1. No category

Chapter 5
Results and discussion
5.1 Preformulation and authentication of drug
A. Differential Scanning Colorimetry
Fig. 5.1 DSC curve for RPG
Fig. 5.2 DSC curve for MTF
The physical state of RPG and MTF was confirmed by DSC. (Fig. 5.1 &
5.2) The DSC curve for RPG and MTF showed sharp endotherm at
136.69oC and 234.59oC, confirming samples were in pure state.
Ph.D. Thesis
61
Chapter 5
Results and discussion
B. Ultra Violet Spectrum
Table 5.1 max of RPG and MTF in different solvents.
max (nm)
S. No. Solvent
RPG
MTF
1
Methanol
236.0
237.5
2
Distilled water
240.5
234.0
3
0.1 N HCl
237.5
233.0
4
Phosphate buffer (PH 6.8)
236.0
238.5
5
Octanol
240.5
237.0
Ph.D. Thesis
62
Chapter 5
Results and discussion
C. Fourier Transform Infra Red Spectroscopy (FTIR)
Fig. 5.3 FTIR spectrum of RPG
Ph.D. Thesis
63
Chapter 5
Results and discussion
Fig. 5.4 FTIR spectrum of MTF
RPG and MTF were identified on the basis of UV and FTIR analysis.
The drug samples exhibited absorption maxima in the range of 200-400
nm, which was same as mentioned in literature. The FTIR of the drug
Ph.D. Thesis
64
Chapter 5
Results and discussion
confirmed characteristic absorption bands. On the basis of above studies,
it was proved that drug samples were authentic.
E. pH solubility profile
Table 5.2 Solubility of RPG & MTF at different pH
Conc. (g mL-l)
S. No. Media
RPG
MTF
665.9
893.3
1.
0.1 N HCl (PH 1.2)
2.
Acetate buffer (PH 4.5)
-
904.8
3.
Phosphate buffer (PH 6.8)
-
500.3
4.
Distilled water (PH 7.2)
-
997.4
5.
Borate buffer (PH 8.6)
-
629.9
884.6
-
6.
Ethanolic
phosphate
buffer
(5:5; PH 6.8)
On the basis of the above studies the solubility of MTF was increased, as
pH was increased and showed maximum solubility in alkaline medium.
However RPG was found to be soluble in ethanolic phosphate buffer.
F. Stability at different temperatures
The max was obtained at 240 and 234 nm for RPG and MTF
respectively. This showed that no degradation of the drugs had occurred
at 370C and 80C indicating stability of the drugs.
5.2 Analytical methodology
5.2.1 Repaglinide
High Performance Thin Layer Chromatography (HPTLC)
i) Mobile phase optimization
The TLC procedure was optimized with a view to develop a stabilityindicating assay method. Both the pure drug and the degraded drug
solution were spotted on the TLC plates and run in different solvent
Ph.D. Thesis
65
Chapter 5
Results and discussion
systems. Initially, chloroform/methanol/ammonia was tried. The mobile
phase chloroform/methanol/ammonia (7.5:1.5:0.9, v/v/v) gave good
resolution with RF value of 0.38 for repaglinide but typical peak nature
was missing. Also the spot for repaglinide was slightly diffused.
Addition of 1.5 mL of glacial acetic acid to the above mobile phase
improved the spot characteristics. Finally, the mobile phase consisting of
chloroform/methanol/ammonia/glacial
acetic
acid
(7.5:1.5:0.9:0.1,
v/v/v/v) gave a sharp and symmetrical peak with RF value 0.38 ± 0.02
(Fig. 5.5). Well-defined spots were obtained when the chamber was
saturated with the mobile phase for 30 min at room temperature.
Fig. 5.5 Typical chromatogram of standard RPG (400 ng spot-1); peak 1:
RF: 0.38 ± 0.02, mobile phase chloroform/methanol/ammonia/glacial
acetic acid (7.5:1.5:0.9:0.1, v/v/v/v) at 240 nm.
ii) Validation
A. Linearity
The linear regression data for the calibration curves (n=3) showed a good
linear relationship over concentration range 50–800 ng spot−1 with
respect to the peak area. The regression equation was found to be Y =
Ph.D. Thesis
66
Chapter 5
Results and discussion
12.138 + 74.235 with correlation coefficient (r ± S.D.) of 0.998 ± 0.032.
No significant difference was observed in the slopes of standard curves
(ANOVA, P > 0.05).
B.Precision
Precision was considered at two levels of ICH suggestions i.e.
repeatability and intermediate precision. Repeatability of sample
application was determined as intra-day variation whereas intermediate
precision was determined by carrying out inter-day variation at three
different concentration levels 100, 300 and 500 ng spot-1 in triplicates.
% R.S.D. was found in the range between 0.1648–0.2859% and 0.1285–
0.3539%, respectively (Table 5.3). The low value of % R.S.D. (<1%)
reveals an excellent precision of the method.
Table 5.3 Intra- and inter-day precision of HPTLC method (n=3)
Amt
Intra-day precision
ng
Mean
S.D.
%RSD
Inter-day precision
SE
Mean
S.D.
%RSD
SE
-1
spot
area
100
4738.65
13.55
0.2859
7.8233
4625.43
12.43
0.3539
7.1766
300
15342.95 25.30
0.1648
14.6073
14832.75 14.57
0.2098
8.4122
500
25683.54 55.63
0.2169
32.1189
26893.54 45.44
0.1285
26.2355
area
iii) Accuracy, as recovery
The proposed method when used for extraction and subsequent
estimation of repaglinide from bulk drug and nanoemulsion formulation
formed after spiking with 50%, 100% and 150% of additional drug
afforded recovery of 98.79–99.61%, as listed in Table 5.4
Ph.D. Thesis
67
Chapter 5
Results and discussion
Table 5.4 Accuracy, as recovery studies (n=6)
Excess
Theoretical
Amount
(%)
(%)
S.E.
drug
content (ng)
obtained (ng)
Recovery
R.S.D.
0
200
198.76
99.38
0.486
0.572
50
300
298.84
99.61
0.354
0.584
100
400
396.61
99.15
0.515
1.153
150
500
493.97
98.79
0.286
0.654
added to
analyte
(%)
iv) Robustness
The S.D. of peak areas was calculated for each parameter and RSD was
found to be less than 2%. The low values of % RSD as shown in Table
5.5 indicated robustness of the method.
Table 5.5 Robustness testing (n=6)
Parameters
S.D
% R.S.D.
S.E.
Mobile phase composition (± 0.2 mL)
1.36
1.14
0.785
Amount of mobile phase (±5%)
1.08
0.85
0.623
Temperature (± 5%)
1.34
1.04
0.773
Plate pretreatment (± 5 min)
0.89
0.67
0.513
Relative humidity (± 5%)
1.44
1.21
0.831
Time from spotting to chromatography
0.75
0.63
0.433
0.93
0.74
0.536
( ± 20 min)
Time from chromatography to scanning
( ± 20 min)
v) Specificity
The peak purity of repaglinide was assessed by comparing the spectra at
peak start, peak apex and peak end positions of the spot, i.e. r2 (S, M) =
0.9997 and r2 (M, E) = 0.9979. Good correlation (r2 = 0.9998) was also
obtained between standard and sample spectra of repaglinide.
Ph.D. Thesis
68
Chapter 5
Results and discussion
vi) Sensitivity
The signal-to-noise ratios 3:1 and 10:1 were considered as LOD and
LOQ, respectively. The LOD and LOQ were found to be 0.023 and
0.069 ng spot-1, respectively.
vii) Nanoemulsion analysis for RPG
A single spot at RF 0.38 was observed in the chromatogram of the drug
samples extracted from prepared formulation. There was no interference
from the excipients commonly present in the formulation. The drug
content was found to be 99.39 ± 0.35 % with a % RSD of 0.62 for six
replicate determinations. It may therefore be inferred that degradation of
repaglinide had not occurred in the prepared nanoemulsion formulation
that were analysed by this method. The good performance of the method
indicated the suitability of this method for routine analysis of repaglinide
in pharmaceutical dosage form.
viii) Forced degradation of repaglinide
Fig. 5.6 Forced degradation study; (A) Chromatogram of acid treated
RPG (peak 1: degraded, RF: 0.19; peak 2: repaglinide, RF: 0.38; peak 3:
degraded. RF: 0.43); (B) Chromatogram of base treated RPG (peak 1:
degraded, RF: 0.20; peak 2: degraded, RF: 0.25; peak 3: degraded, RF:
0.29; peak 4: repaglinide, RF: 0.38; peak 5: degraded, RF: 0.51).
Ph.D. Thesis
69
Chapter 5
Results and discussion
A. Acid & base induced degradation
The chromatogram of the acid degraded sample for repaglinide showed
peak at RF 0.19 and 0.43 (Fig. 5.6A). The chromatogram of the base
degraded sample showed peak at RF value of 0.20, 0.25, 0.29 and 0.51
(Fig. 5.6B). The areas of the degraded peaks were found to be lesser than
the area of standard drug concentration (400 ng spot -1) indicating that
repaglinide undergoes degradation under acidic and basic conditions.
B. Photo-UV degradation
The photo degraded sample showed one additional peak at R F 0.22 when
drug solution was left in daylight for 3 days. The drug was degraded
when exposed to UV irradiation for 10 days and showed additional peaks
at RF value of 0.53.
C. Dry & wet heat degradation
The samples degraded under dry heat and wet heat conditions (Figs.
5.7A & 5.7B) showed additional peaks at RF 0.16, 0.46 and 0.51,
respectively. The spots of degraded products were well resolved from the
drug spot.
Fig. 5.7 Chromatograms of (A) dry heat degraded RPG (peak 1:
degraded, RF: 0.16, peak 2: repaglinide, RF: 0.38, peak 3: degraded, RF:
0.45); (B) wet heat degraded RPG (peak 1: repaglinide, RF: 0.38, peak 2:
degraded, RF: 0.51).
Ph.D. Thesis
70
Chapter 5
Results and discussion
D. Peroxide-induced degradation
The sample degraded with 30% v/v hydrogen peroxide showed
additional peaks at RF value of 0.41, 0.46 and 0.62 (Fig. 5.8A). The spots
of degraded products were well resolved from the drug spot.
ix) Detection of the related impurities
The spots other than the principal spot (repaglinide) from the sample
solution was not intense than the principal spot from the standard
solution. The sample solution showed one additional spot at R F0.06 (Fig.
5.8B). However, the peak area of the additional spot was found to be
much less as compared to the peak area of principal spot from the
standard solution.
Fig. 5.8 Chromatogram of (A) hydrogen peroxide treated RPG (peak 1:
repaglinide, RF: 0.38, peak 2: degraded, RF: 0.41, peak: 2 degraded, RF:
0.46, peak 3: degraded, RF: 0.62); (B) impurity profiling (peak 1:
impurity, RF: 0.06. peak 2: repaglinide, RF: 0.38).
x) Degradation kinetics
In basic medium, a decrease in the concentration of drug with increasing
time was observed. At the selected temperatures (50, 70 and 90 0C), the
degradation process followed pseudo first-order kinetics. Apparent first
order degradation rate constant and half life were obtained from the
Ph.D. Thesis
71
Chapter 5
Results and discussion
slopes of the straight lines at each temperature. Data obtained from first
order kinetics treatment was further subjected to fitting in Arrhenius
