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Chapter -4 Experimental &

Results4a. Analytical method development

Preformulation studies

4b. Effect of physical process on solubility and thermodynamic properties

4c. Mucoadhesive, acid buffering and sustained release tablet

4d. Mucoadhesive and thermo-sensitive gel incorporating SLN

4e. Mucoadhesive and thermo-sensitive gel incorporating Nanoemulsion

Experimental & Result Chapter 4

4.1 MATERIALS AND EQUIPMENTS

4.1.1 MATERIALS

1 Itraconazole

2. Potassium dihydrogen orthophosphate

3. Sodium Hydroxide

4. Acetonitrile HPLC and UPLC Grade

5. Methanol HPLC and UPLC Grade

6. Ethanol (Absolute)

7. Chloroform, AR

8. Orthophosphoric acid

9. Potassium bromide

10. Sodium chloride

11. Stearic acid

12. Compritol® 888 ATO

13. Compritol® E ATO

14. Gelucire® 50/13

(stearoyl macrogolglycerides).

15. Glyceryl monostearate

16. Precirol®ATO-5

17. Cetyl palmitate

(glyceryl palmitostearate)

18. Poloxamer-188 (Pluronic® F-68)

19. Sodium taurocholate

20. Mannitol

20. Nutrient agar

21. Sabourad’s dextrose agar

22. Stearylamine

23. Humic acid

24. Fulvic acid

Jubilant Organosys, Noida, India.

Merck, India Ltd., India

Merck India Ltd., India



Changshu Yangyuan Chemical,

China

S.D. Fine chemicals, India

Merck India Ltd. India

S.D. Fine Chem. Ltd, Mumbai,

India

S.D. Fine Chem. Ltd, Mumbai,

India

Qualikems Fine Chem. Pvt. Ltd,

India

Gattefosse, France

Gattefosse, France

Gattefosse, France

Gattefosse, France

Gattefosse, France

Pioneer organics, India

Merck, France

Thomas Baker Ltd., Mumbai, India

S.D. Fine Chem. Ltd, Mumbai, India

Himedia Labs Pvt. Ltd. India

Himedia Labs Pvt. Ltd. India

S.D. Fine Chem. Ltd, Mumbai, India

University of Sao Paulo, Brazil

University of Masaryk, Czech

Republic

PhD Thesis 49 Jamia Hamdard


4.1.2 EQUIPMENTS

1. HPLC system with

Pump

Co.,

UV detector

Column

Software

2. UV-Visible Spectrophotometer

3. FTIR spectrophotometer

4. Differential scanning calorimeter (DSC)

5. pH meter

6. HPTLCLinomat-V

TLC scanner-III

Microlitre syringe

7. Precoated Silica-Gel Aluminium

8. Electronic balance

9. Millipore filtration unit

10. Oven

11. Centrifuge

12. Deep freezer

13. Solvent filtration unit

14. Ultrasonicator

15. Transmission electron microscopy

16. Particle size analyzer

17. Powder X-ray diffractometer (PXRD)

18. Freeze Dryer

19. Dialysis membrane (MWCO =12kDa)

20. Micropipette

21. Stability oven

22. Water bath

23. Automated transdermal diffusion cells

: Waters Alliance e2695e (Waters

MA, USA)

: Photo diode array detector (Waters

2998)

: C18 reverse phase column (25 x

4.6mm, particle size 5 ^m, Merck,

Germany)

: Empower software

: Shimadzu, Japan

: Win-IR, Bio-Rad FTS

spectrophotometer, USA

: Perkin Elmer, Pyris 6 DSC, USA

: HI84240, Microcomputer pH meter,

Italy

: Camag, Muttenz, Switzerland



: Merck, Darmstadt, Germany Plates

(60F-254)

: Mettler, Japan

: Milli Q academic, India

: Widsons Scientific Work, India

: Cooling Centrifuge, C24, REMI Ins.

Ltd. India

: West Frost, India

: Schott Duran, Australia

: PRAMA Instruments Pvt. Ltd. India

TEM TOPCON 002B, UK

Malvern Instruments, UK

PANalytical, Netherlands, PW 3710

Heto Dry winner, Denmark

Sigma Aldrich, USA

Eppendorf, Germany

Nirmal International, India

Metrex, India

Sampling system-SFDC 6, LOGAN

Inc. NJ, USA


4.2 PREPARATION OF STANDARD BUFFERS/SOLUTIONS/CULTURE MEDIA

4.2.1 SIMULATED VAGINAL FLUID (SVF), pH 4.5

Vaginal fluid simulant was prepared by dissolving following constituents in 1000 mL of

purified water. The pH of the final solution was adjusted to pH 4.5 ± 0.05 using 0.1M

HCL (Owen and Katz 1999).


Components Quantity (gL-1)

Sodium chloride 3.510

Potassium hydroxide 1.400

Calcium hydroxide 0.222

Bovine serum albumin 0.018

Lactic acid 2.000

Acetic acid 1.000

Glycerol 0.160

Urea 0.400

Glucose 5.000

Mucin 15.00

Purified water (q.s.) 1000 ml

4.2.2 NUTRIENT AGAR MEDIUM: 20g of the nutrient agar was suspended in 1000

ml of purified water and dissolved by slight warming. The media was sterilized by moist

heat sterilization (autoclaving) at 15 lbs pressure (121° C) for 15 minutes.


Peptone from meat 5.0

Meat extracts 3.0

Agar 12.0

Purified water (q.s.) 1000 ml

4.2.3 PHOSPHATE BUFFER: Accurately weighed 6.80 g of potassium dihydrogen

orthophosphate was dissolved in 1000 ml of purified water and pH of the solution was

adjusted to 4.5 ± 0.05.

4.2.4 SABOURAUD’S DEXTROSE MEDIUM: The media was prepared as per the

instructions of USP; the media was sterilized by moist heat sterilization (autoclaving) 15

lbs pressure (121°C) for 15 minutes. pH after sterilization was in the range of 5.4 ± 0.2.




Dextrose 40 g

Mixtures of equal parts ofPeptic digest of animal tissue and 10 gPancreatic digest of casein

Agar 15 g

Purified water (q.s) 1000 mL


4a. ANALYTICAL METHOD DEVELOPMENT

For routine analysis of Itz, analytical methods were developed with High performance

thin-layer chromatography (HPTLC) and UV spectroscopy. HPTLC is known for its

rapidity, selectivity, economic and overall versatility in QC aspects of pharmaceuticals.

4a.1 HPTLC INSTRUMENTATION, CHROMATOGRAPHIC CONDITIONS AND

OPTIMIZATION OF MOBILE PHASE

Samples were spotted (started from the point, X= 15 mm and Y= 10 mm) in the form of

distinct bands (4 mm in width; 10 mm apart) with a CAMAG 100 ^L syringe using a

Linomat V (CAMAG, Muttenz, Switzerland) sample applicator. Constant application rate

160 nL s-1; slit dimension 3 mm x 0.45 mm and scanning speed 20 mm s-1 were chosen as

optimized equipment parameters. Linear ascending development was carried out in a 20

cm x 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland), previously

saturated with optimized mobile phase for 15 min at room temperature (25 ± 2°C) and

relative humidity (RH) of 60±5% (Fisher scientific). The development includes

chromatogram run of 8 cm, 20 mL of mobile phase and time duration of 10 minutes. The

mobile phase was Toulene : Ethyl acetate: Ammonia (1:5:0.1, v/v) with densitometric

analysis at 266 nm in absorption mode with CAMAG TLC scanner III, using tungsten

lamp as a radiation source and operated by win CATS software (Version 1.2.0).

4a.2 PREPARATION OF STANDARD SOLUTIONS AND CALIBRATION PLOTS

10 mg of Itz was dissolved in 50 ml of dichloromethane kept in a volumetric flask and

sonicated for 5 min. The volume was then made up to 100 ml with dichloromethane to

give a final concentration of 100 ^g/ml. This solution was filtered with a membrane filter

(0.22 ^m ) and kept as a stock solution. Different volumes of the stock solution were

applied (n=6) to a plate to furnish 50, 100, 200, 400, 600, 800, 1000 and 2000 ng Itz band-

1 respectively. Peak area data and the corresponding amounts were treated by linear least-

square regression analysis.



4a.3 METHOD VALIDATION

The developed method was validated as per ICH guidelines by determining linearity

range, precision, robustness, limits o f detection (LOD), Limit o f quantification (LOQ) and

recovery.

Precision and accuracy

The precision and accuracy of the system was determined by measuring repeatability of

sample application and measurement of peak areas for three replicates of the bands (200,

400, 600 ng Itz band-1). Intra and inter-day variation for the determination of Itz was

carried out. The inter-day precision (intermediate precision) was studied by comparing

assays performed on three different days. The precision of the system and method were

expressed as relative standard deviation (RSD, %), standard error (SE) of peak area and

coefficient of variation (CV; %). Accuracy was expressed as percent recovery [(Itz found /

Itz applied) x100].

Robustness

Robustness was studied in triplicate at 400 and 800 ng band-1 by making small changes to

mobile phase composition, mobile phase volume, and duration o f mobile phase saturation

and activation of TLC plates. The effects on the results were examined by calculation of

RSD (% ) and SE of peak areas. Mobile phases prepared from Toulene: Ethyl acetate:

Ammonia in different proportions (1.25:4.75:0.1 and 0.75: 5.25:0.1 v/v) were used for

chromatography. Mobile phase volume and duration of saturation investigated were 15 ± 2

mL (13, 17, and 15 mL) and 20 ± 10 min (10, 20, and 30 min) respectively. Further, the

plates were prewashed with methanol and activated at 60±5°C for 2, 5 and 7 minutes.

Limit of detection (LOD) and quantification (LOQ)

To estimate the limits of detection (LOD) and quantification (LOQ), blank methanol was

applied (n=6) and the standard deviation (g) of the analytical response was determined.

The LOD was expressed as 3.3G/slope of the calibration plot for Itz and LOQ was

expressed as 10G/slope of the calibration plot.

Recovery studies

Recovery was studied by applying the method to drug samples to which known amounts

of Itz corresponding to 50, 100, and 150% of the drug label claim had been added. Each



level was analyzed in triplicates. This was to check the recovery of Itz at different levels in

the formulations.

4a.4 FORCED DEGRADATION STUDIES

A stock solution of 1000 ^g mL-1 in methanol was prepared as method described in section

4a.2 and left for below mentioned forced degradation conditions. Different peaks

obtained in the chromatogram were scratched and analyzed by NMR and Mass

spectroscopy for evaluation of probable degradation products.

Hydrogen peroxide induced degradation

To 5 ml of methanolic stock solutions of Itz, 5 ml of hydrogen peroxide (H 2O2; 30.0%,

v/v) was added. The solution was refluxed for 3 h at 80°C in dark (to exclude the possible

degradation effect of light). 2 ^L of the resultant solution were spotted on the TLC plate

and the chromatograms were run as described.

Acid and Alkali induced degradation

To each 5ml of methanolic stock solutions, 5 ml of 2N NaOH (alkali) and 2N HCl (acid)

was added separately and the resultant mixtures were refluxed separately for 3 h at 80°C

in dark (to exclude the possible degradation effect of light). 2 ^L (1000 ng spot-1) of the

resultant solutions were gently applied on the TLC plate and the chromatograms were run

as described earlier.

Photochemical and UV induced degradation

To 5 mL of methanolic stock solutions of Itz, 5 ml of methanol was added and the solution

was exposed to direct sunlight for 3 consecutive days (GMT: 09:00-17:00 h at 30°C; total

24 h) and UV irradiation at 254 nm for 8 h in a UV chamber. 2 ^L of the resultant

solutions were applied on the TLC plate and the chromatograms were run as described

previously.

Wet heat degradation

5 ml of the methanolic stock solution of Itz was diluted with 5 ml of methanol and

refluxed for 3 h at boiling water bath placed in dark to study the wet heat degradation. 2 ^l

of the resultant solutions were applied on TLC plate and the chromatograms were run as

described previously.



Dry heat-induced degradation-

The powdered Itz (50 mg) was stored at 100° C for 8 h under dry heat conditions (in dark)

to study the inherent stability to dry heat-induced degradation. The methanolic stock

solution of this dry heat exposed drug was prepared as described in section 4a.2 and 5 ml

of the prepared stock solution was diluted to 10 ml with methanol. 2 ^l of the resultant

solutions were applied on TLC plate and the chromatograms were run as described.

RESULT AND DISCUSSION

1. OPTIMIZATION OF MOBILE PHASE

The TLC procedure was optimized with a view to develop a stability indicating assay

method to quantify the Itz in bulk drug and formulations. Different solvent systems were

tried for the separation of pure drug as well as degraded products on the TLC plates.

Initially, Toulene-Ethyl acetate-Methanol-Ammonia (1:4:0.5:0.1, v/v) emerged as a better

solvent system with Rf value = 0.82. But to bring the spot to the middle and get better

resolution methanol was removed and mobile phase was optimized as Toulene-Ethyl

acetate-Ammonia (1:5:0.1, v/v) with Rf = 0.77±0.02 (Fig 6). Other parameters optimized to

get the well defined spots were activation o f TLC plates (60°C for 30 minutes) and

saturation of development chamber with mobile phase (30 minutes at room temperature).

Thus on an average it takes 2.5 hrs to complete the whole analytical procedure.

2. PREPARATION OF STANDARD SOLUTIONS AND CALIBRATION PLOTS

The linear regression data for the calibration curves (n=3) showed a good linear

relationship over concentration range 50-2000 ng spot-1. Graph was plotted between

concentration (ng/spot) and peak area. There was no significant difference between the

slopes of the calibration curves. Different linear regression data are shown in Table 7.

No significant difference was observed in the slopes o f standard

curves (ANOVA, P > 0.05).




Figure 6: Representative Peak (Rf = 0.77) of Itz, mobile phase Toulene: Ethyl acetate:Ammonia (1:5:0.1 v/v).

Table 7: Linear regression data for calibration plots of Itz (n=3)

Parameters Values

Linearity range (ng per spot) 50-2000

Regression equation 3.244x + 117.2

Correlation coefficient (r±SD) 0.996±0.0058

Slope ± SD 3.244± 0.01

Standard error of slope 117.2±1.422

Confidence limit of slope 3.234-3.254

Intercept ± SD 117.2±1.422

Standard error of intercept 0.58

Confidence limit of intercept 115.71-118.69

3. METHOD VALIDATION

3.1 PRECISION AND ACCURACY

The % R.S.D. and S.E. for repeatability of sample application was done at three

concentration levels (200, 400 and 600 ng spot-1). All the three concentrations were



applied in triplicate and the studies included both intraday and inter-day analyses. Peak

area for the developed chromatogram was measured and statistical analysis was done for

the inter-day and intraday variation.

Table 8a: Intraday and Inter-day precision of the method

uep

i:g ^

(. p c sso

C

Repeatability (intraday precision) Intermediate precision (interday)

Mean area ±

SD

Standard

E rro r (SE)

RSD

(% )

Mean area ±

SD

Standard

E rro r (SE)

RSD

(% )

200 648.33 ± 1.94 1.12 0.30 695.78± 2.83 1.63 0.40

400 1385.78 ± 4.0 2.31 0.28 1350.78± 5.6 3.23 0.41

600 2016.27 ± 5.55 3.20 0.27 2007.67±5.44 3.14 0.27

Table 8b: Precision and accuracy of the HPTLC method

Concentrationapplied

(ng spot-1)

Concentration

founda

(ng spot-1)

Precision

(CV; % )

Accuracy

(% )

Intraday

200 163.7269 1.18 81.86

400 391.0543 1.02 97.76

600 585.41 0.94 97.56

Inter-day

200 178.3539 1.58 89.17

400 380.2651 1.47 95.06

600 582.7589 0.93 97.12

a Mean of six determinations (n = 3).

b Precision as coefficient of variation (CV; %) = Std. deviation divided by concentration found x

100.

c Accuracy = conc. found / conc. applied x 100.

The measurement o f the peak area at three different concentration levels showed low

values of S.E. (<4) and also low values of the % R.S.D. (<0.5%) for inter and intra-day

variation, which suggested a good precision of the method (Table 8a). In terms of


coefficient of variation (CV; %), it was observed <1.08% for intraday and <1.58% for

inter-day (Table 8a).

The intra-day and inter-day accuracy were in the range of 81.86- 97.76% and 89.17 -

97.12 % respectively (Table 8b).

3.2 ROBUSTNESS

Table 9 describes the robustness of the proposed method. The % RSD of the peak areas

was calculated for the change in mobile phase composition, mobile phase volume,

duration of saturation and activation of prewashed-TLC plates at concentration levels of

400 ng spot-1 and 800 ng spot-1 (in triplicate). The result was slightly different for different

concentration of spots. At low concentration of Itz (400 ng spot-1) statistical parameters

observed were SD (<1.76), % RSD (< 0.119) after introducing small deliberate changes in

developed HPTLC method. While at higher concentration (800 ng spot-1) the parameters

were SD (< 6.68) and % RSD (<0.72). There was no significant variation in the results

obtained (ANOVA; P>0.05) in the different robustness parameters. But the only

substantial variation in results was obtained when the composition of mobile phase was

varied. This variation was more prominent in spot with higher concentration (800 ng spot-

1) especially with higher proportion of Ethyl acetate. Ethyl acetate in the mobile phase is

responsible for lowering of pH. Since lowering of pH is found to increase the solubility of

Itz (pH dependent solubility of Itz is well reported in literature), spots respond little bit

variably. Thus, variations in results were more prominent in spots with higher amount.

3.3 LIM IT OF DETECTION (LOD) AND QUANTIFICATION (LOQ)

The signal-to-noise ratio of 3:1 and 10:1 were considered as LOD and LOQ and were

found to be 14.29 and 43.31 ng spot-1. It indicates the adequate sensitivity of the method.

The detection limit of 10 ng ml-1 (Gubbins et al., 1998) has been reported in literature for

HPLC (UV detector). Similarly, corresponding limit of quantification reported for Itz was

25 ng ml-1.




Table 9: Robustness of the method, n=3. Parameter was carried out at two different levels

(400 ng spot-1 and 800 ng spot-1)

Parameters

400 ng 3er spot 1 800 ng per spot

S.D. ofpeakarea

%R.S.D

S.ERfvalue for Itz

S.D ofpeakarea

%R.S.D

S.E.Rfvalue For Itz

Mobile phase composition 1.25:4.75:0.1 v/v/v 0.75: 5.25:0.1 v/v/v

1.131.31

0.0820.095

0.650.76

0.680.76

3.726.68

0.130.24

2.153.85

0.690.76

Mobile phase volume (13, 15 and

17 ml)

1.561.481.40

0.1120.1070.101

0.9010.8590.811

0.730.730.73

3.761.752.15

0.130.060.72

2.171.0112.2

0.730.730.74

Duration of saturation (10, 20 and 30 min)

1.631.64 1.05

0.1190.1180.075

0.9420.9480.607

0.720.730.73

4.593.362.15

0.160.120.07

2.651.941.24

0.720.720.73

Activation of prewashed TLC plates (2,

5 and 7 min)

1.161.761.35

0.080.120.09

0.671.020.78

0.710.710.72

1.401.490.98

0.050.050.03

0.800.860.56

0.710.720.73

3.4 RECOVERY STUDIES

The proposed method when used for estimation of Itz after spiking with 50, 100 and 150%

of additional drug afforded recovery ranging from 99.65-100.23% (Table 10). The %

RSD of Itz was in the range of 0.25-0.53.

Table 10: Recovery studies (n=3).

