients. It is important
to evaluate excipients, since they are usually present in a majority of the dosage form and often play an important role
in determining the overall characteristics and stability of the drug product. Analysis of excipients should, therefore,
ensure the suitability of the materials for their intended use. Tests should be employed to confirm the identity, chemical
and microbiological purity, assay result, and critical physical characteristics of the excipient.
Excipient testing must also satisfy regulatory requirements to support the use of a drug product in various countries
throughout the world. Compendial excipients, such as lactose, microcrystalline cellulose, magnesium stearate, and
hydroxypropyl cellulose, have official monographs consisting of the test methods, and acceptance criteria. Although
many countries including Britain, France, Germany, India, and China have their own compendia, it is generally recognized
that the major compendia’s are the United States Pharmacopoeia - National Formulary (USP-NF), European Pharmacopoeia
(EP), and Japanese Pharmacopoeia-Japanese Pharmaceutical Excipients (JP-JPE). One of the many challenges being faced
by global pharmaceutical manufacturers is ensuring compliance with multi-compendial testing requirements for excipients,
since there may be significant differences in the monographs for the same excipient in the USP-NF, EP, and JP-JPE.
INTRODUCTION
The issues of harmonization started in September
1989, where 3 pharmacopoeias viz USP, JP and EP made a
voluntary alliance called Pharmacopoeial Discussion
Group (PDG) to work on harmonization of excipients,
including their standards and test methods. These
pharmacopoeias conducted 2 surveys in May 1990 and
May 1992 and selected the most important 10, and
subsequently 25 excipients for harmonization. PDG ranked
the excipients and established a lead pharmacopoeia
system to bring about harmonization among the 3
pharmacopoeias.1
Full harmonization means identical standards and
test methods. The most important excipients selected for
retrospective harmonization were used in numerous drug
products worldwide. These products were approved by
regulatory authorities based on old standards in each of
the three compendia.2
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Excipients are derived from various sources like
· Natural
· Natural modified
· Synthetic
· Organic
· Inorganic
Differences exist in natural materials because of their
origin and source. Sucrose produced from sugarcane and
beetroot using different purification process results in
different impurities and different levels. Modified and
synthetic excipients use different sources of materials,
different methods of manufacturing, and different
inprocess controls that may result in different purity
profiles. Equally important are the test methods, which
vary from qualitative to semi-quantitative to quantitative.
Without harmonizing test methods, harmonization of
standards has very little meaning. The figure 1 outlines
the components of harmonization.
93
Material origin
Starting Material
MANUFACTURING PROCESS
IN-PROCESS CONTROLS
Natural
Natural modified
Synthetic
Organic
Inorganic
EXCIPIENTS
EXCIPIENTS
DRUG
SUBSTANCES
DRUG
SUBSTANCES
TEST METHODS
MONOGRAPH TO ENSURE IDENTITY, QUALITY & PURITY
The overall approach of harmonization is
Ø Objective comparison of the monographs, i.e.
requirements in the relevant pharmacopoeia e.g.
USP, EP & JP.
Ø Starting from ground zero and trying to find out a
scientific ratio for standards and test methods.
Ø The lead pharmacopoeia (now called Coordinating
Pharmacopoeia) makes a proposal and explains the
scientific basis of the same.
Ø Continuous communication among the compendia
in turn publishing the proposals at different stages
in its respective forum for comments.
Ø Discussing the differences in the standards and
test methods with other compendia and reaching
the consensus.
Harmonization is essentially required among
pharmacopoeias for product registration exercises. That
is, product development can proceed as is, without the
need for repeating of studies or testing to support
registration in other than the original region. The primary
beneficiaries, thus, would be international companies. But
harmonization has an independent value in facilitating
international commerce of excipients as well as finished
drug products. International companies would prefer to
market a minimum number of formulations worldwide,
because product registration of formulations is a very
complex area, and the pharmacopoeias is one evidence
of the fact that different formulations of same action may
require different test methods. Thus, harmonization must
evolve monographs that are acceptable to the registration
authorities in different regions. Differences in
pharmacopoeial standards could be avoided and technical
barriers to trade could be minimized and abolished.
Thus, it is a process involving analysis of every
standard and test method and reaching a consensus that
is scientifically, functionally and economically justifiable.3
The process of harmonization is quite stretched and
tedious, which involves 7 steps. The 5th step is divided
into two substeps. The details of the steps are shown in
table 1.4
Table 1: Steps in Harmonization
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5A
Stage 5B
Stage 6
Stage 7
94
Identification of the Coordinating Pharmacopoeia among the participating
group of participating Pharmacopoeia for a subject selected for harmonization
Investigation: Preparatory work by the Coordinating Pharmacopoeia
Publication of Proposal stage draft in the forum of each pharmacopoeia in the
style of the Coordinating Pharmacopoeia for public comments
Publication of Official inquiry stage draft after addressing the harmonization
issues and the draft is published by each pharmacopoeia in its own style
Publication of Provisional Harmonization text by each pharmacopoeia. If
consensus is not reached, the Coordinating Pharmacopoeia prepares another
version of text
Approval of Consensus Text, signing-off, and publication in the forum if major
revision takes place
Adoption
Implementation
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
The article will focus on some of the test procedures and
specifications adopted by the three participating
pharmacopoeias. These will be compared with our Indian
pharmacopoeia.
A. TESTS
1. Microbial Limit Test
Major source of contamination in non-sterile dosage
form is the bio-burden in excipients. Therefore, its control
of bio-burden is important in assessing the
microbiological quality of a drug product. Excipients that
are obtained by chemical synthesis are least contaminated,
because they do not possess the necessary conditions
that allow the growth of microorganism. Excipients derived
from minerals are also less likely to get contaminated
with microorganism. But there are certain exceptions to it
e.g. talc, bentonite, Kaolin, Aluminium and magnesium
salts may be contaminated with pathogenic
microorganism such as Pseudomonas aeruginosa ,
Staphylococcus aureus, Salmonella species, Clostridium
prefringens.5-7 Excipients of natural origin (animal and
botanical) have the highest risk of contamination by
microorganisms. These may be due to their specific
collection, extraction and storage process. Gram negative
bacteria (Pseudomonas), Gram positive bacteria
(Lactobacillus, Streptococcus, Bacillus) and molds
(Penicillium and Aspergillus) can be found in raw
materials though they are not usually pathogenic agents.
The excipients derived from animal origin present a very
high risk of contamination, mainly due to non-pathogenic
Enterobacteria, though pathogenic strains of Salmonella
and Shigella have also been found.8 De. La. Rosa, et.al,
proposed that each article comply with a different
microbiological pattern with regard to total number of
microorganisms and absence of specified pathogens.
Furthermore the Fungi limits must be extended to every
excipient, because this type of contamination can produce
problems like changes in organoleptic properties and
injuries to human health due to Mycotoxin.9-10
Table 2: Comparison of microbial requirements in different Pharmacopoeias
Excipient
USP/NF Microbial Limit
Total
Combined
Absence
Aerobic
yeast and
of
count
moulds
Lactose
monohydrat
100
50
e /anhydrous
Magnesium
1000
500
Stearate
MCC/
Powdered
1000
100
cellulose
Sucrose
NA
NA
A=S.aureus B= P.aeruginosa
EP Microbial Limit
JP Microbial Limit
Total
Combined
Total
Combined
Absence Aerobic
Absence
Aerobic
yeast and
Aerobi
yeast and
of
of
count
moulds
ccount
count
moulds
C
100
NA
C
100
50
C, D
C, D
NA
NA
NA
1000
500
C, D
A, B, C, D
1000
100
A, B, C,
D
1000
100
A, B, C, D
NA
NA
NA
NA
NA
NA
C= E.coli D= Salmonella species NMT = Not more than NA: Not applicable
NA
Table 3: Limits for some Excipients in Indian Pharmacopoeia11-12
Excipients
Lactose Monohydrate/anhydrous
Magnesium stearate
Talc
MCC
Sucrose
Microbial limits NMT/Gram
100
None
None
None
None
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Absence of microorganisms
E.coli, Salmonella species
None
None
None
None
95
Table 4: Comparison of microbial limit before and after harmonization in USP/NF
USP/NF Microbial Limit Before
Harmonization
Total
Aerobic
Absence of
count
NA
Salmonella, E.Coli
Total Aerobic
count
Combined yeast
and moulds
100
50
E.coli
1000
E.coli
1000
100
Salmonella sp, E.coli.
NA
NA
1000
100
Sucrose
NA
NA: Not applicable
NA
NA
NA
Salmonella sp, E.coli,
S.aureus, P.aeruginosa
NA
Excipients
Lactose
Monohydrate/
Anhydrous
Magnesium
stearate
MCC
USP/NF Microbial Limit After
Harmonization
2. Organic Volatile Impurities
The USP sub-committee on chemical purity is
concerned with solvent residues and volatile
contaminants resulting from the synthesis, processing
and transfer of materials among containers. In October
1986, proposal was made for new chapter <467>, Organic
Volatile Impurities (OVI). The requirements for testing
OVI would be proposed for monographs on substance
that are generally administered for the long term systemic
treatment for chronic conditions. Chronic was defined as
30 days or longer. The initial focus on the OVI testing
was on widely used Organic Volatile Liquids that are
known to be toxic. The limits were expressed as maximum
daily exposure per patient per day as mg of each OVI per
gram of formulation. In 1988 a proposal was made to
consider solvents that cause irreversible toxic effects such
as carcinogenicity, teratogenicity, and mutagenicity.
Consequent to these, newer limits of safety factor as
high as 105 were proposed based on tumor-dose bioassay
studies.13
The International Conference on Harmonization
(ICH) on 7th November 1996 released its draft consensus
Absence of
guidelines, “Impurities: Guidelines for Residual Solvents”,
for consultation at step 2 of ICH process.14 The Residual
Solvents in pharmaceuticals were defined as Organic
Volatile chemicals that are used or that get produced in
the synthesis of drug substances or excipients or during
formulation, which are not removed completely by the
conventional practical manufacturing techniques. Thus,
these guidelines emphasize that all residual solvents
should be removed to the extent possible to meet product
specifications, good manufacturing practices, or other
quality based requirements. The product should contain
no higher levels of residual solvents that can be
supported by safety data.
The residual solvents are classified into four
categories
i. Class 1 solvents, which are, know to cause
unacceptable toxicities and should not be used
ii. Class 2 solvents, which should be limited to protect
patients from potential adverse effects.
iii. Class 3 solvents, which can be used.
iv. Additional solvents for which no toxicological
data have been found.
Table 5: Limits of Organic Volatile Impurity Before and After Harmonization
Organic Volatile impurity
Benzene
Chloroform
1,4-Dioxan
Methylene chloride
Trichloroethylene
96
USP/NF Limits before
2003 (ppm)
100
50
100
500
100
USP/NF 2003 Limits (ppm)
Not specific (individual Monograph)
60
380
600
80
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
B. MONOGRAPHS
1. Magnesium Stearate
Harmonization efforts on magnesium stearate began
in 1990. Originally there were lots of differences in the
monographs, including the identification test, sulfate,
heavy metals, microbial contamination etc. This is
described briefly in Table 6. In 1990, for the purpose of
harmonization the limit of sulfate was proposed to be
1%. In 1991, it was proposed to be 0.3%. But
Pharmacopoieal Forum (PF) did not include test for
sulfate. This was based on the comments that test failures
were not indicative of poor production process. The 1%
limit was considered appropriate based on daily sulfate
intake from dosage forms containing only 0.2-2.0%
magnesium stearate. But E.P responded that their 0.5%
limit had not elicited any criticism so far. E.P also stated
that the limit for sulfate could be set at 0.5%. So as a part
of harmonization efforts, subcommittee again reviewed
the proposals regarding the sulfate limit. Thus, on August
13 1998 it was made sure that sulfate limit would be kept
at 0.5%. Thus, we learn that the standard of USP was
raised. Similarly if Indian Pharmacopoeia would participate
in such harmonization exercise it could improve its own
as well as standards other pharmacopoeias. IP’s limit for
sulfate is 0.6%, while that of USP’s is still 1%. Thus it
can bring down their sulfate limit which will lead to more
standardized drugs.
Table 6: Limits for Magnesium Stearate in USP (2003) & IP (1996)
Test
I.P 1996 (Present Or Absent) If
Present, The Limit
U.S.P 2003 (Present Or Absent) If
Present, The Limit
Microbial count
NS
1000 (Aerobic), 100(combined fungi,
mold), Salmonella sp and E.coli absent
LIMITS
Chloride
Sulfate
250 ppm
0.6%
0.1%
1.0%
OVI
NS
As given in table 5
NS : Not specified
2. Talc
Harmonization proposed the following limit. Total
Aluminium: NMT 2.0%, total Calcium: NMT 0.9%, total
Magnesium: NMT 17.0-19.5% and total Iron: NMT 0.25%.
Another issue to be resolved is the appropriateness and/
or necessity for the inclusion of a proposed pH-range
specific test in the monograph. But then the basis for
adding a pH test to the harmonized monograph must be
fully understood before proceeding. Thus it was decided
that unless this test serves some real performance
property measurement or public health purpose, it should
not be added to the monograph.
This monograph would tell us that Indian
pharmacopoeia has missed out on some important limits
i.e. arsenic, lead, and heavy metal. But these limits are
very essential for public health. So, such considerations
are very important for Indian market and International
usage.
Table 7: Limit for Talc in USP & IP
Test/ Limits
Microbial count
Arsenic
Iron
Lead
Heavy metal
NS : Not specified
I.P 1996 (Present or Absent) If Yes, The
Limit
NS
NS
10 ppm
NS
NS
Similarly there are other excipients that are still going
through the process of harmonization. Tables 8 and 9
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
U.S.P/Nf 2003 (Present or Absent) If
Yes, The Limit
1000 (Aerobic), None (combined fungi,
mold), None
3 ppm
Colour reaction
0.001%
0.004%
will show the limits for harmonized excipients in USP and
their comparison with IP.
97
Table 8: Limit for Microcrystalline cellulose in USP (2003) & IP (1996)
Test
Microbial count
I.P 1996(Present or Absent) If Yes,
The Limit
NS
Conductivity
NS
Water soluble
NMT 0.2%
substance
OVI
NS
LOD
6.0%
pH
5.0-7.5
NS : Not specified NMT : Not more than
U.S.P 2003(Present or Absent) If Yes, The
Limit
1000 (Aerobic), 100 (combined fungi,
mold), Salmonella sp and E.coli, S.auerus,
P.aeruginosa absent
75 µs per cm.
NMT 0.24%
Given in Table 5 (USP Limits 2003)
7.0%
NS
Table 9: Limit for Lactose Monohydrate in USP (2003) & IP (1996)
Test
I.P (Present or Absent) If Yes, The
Limit
Total Microbial count NMT 100/gram.
Absence of Salmonella sp and E.coli.
+54.40 to +55.9 0
Microbial count
Specific rotation
Water soluble
NMT 0.24%
substance
Water
4.5 to 5.5%
Heavy metals
NMT 5 ppm
NMT : Not more than
There is an obverse to harmony and that is
disharmony. An example of disharmony is the need to
repeat tests using rabbits for pyrogen, where testing with
bacterial endotoxin is otherwise prescribed. This
apparently represents the most extreme disharmony of
methods. But there is a greater disharmony; i.e., reaching
different conclusions whether to pass/fail the specimen!
In this case, the quality control professionals must make
a judgment whether or not this material can be sold in
one or more regions. Functionally equivalent to
harmonization is the absence of disharmony. Because of
differences in policies, pharmacopoeias may differ on
adoption of a test. If certain tests are considered
necessary by one pharmacopoeia in order to protect the
consumer, then it is appropriate for that pharmacopoeia
to adopt the test without reference to any other region.
Most discussions on harmonization revolve around
excipients or general tests and assays. But the
performance of even a harmonized method using different
reference standards is not an optimal situation. In fact,
harmonization of reference standards preceded many of
98
U.S.P (Present or Absent) If Yes, The
Limit
100 (Aerobic), 50 (combined fungi,
mold), Absence of E.coli.
+54.4 0 to +55.90
NMT 0.24%
4.5 to 5.5%
5 ppm
the harmonization efforts of the last 10 years.
Pharmacopoeias and the World Health Organization
(WHO) have, in the past, shared bulk materials to create
their individual reference standards, where a drug exists
as a highly purified crystal, the difference in
pharmacopoeial reference standards is then administrative
and legal and that no difference in results in laboratories
is to be seen. This is not the case with mixtures, such as
an antibiotic reference standard, which may be
established based on different microbiological assays.
Pharmacopoeial harmonization is challenging, as
differences exist, because of the background of the
pharmacopoeias. There are many factors. The most
obvious are: content, language, legalities, speed, the
audiences for the standards and socioeconomic status
of the region. For example USP applies harmonization to
the practice of pharmacy, both in community pharmacy
and hospital in tune with the environment prevailing in
the country.
Thus, for complete success of harmonization there
should be support at both the national and international
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
levels. There should be support both for harmonization
of excipients, general tests and assays. In doing so, one
should prefer meaningful standards, not necessarily the
lowest common denominator or the most stringent. Major
support for pharmacopoeial harmonization would come
from increased cooperation and contribution of
Pharmacopoeias.
Thus, harmonization could be viewed to be having
three essential values. The first is the facilitation of
international commerce. The second is the facilitation of
product registration processes among nations. The third
is to reduce duplicative testing costs. The registration
for any new molecular entity is a onetime event for the
relevant country; whereas in international commerce,
reduction in the duplicative testing remains during the
lifetime of that product.
3.
Chowhan ZT. The long, difficult, and frustrating
process of Harmonization of Excipient Standards &
Test Methods. Pharmaceutical technology. 1997;
21(March): 76, 78, 80, 82, 84, 86 & 88.
4.
Chowhan ZT. Progress and Impediments in the
Harmonization of Excipient Standards & Test
Methods, Part II. Pharmaceutical technology. 1999;
23(April): 50, 52, 54, 56 & 58.
5.
Buhlmann X. Microbiological Quality of
Pharmaceutical Preparations. American Journal of
Pharmacy. 1972; 144:165-185.
6.
Kruger D. Ein Beitragzum Thema der mikrobiellen
Kontamination von Wirkund Hilfsstoffen. Pharm Ind.
1973; 35:569-577.
7.
Sykes G. The total control of microbiological
contamination in Pharmaceutical Products for Oral
& Topical use. J Mond Pharm. 1971; 14:78-81.
8.
Underwood E. Ecology of microorganisms as it
affects the Pharmaceutical Industry. In: Hugo WB,
Russel AD, editors. Pharmaceutical Microbiology.
Oxford: Blackwell; 1992. p.353-368.
9.
Hiticoto H. Fundamental contamination and
Mycotoxin Detection of Powdered Herbal Drugs.
Applied Environmental Microbiology. 1978; 36:252256.
CONCLUSION
Thus to conclude it may be stated that Indian
Pharmacopoeia should make strong moves in improving
the test methods, limits and assay procedures. Though it
may be expensive, time consuming and difficult, such
moves would:
·
Ensure the quality and purity of excipients.
·
Enhance public health status
·
Make industry strongly rely on Indian
Pharmacopoeia for International trade.
Though our country is making world class
pharmaceuticals many of us are compelled to refer
Pharmacopoeias of other countries. Surely it’s not a
pleasant scene! This should be changed and the earlier
the better.
References
1.
2.
Chowhan ZT. Harmonization of Excipient standards
and test methods: A progress update on
development of test methods and standards, Part I.
Pharmaceutical technology. 1994; 18(October):150,
152, 154, 156, 158, 160-165.
Chowhan ZT. Progress and Impediments in the
Harmonization of Excipient Standards and Test
Methods, Part I. Pharmaceutical technology. 1999;
23(March): 64, 66, 68, 70, 72 & 74.
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10. Fernandez GS, Ylla-Catala M. The formation of
Alfatoxins in different types of starches for
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451, 494.
12. Indian Pharmacopoeia. 1996. (Vol 2):736 and 737.
13. Chowhan ZT. Excipient Harmonization Issue: An
Overview. Pharmaceutical technology. 1997;
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14. ICH Steering Committee. Impurities: Guidelines for
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15. The United States Pharmacopoeia (26) & The
National Formulary (21). Asian Edition. Jan 2003.