equation; [K = Ae-Ea/RT], Where K is rate constant, A is frequency factor,
Ea is energy of activation (Cal mol–1), R is gas constant (1.987 cal deg-1
mol-1) and T is absolute temperature (oK). A plot of (2 + log Kobs) values
versus (1/T × 103) the Arrhenius plot was obtained (Fig. 5.9), which was
found to be linear in the temperature range of 40–90 0C. The degradation
rate constant at room temperature (K250) was obtained by extrapolating
the resulting line in Arrhenius plot to 25 0C (Fig. 5.10) and was found to
be 12.4 × 10−2 h−1 and calculated t1/2 and t0.9 were 5.59 and 0.848 h
respectively.
Fig. 5.9 Pseudo first-order plot for the degradation of RPG with 1M
NaOH at various temperatures using HPTLC method. Ct, concentration
at time t, C0, concentration at time zero.
Ph.D. Thesis
72
Chapter 5
Results and discussion
Fig. 5.10 Arrhenius plot for RPG degradation in 1M NaOH and its
extrapolation for predicting the degradation at room temperature (25 °C).
5.3 Formulation development and optimization of repaglinide
nanoemulsion
A. Screening of components
i. Oils
Modified or hydrolysed vegetable oils have been widely used since these
excipients formed good emulsification systems with large number of
surfactants approved for oral administration and exhibited better drug
solubility properties (Kimura et al, 1994, Constantinides, 1995, Hauss
et al, 1998). They offer formulative and physiological advantages and
their degradation products resemble the natural end products of intestinal
digestion.
ii. Surfactants
An important criterion for selection of the surfactants is that the required
HLB value to form o/w nanoemulsion was >10 (Kommuru et al, 2001).
The surfactant chosen must be able to lower the interfacial tension to a
very small value to aid dispersion process during the preparation of the
nanoemulsion, provided a flexible film that can readily deform the
Ph.D. Thesis
73
Chapter 5
Results and discussion
correct curvature at the interfacial region for the desired nanoemulsion
type.
iii. Cosurfactants
Transient negative interfacial tension is rarely achieved by the use of
single surfactant, usually necessitating the addition of a cosurfactant.
Fluid interfacial film is again achieved by the addition of a cosurfactant.
B. Phase solubility studies
Phase solubility studies were done to determine the most suitable oil for
the preparation of nanoemulsion for repaglinide.
Table 5.6 Phase solubility determination (n=3)
S. No.
Oil
Average ± S.D. (mg mL-1)
1
Sefsol 218
182.06 + 0.04
2
Sefsol :Tween 80 (2:1)
94.23 + 0.08
3
Sefsol :Tween 80 (1:1)
89.98 + 1.06
4
Caproyl 90
45.7 + 0.75
5
Hydrogenated castor oil
64.37 + 9.35
6
Triacetin
35.85 + 2.20
7
Labrafac
23.54 + 1.22
8
Tween 80
98.73 + 8.39
9
Lauroglycol
15.68 + 3.59
10
Tween 20
43.48 + 7.12
Ph.D. Thesis
74
Chapter 5
Results and discussion
200
180
160
solubility (mg/mL)
140
120
100
80
60
40
20
0
SF 218 S:T 80 S:T 80 T80 H C O CP 90 T20
oils
(2:1) (1:1)
TCN
LBF
LRG
IPM
OA
Fig. 5.11 Bar diagram showing the highest solubility of repaglinide in
sefsol 218, HCO-Hydrogenated castor oil, CP90 -Caproyl 90, SF218 Sefsol 218, T80 -Tween 80. T20 -Tween 20, TCN - Triacetin, LBFLabrafac, LRG - Lauroglycol, IPM -Isopropyl myristate, OA - Oleic
acid, S:T 80 (2:1)- Sefsol:Tween 80 (2:1) , S:T 80 (1:1) - Sefsol:Tween
80(1:1).
Oil represents one of the most important excipients in the nanoemulsion
formulation, which can solubilize marked amounts of the lipophilic drug
and also because it can increase the amount of lipophilic drug
transportation (Holm et al, 2002). Sefsol 218 was found to solubilize
Ph.D. Thesis
75
Chapter 5
Results and discussion
maximum quantity of repaglinide i.e. 182.06 ± 7.68 mg/mL for the
preparation of nanoemulsion (Fig. 5.11). Therefore, it was selected as
the oil phase for the development of nanoemulsion. Higher oil solubility
of a poorly water soluble drug will favor an overall stability of the
formulation with effective dose optimization leading to cost effective
delivery system for repaglinide. Tween 80 was selected as the surfactant
and transcutol as the co-surfactant. Surfactant lowers the interfacial
tension to a very small value to aid dispersion process and provide a
flexible film that can readily deform around the droplets. The presence
of co-surfactants allows the interfacial film sufficient flexibility to take
up different curvatures required to form nanoemulsion over a wide range
of composition (Gosh, Murthy, 2006). Milli-Q water was taken as the
aqueous phase. All the selected excipients for the preparation of
formulations were under the GRAS (Generally Regarded as Safe)
category (Table 5.6).
C. Phase diagram construction
Pseudoternary phase diagrams were developed using the aqueous
titration method. Slow titration with the aqueous phase was performed
for each combination of oil and Smix, separately. The amount of aqueous
phase added was varied to produce a water concentration in the range of
5% to 95% of total volume at around 5% intervals. The phase behavior
of nanoemulsion system comprising oil, water and S mix ratio can be
studied with the aid of ternary phase diagram in which each corner of the
diagram represents 100% of that particular component. Special care was
taken to ensure that observations are not made on metastable systems
(Gosh, Murthy, 2006). The pseudoternary phase diagrams were
constructed using sefsol-218 as oily phase, Smix ratio (Tween 80 as a
surfactant and transcutol as a co-surfactant) and water. In the phase
diagrams, only o/w nanoemulsion region is shown, other phases are not
Ph.D. Thesis
76
Chapter 5
Results and discussion
shown due to overcrowding of the diagrams. Pseudoternary phase
diagrams were constructed separately for each Smix ratio (Fig. 5.12A–F).
In Fig. 5.12A, (Smix ratio 1:0) surfactant was used alone without cosurfactant and observed that a low amount of oil (27%, v/v) was
solubilized at high concentration of surfactant (48% v/v). Oil
solubilization was decreased as the concentration of surfactant was
increased. On addition of co-surfactant, solubilization of oil was
increased at lower concentration of Smix (1:1) and the region for
nanoemulsion in phase diagram was increased, as shown in Fig. 5.12B.
With slight increase in the concentration of co-surfcatant (Smix ratio 2:1),
no marked difference in nanoemulsion region in phase diagram, Fig.
5.12C was observed. In Smix ratio 3:1, (Fig. 5.12D), there was an
increrement in the nanoemulsion region with increasing concentration of
co-surfactant. But as the concentration of surfactant increasing in S mix 1:2
and 1:3, the region for nanoemulsion was decreasing due to decreasing
oil solubilization (Fig. 5.12E & F). This indicates that the proper ratio of
Smix is important for a wide range of nanoemulsion region in phase
diagram. Different formulations having less than 27% of the oily phase
and minimum quantity of Smix were selected from phase diagrams for
further studies. This may be attributed to the fact that the addition of cosurfactant may lead to greater penetration of the oil phase in the
hydrophobic region of the surfactant monomers thereby further
decreasing the interfacial tension, which will lead to increase in the
fluidity of the interface and thus increasing the entropy of the system
(Gosh, Murthy, 2006). While studying the phase diagrams (Fig. 5.12
A–5.12 F), it can be seen that transient negative interfacial tension is
rarely achieved by the use of single surfactant, usually necessitating the
addition of a cosurfactant. Fluid interfacial film is again achieved by the
addition of a co-surfactant. In the absence of co-surfactant, a highly rigid
Ph.D. Thesis
77
Chapter 5
Results and discussion
film is formed by the surfactant and thus it produces nanoemulsion over
only a very limited range of concentration (Lawrence, Rees, 2000).
Fig. 5.12 Pseudo-ternary phase diagrams [Smix ratio = 1:0 (A), 1:1 (B),
2:1 (C),3:1 (D), 1:2 (E), 1:3 (F)].The dotted area represents o/w
nanoemulsion region.
D. Thermodynamic stability
Nanoemulsions are thermodynamically stable systems with no phase
separation, creaming or cracking. Therefore, the selected formulations
were subjected to thermodynamic studies (i.e. heating cooling cycle,
centrifugation
and
freeze–thaw
cycle).
The
observation
for
thermodynamic stability studies are given in Table 5.7. Formulations,
which did not pass the thermodynamic tests, were dropped out and the
remaining
were
subjected
to
dispersibility
test.
In
case
of
macroemulsions, the interfacial energy is much larger than the entropy
and hence the process of emulsification is non-spontaneous i.e. energy is
needed to produce the emulsion by the use of high-speed mixture,
whereas in case of nanoemulsion the interfacial tension is made
Ph.D. Thesis
78
Chapter 5
Results and discussion
sufficiently low so that interfacial energy become comparable or even
lower than the entropy of dispersion, and hence the free energy of the
system becomes zero or negative. This explains the thermodynamic
stability of nanoemulsion (Razdan, Deverajan, 2003).
E. Dispersibility tests
The use of gastro-intestinal fluids for dilution of nanoemulsion may
result in the gradual desorption of surfactant located at the globule
interface leading to precipitation of the drug or phase separation of the
nanoemulsion making the formulation useless. The dispersibility test was
carried out to assess the efficiency of nanoemulsion and the results are
demonstrated in Table 5.7 Formulations, which failed dispersibility test
i.e B, C, D and E were discarded.
Ph.D. Thesis
79
Chapter 5
Results and discussion
Table 5.7 Observations for thermodynamic stability study and dispersibility test of repaglinide nanoemulsion formulations.
Smix
Formulation
ratio
code
1:0
1:1
2:1
% Smix
% Oil
%
Observations
based
on Observations
Water
themodynamic stability test
HCa
Centb
Fc
based
dispersibility test
Distilled
H2 O
on
Inference
0.1 N HCl
RN 1
20
5
75
x
–
–
–
–
Failed
RN 2
25
10
65
x
–
–
–
–
Failed
RN 3
25
5
70
x
–
–
–
–
Failed
RN 4
30
20
50
x
–
–
B
B
Failed
RN 5
30
10
60