Excess drug added to analyte (% )

Theoretical content (ng)

Average amount found (ng)

Recovery(%,)

%RSD

SE

0 400 398.33 99.73 0.53 1.23

50 600 599.14 99.65 0.25 0.87

100 800 897.45 99.97 0.31 1.45

150 1000 1010.65 100.23 0.34 1.99

4. FORCED DEGRADATION STUDIES

The NMR spectra of Itz (Fig 7) showed characteristic peaks of various protons present in

the structure. The terminal methyl group gave a triplet at 5 0.759-0.796 ppm. Whereas the

methyl group showed a doublet at 5 1.260-1.277 ppm. The eight piperazine protons were



observed as a singlet at 5 3.164 ppm. The aromatic region of the spectra showed the

integration for 14 protons in the range 5 6.826-8.390 ppm for the three phenyl rings and

the two triazole moieties for various methylene and methyne protons.

Figure 7: NMR spectra of Itz

The mass spectrum (Fig. 8) showed the molecular ion peak

(m/z=705.22) as m+1 corresponding to molecular weight of Itz. A diminished peak was

also obtained at m/z= 704.24. Except photochemical and thermal treatment all the forced

degradation studies showed almost complete degradation of Itz. Detailed results of the

degradation studies have been presented in Table 11.

4.1 HYDROGEN PEROXIDE INDUCED DEGRADATION

The peroxide degradation of Itz was performed and the 1HNMR and mass spectrum were

taken. The molecule degraded completely in reaction vessel and when chromatogram was

run (Fig 9), four very diminished spots were observed at Rf values 0.57, 0.60, 0.66 and

0.69. When the 1HNMR was studied, it was found that all the characteristic peaks of Itz,

that were observed earlier, were absent. Two singlets at 5 3.186 and 4.133 were the only

peaks indicating the complete degradation of the molecule (Fig 10).



Figure 8: Mass spectra of Itz (parent ion at m/z= 705.229 and other daughter ions)

Table 11: Degradation studies of Itz at different stress conditions. Multiple degradation

products were present after hydrochloride, alkali and peroxide degradation. Parent moiety

was detectable only at heat and illumination conditions.

S. NoStressconditions

Time

(h )

No. of degradation

products (R f values)

Drug remained (ng/2000 ng ) (±SD., n=3)

Recovery

(% )

1HCl (5N), refluxed

3 0.65, 0.70, 0.73Degradedcompletely

Notquantified

2NaOH (5N), refluxed

30.57, 0.60, 0.66,0.68

Degradedcompletely

Notquantified

3H2O2 (30%, v/v), refluxed

30.57, 0.60, 0.66, 0.69

Degradedcompletely

Notquantified

4 Daylight 24 Not detected 1978.8±6.80 98.9

5 UV (254 nm) 8 Not detected 1972.02± 5.07 98.6

6Dry heat (100 °C)

8 0.67 1912.6± 4.52 95.63

7Wet heat (100 °C)

3 0.68 1943.2± 3.66 97.16



Figure 9: Chromatogram of peroxide (30.0% v/v H2O2, refluxed for 3 hrs at 80°C) degraded

product, four peaks (Rf = 0.57, 0.60, 0.66 and 0.69) were observed.

Figure 10: NMR spectra of spots developed after peroxide degradation (two distinct siglets

were at 8 3.186 and 4.133).

The mass spectrum (Fig 11) showed a number of fragment peaks with no distinct

molecular ion peak. Base peak (m/z=303.022), together with other fragment ion e.g.

149.9663 and 415.8634 revealed a complex mixture of different compounds but no peak

corresponding to Itz indicating the complete degradation of the same.



Figure 11: Mass spectra of spot developed after peroxide degradation, prominent peaks were

observed at m/z 303.022, 149.9663 and 415.8634

4.2 ALKALI INDUCED DEGRADATION

When Itz was subjected to NaOH degradation studies, four degraded peaks (Fig. 12) were

observed at Rf values 0.57, 0.60, 0.66 and 0.68 and parent molecule degraded completely.

Figure 12: Chromatogram of NaOH degraded (2N NaOH, refluxed for 3 hrs at 80°C)

product.



Interestingly, in this case also, the 1HNMR spectra (Figure 13) showed similar kind of

pattern and two distinct singlets were observed at 5 3.648 and 5 4.131 ppm. These two

peaks were not present in the 1HNMR spectra of the Itz that showed the formation of a

new compound completely different from the present drug. New peaks were observed in

the aromatic region indicating the cleavage o f phenyl and triazole rings during the

degradation process.

The mass spectrum showed the typical basic degradation of Itz as the base

peak was observed at m/z 303.559. No other prominent peaks were present except some

smaller peaks that may have resulted from the presence o f more than one component.

Figure 13: NMR spectra (two distinct singlets) of spot developed after NaOH degradation

4.3 ACID INDUCED DEGRADATION

Three peaks (Fig 14) were observed at Rf values 0.65, 0.70 and 0.73 emphasizing the

complete degradation o f parent molecule.

The 1HNMR spectrum again revealed the complete absence of all

the characteristics peaks of Itz. Two singlets at 5 3.177 and 5 4.237 ppm were observed in

this case also that were entirely different from the peaks o f Itz indicating complete

degradation of the peak in acidic medium (Fig 15).



Figure 14: Chromatogram of HCl degraded (very closely placed three peaks) product

Figure 15: NMR spectra of spot developed by HCl degradation

The mass spectrum showed a range of medium to prominent peaks of

various fragments that were formed due to degradation of the drug. The molecular ion

peak at m/z 705.22 of parent molecule was absent and the base peak was present at m/z

493.319 and m/z 303.035 corresponding to the fragments resulting from the degradation

of the drug. No distinct molecular ion peak was observed in this case. This may be due to

presence of a mixture of various products (Fig 16).



Figure 16: Mass spectra of spot developed after HCl degradation

4.4 PHOTOCHEMICAL AND UV INDUCED DEGRADATION

The chromatogram o f the sample exposed to photochemical degradation and ultraviolet

(UV) light at 254 nm and more than 98.5 % of the drug was recovered during the

development o f chromatogram. It showed no additional peak other than the standard peak

(Fig 17) of Itz at Rf = 0.79±0.03. This indicates that the drug is stable towards the

photochemical and UV irradiations for the exposure period under study. The NMR and

mass spectroscopy did not show any significant difference.

Figure 17: Chromatogram of Itz after photochemical induced degradation studies.



4.5 DRY HEAT AND WET HEAT DEGRADATION

Itz was quite stable at heating conditions. At dry heat and wet heat conditions it produced

negligible peaks. In dry heat condition a very small additional peak was observed at Rf

value of 0.78 and more than 95% of the drug was recovered in the chromatogram (Fig 18).

The chromatogram developed from wet heat condition also showed a small

additional peak at Rf value 0.78 (Figure 19). The new peaks were not well resolved from

the peak of Itz. The NMR and mass spectroscopy did not show any significant difference.

The developed HPTLC technique is precise, specific, accurate and stability indicating for

the determination of Itz. As per the statistical analysis the method is reproducible and

selective for the quantitative analysis of Itz as bulk drug as well as vaginal formulations.

Figure 18: Chromatogram of Itz after dry heat induced degradation studies



Figure 19: Chromatogram of Itz after wet heat induced degradation studies

CONCLUSION AND INFERENCES WITHDRAWN

The developed HPTLC technique is precise, specific, accurate, and stability indicating for

the determination of Itz. As per the statistical analysis, the method is reproducible and

selective for the quantitative analysis o f Itz as bulk drug, marketed formulation, and

developed vaginal formulation. It can also be used for the study of in-vitro release pattern

from formulations. Chromatographic, NMR and Mass spectroscopic analyses of bulk drug

and forced degradation samples give further insight into the probable degradation

products.



4a.5 PREFORMULATION STUDIES

A preformulation study is carried out to find out the physical characteristics of the

molecule. It paves the way for the formulation development.

4a.5.1 Physical characterization of Itz

The sample of Itz was characterized on the basis of its physicochemical properties such as

color, odor, taste, solubility in water and other solvents.

Appearance: White crystalline powder

Loss on drying: <0.5%

Residue on ignition: 0.2%

Melting point: Determined by melting point apparatus and it was found to be

170°C (reported 166-170°C)

Solubility: Specific amount of Itz was taken in a 250 ml volumetric flask containing a

specified volume of solvent. It was sonicated for an hour to facilitate the solubilization of

the drug in the solvent at room temperature and then solution was observed for a clear

transparent solution. If transparent solution was not formed more solvent was added,

procedure was repeated until a clear solution obtained.

Table 12: Solubility of Itz in different solvents

Solvents Solubility

Water Practically insoluble

Tetrahydrofuran Sparingly soluble

Ethanol Slightly soluble

Dichloromethane Freely Soluble

4a.5.2 UV-Visible spectral analysis

Spectrophotometric analysis was carried out by scanning 10 ^g/ml methanolic solution of

Itz in the UV absorption range of 200-400 nm. The scan is depicted in figure 20.

4a.5.3 FT IR spectral analysis

The Fourier transform infrared spectroscopy (FT-IR) of samples was recorded on the

Perkin Elmer calorimeter using the potassium bromide (KBr) disc technique. 5 mg of


previously dried sample was mixed with 100 mg KBr and compressed into a pellet on an

IR hydraulic press. Base line was corrected and scanning was done from 4000 to 400 cm-1.

4a.5.4 DSC analysis

DSC thermo grams were obtained under a nitrogen gas flow of 50 ml/min. Calibration of

the DSC instrument (DSC-7, Perkin Elmer Pyris 6 instrument, USA) was carried out using

indium as a standard. Sample powders (5 mg) were crimped in an aluminum pan and

heated from 30 to 350 °C (highest melting points of organic substances) at a rate of 10 K

min-1. Generally, scan rates are taken between 1 and 10 K.min-1. Low scan rates are

preferable in terms o f peak resolution and investigation o f the sample having close peaks

while high scan rates increase the sensitivity o f the measurement as they lead to the

exchange o f heat within a comparatively short time period. Further, scan rate may also

influence the course o f temperature related processes within the sample.

4a.5.5 Wide angle X-Ray (WXRD) study

XRD samples were studied using X-ray diffractometer (PW 1830, Phillips). The samples

(1000 mg) on XRD plates were rotated during data collection to reduce orientation effects

of particles. XRD patterns of all the samples were recorded between 29 = 5° and 70° at 35

kV and 30 mA, respectively. Spectra obtained were analyzed for their physical states

(crystalline or amorphous).

4a.5.6 Mass spectroscopy

The samples were dissolved in Milli-Q water to make a stock of 1 mg/mL and then further

dilution was prepared in Milli-Q water: Methanol (50:50 v/v). Finally a concentration of

100 ng/mL was prepared and injected into the mass (Synapt Mass Spectomery, Q-TOF

with UPLC) on Electro spray ionization with positive mode. Capillary, sampling cone, and

extraction voltages were 2.51,21, and 5.3 units respectively. Source and desolvation

temperatures were 80°C and 250° C respectively. Nitrogen gas was used as cone and

desolvation gas at 50 and 600 L/h respectively. Trap collision energy was used (6.0 units).

The system was from Waters bearing serial No. JAA 272 Waters, USA. Software used

was MassLynx V 4.1 Waters.



4a.5.7 Compatibility studies

The careful selection of excipients to fabricate a suitable drug delivery system would

result into a stable, effective and reproducible release profile. Excipients

compatibility/interaction studies are, therefore, conducted in preformulation studies to

obviate the incompatibility problems. The compatibility of Itz with different excipients

used in the formulation was conducted using a technique of thermal stress. Storage at

50°C for 4 weeks or at ambient temperature (25 ± 2°C) for 12 weeks is generally

recommended for drug excipients compatibility studies. The drug samples along with the

excipients in a 1:1 ratio were kept at both the conditions along with and without added

moisture. Samples were periodically evaluated for any usual colour change and

instrumental analysis like UV spectroscopy, DSC and HPTLC.

After a storage for specified time, samples were withdrawn and

analyzed by validated HPTLC method for qualitative (presence of any degradant and

drug) and quantitative analysis. Sample solutions were prepared in methanol by sonication

for about 15 minutes and then filtered through 0.22 ^m nylon membrane filter. The filtrate

was suitably diluted with methanol to obtain 200 ^g/ml of concentration and applied in

triplicate mode (bands of 1000 ng/spot-1) as described in section 4a.1. Qualitative analysis

of drug solution by UV spectroscopy gives an idea about the shifting of chromophore in

the given media after the storage period.

For thermal analysis, samples withdrawn, pure drug and excipients

were analyzed according to the method described in section 4a.5.4 and evaluated for any

shifting, appearance or disappearance of peak or any characteristic change in thermo

gram.

4a.5.8 Solubility studies of Itz

4a.5.8 A. In different solvent and pH (buffers)

The solubility of the drug was determined by the equilibrium solubility method, which

employs a saturated solution of the material, obtained by stirring an excess of material in

the solvent for a prolonged period until equilibrium is achieved. Solubility was determined

in following media- Acetate buffer (pH 4.5), Phosphate buffer (pH 6.8), Aqueous solution

(pH 1.2), Aqueous solution (pH 2.1), Methanol, Ethanol, n-propanol, n-Butanol, n-

Octanol, n-Dodecanol, PEG 200 and PEG 400.



2 gm of Itz was transferred in different 50 mL volumetric flasks each

containing 30 mL of different media. The flasks were sealed and shaken in a mechanical

shaker at 25°C for 48 hours at 100 rpm. On the completion of the study period the flasks

were removed and the saturated solution was passed through 0.22 ^m Nylon member

filter. The solution obtained was suitably diluted and the solubility was determined by the

developed HPTLC method using linear regressed equation.

4a.5.8 B Solution stability studies

The solution stability of Itz was determined in Acetate buffer (pH 4.5) and Phosphate

buffer (pH 6.8) at 4°C, 25°C, and 40°C by measuring initial absorbance of drug and

absorbance after 24 hrs. These are the approximate pH values o f the vaginal milieu in

normal healthy condition and diseased condition respectively.

4a.5.8 C Preparation o f standard plots (pure solvents and buffers)

0.0002% w/v solution of Itz was prepared in methanol and was scanned for UV

absorbance in the wavelength of 200-350 nm. Calibration curves in different media were

prepared, like in the following manner:

1. Preparation of standard plot in methanol

10 mg of Itz was dissolved in 100 mL of methanol by using bath sonicator for 10 minutes

to form a stock solution of 100 ^g/mL. A series of dilutions (1 to 10 ^g/mL) were

prepared using stock solution. Absorbance o f each concentration was recorded by UV

spectrophotometer at 262 nm against blank and calibration curve was plotted.

II. Preparation of calibration curve in Dichloromethane

10 mg of Itz was dissolved in 100 mL of Dichloromethane by using bath sonicator for 5

minutes to form a stock solution of 100 ^g/ml. A series of dilutions (2 to 18 ^g/ml) were



III. Preparation of calibration curve in Acetate buffer (pH 4.5)

10 mg of Itz was dissolved in 100 mL of acetate buffer by using bath sonicator for 15

minutes to form a stock solution of 100 ^g/ml. A series of dilutions (2 to 18 ^g/ml) were






RESULTS AND DISCUSSION

1. UV-Visible spectral analysis

UV scan of Itz in methanol is given in Figure 20. A prominent Xmax has been observed at

263 nm. Observed Xmax varied slightly with the variation of solvents (like

dichloromethane, acetone, ethanol etc).

Figure 20: UV scan of Itz in methanol

2. FT IR spectral analysis

It is a common technique used to characterize drugs. Fourier transform infrared (IR)

spectroscopy is sensitive to the structure, conformation and environment of organic

compounds and remains a sensitive and useful characterization tool for pharmaceutical

substances.

The characteristic peaks of Itz occurred at 3381, 3126, 3069, 2962,

1697, 1510, 1450, and 418 cm-1. The absorption of the NH2 groups, are located in the

bands at 3381, 3126, 3069, cm-1. The first band assigned is due to stretching vibrations of

the free NH2 group in the molecule of the pure drug. The wave numbers observed at 1609

cm-1 and 1425 cm-1 may be assigned to the C=N and C-N bonds respectively and the

sharp peak occurred at 1697 cm-1 is due to C=O of the drug (Figure 21).



Figure 21: FT IR spectra of bulk Itz.

3. DSC analysis

DSC is a tool to investigate the melting and recrystallization behavior of crystalline

materials. Organic substances usually show a melting range. An increased melting range

could be correlated with impurities or less ordered crystals but the various instrumental

analyses used in the current work have ensured lowest levels o f impurities. An

endothermic peak was observed at 170.124°C with an enthalpy of 105.073 j/g (Figure 22).

Higher melting enthalpy value is indicative o f higher ordered lattice arrangement.

Observed peak width for the sample is 6.62, which indicates the narrow particle size

distribution o f the sample in nanometric range

Although DSC is able to monitor and quantify even minute thermal events in the sample

(depending on the sensitivity o f the instrument) and to identify the temperatures at which

these events occur. But it does not directly reveal the cause of a thermal event. The exact

nature o f the thermal transitions has to be determined with complementary methods such

as microscopic observations, X-ray diffraction or spectroscopic techniques to distinguish,

for example, between melting, polymorphic transitions, loss o f water from hydrates or

decomposition o f the substance.



Figure 22: Representative Differential scanning calorimetry profile of Itz.

4. WXRD study

Use of these two techniques (DSC and XRD) often lead to complementary information on

the systems of interest and data evaluation from these methods is usually straightforward.

Such a plot (29 Vs % Intensity) can be considered a fingerprint of the crystal structure and

to differentiate with different crystallographic status of the system. One peak will be

exhibited for all repeating planes with the same spacing. By contrast, an amorphous

sample will exhibit a broad hump in the pattern called an amorphous halo. These patterns

are representative of the structure but do not give positional information about the atoms

in the molecule. Itz was showing a characteristic crystalline pattern exhibiting intense

peaks at 20.38 (100% ), 20.40 (97.59% ), 20.36 (96.53% ), 20.34 (88.53% ), 20.32 (80.66% )

and 17.5 (77.9% ) etc. With the help of Bragg's equation (n X = 2d sin 9), d value was also

calculated (2.133) at an angle at which the samples give a peak of maximum intensity

(Figure 23).



Figure 23: Wide angle X- Ray powder diffraction patterns of Itz

5. Mass spectroscopy

The mass spectrum (Figure 24) showed the molecular ion peak (m/z=705.22) as m+1

corresponding to molecular weight of Itz. Diminished peaks were also obtained at m/z=

704.24.

Figure 24: Representative Mass spectra of Itz

6. Compatibility studies

Interaction studies were carried out to ascertain any kind o f interaction with the excipients

used in different formulations. This initial effort helps in rejection of components at the

earlier stage and to obviate the stability issues in later stages of development. Thermal,


chromatographic and spectroscopic analysis help in detection of any minute

physicochemical interaction of the drug (Itz) with other excipients. Pure Itz shows an

endothermic peak at 170.124°C which is well supported by literature. A maximum of ±

2.5°C change in the peak was observed in different compatibility studies samples. Any

small change in the peak shape or area and enthalpy can also be attributed to the quality of

the material used, as quality of material is affected by mixing of two or more components

(Marini et al., 2003). Excipients used in the formulations have also low melting points, so

chances are there that they may depress each other’s melting points.

Another mode of assessing compatibility study was to offer

isothermal stressed conditions (temperature, humidity and time) to physical mixtures and

to evaluate Itz qualitatively and quantitatively by HPTLC and UV spectroscopy. HPTLC

helps in both qualitative and quantitative analysis while UV spectroscopy was used only

for qualitative analysis. The quantitative evaluations are given in table 13 and 14 which

explains the stability of Itz at the given conditions and minimal loss of the molecule. Heat

stable nature of Itz is also well supported by literature (Weuts et al., 2003). Qualitatively

the characteristic chromatogram of Itz also remained significantly unchanged (Rf =

0.77±0.06). Also minimal interference was observed in UV spectroscopy and variation in

Xmax was observed to be ± 4. In some of the stability study samples qualitative evaluation

by UV spectroscopy was not an appropriate option since solubility profile of drug and

excipients were different from each other. Itz was hydrophobic in nature while humic acid,

fulvic acid, P-CD, HP-P-CD and caffeine were hydrophilic in nature. Minimum interaction

between these excipients was itself expected in solution state.