99
Research Article
Pharm. Analysis
Stability-indicating HPTLC Determination of
Donepezil HCl in Pharmaceutical Dosage Form
Vinay Saxena1, Zahid Zaheer2 and Mazhar Farooqui3
1
Maulana Azad College, Aurangabad 431001
Kamla Nehru College of Pharmacy, Aurangabad 431001
3Aurangabad College for Women, Aurangabad 431001
2
Vinay Saxena
Received on 08.06.2005
Accepted on 22.07.2005
ABSTRACT
A simple, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis
of Donepezil HCl in pharmaceutical dosage form was developed and validated. The method employed TLC aluminium
plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of ethyl acetate methanol - toluene - ammonia (8:1.5:8.5:0.4, v/v/v/v). This system was found to give compact spots for Donepezil HCl (Rf
value of 0.57). Donepezil HCl was subjected to acid and alkali hydrolysis, oxidation, photochemical degradation and
thermal degradation. Also, the degraded product was well separated from the pure drug. Densitometric analysis of
Donepezil HCl was carried out in the absorbance mode at 316 nm. The linear regression analysis data for the calibration
plots showed good linear relationship with coefficient of regression value, r2 = 0.9976 in the concentration range 41.20123.60 ng per spot. The value of correlation coefficient, slope and intercept were 0.9987, 26.95 and 272.43, respectively.
The method was validated for precision, recovery, ruggedness and robustness. The limits of detection and quantitation
were 5.0 and 20.6 ng per spot, respectively. The drug undergoes degradation under acidic, basic, oxidation and
photochemical degradation conditions. All the peaks of degraded product were resolved from the active pharmaceutical
ingredient with significantly different Rf values. The sample degraded with thermally showed no additional peak. This
indicates that the drug is susceptible to acid-base hydrolysis degradation, oxidation and photochemical degradation.
Statistical analysis proves that the method is reproducible and selective for the estimation of said drug. As the method
could effectively separate the drug from its degradation product, it can be employed as a stability-indicating one.
INTRODUCTION
Donepezil HCl, chemically, 2,3-Dihydro-5,6dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl] methyl]-1Hinden-1-one; (Figure1) is an Nootropic drug. The drug is
listed in Merck index. 1 Literature survey reveals that there
are capillary electrophoresis method2-3 HPLC method4-5
for quantitation of donepezil. A HPLC - electrospray
tandem mass spectrometry is reported for evaluation of
donepezil HCl in blood plasma.6 But there is no stability
indicating HPTLC method for determination of donepezil
HCl from its tablets, as its pharmaceutical dosage form.
stability-indicating method is one that quantifies the drug
per se and also resolves its degradation products. A very
viable alternative for stability-indicating analysis of
Donepezil HCl is high-performance thin-layer
chromatography (HPTLC). The advantage of HPTLC is
that several samples can be run simultaneously by using
a small quantity of mobile phase unlike HPLC, thus
lowering analysis time and cost per analysis.8-10
The aim of the present work was to develop an
accurate, specific, reproducible, and stability indicating
method for the determination of low levels of Donepezil
HCl in the presence of its degradation products and
related impurities as per ICH guideline.11
2. METHODOLOGY
Figure 1: Chemical Structure of Donepezil HCl
The International Conference on Harmonization
(ICH) guideline entitled ‘Stability Testing of New Drug
Substances and Products’ requires the stress testing to
be carried out to elucidate the inherent stability
characteristics of the active substance.7 Susceptibility to
oxidation is one of the required tests. The hydrolytic and
the photolytic stability are also required. An ideal
100
2.1.Materials : Donepezil HCl was supplied by Sun
pharma India Ltd. and tablets (Label Claim:5 mg/ tablet,
Product Name : Donep 5 and Manufacturer: Hetro Drugs
Limited) were procured from the market. All chemical and
reagents used were of analytical grade and were
purchased from Merck Chemicals, India.
2.2.HPTLC Instrumentation: The samples were spotted
in the form of bands of width 5 mm with a Camag
microlitre syring on precoated silica gel aluminium plate
60F-254 (20 cm x 10 cm with 250 mm thickness; E.merck,
Germany) using a Camag Linomat IV (Switzerland). A
constant application rate of 0.1 µl/s was employed and
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
space between two band was 5 mm. The slit dimension
was kept at 4 mm x 0.5 mm and 20 mm/s scanning speed
was employed. The mobile phase consisted of ethyl
acetate - methanol - toluene - ammonia (8:1.5: 8.5:0.4, v/v/
v/v). Methanol - chloroform (1:1, v/v) was used as diluent
for standard and sample preparation. Linear ascending
development was carried out in twin trough glass chamber
saturated with the mobile phase. The optimized chamber
saturation time for mobile phase was 30 min. with filter
paper at room temperature. The length of the
chromatogram run was 7 cm. Subsequent to the
development, TLC plates were dried in a current of air
with the help of an air-dryer. Densitometric scanning was
performed on Camag TLC scanner III in the absorbance
mode at 316 nm. The source of radiation utilized was
deuterium lamp.
2.3.Calibration Curves of Donepezil HCl: A stock
solution of Donepezil HCl (10.3 mg/ml) was prepared in
diluent. Different volumes of stock solution 4, 6, 8, 10
and 12 m L, were spotted on TLC plate to obtain
concentration of 41.20, 61.80, 82.40, 103.00 and 123.60 ng
per spot of Donepezil HCl, respectively. The data of peak
area versus drug concentration were treated by linear
least-square regression analysis.
2.4 Method Validation
2.4.1.Precision: Precision was measured in terms of
repeatability of application and measurement.
Repeatability of standard application were carried out
using six replicates of the same spot (80 ng / spot for
standard application). Repeatability of sample
measurement were carried out in six different sample
preparation from same homogenous blend of marketed
sample (80 ng / spot for sample application). It showed
very low % relative standard deviation (% RSD) of peak
area of donepezil HCl.
2.4.2 Ruggedness and Robustness: Method ruggedness
and robustness was determined by analysing same
sample blend at normal operating conditions and also by
changing some operating analytical conditions such as
development distance, mobile phase composition,
injection volume, chamber saturation time and analyst.
2.4.3 Limit of detection and limit of quantitation: In
order to estimate the limit of detection (LOD) and limit of
quantitation (LOQ), blank diluent was spotted in replicates
following the same method as explained in section 2.2.
The signal to noise ratio was determined.
2.4.4 Recovery Studies: Recovery study was performed
by spiking 30%, 50% and 70% of Donepezil HCl working
standard to a preanalysed sample. The preanalysed
sample is to be weighed in such a way that final
concentration is half or 50% of the sample preparation
before spiking. The percentage sum level of preanalysed
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
sample and spiked amount of drug should be 80%, 100%
and 120% of simulated dosage nominal or target
concentration of sample preparation. The accuracy of the
analytical method was established in duplicate across its
range.
2.5. Analysis of marketed formulation: Weigh and finely
powder not less than 20 tablets. Transfer blend equivalent
to 8 mg of Donepezil HCl to a 100 ml volumetric flask.
Add about 60 ml of diluent and sonicate for 15 minutes
and make up volume with diluent. Mix well and centrifuge
the solution at 2500 rpm for 10 minutes. Spot the clear
supernatant solution in the form of bands on the specified
TLC plate followed by development and scanning as
described in Section 2.2. The analysis was repeated in
triplicate. The possibility of excipient interference in the
analysis was studied.
2.6 Forced degradation of Donepezil HCl
2.6.1 Preparation of acid and base- induced degradation
product: Tablet powder equivalent to 8 mg of Donepezil
HCl was transferred to 100ml volumetric flask. To it, 25
ml of diluent was added and sonicated for 10 minutes
with intermittent shaking. To it, 1ml of 5N HCl was added
and 5ml of 1N NaOH were added separately. The sample
was heated on a boiling water bath for 5 minutes. After
cooling, to room temperature it was diluted to volume
with diluent and mixed. This solution was centrifuged at
2500 rpm for 10 minutes. This acidic and basic forced
degradation was performed in the dark in order to exclude
the possible degradative effect of light. The resultant
supernatant solution was applied on TLC plate and the
chromatograms were run as described in Section 2.2.
2.6.2.Preparation of hydrogen peroxide- induced
degradation product: Tablet powder equivalent to 8 mg
of Donepezil HCl was transferred to 100ml volumetric
flask. To it, 25 ml of diluent was added and sonicated for
10 minutes with intermittent shaking. To it, 1ml of 3.0%
H2O2 was added. The sample was heated on a boiling
water bath for 5 minutes. After cooling, to room
temperature it was diluted to volume with diluent and
mixed. This solution was centrifuged at 2500 rpm for 10
minutes. The resultant supernatant solution was applied
on TLC plate and the chromatograms were run as
described in Section 2.2.
2.6.3.Photochemical degradation product: Tablet powder
equivalent to 8 mg of Donepezil HCl (previously kept in
UV light for 24 hours), was transferred to 100ml volumetric
flask. To it, 25 ml of diluent was added and sonicated for
10 minutes with intermittent shaking, diluted to volume
with diluent and mixed. This solution was centrifuged at
2500 rpm for 10 min. The resultant supernatant solution
was applied on TLC plate and the chromatograms were
run as described in Section 2.2.
101
2.6.4. Thermal degradation product: Tablet powder
equivalent to 8 mg of Donepezil HCl was transferred to
100ml volumetric flask. To it, 50 ml of diluent was added
and sonicated for 10 minutes with intermittent shaking.
This sample was heated on a boiling water bath for 5
minutes. After cooling to room temperature it was diluted
to volume with diluent and mixed. This solution was
centrifuged at 2500 rpm for 10 minutes. The resultant
supernatant solution was applied on TLC plate and the
chromatograms were run as described in Section 2.2.
2.7.Detection of the related impurities: Weigh and finely
powder not less than 20 tablets. Transfer blend equivalent
to 8 mg of Donepezil HCl to a 100 ml volumetric flask.
Add about 60 ml of diluent and sonicate for 15 minutes
and make up volume with diluent. Mix well and centrifuge
the solution at 2500 rpm for 10 minutes. Spot the clear
supernatant solution in the form of bands on the specified
TLC plate and the chromatograms were run as described
in Section 2.2.
3.RESULTS AND DISCUSSION
3.1.Development of the optimum mobile phase: TLC
procedure was optimized with a view to develop in
stability- indicating assay method. Both the pure drug
and the marketed products were spotted on TLC plates
and run in different solvent system where bands closer
to the solvent front and diffused bands were observed.
Finally, the mobile phase of ethyl acetate - methanol toluene - ammonia (8:1.5:8.5:0.4, v/v) gave good sharp
and symmetrical peak with Rf value of 0.57 for Donepezil
HCl. Well defined spots were obtained when the chamber
was saturated with the mobile phase for 30 min with filter
paper at room temperature.
3.2.Calibration curves: The linear regression data for the
calibration curves indicate that the response is linear over
the range 41.20 to 123.60 ng/spot for Donepezil HCl with
coefficient of regression, r2, value as 0.9976. The value of
correlation coefficient, slope and intercept were 0.9987,
26.95 and 272.43, respectively.
3.3.Validation of the method
3.3.1.Precision: The % RSD for repeatability of standard
application is 0.65%. Whereas, the % RSD for repeatability
of sample preparation is 0.62%. This shows that precision
of the method is satisfactory as % relative standard
deviation is not more than ±2.0% and mean recovery
between 98.0 to 102.0%. Table 1 represents the precision
of method.
Table 1: Method precision of Donepezil HCl
Sample
Preparation
% Assay
% Deviation from
Mean Assay Value
1
2
3
4
5
6
Mean
± SD
%RSD
98.32
99.92
98.87
98.20
98.79
98.72
98.80
0.61
0.62
-0.49
1.13
0.07
-0.61
-0.01
-0.08
3.3.2. Ruggedness and Robustness of the method: The
parameters and results of normal operating condition
(original) against changed conditions are indicated in
Table 2. The low value of % RSD obtained after
introducing the deliberate changes in parameters alters
the results of Donepezil HCl to -0.46% of method
precision study, which is not a significant change. The
ruggedness and robustness of the method is established,
as the % deviation from mean assay value obtain from
precision study is less than ±2.0%.
Table 2: Ruggedness and Robustness of Donepezil HCl
Parameter
Development Distance (from line
application)
Mobile Phase Composition (% v/v/v)
Normal (Original)
of
70%
Changed conditions
75%
Ethyl Acetate: Methanol
Ethyl Acetate:
Toluene: Ammonia
Methanol Toluene:
(8:1.5:8.5:0.4)
Ammonia (8:2:8.0:0.4)
Injection Volume
1 µL
2 µL
Chamber Saturation time with filter paper
30 Minutes
15 Minutes
Analyst
Vinay
Analyst II
% assay, Donepezil HCl
98.80%
98.34%
% RSD from mean assay value obtain in method precision studies is -0.46% of Donepezil HCl
102
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
3.3.3. LOD and LOQ: The signal-to-noise ratios of 3
and 10 were considered as LOD and LOQ, respectively.
The LOD and LOQ for Donepezil HCl is 5.0ng/spot and
20.6ng/spot respectively.
3.3.4
The results of recovery are shown in Table 3. The
results indicate that the individual recovery of Donepezil
HCl ranges from 97.96% to 100.31% with mean recovery
of 98.91% and % relative standard deviation of 0.87%.
The recovery of Donepezil HCl by proposed method is
satisfactory as % relative standard deviation is not more
than ±2.0% and mean recovery between 98.0% to 102.0%.
Recovery studies
% Recovery = % Amount Recovered x 100
% Sum Level
Table 3: Recovery of Donepezil HCl
Sample Preparation
% Simulated
Dosage
Nominal
% Sum
Level
% Amount
Recovered
88.25
84.00
102.75
99.25
114.25
113.00
86.82
84.62
101.88
98.62
111.92
111.12
Pre-analysed Sample
1
80
2
80
1
100
2
100
1
120
2
120
Mean
+ Standard Deviation
% Relative Standard Deviation
3.4.Analysis of the marketed formulation: A single spot
at Rf 0.57 was observed in the chromatogram of the drug
sample extracted from tablets. There was no interference
from the excipients commonly present in the tablets. The
drug content was found to be 99.04% with a % RSD of
0.82%. It may therefore be inferred that degradation of
Donepezil HCl was not occurred in the marketed
formulation that were analyzed by this method. The low
% RSD value indicated the suitability of this method for
routine analysis of Donepezil HCl in pharmaceutical
dosage form.
3.5.Stability- indicating property: The % assay and %
degradation with stress conditions are shown in Table 4
with respective figures in Figure 2. The no treatment
sample (as control) had been evaluated relative to the
standard concentration where as rest of the stressed
condition samples (Sr.No.2 to 6) were evaluated relative
to the control sample with respect to the % assay and %
degradation. The percentage degradation results were
%
Recovery
98.80
98.38
100.31
99.15
99.35
97.96
98.33
98.91
0.86
0.87
calculated by area normalization method. The
chromatogram of the acid degraded sample for donepezil
showed additional peak at Rf value of 0.23 and 0.49
respectively. The chromatogram of the alkali degraded
sample for donepezil showed additional peak at Rf value
of 0.08 and 0.72 respectively. The sample degraded with
hydrogen peroxide showed additional peak at Rf value of
0.14. The spot of degraded product was well resolved
from the drug spot. The photochemical degraded sample
showed additional peak at Rf value of 0.05. Whereas
thermally degraded sample showed no additional peak.
In each forced degradation sample where additional peak
were observed, the response of the drug was changing
from the initial control sample. This indicates that the
drug is susceptible to acid-base hydrolysis, oxidation and
photo chemical degradation. The lower Rf values of the
degraded components indicated that they were less polar
whereas higher Rf values of the degraded components
indicated they were more polar than the analyte itself.
Table 4: Stressed study data of Donepezil HCl
Sr.
No.
1.
2.
3.
4.
5.
6.
Condition
% Assay Donepezil HCl
No treatment (control sample)
Acid
Alkali
H 2O 2
UV
Thermal
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
98.80
87.71
93.94
94.58
97.74
98.30
% degradation
Single maximum
nil
8.87
3.05
4.55
1.94
nil
Total
nil
12.28
5.77
4.55
1.94
nil
103
Figure 2 : Stress Condition Data of Donepezil HCl
AU
AU
(d)
(a)
Rf
Rf
AU
AU
(b)
Rf
AU
(e)
Rf
AU
(f)
(c)
Rf
Rf
Figure 2 chromatograms of Donepezil HCl and its degraded products : (a) Pure drug : Peak1 (Rf 0.57) is of Donepezil
HCl (b) Acid induced : Peak1 (Rf 0.23) is of degraded product, peak2 (Rf 0.49) is of degraded product, peak3 (Rf 0.57) is
of Donepezil HCl. (c) Based induced : Peak1 (Rf 0.08) is of degraded product, peak2 (Rf 0.57) is of Donepezil HCl, peak3
(Rf 0.72) is of degraded product. (d) Hydrogen peroxide induced : Peak1 (Rf 0.14) is of degraded product, peak2 (Rf 0.57)
is of Donepezil HCl. (e) Photochemical degradation : Peak1 (Rf 0.05) is of degraded product, peak2 (Rf 0.57) is of
Donepezil HCl. (f) Thermal degradation : Peak1 (Rf 0.57) is of Donepezil HCl.
104
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
3.6.Detection of the related impurities: The sample
solution showed no additional spots other than principal
spot. Hence no related impurities are present in the market
sample.
4.0. CONCLUSION
The developed HPTLC technique is precise, specific,
accurate and stability indicating. Statistical analysis
proves that the method is reproducible and selective for
the analysis of Donepezil HCl in pharmaceutical dosage
form. The method can be used to determine the purity of
the drug available from various sources by detecting the
related impurities. As the method separates the drug from
its degradation products, it can be employed as a stability
indicating one.
4.
Andrisano V, Bartolini M, Gotti R, Cavrini V, Felix G.
Journal of Chromatography-B: Biomedical
Applications. 2001; 753(2): 375-383.
5.
Oda YS. 2000; 18(6): 508-517.
6.
Matsui K, Oda Y, Nakata H, Yoshimura T. Journal of
Chromatography-B: Biomedical Applications. 1999;
729(1-2): 147-155.
7.
ICH. Q1A Stability Testing of New Drug Substances
and Products. International Conference on
Harmonization, Geneva; 1993 October.
8.
Sethi PD. High performance Thin-Layer
chromatography: quantitative analysis of
pharmaceutical formulations. New Delhi: CBS; 1996.
p.3-62.
9.
Fried B, Sherma J. Thin-Layer chromatography:
Techniques and application. 3rd ed. New York: Marcel
Dekker; 1994. p.11-22.
Acknowledgements
The authors thank Sun Pharma India Ltd for gift
sample of Donepezil HCl and Anchrom Labs., Mumbai,
for their instrumental facilities for the project.
References
1. The Merck Index. Monograph No. 3453. 13th ed. NJ:
Merck and Co; 2001. p.602.
2.
3.
Gotti R, Cavrini V, Pomponio R, Andrisano V. Journal
of Pharmaceutical and Biomedical Analysis. 2001;
24(5-6): 863-870.
Katayama H, Ishihama Y, Asakawa N. Journal of
Chromatography-A. 1997; 764(1): 151-156.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
10. Elke Hahn – Dienstrop. Applied Thin-Layer
chromatography: best Practice and avoidance of
mistakes. Germany:Wiley-VCH; 2000. p.67-109 and
201-207.
11. ICH. Q2B Validation of Analytical Procedure:
Methodology. International Conference on
Harmonization, Geneva; 1996 March.