C
C
Failed
RN 6
30
5
65



A
A
Passed
RN 7
35
15
50


x
B
B
Failed
RN 8
35
10
55


x
C
C
Failed
RN 9
35
5
60


x
C
C
Failed
RN 10
40
10
50

x
–
B
B
Failed
RN 11
40
5
55

x
–
B
B
Failed
RN 12
45
5
50



A
A
Passed
RN 13
30
5
65



A
A
Passed
RN 14
35
5
60

x
–
B
B
Failed
Ph.D. Thesis
80
Chapter 5
3:1
1:2
1:3
Results and discussion
RN 15
40
10
50

x
–
B
B
Failed
RN 16
40
5
55



A
A
Passed
RN 17
45
5
50



A
A
Passed
RN 18
40
5
55



A
A
Passed
RN 19
40
10
50



A
A
Passed
RN 20
40
10
50


x
C
C
Failed
RN 21
30
5
65


x
B
B
Failed
RN 22
30
10
60


x
B
B
Failed
RN 23
35
5
60


x
B
B
Failed
RN 24
40
5
55


x
C
C
Failed
RN 25
40
10
50


x
D
D
Failed
RN 26
45
5
50


x
B
B
Failed
RN 27
30
20
50
x
–
–
D
D
Failed
RN 28
35
15
50
x
–
–
E
E
Failed
RN 29
40
10
50


