1. No category

-
Mucosal Tissue
Dermatology 2002;204(suppl 1):86-91
New Aspects of the Tolerance of the
Antiseptic Povidone-lodine in Different
ex viv0 Models
A. Kramer H. Below W. Behrens-Baumann G. Müller P. Rudolph
K. Reimer
Institute of Hygiene and Environmental Medicine, Ernst Moritz Arndt University, Greifswald, Germany
Key Words
Povidone-iodine lrritating potency Tissue toxicity
Eye resorption Nasal tolerance Cartilage tolerance
M e thici IIin-resista nt Staphylococcus aureus
-
Objective
-
In the German-speaking countries, the following fields
of application have established themselves for povidoneiodine (PVP-I): hand disinfection in cases of intolerance
of alcohols, antisepsis of the skin, antisepsis of the oral
cavity with the Special indications of prevention of mucoAbstract
lnvestigating n e w possibilities for the application of 1 YO sitis and application prior to tooth extraction in the sulcus
(v/v) iodophors, povidone-iodine (PVP-I) was better toler- gingivae to prevent bacteremia, antisepsis of the Vagina
ated in the HET-CAM or explant test than 1% (w/v) silver and orificium urethrae, wound antisepsis except bums
nitrate or tetracycline. After application t o the eye, at after transplantation, antiseptic irrigation of body cavities
least 2.6% o f used iodine were adsorbed. Therefore PVP- except Peritoneum and intraoperative application to the
I is more effective than silver nitrate or erythromycin,
bowel before surgical anastomosis [I]. Given the wide
meaning a possible alternative for the prevention o f oph- therapeutic scope of the iodophors, their clinical efficacy,
thalmia neonatorum. PVP-I is more active against methi- their high therapeutic range (table I), their good tolerance
cillin-resistant Staphylococcus aureus (MRSA) in a hu- for wounds [2] and their inhibition of bacterial mediators
m a n ex viv0 Skin model, which results in a complete of inflammation as well as of mediators of the host cells
eradication of S. aureus in the nasal cavity o f volunteers [3], we carried out supplementary studies on the field of
after 2 daily applications and will be better tolerated by application of the eye and examined the suitability of
human nasal cilial epithelium than chlorhexidine. Having iodophors for the biotopes nasal cavity and cartilage
the Same clinical tolerance as mupirocin, PVP-I is a useful tissue.
alternative for the antiseptic therapy of germ Carriers o f
MRSA. The synthesis o f proteoglycans in articular cartilage of bovine sesamoid bones was increased after
application of 5% (v/v) PVP-I without any increase in
catabolism revealing possibilities for the use as irrigation
Solution in the joint.
Copyright02002 S. Karger AG, Basel
KARG ER
Fax+4161306 1234
E-Mail [email protected]
www. karger.com
02002 S. Karger AG, Basel
1018-8665/02/2045-0086$18.50/0
Accessible online at:
www. karger. com/joumals/drm
Prof. Dr. Axel Kramer
Institute of Hygiene and Environmental Medicine
Emst Moritz Amdt University Greifswald, Hainstrasse 26
D- 17487 Greifswald (Germany)
Tel. +49 3834 83 46 10, Fax +49 3834 83 46 00, E-Mail [email protected]
Studies to Define the Ways of Using PVP-I as
Prophylaxisagainst Ophthalmia neonatorum
HET-CAM (Irritating Potency)
Method. The study was carried out in the classical
manner [4] by way of an objective quantification of the
response. For this purpose, the reaction at the chorioallantoic membrane was recorded with an S-VHS-C video
camera (Panasonic NV-S 70 E) and the image evaluated
by morphometry using the image analyzer Kontron IBAS
2000. In effecting a comparison with the stereomicroscopic assessment [4], an area of < 128% (median) could be
ascertained as tolerable range for the application of antiseptics to the eye.