Table 13: Drug content in drug+excipient physical mixture in absence of moisture

Concentration (% w/w of drug

Drug+excipientscombinations Initial conc'

Drug content after 4 weeks at stressed

Drug content after 12 weeks at

condition ambient condition

Itz 101.14 100.02 99.96

Itz + compritol 888 99.97 99.64 99.13

Itz + compritol E ATO 100.02 99.23 98.49

Itz + Stearic acid 99.86 99.81 99.22

Itz + Poloxamer 188 98.99 98.63 98.12

Itz + Sod. Taurocholate

100.08 99.19 98.47

Itz + Mannitol 100.11 99.73 99.61

Itz + Humic acid 99.91 99.68 98.43

Itz + Fulvic acid 99.56 99.26 99.05

Itz + p CD 98.67 98.59 98.14

Itz + HP+ p CD 99.06 98.66 98.43

Itz + Caffeine 100.82 99.44 99.32

Table 14: Drug content in drug+excipient physical mixture in presence of moisture

Drug+excipientscombinations

CConcentration (% w/w) of drug

Initial conc. Drug content after 4 weeks at stresses

condition

Drug content after 12 weeks at ambient

condition

Itz 101.14 99.98 99.26

Itz + compritol 888 99.97 98.42 98.13

Itz + compritol E ATO 100.02 99.16 98.41

Itz + Stearic acid 99.86 98. 11 97.33

Itz + Poloxamer 188 98.99 97.48 97.26

Itz + Sod.Taurocholate 100.08 98.77 97.60

Itz + Mannitol 100.11 98.55 98.32

Itz + Humic acid 99.91 98.75 98.03

Itz + Fulvic acid 99.56 99.07 97.84

Itz + p CD 98.67 98.06 97.42

Itz + HP- p CD 99.06 97.68 97.23

Itz + Caffeine 100.82 98.97 98.66



7. Solubility studies of Itz

7 A. In different solvent and pH (buffers)

The solubility data of Itz in different solvents and buffers are given in Table 15. pH

dependent decrease in solubility (decrease in solubility with the rise in pH) was observed.

Solubilizers (PEGs) were also found to have profound effect in the solubility of the drug.

With increasing chain length of the alcohol solubility was found to be decreasing. Using

low viscosity solvents (at room temperature), having resonance stabilized phenol ring and

hydroxyl group may leave positive effects on the solubility of Itz (Rhee et al., 2007).

7 B. Solution stability studies

Not much change in the initial absorbance reading and final reading of standard solutions

of Itz in different media at different temperature after 24 hrs was observed. The

absorbance was within the range of 98-100%. Hence the solutions were found to be quite

stable for 24 hrs at above temperature. Results obtained are given in Table 16.

Table 15: Determination of solubility of Itz in different solvents

S. No Media Solubility (^g/m l)

1. Methanol 641.23± 4.2

2. Ethanol 304.30 ± 6.8

3. n-Propanol 395.42 ± 5.6

4. n-Butanol 327.17 ± 5.8

5. n-Octanol 181.62 ± 4.4

6. PEG 200 1615.18 ± 9.8

7. PEG 400 2206.37 ± 8.4

8. n-Dodecanol 96.33 ± 2.6

9. Acetate buffer (pH 4.5) 1.81 ± 0.12

10. Phosphate buffer (pH 6.8) 0.298 ±0.02

11. Aqueous solution (pH 1.2) 6.9 ± 5.4

12. Aqueous solution (pH 2.1) 3.73 ± 2.4



Table 16: Solution stability studies of Itz at different media and temperatures

e

cC

TemperaturesInitial

AbsorbanceAbsorbance after

24 hrs

4°C 0.486 0.485

25°C 0.484 0.486

40°C 0486 0.484

r4°C 0.492 0.490

s _

STte .6

VIo

.aCIh

25°C 0.493 0.492

40°C 0.494 0.489

7 C. Preparation of standard plots (pure solvents and buffers)

For routine analysis calibration curves of Itz were prepared in different media. Some of

them are given as follows,

(Uo

Concentration (^g/mL)

Figure 25: Calibration curve of Itz in methanol



(Uo

Concentration (^g/mL

Figure 26: Calibration curve of Itz in dichloromethane

Figure 27: Calibration curve of Itz in acetate buffer (pH 4.5)


Experimental Chapter4

4b. Effect of physical processes (differential heat treatments)

on

solubility and thermodynamic properties of Itraconazole

PhD thesis Jamia Hamdard

4b. EFFECT OF PHYSICAL PROCESSES (DIFFERENTIAL HEAT

TREATMENTS) ON SOLUBILITY AND THERMODYNAMIC PROPERTIES OF

ITRACONAZOLE

Itz shows minimal solubility in oily/lipidic substances in spite of having appreciable

lipophilic nature (log P ~6.2). Keeping the concept of like dissolves like, it is expected

that this lipophilic solute will get dissolved into nonpolar solvents with similar internal

pressures through induced dipole interactions but the studies deduced that the solubility of

Itz was <100 ^g/mL for most of the oils examined (Rhee et al., 2007) with the only

exception being MCT (medium chain triglycerides), in which Itz showed a solubility of

141.79±8.10 ^g/ml. Although Itz has elicited hydrophobic properties but it is not

lipophilic at room temperature. Studies using low viscosity solvents (at room temperature)

having resonance stabilized phenol ring and hydroxyl group have concluded that they can

dissolve Itz in higher quantities (e.g., 2-phenethyl alcohol ~ 68.95±1.47 ^g/ml and 3-

phenyl-1-propanol ~ 55.60±0.29 ^g/ml). Earlier experiments in our lab concluded that this

thermo-stable molecule when heated above its melting point, elicits different solubility.

Probably based on these peculiarities, the molecule has gained the popularity as

“practically impossible to apply to the body” (Barrett et al., 2008). So, we hypothesized

that the crystalline nature of the drug must be playing a decisive role in determining its

solubility; an experiment was designed to generate different solid states of the substance.

An exhaustive solubility analysis was carried out to evaluate thermodynamic parameters

(AH, AS and AG) and to identify the factor that represents itself as a driving force in

solubilisation. Similarly, partitioning study between acetate buffer (pH 4.5) and n-octanol

was carried out to elucidate the driving force for absorption when the drug is formulated

into a VDDS. Also, analysis of earlier similar studies have shown that Itz has not been

described in the literature with point of view studying thermodynamics of solubility,

solvation processes, partitioning into oppositely natured solvent and drug delivery.

Following strategy was envisaged to complete the proposed study:-

4b.1 PHYSICAL TREATMENTS TO BULK DRUG

Bulk drug was subjected to the following treatments to create changes in the crystal lattice

of Itz.



Sample A. Bulk Itz. It remained untreated and served as a control.

Sample B. 5 grams of Itz were exposed to hot metal plate at 180°C (above the melting

point) for 15 minutes. Then it was allowed to cool at room temperature and passed from

100 mesh sieve to get a fine powder.

Sample C. 5 gram of Itz was dissolved in 100 ml of dichloromethane in a conical flask

and sonicated for 5 minutes. It was poured immediately over metallic plate (previously

heated above 100° C) for 30 seconds and then placed immediately inside refrigerator at -

20° C. After 5 minutes it was removed from the refrigerator and placed in vacuum oven at

40°C to remove the moisture adsorbed. Then the solid residue (lump) was scrapped and

passed from 100 mesh sieve to get a fine powder.

Sample D. 5 gram of Itz was dissolved in 100 ml of dichloromethane in a conical flask

and sonicated for 5 minutes. It was poured over china dish and placed over water bath that

allows the solvent to evaporate slowly. Obtained solid sticky mass was allowed to cool at

room temperature and then passed from 100 mesh sieve to get a fine powder.

The product was stored in desiccators at 25± 3 °C and 0% RH conditions

over phosphorus pentoxide. Different instrumental analysis and evaluation parameters

were carried out to check the effects of three different physical treatments.

4b.2 CHARACTERIZATION OF DIFFERENT SOLID STATES

Following instrumental analysis were carried out to characterize the samples obtained by

different physical treatments.

a) Differential scanning calorimetry

DSC thermo grams were obtained by the method described in section 4a.5.4 and the

results are shown in Table 17 and Fig 28.

b) Powder X ray diffractionXRD samples were studied using X-ray diffractometer by the method described in section

4a.5.5 and the results are shown in Fig 29.

c) Molecular dynamics simulation studies

The conformational studies and calculation of total energy were performed using Macro

Model module of Schrodinger 9 (MacroModel, version 9.7, NY 2009). Molecular

dynamics simulations in Macro Model use classical mechanics (Newton’s equations of

motion) to mimic how the system would behave as a function of time, typically at or close



to the temperature of interest. The present study was performed at different temperatures

like -20°C, 0°C, 25°C and 180°C. For the simulation studies OPLS_2005 force field was

used and water was taken as solvent media. All the parameters were kept default except

the simulation temperature which was changed.

d) Fourier transform infrared spectroscopy

The Fourier transform infrared spectroscopy (FT-IR) spectra of samples were recorded by

the method described in section 4a.5.3 and result has been given in Fig 30.

e) Mass spectroscopy

The mass spectroscopy was carried out by the method described in section 4a.5.6.

f) Determination of residual solvent content

The residual solvent content in samples C and D was quantified by GC-MS system

[Agilent 7890A series (Germany)] equipped with split-splitless injector and CTC-PAL

auto sampler attached to an apolar HP-5MS capillary column (30 m x 0.25 mm i.d. and

0.25 ^m film thickness) and fitted to a mass detector. Carrier gas flow rate (Helium) was

1ml/min, split: splitless ratio 1:100, injector temperature was 70°C, detector temperature

250°C, while column temperature was kept at 60°C for 2 min followed by linear

programming from 70 to 230 °C (at rate of 5°C/min), and then kept isothermally at 230 °C

for 2 min. Transfer line was heated at 280 °C. Split ratio was kept at 1:100. Mass spectra

were acquired in EI mode (70 eV); in m/z range 30-400. The amount of sample was

injected through head space. The residual solvent of the formulation were identified by

comparison of their mass spectra to those from Wiley 275 and NIST/NBS libraries, using

different search engines.

4b.3 SOLVATION CHARACTERISTICS

Formation of a high energy amorphous or semi crystalline state can increase the predicted

solubility, in many cases up to 100- times that of its crystalline form (Hancock and Parks,

2000; Gupta et al., 2004). Drug transport and delivery in the body, if restricted to passive

processes, comprises, absorption of drug molecules, their distribution between different

tissues, redistribution from deep compartments, and finally excretion. These are

determined by the physicochemical characteristics and solvation abilities of the drug

molecules in different environments (solvents). Hence, an exhaustive solubility study in

aqueous media and on different pH was carried out to have views on solubilization and the



different mechanisms involved in it. Further, a partition behavior of samples describes

much about the thermodynamics involved during the transfer of molecule from the release

media to biological membranes.

4b.3.1 Aqueous solubility

Excess of Itz (Samples A, B, C and D) was added to 10 ml aqueous solution (pH 1). The

pH was maintained by drop wise addition of concentrated HCl. The samples were shaken

on an orbital shaker incubator (Metrex scientific instrument Ltd, New Delhi) for 3 days at

25°C, centrifuged at 5000 rpm for 10 min in a high speed refrigerated micro centrifuge

(Tomy MX-305) and filtered through a membrane filter (0.45 ^m). The concentration of

Itz in the resulting solutions was then analyzed by slight modification into reported LC-

MS/MS method (Bharathi et al., 2008) with an accuracy of 95.5% and a precision of

6.7%. Description of instrumentation was Acquity UPLC BEH C8 (2.1^100, 1.7^m)

column with a mobile phase composed of 0.2% (v/v) ammonia solution: acetonitrile

(20:80, v/v) at a flow rate of 0.40 mL/min. Analytes were determined by electro spray

ionization synapt mass spectrometry in the positive ion mode.The resulting values are the

average of at least three replicated experiments.

The standard solution Gibbs energies were calculated using following equation:

AG°sol = -R T ln X2,

Where, X2 is the molar fraction of a solute in a saturated solution.

The standard solution enthalpies were derived from temperature dependent drug

solubilities expressed in molar fractions (van’t Hoff equation):

d h^X2 _ A Jf^ i

For rightful use of above equations following assumptions were made: (a) the activity

coefficients of dissolved drugs do not deviate from unit and (b) the solution enthalpies do

not depend on concentration. The solution heat capacities are considered to be constant

within studied temperature range, since the temperature dependence of solubility is

described by linear equations.

4b.3.2 Determination of driving forces of solubilisation

To find out the driving force for solubilisation among different thermodynamic parameters

(Gibb's free energy, Entropy and Enthalpy), following study was carried out. Different

aqueous solutions with variable pH (1-5) were made by addition of drops of concentrated



HCl into 100 ml of Milli Q water and adjusting the reading with pH meter (Decibel 1011).

Excess amount of all different samples (A, B, C and D) were added into different solution

and solubility studies (25°C) were carried out by the method described in section 4b.3.1.

Here aqueous solutions of variable pH were selected instead of buffer solution. Since in a

study (Perlovich et al., 2003), effect of composition of buffer was found to be profound in

deciding the thermodynamic force for solubility enhancement.

4b.4 DETERMINATION OF PARTITION COEFFICIENT

There is not enough information available to propose suitable mechanisms for the transfer

process of Itz between immiscible liquid phases, and between aqueous media and

biological membrane models. Hence, the following classical partition coefficient study

was carried out to mimic the transfer process between release media and biological

membrane. Partitioning between acetate buffer (pH 4.5)/octanol was determined for

different samples (A, B, C and D). It was pre-saturated with each other for at least 24 h

prior to the experiment. Samples in acetate buffer (pH 4.5) were prepared at a

concentration of 30 ^g/ ml (by sonication and vortexing) and 5 mL of this solution was

used. After the addition of an equal volume of octanol, the tubes were stopper and shaken

for 72 h in orbital shaker incubator (Metrex scientific instrument Ltd, New Delhi) at 25°C.

After shaking, samples were taken from both the media for assaying. The partition

coefficients were then calculated as the ratio of Itz concentration in octanol to buffer. The

fraction of moles in both the immiscible fluids was taken to calculate various

thermodynamic parameters.

4b.5 APPLICATION OF PHYSICAL TREATMENTS IN DRUG DELIVERY

4b.5.1 Increased lipophilicity by changing physical state and heat treatment

In the support of hypothesis that it is crystallinility of Itz that creates an impediment for it

to get into lipophilic substances (lipids), following study was carried out. A homogenous

binary mixture (stearic acid and Itz) was made and given a heat treatment. Temperature of

the mixture is raised above the melting point of drug (170°C) and allowed to cool. After

attaining the room temperature it is again heated above the melting point of lipid (69°C)



and then solubility of Itz in the lipid was observed. Effect of temperature changes integrity

of molecule and extent of interaction between drug and lipid was also studied by different

techniques like- DSC, UV spectroscopy and NMR spectroscopy. DSC was carried out

according to method described in 4.3.5.4. Proton Magnetic Resonance ( 1H NMR)

spectra were recorded on Brucker Model DRX-300 NMR spectrometer in CDCl3

using tetramethylsilane (TMS) as the internal standard. Chemical shift s are reported

in parts per million (ppm, 5 ) and signals are described as a singlet (s), doublet

(d), triplet (t), quartet (q) and multiplet (m).

4b.5.2 Ex vivo permeation across vaginal tissues

Ex vivo permeation studies were performed using a Franz diffusion cell with an effective

diffusion surface area of 7.16 cm2 and 37 ml of receiver chamber capacity using excised

and defatted rat vaginal tissues. The tissue was stored in the deep freezer at -21°C till

further use. During use, it was brought to room temperature and mounted between donor

and receiver compartment of the Franz diffusion cell; the lumen side of the vaginal tissue

was facing the donor compartment and the opposite side was facing the receiver

compartment and stabilized with the simulated vaginal fluid. For this, receiver chamber

was filled with SVF and stirred with a magnetic rotor at a speed of 100 rpm in hot air oven

maintaining temperature at 37±1°C. The whole media was replaced with a fresh one after

every 30 min to stabilize it. After running the 6 cycle of stabilization, 1 mL of sample

(200 ^g/ml) solutions (A, B, C and D) in acetate buffer (pH 4.5) were placed into donor

compartment. The receptor compartment was having 20 ml phosphate buffer (pH 4.5)

solution. The samples were withdrawn at regular interval (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

12, 14, 16 and 24 h), filtered through 0.45 ^m membrane filter and analyzed for drug

content by the method described in 4b.3.1. The cumulative amount of drug permeated

through the membrane (^g/cm2) was plotted as a function of time (t) for each formulation.

All the permeability parameters were determined according to the methods reported in

literature (Shakeel et al., 2007).





1. CHARACTERIZATION OF DIFFERENT SOLID STATES

The generated different solid states were characterized on the following basis.

1A. DIFFERENTIAL SCANNING CALORIMETRY

Melting enthalpy (AH), expressed in joule/gram (J/g) is characteristic of the crystal order

if the impurities influence could be ignored. For the less ordered crystal or amorphous

state, the melt of the substance does not require or just requires less energy than the

perfect crystalline substance which needed to overcome the lattice force. As a result, the

higher melting enthalpy values should suggest higher ordered lattice arrangement and vice

versa. Peak width (i.e. difference between onset and maximum) is another parameter to

examine the thermogram critically. Values of AH and peak width are given in Table 17.

Table 17: DSC data of different samples

Samples AH (j/g) Maximum peak (°C) Peak width

A 105.063 170.408 6.62

B 48.055 167.787 12.23

C 33.502 166.124 16.54

D 78.033 165.042 13.11

Sample B, C, D did not show much variation in peak width but

varied substantially with A. This difference could be attributed to the size effect and can

be explained by Thomson equation (Westesen et al, 1997). The particle size of

unprocessed drug (sample A) is around 152.3 nm while the other samples (B, C and D)

were of larger size and having a broader particle size distribution as they were passed

through sieve no.100. DSC thermograms of all the samples are given in Fig. 28.

The endotherms of the processed sample (B, C and D) are also

shifted to lower temperatures. This phenomenon can be explained by Gibbs-Thomson

equation;

Where, T T0

YslVs

Melting temperature of a particle with radius rMelting temperature of the bulk material at the same external pressureInterfacial tension at the solid-liquid interfaceSpecific volume of the solid

AHfus Specific heat of fusion



Figure 28: Representative Differential scanning calorimetry profiles of samples (A, B, C and D). The alphabets (B, C and D) are indicative of processes carried out in section 4b.1. Sample A is unprocessed Itz.

It reflects the decrease in melting temperature for a particle of given size compared to the

bulk material and becomes particularly more pronounced in the lower nanometer size

range. Further the processed samples (B, C and D) are usually polydispersed (it was

passed through mesh size 100) the melting transition is not only shifted to lower

temperatures but is also broadened since the fractions of different particle sizes melt at

different temperatures.

Previous experimentations have also shown that these observations

occur irrespective of the heating and cooling rates used.

1B. POWDER X RAY DIFFRACTION

A plot (29 Vs % Intensity) can be considered a fingerprint of the crystal structure and to

differentiate with different crystallographic status of the system. One peak will be

exhibited for all repeating planes with the same spacing. By contrast, an amorphous

sample will exhibit a broad hump in the pattern called an amorphous halo. These patterns

are representative of the structure but do not give positional information about the atoms

in the molecule. To have a close look in the change in conformation and energy states of



the molecule a software analysis was done as described in section 4b.2.C. It was observed

that with the increase of temperature, total energy of the molecule was increasing and this

energy change was more when temperature is above room temperature (Table 18). High

energy state of molecule offers substantial conformational changes in the structure and

also provides useful energy to the molecule to interact surrounding matrix.