105
Research Article
Pharmaceutics
Chitosan-Based Nanoparticles for Delivery of
Proteins and Peptides
V.J.Mohanraj1 , Y.Chen2 and B.Suresh3
1Orchid Chemicals & Pharmaceuticals Limited, Chennai
School of Pharmacy, Curtin University of Technology, Perth, Australia
3J.S.S. College of Pharmacy, Ooty
2
V.J. Mohanraj
Received on 08.06.2005
Accepted on 05.08.2005
ABSTRACT
A simple and effective method was developed to formulate biodegradable nanoparticles for the delivery of a model
protein-bovine serum albumin (BSA) and an angiogenesis inhibitor, arginine-rich hexapeptide (ARH peptide). Major
factors, which determine nanoparticle formation and loading of the protein and the peptide as well as the underlying
mechanisms controlling their incorporation and release characteristics, were investigated. The preparation technique,
based on complex coacervation process, is extremely mild and involves the mixture of two aqueous solutions (chitosan
and dextran sulphate) at room temperature. The formation of nanoparticles is dependent on the concentrations of
chitosan (CS) and dextran sulphate (DS); particles with size, of 257 to 494nm and zeta potential from -34.3mV to +52.7mV
obtained with 0.1%w/v solutions of CS and DS, can be modulated conveniently by varying the composition of the two
ionic polymers. Furthermore, using both BSA and the ARH peptide it was shown that these nanoparticles have a great
protein loading capacity (incorporation efficiency up to 100% and 75.9% for the protein and peptide respectively) and
provided a continuous release for up to 7 days for both the macromolecules with a significant difference in the release
behaviour, which could be due to the effect of molecular size of the compounds and their interaction with the polymer
matrix of the nanoparticle.
INTRODUCTION
The development of appropriate vehicles has
become a meaningful challenge for pharmaceutical
scientists to deliver unstable macromolecule compounds
like peptides, proteins, oligonucleotides and genes. As
their efficacy is highly limited by their ability to cross the
biological barriers and reach the target site along with
their need for protection from degradation in the biological
environment, its future as therapeutic agents clearly
depends on the design of an appropriate vehicle for their
delivery to the body.1
Several strategies have been explored so far, among
which the development of nanoparticles from hydrophilic
polymers as drug carriers has received considerable
attention in the last few years. The nanoparticulate
system has one advantage that is if the drug particles
are unstable physicochemically or biochemically in a
particular environment, the solid nanoparticles can be
converted to polymeric nanoparticulate systems in which
the drugs get encapsulated in the form of a solid solution
or dispersion or adsorbed on the surface or chemically
bound. Such a drug-polymer hybrid or combined system
offers the loaded drug more delivery properties.2,3
The particle size and distribution, surface charge,
and hydrophilicity determine the functions of
nanoparticles, which can also be modified by selecting
appropriate polymer materials. Away from that the
particulate drug delivery systems have also shown to
influence the pharmacokinetics of drug molecules in the
body, nanoparticulate system was chosen to provide the
106
controlled delivery of the protein.2,3 Recently, the use of
nanoparticles made up of CS has drawn much interest
due to their association and delivery of labile
macromolecular compounds.
CS, [b -(1® 4)-2-amino-2-deoxy-D-glucose] is a
deacetylated form of chitin, an abundant polysaccharide
widely distributed in nature as a principal component of
crustaceans shells and insects. 4 This cationic
polysaccharide CS has favourable properties as a
pharmaceutical material because of the important
properties such as mucoadhesivity, biocompatibility and
low cytotoxicity which is related to the nature of the salt
used and the molecular weight of the polymer.5
In addition, CS has shown the special character of
gelling on contact with anions to form beads under mild
conditions. These CS beads showed a pH- dependent
swelling and solubilising behaviour, which makes them
appropriate for the delivery of drugs in the gastric cavity
or to acidic, compartments such as endosome or
lysosome. 6,7 The latter is a quality that may give
advantages to these nanoparticles in the field of gene
therapy.8
In our study we have modified a method reported
by calvo et al9 to create a new type of hydrophilic
nanoparticles and evaluate their efficacy for the
incorporation and controlled release of an angiogenesis
inhibitor, arginine-rich hexapeptide (ARH peptide). Initially
the study was done using a model protein Bovine serum
albumin (BSA).
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
MATERIALS AND METHODS
Materials
The polymer CS (medium molecular weight: 400,000
and Catalog no: 22742) was purchased from Fluka /
Sigma-Aldrich, Australia. ARH peptide (Mw: 927, code
no: CS and Batch no: M10384, Purity 95%) was
synthesized by Auspep Pty Ltd, Australia. BSA (Fraction
V, Code no: A-7906); sodium salt of DS (Mw: 10,000 and
Catalog no: D6924), sodium phosphate dibasic (Mw:
141.96); Bradford reagent (Fraction Code no: B-6916) and
fluorescamine (Code no: F-9015) were purchased from
Sigma Chemical Co. (St.Louis, Missouri, USA).
Fluorescamine was dissolved in HPLC-grade acetonitrile.
All other solvents and materials were of analytical grade.
Deionized water (Milli-Q water) was used in the
preparation of buffers and standard solutions of peptide.
Chemicals and reagents used in this study if not specified
were all used as received.
Preparation of Empty and BSA or ARH Peptide Loaded
Nanoparticles
The hydrophilic nanoparticles were prepared using
the complex coacervation of CS and DS. 0.1%w/v CS
solution was prepared by dissolving CS in aqueous acetic
acid (0.175%w/v). DS was dissolved in deionized water
to obtain the same concentration as CS (0.1%w/v).
Finally, variable volumes of the DS solution (3, 5, 8.5 and
10ml) were added to 5mL of the CS solution with magnetic
stirring at room temperature for 15 minutes. The pH of
solutions was then measured and correlated to the
formation of nanoparticles.
Incorporation of the BSA into the nanoparticles was
performed by dissolving BSA in either the polycation CS
or in polyanion DS to obtain a BSA concentration of
1mg/mL in the polymer before mixing with oppositely
charged polymer. The BSA-loaded nanoparticles were
formed spontaneously upon addition of variable volume
of 3mL, 5mL, and 8.5mL (0.1% w/w) of the DS aqueous
solution to 5ml of the CS acidic solution (0.1% w/w) with
magnetic stirring for 15 minutes. The ARH peptide-loaded
nanoparticles were prepared in same fashion as that of
BSA-loaded nanoparticles except the ARH peptide was
dissolved in DS solution instead of in the CS solution,
with its concentration varied from 0.27 mg mL-1 to 0.36
mg mL-1. However, the final concentration of ARH peptide
in the mixture of CS/DS was always kept between 0.18mg
mL-1 to 0.20 mg mL-1.
Determination of Nanoparticle Yield
The yield of nanoparticles was determined by
decanting the supernatant after the separation of
nanoparticles from aqueous suspension medium by
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
centrifugation of solution at 15,000g for 15 min at 4°C
and drying the nanoparticles at 60°C to a constant weight.
The same volume of water was used as a control.
Physicochemical Characterization of CS-Based
Nanoparticles
The morphology examination of nanoparticles was
performed using a transmission electron microscopy
(TEM). Samples were prepared by dispersing dried
nanoparticles powder in distilled water and a drop of
sample solution was placed on the top of the copper
grids, allowed to air dry and then coated with carbon.
TEM pictures were taken in CM 12 Philips transmission
electron microscope.
Measurements of particle size and zeta potential of
the nanoparticles were performed by photon correlation
spectroscopy and laser Doppler anemometry, respectively
using a Zetasizer 3000HS (Malvern Instrument, UK). The
particle size measurements were performed using a quartz
cell in the automatic mode. The particle size analysis of
each sample was performed at 25°C with a detection angle
of 90º and ten repeat measurements,10 the raw data were
subsequently correlated to Z average mean size by
cumulative analysis, performed by the zetasizer 3000 HS
software package. The zeta potential measurements were
determined by diluting the samples in deionised water
and measured in automatic mode using a Malvern
Zetasizer.
Evaluation of BSA or ARH Peptide Loading Capacity of
Nanoparticles
The amount of BSA or ARH peptide entrapped in
the nanoparticles was calculated by the difference
between the total amount protein/peptide added into the
nanoparticle formation medium and the amount of nonentrapped protein/peptide remaining in the aqueous
supernatant. This latter amount was determined following
the separation of protein/peptide loaded nanoparticles
from the aqueous suspension medium by ultra
centrifugation at 15,000rpm and 40c for 15min.1 The amount
of free protein in the supernatant was determined by
Bradford protein assay 11 whereas of peptide by
fluorescence assay method. The protein/peptide loading
of the nanoparticles and their incorporation efficiency
(IE) were calculated from the equation below.
Total amount of protein / peptide (mg) – Free
amount of protein / peptide (mg)
Loading (%) =
x 100
NS weight (mg)
Total amount of protein / peptide – Free
amount of protein / peptide (mg)
IE (%) =
x 100
Total amount of protein / peptide (mg)
107
In vitro BSA or ARH peptide release from CS – Based
nano-particles
A known quantity of protein loaded nanoparticle
suspension was centrifuged at 15,000 rpm for 30 min at
4ºC. The supernatant solution was decanted and the
collected nanoparticles were then re-suspended and
incubated in 5mL of an aqueous 10mM phosphate buffer
pH 7.4 or water with controlled agitation at 37ºC. The
quantity of nanoparticles was adjusted to obtain a BSA
concentration of 1mg mL –1 per release study. At
designated time intervals samples were centrifuged (15,000
rpm) and 5mL of the supernatant were removed and
replaced by an equal volume of the fresh medium (10mM
phosphate buffer pH 7.4). The amount of BSA released
at various time intervals was determined using the
Bradford protein assay method. BSA calibration curves
were made with fresh BSA dissolved in the incubation
medium, whereas, for ARH-peptide a known quantity of
nanoparticles was collected by centrifugation at 12,000
rpm for 30 min at 4ºC. The nanoparticles were
resuspended and incubated in 1.45 mL of 10mM
phosphate buffer (pH 7.4) or water at 37ºC with controlled
agitation. The quantity of nanoparticles was adjusted to
obtain a final peptide concentration of 0.2mg mL-1(instead
of 1mg mL-1 as per BSA release study) because of the
limited availability of the ARH peptide and its highly
sensitive fluorescence assay. At varying time intervals,
supernatants were isolated by centrifugation at 12,000
rpm for 30 min at 4ºC and measured by the fluorescence
assay method. Peptide calibration curves were made with
water or 10mM phosphate buffer pH 7.4. The
centrifugation speed of 12,000 rpm used for peptide
release study is different from that of BSA study, in which
15,000 rpm was used. This is due to the high cost of
peptide and we used small volumes of peptide-loaded
nanoparticle suspension for the release study,
consequently we used a micro-centrifuge (Minispin
Eppendorf, Germany), which has a maximum speed of
12,000 rpm, for separation of nanoparticles from the
release medium. In both the cases measurements were
performed in triplicate.
RESULTS AND DISCUSSION
In this study the nanoparticles are composed of
cationic and anionic polymers (CS and DS) to incorporate
BSA or ARH peptide via ionic interaction. Though CS
and DS matrices have been used in the pharmaceutical
industry for many years, to our knowledge little
information concerning the interaction between DS and
CS has been reported. Sufficient charge numbers are
necessary for anions to cross-link CS by electrostatic
force. DS is a polyvalent anion and carries 65 negative
charges per mole, equivalent to 6.5 x 10-3 sulphate groups
per gram DS. On the other hand, CS used in our
108
experiment is a weak poly base with 2259 positive charges
per mole, equivalent to approximately 5.6 x 10-3 amino
groups per gram CS. However, the net charge number of
the DS and CS are mainly controlled by the solution pH.12
CS, at a low solution pH (especially less than pKa of CS
6.3), has a high degree of ionization due to the amine
groups. In this study, pH of CS (3-4) is well below its
pKa, hence, degree of ionization of amino groups of CS
is expected to be greater than 99%, whereas, DS’sulphate
group is negatively charged, thereby resulting in ionic
interaction between CS and DS.
Incorporation of BSA-loaded CS-Based nanoparticles
BSA-loaded nanoparticles were obtained
spontaneously upon addition of variable volumes (3mL,
5mL and 8.5mL) of the DS aqueous solution (0.1%w/v)
to 5mL of the CS solution (0.1% w/v) with magnetic
stirring. The incorporation of BSA in the CS/DS
nanoparticles was achieved by dissolving the protein
either in the CS solution before mixing with DS or in the
DS solution prior to mixing with CS solution. The
influence of the matrix materials ratio on the physicochemical characteristics of empty and BSA–loaded
nanoparticles is presented in Table 1. The change of zeta
potential in both sign and magnitude, when comparing
empty to the BSA loaded nanoparticles prepared from
CS/DS ratio of 5mL:3mL (1.67:1) to 5mL:8.5mL (0.5:1),
clearly supports the hypotheses that BSA association
with nanoparticles can be modulated by its ionic
interaction with oppositely charged ionic polymer (DS in
our case) in the nanoparticles.
The increase in particle size, with corresponding
reduction of zeta potential (Table 1) of these BSA-loaded
nanoparticles, compared to the empty ones, is also a good
indication of the incorporation of BSA in the
nanoparticle’s structure. The empty nanoparticles
prepared from the CS/DS ratio 5mL:3mL (1.67:1) to
5mL:8.5mL (0.59:1) showed a change in surface charge
from positive to negative as the volume of DS was
increased. This change in charge is attributed to the
presence of negatively charged DS at the particle surface
as there are more sulphate groups than amino groups in
the formulation, when CS/DS ratio increases to 5mL:8.5mL
(0.59:1) based on molar ratio calculation of the CS and
DS molecules. On the other hand, the increase in size
was observed when BSA was incorporated suggests that
BSA might have formed close association with
nanoparticle matrix material. BSA-loaded nanoparticles
had a greater reduction in zeta potential than
corresponding empty nanoparticles as the CS/DS ratio
increases from 5mL:3mL (1.67:1) to 5mL:8.5mL (0.59:1)
(Table 1). This difference could be due to the various
binding / association mechanisms BSA involved with
nanoparticles at different CS/DS ratio.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
In order to assess BSA mixing, characteristics and
incorporation efficiency of nanoparticles, we compared
BSA-loaded nanoparticles prepared by either dissolving
the BSA in CS or in DS. No significant difference was
found in most of their physico-chemical characteristics
of the CS/DS nanoparticles with exception of zeta
potential (Table 2).
Results of the incorporation efficiency and protein
loading capacity of different ratios of nanoparticles are
displayed in Table 3. It was seen that the BSA
incorporation efficiency was affected by the ratio of DS,
the higher the DS ratio the higher the incorporation
efficiency. The maximum incorporation efficiency 100%
was achieved with CS/DS ratio of 5mL:8.5mL (0.59:1).
However, the protein loading capacity reached maximum
when CS/DS ratio was 5mL:5mL (1:1) with 33.7 mg of
BSA incorporated in 100mg of nanoparticles. This
loading is best when compared to our calculation of
theoretical loading (i.e. the highest loading capacity).
Comparing to results reported by Calvo et al.13 we
obtained a much higher incorporation efficiency (53.2100%) at the equivalent pH conditions (Table 3). It is
worth pointing out that incorporation efficiency was
determined directly whereas the loading capacity value
was calculated based on the nanoparticle yield and
incorporation efficiency.
Table 1:
Table 2:
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
109
Protein association studies carried out by the
Calvo’s group at different pH values 13 indicated that,
there was significant BSA incorporation at all pH values
but the greatest loading efficiency was obtained when
the protein was dissolved at a pH above its isoelectric
point, (i.e. when BSA was predominantly negatively
charged). This suggests the major factor that lead to the
association of protein to the nanoparticle might be the
protein-polysaccharide electrostatic interaction as well as
other forces including hydrogen bonding, hydrophobic
interactions and the reduction of the solubility of BSA
near its isoelectric point. In our case, BSA was positively
charged when it was dissolved in CS solution (pH 3.573.63), therefore capable of establishing ionic interaction
with negatively charged DS during the formulation
process. The fact that the incorporation efficiency
increased with the DS ratio and the magnitude of negative
charge of nanoparticles strongly supports that ionic
interaction is the major factor contributing to the
incorporation of BSA with nanoparticles prepared by
dissolving BSA in CS, whereas, a cloudy preparation was
obtained when BSA (concentration 1mg mL-1 ) was
incorporated into a DS solution (pH 4.6-4.8). Taking into
account that the isoelectric point of BSA is 4.8, and at
pH near 4.8, the BSA is expected to have minimum charge
and therefore the lowest solubility, which might have
resulted in a cloudy preparation when it was mixed with
DS suspension. It is possible, in this case, that in addition
to electrostatic interaction, other mechanisms such as
hydrophobic interactions, hydrogen bonding, and other
physicochemical forces including physical incorporation
could also be responsible for the incorporation and
association of BSA with nanoparticles.
Incorporation of ARH peptide loaded into
CS-Based nanoparticles
For incorporation of ARH peptide into nanoparticles,
we selected three different ratios of CS/DS to formulate
nanoparticles; 5mL:5mL (1:1), 5mL:8.5mL (0.59:1) and
5mL:10mL (0.5:1). This selection is based on our
observation that when the ratio of CS/DS is 5mL:5mL
(1:1) or above (i.e. even higher proportion of DS) in the
formulation, the surface charge of the empty nanoparticles
is negative, which is consistent with our prediction based
on the calculation of charge ratio between the two. Since
the ARH peptide is positively charged, we chose these
three ratios, with an aim to achieve a strong ionic
interaction between the peptide and CS/DS
nanoparticules, which in turn might encapsulate
appreciable quantities of ARH peptide.
ARH peptide-loaded CS/DS nanoparticles were
prepared by the same protocol as described for the BSA
study. In all cases, however, the peptide was dissolved
in the DS solution. This is because dissolving the peptide
110
in CS solution may produce a repulsion between CS and
the peptide, owing to their positive charges. From our
BSA study, it is noted that dissolving BSA in either of
the polymer solutions, does not have a great impact on
the results. However, it was envisaged that electrostatic
interactions between the amino groups of the peptide
and the sulphate groups of DS might facilitate the
association of peptide to the CS/DS nanoparticles. Table
1 compares the size and surface charge of peptide-loaded
CS/DS nanoparticles prepared from different CS/DS ratios.
Similar to the findings in the BSA study,
incorporation of peptide into nanoparticles also resulted
in augmentation of both size and zeta potential of
nanoparticles (Table 1). The increase in particle size and
change in the magnitude of zeta potential (Table 1) of
these nanoparticles is a good indication of the
incorporation of peptide in the nanoparticle’s structure.
These results are in consistent with the BSA study. As
stated earlier, it is not surprising that an increase in
concentration of DS led to the reduction of the size of
the nanoparticles. The increase in the magnitude of zeta
potential leads to more stable colloidal dispersion. Results
presented in Table 1 indicate that the particle size is, as
previously reported,9 dependent upon the CS
concentration, the minimum size of nanoparticles
corresponding to the lowest CS concentration. This could
be due to the reduction of viscosity of CS solution after
addition of DS. The CS/DS empty nanoparticles with CS/
DS ratio 5mL:5mL (1:1) to 5mL:10mL (0.5:1) showed an
increase in surface charge towards more negative as the
ratio of DS was increased. This negative charge could
only arise from the excess DS adsorbed on the surface of
particles. On the other hand, increase in size was observed
when peptide was incorporated, suggesting that peptide
has formed close association with nanoparticle matrix
material. The charge of peptide-loaded nanoparticles,
changed from positive to negative as the ratio of the DS
in the formulation increased, leads to believe that almost
all the ARH peptide might have been incorporated into
the nanoparticles. But these speculations are not
supported by the ARH peptide loading and entrapment
efficiency data (Table 3).
The loading capacity of peptide for 5mL:5mL (1:1)
and 5mL:8.5mL (0.59:1) CS/DS nanoparticles ratios is
12.3% w/w and 13.4% w/w respectively, with entrapment
efficiency of 72.9% w/w and 75.9%w/w (Table 3). These
results were different from those obtained in the BSA
study. Based on the BSA study, one would expect the
higher the concentration of DS, the higher the
encapsulation efficiency. In fact this hypothesis failed in
the case of 5mL:10mL (0.5:1) CS/DS ratio. The loading
capacity as well as incorporation efficiency (10.9%
w/w and 36.3% w/w respectively) of 5mL:10mL
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Table 3:
Table 4:
(0.5:1) ratio nanoparticles is lower than the other two
ratios studied, despite the fact that a larger amount of
DS was used. It is speculated that this decrease in loading
level might be due to the smaller initial amount of ARH
peptide added to DS solution, resulting in lower
theoretical loading as expected. However, the much lower
incorporation efficiency (36.3% w/w) is unexpected. The
duplicate preparation confirmed this level (Table 4),
suggesting factors other than experimental error may be
the cause. One possible explanation is the formation of
soluble ionic complex between ARH peptide and DS. It
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
can be noted from the results in Table 3 that there is no
clear trend between the CS/DS ratio and incorporation
efficiency. Though the maximum incorporation efficiency
75.9% w/w was obtained with 5mL:8.5mL (0.59:1) ratio of
CS/DS nanoparticles, this level is much lower when
compared to the BSA study which achieved 100% w/w
with the same formulation. When the loading and
incorporation efficiency of ARH peptide was compared
with that of BSA study, it can be seen the actual loading
of peptide appeared much lower than that of BSA, largely
due to a smaller amount of ARH peptide added (only
111
about half of BSA quantity was used) to the starting DS
solution. The loading efficiency, however, is consistently
at 80 to 81% level, suggesting the actual loading is
affected more by the initial amount of ARH peptide in
DS solution, rather than CS/DS ratio, which is contrary
to what is seen with BSA incorporation into nanoparticles.