Results und Discussion. With an exposure time of 30 s,
1Oo/ PVP-I meets the requirements for tolerance of an eye
Table 1. Calculated therapeutic range as quotient of oral LD50 for
rats (mmol/kg) and minimal microbicidal concentration at 5 min
exposure
Agent
Quotient
S.aureus
Chlorhexidine
Octenidine
Benzalkonium Chloride
PVP-I
Table 2. Growth rate
(O/O)
0.9
3.2
8
500
P.aeruginosa
0.9
3.2
8
1,000
in explant test with prepared peritoneal
tissue
Agent
Goncentration Exposure
min
Growth rate, YO
(control = 100%)
PVP-I
10%
1
63
30
1
40
Tetracycline
30 mg
Table 3. Iodine resorption after
conjunctival and periorbital application
of PVP-I on one eye
0
PVP-I
concentration, %
10
1.5
Tolerance of PVP-I
antiseptic with a median of 128%, whereas 1% AgN03
induces complete lysis and hence fails to meet the requirements of tolerance.
Explant Test (Tissue Tolerability)
Method. 1 mm x 1 mm pieces of Peritoneum of neonatal rats (inbreeding Lew 1 A) were exposed to the agents,
thereafter washed with Ringer’s Solution and cultivated in
24-well culture plates for 10 days [2].
Result und Discussion. Tetracycline proved to be highly toxic to tissue, as compared to PVP (table 2), although
the latter was undiluted (i.e. 10% PVP-I).
Resorption by Eye Antisepsis
Method. Prior to the extraction of the lens and subsequent implantation of a posterior chamber lens, antisepsis
of the conjunctiva and the periorbital skin was carried
out, each by way of a swab freshly impregnated with PVPI Solution (calculated average application 0.4-0.8 ml). For
the antisepsis of the conjunctiva, the PVP-I Solution was
applied on an Ethikeil swab, to wipe the conjunctiva for
about 10 s. Prior to surgery and after 24 h, the iodide in
the Urine was determined by the modified Wawschinek
method [5]. Creatinine was analyzed by the Jaffk method
using system packs from Boehringer Mannheim and a
Hitachi 7 17 device.
Results und Discussion. The elevated excretion of iodine is clearly below the theoretic maximum of full
resorption (table 3). In no case was any iodine contamination (for Germany, quotient > 300) ascertained; in fact,
the values were within the desirable range of the WHO
classification [61.
2.5% PVP-I on the eye causes severe burning so that
we recommend I .25% PVP-I on the grounds of our findings on tolerance and efficacy. With this concentration,
Neisseria gonorrhoeae is reduced >5 log within 15 s,
Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa within 30 s and adenoviruses type 2 are inactivated within 5 min by >4 log [7]. Chlamydia trachomatis
strain D is even destroyed by 0.1 13% PVP-I within 30 s
by >5 log [7]. By contrast, AgN03 and erythromycin are
Renal iodine excretion 24 h after surgery, pg I-/g creatinine
resorbed I%
calculated
increase
determined
increase
total
excretion
5,000
750
70
20
160
1.4
130
2.6
Dermatology 2002;204(suppl 1):86-9 1
87
4.5
4.0
3.5
1
After 15 s
0After 1 min
[7 After 10 min
3.36
3.0
0
$
2.0
Chlorhexidine 1.5%
PVP-I 10%
2-Propan01 70%
Fig. 1. Efficacy of PVP-I against MRSA on
human ex viv0 skin.
without effect against the chlamydial stem in a period of
15 min (reduction 0.9 and 1.7 log, respectively) [ 81.
Conclusion
Hence I .25% PVP-I for prophylaxis against ophthalmia neonatorum is a clearly more effective and efficient
alternative to local antibiotics and likewise silver nitrate.
For the Same reasons, 1.25% PVP-I is the treatment of
choice for preoperative eye antisepsis and 0.5 O/o PVP-I
with 5 min exposure for the prevention of infection in corneal transplants [9].