Table 18: Total energy calculations of molecule at different temperatures

S. No Temperature (°C )

-20

25

180

Average Total energy

635.36 kJ/mol

670.10 kJ/mol

734.82 kJ/mol

1070.95 kJ/mol

Itz (Sample A) was showing a characteristic crystalline

pattern exhibiting intense peaks at 20.38 (100% ), 20.40 (97.59% ), 20.36 (96.53% ), 20.34

(88.53% ), 20.32 (80.66% ) and 17.5 (77.9% ) etc. All the characteristic peaks were highly

diminished (Figure 29) in all the samples (B, C and D) and the sharpness was lost fully in

sample C. Obtained XRD pattern first obliterates the doubts of impurity as was

hypothesized in DSC interpretation and also indicates minimal attainment of crystalline

pattern in all the samples (B, C and D). The peaks of sample C were highly dispersed

which is an indicative of minimum crystal growth. The spectra revealed that all the

samples had a tendency to regain their lost crystal pattern, as almost all the diminished

peaks are at nearly same 29 values. An interesting pattern was observed in sample D. It

gained a slightly different lattice arrangement. Peaks around 29 = 17 almost disappeared

and another peaks appeared around 35°. Although the exact reason for this is not

investigated but it is hypothesized that molecular dispersion into dichloromethane,

presence of thermal energy (higher Brownian movement) and comparatively slow

evaporation offered new, high energy arrangements to the bulk drug.


2 0

3

4


Figure 29: X- Ray powder diffraction patterns of Samples. A is unprocessed Itz and samples B, C and D are processes carried out in section 4b.1

A remarkable change in the inter-atomic spacing (d value) was also

observed among different samples. With the help of Bragg's equation (n X = 2d sin 9), d

values were calculated at an angle at which the samples give a peak of maximum

intensity. Calculated values of d were 2.133, 1.734, 2.285 and 1.304 for A, B, C and D

respectively. Thus in cases except C inter-atomic distance in lattice was found to be

decreasing and sample C was comparable to the bulk drug (Sample A). We could

conclude that, with the treatment followed by C, sample almost gained the lattice spacing.

Different characterization and analysis were also carried out after a

three month storage period. An insignificant change in results (only in B and D) was

obtained, thus obliterating the qualms of time dependent regain of crystallanity.

An empirical equation known as the Vogel-Tammen- Fulcher (VTF) is the

most commonly used to quantitatively describe the temperature (T) dependence of

molecular motion in amorphous material (Hancock, 1998)


Where, A, B and C are arbitrary fitting parameters. A is usually considered to represent the

time needed for a molecule to move in an open space (T0) (10‘14 sec) (Hancock, 2000). B is

related to the temperature dependence of t at Tg (also known as fragility of the amorphous

material), and C is said to be the temperature at which the average molecular mobility

approaches zero (To) (~ 50 K below Tg). Alternative equations and more detailed

interpretations of the VTF constants have been proposed (e.g. Angell's D parameter)

(Angell et al., 1996), however none provides any better or more widely applicable

physical interpretation of the temperature dependence of molecular motions in the various

amorphous region.

1C. FOURIER TRANSFORM INFRARED SPECTROSCOPY

The characteristic peaks of Itz occurred at 3381, 3126, 3069, 2962, 1697, 1510, 1450, and

418 cm-1. The absorption of the NH2 groups are located in the bands at 3381, 3126, 3069,

cm-1. The first band is assigned to be due to stretching vibrations of the free NH2 group in

the molecule of the pure drug. The wave numbers observed at 1609 cm-1 and 1425 cm-1

may be assigned to the C=N and C-N bonds respectively and the sharp peak occurred at

1697 cm-1 is due to C=O of the drug (Figure 30).

After the different treatments no major changes were observed

except at certain points. Some band broadening was observed in the region of 2900-3300

cm-1. A variable depression was also observed at 1700 cm-1. The reason again indicates

the absence or distortion of crystalline pattern. Crystalline organic solids are made up of

molecules that are packed or ordered in a specific arrangement. These molecules are held

together by relatively weak forces, such as hydrogen bonding and van der Waals

interactions. The bands (3381 - 3069 cm-1) which dispersed in different processes (B, C

and were actually the fractions (amino group) which are involved in intermolecular, intra

molecular H-bonding and electrostatic interaction which usually lead to strengthening of

crystal pattern.




Figure 30: FT IR spectra of samples (A, B, C and D). Minor changes in B, C and D are noticeable as compared to unprocessed Itz (A).

ID. MASS SPECTROSCOPY

No changes were observed in the mass spectra of different samples (B, C and D) when

compared with bulk drug (A). It was also expected from the process applied (only

physical changes), since no chemical changes were applied to the samples. The mass

spectrum (Figure 24) showed the molecular ion peak (m/z=705.22) as m+1 corresponding

to molecular weight of Itz. Diminished peaks were obtained at m/z= 704.24.

IE. DETERMINATION OF RESIDUAL SOLVENT CONTENT

Dichloromethane was found to be absent in the samples C and D. Recommended limit of

ICH Q3C guideline (ICH, 1997) in case of Dichloromethane is 6 mg/day . And the

residual amount limit in USP XXIII is 6 mg/day.



2. AQUEOUS SOLUBILITY

From a pharmaceutical perspective, the interest in the amorphous state stems from its

higher apparent solubility as compared to crystalline counterparts (Kaushal et al., 2004).

The saturated solubility of different samples at pH 1 is given in Table 19. pH of aqueous

medium was selected as 1 because Itz is shown to have a pH dependent solubility and it

increases with decreasing pH. Obtained solubility of plain bulk drug is in accordance with

the earlier reporting (Prasad et al., 2010). Other thermodynamic parameters (AG298sol,

AH298sol, TAS298sol and A^^9*sol) that may play a determining role in solubilization are also

given in the table. Highest increase in solubility as compared to bulk drug (sample A) was

observed with C (~2.5 times). Negligible increase in solubility was observed with sample

D. This means that it had regained its crystalline lattice after the processing. Substantial

amorphous pattern developed with the treatment B as it was also evident from the

different spectroscopic analyses. Thus, different treatments elicited different modes of

action and heat treatment of the sample post dispersion into dichloromethane played a

crucial role in developing the amorphous form. The solution enthalpies (AH298sol) for

every sample have positive values, and it means that the crystal lattice energy overweighs

the solvation energy.

298 298

2. 1 DETERMINATION OF DRIVING FORCES OF SOLUBILISATION

Itz is a weak base with four ionisable nitrogen atoms. Two of the p.^a values are 4 and

1.5-2 whereas the other ionizable nitrogens are not protonated between pH 2 and 10

(Peeters et al., 2002). This explains the drop-in solubility above pH 3 and why changes in

pH profoundly influenced both solubility and dissolution (Table 20).

Table 19: Solubility thermodynamic functions of different forms of Itz (A, B, C and D) at pH 1 (at 298 K)

Sample Solubility ( ^g/ ml>

X2 (molar fraction)

A <-298AG sol

(k J mol-1)

ATt298 AH sol(k J mol-1)

T A c 298T AS sol (k J mol-1)

AS^98„l (J mol 1 K-1)

A 24.17±2.3 0.618X 10'6 35.42 55.3±1.5 19.88 66.7

B 35.97± 1.9 0.927X10'6 34.43 40.2±1.9 5.77 19.3

C 57.23± 3.4 1.47X10'6 33.21 48.8±1.1 15.59 52.3

D 28.26± 3.6 0.728X10'6 35.02 56.2±2.1 21.18 71.1



Table 20: Thermodynamic parameters of solubilisation process of A, B, C and D in aqueous solution at variable pH (1, 2 and 4) at 25° C.

T7^298---AG sol(k J

mol-1)

* t t 298---AH sol(k J

mol-1)(J mol

K-1)

Samples pH Solubility ( ^g/ ml)

MoleFraction 298

T AS sol (k J mol-1)

sol-1

A24.17±2.3 0.618X 10 55.42 55.3±1.5 19.88

0.55±4.1 0.014X 10 44.79 35.6±1.5 -9.19

0.13±3.7 0.0033X 10 -̂6“ 48.33 44.5±0.8 -92.83

66.71

-30.83

-311.51

B35.97± 6.9 0.927x10 34.40 40.2±1.9 5.8

2.95±2.4 0.0760X 10 40.60 55.1±1.1 10.92

0.695±5.1 0.0179X 10

1.47x10'6

44.18 37.3±0.9 -6.88

19.46

36.64

-23.08

C57.23± 3.4 33.26 48.8±1.1 15.547.76±5.6 0.2001X 10 38.20 31±0.9 -7.2

2.49±6.4 0.0642X 10

0.728x10'6

41.02 47.3±0.7 6.28

52.14-24.16

21.07

D

4

28.26± 3.6 34.83 56.2±2.121.37

9.71±5.30.2504X 10-6 37.65 29.2±1.8 -8.45

0.93±2.4 0.0239X 10-6 43.46 28.8±1.3 -14.66

71.71

-28.35

-49.19

Due to the preferential ionization near pH 2 there has been a

shifting of driving factor at this point also for different samples the factors were varying.

Except for sample C, it is the entropy which provided the force to the molecules to bring

into the solution as the pH gets down (pH 4 to pH 2). For sample A, B and D although the

values of entropy were negative, but were showing positive change in tendency and the

magnitude of change are highest among all the variables. For the pH values 2 to 1, it is

again entropy driven process, except sample B for which it is driven by AG, as negative

change in Gibb's free energy is minimal for it. In this case values of AH and AG are about

6 times the value of entropic terms (TAS). When we have a look on a broad range of pH

change (4 to 1) it is found to be entropy driven only as the change in entropy is highest

and positive. So far as the spontaneity is concerned, the values of AG is positive in all

cases but with the drop of pH, spontaneity was increasing (AG was showing negative

tendencies). Among all the samples, no major differences in free energy changes were

observed for different samples. For all the processes and samples, values of AG and AH

are positive, thus we could conclude that the process is non spontaneous at low

temperature while spontaneous at high temperature. Here, terms low temperature and high


12

4

2

4

12

4

12

temperatures are relative terms. For a particular reaction, high temperature could mean

room temperature also. Results of solvation enthalpy (AH) for ionized and non ionized

molecules of different samples were not conclusive. In authors’ point of view, here

formation of solvation energy of ionized and non ionized moieties and crystal lattice

energy are creating some interdependent and complicated phenomena in case of different

samples.

3. DETERMINATION OF PARTITION COEFFICIENT

Water-octanol system (and the partition coefficient in the form of log P as a measure of

drug lipophilicity), is most widely used model to describe biological membranes with

respect to the estimation of passive transport properties (Rogers and Wong, 1980). But, it

is still an open question whether the octanol-water system is at all suitable to describe

partitioning/distribution processes in terms of thermodynamic aspects. The reason

attributed to this contention is that the octanol-water system is classified as an enthalpy

driven process, whereas the lipid phase-water system is entropy driven (Rogers and

Wong, 1980).

On the other hand if drug molecules show different degrees

of ionization with variable pH (like Itz), presence of non dissociated and differently

dissociated drug molecules in various compartments of the body will not only affect the

rate of absorption of per orally administered drugs from different sections of the gastro-

intestine, but also distribution and excretion. Thus the nature of the driving forces of the

processes as well as the ratio between enthalpic and entropic terms is determined by an

eventual charge of the drug molecule, and the energetic state of this molecule within the

respective phase. It is therefore imperative that biopharmaceutical characteristic of the

molecule should be established considering variable compartments (pH) of the body with

relevance to drug molecule. Thus, to mimic the actual environment of delivery site,

acetate buffer (pH 4.5) -n-octanol system was selected and different energetic parameters

were determined. The values of Log P calculated for different samples were as 2.21, 1.94,

0.801 and 1.048 for A, B, C and D respectively taken at room temperature.

In order to transfer a molecule from one phase (buffer) to

another (octanol) it needs to overcome a potential barrier, which is “hypothetical” and




equals to the solvation enthalpy in the buffer. The resolvation process is a complicated

process where the old solvation shell is destroyed simultaneously as the new one is

created. As a consequence of this competition, the height of the activation barrier

decreases considerably. The value of the activation barrier may be estimated from kinetic

parameters of the partitioning/distribution process, which may in the future be helpful for

further characterization of biopharmaceutical properties of drug molecules.

In general two types of the transfer processes (non

dissociated molecule to octanol phase, and dissociated molecule to octanol) are basically

different regarding their respective driving force. In most of the cases, partitioning is a

typically enthalpy driven process, whereas the second case, i.e. distribution of the charged

form of the molecules, in contrast, is entropy driven (Tomlinson, E., 1983). In our

experimental conditions (pH 4.5) ionizable nitrogens (P.^a1 and P.^a2) are not protonated,

effect of which can be seen in Table 21. These nitrogens remain non protonated in the pH

region of 2-10. So, a sharp increase in solubility was observed as the pH goes below 2

since after protonation it interacts with aqueous media in a significant strength.

Figure 31: Schematic representation of driving forces during partitioning between Acetate buffers (pH 4.5) to n-octanol. Experiment was carried out at room temperature (298K) and in a triplicate mode. Thermodynamic behavior of samples was observed in a group of two. In one group A and D were matching while in another group B and C were same.

Transfer enthalpy is positive only in the case of sample A. It may

be assumed that in sample A, drug molecule interacts more strongly with the solvate shell

in the water phase in comparison with the octanol. Therefore there is a high probability of

transferring of the drug molecule together/partly with the solvation shell (water

molecules). In this case the substructure transferring unit is a drug molecule + solvation



shell. At AH°tr < 0 the opposite picture is observed (B, C and D). The total resolvation of

drug molecule in water phase occurs at the transferring process. Thus the substructure

transferring unit is just a drug molecule. This information can be useful to analyze

diffusion processes of drug molecules through biological barriers (passive transport),

because the size of the substructure unit plays a key role and determines coefficient

diffusion values and mechanism of it.

In case of Sample A and D the transfer process appears to be

entropy driven but in the cases of B and C the factor appears to be enthalpy driven (Fig.

31). Reason behind the shifting of force from entropy to enthalpy may be higher energy

states and lesser crystalline nature of the substance that leads to lesser distinguishable

interaction with the solvents (Table 21).

Table 21: Transfer thermodynamic characteristics of the transfer process from acetate buffer (pH 4.5) to n-octanol of samples studied at 298K.

Parameters A B C D

IS

01n

AG298sol (kJ mol-1)36.63 36.39 35.84 36.10

AH298sol (kJ mol-1)52.3±1.3 30±0.8 41.1±0.8 42±0.5

T AS298sol (kJ mol-1) 15.67 -6.39 5.26 5.9

AS"9'sol (J mol-1 K-1)52.58 -21.44 17.65 19.79

Uh

ff )

c

AG298sol (kJ mol-1)49.16 47.49 40.41 42.09

AH298sol (kJ mol-1)43.4±0.5 46±1.2 53±0.5 41.7±0.7

T AS298sol (kJ mol-1)-5.76 -1.49 12.59 -0.39

AS"9'sol (J mol-1 K-1)-19.32 -5.0 42.24 1.30

Transfer

Octanol

tTo

Acetate buffer (4.5)

AGbuff ^ AGoct -12.53 -11.1 -4.57 -5.99

AHbuff ^ AHoct 9.25 -16 -11.9 0.3

TASbuff ^TAS°ct 21.43 -4.9 -7.33 6.29

ASbuff ^A S°ct 71.9 -10.3 -24.59 18.49


4. APPLICATION OF PHYSICAL TREATMENTS IN DRUG DELIVERY

4 A. INCREASED LIPOPHILICITY BY CHANGING PHYSICAL STATE AND

HEAT TREATMENT

A remarkable change in behavior of binary mixture is observed. When the temperature

was raised above the melting point (69°C) of lipid, a substantial part of the drug remained

settled at the bottom of molten lipid and only 20 mg of drug was able to get solubilized. It

was due to inherent solubility of Itz into lipid at that temperature. As temperature

approaches the melting point of the drug (i.e. 169°C) more Itz starts solubilizing into the

lipid. If further drug is added into lipid melt it easily gets solubilized. It means at this

stage, enough energy was available to the lattice of highly crystalline Itz to break down its

lattice and it became amorphous. The ambivalence (lattice Vs lipophilicity) vanishes away.

Now, Itz behaves solely according to its lipophilicity. Even, more than 2 g of Itz

dissolved smoothly into the lipid melt at this point of temperature. The color change

(turning into brown) in binary mixture was also observed. To have a clearer picture of the

phenomenon occurring, the lipid melt was allowed to cool to room temperature and then

re-heated up to melting point of lipid. Surprisingly there was a single phase and no un

dissolved drug particles were observed. It means that once the drug is made amorphous

and remained amorphous (e.g., by dispersion into lipid melt here) it shows solubility to an

appreciable extent. Thus, it is quite evident from the study that once the problem of

crystallinity is overcome in the case of Itz, solubility no longer remains a problem.

Probability of interaction between Itz and molten lipid was evident through the change in

color of binary mixture. To investigate the interaction between these two chemical

moieties, differential scanning calorimetry, linearity of chromophore of molecule in UV

spectroscopy and NMR spectroscopy was evaluated (Figure 32, 33 & 34).


PhD Thesis 100 Jamia

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Figure 32: DSC thermogram of mixture of stearic acid and Itz, previously heated at 180°C.

Only the peak of stearic acid was found at 60.260°C.

In DSC study there was no sign of chemical interaction

between Itz and stearic acid. Only a singular peak at 60.26°C corresponding to the melting

point of lipid was shown (Figure 32) while the presence of amorphous form of Itz became

evident as there was no peak near the melting range of Itz (169°C), although there was a

hump in this region. Thus, crystalline pattern of Itz was almost lost as it is dispersed in the

matrix of stearic acid. Similarly, in UV spectroscopy, Xmax was found nearly at the same

point (±3 nm) where the chromophore of Itz (260 nm) was found during scanning in pure

solvent. Linearity of the absorption Xmax was found to be between 0.5-40 ^g/ml (Figure

33). Although some noises in the spectra were also observed in the region of 220-230 nm.

Presence of noise was much expected as it was a multi-component media.

NMR spectra showed characteristic peaks of various protons

present in the structure (Figure 34). The terminal methyl group of Itz gave a triplet at 5

0.759 - 0.778 ppm. Whereas the methyl group showed a doublet at 5 1.260 - 1.277 ppm.

The eight piperazine protons were observed as a singlet at 5 3.164. The aromatic region of

the spectra showed the integration for 14 protons in the range of 5 6.826 - 8.390 ppm for

the three phenyl rings and the two triazole moieties for various methylene and methyne

PhD Thesis

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protons. So far the protons of stearic acid is concerned, the terminal methyl group of

stearic acid gave triplet at 5 0.778 - 0.796 ppm. Whereas, 30 methylene protons showed

multiplets at 5 1.277-2.485 ppm. Carboxylic -O H protons showed singlet at 5 8.314.

4 B. EX VIVO PERMEATION ACROSS VAGINAL TISSUES

The vaginal tissue permeation profiles of samples (B, C and D) were little improved than

control (A) and these are comparable to each other (Table 22). The highest drug flux (Jss)

was observed with sample B (0.0077 mg/cm2/h). Samples C and Sample D were

comparable (0.0051 and 0.0056 mg/cm2/h) to each other. Thus it is observed that a

decrease in the crystalline state of bulk drug leads to its interaction with lipophilic

biological membrane based on its inherent lipophilicity and showed a better profile.

Figure 33: Linearity of absorption spectra of mixture of stearic acid and Itz by UV spectroscopy. Substantial noises were found near 220 nm. Little noises near 200-220 nm were also present when UV scan of pure Itz was taken.