It is suspected that the huge difference in the molecular
size of BSA and ARH peptide may had an impact on the
incorporation. The molecular size of BSA (Mw 60,000) is
much larger than that of ARH peptide whose molecular
weight is only 927, and these small molecules could have
readily diffused out through the polymer matrix of
nanoparticles to establish the equilibrium and also form
soluble ionic complex with DS. This in turn could have
resulted in a low ARH peptide loading.
Pan et al. reported the insulin loading capacity and
their incorporation efficiency were affected by
concentration of insulin in the opposite charged
polyanion tripoly- phosphate and the amount of insulin
added,14 with increasing ratio of insulin to CS resulted in
slight decrease of entrapment efficiency but a great
increase in loading capacity. In our case 5mL:5mL (1:1)
ratio showed a slight increase in loading and entrapment
efficiency compared to that of 5mL:8.5mL (0.59:1), it might
be due to relatively higher initial concentration of ARH
peptide (0.32mg mL-1) in DS solution with 5mL:8.5mL
(0.59:1), whereas only 0.27mg mL-1 ARH peptide in DS
solution was used with 5mL:10mL (0.5:1) ratio, despite
the final concentration of ARH peptide in the CS/DS
mixture was very similar (0.20 and 0.18mg mL - 1
respectively). This suggests that for the 5mL:10mL (0.5:1)
CS/DS ratio, the loading capacity and incorporation
efficiency, may be affected by the initial concentration of
ARH peptide in the DS solution. It could be more
comparable if the initial level of ARH peptide
concentration was kept the same. Due to high cost and
availability of ARH peptide, we had to modify the
quantity in the study.
Morphology of empty, BSA/ARH Peptide-loaded CS/DS
nanoparticles
Transmission electron microscopy (TEM) images of
BSA and peptide-loaded CS/DS nanoparticles prepared
from the 5mL: 8.5mL (0.59:1) ratio are shown in the figure1.
ARH peptide-loaded CS/DS nanoparticles exhibited a
solid structure but different from that of BSA-loaded
nanoparticles in shape. They appear to be dense, nonsmooth and non-spherical structure.
In-Vitro release of BSA from CS-Based nanoparticles
Albumin nanoparticle formulations, prepared under
the experimental conditions were tested for in vitro
0
release at 37 C. Figure 2 shows the cumulative percentage
release of BSA in vitro from nanoparticles prepared with
various ratios of CS/DS, as a function of time. All data
112
Figure 1: Electron transmission microphotography of (a)
BSA loaded CS/DS nanoparticles [(CS/DS 5mL:8.5mL)
(b)ARH peptide loaded CS/DS nanoparticles.
Figure 2: Cumulative release of BSA loaded CS/DS
nanoparticles in: (a) Water; (b) 10mM phosphate buffer
(pH 7.4)
shown are the mean + standard deviation (n=3).
It is seen that the release rate is highly affected by
the nature of the interactive forces between the associated
BSA macromolecule and the matrix material as well as by
the ionic nature of the release medium. The interesting
observation was that a consistent low portion of BSA
(less than 13% loaded BSA) was released in water over
the 7-day period. The majority release occurred in the
first two time points (12 and 24hrs) irrespective of the
ratio of CS/DS (Figure 2a). In comparison, the same batch
of nanoparticles, when exposed to 10mM phosphate
buffer pH 7.4, showed continuous release of BSA over 7
days (Figure 2b). There was an initial small burst release,
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
corresponding to the level of release of BSA in water,
suggesting that the burst release may arise from the
desorption of those loosely attached BSA from the
surface of the matrix polymers. Though dissociation
appears to be the principal mechanism, other factors, such
as diffusion of physically entrapped BSA, may also have
a role in the release process.
It is noted, when 10mM phosphate buffer pH 7.4,
was used as a release medium, that BSA released at a
relatively continuous rate for 5:5 (1:1) and 5:8.5 (0.59:1)
ratios of CS/DS nanoparticles. It is seen that 31% and
40% of BSA were released in 7 days for 5mL:8.5mL (0.59:1)
and 5mL:5mL (1:1) ratio of CS/DS nanoparticles,
respectively. It appears that 5mL:5mL (1:1) ratio
nanoparticles released BSA faster than that of the
5mL:8.5mL (0.59:1) system. It depicts the higher the
concentration of DS in the nanoparticles preparation
medium, the slower was the release of BSA from the
nanoparticles. The dissociation of BSA from
nanoparticles via ionic-exchange appears to govern the
release process in the second phase i.e. from day 2
onward.
The small size of the nanoparticles is also a major
factor, which influences the release rate. These
nanoparticles have a large surface area due to their small
size; therefore a significant portion of the BSA will be at
or near the particle surface and can be readily released.
Furthermore, the diffusion distances encountered in the
particles are small which allows the release medium and
the ions to diffuse in readily to exchange with BSA.
However, due to the large molecular size of BSA, it is
expected to diffuse out slowly even when it becomes
dissociated.
It is noted that the in vitro release rate behavior of
BSA-loaded CS/DS nanoparticles was affected by their
level of loading. Calvo et al.9,13 observed the percentage
in vitro release of BSA from CS/DS nanoparticles was
greater for those formulations containing a higher protein
loading; our finding is consistent with their observation.
In our study the BSA loading capacity of 5mL:8.5mL
(0.59:1) ratio CS/DS nanoparticles is 24.1% w/w compared
to that of 5mL:5mL (1:1) which is 33.7% w/w, and the
release rate of 5mL:5mL (1:1) ratio of CS/DS nanoparticles
is faster than that of 5mL:8.5mL (0.59:1) ratio in 10mM
phosphate buffer pH 7.4. This might also be attributed to
the higher proportion of DS in nanoparticles, in other
words, a larger proportion of positively charged BSA
might be bound to the negative DS in 5mL:8.5mL CS/DS
nanoparticles via ionic interaction, therefore, released
slowly. Overall these results suggest that the level of
BSA released from nanoparticles can be modulated,
simply by adjusting the composition of the CS/DS in
nanoparticles or the protein loading.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
In-vitro release of ARH peptide from CS-Based
nanoparticles
ARH peptide nanoparticles, prepared under the
experimental conditions described in Table 3 & 4, were
0
evaluated for in vitro peptide release at 37 C. Figure 3
show the plot of the release data expressed as cumulative
percentage of ARH peptide in vitro from the CS/DS
nanoparticles against time.
In most cases, the peptide appeared to be released
in a biphasic fashion, characterized by an initial rapid
release period followed by continuously slower release.
Different levels of initial release or burst effect were
observed for different ratios of CS/DS nanoparticles
(Figure 3a) in10mM phosphate buffer pH 7.4, which is
different from the results obtained from the initial BSA
study.
On the other hand, when deionised water was used
as the release media, 5mL:5mL (1:1) CS/DS ratio
nanoparticles showed a level of release less than 10% in
7 days, which was consistent with the BSA study results,
whereas, 5mL:10mL (0.5:1) ratio CS/DS nanoparticles
exhibited an initial burst release of 28% peptide release
in 12 hours (Figure 3b). After this initial release, the ARH
Figure 3: Cumulative release of ARH peptide loaded CS/
DS nanoparticles (a) in 10mM phosphate buffer (pH 7.4)
(b) in water.
113
peptide was released in a much lower level for up to 7
days reaching percentage of cumulative release close to
58% for 5mL:10mL (0.5:1). The same trend is also seen in
the buffered media. This is in total contrast to the initial
BSA study, which showed a very much lower level of
BSA release in water than that in buffered media. This
finding suggests disassociation of peptide from CS/DS
nanoparticles may not be controlled by ion-exchange
process.
In 10mM phosphate buffer pH 7.4, the ARH peptide
released at various rates from the three different ratios of
CS/DS nanoparticles (Figure 3a): 22%, 23% and 68% of
ARH peptide released in 7 days by 5mL:5mL (1:1),
5mL:8.5mL (0.59:1), and 5mL:10mL (0.5:1) ratio CS/DS
nanoparticles respectively. It demonstrates the higher the
ratio of DS in the formulation, the faster the release of
peptide from the nanoparticles, which is contrary to the
findings in the BSA study. From the initial BSA study,
we noted that the higher the DS ratio, the slower the
release of BSA in buffered media. However, the ARH
peptide-loaded nanoparticles prepared from the 5mL:10mL
(0.5:1) ratio of CS/DS produced an unexpected result. To
confirm the finding, a separate batch of nanoparticle
formulation was prepared using 5mL:10mL (0.5:1) ratio
and its release was assessed. The results obtained are
the same. It appears that factors other than experimental
error may be the cause. We speculate that the release
behaviour of the three types of nanoparticulate
formulation might be influenced by their particle size, zeta
potential and its related high dispersibility and stability
of nanoparticles. From Table 4, it can be seen that an
increase in concentration of the DS results in a decrease
in size of the nanoparticles and an increase in magnitude
of zeta potential. It is well known that the small size of
nanoparticles has a major influence on the release rate.15
These particles have a high surface area per volume;
therefore most of the peptide will be at or near the particle
surface and can be readily released. Furthermore, the
diffusion distances encountered in the particles are small
which allows drug trapped in the core to rapidly diffuse
out and also for the release medium to diffuse in.
Comparing the system of 5mL:10mL (0.5:1) with that of
5mL:8.5mL (0.59:1), although both showed equivalent
particle size, the latter did have a much smaller zeta
potential. This suggest that the nanoparticles of
5mL:8.5mL (0.59:1) ratio of CS/DS may not be very stable
as a dispersion and have a high tendency to coagulate,
therefore increasing particle size. Indeed, it was noticed
that the system of 5mL:10mL (0.5:1) ratio is more readily
dispersed whereas the system of 5mL:8.5mL (0.59:1) tends
to aggregate during the study.
114
CONCLUSIONS
In this study we developed a novel biodegradable
nanoparticle system solely made of hydrophilic polymers,
i.e. CS and DS, for the delivery of BSA and ARH peptide.
Major advantages of this nanoparticle technology include
that the process is very simple, and nanoparticles can be
prepared in a short time and under extremely mild
conditions with ionic cross-linkage, without involving
high temperatures or sonication. The physicochemical
characterization of these nanoparticles revealed that the
particle size and surface charge are dependent on the CS
and DS concentration and ratio. They can be modulated
by varying the ratio of two ionic polymers. A high loading
capacity and a continuous release of a protein over
extended periods of time can be achieved readily. For the
small molecule ARH peptide, a reasonable incorporation
with entrapment efficiency of 75% was also achieved with
the optimum CS/DS ratio being 5mL:5mL (1:1) to
5mL:8.5mL (0.59:1). The release of BSA from the
nanoparticles was slow and relatively constant, whereas,
ARH peptide showed a similar slow release with 5mL:5mL
(1:1) and 5mL:8.5mL (0.59:1) ratio nanoparticles but at
high DS ratio of 5mL:10mL (0.5:1) the release was in a
biphasic fashion with an initial large burst release followed
by a very slow rate up to 7 days in 10mM phosphate
buffer pH 7.4. It was found that the slow release of BSA
was correlated to the high DS ratio in nanoparticles
formulation, whereas, the ARH peptide only showed the
slow release at CS/DS ratio of 5mL:5mL (1:1) and
5mL:8.5mL (0.59:1) but not 5mL:10mL (0.5:1). The
differences observed with BSA and ARH peptide release
behaviour could be possibly due to the differences in
their molecular size, their entanglement with polymer
matrix structure and solubility of ionic complex formed.
A better understanding of the release mechanism of
proteins and peptides from these CS/DS nanoparticles
obviously is required in the future study. This will provide
a basis for their further optimization, thus opening more
exciting opportunities for improving the administration
of Biomolecules. But all other interesting features render
this novel nanoparticle system as a very promising vehicle
for the formulation of therapeutic proteins and peptide.
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115
Pharmacology
Rhabdomyolysis
Milind Parle, Mamta Farswan and Arvind Semwala
Pharmacology Division, Dept. of Pharm. Sciences, Guru Jambheshwar University, Hisar 125 001
a Torrent Research Center, Ahmedabad
Received on 04.05.2005
Accepted on 29.06.2005
The term rhabdomyolysis denotes disintegration of
striated muscles causing exudation of muscular cell
contents into the extra cellular fluid and blood, and
consequently resulting in excretion of myoglobin in the
urine. About 10-40% of patients with rhabdomyolysis
develop acute renal failure. Rhabdomyolysis is known
since ancient times.1 Long long ago, the Israelites
developed myolysis during their departure from Egypt
due to abundant consumption of quails. Myolysis due
to quails is now well known in the Mediterranean region.
This myolysis is the result of intoxication by Hemlock
herbs, which are eaten by quails during their spring
migration.2 In Modern English Literature, Bywaters and
Becall reported the first case of myolysis followed by
acute renal failure in four war victims during the bombing
of London and the battle of Britain in 1940.3 In 1970,
nontraumatic cases of rhabdomyolysis were recognized
as a potential cause of acute renal failure.4 Patients with
additional risk factors (such as concomitant diabetes, old
age, hypothyroidism, liver or renal disease) need close
monitoring as they may be more at risk of
rhabdomyolysis.5 Myalgia is clinically manifested by
muscle weakness, tenderness or pain in a muscle either
in a proximal or regional pattern. Patients often describe
of a feeling of cramps in the muscles.6 The severity of
rhabdomyolysis may range from a sub clinical rise of
creatine kinase to a medical emergency comprising of
interstitial and muscle cell edema followed by
myoglobinemia and pigment induced acute renal failure.7
CAUSES OF RHABDOMYOLYSIS
Rabdomyolysis is a result of wide array of causes
and habits. The most important causes are alcohol
dependence, muscle compression, seizures, hereditary
metabolic derangements, drug abuse and infections.
1. Trauma and compression: Traumatic rhabdomyolysis
occurs mainly as a result of traffic or occupational
accidents. Compression of muscles may also be
induced by torture or long-term confinement in the
same position.
2. Occlusion of muscular vessels: Thromboembolism,
cramping of blood vessels during surgical procedures
may all result in muscle cell necrosis.8
3. Strenuous exercise of muscles: Strenuous muscular
exercise may cause myolysis in individuals exercising
under extremely hot or humid conditions. Muscular
necrosis more frequently occurs after downhill
116
walking rather than after uphill climbing.9
4. Electric current: High voltage electric shocks and
lightening strikes cause rhabdomyolysis in at least
10% of the subjects surviving the primary accident,
even if the wounds are small. Myolysis is attributable
to thermal injuries, or to electrical disruption of
sarcolemmal membrane.10
5. Hyperthermia: An excessive body temperature may
result in muscle damage. Rhabdomyolysis also results
from neuroleptic malignant syndrome, which is
characterized by high fever in-patients treated with
phenothiazines or haloperidol.11
6. Metabolic myopathies: Hereditary defects in glucose,
lipid, or nucleoside metabolism constitute one of the
rare causes of rhabdomyolysis. In most of the cases,
the final common pathway leading to skeletal muscle
cell disintegration is deficient delivery of ATP
(adenosine tri phosphate), challenging the cell
integrity.12
Abnormal Carbohydrate Metabolism:
Myophosphorylase deficiency, Phosphofructokinase
deficiency,
Phosphorylase
deficiency,
Phosphoglycerate kinase and Phosphoglycerate
mutase deficiency, Lactate dehydrogenase deficiency
(LDH)
Abnormal lipid metabolism: Carnitine deficiency,
Carnitine palmityl transferase deficiency
7. Electrolyte
abnormalities:
Hypokalemia,
hypocalcemia, hyponatremia, hyperosmotic
conditions are associated with rhabdomyolysis.
Myotoxicity due to alcohol is linked with electrolyte
abnormalities.13
8. Drugs: Following categories of drugs can induce
rhabdomyolysis
Antipsychotics and antidepressants: Amitriptyline,
Amoxapine, Doxepine, Haloperidol, Lithium, Loxapine,
Phenelzine.14
Sedatives and hypnotics: Benzodiazepines, Diazepam,
Nitrazepam, Lorazepam.
Lipid lowering agents: Lovastatin, Pravastatin,
Simvastatin, Gemfibrozil, Bezafibrate Clozafibrate,
Nicotinic acid.15,16
Drugs having addiction liability: Heroin, Cocaine,
Amphetamine, Alcohol, Narcotics.17
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Miscellaneous: Amphotericin B, Isoniazid, Diuretics,
Laxatives, Emetics, and Antimalarials.
9. Infections leading to rhabdomyolysis
Locally invasive infections of muscles (pyomyositis),
septicemia and other infections with microbes
causing toxic shock syndrome.
Bacterial infections due to E.coli,Staphylococcus
aureus, Shigella, Salmonella, Leptospira, Legionella.
Viral infections due to Epstein Barr virus, Influenza
virus
PATHOPHYSIOLOGY OF RHABDOMYOLYSIS
Large number of drugs can cause rhabdomyolysis
through various mechanisms. Any drug, which directly
or indirectly impairs the production or utilization of ATP
by skeletal muscles or increase energy requirement so as
to exceed ATP production, can cause rhabdomyolysis
(Figure: 1) Ischemia, prolonged immobilization of a muscle,
over-dosage with CNS depressants, drug induced
delirium, choreoathetosis, dystonic reactions and seizures
can all lead to rhabdomyolysis. Drugs can also be directly
toxic to the skeletal muscle cells interfering with the
production or utilization of ATP.
In rhabdomyolysis, a change in the viscosity of
sarcolemma is caused by activation of phospholipaseA2 as well as various vasoactive molecules and
proteases.18,19 It results in increased permeability of
sarcolemma, permitting leakage of intracellular contents,
as well as increase in the influx of sodium ions into the
cell. The increased intracellular sodium conc. activates
Na + -K+ -ATPase, a process that requires energy. This
eventually exhausts the supplies of ATP and thus,
impairs cellular transport. 20 The increase in the
intracellular conc. of Na + leads to an increase in the conc.
of intracellular Ca ++ ions. This enhances the activity of
the intracellular proteolytic enzymes, leading to further
destruction of intracellular structures. Stretching or
exhaustive work of muscle cells (Figure:1) increase
sarcoplasmic influx of Na + , chloride and water, which
result in cell swelling and auto destruction.21 Large
quantities of Ca ++ inside the muscles trigger persistent
contraction, causing energy depletion and cell death.22
Furthermore, calcium activates phospholipase A2, as well
as various vasoactive molecules and proteases, which
lead to the production of oxygen free radicals. These
free radicals, neutrophils, and proteases produce an
inflammatory, self-sustaining, myolytic reaction rather
than pure necrosis. Severe hypokalemia associated with
significant reduction in intracellular K+ content has been
implicated in the pathogenesis of rhabdomyolysis. In
hypokalemic states glycogen synthesis is impaired
leading to reduction in energy production during
sustained muscle contraction.23 In a normal person,
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
muscle contraction leads to the release of potassium ions
into intracellular spaces. Potassium has a vasodilatory
effect, which enhances blood flow to the muscles during
exercise. In hypokalemic states, there is no release of
potassium from the contracted muscles, and hence no
increase in the blood flow. Continued muscle contraction
in hypokalemic states leads to ischemia and muscle
necrosis consequently.24
Alcohol directly injures the sarcolemma and
increases sodium permeability. These in turn increase the
activity of the Na + -K+ -ATPase pump, with the eventual
exhaustion of energy stores. Analysis of skeletal muscles
from chronic alcoholics and experimental animals fed with
ethanol showed a marked depletion of potassium,
phosphorus, magnesium and elevated sodium, chloride,
calcium and water contents.25 Uniform muscle necrosis,
leukocyte and macrophage invasion of degenerated
muscle fibers is observed during rhabdomyolysis. Ultra
structural changes include the separation of myofibrils
and other cellular elements by clear spaces.26 Severe drug
poisoning is associated with rhabdomyolysis. In the case
of statins rhabdomyolysis is associated with CoQ10
deficiency, which leads to statin toxicity. 27,28 The
mechanism involved varies with the type of statin. CNS
acting drugs cause rhabdomyolysis by pressure induced
ischemia due to prolonged immobilization (Figure: 1).