Nasal Cavity
Task
For two reasons, there is a need for an alternative to
mupirocin, currently used as Standard for MRSA Carriers
in nasal antisepsis:
because of the effect that is only microbiostatic, a failUre of therapy in up to 27% is observed [ 101;
for about 10 years, an increase has been ascertained in
methicillin-resistant S. aureus (MRSA) strains resistant to
mupirocin [ 113.
Contrary to mupirocin, PVP-I has a microbicidal effect without the risk of developing resistance [ 121.
Prior to testing PVP-I for tolerance in the nasociliary
epithelium, we checked its efficacy against MRSA in an
ex viv0 model on amputated skin, as requirement for the
application of PVP-I to eliminate MRSA Carriers.
88
Dermatology 2002;204(suppl 1):86-9 1
Antiseptic Efficacy of PVP-I against MRSA O n Human
ex viv0 Skin
Method. Testing was carried out on skin areas from
freshly amputated Skin of the thigh carried out for surgical
indications. Othenvise the test was carried out in the Same
way as the testing of skin antiseptics in volunteer trials
[ 131. Nevertheless, instead of contaminating the resident
flora as marker for efficacy, the amputated Skin was contaminated with an MRSA Patient strain [ 108CFU/ml].
Results und Discussion. 10% PVP-I is significantly
more effective than chlorhexidine and achieves practically the Same effect as 2-propanol (fig. 1). Since PVP-I is
also highly effective on oral and genital mucosa [ 14, 151, it
would seem to be promising, in view of the evidence of
tolerance, for the antisepsis of nasal cavities.
Tolerancefor the Ciliated Epithelium Cells of the
Nasal Mucosa
Method. Ciliated epithelium was taken out from the
concha nasalis inferior with a bronchoscopic brush without application of local anesthetics. The brushes were
washed out in 100 p1 DMEM at 37 "C. The activity of the
cilia was measured with a photocell under a Special microscope. The Signals were analyzed with the PC program
Turbolab [ 161.
Results und Discussion. No suppression of ciliary activity was caused by 1.25% PVP-I. By contrast, 0.2% chlorhexidine induced inhibition so that the activity of the cilia
was reduced to below the range of the physiological minimum (fig. 2).
Since chlorhexidine - despite the lack of a tolerance
test for ciliated epithelium - was used, albeit with unsatisKramer/Below/Behrens-Baumann/Miiller/
Rudolph/Reimer
..
PV?4 2.5%
PVP-I 1.25%
i
I
I
1
I
0
20.0
40.0
60.0
l
I
I
Ringer's
solution
0
100.0
l20.0
Fig. 2. Frequency (median) of cilia (100
celldagent).
Sesamoid bones from metacarpophalangeal joints of
adult animals were used [20]. Medial sesamoid bones
were dissected within 2-4 h after slaughter under aseptic
conditions. The cartilage from the medial sesamoid bones
of a particular animal is uniform with respect to glycosaminoglycan synthesis, glycosaminoglycan content and
proteoglycan composition.
The sesamoid bones were rinsed with 10 ml of sterile
Ringer's solution for 10 min, incubated, respectively, in
10 ml of sterile Ringer's solution (control), 10°/o PVP-I,
1O!o PVP-I, 0.1 Q!o Octenisept@ and 0.0 1O/o Octenisept in
Ringer's Solution at 37 "C for 2 h in a humidified atmo-
sphere with 5% CO2 and subsequently rinsed again for
5 x 5 min in 10 ml Ringer's Solution in each case. Thereafter sesamoid bones were incubated in 10 ml Ham's
F- 12 medium supplemented with antibiotics for 7 days at
37 "C in a humidified atmosphere with 5% CO2 changing
the medium daily. On day 6, 1.85 MBq 35S-sulfatewas
added to the medium to labe1 newly synthesized proteoglycans. After the labeling experiments, nonincorporated
35S-sulfatewas largely removed from the cartilage matrix
by way of a 5-fold washing process of the sesamoid bones;
10 ml Ringer's Solution was used for each wash for 5
min.