PhD Thesis

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Figure 34: NMR spectra of mixture of stearic acid and Itz

Table 22: Results of ex vivo permeation studies. Here, sample A was taken as control, since it is untreated. Jss was calculated from the slope of the linear portion of graph. Kp was calculated by dividing Jss with the concentration of the drug in donor cell (Co). Er was calculated by dividing the Jss of respective formulation with Jss of control formulation.

Samples Jss±SD (mg/cm2/h)a Kp ±SDa Enhancement ratio (Er)

A 0.0047±0.057 0.0235±0.12 -

B 0.0077±0.068 0.0385±0.11 1.63

C 0.0051±0.039 0.0255±0.21 1.08

D 0.0056±0.096 0.028±0.14 1.19

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CONCLUSION AND INFERENCES DRAWN

Limited solubility of any compound is considered to be a major impediment in delivering

it across biological membranes. Analysis of a drug’s crystal structure has been long used

as a tool to understand its solubility, partitioning, distribution as well as other

biopharmaceutical effects. Often a change in crystal lattice leads to conspicuous changes

in solubility behaviour. The current research work ensures the formation of an amorphous

state of a highly crystalline but thermo-stable substance by employing new controlled heat

methods without using any other excipients. Such systems were then evaluated for

solubility and partition coefficient.

The methods used for preparing them were also compared. These

parameters were also studied in terms of thermodynamics (AG, AH and AS). Driving

forces of different solid states prepared by the various physical processes were found to be

changing according to their chemical potential and free energy available. As outlined in

the present work, the newly engineered particles were further tested for their feasibility in

drug delivery wherein these elicited better lipophilicity and ex-vivo permeation across

vaginal tissue providing an additional mechanistic insight. The in vitro toxicity studies

also did not show any adverse effect emphasizing that the conversion of Itz into an

amorphous form does not compromise its safety.

PhD Thesis 104 Jamia

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4c. Formulation development and evaluation of a mucoadhesive, acid bufTering and

sustained release tablet using inclusion complexes of Itraconazole


4c. MUCOADHESIVE, ACID BUFFERING AND SUSTAINED RELEASE

TABLET USING INCLUSION COMPLEXES OF ITRACONAZOLE

One of the approaches to improve the solubility is complexation which in tern also

increases the bioavailability of poorly water soluble drug. These complexing agents

(Figure 35) include; humic acid (HA), fulvic acid (FA), P-cyclodextrin (P-CD), 2-

hydroxypropyl-P-cyclodextrin (HP-P-CD), caffeine (Caff) and so on. Since, the

development of inclusion complexes of Itz was well proven; this study explored the

potential of various complexing agents from different sources to address the solubility

problem of the molecule. Newly introduced humic acid and fulvic acid belong to natural

organic matter which forms inclusion complexes with therapeutic molecules (Mirza et al.,

2011). Cyclodextrins are torus-shaped oligosaccharides obtained from the enzymatic

degradation of starch by bacteria (Szejtli, J., 1998) which readily form inclusion

complexes with many lipophilic organic molecules both in solution and in the solid-state.

Caffeine is a natural xanthine alkaloid compound that acts as a central nervous system

stimulant and reportedly forms complexes by a charge transfer mechanism (Fouad et al.,

2010). This section aimed at identifying the best inclusion complex to be used in the case

of vaginal candidiasis and then converting it into a final dosage form.

4c.1. EQUILIBRIUM PHASE SOLUBILITY STUDIES

Phase solubility studies were carried out at room temperature (25°C) in triplicate

according to the method reported by Higuchi and Connors (Vlachou and Papai'oannou,

2003). Excess amount of Itz was added to distilled water containing various

concentrations (0.2- 2% w/v) of different complexing agents humic acid (HA), fulvic acid

(FA), P-cyclodextrin (P-CD), 2-hydroxypropyl-P-cyclodextrin (HP- P-CD) and caffeine

(Caff) in a series of stopper conical flasks (100 ml) and shaken for 48 h on a rotary flask

shaker. The suspensions were filtered (0.45 ^m). The solubility and chemical stability of

Itz was also assessed by a slight modification of a validated HPLC method (Yoo et al.,

2000), with an accuracy of 95.5% and a precision of 6.7%. The analysis was carried out

on a Waters Alliance e2695 separating module (Waters Co., MA, USA) using photo diode

array detector (Waters 2998) with auto sampler and column oven. The instrument was

controlled by use of Empower software installed with the equipment for data collection



http://jba.sagepub.com/search?author1=George+Papa%C3%AFoannou&sortspec=date&submit=Submit


and acquisition. Compounds were separated using a Ci8 reverse phase column (25 x

4.6mm, particle size 5 ^m, Merck, Germany) maintained at room temperature. The mobile

phase consisted of acetonitrile, 0.05% diethylamine in deionized water (7: 3, v/v), with the

pH adjusted to 7.0 by drop wise addition of phosphoric acid. The flow rate was 1 ml/min

and the detection wavelength was 260 nm. 10 ^l of the sample was injected into the

column. Rt of Itz was observed at 8.25 min.

Figure 35: Molecular structure of Itz and different complexing agents (P- cyclodextrin, HP- P- cyclodextrin, Caffeine, Fulvic acid and Humic acid).

4c.2. PREPARATION OF COMPLEXES

The required stoichiometric (1:1, 1:2 or 1:3) quantity of drug was added to an aqueous

solution of the complexing agent and was agitated on a magnetic stirrer for 24 h. Sucrose

solution (2%, w/v) was added as a cryoprotectant. The resulting solutions were frozen at


(-70°C) in a deep freezer for overnight. This was then lyophilized (Dry Winner, DW-8-85

Heto Holten, Denmark) to obtain a dried mass which was further powdered in a glass

mortar and pestle and passed through a 100-mesh sieve to obtain a uniform-size fine

powder. The samples were then transferred into vacuum desiccators and dried over silica

gel under vacuum for at least 24 h. A similar batch of complex, without cryoprotectant

was also developed to carry out instrumental analyses like DSC and XRD.

4c.3. CHARACTERIZATION OF COMPLEXES

DSC, XRD and Mass spectroscopy were carried out by the methods described in section

4a.5.

4c.3.1. Proton nuclear magnetic resonance ( 1H NMR)

Proton Magnetic Resonance ( 1H NMR) spectra were recorded on Brucker Model

DRX-300 NMR spectrometer in CDCls using tetramethylsilane (TMS) as the

internal standard. Chemical shifts are reported in parts per million (ppm, 5) and

signals are described as a singlet (s), doublet (d), triplet (t), quartet (q) and

multiplet (m).

4c.4. DETERMINATION OF COMPLEXATION EFFICIENCY

The ability of different agents to complex Itz was determined quantitatively by UV

spectrophotometer (Hassan et al., 2007). Acetonitrile was used for the determination of

free Itz content since complexing agents taken, had poor solubility in acetonitrile and the

inclusion complex was also expected to have low solubility in acetonitrile. To estimate

free Itz content, 10 mg of the sample was dissolved in 100 ml of acetonitrile. This solution

was then sonicated for 2 min, filtered on a 0.45 ^m filter (Millipore) and analyzed using

UV spectroscopy (UV 1601, Shimadzu, Japan) at 260 nm. The free Itz content was

determined in triplicate. Following equation was used to determine the complexation

efficiency,

% inclusion efficiency = Total Itz content - Free Itz content X 100

Total Itz content



4c.5. SATURATION SOLUBILITY OF COMPLEXES AT VAGINAL pH

Excess amount of different complexes were added to 10 ml of an aqueous solution (pH

4.5). They were shaken in a water bath (Ray Scientifics Instruments, India) for 5 days at

37°C, centrifuged (Tomy MX-305, Japan) at 3000g for 10 min and filtered (0.45 ^m). The

concentration of Itz in the resulting solutions was then analyzed by the method described

in section 4c.1. The resulting values were the average of at least three replicates.

The standard solution Gibbs energies were calculated using following equation:

AG°sol = -R T ln X2

Where, X2 is the molar fraction of Itz in a saturated solution.

The standard solution enthalpies were derived from temperature dependencies of drug

solubilities expressed in molar fractions (van’t Hoff equation):

d InJ:; _ AJf^i

For use of the above equation following assumptions were made: (a) the activity

coefficients of dissolved drugs do not deviate from unity and (b) the solution enthalpies do

not depend on concentration. The solution heat capacities are considered to be constant

within studied temperature range, since the temperature dependence of solubility is

described by linear equations.

4c.6. DETERMINATION OF DRIVING FORCE OF SOLUBILIZATION

To determine the driving force for solubilization of the bulk drug and complexes among

different thermodynamic parameters (Gibb's free energy, Entropy and Enthalpy), the

following study was carried out. Different aqueous solutions with variable pH (1 and 4)

were prepared by the drop wise addition of concentrated HCl into 100 ml of Milli Q water

and the required pH was confirmed using a pH meter (Decibel 1011, Chandigarh, India).

Excess of each sample was added to different pH solutions and the solubility was

determined by the method described in section 4c. 1. Here aqueous solutions of variable

pH were selected instead of buffer solution as in an earlier study (Perlovich et al., 2003)

composition of buffer was found to have pronounced effect in determining the

thermodynamic driving force for solubility enhancement during the complexation studies.



4c.7. IN VITRO RELEASE STUDY

Drug release study of active pharmaceutical ingredient (100 mg Itz suspension) and

inclusion complexes (equivalent to 100 mg Itz) was performed using USP II dissolution

apparatus (Hanson Research SRS, Chatsworth, CA, USA) in 900 ml of simulated vaginal

fluid containing 1% sodium lauryl sulphate at 50 rpm and 37°±2°C. The concentration of

Itz was determined using HPLC method described in section 4c.1. The study was carried

out by transferring the constituted suspension (5 mL) in dialysis bag (Spectra-Por dialysis

bag supplied by Sigma Aldrich, St. Louis, MO, USA with cutoff 12,000- 14,000 Da). All

dissolution studies were carried out in triplicate.

4c.8. IN VITRO CELL TOXICITY STUDIES OF COMPLEXES

An MTT assay was used to assess the cytotoxicity of the free drug as well as the control

and the complexes. The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium

bromide) assay is based on conversion of yellow water soluble tetrazolium dye to a water-

insoluble purple formazan by living cells. The amount of formazan generated is directly

proportional to the number of viable cells.

MCF-7 cells (ATCC, USA grown in NII, New Delhi) were grown (37° C, 5

% CO2 in water jacketed incubator shell) using DMEM media (Dulbeccos modified eagle

medium for cell culture growth, Gibco Invitrogen, USA) with 10% FBS and seeded on a

single 96 well plate (Corning costar, USA) and allowed to adhere for MTT assays.

Following this a concentration of free drug as well as the control and complexes ranging

from 50 ^g/ml to 500 ^g/ml having equivalent concentration were added in duplicates to

the single 96 well tissue culture plate (Falcon Plate, Corning costar, USA). After 24 hours

of treatment, the MTT assay was performed to check cell viability (Mosmann, T., 1983).

For the MTT assay, the media was removed from all the wells, 10 ^l of MTT reagent

(Chemicon International, USA) per well from a working stock (5 mg/ml) was added and

the plates incubated (37° C and 5 % CO2) for 2-3 hrs, after that the reagent was removed

and the crystals were solubilised using isopropyl alcohol (IPA). This IPA extract

(Isopropanol with 0.1 N HCl) was then transferred to 96 well-plates. The HCl converts the

phenol red in the tissue culture medium to a yellow colour that does not interfere with the

MTT formazan measurement. The Isopropanol dissolves the formazan to give a



homogeneous blue solution. The absorbance was measured at a test wavelength of 570 nm

and reference wavelength of 630 nm using an ELISA plate reader, LMR-340 M (Labexim

International, Austria). The value of absorbance is a measure of the number of live cells.

4c.9. PREPARATION AND OPTIM IZATION OF TABLETS (mucoadhesive,

sustained release and acid buffering)

From the comparative studies of different complexes, it was quite evident that FA-Itz

complex was performing well in different studies. Furthermore, a composition comprising

more than 40 % (w/w) of FA was also found to exhibit useful mucoadhesion properties.

Being macromolecular and negatively charged together with presence of poly-functional

groups (such as carboxylic, amino and phenolic moieties) also paves the way to think for

the exploration of this excipient as an acid buffering agent also. Hence this complex was

carried forward for the development of above said tablet. To study the effect of FA on the

complexation of Itz, complexes were developed in two ratios (1:1 and 1:2). Before

moving for further formulation development, FA-Itz complexes were further characterized

by following studies:-

4c.10 FT IR ANALYSIS

It was carried out by the method described in section 4a.5.3.

4c.11. CONFORMATIONAL ANALYSIS BY COMPUTATIONAL METHOD

The 3D-molecular structures were generated and optimized with Chem 3D-Ultra 8.0

software (CambridgeSoft Corporation, USA). All calculations used are for geometric

optimization. All the energy minimizations were carried out until the RMS gradient was

less than 0.08. Optimized molecular structures and partial atomic charges were used for

the molecular modeling of FA, Itz and its complex. H-bonding analysis was based on

ORTEP III (v1.0.3, L.J. Farrungia, University of Glasgow, UK).

4c.12. PREPARATION OF TABLETS

Different batches of tablets (500 mg each) were manufactured according to Table 23 by

direct compression. Itz complex, fulvic acid and MCC PH 102 were mixed together using

a mortar and spatula. Magnesium stearate and silica gel were then added and mixed

further. The tablet compositions were compressed on a 16-station rotary tablet machine

using 16-mm flat-faced round punches (4-tons force for 10 s). Tablets T 1 to T 4




contained the 1:1 complex while T 5 to T 8 contained the 1:2 complexes. The excipients

other than FA used for the formulations were approved, or GRAS listed and suitable for

use in a VDDS.

Table 23: Tablet composition of different batches. Regulatory statuses of excipients by

different regulatory agencies are also given. The amount used for the study was within

prescribed limits.

Ingredients T-1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 Regulatory status ofexcipients for VDDS

Itz 100 100 100 100 100 100 100 100

Fulvic acid 320 345 320 345 320 345 320 345

MCC PH

102

75 50 75 50 75 50 75 50 G, 3, 6, 7, 9, 10,

11, 12, 13, 14, 15, 20, 21

Silica gel 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 I

Mg stearate 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22.

G: GRAS, I: inactive ingredients guide, Pharmacopeias, 1. Austrian, 2.Belgian, 3.British,4. Chinese, 5. former Czechoslovakian, 6. European, 7. French, 8. German, 9. Greek, 10. Hungarian, 11. Indian, 12. Italian, 13. Japanese, 14. Mexican, 15. Netherlands, 16. Nordic, 17. Portugese, 18.Romanian, 19. Russian, 20. Swiss, 21. United States, 22. Former Yugoslavian

4c.13. OPTIMIZATION OF TABLETS

The acid-buffering bioadhesive tablets were optimized on the basis of hardness,

mucoadhesion and release profile. The release profiles of the tablets were studied in 900

ml of simulated vaginal fluid containing 1% sodium lauryl sulphate using USP dissolution

apparatus II at 50 rpm and 37°C. The concentration of Itz was determined by UV

spectroscopy (UV 1601, Shimadzu, Japan) at 260 nm.

In vitro mucoadhesion studies were carried out by slightly

modifying a published method (Nakamura et al., 1996). Briefly, an agar plate (1%, w/w)

was prepared in pH 4.5 citrate-phosphate buffer. A tablet was placed at the center of plate.


After 5 min, the agar plate was attached to a USP disintegration test (model 1901,

Electronics India, India) apparatus vertically and moved up and down (10±1 cycle per

minute) in pH 4.5 citrate phosphate buffer at 37±1 °C. The sample on the plate was

immersed into the solution at the lowest point and was out of the solution at the highest

point. The residence time of the tablet on the plate was noted visually. Hardness of the

tablet was determined using a Pfizer type hardness tester (KIP 2072, Kshitij Innovation,

India).

4c.14. EVALUATION OF OPTIMIZED TABLETS

4c.14.1. Determination of acid buffering capacity

The pH of the tablets was determined after being in contact with 2 ml of distilled water for

2 minutes. The pH of the swollen tablet was measured by touching the tip of the electrode

to the wet mass of the tablet. For buffering capacity, one tablet was allowed to dissolve in

10 ml of 0.9% (% w/v) NaCl (normal saline) solution. Sodium hydroxide (1.0 N) was

added in 20 ^l increments under constant stirring. The pH was measured with a standard

combination electrode, 30 sec after each addition. Stirring was stopped during the pH

measurements. This procedure was repeated until the pH rose above 7.0. The titrations

were performed in triplicate for each formulation. Curves were used to calculate the

amount of NaOH required for bringing the pH of each solution to 5.0. This was taken as a

measure of the buffering capacity of the tablet.

4c.14.2. Tablet swelling and spreadability studies

The swelling characteristics of the bioadhesive tablets were evaluated using dynamic

swelling studies. Each sample was weighed and then placed in 10 ml sodium

citrate/Hydrochloric acid buffers at pH 4 in a glass vial at 37°C ± 2°C. The samples were

periodically weighed after removing excess water on the surface with a filter paper and

swelling was calculated. For the spreadability study, the tablets were allowed to swell in 2

ml of distilled water for 1 minute; the swollen mass was gently transferred to the center of

a glass plate and compressed under several glass plates (100 g each minute). The spread

diameters were recorded each time and compared with a marketed vaginal gel formulation

used as a reference formulation (Candid-V).




4c.14.3. Ex Vivo Mucoadhesion studies

A modified Setnikar and Fantelli apparatus (Setnikar and Fantelli, 1962) was used for the

study of mucoadhesion (Figure 36). Excised and cleaned buffalo vaginal tube was everted

on the upper and lower ends of the glass cell and crimpled using rubber bands. Water was

circulated in the cell to maintain temperature and also to keep the tissue moist. The

bioadhesive tablet was inserted into the tube using a pair of blunt forceps. A preload time

of 5 min was allowed to help the formulation to adhere properly to vaginal walls.

Simulated vaginal fluid (Owen and Katz, 1999) was allowed to fall drop wise (3 ml/h) into

the vertically suspended vaginal tube. The time for expulsion of the formulation from the

lower end of the cell was recorded.

Figure 36: Modified Setnikar and Fantelli apparatus. Water flows through the crimped vaginal mucosa at fixed rate. Temperature of the media is controlled by thermometer and

heating devise attached.

4c.14.4 In vivo antifungal studies of optimized tablet

Following rat infection model was used to study the complexes in v^vo (De Bernardis et

al., 1997). Fifteen female wistar rats were divided in groups of three for the application of

T4, T8, and control. To be applied into vaginal cavity, all the complexes were converted

into suspension, keeping final concentration of Itz 2% w/w, since in a clinical study 5 g of

2% Itz cream was found to be very well tolerated and was not absorbed systemically. Six

days prior to inoculation of infection, all animals were maintained under pseudoestrus by

subcutaneous administration of estradiol benzoate. Into these oophorectomized rats C.

albicans (108 cells per ml of saline) were inoculated. Then after, different suspensions (0.5

mL) were applied. Control was not having any complex or Itz, it was a placebo. During

specified time intervals 1 ^L of the fluid from vaginal cavity was withdrawn, diluted and


spread on Sabouraud agar containing chloramphenicol (50 mg/ml). It was then incubated

at 32°C in the BOD incubator (LHC-78-Labhospmake, India) for 48 hrs and CFUs were

counted. A rat was considered infected when at least 1 CFU was present in the vaginal

sample (i.e., a count of >103 CFU/ml). A graph was plotted between time (days) and no. of

CFU present in vaginal fluid and different tablets studied comparatively.


1. PHASE SOLUBILITY STUDIES

Phase solubility analysis of Itz in presence of different complexing agents are given in

Fig. 37. Here different natures of interaction between complexing agents and Itz have been

observed. Broadly, two types of interactions were observed. In one category curve fitting

line was inclined towards Y-axis while in other category, it was inclined towards X-axis.