Drugs such as LSD, sympathomimmetics, phencyclidine
and phenothiazines lead to increased ATP demand and
eventual exhaustion of its stores. HMG- CoA reductase
inhibitor causes rhabdomyolysis by the same
mechanism. 29,30
Hyperthermia also increases the energy requirement
of muscles and contributes to its damage. Cocaine and
salicylate induced rhabdomyolysis is due to hyperthermia.
In ischemic tissue injury (e.g. myocardial infraction, ARF)
most of the damage occurs after the blood flow into the
damaged tissue is restored (reperfusion injury).
Leukocytes migrate into the damaged tissue only after
reperfusion has started, and production of free radicals
starts only, when oxygen is fully available.31
CLINICAL SIGNS AND SYMPTOMS
Muscular symptoms: include muscle pain, weakness,
tenderness, stiffness and cramps. Usually large muscles
of the thighs and lower back are affected in around 50%
of patients showing muscular symptoms. The affected
muscles become swollen and tender on palpation.33
Urinary findings: Most significant diagnostic feature is
the colour of urine. Dark urine, typically brown in colour
is the first clue of rhabdomyolysis. This colour change is
due to the presence of myoglobin in urine. Screening of
rhabdomyolysis includes urine dipstick tests along with
urine microscopy.34 Myoglobin is also intrinsically
nephrotoxic and can precipitate acute tubular necrosis
117
a
118
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
through iron dependent inhibition of oxidative
phosphorylation and iron independent inhibition of
gluconeogenesis.35
Constitutional symptoms: These symptoms vary
depending on the cause of rhabdomyolysis. The most
common symptoms are generalized malaise, fever,
tachycardia, nausea and vomiting. Electrolyte
disturbances can lead to agitation, confusion, disturbed
mental status and low urine output.
Biochemical features: Rabdomyolysis results in the
release of myoglobin, creatine phosphokinase as well as
other cellular contents into the plasma. An increase in
serum myoglobin, creatine phosphokinase, aldolase, lactic
dehydrogenase, potassium, phosphate, purines, uric acid
and aspartate aminotransferase (AST) are all pathological
features observed in rhabdomyolysis. Following are the
pathological features of rhabdomyolysis.
· Elevated creatine phosphokinase: Elevated CPK level
is one of the most important diagnostic features of
the rhabdomyolysis.36
· Hyperkalemia: It is a major cause of morbidity and
mortality in rhabdomyolysis.
· Increased anion gap: The serum anion gap in patients
with rhabdomyolysis is higher than in non-rhabdo
patients. It may be due to unidentified organic acid.
Pathophysiology of acute renal failure:
1)
Myoglobin (pigment) induced intrarenal
vasoconstriction,
2)
Scavenging of the vasodilator nitric oxide in
renal microcirculation by myoglobin.
3)
Tubular obstruction by myoglobin casts or uric
acid crystals.
4)
Direct nephrotoxicity of myoglobin or indirect
toxicity by its metabolite ferrihemate.
5)
Release of vasoactive kinins from muscles may
also add to the damage of the kidneys.38
B) Compartmental Syndrome: This complication is
more common in crush injury. Most striated muscles
are contained within rigid compartments formed by
fasciae, bones and other structures. If, the energy
dependent transcellular energy system fails in the
traumatized tissue, the muscle cells swell. As a result
of the swelling, intracompartmental pressure rises
and produces additional damage and necrosis.
Because, such compartments form a noncommunicating closed system, the only way to
decrease the pressure is to decompress the facial
system surgically by fasiciotomy.
· Elevated blood urea, nitrogen and creatinine
C) Other complications include: Hyperkalemia,
Metabolic acidosis, Respiratory failure, and Cardiac
arrhythmia,39 disseminated intravascular coagulation.
· Elevated calcium and phosphate: Early hypocalcemia
and late hypercalcemia along with hyper
phosphatemia have been demonstrated in patients
suffering with rhabdomyolysis.
Emergency Treatment:The severity of symptoms and
laboratory results will guide treatment decisions. The goal
of initial management is to minimize injury to the renal
tubules with fluids and buffering.
· Elevated uric acid: Marked hyper uricemia is
commonly seen in patients suffering with
rhabdomyolysis. It is due to hepatic conversion of
the purines released from damaged muscle cells.
Saline infusion: In order to prevent renal complications,
early and aggressive saline infusion is advocated. Large
quantities may be required to achieve a urinary output of
200 to 300 ml/hr. Fluids are given in an attempt to i)
prevent injury to the renal tubules, ii) increase renal
perfusion pressure, iii) dilute toxic substances and iv-)
facilitate excretion. In the elderly or those with
compromised cardiovascular or renal function, central
hemodynamic monitoring should be used to prevent fluid
overload. If the desired urinary output cannot be
achieved with fluid administration alone, intravenous
furosemide (40 mg) or 20% solution of mannitol (15 ml/
min) should be considered to aid in diuresis.
Diagnosis: Diagnosis is based on the biochemical
features. Estimation of myoglobin in serum and urine is
useful for the diagnosis of rhabdomyolysis, particularly
in the early phase of the disease. Elevated creatine
phosphokinase is the hallmark of rhabdomyolysis.
Increased level of carbonic anhydrase and hyperkalemia
are the other diagnostic features of rhabdomyolysis.
Rhabdomyolysis is an etiological factor in about 8% of
cases of acute renal failure.37
COMPLICATIONS OF RHABDOMYOLYSIS
A) Acute renal failure (ARF): It is one of the most
frequently
reported
complications
of
rhabdomyolysis:
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Buffering: Alkalinization of the urine is often used in
the treatment of rhabdomyolysis, because myoglobin and
urate are toxic to the tubules in an acidic environment.40
Alkalinization of the urine may be achieved by adding 2
119
ampoules of bicarbonate per liter of saline. The goal is to
keep the urine pH level at 7.5 or higher. If, there happens
to be a problem of renal compromise dialysis is warranted.
includes range-of-physical exercises, both, active and
passive. These may be started, once the patient has been
cleared of the pathological signs.
Blood Tests: Laboratory reports should be obtained as
early as possible during the course of the treatment,
including levels of electrolytes, blood urea nitrogen,
calcium, magnesium, phosphorus, creatinine, and
transaminases, in addition to complete blood count,
prothrombin time, and partial thromboplastin time. Of the
electrolyte abnormalities, hyperkalemia is the most lethal;
therefore, early recognition and treatment are warranted
to avoid cardiac dysrrhythmias.41
CONCLUSION
Drugs: Dantrolene sodium used for 4 days postrhabdomyolysis has been reported to produce
satisfactory relief. As noted earlier, patients who have
rhabdomyolysis have an increased intracellular calcium
level caused by calcium release from the sarcoplasmic
reticulum of the damaged cells.42 In one study, dantrolene
reduced the intracellular calcium level by 83% and was
associated with clinical improvement of muscle pain,
stiffness and rigidity. The site of action of dantrolene is
on the sarcoplasmic reticulum in skeletal muscles.
Dantrolene interferes with excitation contraction coupling
in the muscle fibers. The normal contractile response
involves release of activator Ca ++ from its stores in the
sarcoplasmic reticulum of the sarcomere. This activator
Ca++ brings about the tension generating interaction of
actin with myosin. The activator Ca ++ , normally exits the
sarcoplasmic reticulum via a ryanodine receptor channel.
Dantrolene blocks this ryanodine channel and locks it in
the open position thereby obstructing the exit of activator
Ca++ . Dantrolene is administered intravenously at 2.5 mg/
kg of body weight on day one and orally at 2.0 mg/kg on
days 2 through 4. Dantrolene is the medication of choice
for patients who have a history of malignant hyperthermia
and a clinical picture of rhabdomyolysis. The drug is
administered by continuous intravenous push, starting
at 1 mg/kg and continued until symptoms subside or the
maximum cumulative dose of 10 mg/kg of body weight
has been reached.
Sub-Acute Treatment
Patients, who have milder symptoms with little more
than muscle soreness and a slightly elevated creatine
kinase level may be treated less aggressively in an
outpatient setting. It is important to ensure that the patient
has no difficulty in passing urine and the urinary sediment
is reasonably clear. The patients should be encouraged
to drink large amounts of fluids. An often-overlooked
aspect of treatment is aggressive physical therapy. This
120
Rhabdomyolysis is basically a disease of skeletal
muscles causing exudation of muscular cell constituents
into the extra cellular fluid and blood. Around 10-40% of
patients suffering from rhabdomyolysis develop acute
renal failure. Elevated levels of creatine phosphokinase,
myoglobin, lactate dehydrogenase, purines and uric acid
constitute major biochemical features of rhabdomyolysis.
The mainstay of preventing acute renal failure is through
aggressive saline infusion. Dantroline sodium is the drug
of choice in the treatment of rhabdomyolysis and is found
to reverse most of the clinical signs and symptoms, when
administered intravenously. However increased physical
activity and regular, but modest exercise have yielded
promising results.
References
1.
Rutecki, GW, Ognibene AJ, Geib JD.
Rhabdomyolysis in antiquity: From ancient
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Rizzi D, Basile C. Clinical Spectrum of accidental
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Bywaters EGL, Becall D. Crush injury with
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4.
Koffler A, Friedler RM, Massry SG. Acute renal
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Australian Adverse Drug Reactions Advisory
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6.
Tobert JA. Efficacy and long-term adverse effect
pattern of statins. AMJ Cardiol. 1998; 62: 28-34.
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Sinzenzer. Atherosclerosis research group (ASF),
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Adiseshiah M. Reperfusion injury in skeletal
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revascularization. Br J Surg. 1992; 1026-1029.
9.
Knochel JP. Catastrophic events with exhaustive
exercise: White color rhabdomyolysis. Kidney Int.
1990; 38:707 - 719.
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10. Brumback JP, Feeback OL, Leech RW.
Rhabdomyolysis following electrical injury. Semin
Neurol. 1995; 15:329-334.
11. Abraham B, Cahana A, Krivosic-Horber RM, Perel
A. Malignant hyperthermia susceptibility:
Anaesthetic implications and risk stratification. Q J
Med. 1997; 90:13-19.
12. Felig P, Wahren J. Fuel homeostatis in exercise. N
Engl J Med. 1975; 293:1078-1084.
13. Jermain DM, Crismon M L. Psychotropic drug-related
rhabdomyolysis. Ann Pharmacoether. 1992; 26:948954.
14. Gladding P. Potentially lethal interaction between
diltiazem and statins [Letter]. Ann Int Med 2004;
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15. Cristopher P, Holstege. Cocaine related psychiatric
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27. Ann Pharmacother. 2002; 36:1957- 60.
28. Bruce M, Psaty MP. Use of cerivastatin and risk of
rhabdomyolsis. JAMA. 2004; 292:2622-2631.
29. Chang JT, Staffa JA, Parks M, Green L.
Rhabdomyolysis with HMG-CoA reductase
inhibitors and gemfibrozil combination therapy.
Pharmacoepidemiol Drug Safety. 2004; 13:417-426.
30. Mavais GE, Larson KK. Rhabdomyolysis and acute
renal failure induced by the combination lovastatin
and gemfibrozil therapy. Ann Intern Med. 1990; 112:
228-230.
31. Woodrow G, Brown John AM. Clinical and
biochemical features of acute renal failure due to
rhabdomyolysis. Intern Med. 1999; 223-239.
16. Knochel JP. Hyperthermia and rhabdomyolysis. Am
J Med. 1992; 92:455-457.
32. Bill DR. Radionuclide imaging of non-neoplastic soft
tissue disorders. Seminars in nuclear medicine, 1981;
277-288.
17. Graham DJ. Incidence of Hospitalized
Rhabdomyolysis in Patients Treated With LipidLowering Drugs. JAMA. 2004; 292:2585-2590.
33. Sauret JM, Marinides G, Wang GK.
Rhabdomyolysis. Am Fam Physician. 2002; 65(5):907912.
18. Omar AM, Wilson JP. FDA adverse reports on
statin-induced rhabdomyolysis. Ann Pharmacoether.
2002; 65:907-912.
34. Minigh JL, Valentovic MA. Characterisation of
myoglobin toxicity in renal cortical slices from
Fischer 344 rats. Toxicology. 2003; 184:113-23.
19. Goldman JA, Fishman AB. Clin Invest Med. 2001;
24:2258-72.
35. Word MM. Factors predictive of acute renal failure
in rhabdomyolysis. Arch Intern Med. 1998; 148:155157.
20. Rubin B. Prolonged adenine nucleotide synthesis
and reperfusion injury in post-ischemic skeletal
muscles. Am J Physiology. 1992; 262.
21. Saurat JM, Marinides G, Wang GK.
Rhabdomyolysis. American family physician. 2002;
65: 907-912.
22. Armstrong R, Warren G, Warren J. Mechanism of
exercise induced muscle fibre injury. Sports Med.
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23. Larner AJ. Potassium depletion and rhabdomyolysis.
BMJ 1994; 308-309.
24. Knochel J, Schlein E. Mechanism of rhabdomyolysis
in potassium depletion. J Clin Invest. 1972; 51:17501758.
25. Victor M, Toxic and nutritional myopathies. Vol.2.
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36. Ray WA. Population-based studies of adverse drug
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38. Allison RC, Bedsole DL. The other medical causes
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39. Hamer R. When exercise goes awry, exertional
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121
Pharmacy Practice
Protease Inhibitors – Spanking Agents for Respiratory Disorder
S. Ponnusankar, R. Senthil, Sandip Kumar Bhatt and B. Suresh
Drug and Poison Information Centre, Department of Pharmacy Practice,
JSS College of Pharmacy, Ooty – 643 001
Received on 21.10.2004
Modified on 12.07.2005
Accepted on 19.08.2005
ABSTRACT
Disruption of the natural equilibria between proteases and their cognate inhibitors is a common feature of inflammatory
disease. When this occurs in the lung, the effects can lead to irreversible impairment of pulmonary function. Although
protease inhibition was used in hereditary emphysema, recent research shows the use in respiratory disorders. In
addition to current anti-inflammatory respiratory therapeutics, certain small molecule and protein protease inhibitors also
have the capacity to inhibit directly the chronic air-way remodeling and lung degeneration mediated by uncontrolled
proteolytic activity.
INTRODUCTION
Respiratory disease is a significant cause of morbidity
and mortality in individuals of all ages. It may have severe
acute manifestations or can involve long term chronic
symptoms, each of which can severely and adversely
affect lung function. Indeed, many lung diseases are of
such potential severity that they can lead, in some
instances, to the premature death of the affected
individual.1 For example, in the neonate, infant and young
adult, respiratory distress syndrome (RDS), cystic fibrosis
and in rarer cases, asthma are potentially fatal diseases.
In the higher age groups, the debilitating effects of
chronic obstructive pulmonary disease (COPD) and
chronic bronchitis represent significantly higher causes
of morbidity and mortality.
Respiratory disease: a large and growing market
The National Heart, Lung and Blood Institute has reported
that, with around 1,19,000 adults of age 25 and older
dying of chronic obstructive pulmonary disease in 2000
(US data).1 Of the indications that can likely be impacted
by protease inhibitors (Table 1), the overlapping families
of symptoms caused by asthma, COPD and chronic
bronchitis are clearly the most prevalent, with many tens
of thousands of individuals affected by one or more of
these disorders.
Table 1
Respiratory Disease
Neonatal respiratory distress syndrome
Chronic lung disease of prematurity
Airway hyper responsiveness
Cystic fibrosis
Hereditary emphysema
COPD/Chronic bronchitis
Recent drug launches namely Singulair® (orally
active leukotriene receptor antagonist), Advair® (an
inhalable steroid and long acting bronchodilator
combination product), Spiriva® (once-a day long acting
M3 muscarinic receptor antagonist) are the successful
respiratory therapeutic product in the market for the
treatment of COPD.
Despite this, however, neither Advair® nor Spiriva®
122
Protease(s) involved in disease etiology
and progression
Neutrophil elastase
Tissue kallikrenin
Mast cell chymase
Mast cell tryptase
Cathepsins
ADAM33
Neutrophil elastase
Neutrophil elastase
Neutrophil elastase
Matric metalloproteases
TNF-alpha converting enzyme
address the underlying pathology inherent in COPD. The
eosinophilic inflammation in asthma is markedly
suppressed by corticosteroids, but they have no
appreciable effects on the inflammation in COPD,
consistent with the failure of long term corticosteroids to
alter the progression of COPD.2 Similarly, bronchodilation
provides substantial palliative relief of symptoms, but
does not address the long-term destruction of lung tissue
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
arising from the protease/protease inhibitor imbalances,
the chronic inflammation and the frequent exacerbations
seen in COPD.
PROTEASES AND PROTEASE INHIBITORS IN
RESPIRATORY DISEASE
In asthma, members of the cysteinyl protease family
of Cathepsins have been implicated.3 Also, more recent
human genetic studies have shown a membrane-anchored
metalloprotease, ADAM33, to be associated with asthma
and bronchial hyperresponsiveness and therefore to be
a novel therapeutic target.4 To date, however, the main
thrust of protease inhibitors for the treatment of asthma
has been towards mast cell proteases, and particularly
tryptase.5 Mast cell chymase and tryptase are each known
to contribute to airway inflammation in asthma. Tryptase
also induces the proliferation of human airway smooth
muscle cells through activation of the tethered-ligand Gprotein-coupled receptor PAR-2 (protease activated
receptor-2). Accordingly, this target has come under
intense scrutiny as a target for protease inhibition
therapy, with specific inhibitors reaching Phase II human
clinical trials.1
In contrast to asthma, where morbidity is huge but
mortality is comparatively rare, COPD is a pulmonary
disease in which both morbidity and mortality are
enormous. To address COPD using protease inhibitors,
it has been instructive to take advantage of the finding
that some individuals who are deficient in circulating
levels of Alanine amino transferase (AAT) are
predisposed to early onset emphysema, and that this
condition is exacerbated significantly by cigarette
smoking. Also, the finding of a clear role for matrix
metalloproteases in the development of smoking-related
emphysema, and that these metalloproteases, along with
neutrophil elastase and their endogenous inhibitors are
all intimately related with respect to the modulation of
their activities, has verified the validity of each of these
protease classes as targets for COPD therapeutics.
AAT DEFICIENCY AS A PARADIGM FOR COPD
THERAPY
AAT is an endogenous acute phase plasma protein
that has multiple functions. Produced at high levels in
the liver in unaffected individuals, AAT plays a critical
role in protecting against protease-mediated tissue
degradation during periods of inflammation. Genetic
deficiencies that lead to decreased levels of circulating
AAT are linked to the onset of a number of diseases,
most prominent among them being hereditary emphysema
(HE). In HE, the pathology of the disease is linked to
degradation of pulmonary tissue elastin, mediated by
unrestricted human neutrophil elastase (HNE) activity.
This ultimately results in decreased lung elasticity and
the presentation of clinical disease.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
A commercially available form of alanine amino
transferase (AAT) fractionated and purified from pooled
human plasma was approved in 1987 for chronic
replacement therapy of individuals having congenital
AAT deficiency with clinically demonstrable emphysema.
Prolastin ®, AralastTM and Zemaira TM are now marketed
for the treatment of HE. Despite the utility of these
products, however, their availability still does not make
up the shortfall between the current availability of
Prolastin and diagnosed population suffering from the
consequences of AAT deficiency. Accordingly, a
recombinant form of AAT (rAAT), manufactured in
genetically-engineered yeast cells, is currently being
tested as an inhaled therapeutic in heredity emphysema
patients.6
Chronic Obstructive Pulmonary Disease (COPD) is
a group of degenerative lung diseases that usually
develop after many years of assault on lung tissues from
cigarette smoke or other toxins and particles that pollute
the air. These environmental insults to the lung destroy
the alveoli that stretch as they transport oxygen from the
air to the blood and then shrink as they force out carbon
dioxide. As a result, the damaged lungs lose their
elasticity, exhaling becomes severely compromised and
they cannot effectively exchange trapped air with fresh
air.