Cartilage plugs of 2.8 mm in diameter were carefully
removed and extracted thereafter in a stepwise manner
[21, 221. The first extraction was effected in 0.5 ml of
0.15 M Na-acetate buffer, pH 6.8, in the presence of protease inhibitors, by shaking 1.5-ml Eppendorf vials at 4 " C
for 24 h (iso-osmolar conditions). The second extraction
of cartilage plugs was carried out in 0.5 ml of 4 M guanidinium hydrochloride, 50 mM Na-acetate buffer, pH 5.8,
containing Protease inhibitors for 2-3 days at room temperature (dissociative conditions). The doubly preextracted plug was digested in 0.5 ml tris(hydroxymethy1)aminomethane hydrochloride buffer, pH 8.0, containing 1 mg pronase per milliter for 24 h at 56 "C (remaining
proteoglycans).
The culture media of sesamoid bones and extractsl
digests of cartilage plugs were used for the quantitative
determination of sulfated glycosaminoglycans by way of
the dimethylmethylene blue method [23]. Extracts and
digests of cartilage plugs were used for the estimation of
3%-sulfate incorporation into cartilage matrix by way of
Tolerance of PVP-I
Dermatology 2002;204(suppl 1):86-9 1
factory results on the MRSA colonization [ 171, we used
PVP-I on volunteers with S. aureus colonization. After
twice daily application for 3 days, total eradication was
achieved [ 181. Hence PVP-I is a more effective alternative
to mupirocin, with the Same clinical tolerance as mupirocin [ 181.
Cartilage
Task
After Kallenberger et al. [ 191had furnished evidence of
inhibited growth with fetal cartilage and the release of glycosaminoglycan in the medium, iodophors were deemed
to be unsuitable for use on the joint. Since, however, this
model does not relate to the status of the adult cartilage,
the issue should be taken up again with a sensitive relevant in vitro test.
Method
89
Table 4. Metabolism marker of cartilage
after 2 h exposure with test substances and
7 days cultivation in vitro
Agent
Concentration
Differences to the control (Ringer’s solution)
proteoglycans
p g h g wet weight cartilage
PVP-I
Octenidine
10
1
0.1
0.0 1
COMP
pg/mg wet weiht cartilage
-2.13
+1.79
- 16.44*
- 1.69
+2.62
+0.25
-1.35
+ 1.02
* p < 0.01. COMP = Cartilage oligomeric matrix protein.
Table 5. Incorporation rate of 35S-sulfate
into the proteoglycan fraction
Agent
PVP-I
Octenidine
Differences of proteoglycansto the control (Ringer’s Solution)
conc.
96
iso.
cpmlmg
diss.
cpmlmg
remaining
cpdmg
total
cpmlmg
10
1
0.1
0.0 1
+53
-6
-25
-35
+691
+1,013
- 1,432
-1,167
-384
-417
- 1,950
- 1,970
+ 307*
+597*
-3,381*
-3,137*
* p < 0.001. conc. = Concentration; iso. = extraction under iso-osmolarconditions; diss. =
extraction under dissociative conditions.
scintillation ß-counting. In the culture media of the sesamoid bones and in extraction solutions of the cartilage
plugs, the content of the cartilage oligomeric matrix protein was determined by way of a quantitative ELISA
method [24].
Results und Discussion
PVP-I as well as octenidine did not induce catabolic
metabolism with an increased loss of proteoglycans (table 4).
Under the influence of PVP-I, the incorporation rate of
35S-sulfatewas increased by 10 and 20%, respectively (table 5). Until now, this phenomenon, the Stimulation of
proteoglycan synthesis, has not been reported in the literature for antimicrobials. By contrast, corresponding to the
eluated amounts of proteoglycan and cartilage oligomeric
matrix protein, octenidine nearly completely inhibits the
synthesis of proteoglycan, depending on the concentration of octenidine (table 5).