This tendency may be used to predict the comparative orders of complexation. With

respect to complexing agents, HA and FA were exhibiting lower order of complexation as

compared to HP P CD, CD and Caffeine. The total solubility (^xotal) of a drug as a function

of concentration of different complexing agents is given by any of the following equations

which also give an estimate to predict the stoichiometry of the complexation. For single

order complexation, solubility relationship is expressed by:

STotal = S0 + m [DmCA]

Where, S0 is the drug concentration is the absence of complexing agents and m refers to

the stoichiometry of the complexation. Here, one molecule of the drug interacts with one

molecule of complexing agents. Thus, solubility of Itz can be assumed linear and

proportional with complexing agent when stoichiometry of complexation is 1. This is the

case with HA and FA. For second order complexation (one drug molecule interacts with

two molecules of complexing agents), fitting the data to a quadratic model is appropriate.

It is expressed by:

STotal = S0 + K1:1S0 [CA] + K1:1K1:2S0 [CA] 2

A positive deviation from the linearity for the phase-solubility diagram and deconvolution

of the curve is observed with higher order complexes. In third order complexation, one

molecule of the drug interacts with three molecules of complexing agents. To account for

the concentration of complexing agents in these cubic relationships, (Peeters et al., 2002)




suggested a non-linear optimization technique based on Nelder-Mead approach. Higher

ordered complexation is observed with remaining complexing agents. So, cyclodextrin

molecules (P-CD and HP p CD) are considered to interact in a fashion of second order. On

the other hand caffeine molecule follows a third order pattern which is also evident from

the literature.

Figure 37: Phase solubility studies of Itz at room temperature (25°C) in triplicate mode.

Curve fitting line appears to be divided into two groups.

A phase solubility graph is also used to determine the binding

constant and other thermodynamic (AG, AH and AS) parameters (Yadav et al., 2009). The

binding constant was calculated according to the formula:

^ ^ slope ̂ S 0 (1 - slope)

Where, S0 is the solubility of Itz without complexing agents.

The binding constant for different complexes was found to be

varying like Itz-FA (1104.26 M-1), Itz-HA (1024.65M '1), Itz-HP-P-CD (1012.44M '1), Itz-

P-CD (993.47M '1) and Itz- Caffeine (568.21 M'1) . Stability constant (Ks) values lying

between 200 and 5,000 M-1 are considered as most suitable for the improvement of



solubility and stability of a poorly soluble drug (Patel and Patel, 2007). To investigate the

spontaneity and feasibility of the entrapment by the thermodynamic approach, changes in

Gibb’s free energy (AG) were also calculated (at constant temperature and pressure). It is

the net energy available for useful work.

AG°sol = -R T ln X2

Where, X2 is the molar fraction of Itz in a saturated solution. Results obtained are given in

Table 24. Other thermodynamic parameters are obtained by using Van't Hoff equation. Ks

is calculated at different temperatures and AH is calculated according to the equation

given below,

Or,

Thus, if a plot is drawn between 1/T and Log K, slope of the plot will be AH. Entropy may

be calculated using the equation, AG = AH - T AS. Thermodynamic parameters calculated

are given in Table 24.

Table 24: Saturation solubility and thermodynamic parameters of complexes at vaginal pH

(4). Experiment was carried out in a triplicate mode.

Substances Solubility (^g/m l) at pH 4

X2 (molar fraction)

A /"'’298sol

(k Jmol-1)

AH298sol(k Jmol-1)

T

AS298 sol (k J mol-1)

AV298sol (J mol 1 K-1)

1:1 Itz-FA 5.81±1.4 0.149X10'6 38.94 48.3±0.5 9.36 31.4

1:1 Itz- HA 3.11±0.3 0.08X10'6 40.47 52.1±0.5 11.63 39.02

1:2 Itz- HP-P-

CD0.221±0.02 0.005X10'6 47.34 47.2±0.7 -0.14 -0.469

1:2 Itz- P-CD 0.179±0.004 0.0046X10'6 47.55 46±0.9 -1.55 -5.20

1:3 Itz- Caff 0.156±0.023 0.00402X10'6 47.88 46.1±1.1 -1.78 -5.97

Itz 0.13±3.7 0.0033X10'6 48.46 44.5±0.8 -3.96 -13.28


2. CHARACTERIZATION OF COMPLEXES

2. a. DIFFERENTIAL SCANNING CALORIMETERY

The DSC analysis showed a sharp peak of Itz at 170 °C. None of the complexing agents

showed any sharp peak except Caffeine at 198 °C. All the complexes exhibited broad

humps at variable temperature ranges (Figure 38). It may be inferred from the thermo

grams that Itz may have lost its crystalline pattern and thus is present in its amorphous

form. The details from the thermo grams of Itz and its complexes are given in Table 25.

Melting enthalpy (AH), expressed in joule/gram (J/g) is a

characteristic of the crystal order if the influence of impurities can be ignored. The higher

enthalpies were noted with single entities like Itz (105.035 j/g), FA (142.075 j/g), P-CD

(117.96 j/g) and Caffeine (124.630 j/g). So far the complexes are concerned, AH values

were comparatively lower. Thus, it may be inferred from the results that complexes have

more disordered structures. This decrease in disorder is created due to positive interaction

between two entities (drug and complexing agents).

2. b. X-RAY DIFFRACTION

Itz was showing a characteristic crystalline pattern (Figure 39) exhibiting intense peaks at

20.38 (100% ), 20.40 (97.59% ), 20.36 (96.53% ), 20.34 (88.53% ), 20.32 (80.66% ) and 17.5

(77.9% ) etc. Few sharp peaks were present only in caffeine (100% at 11.24, 49.56% at

27.32, and 47.64% at 28.42) and HP-P-CD (100% at 12.34, 39.40% at 23.68, 42.52% at

25.34 etc). Other complexing agents (FA, HA and P-CD) were almost amorphous in

nature (Figure 40). XRD of Itz-HA and Itz-FA showed amorphous nature and some

diminished peaks were observed in Itz- HP-P-CD, Itz-P-CD and Itz- Caffeine complexes.

These peaks were not from the fingerprint regions of Itz but from the complexing agents

itself. Thus, the XRD of inclusion complexes showed their amorphous nature, indicating

entrapment of drug and, peaks if any were appreciably diminished.




Figure 38: Representative differential scanning calorimetric profiles of Itz, Complexing

agents and different complexes. Sharp peak was obtained only with neat drug and caffeine.

Marked differences in developed complexes are observed.



Table 25: Differential scanning calorimetric data of pure Itz, complexing agents and

complexes. Peak width was calculated manually.

Samples AH (j/g) Maximum peak (°C) Peak width

Itz 105.035 170.408 6.62

FA 142.075 162.387 11.13

Itz-FA 78.502 136.214 26.44

HA 44.236 77.365 44.63

Itz-HA 31.461 163.628 16.50

HP-P-CD 33.768 119.876 1.42

Itz- HP-P-CD 69.119 161.452 11.22

P-CD 117.96 127.625 41.26

Itz- P-CD 49.825 147.473 8.32

Caffeine 124.630 198.524 7.46

Itz- Caff 69.215 81.213 4.29

2. c. MASS SPECTROSCOPY

A representative spectrum is given in Fig. 41. Here only the spectra of fulvic acid complex

have been given, as this complex was found to evolve better in earlier studies. In all the

mass spectra some peaks were common (with m/z variation ~ 5) that were either non-

complexed drug (Itz), non-complexed fulvic acid (avg molecular wt ~ 1200) or 1:1 and

1:2 complexes. Extensive noise was also present. It was evident from the result that in any

complex (irrespective of the ratio) some fraction of uncomplexed drug, uncomplexed

fulvic acid and 1:2/ 1:1 complexes were present. Their relative abundances depended upon

the ratio (1:1 or 1:2) taken.



Figure 39: X ray powder diffraction of Itz and complexes. Although diminished but peaks of

Itz were observed in Caffeine, P- cyclodextrin and HP- P- cyclodextrin complexes.



Figure 40: X ray powder diffraction of complexing agents. Crystallinity was observed only with Caffeine and HP-P- cyclodextrin. Some of these peaks were also present in developed complexes.

Figure 41: Mass spectra of Itz-FA complex. Peaks of uncomplexed drug, uncomplexed FA.



2. d. PROTON NUCLEAR MAGNETIC RESONANCE (1H NMR)

The 1H NMR spectra of Itz (Fig. 42) showed singlet protons at 5 8.29 and 8.37 for triazole

nucleus. While 1H NMR spectra of Itz-P-CD complex showed two singlet peaks at 5 8.31

and 8.39 which are slightly downfield shifted peaks. All the other peaks of both P-CD and

Itz remain almost unchanged. This clearly indicates the inclusion phenomena of the

triazole nucleus into the P-CD cavity (Spulber et al., 2008). Nearly same phenomena was

observed for inclusion complex of Itz with FA, HA and HP- P-CD. The downfield shift

values observed were 5 8.32 and 8.44 for Itz-FA, 5 8.34 and 8.42 for Itz-HA, 5 8.30 and

8.40 for Itz- HP- P-CD. 1H NMR spectra of Itz-caffeine complex also showed changes in

both the protons and shifting pattern of the proton in Itz-and caffeine. H8 proton of

caffeine was observed at 5 7.90 where as Itz-caffeine complex showed H8 peak as up

folded doublet at 5 7.84. Also triazole nucleus of Itz showed one singlet at 5 8.28 and

doublet at 5 8.34. This clearly indicates the introduction of H 8 (caffeine) with H3 of

traizole. Since, it was difficult to develop an overlay diagram of NMR spectroscopy only

the data obtained are discussed here.

Figure 42: NMR spectra of Itz



2. e. DETERMINATION OF COMPLEXATION EFFICIENCY

The results obtained for complexation efficiency studies are given in Table 26. Except Itz-

Caffeine complex (~ 47.54% ) all other complexes were acceptable. In author's point of

view this may not be a very appropriate method to determine the complexation efficiency.

High sonication energy may result into dissociation of no bond complexes (inclusion

complex) since with small gastric movement we expect the release of molecule from the

inclusion complex. However, due to unavailability of any appropriate method, authors had

to follow the reported method.

Table 26: Complexation efficiencies of different complexes. Inclusion of drug appears to be a better approach than charge-transfer mechanism to associate a drug with inert excipients.

Samples

1:1 FA-Itz

1:1 HA-Itz

1:2 Itz- HP-P-CD

1:2 Itz- P-CD

1:3 Itz- Caff

% Complexation efficiency ±SD (n=3)

67.32±2.6

59.21±1.9

61.48±3.3

58.31±1.4

47.54±2.7

3. SATURATION SOLUBILITY OF COMPLEXES AT VAGINAL pH

In a 5 day study all the samples became saturated. The maximum solubility was observed

with 1:1 Itz-FA complex (5.81±1.4 ^g/ml). Decreasing order of solubility is given in

Table 27. Thermodynamic parameters (AG, AH and AS) calculated for the samples are

also given in the same table. Positive signs of enthalpy of solublisation (AH) indicate that

it is an endothermic process at room temperature and crystal lattice energy of the

substance outweighs the solvation energy. Positive values of AG also indicate that the

process is non-spontaneous at room temperature and is driven by some energy source.

Nature of change of disorder (AS) is different in all systems (complexes). In case of HA

and FA complexes, it is increasing (positive values) while in other cases (HP-P-CD, P-CD

and Caffeine) it is decreasing (negative values). With increasing disorder of the system,

we may expect better encapsulation of one moiety into another.


4. DETERMINATION OF DRIVING FORCE OF SOLUBILISATION

Thermodynamic parameters calculated for different samples depict a clear picture about

the driving forces involved when the pH is varied in acidic range (4 to 1). Vaginal milieu

is acidic at normal physiological conditions but often rises to alkaline during infections.

Marketed formulations like Aci-gel, Buffer gels etc are often prescribed to maintain

vaginal acidity which eventually curtails the vaginal floral growth. Conclusively, there is a

significant pH variation in the vaginal cavity. Itz also elicits variation in ionic state at

different pH which translates into differential interaction with the complexing agents. Itz

is a weak base with four ionizable nitrogen atoms. The two pKa values are 4 and 1.5-2

whereas the other ionizable nitrogens are not protonated between pH 2 and 10 (Peeters et

al., 2002). Also the pKa of the nitrogen atom of the triazolone moiety is 4.0. This explains

the sudden drop in solubility above pH 3 and why changes in pH profoundly influenced

both solubility and dissolution. It also explains non linear (ascending or descending)

pattern for any thermodynamic parameter during complexation. The sudden drop in

solubility of Itz above pH 3 leads to a greater hydrophobic interaction of guest and host

molecules and the Itz penetrates deep into the host molecule thus creating Van der Waals

interaction and hydrogen bonds thereafter. For the complexes and API the values of AG

and AH are positive (AS were found to be both positive and negative). Thus we can

conclude that complexation of Itz with fulvic acid is non-spontaneous at low temperature

while spontaneous at high temperature. Here, terms low temperature and high

temperatures are relative terms. For a particular reaction, high temperature could mean

room temperature also.

Change in entropy (as the pH of the solution was moved from 4 to

1) was observed to be the main driving force as it increased substantially. Although it was

negative also in case of 1:1 Itz-FA, but other factors (AG and AH) were more negative as

compared to AS (Table 27). At pH 1 Itz molecule has a solvation shell, which consists of

entropic ally ordered molecules of the medium in the space next to the ionized atoms, and

a more disordered part of the solvation shell is located around other parts of the molecule

(hydrophobic effect). Thus during complexation the ordered shell portion along with some

other hydrophobic portions finds its way into the cavity leaving behind disordered

portions.




Table 27: Thermodynamic parameters of solubilisation process of pure Itz, 1:1 FA-Itz and

1:1 HA-Itz, 1:2 Itz- HP-P-CD, 1:2 Itz- P-CD and 1:3 Itz- Caff complexes in aqueous solution

at variable pH at 298 K.

Samples pHSolubility

( ^g / ml)

X2 (molar fraction)

A ^298AG sol

(k J mol-1)

ATt298AH sol(k Jmol-1)

298T.AS sol (k J mol-1)

A C298 AS sol

(Jmol 1

K-1)

API (Itz)

24.17±2.3 0.618X 10 35.42 55.3±0.71 19.88

0.13±3.7 0.0033 X 10

2.02X10'6

48.46 44.5±0.8 -3.96

66.71

-13.28

1:1 FA-Itz

78.46±2.6 32.48 38.3±0.6 5.82

5.81±1.4 0.149x10 -68.94 48.3±0.5 9.4

-6

19.5

1.4

1:1 HA-Itz

33.26±1.3 0.857x1034.60 58.1±1.8 23.5

3.11±0.3 0.08x10 -640.47 52.1± 0.5 11.63

-6

78.85

39.02

1:2 Itz- HP-P-CD

92.56 ±1.7 2.38x1032.07 42.3±0.7 10.23

0.221±0.02 0.005x10-' 47.34 47.2±0.7 -0.14

-6

34.32

-0.47

1:2 Itz- P-

CD

83.17 ±3.2 2.14x1032.33 40.6±0.4 8.27

0.179±0.004 0.0046x10 -647.55 46±0.9 -1.55

-6

27.75

-5.2

1:3 Itz- Caff

56.48 ±2.4 1.46x1033.28 48.5±0.8 15.22

0.156±0.023 0.00402x10 -647.88 46.1±1.1 -1.78

51.07

-5.97

5. IN VITRO RELEASE STUDY

The results obtained after dissolution study are given in Fig. 43. Bars for standard

deviation have been removed to have a clear picture of the release profiles. To analyze the

in vitro release data various kinetic models were used to describe the release kinetics. The

zero order rates describe the systems where the drug release rate is independent of its

concentration. The first order describes the release from system where release rate is

concentration dependent (order is one). Higuchi described the release of drugs from

insoluble matrix as a square root of time dependent process based on Fickian diffusion.


4

14

4

4

4

4


Figure 43: In vitro drug release profile of complexes. In a 24 hrs study, maximum drug release was observed with Itz -Caff complexes.

Figure 44: MTT assay results of pure drug and complexes.

,-V*'Figure 45: Ball and stick model (energy minimized) of Itz



Whereas, Peppas Korsemeyer describes the log fraction of drug release with respect to log

time. Release order of all the complexes appears to be first order (Table 28). To evaluate

the mechanism of drug release from the preparation, data of drug release may be plotted in

Korsmeyer and Peppas equation,

It is often used to describe the drug release behavior from polymeric systems. Where Mt is

the amount of drug release at time t, Mf is the amount of drug release after infinite time; k

is a release rate constant incorporating structural and geometric characteristics of the

dosage form, n is the diffusion exponent indicative of the mechanism of drug release. The

log value of percent drug dissolved is plotted against log time for each formulation

according to the equation. For a cylinder shaped matrix the value of n < 0.45 indicates

Fickian release; > 0.45 but < 0.89 for non-Fickian (anomalous) release; and > 0.89

indicates super diffusion type of release. It generally refers to the erosion of the polymeric

chain and anomalous transport (Non-Fickian) refers to a combination of both diffusion

and erosion controlled drug release. In all five complexes release appears to be Fickian

diffusion (n < 0.326).

Table 28: Release kinetics and mechanism involved with different complexes. Release pattern with highest r2 value is used to predict the kinetics involved.

Zero-order

First-order

Higuchimatrix

Korsmeyer-peppasBest fit model

FormulationsRel. Expo.

(n)r2 r2 r2 r2

1:1 Itz-FA 0.967 0.995 0.974 0.960 0.326 First order

1:1 Itz- HA 0.967 0.982 0.932 0.960 0.319 First order

1:2 Itz- HP-P-

CD0.967 0.988 0.963 0.960 0.326 First order

1:2 Itz- P-CD 0.967 0.968 0.946 0.960 0.326 First order

1:3 Itz- Caff 0.967 0.996 0.990 0.960 0.326 First order

6. IN VITRO CELL TOXICITY STUDIES OF COMPLEXES

The corresponding values for optical density for the control, neat drug and the complexes

are shown in Fig. 44. The Itz was found to have some cytotoxic effect. At the lowest


concentration (50 ^g/ml) relative difference of cell viability was not distinguishable with

each other, although they were not interfering with cell growth. At higher concentrations

(100 ^g/ml, 200 ^g/ml and 500 ^g/ml) some conclusive results were obtained. HP-P-CD

and P-CD complexes showed some negative pattern in cell growth as compared to other

complexing agents However, for the complexes the toxicity did not increase at different

concentrations. So far the humic substances are concerned; they are also reported to be

safe in cytotoxicity studies (Yamada et al., 2007). Thus, the reports of in vitro cell toxicity

study did not indicate any serious cellular toxicity.

In continuation with previous section following studies were further

carried out to convert a best suited complex (FA-Itz) into an optimized tablet formulation.

7. FOURIER TRANSFORMS INFRARED SPECTROSCOPY

FT-IR spectrum of the drug shows the stretching and vibrational peaks in the fingerprint

region (Figure 46) that are characteristic of the molecule, and which are overlapped,

diminished or dispersed in inclusion complexes. FT-IR spectroscopy is sensitive to the

structure, conformation and environment of organic compounds. The characteristic peaks

of Itz occurred at 3381, 3126, 3069, 2962, 1697, 1510, 1450, and 418 cm-1. The

absorptions due to the NH2 groups are located in the bands at 3381, 3126, 3069, cm-1. The

first band is assigned to stretching vibrations of the free NH2 of the pure drug. The peaks

observed at 1609 and 1425 may be assigned to the C=N and C-N bonds respectively and

the sharp peak occurred at 1697 cm‘1is due to the C=O group of the drug. FT-IR

absorption band for fulvic acid extracted from shilajit were found to be in accordance with

those reported in literature. Interactions between the carbonyl peak of Itz and the

carboxylic group of FA, and also between the stretching vibration of N-N of Itz and the O

H vibration of FA were observed. Olefinic and carbonyl peaks of the drug are widespread

and dispersed indicating weak interaction with similar bands in the complexing agent.