x
E
E
Failed
RN 30
45
5
50


x
D
D
Failed
a
Heating–cooling cycle, bCentrifugation, cFreeze–thaw cycle.
Ph.D. Thesis
81
Chapter 5
Results and discussion
F. Formulation of drug containing nanoemulsion
Six formulations (RN12, RN13, RN16, RN17, RN18 and RN19) were
selected on the basis of above studies, which were subjected to further
studies after addition of drug. The composition of selected formulations
is given in Table 5.7
G. Characterization of repaglinide nanoemulsion
i) Visual observation
The nanoemulsion was clear transparent, easily flowable liquid whereas
the macroemulsion was opaque and milky/cloudy white in appearance.
ii) Surface morphology
Morphology and structure of the nanoemulsion droplets were determined
by Transmission electron microscopy (TEM). The surrounding was
bright and the nanoemulsion appeared dark (Fig. 5.13A). A “positive”
image was seen using TEM. It is capable of point-to-point resolution;
therefore, droplet sizes were measured using TEM.
iii) Droplet size analysis (particle size distribution)
The RN13 formulation was presented minimum droplet size (76.23 ±
4.14 nm) whereas RN17and RN18 showed increase in the droplet size
due to increased concentration of oil (Table 5.8). In formulation RN13,
the distribution of droplets was in the range of 76–89 nm and the
maximum droplets (82%) were below a size of 90 nm (Fig. 5.13B). The
formulation showed nano droplets with low values of polydispersity
indicating
uniformity
in
the
nanoemulsion
formulation.
The
polydispersity values were 0.214, 0.183, 0.195, 0.229, 0.407 and 0.198
for different formulations RN12, RN13, RN16, RN17, RN18 and RN19
respectively (Table 5.8).
Ph.D. Thesis
82
Chapter 5
Results and discussion
Fig. 5.13 Transmission electron microscopic positive image of optimized
repaglinide nanoemulsion (Formulation RN13). B. Size distribution by
intensity of optimized nanoemulsion (Formulation RN13)
iv) Viscosity determination
The viscosities of the nanoemulsions (RN12, RN13 RN16, RN17, RN18
and RN19) are given in Table 5.8. The viscosity of nanoemulsion
formulation was very low as expected as one of the characteristic. It was
observed from the Table 5.8 that viscosity of all the formulations was
less than 24 cps. Formumlation RN13 has the minimum viscosity i.e.
Ph.D. Thesis
83
Chapter 5
Results and discussion
21.447 ± 0.215 cps. Results also revealed that the viscosity is directly
proportional to the concentration of oils and surfactants used in the
formulation. It can be observed that, in general, viscosity of all
formulations was very low.
v) Refractive index
Refractive index (RI) being an optical property is used to characterize
the isotropic nature of the nanoemulsion. It was observed from the Table
5.8 that the selected nanoemulsion formulations were chemically stable
and remained isotropic in nature, thus having no drug excipient
interactions. The observation table shows that as the concentration of the
oils increases in the formulation, the RI increases (RN19) (Table 5.8).
vi) Electrical conductivity
Type of nanoemulsion (o/w or w/o) and the stability of the nanoemulsion
(phase inversion on storage) can be determined by electrical conductivity
(σ). The conductivity of the formulations is given in the Table 5.8 The
lowest σ was found 403.213 ± 1.181μS/cm for RN17 and the highest was
527.106 ± 2.125μS/cm for RN12. This indicated that the formulation
was o/w type. Electrical conductivity is directly proportional to the
percentage of water. Higher the electrical conductivity more will be the
percentage of water, which allows more freedom for mobility of ions.
Ph.D. Thesis
84
Chapter 5
Results and discussion
Table 5.8 Droplet size, polydispersity index, viscosity, refractive index and electrical conductivity of selected repaglinide
nanoemulsion formulations.
Formulation
Droplet size ± S.D.
Polydispersity indexa ±
Viscosity ± S.D.
Refractive index ±
Conductivity
code
(nm)a
S.D.
(cps)a
S.Da
(μS/cm) ± S.Da
RN 12
92.18 ± 8.31
0.214 ± 0.013
23.195 ± 0.342
1.6852 ± 0.008
527.106 ± 2.125
RN 13
76.23 ± 4.14
0.183 ± 0.011
21.447 ± 0.215
1.6605 ± 0.004
512.413 ± 3.157
RN 16
89.75 ± 6. 43
0.195 ± 0.014
22.412 ± 0.435
1.6962 ± 0.008
458.416 ± 4.685
RN 17
91.24 ± 11.82
0.229 ± 0.016
21.984 ± 0.463
1.6756 ± 0.005
403.213 ± 1.181
RN 18
96.19 ± 14. 97
0.407 ± 0.013
23.056 ± 0.216
1.7912 ± 0.007
452.415 ± 1.105
RN 19
97.15 ± 9.16
0.198 ± 0.017
23.857 ± 0.541
1.8502 ± 0.008
501.183 ± 5.764
a
Mean ± S.D., n = 3
Ph.D. Thesis
85
Chapter 5
Results and discussion
5.4 In vitro drug release for repaglinide
Dissolution studies were performed to compare the release of drug (2 mg
repaglinide) from six different formulations (RN12, RN13, RN16,
RN17, RN18, RN19) and marketed formulation i.e. Repaglinide tablet
(REGAN) manufactured by Ranbaxy India Pvt. Ltd., India.
The concentration was determined by extrapolation of calibration curve
and graph was plotted between time and percent cumulative release (Fig.
5.14). The pattern of drug release in distilled water and simulated gastric
fluid was found very similar to each other in all formulations. The
highest release i.e. 98.22% was obtained in case of RN13. The minimum
release was observed in RN19 formulation (61.90%), this may be due to
bigger globule size, which may slow down the release of the drug from
nanoemulsion formulation. All the nanoemulsion formulations showed
better results as compared to conventional marketed formulation, (i.e.
tablet) because of small globule size, low viscosity and low
polydispersity values. Release of drug from RN19 (10% v/v, oil) was
lower than that from RN12, RN13, RN16, RN17 and RN18 (5% v/v, oil)
because of higher oil concentration and bigger droplet size. In addition to
this, the higher oil concentration may restrain the release of drug into the
medium due to lipophilic character of repaglinide as the partitioning of
drug will be more towards the oil.
Ph.D. Thesis
86
Chapter 5
Results and discussion
120
% Drug release
100
80
60
RN12
RN13
40
RN16
RN17
20
RN18
RN19
CF
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (Hrs)
Fig. 5.14 Comparative in vitro release profile of different formulations
of repaglinide.
The complete dissolution of repaglinide in oily phase showed maximum
release because of small droplet size, and eventually higher surface area,