In the meantime, the tolerability of 0.5% PVP-I is confirmed in the Synovia and hyaline cartilage of the rabbit
joint [25].
90
Dermatology 2002;204(suppl 1):86-9 1
Since, in contrast to the comparatively tested mucosal
antiseptics of chlorhexidine, polyhexanide, hexetidine
and cetylpyridinium Chloride, PVP-I loses none of its efficacy in vitro from 0.25% chondroitin Sulfate [26], the
requirements for efficacy in the joint are given.
Conclusion
In evaluating the test results and in light of the status of
knowledge on PVP-I, the following new application fields
can be Seen for iodophors: the prevention of ophthalmia
neonatorum, already empirically successful in Kenya
[27], the antisepsis of nasal cavities, especially with
MRSA colonization, and the antisepsis of joints.
Kramer/Below/Behrens-Baumann/Miiller/
Rudolph/Reimer
References
1 Kramer A: Antiseptika und Händedesinfektionsmittel; in Korting HC, Sterry W (eds):
Therapeutische Verfahren in der Dermatologie. Berlin, Blackwell, 2001, pp 273-294.
2 Kramer A, Adrian V, Rudolph P, Wurster S,
Lippert H: Explantationstest mit Haut und
Peritoneum der neonatalen Ratte als Voraussagetest zur Verträglichkeit lokaler Antiinfektiva
fur Wunden und Körperhöhlen. Chirurg 1998;
69:840-845.
3 König B, Reimer K, Fleischer W, König W:
Effects of betaisodona On Parameters of host
defense. Dermatology 1997;195(suppl 2):242248.
4 Kramer A, Adrian V, Rudolph P, Kühl H: Invitro-Prüfung der Verträglichkeit ausgewählter
antiseptischer Wirkstoffe bzw. Präparate; in
Kramer A, Wendt M, Wemer H-P (eds): Möglichkeiten und Perspektiven der klinischen Antiseptik. Wiesbaden, mhp, 1995, pp 41-48.
5 Zöllner H, Kramer A, Hampel R: IodmangelScreening. GIT Labormed 1995;18:330-332.
6 WHO, UNICEF, ICCIDD: Indicators for Assessing Iodine Deficiency Disorders and Their
Control through Salt Iodization (unpubl document WHO/NUT/94.6). Geneva, WHO/NUT,
1994, pp 28-36.
7 Wutzler P, Sauerbrei A, Klöcking R, Burkhardt
J, Schacke M, Thust R, Fleischer W, Reimer K:
Virucidal and chlamydicidal activities of eye
drops with povidone-iodine liposome complex.
Ophthalmic Res 2000;32:118-125.
8 Kramer A, Aspöck C, Below H, Behrens-Baumann W, Fusch C, Reimer K, Rudolph P,
Wutzler P: Prevention of ophthalmia neonatorum; in Kramer A, Behrens-Baumann W (eds):
Antiseptic Prophylaxis and Therapy of Ocular
Infections. Basel, Karger, in press.
9 Wilhelm F, Jendral G, Bredehom T, Duncker
G, Wilhelms D, Kramer A: Antimikrobielle
Dekontamination von Homhautspendermaterial. Ophthalmologe 2000;98: 143-1 46.
Tolerance of PVP-I
10 Bommer J, Vergetis W, Hingst V, Lenz W:
Elimination of Stuphylococcus uureus in dialysis patients by pseudomonic acid nasal ointment. Am SOCNephrol NY, November 17-20,
1991.
I 1 Layton MC, Patterson JD: Mupirocin resistance among consecutive isolates of oxacillinresistant and borderline oxacillin-resistant
Stuphylococcus uureus at a university hospital.
Antimicrob Agents Chemother 1994;38:16641667.