Peaks from the fingerprint regions (1300 - 400 cm’1) are more diminished, indicating

interaction between the drug and the complexing agent.




8. CONFORMATIONAL ANALYSIS BY COMPUTATIONAL METHODS

Molecular modeling has shown that complexes of Itz-FA are stable. A ball and stick

model figure of Itz is given in Fig 45. The molecular modeling showed that FA has the

ability to form the inclusion complexes with Itz. The intra-molecular hydrogen bonds

observed for FA contribute to the stability of the molecule (figure not shown). This

structure shows at least five intra-molecular H-bonds. Three out of five intra-molecular H-

bonds are OH-O type which means that these are strong H-bonds. These hydrogen bonds

are supposed to increase the stability of the molecule. Drug complex optimization with FA

shows that the Itz is stabilized by a strong NH-N interaction with FA. Total potential

energy of the FA using Chem 3D-Ultra 8.0 software comes to -38.8716 Kcal/mol while a

complex of Itz and the FA was stabilized at -26.283, which is more stable than Itz alone.

The energy optimization of Itz gave -30.84 Kcal/mol.

9. OPTIMIZATION OF THE TABLET FORMULATION

On the basis of hardness, mucoadhesion and release profile, T 4 and T 8 were found to be

good candidates for further investigation. T 8 showed a better sustained action compared

to T 4. The results obtained are given in table 29.

Figure 46: FT IR spectra of Itz, fulvic acid and complexes.



Table 29: Optimization of the tablet formulations. Parameters are given in the order of their

priority. Drug release behavior was given highest priority while Hardness was given lowest.

The formulae which performed best in the first two tests were selected as optimized

preparations.

Parameters T-1 T-2 T-3 T-4 T-5 T-6 T-7 T-8

1:1 complex 1:2 complex

% drugreleased after 24 h

68.3±3.2

71.2±2.6

73.5±1.9

76.1±2.4

72.4±2.6

74.8±4.0

77.9±3.7

79.8±3.3

Mucoadhesion(seconds)

90±2 95±3 96±3 102± 102±6 96±6 98±8 100±7

Hardness (Kg) 6.2±0.5

5.4±0.7 4.6±0.8 3.7±0.7

6.3±0.6

5.5±0.5

4.6±0.6

4.0±0.6

10. EVALUATION OF OPTIMIZED TABLETS

10.1 DETERMINATION OF ACID BUFFERING CAPACITY

The pH of the optimized tablets was found to be acidic (pH 4.8±0.068, n=2) for T-4 and

4.9±0.076, n=2 for T8, very similar to the required pH of the vaginal tract (US Patent

4,551,148). The pH of the reference marketed formulation was also acidic (Infa-V= 5.19 ±

0.26, n=2). The amount of NaOH required to bring 1g equivalent of tablets to pH 5.0 was

about 0.052 mEq. This data was comparable to the literature report where an amount of

NaOH required bringing 1 g equivalent of Advantage 24 to a pH 5 was reportedly 0.080

mEq (Haineault, Gourde et al. 2003). C. albicans adheres to vaginal tissues with

considerably higher affinity at pH 6 than pH 4 (Galask RP, 1988), the formulations with

their pH adjusted around 4.0 may have additional advantages of reducing fungal

adherence and growth at the site of infection. Moreover, the acidic formulations may help

to restore the physiological acid pH of vagina.


10.2 TABLET SWELLING AND SPREAD-ABILITY STUDIES

Increasing the amount of fulvic acid produced tablets with higher swelling rates. The

equilibrium swelling of T 4 after 15 min was 66.2% while T8 swelled up to 48.6%. After

maximum swelling, disintegration started. Being a gel, Candid-V spreads smoothly and

covers a large area at any point of time. T4 spread a little better than T 8 but was

comparable to Candid-V (Figure 47) in spread-ability.

10.3 EX VIVO MUCOADHESION STUDIES

Tablets (T 4 and T 8) showed very good retention times (>40 hrs)in the ex-vivo

experiment. They swelled inside the tube and adhered to the wall. Tablet T 8 showed

maximum adherences (>48 hrs). While the Infa -V (reference product) disintegrated inside

the vaginal tube and started leaking after 5-8 hrs.

10.4 IN VIVO ANTIFUNGAL STUDIES

Results of 21 days in vivo studies are given in Fig. 48. Negligible reduction in CFUs was

observed with placebo. Optimized tablets exhibited substantial reduction in fungal

colonies. T8 reduced the colonies > 1.7 times in comparison with T4 after 21 days. The

fact may be attributed to better solubilization and release behavior of T8; as the drug

contents were same in both the tablets.




Weight applied

Figure 47: Spread-ability studies of tablets when compared with marketed gel (Candid-V)

Figure 48: Outcome of vaginal infection by C. albicans in oophorectomized, Estradiol-reated

rats after first infections with 108 C. albicans cells. Each curve represents the mean of the

fungal CFU of three rats.



Thus, Itz can be complexed with Fulvic acid and formulated as a vaginal drug delivery

system with an efficacy comparable to existing topical formulations on the market. The

complexation was found to be largely entropy driven with the potential energy of the

complex being comparable to that of Itz at pH values where the API was non-ionizable.

Fulvic acid can be explored in three very new roles namely for acid buffering,

mucoadhesion and sustained release in vaginal drug delivery systems. The proposed

formulation used in this study suggests that further research should be carried out to

evaluate fulvic acid as a potential excipient for VDDS.




4d. Formulation development and evaluation of mucoadhesive and

thermo-sensitive gel incorporating SLN of Itraconazole


4d. MUCOADHESIVE AND THERMOSENSITIVE GEL INCORPORATING SLN

OF ITRACONAZOLE

As discussed in the rationale and based on the literature review, it was proposed to

develop a mucoadhesive and thermo-sensitive gel incorporating SLN of Itz. SLNs are

amenable to commercialization and hence had an added advantage. The final dosage form

is proposed to consist of a thermo-sensitive gel base with attributes of mucoadhesion and

probable application. The research plan focussed on selection of suitable excipients and

their further conversion into purported delivery system.

4d.1 SELECTION OF LIPIDS FOR DEVELOPMENT OF SLN

Lipids for the development of SLN were sorted out on the basis of following two studies:-

4d.1. A. Interaction with bovine serum albumin

The general criterion of lipid selection for the development of SLN is drug solubility in a

particular lipid. To obtain a better bio-distribution, stabilizers (surfactants) are selected

after studying interaction with proteins (Goppert and Muller, 2005) which results into

maximal in vivo residence time. But stabilizers, if not bounded chemically are loosely

attached to the colloidal surfaces. Also, their micellar network is concentration dependent

and it is expected that after sufficient dilution they may leave the surface. On the other

hand chemical and enzyme-catalyzed processes are the main forces which may lead to

degradation of stabilizers. The enzymatic degradation of such polymers depends on a

number of factors (apart from those related to the polymer chemical structure) like,

temperature, pH, electrolyte concentration, presence of enzyme inhibitors, presence of

other organic compounds (e.g., urea), cations (certain systems), and other co-solutes.

Thus, when the stearic hindrance by the stabilizers is minimized around colloidal particles

(e.g., solid lipid nano particles), it is the core of particle (lipid in case of SLN) which

interacts with body fluids. So, an attempt has been made here to study the preferential

adsorption of a protein over different solid lipids available. Bovine serum albumin (BSA)

has been chosen for study, considering the fact that it is the only protienous constituent of

simulated vaginal fluid (Owen and Katz, 1999) which is used widely in several in vitro

studies and is widely studied protein. Once lipid is without the coatings of stabilizers, it is

its inherent affinity of lipids with body fluid / proteins that allow it to remain dispersed



http://www.sciencedirect.com/science/article/pii/S0378517305004540

http://www.sciencedirect.com/science/article/pii/S0378517305004540

within body. Otherwise it may precipitate out. For the above hypothesis, following

experimental set up was run.

1.5 grams of selected lipids (lipids showing maximum drug solubility) were

passed through sieve number 80 and stored in desiccators. In a 15 ml glass syringe

(inverted) lipids were put by blocking the nozzle with a filter paper of same internal

diameter and putting a cap also. 10 ml of freshly prepared BSA solution (2 mg/ml) was

added into the syringe. The assembly was left undisturbed at room temperature (25°C) for

an hour. Then the cap was removed and samples were collected, samples were analyzed

by UV spectrophotometer (Shimadzu, UV 1601) using a calibration plot (R 2= 0.996)

made with BSA at 279 nm.

4d.1. B. On the basis of maximum crystal lattice space

After sorting out the lipids on the basis of preferential adsorption with bovine serum

albumin, we developed combinations of them to generate maximum lattice space within

the bulk. By this approach we may increase the drug loading in the colloidal particles. All

the lipids either single or in mixture (1:1 ratio) were dissolved in chloroform and then

were dried slowly. They were pulverized to get uniform size of mass. The samples (1000

mg) on XRD plates were rotated during data collection to reduce orientation effects of

particles. XRD patterns of all the samples were recorded between 29 = 8° and 88° at 35

kV and 30 mA, respectively. Spectra obtained were then analyzed for their physical states

(crystalline or amorphous) and also for the d values (inter-atomic distances) in different

solids.

4d.2 SELECTION OF SURFACTANT

A high surfactant concentration favors a lower particle size, a narrower particle size

distribution and a better long-term stability of lipid nanoparticles but simultaneously

increases the toxicological potential. Hence, a balance needs to be found to have sufficient

surfactant present to ensure a small particle size and a good physical stability of the carrier

system as well as avoiding free surfactant in the formulation as binding surfactants to the

surface of lipid nanoparticles markedly reduces the toxicological potential. In this section

we select only the type of surfactant, strength will be optimized in section 4d.3. To select



the surfactant (or combination), placebo SLNs were made and percentage transmittance

were determined. % Transmittance was also determined after 3 days to see the effect of

stabilizers on particle growth (size ripening). Stearic acid and compritol E ATO (1:1 ratio)

was taken as a lipidic phase. Method described in section 5b.3 was followed for the SLN

development. Organic aqueous phase ratio was taken 10:50. Stabilizers selected (in

strength of 4% ) were of high HLB values, considering the formation of o/w type of

emulsion.

4d.3. DEVELOPMENT OF SLN

The solid lipids (Stearic acid, Compritol 888, Compritol E ATO and their combinations)

were dissolved in small quantity of dichloromethane; bath sonicated for 2 minutes and

total volume was made up to 10 ml. It was considered as an organic phase. Aqueous phase

was made in such a manner that after addition of organic phase, total concentration of

surfactant follows the values shown in Table 31. Sodium taurocholate was taken as a co

surfactant to generate sufficient negative charge over the SLNs and was taken in a fixed

quantity (0.5 % w/v). Poloxomer 188 was taken as a stearic stabilizer in a variable

quantity (2-5% w/v). Organic phase was then added drop wise (with the help of syringe)

into aqueous surfactant solutions and simultaneously it was given high energy with the

help of probe sonicator for total 3 minutes with a pause of 10 seconds after every minute.

Ratio of organic to aqueous phase was taken 10:50. The colloidal suspension was then

filtered through Whatman filter papers and left for stirring (1500 rpm) for 4 hrs to remove

the chloroform. Higher stirring rates don’t significantly change the particle size, but

slightly improve the polydispersity index. The suspension was frozen for 24 h in a Lyph-

lock apparatus and then freeze dried (Dry winner, DW-8-85 Heto Holten, Denmark) for 12

h. Mannitol was (1% w/v) added as a cryoprotectant. A batch of optimized formulation ( T

10) was also freeze dried without cryoprotectant for different characterizations.

There were two main reasons to adopt this method for SLN production and

avoiding well established methods like high pressure homogenization, hot

homogenization and others. Increased temperature and use of higher amount of surfactant

allowed partitioning of drug into water phase and subsequent re-partitioning to the

organic phase; consequently the drug concentrates in the outer shell and/or on the surface



of the particle. The molecules that accumulate in the outer core are responsible for the

phenomena of ‘burst release’. Hence to have a more sustained action (and to accumulate

the molecule in core) of the final formulation we opted abovementioned method.

4d.4. CHARACTERIZATION OF SLN

SLN based on single lipids exhibits limited drug payloads and drug expulsion from the

crystal lattice as the lipids have a tendency to re-crystallize in due course of time. During

re-crystallization, the surface area of the particle would increase remarkably which add

instability to the colloidal system. Cryoprotectors are space holders which prevent the

contact between discrete lipid nanoparticles. Furthermore, they interact with the polar

head groups of the surfactants and serve as a kind of ‘pseudo hydration shell’ (Mobley

and Schreier, 1994). Following characterizations were performed for the developed

SLNs.

4d.4.1. Particle size and zeta potential (^ ) analysis

Particle size was determined using a photon correlation spectrometer (PCS; Zetasizer-

1000 HAS, Malvern Instruments, UK) based on the laser light scattering phenomenon,

which analyzes the fluctuations in light scattering. Freeze dried powders were dispersed

in deionized water and 0.1 N HCl in a drop-wise manner at 25°C under gentle shaking

and filtered through 0.22 ^m membrane filter in order to eliminate multi-scattering

phenomena and experimental errors. Average particle diameter ( J dm), polydispersity

index (p^) and zeta potentials (Z) were recorded. The values were calculated from

electrophoretic mobility using Henry’s equation:


Where, Ue = electrophoretic mobility

z = Zeta potential

8 = Dielectric constant

n = Viscosity

/ ( K a ) = Henry’s function

All measurements were done in triplicate using disposable polystyrene cuvettes (Malvern

Instruments, UK).


4d.4.2. Differential Scanning calorimetry

DSC was performed according to the method described in section 4a.5.4.

4d.4.3. Powder X ray diffraction

X ray diffraction study of SLN was performed according to the method described in

section 4a.5.5.

4d.4.4. Surface Morphology studies

Surface morphology of SLN was studied with the help of transmission electron

microscope (TEM) (TOPCON 002B) operating at 200 KV, capable of point to point

resolution. Properly diluted samples (1/100 in water) of samples were used for TEM

observations. A drop of diluted suspension of SLN was allowed to deposit directly on the

circular copper film grid and observed after drying. Combination of bright field imaging

at increasing magnification and of diffraction modes were used to determine the form and

size of the particles.

4d.4.5. % Entrapm ent efficiency

The % entrapment efficiency (% EE) of Itz into SLN was determined by ultra filtration

method using centrifugal filter tubes with a molecular weight cut-off of 30 kDa (Amicon

Ultra, Millipore, Ireland). The concentration of Itz in SLN and in the ultra-filtrate (free

drug) was analyzed by the method described in section 5a.1. Rt of Itz was observed at

8.25 min. The E.E. was calculated using the following equation:

T otal am o u n t of Itz -A m o u n t of free Itz% E E = --------------------------------------------------------X 100

T otal am o u n t of Itz

Possible lipid interferences with Itz were also investigated by

comparing the standard curves of drug alone and in the presence of lipids. The

differences observed between the standard curves were within the experimental error,

thus inferring that no lipid interference occurred.

4d.4.6. Determination of residual solvent content

The residual solvent content in samples were quantified by GC-MS system [Agilent

7890A series (Germany)] equipped with split-split less injector and CTC-PAL auto

sampler attached to an apolar HP-5MS capillary column (30 m x 0.25 mm i.d. and 0.25



^m film thickness) and fitted to a mass detector. Carrier gas flow rate (Helium) was

1ml/min, split: split less ratio 1:100, injector temperature was 70°C, detector temperature

250°C, while column temperature was kept at 60°C for 2 min followed by linearly

programming from 70 to 230 °C (at rate of 5°C/min), and then kept isothermally at 230

°C for 2 min. Transfer line was heated at 280 °C. Split ratio was kept at 1:100. Mass

spectra were acquired in EI mode (70 eV); in m/z range 30-400. The amount of sample

was injected through head space. The residual solvent of the formulation were identified

by comparison of their mass spectra to those from Wiley 275 and NIST/NBS libraries,

using different search engines.

4d.4.7. Lipid stability by GC-MS study

For successful formulation of drugs in SLN, the chemical stability of excipients used for

particle production is a prerequisite. Chemical analysis of lipids was performed by gas

chromatography using a GC-MS system [Agilent 7890A series (Germany)] equipped

with split-split less injector and CTC-PAL auto sampler as described previously

(Radomska et al., 1999). Determination was performed via the corresponding methyl

esters of the fatty acids of the lipids. Transmethylation of the lipids was made according

to the method described (Garces and Mancha, 1993). In order to separate the lipid from

the SLN dispersions, the formulations were centrifuged at 17,000 rpm for 30 min. 50 mg

of the obtained lipid phase were mixed with methylating mixture containing

methanol/toluene/dimethoxypropan/H2SO4 (39:20:5:2, by volume) in tubes with teflon

caps. The quantity of methylating liquid was 3.3 ml, heptane was added to a total volume

of 5 ml. This mixture was incubated in a water bath at 80 °C for 90 min. The tubes were

cooled to room temperature and shaken, two phases were formed. The upper phase

contained the fatty acid methyl esters. The fatty acid composition was analyzed using a

silica capillary column with high polarity (Supelkowax™10, 30 m ^0.32 mm ID, 0.25 ^m

film thickness, Supelco, Bellefonte, USA). It operated at a constant temperature. The

temperature of the injector and the detector (FID) were 200° C and 220 °C, respectively.

The carrier gas was helium at the flow rate of 1.5 ml/min.



4 d . 5 D E V E L O P M E N T O F M U C O A D H E S I V E A N D T H E R M O S E N S I T I V E G E L

I N C O R P O R A T I N G S L N

It is widely reported that amongst the various vaginal dosage forms patients are known to

better tolerate gels than than inserts or ointments (Edsman et al., 1998). Additionally, it

is advisable to have a prolonged effect which could be obtained via mucoadhesion. In

order to substantially enhance the sustain drug release profile; SLNs were further

converted into gels. Carbopols are known to have mucoadhesive properties and have

been used in the formulation of vaginal delivery systems (Wang and Lee, 2002). Also

carbopol is reported to have highest bioadhesion potential at vaginal pH (Repka and

McGinity, 2001). Poloxamer 407 (Pluronic F127) has been used as a thermo-sensitive

polymer which shows excellent water solubility, good drug release characteristics, low

toxicity and irritation, and has compatibility with other excipients (Zaki et al., 2007).

Thermo sensitive and mucoadhesive gel formulations were prepared

according to the cold method (Choi et al., 1998). Briefly, mucoadhesive polymer (CP

934) (0.2% w/v) was slowly added to citrate phosphate buffer (0.1 M, pH 4.0) at 4° C

with gentle mixing for an hour and allowed to hydrate. Then it was centrifuged at 3000

rpm for 15 min to remove the air bubbles. Care was taken to allow the minimum air

bubbles level during handling. Pluronic® F 127 (15%, 18% & 20%, w/v) was then added

to CP 934 solution and allowed to dissolve overnight at 4° C. Optimized SLN (T 10) was

dispersed in Pluronic solution in such a way that total strength of Itz in gel remains 1%

w/v. Developed gels were studied for following parameters;

4 d . 6 M E A S U R E M E N T O F S O L - G E L T R A N S I T I O N T E M P E R A T U R E ( T sol-g el )

In a 20 ml transparent vial containing a magnetic bar in 5 ml gel was placed in a water

bath, which was heated at a rate of 2° C/min with constant (150 rpm) stirring. When the

magnetic bar stopped moving due to gelation, the temperature displayed on the thermistor

was determined as the gelation temperature.