A potential role for AAT in the treatment of
emphysema caused by cigarette smoking has been known
for more than two decades. Ever since the elucidation of
the molecular mechanism of hereditary emphysema, it has
been generally accepted that AAT would exert protective
effects against proteolytic lung degradation in COPD.
Proponents of early hypotheses concerning smokingrelated emphysema considered cigarette smoke as an
agent that could directly oxidize, and thereby inactivate,
natural AAT in the lung. More recently, it became clear
that reduced levels of AAT activity in the lung were
more likely related to the indirect oxidation of AAT
through generation of reactive oxygen species by
activated neutrophils. Furthermore, it has also been
shown that the macrophage-derived metalloproteases can
inactivate AAT by proteolytic cleavage. Macrophages
are extremely prominent in the smoker’s lung, typically
reaching steady state levels five-to-ten fold higher than
those seen in the non-smoker’s lung. More recently, it
has been demonstrated that macrophage-derived
metalloproteases activate tumour necrosis factor alpha
(TNF-alpha) further enabling the recruitment of
neutrophils that are key to the development of cigarette
smoke-induced emphysema via HNE-mediated
degradation of lung elastin.7 Thus, although there is a
clear involvement of macrophages and macrophagederived metallo-proteases in smoking-induced
emphysema, the neutrophil-derived protease HNE is still
123
considered to be the key mediator of lung degradation in
COPD.
The limited supply of AAT isolated from human
plasma has not allowed efficacy studies in human COPD,
nor would this expensive therapy be economically
available. A recent study published a result that, Injected
Prolastin was capable of reducing lung degeneration by
63% over a six month period in a mouse model system
for cigarette smoke-induced emphysema. The availability
of inhalable forms of the less expensive recombinant AAT
and the results of its testing in HE patients will allow its
evaluation in the more prevalent forms of COPD induced
primarily by cigarette smoking.
CURRENT DEVELOPMENTAL STRATEGIES THAT
ADDRESS COPD USING PROTEASE INHIBITORS
Several literatures support the potential use of small
molecule elastase inhibitors in respiratory disease.8 Of
the numerous such molecules that have been tested, only
one has been approved to date. Sivelestat (ONO-5046) is
currently used in Japan for the treatment of acute lung
injury.8 Although the potent elastase-inhibitory activity
of this molecule has suggested its potential use in COPD,
chronic application of this and other small molecule
elastase inhibitors have suffered from toxicity issues when
introduced into human clinical trials. The animal model
study using Prolastin has, however, suggested strongly
that AAT has the capacity to inhibit lung degeneration
induced by cigarette smoke, even when delivered by IP
injection. Thus it is likely that a recombinant form of
AAT, when administered more effectively through a
modern high efficiency delivery device, will have similarly
positive effects, without the toxicity issues.
In a second approach, based on the molecular
principles described above, animal model studies for
smoking-related emphysema using MMPIs have been
extremely encouraging. Two studies demonstrated that
orally or subcutaneously administered broad-spectrum
MMPIs were capable of reducing dramatically the lung
degeneration in cigarette smoke-treated mice and guinea
pigs respectively. Because of the potential toxicity issues
of such therapeutic regimens in humans, a further
significant advance was recently reported. In this study,
it was shown that nebulised Ilomastat, the prototypic
broad-spectrum MMPI, was capable of inhibiting alveolar
degeneration by 96% when administered by nebulisation.
Very low doses were required to achieve this effect in
mice that were treated daily with cigarette smoke over a
six-month period.9 Ilomastat has sub-nanomolar Kis
against many of the MMPs known to be involved in the
development of emphysema. In human COPD, the main
contributors to the excess metalloproteases load in the
lung are generally believed to be MMP-9 and MMP-12,
with MMP-12 (murine macrophage elastase) also being a
124
key factor in lung destruction in the smoke-treated mouse.
The ability of ilomastat to bind tightly at the active site
of human MMP-12 has been shown by molecular
modeling. Further drug development work will be required
to analyze the relative contributions of inhibition of each
member of the MMP family towards amelioration of both
direct extracellular matrix destruction and also towards
blocking the activation of inflammatory proteins, such as
TNF-alpha, that cause further direct lung tissue damage
by recruitment of neutrophils, and generation of excessive
HNE activity.
CONCLUSION
It is interesting that the finding about the blood
deficiency of a natural protease inhibitor leads to early
onset emphysema was first reported in 1936. A protease
inhibitor therapeutic, in the form of the plasma-derived
Prolastin, was first approved to treat the underlying
manifestations of the genetic form of emphysema in 1987,
a gap of 24 years. Despite this approval, however, and
because of the undersupply of the product, individuals
suffering from other forms of chronic lung degeneration
have had to endure another extremely barren period of
disease modifying drug discovery since then. The latest
findings, described above, have shown that both
recombinant protein and small molecule protease
inhibitors are capable of disease-modifying effects in
these additional chronic lung disorders.
Emphysema patients can now look forward to the
prospect of therapeutic protease inhibitors that will add
to the current armamentarium of corticosteroids and
bronchodilators by providing long-term protective effects
over and above the symptom-treating drugs that are
currently available.
Therapies aimed towards sustained amelioration of
the destruction of lung tissue architecture will likely have
highly beneficial effects on oxygen transfer capacities,
cardiovascular sequelae and other symptoms that are the
ultimate cause of death in individuals suffering from
severe respiratory disease. Therapeutic agents that can
address these issues will generate substantial revenues
that will also grow enormously as the population ages,
and as the incidence of chronic degenerative lung disease
increases.
References
1. Barr PJ. Protease inhibitor therapeutics for respiratory
disease. Drug Discovery World. 2003; 4: 13.
2. Barnes PJ. Mechanisms in COPD – differences from
asthma. Chest. 2000; 117: 10S-14S.
3. Barnes PJ. Chronic Obstructive Pulmonary Disease.
N Eng J Med. 2000; 343: 269-280.
4. Van Eerderwegh P, Little RD, Dupuis J, Del Mastro
RG, Falls K, Simon J. Association of the ADAM33
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
gene
with
asthma
and
bronchial
hyperresponsiveness. Nature. 2002; 418:426-430.
5.
Burgess LE. Mast cell tryptase as a target for drug
design. Drug News Perspect. 2000; 13 (3): 147 – 157.
6.
www.arrivapharma.com accessed on 10.08.2004
7.
Churg A, Wang RD, Tai H, Wang X, Xie C, Dai J,
Shapiro SD, Wright JL. Macrophage metalloelastase
mediates acute cigarette smoke-induced inflammation
via TNF-alpha release. Am J Respir Crit Care. 2003;
167(8):1083-1089.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
8.
Ohbaysahi H. Neutrophil elastase inhibitors as
treatment for COPD. Expert Opin Investig Drugs.
2002; 11 (7): 965-980.
9.
Pemberton PA, Cantwell JS, Kim KM, Sundin DJ,
Kobayashi D, Fink J, Shapiro SD, Barr PJ. An inhaled
MMP inhibitor blocks cigarette-induced lung
damage in the smoking mouse model.In: Proceedings
of European Respiratory Society Annual Congress;
2003.
125
Pharmacy Practice
Pharmacist – Against Drug Abuse
P. G. Yeole
Institute of Pharmaceutical Education and Research, Wardha – 442 001
Received on 28.10.2004
Modified on 04.07.2005
Accepted on 01.09.2005
ABSTRACT
Drug addiction is a devastating disorder that has severe health costs to both the individual and the public.
Although most patients use medications as directed, abuse of prescription drugs is public health problem for many
Indians. Addiction rarely occurs among those who use drugs as prescribed; however, the risk exists when drugs are
used in ways other than as prescribed. Pharmacists can play a key role in preventing drug abuse by providing clear
information and advice about the effects of medications. However, pharmacy education has failed to recognize the
potential for pharmacists in public health as well as to acquaint pharmacy students and practitioners with role models in
public health. Thus pharmacy will lose out, if it continues to take a parochial view.
INTRODUCTION
-
Drug nutrient interactions
In 1982 a national campaign was launched against
drug abuse in the USA. It was considered that the
community pharmacist would play a key role in this
campaign. This programme was appropriately called as
PADA “Pharmacists against Drug Abuse”. The
Pharmacists were chosen as the key persons because of
their education, their accessibility and their
trustworthiness. Similar programmes were launched in
other countries and India should not be an exception.
-
Self Medication with OTC / prescription drugs
Want of facilities in rural areas
A pharmacy is not a drug store only where a patient
can go and shop with his prescription as a shopping list.
A pharmacist can offer much more than dispensing and
ensure that patients are provided right medication, for
the right condition, in right dosage forms and in right
doses, in other words, this amounts to prevention of
drug abuse. By virtue of his professional knowledge and
practical experience a pharmacist can help in rational drug
therapy and prevention of drug abuse.
DRUGS ABUSE IN A BROADER BUT PRACTICAL
SENSE
The dictionary meaning of abuse is to ‘use wrongly’
or ‘improperly’ or ‘to misuse’. 1 The forthcoming
discussion is about drug abuse, patient’s misuse of any
drug, belonging to any therapeutic category originating
from prescriptions or self-medications. This is a more
practical approach to define drug abuse and to come out
with remedial measures. The abuse of drugs may arise
under circumstances such as
Wrong diagnosis
Inappropriate drug selection
Wrong dosage schedules
Situations where drugs are not indicated
Unwarranted prophylactic use
Polypharmacy and drug interaction
Adverse reaction
126
The two important areas of origin of drug abuse in
which pharmacists can be of help to the patients are (a)
OTC drugs and (b) Prescription drugs.
The OTC drugs are bought and ingested as casually
as eatables by the patients. Pain-killers which may contain
Aspirin, Paracetamol or Phenazone. Cold remedies 2
(Caffeine or sympathommetics); cough mixtures and
topicals are some of the commonly abused OTC drugs.
These drugs are not mimetics as for example Aspirin can
cause gastrointestinal hemorrhage, Paracetamol overdose
can cause liver damage and sympathomimetics can cause
hypertension. By counseling the patients on OTC drugs,
a pharmacist can contribute to prevent abuse.
PRESCRIPTION DRUGS AND THEIR ABUSE
Prescription drugs are even more liable for selfmedication abuse. Ignorance, ill acquired and incomplete
knowledge about drug effects are important factors for
prescription drug abuse. Self-medication is very common
and need prevention with
(i) decongestants causing hypertension
(ii) thyroid hormones for weight reduction inducing
hyperthyroidism
(iii) illicit use of anabolic hormones by the athletes
causing liver damage, menstrual abnormalities etc.
(iv) self use of potent antibiotics for example
Chloramphenicol for trivial conditions leading to
bone marrow depression and aplastic anemia or
development of bacterial resistance and
(v) analgesics causing ulcer, renal damage etc.
This type of self-induced misuse is particularly
prevalent in the educated class. A pharmacist can play a
crucial role in breaking this type of drug abuse because
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
ultimately the drugs are to be channeled through the
pharmacy counter and Pharmacist’s hands.
·
To compensate for feelings of inferiority,
shyness, insecurity
DRUG ADDICTION: A FEW EMERGING TRENDS3
·
To relieve boredom
Abuse of pharmacological medicines is a serious
global problem. Valium (diazepam), Phensedyl cough
syrup and other medical drugs have found their way in
to the illicit drug market the world over. The International
Narcotic Control Board has seriously recommended to
governments all over the world that they should make
efforts to raise the awareness of pharmaceutical
manufacturers and wholesale and retail distributors about
methods of diversion used by traffickers and should
encourage their co-operation with the competent
authorities.
SHORT-TERM EFFECTS
Cough mixtures and medicines are being abused by
a large number of addicts in the North East part of India.
The common brands abused by addicts are Phensedyl,4
Corex, Valium, Phenargan, Nitrazepam, etc.
Buprenorphine, 5
trihexylphenidyl6
and
dextropropoxyphene are also abused in several parts of
the country. Data reveals that 658 patients of the 1709
(38.5%) admitted at National Addiction Research centre
have been abusing medicinal drugs along with Brown
sugar and/or Cannabis. Fifty three patients who have
been abusing Nitravet have also found dependent on
tablet Valium. 77.05% of those who have been addicted
to pharmaceutical drugs reported to have been dependent
on nitrazepam, a benzodiazepine. 97.8% of those who
have reported to be dependent on nitrazepam were known
to abuse Nitravet. Nitravet is the most preferred medical
drug of abuse among most of the addicts in Mumbai.
Serious rethinking among de-addiction professionals,
health policy makers and other concerned personnel in
the light of the changing typology and emerging trends
needs to be carried out on a war footing.
Weight loss, fatigue, electrolyte, imbalance, sore
nose and mouth pallor. Tolerance can develop over a
period of time. Depression, irritability, hostility and feeling
of persecution can occur.
ABUSE OF SOLVENTS
Inhalant abuse is a prevalent and most overlooked
form of substance abuse in adolescents. Survey results
consistently show that nearly 20 percent of children in
middle and high school have experimented with inhaled
substances.7 Solvents include almost any household
cleaning agent, plastic cement (Hexane8), model airplane
glue,7 lacquer thinners (eg.Toluene, xylene), nail polish
remover (acetone), lighter fluid (eg. Napha), cleaning fluid
(benzene, trichloroethane, gasoline, toluene, xylene).
Inhalant abuse can cause a euphoric feeling and
can become addictive. Solvents act similar to anesthetics
and slow down body’s functions.
REASONS FOR USE
·
Curiosity and social pressure
·
To reduce anxiety and / or depression
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Immediately, they cause nausea, sneezing, coughing,
nosebleeds, bad breath, lack of co-ordination, and
appetite loss.
Inhaling high doses can result in losing touch with
one’s surrounding, loss of self-control, violent behavior,
unconsciousness or death. Sniffing highly concentrated
amount of solvents or aerosol sprays can produce heart
failure and death, the first time or any time.
LONG-TERM EFFECTS
TREATMENT
There appears no specialized treatment for inhalants
abuse or dependence. Intoxication is managed
symptomatically, supportive management is generally
sufficient.
SUGGESTED ACTIONS TO CURE ABUSE OF
CONTROLLED DRUGS
(1) No drugs like Barbiturates, Opioids, Diazepams,
Inhalants or Amphetamines should be dispensed
without a proper prescription.
(2) The refill for such drug should be done only if the
pharmacists are sure about the need or on a fresh
prescription only.
(3) A long and recurring supply should not be given.
(4) Proper records of controlled substances should be
maintained.
(5) Patient counseling about hazards of misuse of such
substance.
(6) Displaying patient awareness charts, posters or
pamphlets.
PREVENTING ABUSE OF DRUGS
Abuse of drugs can be prevented if a health care
system becomes a partnership between the public and
the health professional to increase the likelihood that
people will stay well and that, when ill, they will take
appropriate action in an appropriate system. Such system
is possible with improvement in following arena.
·
Pharmacist - physician Interaction
·
Pharmacist - consumer Interaction
·
Prescription Drugs and their abuse
127
·
Counseling on drug abuse
·
Quack medical products and the Pharmacist
·
The role of Community Pharmacist in
preventing drug abuse in the rural setting
CONCLUSION
Scientist who discover miracle drugs to conquer
disease that have plagued the mankind for many centuries
are also responsible for discovering powerful and potent
drugs, which have caused the problems of drug addiction
and drug abuse. Drugs have confined on mankind
immense benefits and also immense harm. If Pharmacist
do not rise to the occasion and accept these challenges
to save mankind from the drug abuse and addiction, they
will be blamed forever for the disastrous results. The
teenagers and the students grossly misuse the new
amphetamines, tranquilizers and psychedelic drugs.
SUGGESTIONS
(i) Constitution of training centers
(ii) Effective research
(iii) Central documentation and
(iv) Systematic elimination of such drugs from the
range of medical field in the treatment of
diseases
The pharmacist must therefore search his heart and
come out of the second line position attributed to him in
the health administration. He must accept these
challenges boldly and courageously. He cannot be a
128
helpless spectator. If other professions in the health
team have failed the nation, let the pharmacy profession
save it in the end. This is a pledge, which every pharmacist
must take.
References
1. Jaffe JH. Drug addition and drug abuse. In:
Goodman Gilman A, Rall TW, Nias AS, Taylor P,
editors. The Pharmacological Basis of Therapeutics.
8th ed. Vol.1 New York: Pergamon; 1990. p: 522-573.
2. Borde M, Nizamie SH. Dependence on a common
cough syrup. The Lancet. 1988; 760-761.
3. Shetty H, Nimker S, Pinto D, Brahmachari P. Drug
Addiction: A Few Emerging Trends. Ind J Psychiat.
1990; 32(2): 46.
4. Nizamie SH, Debashis R. Phensedyl – Will the
iceberg melt? Indian Journal Psychiat. 1991; 33(3):
212-215.
5. Nizamie SH. Buprenorphine Abuse: A case study.
Indian Journal Psychiat. 1990; 32 (2):198-200.
6. Michael A. Trihexyphenidyl Dependence – Report
of two cases. Indian Journal Psychiat. 1984; 26(2):
178-179.
7. Anderson CE, Loomis GA. Recognition and
prevention of inhalant abuse. Am Fam Physician.
2003; 68(5): 869-74.
8. Kuwabara S, Kai MR, Nagase H, Hattori T. n- Hexane
neuropathy caused by addictive inhalation: clinical
and electrophysiological features. Eur Neurol. 1999;
41(3):163-7.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Pharm. Education
Bibliometric Analysis for Identifying the Spectrum of
Cardiovascular Pharmacological Research and Leading Nations
P.M.K. Reddy, M. Jayanthi, K.M. Ravindra, R. Kesavan and S.A. Dkhar
Department of Pharmacology, JIPMER, Pondicherry – 605 006
P.M.K. Reddy
Received on 29.10.2004
Modified on 21.04.2005
Accepted on 15.10.2005
ABSTRACT
Recently, the bibliometric studies have gained significance in characterizing subject fields and leading nations. Our
earlier study indicated that cardiovascular pharmacology as one of the three leading fields of pharmacological research
and British Journal of Pharmacology published highest number of papers in such field when compared with at least two
reputed pharmacology journals. Further, it is of interest to know the pattern of cardiovascular research and the geographical
contributors in the same journal. Twenty four issues of British Journal of Pharmacology 2002 published around 481
research papers covering various fields of pharmacology. The following sub fields reported highest number of papers,
cardiovascular (30.5%), neuro (22.6%) and molecular pharmacology (20.5%). About 145 papers published in cardiovascular
pharmacology section were evaluated for the spectrum of research and country of origin. Many papers were published
on vasomodulation (51%), ion channels (16.5%) and cellular injury (6.2%) and the same were characterized to be popular
subfields of cardiovascular pharmacology. The UK, France and Germany (13, 12.4 and 11%) accounted for publishing
majority of papers and were presumed to be favourable nations for carrying advanced research in cardiovascular
pharmacology.
INTRODUCTION
MATERIAL AND METHODS
Recently, bibliometric studies have gained
importance in characterizing subject fields and nations
leading the research.1 Such quantitative analysis of
subject fields indicate the booming fields of research and
scientific productivity of nations.2,3 The young aspirants
wish to know the latest trends in research and nations
involved in frontline research in pharmacology to advance
in their field of interest. The research papers published
in any reputed pharmacology journal indicates the latest
trends of various specialities and leading nations in such
specialities of research.
This study is a retrospective library based hard copy
of 24 issues of British Journal of Pharmacology 2002.
About 145 research papers published under
cardiovascular pharmacology section were evaluated
manually for the spectrum of research and country of
origin.