which permits faster rate of drug release. The RN13 formulation (for
each dose i.e. R1, R2 & R3) was selected for in vivo studies because it
offered highest drug release (98.22%), optimum globule size (76.23 nm),
minimum polydispersity value (0.183), lower viscosity (21.45 cps) and
30% v/v of Smix (tween 80 as surfactant, transcutol as the co-surfactant).
5.5 Biochemical evaluation of repaglinide
The rats were divided into six groups, each containing six animals. A
treatment schedule is given in (Table 5.9).
The time course profile of blood glucose response in rats is shown in
Table 5.10. The blood glucose level in diabetic rats significantly
(p<0.001) reduced in orally administered drug (2 mg) upon 12 hrs and
was reported as 109.99 ± 7.92 mg/dl from 304.40 ± 20.73 mg/dl, it was
169.02 ± 18.18 mg/dl (p<0.01), 99.74 ± 8.52 mg/dl (p<0.001) and
100.36 ± 9.50 mg/dl (p<0.001) from 301.73 ± 12.53 mg/dl, 302.32 ±
11.21 mg/dl and 309.63 ± 15.21 mg/dl for 12 hrs in case of orally
Ph.D. Thesis
87
Chapter 5
Results and discussion
administered nano-emulsion containing 0.5, 1.0 and 2 mg of
repaglinide.The findings showed nano emulsion (having 1 mg/kg
repaglinide) significantly decreased blood glucose level which was
almost similar to oral conventional formulation of 2 mg repaglinide in
diabetic rats.
Table 5.9 Treatment schedules of repaglinide formulations for
antidiabetic activity in SD rats.
Groups
I
Drugs
Normal
Treatment schedule
Normal saline 10 mL/kg p.o. single dose
control
II
III
Diabetic
Normal saline 10 mL/kg p.o. single dose +
control
STZ (100 µg/g i.p.)
Standard
Conventional formulation of repaglinide 2
mg/kg p.o. single dose given to diabetic rats
IV
R1 (RN13)
Optimized nanoemulsion of repaglinide 0.5
mg/kg p.o. single dose given to diabetic rats
V
R2 (RN13)
Optimized nanoemulsion of repaglinide 1.0
mg/kg p.o. single dose given to diabetic rats
VI
R3 (RN13)
Optimized nanoemulsion of repaglinide 2
mg/kg p.o. single dose given to diabetic rats
Ph.D. Thesis
88
Chapter 5
Results and discussion
Table 5.10 Effects of repaglinide nanoemulsion on glucose level in the control and experimental groups of rat.
Time (h)
0
2
8
12
24
Normal control
Diabetic control
(NC)
(DC)
85.64 ± 4.88
305.24 ± 21.09*
84.93 ± 3.99
305.84 ± 20.95
*
306.51 ± 39.18
*
313.19 ± 20.25
*
323.60 ± 23.93
*
86.13 ± 4.79
84.28 ± 3.47
83.41 ± 4.31
CF
R1
R2
R3
304.40 ± 20.73*
301.73 ± 12.53*
302.32 ± 11.21*
309.63 ± 15.21*
184.98 ± 11.32##
229.84 ± 6.65#
192.11 ± 16.18##
189.00 ± 16.50##
(39%)
(24%)
(36%)
(39%)
110.69 ± 7.43##
186.50 ± 23.54#
99.92 ± 8.68##
99.27 ± 7.14##
(63%)
(38%)
(67%)
(68%)
109.99 ± 7.92##
219.12 ± 35.30#
100.18 ± 8.47##
100.36 ± 9.52##
(64%)
(27%)
(67%)
(68%)
187.97 ± 11.05##
283.51 ± 31.70 ns
177.90 ± 9.16##
181.93 ± 9.61##
(38%)
(6%)
(41%)
(41%)
All values are expressed as Mean ± SEM of 6 rats in each group,
*P<0.001 considered more significant, when compared with NC,
#P<0.01 and ##P<0.001 considered significant, when compared with DC,
P>0.05 considered non significant (ns), when compared with DC,
% = Percentage reduction in blood glucose level,
CF=Conventional formulation (2 mg/kg),
R1, R2 and R3 = Nanoemulsion 0.5 mg/kg, 1 mg/kg and 2 mg/kg respectively.
Ph.D. Thesis
89
Chapter 5
Results and discussion
5.6 Formulation development and optimization of metformin
A. Screening of components
i. Oils
Modified or hydrolysed vegetable oils have been widely used since these
excipients form good emulsification systems with large number of
surfactants approved for oral administration and exhibit better drug
solubility properties (Kimura et al, 1994, Constantinides, 1995, Hauss
et al, 1998). They offer formulative and physiological advantages and
their degradation products resemble the natural end products of intestinal
digestion.
ii. Surfactants
An important criterion for selection of the surfactants is that the required
HLB value to form o/w nanoemulsion is >10 (Kommuru et al, 2001).
The surfactant chosen must be able to lower the interfacial tension to a
very small value to aid dispersion process during the preparation of the
nanoemulsion, provide a flexible film that can readily deform the correct
curvature at the interfacial region for the desired nanoemulsion type.
iii. Cosurfactants
Transient negative interfacial tension is rarely achieved by the use of
single surfactant, usually necessitating the addition of a cosurfactant.
Fluid interfacial film is again achieved by the addition of a cosurfactant.
B. Phase solubility studies
Phase solubility studies were done to determine the most suitable oil for
the preparation of nanoemulsion for metformin.
Oil represents one of the most important excipients in the nanoemulsion
formulation, which can solubilize marked amounts of the lipophilic drug
and also because it can increase the amount of lipophilic drug
transportation (Holm et al, 2002). Hydrogenated castor oil was found to
solubilize maximum quantity of metformin i.e. 124.37 + 8.32 mg/mL for
Ph.D. Thesis
90
Chapter 5
Results and discussion
the preparation of nanoemulsion (Fig. 5.15). Therefore, it was selected
as the oil phase for the development of nanoemulsion. Tween 80 selected
as the surfactant and transcutol as cosurfactant. Surfactant lowers the
interfacial tension to a very small value to aid dispersion process and
provide a flexible film that can readily deform around the droplets. The
presence of co-surfactants allows the interfacial film sufficient flexibility
to take up different curvatures required to form nanoemulsion over a
wide range of composition (Gosh, Murthy, 2006). Milli-Q water was
taken as the aqueous phase. All the selected excipients for the
preparation of formulations were under the GRAS (Generally Regarded
as Safe) category.
Table 5.11 Phase solubility determination of MTF (n=3)
S. No.
1
2
3
4
5
6
7
8
9
10
11
12
Oil
Oleic acid
Isopropyl myristate
Triacetin
Caproyl 90
Hydrogenated castor oil
Transcutol
Labrafac
Tween 80
Lauroglycol
Tween 20
Hydrogenated castor oil :
Tween 80 (2:1)
Hydrogenated castor oil :
Tween 80 (1:1)
Ph.D. Thesis
Average ± S.D. (mg mL-1)
3.67 ± 0.01
11.82 ± 0.04
15.45 ± 1.04
8.15 ± 0.64
124.37 ± 8.32
35.85 ± 1.30
10.04 ± 0.25
87.34 ± 9.36
15.68 ± 1.53
9.12 ± 8.14
85.34 ± 4.32
78.63 ± 3.43
91
Chapter 5
Results and discussion
Fig. 5.15 Bar diagram showing the highest solubility of metformin in