12 Reimer K, Schreier H, Erdos G, König B,
König W, Fleischer W: Molecular effects of a
microbicidal substance on relevant microorganisms: Electron-microscopic and biochemical study on povidone-iodine. Zentralbl Hyg
Umweltmed 1997/98;200:423-434.
13 Christiansen B, Eggers H-J, Exner M, Gundermann K-0, Heeg P, Hingst V, Höffler U,
Krämer J, Martiny H, Rüden H, Schliesser T,
Schubert R, Sonntag H-G, Spicher G, Steinmann J, Thofern E, Thraenhart 0, Wemer HP: Richtlinie fur die Prüfung und Bewertung
von Hautdesinfektionsmitteln. Zentralbl Hyg
Umweltmed 1991;192:99-103.
14 Pitten F-A, Kramer A: Antimicrobial efficacy
of antiseptic mouthrinse solutions. Eur J Clin
Pharmacol 1999;55:95-100.
15 Rudolph P, Fritze F, Reimer K, Klebingat K-J,
Kramer A: Efficacy and local tolerability of
povidone iodine (Betaisodona@)and octenidine hydrochloride Solution (Octenisept@) for
the antiseptic preparation of the orificium urethrae. Infection 1999;27:108-1 13.
16 Rudolph P, Reimer K, Mlynski G, Reese M,
Kramer A: Nose compatibility of local antiinfectives - An in-vitro model using ciliated epithelium. Hyg Med 2000;25:500-503.
17 Illiams JD, Waltho CA, Ayliffe GAJ, Lowburry
GJL: Trials of five antibacterial creams in the
control of nasal carriage of Stuphylococcus uureus: Lancet 1967;ii:390-392.
18 Kramer A, Glück U, Heeg P, Werner H-P:
Antiseptik; in Kramer A, Heeg P, Botzenhart K
(eds): Krankenhaus- und Praxishygiene.
München, Fischer, 2001, pp 252-268.
19 Kallenberger A, Kallenberger C, Willenegger
H: Experimental investigations on tissue compatibility of antiseptics. Hyg Med 1991;16:
383-395.
20 Korver GHV, Van de Stadt RL, Van Kampen
GPJ, Kiljan E, Van der Korst J K Bovine sesamoid bones: A culture system for anatomically
intact articular cartilage. In Vitro Ce11 Dev Biol
1989;25:1099-1 106.
21 Brand HS, Korver GHV, van de Stadt RJ, van
Kampen GPJ: Studies on the extraction of different proteoglycan populations in bovine articular cartilage. Biol Chem Hoppe-Seyler
1990;371:58 1-587.
22 Müller G, Hanschke M: In vitro model of characterizing the effects of compressive loading on
proteoglycans in anatomically intact articular
cartilage. Int J Sports Med 1997;18:438-448.
23 Müller G, Hanschke M: Quantitative and qualitative analyses of proteoglycans in cartilage
extracts by precipitation with 1.9-dimethylmethylene blue. Connect Tissue Res 1996;33:
243-248.
24 Müller G, Michel A, Altenburg E : COMP (cartilage oligomeric matrix protein) is synthesized
in ligament, tendon meniscus, and articular
cartilage. Connect Tissue Res 1998;39:233244.
25 Ganzer D, Völker L, Follak N, Wolf E, Granzow H : Reaktion des hyalinen Gelenkknorpels
und der Synovialis auf eine intraartikuläre Instillation von verschiedenen Antiinfektiva. Arthroskopie 2001; 14:31-44.
26 Müller G, Kramer A: In vitro action of a combination of selected antimicrobial agents and
chondroitin Sulfate. Chem Biol Interact 2000
124177-85.
27 Isenberg SJ, Apt L, Wood M: A controlled trial
of povidone-iodine as prophylaxis against ophthalmia neonatorum. N Eng1 J Med 1995;332:
562-566.
Dermatology 2002;204(suppl 1):86-9 1
91