A large number of global population suffer from any
one of the chronic diseases, such as cardiovascular
diseases, diabetes and psychiatric disorders, which lead
to investigations to mitigate the disease burden by finding
the suitable drug regimens.4 Cardiovascular pharmacology
deals with the action of drugs upon heart and blood
vessels. Generally, pharmacologists involved in
cardiovascular research explore the pharmacotherapy of
hypertension, heart failure, angina pectoris, dysarrhythmia
and reno-vascular functions, respectively. Our recent
study in 2002 (unpublished) indicated that cardiovascular
pharmacology as one of the three leading fields of
pharmacological research and the British Journal of
Pharmacology (BJP) 2001, published the highest number
of papers (30.3%).5 This observation served as an impetus
for mapping various aspects of cardiovascular research
activity and to identify the leading nations through the
papers published in BJP.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
RESULTS AND DISCUSSION
About 481 research papers were published in BJP
reporting about various fields of pharmacological
research. Again the highest number of papers were
published in cardiovascular pharmacology (30.5%)
followed by other specialities namely, neuropharmacology
(22.6%), molecular and cellular pharmacology (20.5%),
immunopharmacology (11.2%), gastrointestinal
pharmacology (9.3%), renal pharmacology (3%) and
diabetes mellitus (2.7%). The papers published in
cardiovascular pharmacology were analyzed and the
different subfields of research were placed in the
descending order of the number of papers published in
each subfield (Table 1). The highest number of papers
published are as follows, 1. Vasomodulators (endothelins,
prostanoids, bradykinins, angiotensin converting enzyme
inhibitors) 2. Ion channels (Ca 2+, K+ , K+ Ca ,, Na + and Ca 2+
exchange) 3. Cellular Injury (myocardial, ischaemia,
antioxidant protection, free radical protection). An
average number of papers were reported on receptors
129
Table 1: Various subfields of cardiovascular pharmacology research and leading nations
as reported in BJP 2002
Subfields of Cardiovascular
pharmacology research
1.
% of papers
(n=145)
Vasomodulators (endothelins,
prostanoids, bradykinins, ACE
inhibitors, etc.,)
51
Nations/Countries*
1.1.UK
UK
% of papers
(n=145)
13
13
2.
Ion channels (Ca2 +, K+, K +Ca, Na+ and
Ca2+ exchange etc)
16.5
2. France
2. France
3. Germany
3.
Cellular Injury (myocardial, ischaemia,
6.2
4. Japan
3. Germany
10
11
5. USA
8.3
4. Japan
6. Canada
10
8
antioxidant protection, free radical
protection, etc.,)
5
4.
Receptors (atypical 5HT2 , a 1, M2 and
intracellular messengers)
5.
Antiarrhythmia
3.5
3.5
6.
Cardiac contractility
3.5
3.5
7. Spain and Brazil
5. USA
8. Australia and China
6. Canada
9. Sweden, Italy
7. Spain and Brazil
7.
Platelet function
3.5
3.5
10.Others
8. Australia and China
8.
Myocardial infarction
3.5
2
9. Sweden, Italy
9.
Atherosclerosis
2
0.6
10.Others
3.5
10. Cardiomyopathy
0.6
4.7
11.
4.7
Miscellaneous
12.4
12.4
11
5 (each)
8.3
3.5(each)
8
2
5 (each)
18.3
3.5(each)
2
18.3
*The % of papers indicates % of papers published by the corresponding nation/country and not about the
corresponding subfields.
(atypical 5HT2, a 1, M2 and intracellular messengers)
antiarrhythmia and cardiac contractility. The maximum
number of papers were contributed by UK, France and
Germany. The minimum number of papers were published
by Argentina, Chile, Denmark, India, Taiwan, Mexico,
Turkey, etc.
The young scientists wish to know the booming
fields of pharmacological research and leading nations
to further advance in their scientific career. One of the
reliable ways to find out such information, is to, survey
the original research papers published in internationally
reputed pharmacology journals. Our recent study in 2002
for identifying the major specialities of pharmacological
research and geographical contributors based on research
papers published during 2001, in peer reviewed and
popular journals of pharmacology namely, European
130
journal of pharmacology, British journal of pharmacology
and Journal of pharmacology & experimental therapeutics
revealed about three major fields of pharmacological
research namely, neuro, cardiovascular and molecular
pharmacology and USA, Japan and UK contributed
significant number of papers.5 The results of this study
further prompted us to map various subfields of
cardiovascular pharmacological research countries
reporting the same. The cardiovascular pharmacology
has the wider horizon and opportunities as the treatment
of cardiovascular ailments, is described in the health
policy of many countries.
The journal selected in this study is internationally
reputed for reporting scholarly work in all the basic
disciplines of pharmacological sciences which includes
interaction of drugs/chemicals with biological systems at
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
molecular and cellular level, nervous and cardiovascular
systems, gastrointestinal, smooth muscle, immunology,
inflammation as well as endocrine, disposition and
toxicology. Indeed, the journal has been traditionally
popular for reporting more papers on cardiovascular
pharmacology, due to its editorial policy. Its latest impact
factor (3.61) is greater than some of the general
pharmacology journals.5 Articles are placed in the
appropriate fields to indicate the research potential. In
this study we undertook hand search of paper version
as it was found to be foolproof over electronic search.7
Around 145 papers published in cardiovascular
pharmacology were analysed, which is fairly a good
number to make the sound quantitative estimations and
derivation of valid assumptions.
Therefore, the UK, France and Germany would be the
favourable nations for the young researchers who wish
to advance further in cardiovascular pharmacology.
This bibliometric study indicated that the large
quantum of papers was reported about basic research of
vasomodulators, ion channels and cellular injury, which
was largely reported by the UK, France, Germany and
Japan. Basic research was flourishing in evaluation of
different aspect of vasomodulators. Many peptide
mediators were evaluated for their role in regulation of
vascular tone, especially their cellular mechanisms of
action. When their role become clear, these mediators
can form the potential targets for pharmacologists who
are on the lookout for these targets to bring out the
newer drugs with high specificity and potency. Some of
the mediators investigated were angiotensin II,
endothelins, and neuropeptides. Among ion channels,
calcium and potassium channels were studied extensively.
These channels are important regulators of membrane
potentials, on which most of cardiovascular responses
depend on. Studies on Na + -K + ATPase and Na + -H +
exchange were well reported and manipulation of these
channels would be an useful option in management of
cardiac failure. Evaluation of cellular injury and therapy
with antioxidants and free radical scavengers were found
to be promising avenues too. The UK, France and
Germany may possess trained manpower, laboratory
facilities and financial resources to tackle such
cardiovascular diseases suffering by their population.
1.
Hansen HB, Brinch K, Henriksen JH. Scientific
publications from departments of clinical physiology
and nuclear medicine in Denmark: A bibliometric
analysis of ‘impact’ in the years 1989-1994. Clin
Physiol 1996; 16: 507-19.
2.
de Jong JW, Schaper W. The international rank order
of clinical cardiology. Eur Heart J. 1996; 17: 35-42.
3.
Frame JD, Narin F. The international distribution of
biomedical publications. Fed Proc. 1977; 36: 179095.
4.
Wagner EH, Groves T. Care for chronic diseases.
BMJ. 2002; 325: 913-14.
5.
Reddy PMK, Tushar T, Ravindra KM, Nartunai G,
Shewade DG, Ramaswamy, Dkhar SA. A bibliometric
analysis for identifying the major specialities of
pharmacological research and geographical
contributors. Communicated to Indian Journal of
Physiology and Pharmacology.
6.
British Journal of Pharmacology [home page on
internet]. London. [accessed on 24 June 2004].
Available from: http:.//www.brjpharmacol.org/misc/
about.shtml.
7.
Lewison G, Grant J, Jansen P. International
gastroenterology research: subject areas, impact,
and funding. Gut 2001; 49:295-02.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
CONCLUSION
The major part of cardiovascular pharmacological
research papers published in BJP 2002 were about
vasomodulation, ion channels and cellular injury,
indicating them as the booming subfields. The UK, France
and Germany accounted for reporting majority of papers
of cardiovascular pharmacology. Hence, these nations/
countries can be reckoned as favourable nations for
carrying out the advanced research in cardiovascular
pharmacology.
References
131
Pharm. Education
TQM of Pharmacy Teachers –Need and Challenges
Neelam Mahajan, MSIP, Delhi-58
Received on 06.01.2005
Modified on 10.08.2005
Accepted on 19.08.2005
ABSTRACT
Pharmacy Teachers are instrumental in producing Quality Fellow Professionals. From 2005 the global scene has
become totally different and education will also be viewed as service industry. Accountability of educational institutions
and their products in global market will depend ultimately upon TQM of pharmacy teachers. The expectations of
students, parents, employers and society have also undergone a big change. Thus individualistic efforts of pharmacy
teachers should match synergistic efforts of institutions to accomplish TQM of pharmacy teachers. The need of multiple
QIP in the perspective of changing global requirements of the customers of pharmacy education and challenges posed
to this aim are discussed here. It is proposed that NIPER should act as National Center for TQM of pharmacy teachers
and existing QIP Programs should be strengthened.
INTRODUCTION
All India council of technical education is doing the
uphill task of promoting the quality of technical education
in the country. At institutional level, this goal can be
reached by producing Quality Fellow Professionals
through Quality Teaching. This in turn demands
competence of global standards from technical teachers,
and pharmacy teachers are no exceptions.
2.
Promote modernization and technology based
investments
3.
Provide periodic staff development programs based
on expected and actual profile of a teacher
TQM OF PHARMACY TEACHERS: NEED
The educational institutions are facing financial
crunch/pressures. Once they have finances they must
prove their academic global worth. They are compelled
to survive on their own with either nil or dwindling
financial support from the government.
Changing requirements of customers
2. Students
With the happening of GATS a product is
considered to be successfully marketed if it satisfies/
meets the customer requirements. Presently with patent
regime having become operational from 2005, only those
pharmaceutical industries which have a global vision will
survive. Hence the pharmaceutical industries as well as
hospitals are striving hard for accreditation. Their quest
for quality can succeed only through inculcation of
Excellent Quality Skills in Pharmacy Professionals.
§
Students have to bear the cost of quality teaching
§
Characteristics of Pharmacy Students and
perspective of teaching – expectations from learning
process are changing
CHALLENGES FOR TQM OF PHARMACY TEACHERS
Constraints/limitations of
1. Institutions: With India having become a
signatory to GATS (General Agreement on Trade in
Services) acquisition of global perspective features has
become a survival compulsion for pharmacy institutes.
Global perspective of Pharmacy Institutes
Global pharmacy institutions should
1.
132
Be academically viable ie. should be
·
Capable of fulfilling the fast changing needs of
economy and
·
Have a vision to produce globally competent
pharmacists
Pharmacy students of today have passed out from
schools where the transfer of academic knowledge had
been through a mind boggling, intriguing interactive
exchange process using multiple stimuli of doing/seeing/
viewing/ practical demonstration/ audio-visual aids/
workshops/ seminars etc. Hence students nicely know
that dissemination of knowledge is not a one-man show
by the teacher and Lecture/notes are not the only source.
3. Employers: Employers are facing an era of shrinking
financial and personnel resources and hence their hiring
decisions have to be very meticulous and calculated.
4. Teachers: Teachers are no longer supposed to
perform a traditional role in pharmacy education. They
have to shoulder the task of guiding and supervising
students. They not only have to produce good pharmacy
professionals but also good human beings.
HOW TO COPE?
Majority of pharmacy colleges/ institutions/
polytechnics may not be able to make adjustments at the
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
pace expected by technological advancements yet they
should face this problem by adopting more viable options
like:
(1) Shifting from outdated systems and regularly
monitoring teaching – learning process.
(2) Making the training of human resources a
continuous process by arranging periodic staff
development programs
(3) Reorienting their vision with a global perspective
(4) Acquiring new capabilities to learn
(5) Realizing the importance of synergistic efforts over
individualistic efforts for growth and sustenance.
(6) Developing the ability to learn faster than their
competitors as an organization, i.e. they should
become learning organizations. This is the only
sustainable competitive edge. A learning
organization has an enhanced capacity to learn,
adapt and change. It is an organization in which
learning processes are monitored, developed,
managed and aligned with improvement and
innovation in goals, its vision strategy, learning and
development and to accelerate systems of levels of
learning.
MULTIPLE QIP: ADAPTING TO CHANGING ROLE OF
PHARMACY TEACHERS
The focus of pharmacy teacher should shift to
transfer of Quality academic knowledge and widening
their spectra of roles which include teaching, instruction,
curriculum development and value education,
communication, instructional material management
development, R&D extension services and consultancy
and in the management of the institution.
Mere acquisition of higher and specialized
qualifications in pharmacy will not do justice to the QIP
needs of faculty of 21st century. A multidimensional
improvement in the quality of faculty is need of hour.
Hence the faculty development methodology and
orientation needs to undergo metamorphosis.
CHALLENGES
The challenges in the path of TQM faculty are many
and are to be tackled with sincerity:
1. Impact of IT/Communication Technology: Computers
and information technology has become essential to
pharmacy education. Computer applications in pharmacy
for maintaining database, patient profile, for new drug
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
design etc. are fast finding its place in curricula. Use of
multimedia packages, hypertext videotext etc. will also
find place in curricula, the teachers have to learn use of
alternate modes of delivering lectures like multimedia and
satellite.
2. Development of Generic Skills: Acquisition of
multiple generic skills of communication, problem solving,
self-learning, analytical attitudes as well as
interdisciplinary knowledge besides pharmaceutical
expertise increases the employability of pharmacy
students in industry. These generic skills should be
taught regularly in classrooms and labs. To achieve this
goal, faculty has to constantly motivate students and
build awareness of the need of imbibing these skills.
3. Industry partnership: For imparting education in tune
with the fast changing pharmaceutical technology, the
faculty must be motivated to get involved in symbiotic
programs\projects with the industry. Provision should
be made to give compulsory industrial exposure to faculty
as per their competency level.
4. Internal Resource Generation: The pharmacy
institutions must appreciate that modern infrastructure
like audio-visual aids and suitable gadgets like OHP, LCD
projectors, video projectors, screen, microphone and
speakers for use in seminars/lectures etc. in an institution
leads to considerable enhancement of quality of education
and hence should invest accordingly. They should
further take interest in the development of labs. The
pharmacy institutions should arrange and invest in latest
infrastructure. But financial capabilities of the institutions
being limited, advisable updating of equipment in
harmony with fast changing technology is not affordable
at the required time intervals. So, in the interest of
pharmacy students and for the growth of pharmacy
institutions, the institutions must learn inter-institutional,
intra-institutional and institute industry sharing of costly
expertise and equipment.
5. Flexibility in Programme offering & Autonomy to
Institutions: Rigid time-bound, fixed entry and exit
courses should become thing of past if we have to
produce pharmacy professionals of global standards. The
pharmacy courses have to be made flexible, modular and
based on credit based approach to teaching learning.
The pharmacy courses should be run on multi point entry
and credit system. To run flexible modular pharmacy
programmes the institutions must acquire academic,
financial and administrative autonomy. This will enable
133
the pharmacy institutions to design, implement and
evaluate programmes and courses, which in turn helps
to incorporate technology based changes in the curricula.
6. Quality Assurance: Quality of Pharmacy Professional
has become critical variable in their marketability therefore
Quality is must. It amounts to developing policies,
attitudes, actions and procedures necessary to ensure
that quality is being maintained and enhanced in the
academic set up.
7. Building up Values & development of Attitudes: To
achieve this goal of quality assurance, faculty has to be
re oriented to build and create an attitudinal change. This
also demands training of faculty for pursuing the goal of
quality assurance continuing education with consequent
improvement in quality of pharmacy teachers has become
paramount to catch up with new technologies and
transfer to classroom. Hence the institution must be liberal
in deputing their staff to short term courses or higher
degrees. The QIP programmes need further strengthening
to facilitate more teachers to acquire higher degrees.
NEED OF A NATIONAL CENTRE FOR TQM OF
TEACHERS
Continuing education should assume national
character before going global. To achieve this aim,
training of pharmacy teachers should have national
perspective so that they can impart pharmacy education
within national framework of expectations.
their own without grant/support from government and to
generate resources from alternate sources. Thus need of
sustenance gets priority over growth. Yet need to grow
in global market scenario cannot be overlooked. Therefore
institutions have to acquire new capabilities and learn
new skills for added accountability.
Pharmacy institutions-the vendors of human
resources with already shrinking financial and personnel
resources may not be able to take big leaps required to
produce competency based professionals. Rather they
will accept restructuring of traditional teaching styles
within their limiting resources and continuous training of
teachers is the most viable option. The institutes should
encourage teachers to participate in continuing education
programs in industrial as well as academic set up as
suggested. NIPER should be entrusted with the job of
TQM of the pharmacy teachers. Let pharmacy colleges,
institutions, policy makers, regulatory bodies and above
all the pharmacy teachers all over the country contribute
by taking individual small leaps so that the giant leap of
TQM of pharmacy teachers for imparting quality
pharmacy education can be accomplished.
References
1.
Sinha HP. Technical Education Policies: Vital
ingredient for National Development. IJTE. 2004;
27(3):9-12.
2.
Srinivasan S, Khambayat RP. A study on present
status of faculty Awareness of Education Quality
Parameters. IJTE. 2004; 27 (3): 57-63.
3.
Vijay Kumar GP. Quality assurance in Technical
Education. IJTE. 2004; 27(3):73-79.
4.
Sangeeta Sahney, Banwat DK, Karunes S. Changing
trends in Education: The relevance of Total Quality
Management. IJTE. 2004; 27(3):101-107.
5.
Neelam Mahajan. Quality in Pharmacy Education in
New Millennium. IJTE. 2004; 27 (4):67-71.
6.
Editorial: GATS and its impact on Higher Education.
IJPE. 2004; 38(3):114-115.
7.
Editorial: Global Standards for Pharmacy education.
IJPE. 2003; 37(4):170.
SUMMARY
8.
Due to liberalisation of economy and reallocation of
priorities to achieve national goals for socioeconomic
upliftment and infrastructural development, the technical
institutions are subject to the pressure of survival on
Sharma JP. Teachers in Technical Institution and
their Professional Development. IJTE. 2000; 23(2):3944.
9.
Krishnamurthy S. Challenges for Faculty
Development. IJTE. 2000; 23(3):11-15.
To achieve this goal, we further require a national
centre for providing and disseminating facilities for
continuing education for updating and upgrading the
knowledge and skills of pharmacy teachers. It is very
heartening to note that we already have such national
level centre of repute i.e. NIPER which has many feathers
to its cap. This institute is already working as an institute
of higher learning in pharmacy and has been perceived
as a centre of excellence of pharmaceutical sciences and
technology, suggest that NIPER should notionally act
as a centre for TQM of the continuing education training
for pharmacy teachers. All the pharmacy teachers as per
their competency requirements should get exposure to
education technology as well as technology in education
at NIPER.
134
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Ph.D. Theses
Title
: Chemotaxonomical Significance of Anthraquinone
Glycosides in Local Cassia Species
Candidate
: Mohib Khan
Institute
: Shri Bhagwan College of Pharmacy Aurangabad
University
: Dr. Babasaheb Ambedkar Marathwada University, Aurangabad
Guide
: Dr. M. S. Shingare, Dr. B. A. M. University, Aurangabad, (M. S.)
Year
: November 2003
ABSTRACT
vitro) on own blood and antispasmodic activity on rectus
The genus Cassia belongs to family Leguminosea
abdominus muscle of frog were performed4.
and subfamily Caesalpiniae. There are about 580 species,
Antimicrobial activity, on fungi where gresiofulvin
out of which 23 species are found in India. It has been
was used as standard antifungal agent and on bacteria
reported that all the Cassia species contain anthraquinone
where streptomycin was used as standard antibacterial
glycosides 1.
agent were also performed5.
The phytochemical investigation has been carried
In the phytochemical investigation, extraction,
out on these 23 species but no systematic work has been
isolation
and quantitative estimation of three aglcone
undertaken to find out the occurrence of exact amount
chrysophanol,
emodin and physcione in the bark, flower,
anthraquinone derivatives in each part of these species.
leaf,
seed,
pericarp
and stem of C. auriculata, C. fistula,
Hence it has not been possible so far to know exactly
C.
javanica,
C.
roxburghii
and C. siamea were carried
the chemotaxonomical significance of anthraquinone
2
out.