Hydrogenated castor oil. HCO-Hydrogenated castor oil, TRS-Transcutol
CP90 -Caproyl 90, T80 -Tween 80. T20 -Tween 20, TCN - Triacetin,
LBF- Labrafac, LRG - Lauroglycol, IPM -Isopropyl myristate, OA Oleic acid, HCO:T 80 (2:1) , HCO:Tween 80(1:1).
Ph.D. Thesis
92
Chapter 5
Results and discussion
Table 5.12 Observations for thermodynamic stability study and dispersibility test of metformin nanoemulsion formulations.
Formulation
Smix ratio
%
Smix
%
Oil
%
Water
Observations based on
Observations based on
themodynamic stability test
dispersibility test
Cen
HCa
b
t
Fc
Distilled H2O
Inference
0.1 N HCl
–
–
×
–
–
–
70
×
–
–
–
–
Failed
20
50
×
–
–
B
B
Failed
30
10
60
√
√
√
C
C
Failed
MN6
30
5
65
√
√
√
B
B
Failed
MN7
35
15
50
√
√
×
B
B
Failed
MN8
35
10
55
√
√
×
C
C
Failed
35
5
60
√
√
×
C
C
Failed
40
10
50
√
x
–
B
B
Failed
MN11
40
5
55
√
√
√
A
A
Passed
MN12
45
5
50
√
√
√
A
A
Passed
30
5
65
√
√
–
B
B
Failed
35
5
60
√
x
–
B
B
Failed
MN1
20
5
MN2
25
10
65
25
5
30
MN5
MN3
MN4
MN9
MN10
MN13
MN14
Ph.D. Thesis
1:0
1:1
2:1
75
×
93
–
–
–
Failed
Failed
Chapter 5
MN15
40
10
50
√
x
–
B
B
Failed
MN16
40
5
55
√
√
√
A
A
Passed
MN17
45
5
50
√
√
√
A
A
Passed
MN18
40
5
55
√
x
x
C
C
Failed
40
10
50
√
√
√
A
A
Passed
MN20
40
10
50
√
√
x
B
B
Failed
MN21
30
5
65
√
√
√
A
A
Passed
MN22
30
10
60
√
√
×
B
B
Failed
35
5
60
√
√
×
B
B
Failed
40
5
55
√
√
x
C
C
Failed
MN25
40
10
50
√
√
x
D
D
Failed
MN26
45
5
50
√
√
x
B
B
Failed
MN27
30
20
50
×
–
–
D
D
Failed
35
15
50
×
–
–
E
E
Failed
40
10
50
√
√
x
E
E
Failed
45
5
50
√
√
x
D
D
Failed
MN19
MN23
MN24
MN28
MN29
MN30
a
Results and discussion
3:1
1:2
1:3
Heating–cooling cycle, bCentrifugation, cFreeze–thaw cycle.
Ph.D. Thesis
94
Chapter 5
Results and discussion
C. Phase diagram construction
The pseudoternary phase diagrams were constructed using hydrogenated
castor oil as oily phase, Smix ratio (Tween 80 as a surfactant and
transcutol as a co-surfactant) and water. In the phase diagrams, only o/w
nanoemulsion region is shown, other phases are not shown due to
overcrowding of the diagrams. Pseudo ternary phase diagrams were
constructed separately for each Smix ratio (Fig. 5.16A–F). In Fig. 5.16A,
(Smix ratio 1:0) surfactant was used alone without co-surfactant and
observed that a low amount of oil (20%, v/v) was solubilized at higher
concentration of surfactant (30% v/v). Oil solubilization was decreased
as the concentration of surfactant was increased. On addition of cosurfactant, solubilization of oil was increased at lower concentration of
Smix (1:1) and the region for nanoemulsion in phase diagram was
increased, as shown in Fig. 5.16B. With slight increase in the
concentration of co-surfcatant (Smix ratio 2:1), no marked difference in
nanoemulsion region in phase diagram, Fig. 5.16C was observed. In Smix
ratio 3:1 and 1:2 (Fig. 5.16D & 5.16E), there was an increrement in the
nanoemulsion region with increasing concentration of co-surfactant. But
as the concentration of surfactant increasing in Smix 1:3, the region for
nanoemulsion was decreasing due to decreasing oil solubilization (Fig.
5.16F). This indicates that the proper ratio of Smix is important for a wide
range of nanoemulsion region in phase diagram. Different formulations
having less than 25% of the oily phase and minimum quantity of S mix
were selected from phase diagrams for further studies. This may be
attributed to the fact that the addition of co-surfactant may lead to greater
penetration of the oil phase in the hydrophobic region of the surfactant
monomers thereby further decreasing the interfacial tension, which will
lead to increase in the fluidity of the interface and thus increasing the
entropy of the system (Gosh, Murthy, 2006). While studying the phase
Ph.D. Thesis
95
Chapter 5
Results and discussion
diagrams (Fig. 5.16A–5.16F), it can be seen that transient negative
interfacial tension is rarely achieved by the use of single surfactant,
usually necessitating the addition of a cosurfactant. Fluid interfacial film
is again achieved by the addition of a co-surfactant. In the absence of cosurfactant, a highly rigid film is formed by the surfactant and thus
produces nanoemulsion over only a very limited range of concentration
(Lawrence, Rees, 2000).
Water
Water
A
Water
C
B
Oil
Smix
Oil
Smix
Water
Water
Smix
Oil
Smix
Water
F
E
D
Oil
Oil
Smix
Smix
Oil
Fig. 5.16 Pseudo-ternary phase diagrams [Smix ratio = 1:0 (A), 1:1 (B),
2:1 (C),3:1 (D), 1:2 (E), 1:3 (F)]. The dotted area represents o/w
nanoemulsion region.
D. Thermodynamic stability studies
Nanoemulsions are thermodynamically stable systems with no phase
separation, creaming or cracking. Therefore, the selected formulations
were subjected to thermodynamic studies (i.e. heating cooling cycle,
centrifugation
and
freeze–thaw
cycle).
The
observation
for
thermodynamic stability studies are given in Table 5.12 Formulations,
which did not pass the thermodynamic tests, were dropped out and the
Ph.D. Thesis
96
Chapter 5
remaining
Results and discussion
were
subjected
to
dispersibility
test.
In
case
of
macroemulsions, the interfacial energy is much larger than the entropy
and hence the process of emulsification is non-spontaneous i.e. energy is
needed to produce the emulsion by the use of high-speed mixture,
whereas in case of nanoemulsion the interfacial tension is made
sufficiently low so that interfacial energy become comparable or even
lower than the entropy of dispersion, and hence the free energy of the
system becomes zero or negative. This explains the thermodynamic
stability of nanoemulsion (Razdan, Deverajan, 2003).
E. Dispersibility tests
The use of gastro-intestinal fluids for dilution of nanoemulsion may
result in the gradual desorption of surfactant located at the globule
interface leading to precipitation of the drug or phase separation of the
nanoemulsion making the formulation useless. The dispersibility test was
carried out to assess the efficiency of nanoemulsion and the results are
demonstrated. Formulations, which failed (grade B, C, D and E)
dispersibility test, were discarded for further studies. (Table 5.12)
F. Formulation of drug containing nanoemulsion
Six formulations (MN11, MN12, MN16, MN17, MN19, and MN21)
were selected and were subjected to further studies after addition of MTF
(125 mg added to each selected formulation). The composition of
selected formulations is given in Table 5.12.
G. Characterization of metformin nanoemulsion
i. Visual observation
The nanoemulsion was clear transparent, easily flowable liquid whereas
the macroemulsion was opaque and milky/cloudy white in appearance.
ii. Surface morphology
Morphology and structure of the nanoemulsion droplets were determined
by Transmission electron microscopy (TEM). The surrounding was
Ph.D. Thesis
97
Chapter 5
Results and discussion
bright and the nanoemulsion appeared dark (Fig. 5.17A). A “positive”
image was seen using TEM. It is capable of point-to-point resolution;
therefore, droplet sizes were measured using TEM.
iii. Droplet size analysis
Droplet size measurement is the important parameter to optimize the
nanoemulsion formulation as well as to distinguish between the
nanoemulsion from microemulsion. Polydispersity is the ratio of
standard deviation to the mean droplet size and denotes the uniformity of
droplet size within the formulation. The lower the polydispersity value,
higher is the uniformity of the droplet size in the formulation.
The MN21 formulation presented minimum droplet size (92.25 ± 3.54)
nm whereas MN19 showed increase in the droplet size due to increased
concentration of oil (Table 5.12). In formulation MN21, the distribution
of droplets was in the range of 92–98 nm and the maximum droplets
(80%) were below a size of 96 nm (Fig. 5.17B). The formulation showed
nano droplets with low values of polydispersity indicating uniformity in
the nanoemulsion formulation. The polydispersity values were 0.198,
0.185, 0.187, 0.196, 0.204 and 0.172 for different formulations MN11,
MN12, MN16, MN17, MN19 and MN21, respectively (Table 5.12).
iv. Viscosity determination
The viscosites of the nanoemulsions (MN11, MN12 MN16, MN17,
MN19 and MN21) are given in Table 5.13. The viscosity of
nanoemulsion formulation was very low as expected as one of the
characteristic. It was observed from the Table 5.13 that viscosity of all
the formulations was less than 24 cps. Formumlation MN21 has the
minimum viscosity i.e. 22.124 ± 0.327 cps. Results also revealed that the
viscosity is directly proportional to the concentration of oils and
surfactants used in the formulation. It can be observed that, in general,
viscosity of all formulations was very low.
Ph.D. Thesis
98
Chapter 5
Results and discussion
v. Refractive index
Refractive index (RI) being an optical property is used to characterize
the isotropic nature of the nanoemulsion. It was observed from the Table
5.13 that the selected nanoemulsion formulations were chemically stable
and remained isotropic in nature, thus having no drug excipient
interactions. The observation table shows that as the concentration of the
oils increases in the formulation, the RI increases (MN19) (Table 5.13).
vi. Electrical conductivity
Electrical conductivity (σ) was determined to check not only the type of
nanoemulsion (o/w or w/o) but also the stability of the nanoemulsion
(phase inversion on storage). The conductivity of the formulations is
given in the Table 5.13. The lowest σ was found 445.278 ± 1.142 μS/cm
for MN11 and highest conductivity was 532.118 ± 2.451 μS/cm for
MN21. This indicated that the formulation was o/w type. Because the
current was passed through the water and the diffraction was seen.
Electrical conductivity is directly proportional to the percentage of
water. Higher the electrical conductivity more will be the percentage of
water, which allows more freedom for mobility of ions.
Ph.D. Thesis
99
Chapter 5
Results and discussion
Fig. 5.17 Transmission electron microscopic positive image and size
distribution by intensity of optimized nanoemulsion of optimized
metformin nanoemulsion formulation MN21).
Ph.D. Thesis
100
Chapter 5
Results and discussion
Table 5.13 In vitro characterization of the selected metformin nanoemulsion formulations.
a
Formulation
Droplet Size
Polydispersity
Viscosity ± S.D.
Refractive Index
Conductivity
Code
± S.D. (nm)a
Indexa
(cps)a
± S.Da
(μS/cm) ± S.Da
MN11
96.21 ± 3.42
0.198 ± 0.018
23.192 ± 0.332
1.6926 ± 0.007
445.278 ± 1.142
MN12
95.14 ± 7.38
0.185 ± 0.014
23.459 ± 0.269
1.6698 ± 0.009
502.412 ± 2.198
MN16
93.45 ± 4.52
0.187 ± 0.017
22.956 ± 0.389
1.6529 ± 0.006
487.478 ± 4.453
MN17
98.19 ± 7.46
0.196 ± 0.013
23.423 ± 0.423
1.6851 ± 0.005
468.196 ± 4.429
MN19
94.59 ± 5.26
0.204 ± 0.014
23.054 ± 0.425
1.8252 ± 0.007
458.413 ± 1.123
MN21
92.25 ± 3.54
0.172 ± 0.013
22.124 ± 0.327
1.6421 ± 0.006
532.118 ± 2.451
Mean ± S.D., n = 3
Ph.D. Thesis
101
Chapter 5
Results and discussion
5.7 In vitro drug release study of metformin
The composition of selected formulations used for in vitro release is
given in Table 5.12 Dissolution studies were performed to compare the
release of drug from six different formulations (MN11, MN12, MN16,
MN17, MN19, MN21) and marketed formulation i.e. metformin tablet
(GLUMET ®). The concentration was determined by extrapolation of
calibration curve and graph was plotted between time and percent
cumulative release (Fig. 5.18). The pattern of drug release in distilled
water and simulated gastric fluid was found very similar to each other in
all formulations. The highest release i.e. 98.70% was obtained in case of
MN21. The minimum release was observed in MN19 formulation
(76.41%), this may be due to bigger globule size, which may slow down
the release of the drug from nanoemulsion formulation. All the
nanoemulsion formulations showed better results as compared to
conventional marketed formulation, (i.e. tablet) because of small globule
size, low viscosity and low polydispersity values. Release of drug from
MN19 (10% v/v, oil) was lower than that from MN11, MN12, MN16,
MN17 and MN21 (5% v/v, oil) because of higher oil concentration and
bigger droplet size. In addition to this, the higher oil concentration may
restrain the release of drug into the medium due to lipophilic character of
metformin as the partitioning of drug will be more towards the oil. The
complete dissolution of metformin in oily phase showed maximum
release because of small droplet size, and eventually higher surface area,
which permits faster rate of drug release.
The MN21 formulation (for each dose i.e. M1, M2 & M3) was selected
for in- vivo studies because it offered highest drug release (98.70% %),
optimum globule size (92.25 nm), minimum polydispersity value