A
TLC-Colourimetric
method
for the estimation of
glycosides in Cassia species .
chrysophanol, emodin and physcione in different Cassia
The present research work is carried out only for
species has been used.
the Cassia species, which are available locally, and the
It has been observed that C. auriculata contains
study is only performed for the three aglycones namely
total
free
chrysophanol (0.1%), free emodin (0.14%), free
Chrysophanol, Emodin and Physcione. The free aglycones
physcione
(0.23%) and O-glycosidic physcione (0.06%).
are responsible for the action while the sugar/s present
C.
fistula
contains
total free chrysophanol (0.28%), free
in the combined form is/are responsible to carry the
physcione
(0.27%)
and
O-glycosidic physcione (0.15%).
aglycone at the site of action.
C.
javanica
contains
total
free chrysophanol (0.37%), OThe research work was started with the extraction,
glycosidic
chrysophanol
(0.04%),
free emodin (0.05%),
isolation and characterization of three aglycone
O-glycosidic emodin (0.08%), free physcione (0.14%) and
chrysophanol, emodin and physcione and their
O-glycosidic physcione (0.14%). C. roxburghii contains
quantitative estimation from Indian Rhubarb, which
total free chrysophanol (0.1%), free emodin (0.09%), Oconsists of rhyzomes of Rheum emodii family
glycosidic emodin (0.07%), free physcione (0.1%) and OPolygonaceae3.
glycosidic physcione (0.02%). While C. siamea contains
The extraction was carried out using chloroform as
total free chrysophanol (0.71%), free emodin (0.05%), free
organic solvent, isolation was carried out with the help
physcione (0.23%).
of preparative thin layer chromatography, where
petroleum ether (60-80 OC): Ethyl acetate: Formic acid
(75:25:1) was used as mobile phase while the
characterization was carried out using IR, NMR and Mass
spectroscopic techniques. These aglycones were then
quantitatively estimated on colorimeter at 480 nm against
IN aqueous potassium hydroxide solution. The standard
curves were prepared by plotting optical density against
concentration in ug per 10 ml and found to obey Beer’s
law. The developed aglycones chrysophanol, emodin and
physcione from Indian Rhubarb were used for the next
studies.
In the pharmacological investigation, the preliminary
pharmacological screening of chrysophanol, emodin and
physcione were carried out using experimental models.
The activity on intact frog heart, activity on blood vessels
of frog, analgesic activity on albino mice using acetic
acid induced writhing method, anticoagulant activity (in
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
The quantitative distribution of anthraquinone
glcosides in five cassia species present in the
Aurangabad region shows that bark, flower, leaf, seed,
pericarp and stem of C. auriculata, C. fistula, C. javanica,
C. roxburghii and C. siamea contain chrysophanol,
emodin and physcione in a considerable amount. It is
also observed that not a single plant part from all these
Casia species contain chrysophanol, emodin and
physcione in their C-glycosidic form. The finding of the
higher percentage of chrysophanol, emodin and
physcione in C. siamea (1.01%) reflects that C. siamea is
very important as far as the chemotaxonomical point of
view is concerned6.
Publications
1.
Mohib Khan, Zafar R, Shingare MS. Quantitative
Estimation of Free and Combined forms of Aloe
135
emodin, Chrysophanol, Emodin and Rhein in Cassia
pumila Lamk by TLC-Colourimetric method.
Hamdard Medicus. 2003; 46(3):49-52.
4.
Mohib Khan, Shingare MS. Preliminary
Pharmacology Screening of Chysophanol, Emodin
and Physcione. The Antiseptic. 2004; 101(5):166-168.
2.
Mohib Khan, Zafar R. Preliminary phytochemical and
antibacterial properties of Cassia pumia Lamk. The
Indian Pharmacist. 2005; 4(38):91-92.
5.
Mohib Khan, Shingare MS. Isolation and
Antimicrobial Activity of some Free Anthraquinones.
The Indian Pharmacist. 2003; 2(13): 49-52.
3.
Mohib Khan, Shingare MS. TLC-Colourimetric
Estimation of Free and Combined forms of
Chrysophanol, Emodin and Physcione in Indian
Rhubarb. Indian Journal of Pharmaceutical Sciences.
2003; 65(5):552-553.
6.
Mohib Khan, Maske PV, Angadi SS, Siddiqui AR,
Shingare MS. TLC-Colourimetric Estimation of Free
Combined forms of Chrysophanol, Emodin and
Physcione in some Cassia Species. Indian Journal
of Pharmaceutical Sciences (Under consideration).
Title
: Formulation and Evaluation of Mucoadhesive Drug Delivery
Systems of Some Antiasthmatic Drugs
Candidate
: T.M.Pramod Kumar
Institute
: J.S.S.College of Pharmacy, Mysore.
University
: Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.
Guide
: Dr. H.G.Shivakumar, Vice-Principal, Professor and Head,
Dept. of Pharmaceutics, J.S.S.College of Pharmacy, S.S.Nagar,
Mysore-570 015
Year awarded : February 2006
ABSTRACT
The aim of the present study was to formulate
buccoadhesive compacts (BC’s) and films of salbutamol
sulphate, terbutaline sulphate and salmeterol xinafoate in
different concentrations by direct compression method
using polymers like carbopol 934P and hydroxy propyl
methyl cellulose 4KM(HPMC 4KM) at the ratios of 1:0,
1:1, 1:2 and 0:1 respectively. The buccoadhesive films
were prepared by solvent evaporation method using
chitosan
and
hydroxypropylmethylcellulose.
Preformulation studies such as monographic analysis and
compatibility of drug with polymers were performed. The
compacts and films were evaluated for weight variation,
thickness, hardness, drug content, swelling index, in vitro
mucoadhesive strength and in vitro dissolution studies.
Buccoadhesive compacts prepared by carbopol 934P and
HPMC 4KM in the ratio 1:0 have shown maximum
mucoadhesive strength. Swelling of the compacts
increased with an increase in concentration of HPMC
4KM. In vitro dissolution studies were performed using
modified dissolution apparatus in phosphate buffer pH
6.6 for a period of 6 h. The drug release from
buccoadhesive compacts varied with varying
concentration of drugs i.e increases with increasing
concentration of drugs. The drug release rate followed
first order release kinetics. The obtained data when fitted
into a simple power equation (Mt/M¥=kt n), had shown
that the mechanism of drug release was by non-Fickian
diffusion. From the in vivo studies on albino rabbits, it
was observed that the plasma drug concentration of the
buccoadhesive compacts was comparable with
intravenous bolus injection of the drug. From the studies,
it may be concluded that such systems can be designed
for drugs, which suffers high first pass metabolism.
Title
: Studies on Karnataka state pharmacists’ attitudes and behaviours
towards patient counselling and use of patient information leaflets.
Candidate
: Adepu Ramesh
Institute
: J.S.S.College of Pharmacy, Mysore.
University
: Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
Guide
: Dr. B. G. Nagavi, Professor & Principal, JSS College of Pharmacy, Mysore.
Year Awarded : 2005
ABSTRACT
Patient counselling is the most important component
in the delivery of pharmaceutical care services. Many
scientific studies have proven the patient counseling
136
influence on improved patient medication adherence and
health related quality of life. Internationally, pharmacists
proved themselves as professionals by virtue of patient
counselling. In some countries it is mandatory and also a
remunerative service.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
In India, the pharmacy profession underwent
significant changes since independence. Pharmacy
Council of India (PCI) restructured the curriculum
requirement for Diploma in Pharmacy (D.Pharm) course,
the minimum registrable qualification to practice pharmacy
and also introduced various professional subjects such
as Hospital and Clinical Pharmacy, Health Education and
Community Pharmacy. Despite of considerable efforts
by the various professional associations, patient
counselling remained as a dream and pharmacists are still
behaving as traders than professionals.
A detailed study was conducted in the state of
Karnataka, a South Indian state to assess the attitudes
and behaviours of practicing community pharmacists
towards patient counselling and use of patient information
leaflets. As a part of the study public and prescribers
perception towards pharmacists’ services were also
studied. The responded public opined community
pharmacists as traders and expected sincere and honest
professional services from them. The prescribers opined
that, pharmacists are the right persons to take the
responsibility of patient counseling and providing product
information to ease their professional burden. But in
order to provide these services, prescribers desire that,
pharmacists should have B.Pharm or M.Pharm
qualification.
More than 50 % responding pharmacists mentioned
that, they were not aware about the professional
responsibilities of community pharmacists recommended
by World Health Organisation (WHO) or International
Pharmaceutical Federation (F.I.P). Results of critical review
of community pharmacies of the respondents suggest
that, 4% of the pharmacies do not meet the schedule N
requirement, 9% of the pharmacies do not meet the
schedule C & C1 and only 7% of the pharmacies were
having computers but using only for business operations.
About 19% of pharmacies are having patient counselling
cubicle but not using for the counselling purpose.
Majority of the respondents (78%) agreed that, the
patient counseling and providing information leaflets is
their professional responsibility, but in reality patient
counseling rarely happens in the pharmacies due to
reasons like pharmacist’s inadequate knowledge to offer
counselling, lack of proper infrastructure, Peak work load
and business attitude of the practicing pharmacists, nonlegalisation of patient counseling and lack of
remuneration. Doctor dispensing is also another important
threat to offer patient counseling especially in urban
areas. Majority patient information leaflets (95%)
produced by the pharmacists and manufacturers do not
meet the readability and lay out requirements.
In order to bring a change in the knowledge base, a
six-hour training module was developed. The training was
offered to 258 practicing community pharmacists from
nine district head quarters of Karnataka state namely,
Mysore, Mandya, Tumkur, Udupi, Bidar, Gulbarga, Hubli,
Belgaum, and Mangalore. The respondents were very
much satisfied with the content of workshops and opined
that such workshops will improve their knowledge base
and communication skills and wished to have at least
once in three months.
Title
: Preparation and evaluation of waxes/ fat microspheres loaded
with hydrophilic and lipophilic drugs for controlled release
Candidate
: D.Vishakante Gowda
Institute
: J.S.S.College of Pharmacy, Mysore.
University
: Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
Guide
: Dr. H.G.Shivakumar, Vice-Principal, Professor and Head,
Dept. of Pharmaceutics, J.S.S.College of Pharmacy, S.S.Nagar,
Mysore-570 015
Year awarded : February 2006
ABSTRACT
The objective of the present study is to prepare
and evaluate the waxes/fat microspheres for controlled
release loaded with lithium carbonate, theophylline,
indomethacin and griseofulvin in to gastro resistant,
biodegradable waxes such as beeswax, cetostearyl
alcohol, spermaceti and a fat cetyl alcohol using meltable
emulsified dispersion cooling induced solidification
technique utilizing wetting agents. Sieve analysis data
indicated that the prepared microspheres were in the
ranges of 855 to 115 mm. The angle of repose, % carr’s
index and tapped density were well within the limit,
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
indicating reasonable good flow potential for the prepared
microspheres. SEM photographs and calculated sphericity
factor confirms the prepared formulations are spherical
in nature. DSC studies and FT IR spectra showed that
the encapsulated drug was stable in the prepared
formulations. The prepared formulations were analyzed
quantitatively for the amount of encapsulated drug. It
was observed that, there is no significant release of drug
at gastric pH. Intestinal drug discharge from prepared
formulations were studied and compared with
commercially available controlled release oral
formulations. The release kinetics for all the formulations
137
followed different transport mechanisms. From the results
of the studies carried out, indomethacin loaded in
cetostearyl alcohol has been selected for ageing and in
vivo studies and compared with Microcid SRÒ -75. A
single dose randomized two period cross over study was
conducted to compare the pharmacokinetics and
bioavailability of indomethacin (75mg) from test
formulation (product B – indomethacin loaded in
cetostearyl alcohol) with standard formulation (Product
A – Microcid®SR 75 mg capsule). The observed mean
138
values Tmax , Cmax , AUC0-¥ Ka and t ½ for product A&B
does not show any significant statistical difference. From
the dissolution point and in vivo bioavailability, products
A & B could be considered bio equivalent. The release
performance was greatly affected by the materials used
in microspheres preparations, which allows maximum
absorption in the intestine. Indomethacin loaded with
cetostearyl alcohol microspheres has desirable release
profiles and worthy of further investigation as an oral
controlled release dosage form.
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Book Review
Title
:
Author
Publisher
Price
:
:
:
Pharmaceutical Organic Chemistry
(Part-I- Chemistry of heterocyclic and natural compounds)
Rama Rao Nadendla
Vallabh Publications, Delhi
Rs.190 (in Delhi) & Rs.198(out side Delhi)
This book consists of fourteen chapters. In the
chapter 1, Natural sources of organic medicines are listed.
Also, a brief introduction to the origin of natural products
has been given. The contents of chapter 2 are structural
elucidation, synthesis, properties, uses, medicinal
compounds of moiety of naphthalene, anthracene,
phenanthrene and preparation, properties, uses of
diphenyl methane, triphenyl methane. The contents of
chapter 3 are reactions of unsaturated carbonyl
compounds. The contents of chapter 4 are preparation,
uses of aluminium iso propoxide, aluminium t-butoxide,
sodamide, lead tetra acetate, lithium aluminium hydride,
N-bromo succinimide, uses of poly phosphoric acid,
preparation, properties and uses of sodium borohydride.
The contents of chapter 5 are nomenclature of
geometrical isomers, elements of symmetry, resolution of
racemic modification, nomenclature of optical isomers,
asymmetric synthesis, stereochemistry of biphenyl
compounds, nucleophylic substitution reactions of alkyl
halides, alkene addition reactions, stereo selective and
stereo specific reactions, conformational isomerism in
ethane, cyclo hexane. The chapter 6 titled pericyclic
reactions deals with principal categories of reactions,
orbital symmetry rules. The contents of chapter 7 are
classification, nomenclature of heterocyclic compounds,
chemistry of furan, pyrrole, thiophene, pyrazole,
imidazole, oxazole, isooxazole, pyridine, pyrazine,
pyrimidines, pyridazines, oxepines, azepine, quinoline,
isoquinoline, indole, benzofuran. Also, preparation and
medicinal compounds of acridine, phenothiazines,
benzimidazole. The contents of chapter 8 are classification,
qualitative analysis of carbohydrates, chemistry of
glucose, fructose, sucrose, lactose, maltose, starch and
derivatives, cellulose and derivatives. The contents of
chapter 9 are classification, synthesis, properties,
extraction of glycosides. Also, in brief explained about
Title
Author
Publisher
Price
:
:
:
:
cardiac glycosides, anthraquinone glycosides, salicin,
amygdaline. The contents of chapter 10 are extraction,
properties, importance, determination of acid,
saponification, iodine, peroxide, hydroxyl, acetyl, RM
values of fats and oils. Also a brief introduction to waxes
is given. The contents of chapter 11 are classification,
synthesis, properties of amino acids, classification,
synthesis of peptides, classification, properties, structure,
qualitative tests of proteins, chemistry of insulin, oxytocin.
The chapter 12 deals with classification, functions,
structure of nucleic acids. The contents of chapter 13 are
isolation, properties, classification, structure determination
of alkaloids and chemistry of ephedrine, conine, nicotine,
papaverine, atropine, quinine, reserpine, morphine. The
last chapter deals with chemistry of purines, uric acid,
caffeine, theophylline, theobromine.
In this text book, practice questions are given
chapterwise. This develops proficiency in readers. The
last few pages are index pages. This book has been written
in a simple, systematic and concise way. The language
and explanations are simple. The author Professor Rama
Rao Nadendla deserves to be complimented for presenting
this book. I feel, had page number against practice
questions where answers are given and answer to
remaining practice questions given, it would have been
helpful to readers. Some printed mistakes and errors are
noticed inspite of all care taken by the author. In total,
this book is handy with good get up and useful to
students and teachers. The placement of this book in
libraries is worthy.
Reviewed by:
M.S.Venkatesh
Assistant Professor
J.S.S.College of Pharmacy
Mysore-570015
Theory and Practice Pharmaceutics-II
A.P.Pawar
Career Publications, Nashik
Rs.160-00
The book written by Dr.A.P.Pawar is an innovative
Pharmaceutics-II book emphasizing Pharmaceutical health
care. The author has made an attempt to present the
subject with new dimension using pictograms and
Product Information Leaflet.
The book has one general chapter and 12 chapters
as per the syllabus of D.Pharm-II (according to ER-91).
Indian J.Pharm. Educ. Res. 40(2) April - June 2006
Till today, dispensing pharmacy has been considered as
just lab scale manufacturing of dosage form and it is
targeted in the view of production. In the preface by the
author, he has opined that practice of compounding is
diminished and these are the days of ready to use
manufactured formulations.
139
Chapter 1 speaks in general about the
Pharmaceutical Health care (the title in the content page
and in the chapter is different). Chapter 2 comprises of
Prescription and its details. The author should be
congratulated for writing in detail about the
documentation of patient medication record and
compounding and dispensing record and about patient
information leaflet. I personally have strong reservation
in the definition of prescription “The prescription is an
order written by RMP….”. can it not be rewritten as
request by RMP. Around 15 prescriptions have been
given (some examples of prescriptions in other languages
also ) and good idea of reading it and analyzing it is
given. Chapter 3 related to Pharmaceutical calculation is
written well. Chapter 4 of Posology has been given with
relevant information. Nearly 15 pages are dedicated to
give Table4.1 (Name of the drug, caution, patient
counseling). Chapter 5 of incompatibility encompasses
physical, chemical and therapeutic incompatibility. The
physical and chemical incompatibilities are less common
in modern prescriptions. Hence emphasis is given to
therapeutic incompatibility. Examples of modern medicines
need to be appreciated. Chapter 6 to 11 comprises of
solid, liquid and semisolid dosage forms. Lots of new
examples are quoted. In chapter 12, cosmetics have been
discussed, giving different types of classification,
difference between cosmetics and drugs. Facial, Eye and
Hair cosmetics and Dentifrices are discussed well with
good examples. Chapter 13 explains about Steril e
Title
Author(s)
Publishers
Edition
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The author has given annexure giving the guidelines
for practical section of Pharmaceutics-II. He has given
different outlook for the practical component where ER91 syllabus says that “dispensing of at least 100 products
covering wide range of prescriptions”. The author opines
that student should not only be encouraged to 100
products requiring manipulative skill but prescriptions
should be assigned to understand concept of dispensing
such as therapeutic incompatibility, patient counseling,
drug information services, development of patient
information leaflets in the local languages etc.
The publishers have done good job with neat cover
and clarity in printing and diagrams. Bibliography and
indexing are also done but reference No.15 to 26 needs
clarification.
Although number of books are available for
Pharmaceutics-II by different authors at cheaper price,
this book can be clearly distinguished because of its
content and collection of information. I recommend this
book not only for II D.Pharm students but also for
practicing pharmacists to know about Pharmacist role in
Pharmaceutical health care.
Reviewed by:
Dr. T.M. Pramod Kumar
Assistant Professor,
J.S.S.College of Pharmacy,
Mysore-570 015
A Textbook of Hospital and Clinical Pharmacy
P. C. Dandiya and Mukul Mathur
Vallabh Prakashan, New Delhi
Fourth edition (2005)
The release of the fourth edition of this book reflects
that the authors have been constantly updating this
useful textbook for the students. The relevance of the
book is especially important in India since very few books
are available to the students for studying the professional
practice of pharmacy. And this book does absolute justice
in handling the finer aspects of hospital and clinical
pharmacy.
The present book in its fourth edition is divided
into three parts. While the first part is spread in ten
chapters on the hospital pharmacy practice, the second
chapter on clinical pharmacy is indeed impressive. The
authors have used their expertise to cover the entire
components of clinical pharmacy practice. Personally, as
an educator, I would love to see the chapter on
pharmaceutical care in a still expanded form in the
forthcoming editions.
140
products. The author could have included the LAL test
for pyrogen testing.
The third and the last section consists of nearly
two dozens exercises which are as elementary as
preparation of sterile water for injection IP to as advanced
as creation of a database of patients and physicians. I
am sure the present generation of students, who have
access and expertise on the IT tools, will use the
infrastructure at their colleges to initiate such exercises.
In my opinion, the students will find the book very
useful and cost-effective. I am sure the students opting
for practice might like to keep it as a reference for later
time, too.
Reviewed by:
Dr. P. Tiwari
Associate Prof. and In-Charge,
Department of Pharmacy Practice,
NIPER, S.A.S. Nagar, Punjab
Indian J.Pharm. Educ. Res. 40(2) April - June 2006