(0.172), lower viscosity (22.124 cps) and 30% v/v of S mix (tween 80 as
surfactant, transcutol as the co-surfactant).
Ph.D. Thesis
102
Chapter 5
Results and discussion
Fig. 5.18 In vitro release profile of different metformin nanoemulsions.
5.8 Biochemical evaluation of metformin
The rats were divided into six groups, each containing six animals. A
treatment schedule is given in Table 5.14.
The time course profile of blood glucose response in rats is shown in
Table 5.15. The blood glucose level in diabetic rats significantly
(p<0.001) reduced in orally administered drug (500 mg) upon 8 hrs and
was reported as 96.62 ± 6.62 mg/dl from 392.82 ± 12.51 mg/dl, it was
293.68 ± 17.46 mg/dl (p<0.01), 95.34 ± 10.56 mg/dl (p<0.001) and
96.95 ± 9.84 mg/dl (p<0.001) from 392.93 ± 11.78 mg/dl, 392.96 ±
15.71 mg/dl and 394.83 ± 9.23 mg/dl for 8 hrs in case of orally
administered nano-emulsion containing 35, 70 and 140 mg of
metformin.The findings showed nano emulsion (having 70 mg/kg
metformin) significantly decreased blood glucose level which was
almost similar to oral conventional formulation of 500 mg metformin in
diabetic rats.
Ph.D. Thesis
103
Chapter 5
Results and discussion
Table 5.14 Treatment schedules of metformin nanoformulations for
antidiabetic activity in SD rats.
Groups Drugs
I
Normal
control
II
IV
V
VI
Normal saline 10 mL/kg p.o. single dose
Diabetic Normal saline 10 mL/kg p.o. single dose + STZ
control
III
Treatment schedule
Standard
(100 µg/g i.p.)
Conventional formulation of metformin 500 mg/kg
p.o. single dose given to diabetic rats
M1
Optimized nanoemulsion of metformin 35 mg/kg
(MN21)
p.o. single dose given to diabetic rats
M2
Optimized nanoemulsion of metformin 70 mg/kg
(MN21)
p.o. single dose given to diabetic rats
M3
Optimized nanoemulsion of metformin 140 mg/kg
(MN21)
p.o. single dose given to diabetic rats
Ph.D. Thesis
104
Chapter 5
Results and discussion
Table 5.15 Effects of metformin nanoemulsion on glucose level in the control and experimental groups of rat.
Time (h)
0
2
8
12
24
NC
DC
CF
M1 (MN 21)
M2 (MN 21)
M3 (MN 21)
81.45 ± 2.98
305.24 ± 21.09*
392.82 ± 12.51*
392.93 ± 11.78*
392.96 ± 15.71*
394.83 ± 9.23*
81.17 ± 2.63
305.84 ± 20.95
*
189.65 ± 10.2##
282.1 ± 5.63#
152.41 ± 9.86##
144.47 ± 10.11##
(52%)
(28%)
(61%)
(63%)
306.51 ± 39.18
*
96.62 ± 6.62##
293.68 ± 17.46#
95.34 ± 10.56##
96.95 ± 9.84##
(75%)
(25%)
(76%)
(75%)
313.19 ± 20.25
*
131.79 ± 8.37##
311.68 ± 15.33#
132.87 ± 7.47##
130.77 ± 8.23##
(66%)
(21%)
(66%)
(67%)
323.60 ± 23.93
*
191.24 ± 16.29##
351.83 ± 27.79ns
177.90 ± 9.16##
194.35 ± 14.36##
(51%)
(10%)
(55%)
(51%)
84.22 ± 3.23
80.85 ± 2.75
78.17 ± 2.46
Mean ± SEM., n = 6
*
P<0.001 considered more significant, when compared with Normal control (NC),
#
P<0.01 and ##P<0.001 considered significant, when compared with Diabetic control (DC),
P>0.05 considered non significant (ns), when compared with DC,
% = Percentage reduction in blood glucose level,
CF=Conventional formulation (500 mg/kg),
M1, M2 and M3 = Nanoemulsion 35 mg/kg, 70 mg/kg and 140 mg/kg respectively.
Ph.D. Thesis
105
Chapter 5
Results and discussion
5.9 Stability studies
During stability studies, droplet size, viscosity, drug content, refractive
index and electrical conductivity were determined at 0, 30, 60, and 90
days. As can be seen from Table 5.16 & 5.17, the values of these
parameters were slightly varied with respect to time but the changes in
the values of observed parameters were not found to be statistically
significant (P > 0.05). Stability studies at 8 ± 2 0C, 25 0C /60 % RH and
40 0C /75 % RH predicted highest degradation of 0.99% of repaglinide
and 0.98% of metformin in the optimized formulations (RN13 & MN21)
at 40 0C by the end of 90 days.
.
Ph.D. Thesis
106
Chapter 5
Results and discussion
Table 5.16 Percent drug remaining, droplet size, viscosity, refractive index and conductivity in optimized repaglinide
nanoemulsion formulation (RN13), stored at elevated temperatures for 3 months.
Time
(Days)
Temperature
/RH
%Drug
remained
0
30
60
90
0
30
60
90
0
30
60
90
8 ± 20C
8 ± 20C
8 ± 20C
8 ± 20C
250C/60 % RH
250C/60 % RH
250C/60 % RH
250C/60 % RH
400C/75 % RH
400C/75 % RH
400C/75 % RH
400C/75 % RH
100
99.89
99.53
98.98
100
99.87
99.84
99.86
100
99.97
99.94
98.59
Ph.D. Thesis
Mean
droplet
± SD (nm)
92.25 ± 3.54
92.13 ± 4.58
92.18 ± 3.26
92.20 ± 4.62
92.25 ± 3.54
92.19 ± 7.34
92.16 ± 2.68
92.28 ± 5.36
92.25 ± 3.54
91.86 ± 6.34
92.17 ± 4.30
92.19 ± 3.56
Mean viscosity
± SD (cP)
Refractive
index
Conductivity
(μS/cm) ± S.Da
22.124 ± 0.327
21.356 ± 0.243
21.024 ± 0.249
22.448 ± 0.143
22.124 ± 0.327
22.256 ± 0.418
22.246 ± 0.247
22.578 ± 0.356
22.124 ± 0.327
22.359 ± 0.260
22.356 ± 0.298
22.379 ± 0.431
1.6421 ± 0.006
1.639 ± 0.004
1.667 ± 0.002
1.6398 ± 0.002
1.6421 ± 0.006
1.6580 ± 0.002
1.6528 ± 0.004
1.6389 ± 0.006
1.6421 ± 0.006
1.6578 ± 0.002
1.6543 ± 0.007
1.6389 ± 0.004
532.118 ± 2.451
524.234 ± 3.234
516.128 ± 3.284
522.473 ± 5.263
532.118 ± 2.451
512.241 ± 4.756
506.782 ± 5.248
512.326 ± 3.536
532.118 ± 2.451
509.238 ± 3.86
513.536 ± 5.278
501.486 ± 3.896
107
Chapter 5
Results and discussion
Table 5.17 Percent drug remaining, droplet size, viscosity and refractive index in optimized metformin nanoformulation
(MN21), stored at elevated temperatures for 3 months.
Time
(Days)
Temperature
/RH
%Drug
remained
0
30
60
90
0
30
60
90
0
30
60
90
8 ± 20C
8 ± 20C
8 ± 20C
8 ± 20C
250C/60 % RH
250C/60 % RH
250C/60 % RH
250C/60 % RH
400C/75 % RH
400C/75 % RH
400C/75 % RH
400C/75 % RH
100
99.89
99.53
98.98
100
99.69
99.54
99.24
100
99.95
99.91
98.64
Ph.D. Thesis
Mean
droplet
± SD (nm)
76.23 ± 4.14
76.12 ± 3.19
76.09 ± 6.23
76.12 ± 4.42
76.23 ± 4.14
76.15 ± 2.53
75.42 ± 7.24
75.49 ± 4.18
76.23 ± 4.14
75.89 ± 2.51
76.02 ± 8.36
76.04 ± 4.28
Mean viscosity
± SD (cP)
Refractive
index
Conductivity
(μS/cm) ± S.Da
21.447 ± 0.215
21.356 ± 0.243
21.024 ± 0.249
22.448 ± 0.143
21.447 ± 0.215
21.752 ± 0.168
23.412 ± 0.273
22.129 ± 0.214
21.447 ± 0.215
20.423 ± 0.249
21.021 ± 0.186
21.267 ± 0.243
1.6605 ± 0.004
1.6643 ± 0.003
1.6594 ± 0.006
1.6526 ± 0.008
1.6605 ± 0.004
1.6589 ± 0.005
1.6526 ± 0.009
1.6725 ± 0.003
1.6605 ± 0.004
1.6452 ± 0.003
1.6463 ± 0.008
1.6526 ± 0.006
512.413 ± 3.157
505.256 ± 3.692
502.692 ± 3.683
523.294 ± 4.263
512.413 ± 3.157
509.267 ± 2.726
498.446 ± 4.182
501.786 ± 2.148
512.413 ± 3.157
523.243 ± 3.023
516.436 ± 2.173
525.526 ± 4.186
108