4 d . 7 I N V IT R O D R U G R E L E A S E K I N E T I C S

The volume of the vaginal fluid might be much smaller than the volume of buffer

medium used in vitro. Moreover, the fixed stirring conditions used in vitro may not



exactly reflect the vaginal flux of fluids. Given these differences between in vitro test

conditions and in vivo vaginal conditions, we cannot directly apply the in vitro

dissolution result to the in vivo situation. However, despite the limits of in vitro

dissolution profiles, we may at least extrapolate and predict the relative rates of

dissolution in vivo based on the controlled in vitro data. Dissolution studies were

performed according to our earlier reported method (Chopra et al., 2007). In brief,

Dissolution studies were performed using the USP XXVIII, paddle-rotating method

(Electrolab dissolution tester, Electrolab, India) at 37° C ± 0.5° C and 75 rpm using two

dissolution media: phosphate-buffered saline pH 4.5 (PBS) containing 1% Tween 80. The

gels were first packed into a dialysis bag and placed into the dissolution media.

Dissolution studies were carried out in triplicate, maintaining the sink conditions for all

the formulations. The level of Itz in the media was estimated using the reported HPLC

method in section 4c.1. The cumulative % drug release was calculated. Possible

mechanism of drug release from mucoadhesive polyherbal gels was estimated using

different release models.

4d.8 IN VITRO BIOADHESION STUDY

Mucoadhesion studies were carried out by slightly modifying a published method

(Nakamura et al., 1996). Briefly, an agar plate (1%, w/w) was prepared in pH 4.5 citrate-

phosphate buffer. Small amount of gel was placed at the center of plate. After 5 min, the

agar plate was attached to a USP disintegration test (model 1901, Electronics India,

India) apparatus vertically and moved up and down (10±1 cycle per minute) in pH 4.5

citrate phosphate buffer at 37±1 °C. The sample on the plate was immersed into the

solution at the lowest point and was out of the solution at the highest point. The residence

time of the gel on the plate was noted visually.

4d.9. EX VIVO DRUG PERMEATION STUDY










was filled with SVF and stirred with a magnetic rotor at a speed of 100 rpm in hot air

oven maintaining temperature at 37±1°C. The whole media was replaced with a fresh one

after every 30 min to stabilize it. After running the 6 cycle of stabilization, 1 ml of

sample (200 ^g/ml) solutions (A, B, C and D) in acetate buffer (pH 4.5) were placed into

donor compartment. The receptor compartment was having 20 ml phosphate buffer (pH

4.5) solution. The samples were withdrawn at regular interval (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 12, 14, 16 and 24 h), filtered through 0.45 ^m membrane filter and analyzed for drug

content by the method described in 4c.1. The cumulative amount of drug permeated

through the membrane (^g/cm2) was plotted as a function of time (t) for each

formulation. All the permeability parameters were determined according to the methods

reported in literature (Shakeel et al., 2007).

4d.10. IN VITRO CELL TOXICITY STUDIES OF GEL (G 4)

The cytotoxicity of gel was evaluated by 3-[4-5-dimethylthiazol-2-4]-2, 5-

diphenyltetrazolium bromide (MTT) assay using HeLa-S3 cell lines. HeLa-S3 cell lines

obtained from National Center for Cell Science (NCCS, Pune) and grown under 5% CO2

in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2.0 mM L-glutamine,

and 1.5 mg/mL NaHCO3 . Exponentially growing HeLa epithelial cells are seeded into

96-well plate containing RPMI 1640 medium at a density of 106 cells/well. The cells are

allowed to grow for 24 h at 37°C prior exposure to vaginal gel. On the day of treatment,

RPMI 1640 medium is replaced with fresh medium. Test samples were placed on top of

the cells and allowed to incubate for 24 h at 37°C. After incubation, cells were washed

with phosphate-buffered saline (PBS) to remove the formulation, and 100 ^l of fresh

medium with 10 ^l of MTT solution (5 mg/mL in 0.1 M PBS, pH 7.2) was added to each

well. Cells containing only medium and MTT were considered as negative controls

(without formulation treated). Plates were then incubated for 4 h at 37°C in a CO2

incubator. After incubation, MTT reaction medium was discarded and cells were washed

with PBS. Then, 100 ^l DMSO was added in each well to dissolve blue formazan



crystals, and optical density was measured at 570 nm with a 96-well multiscanner ELISA

reader with DMSO serving as blank. The percent viability was calculated by the

following formula:

OD of test sample% Cell viability = — — -̂-------- — X 10 0

OD of control sample

4d.11. PRECLINICAL EVALUATION OF GELS

4d.11. A. Bioadhesion and irritation tests

In vivo bioadhesion studies were carried out after taking permission from Institutional

Animal Ethical Committee of Jamia Hamdard (proposal number 712). Experimentation

was carried out according to the earlier reported method (Chopra, Motwani et al. 2007).

Briefly, 0.5 g of the gel was mixed with 0.4% trypan blue dye. Resulting dark blue

colored formulation was inserted into the rat vagina with a 2 ml plastic syringe (without

needle). For each group three rats were taken. After 24 h of administration, the rats were

sacrificed and the retention of bioadhesive formulation at the administration site was

visualized by dye intensity. For control group simulated vaginal fluid (pH 4.5) was taken.

Rats have simple, cuboidal or columnar epithelium which is highly

sensitive to mucosal irritation when compared to human vagina. Adult wistar rats (3

months old; 150-180 g) were used for the in vivo toxicity studies. Tap water and food

pellets were available ad libitum throughout the study. Room temperature was

maintained at 25±2°C with relative humidity 50±5%. Strength of each group was taken

three. The rats were treated once daily with 0.5 g of gel for 14 days. Prior to every

administration, rats were evaluated for vaginal or vulval irritation, discharge or bleeding

from vagina. After the completion of 14 days, study period, they were scored for possible

erythema. The mean erythemal scores were recorded (ranging from 0 to 4) depending on

the degree of erythema as follows: no erythema = 0, slight erythema (barely perceptible-

light pink) = 1, moderate erythema (dark pink) = 2, moderate to severe erythema (light

red) = 3, and severe erythema (extreme redness) = 4. Then, they were sacrificed and

transverse section of the vaginal tissue was examined by experienced pathologist for the

severity of epithelial loss and atrophy. Control group was given same volume of SVF.



4d.11. B. In vivo antifungal studies

A rat infection model was used to study the final formulation in vivo (De Bernardis et al.,

1997) after getting approval from Institutional animal ethical committee of Jamia

Hamdard (proposal number 742). Eighteen female wistar rats were divided in groups of

three for the application of optimized gel, Gynazole (2% w/w) and control. Six days prior

to inoculation of infection, all animals were maintained under pseudo estrus by


albicans (108 cells per ml of saline) were inoculated. Then after 1 g of G4 (1% w/w) and

0.5 g of Gynazole (2% w/w butoconazole nitrate) were applied. Control was not having

any dosage form. During specified time intervals 1 ^L of the fluid from vaginal cavity

was withdrawn, diluted and spread on Sabouraud agar containing chloramphenicol (50

mg/ml). It was then incubated at 32°C in the BOD incubator (LHC-78-Labhospmake,

India) for 48 hrs and CFUs were counted. A rat was considered infected when at least 1

CFU was present in the vaginal sample (i.e., a count of >103 CFU/ml). A graph was

plotted between time (days) and no. of CFU present in vaginal fluid.

4d.12. ACCELERATED STABILITY STUDIES FOR DETERMINATION OF

SHELF LIFE

It was carried out for a period of three months. The optimized thermo sensitive and

mucoadhesive gel (G 4) was evaluated for different parameters like physical appearance,

pH, drug content uniformity, in vitro bioadhesion, antimicrobial efficacy and in vitro

drug release profile. Since the temperature chosen for the study was high enough to make

its consistency rigid, the accelerated stability studies were carried out by storing the

samples (15 g) in collapsible aluminum tube at 40° ± 2°C, 50° ± 2°C and 60° ± 2°C.

Tubes were also stored at 2-8°C as control samples. Samples were withdrawn at intervals

of initial (zero day), 30 days, 60 days, and 90 days. For proper withdrawal of samples,

tube were cut in a crosswise manner, opened and observed visually and for analysis. For

the estimation of drug in the sample, HPTLC method described in section 4.3 was used.

The logarithms of percent of drug remained in gels was plotted

against time, slope of which was used to calculate degradation rate constant using the

formula;



S lo p e = - K / 2 . 3 0 3 , where K = degradation rate constant.

An Arrhenius plot was drawn for the formulation plotting logarithms of K values against

inverse of the absolute temperatures and straight lines were obtained. K25 was calculated

using the above graph. Shelf life (at room temperature) of the formulation was calculated

using below mentioned formula;

. _ 0 . 1 0 5 4 /

t io % - / K 2 5


1 . SELECTION OF LIPIDS FOR DEVELOPMENT OF SLN

A. INTERACTION W ITH BOVINE SERUM ALBUMIN

Result obtained with the study is shown in Fig. 49. Total volume of sample recovered was

less than the volume of BSA used. Hence, the amount of BSA adsorbed by the 1.5 gms of

lipid was determined which was in the order of Compritol E ATO > Compritol 888 ATO

> Stearic acid > Precirol Cetyl palmitate > Gelucire (39/01) > Gelucire (50/13). Thus,

lipids selected for further studies were Compritol E ATO, Compritol 888 ATO, and

Stearic acid.

Preferential interaction of BSA with lipidic colloidal

particles is well supported by literature (Almeida and Souto, 2007). Like, a double

hydrogen-bonding mechanism for stearic acid binding to BSA has been described in

literature (Ge et al., 1990). The ability of the acid to form two hydrogen bonds apparently

fixes it more rigidly in the protein, preventing rotation about either single hydrogen bond.

A double-hydrogen bonding mechanism is most consistent with the formation of a salt

bridge between the negatively charged carboxylate of the acid and either a positively

charged guanidino group of arginine, or the positively charged omega-amino groups of

two lysine residues. On the other hand, Compritol 888 ATO (glyceryl behenate) is

chemically a mixture of mono-, di- and triglycerides of behenic acid (C 22) behenic acid. It

is also expected to interact with BSA by formation of some hydrogen bonds.




B. ON THE BASIS OF MAXIMUM CRYSTAL LATTICE SPACE (d value)

Slow evaporation of chloroform provided enough time to develop energy minimized and

stable lattice arrangement. All the processed lipids showed maximum counts near 21°

(29). Using Bragg's equation (nX = 2d Sin 9), d values for all the lipid were calculated at

an angle which shows maximum counts (Table 30).

XRD spectra obtained from the analysis are shown in Fig. 50. Thus

it is clear that the powders are not having long range of crystalline pattern. In a solid

lipidic carrier, it is expected that a solid with less orders in lattice could accommodate

maximum amount of drug. On the other hand maximum d value was obtained in case of

Compritol 888 + Compritol E ATO (4.3581) and Stearic acid + Compritol 888 (4.3502).

Thus, we might expect that these combinations of lipids would accommodate larger

amount of drugs.

Table 30: X-ray diffraction data of different lipids and its combinations. Use of mixture has been found to impart positive effects on the inter-atomic distance of solid lipid.

Lipids d value Angle (20)

Stearic acid (SA) 4.2203 21.025

Compritol 888 (CT 888) 4.2099 21.083

Compritol E ATO (CT E ATO) 4.2007 21.128

Stearic acid + Compritol 888 4.3502 20.398

Stearic acid + Compritol E ATO 4.3209 20.533

Compritol 888 + Compritol E ATO 4.3581 20.353



2.664

CGmpritol Compritol CetylEA TO BBBATO palmitate

11.932

IStearic Precirol

1.6921.3BB

G e lu c ire G e lu c ire(39/01) (50/13)

Figure 49: Differential interaction of BSA with different lipids available. Differential affinity is observed in mg of BSA adsorbed per gm of lipid. Results are given on average scale, omitting standard deviation.

12 16 20 24 28 32 35 40 44 48 52 56 60 64 68 72 76 80 84 88

Aogle (2 theta)

Figure 50: X ray powder diffraction of lipids and its combinations. All the spectra followed

same fashion except the values of % intensity.



2. SELECTION OF SURFACTANT

A nanosuspension with high % Transmittance may be correlated with smaller particle size

and maximum adsorption of surfactants over colloidal particles. Maximum transmittance

was observed with Poloxamers, 82.6 % with Poloxamer 407 and 81.3% Poloxamer 188.

But for further studies a Smix (5% P 188 + 0.5% Na Taurocholate) was chosen because the

combination gave negligible decrease in % Transmittance after 3 days (Figure 51).

Figure 51: % transmittance of a model SLN suspension using different surfactant (s). Values were taken just after preparation and after 3 days.

3. CHARACTERIZATION OF SLN

A. PARTICLE SIZE AND ZETA POTENTIAL (^ ) ANALYSIS

Obtained zeta potentials, exhibit sufficient stability of the SLNs. Lipid particles are

generally negatively charged (Schwarz and Mehnert, 1999) and colloidal system is found

to be stable when ^ (> |30 | mV ) due to electrostatic repulsion (Levy et al., 1994). This is

valid only in case of sole electrostatic stabilization. Electrostatic stabilization and stearic

stabilization effects are additive. Hence for the development of SLN we have taken a

combination of surfactants (Poloxamer and sodium taurocholate). It has also been

suggested that without steric stabilization, a colloidal system would not be stable e ven if ^


value is greater than 30 mV (Tadros and Vincent, 1980). A certain thickness of polymer

layer is necessary (> 10 nm) for efficient and complete stearic stabilization (Washington,

C., 1996). According to the theory of DLVO, system could be regarded as stable if the

electrostatic repulsion dominated the attractive Van der Waals forces. Non ionic

surfactants contribute to the stability by offering stearic stabilization while ionic

surfactants contribute by generation of electric double layer. Increasing amount of stearic

stabilizer (P 188) was also found to increasing zeta potential values (Table 31). Non ionic

surfactants couldn’t ionize like ionic surfactants but demonstrated its role in zeta potential.

The reason might be due to molecular polarization and the adsorption of emulsifier

molecule on the charge in water, it was absorbed to the emulsifier layer of particle/water

interface and electric double layer similar to ionic was found. Like in the case of P 188,

less polar poly (propylene oxide) chain were dissolved and segregated into a hydrophobic

micelle core surrounded by a soft “brush” of highly hydrated flexible poly (ethylene

oxide) chain. Average size of the particles and their size distribution are also given in

Table 31. Mean particle size was bigger when stearic stabilizer was used alone. In the

given set of experiments, we achieved a size range of 250.2 - 546.9 nm which is

acceptable in case of topical drug delivery. From the table it is evident that with the

increment of surfactant, size of the particle is falling sharply. Using lipidic mixture had

also a positive effect on decreasing the size of the particle. The difference in polarity of

the molecules (stearic acid Vs Compritol) should have a minor influence on the particle

size as the interfacial phenomena are supposed to be dominated by the properties of the

emulsifier.




Table 31: Details of different trials and evaluation of SLNs formed in terms of % entrapment efficiency (% EE), mean particle size, particle distribution index (PDI) and change in Gibb’s free energy (AG298sol) at room temperature.

Trials Lipid (gms) Surfactant (% w/v) % EE % Drug Mean Zeta PDI

content particle size

(nm)

potential(mV)

T 1 CT E ATO 5% Plxr188 + 0.5% Sod.Trchl 84.55± 0.08 17.68 ± 0.01 250.2 -41.3±1.8 0.292

T 2 CT E ATO 3% Plxr188 + 0.5% Sod.Trchl 83.23 ± 0.24 17.68 ± 0.03 285.3 -37.1±2.6 0.264

T 3 CT 888 3 % Plxr 188 + 0.5% Sod.Trchl 83.91± 1.03 17.92 ± 0.24 503.1 -36.6±2.2 0.538

T 4 CT 888 5 % Plxr 188 + 0.5% Sod.Trchl 81.21 ± 0.62 17.13 ± 0.67 467.6 -39.8±0.8 0.468

T 5 CT 888 + CT E ATO

3% Plxr 188 + 0.5% Sod.Trchl 87.38 ± 0.31 19.74 ± 0.07 306.4 -37.4±1.7 0.295

T 6 CT 888 + CT E ATO

4% Plxr 188 + 0.5% Sod.Trchl 86.90 ± 0.32 18.76 ± 0.07 309.1 -42.5±0.9 0.466

T 7 SA + CT E ATO 2% Plxr 188 + 0.5% Sod.Trchl 88.01 ± 0.83 20.46 ± 0.20 546.9 -35.4±1.4 0.561



T 10 SA + CT 888 3% Plxr 188 + 0.5% Sod.Trchl 90.63 ± 0.48 20.59 ± 0.11 331.4 -32.6±2.6 0.473




B. DIFFERENTIAL SCANNING CALORIMETRY

DSC thermograms of bulk lipids (single or mixture) and drugs are given in Fig. 52 and of

different SLNs are given in Fig. 53. Itz was showing a sharp melting endotherm at

170.408°C. Melting enthalpy (AH), melting point, crystallinty index and peak width of

bulk materials and SLNs are given Table 32.

It was evident from the thermograms that Itz is not in crystalline state

rather amorphous state whether in core or outer shell of the particle. The amorphous form

of a drug has a higher thermodynamic and chemical potential (Weuts et al., 2003) while

the crystalline state has the lowest total energy content. The higher thermodynamic

activity of the drug can produce supersaturated solutions (Betageri and Makarla, 1995)

and providing an opportunity to enhance solubility. One might also expect that the

complete absence of long-range three-dimensional inter-molecular order associated with

any material might significantly modify the mechanical and physical properties also

(Hancock and Parks, 2000).

Organic substances usually show a melting range. An increased

melting range could be correlated with impurities or less ordered crystals but the various

instrumental analyses used in the current work have ensured lowest levels of impurities. A

manual method of measuring melting range is Peak width (i.e. difference between onset

and maximum). Peak width of SLNs were found to be higher (2 or 3 times) than any of

the bulk material (Itz of bulk lipids). Melting enthalpy (AH) is another characteristic of the

crystal order if the impurities influence could be ignored. For the less ordered crystal or

amorphous state, the melt of the substance does not require or just requires less energy

than the perfect crystalline substance which needed to overcome the lattice force. As a

result, the higher melting enthalpy values should suggest higher ordered lattice

arrangement and vice versa. In this perspective also, the AH value of SLNs were smaller

than the bulk materials from which they have developed.

Another phenomenon we could notice from the

thermograms is reduction of melting temperature of colloidal lipid particles compared to

bulk material which has been attributed inter alia to their small size and the presence of

surfactants. It can be discerned by Gibbs-Thomsan equation which is also a derived from

of Kelvin equation. It reflects the decrease in melting temperature for a particle of given




size compared to the bulk material and becomes particularly more pronounced in the

lower nanometer size range.

Figure 52: Representative differential scanning calorimetry profiles of Itz, lipids and its combinations. Thermograms obtained are changed into colors for better illustrations.

Figure 53: Representative differential scanning calorimetry profiles of SLNs.



Where, T Melting temperature of a particle with radius rT0 Melting temperature of the bulk material at the same external pressureysl Interfacial tension at the solid-liquid interfaceVs Specific volume of the solidAHfus Specific heat of fusion

Broadening of peak possibly results due to presence of particle

fractions with variable size which melt at different temperatures. The loading of the drug

did not provoke any considerable effect in the lipid matrix thermal behavior under these

experimental conditions.

In order to compare crystallinity between the developed formulations, a

useful parameter is % crystallinity index which is defined as the percentage of the lipid

matrix that has recrystallized during storage time. Following equation is used to calculate

% CI:

EnthalpySLN

Thus we see that highest crystallinity was developed by SLN

(Stearic acid + Compritol E ATO) with respect to its bulk lipid while lowest crystallinity

was developed by SLN (Compritol 888) with respect to its bulk lipid.

Although DSC is able to monitor and quantify even minute thermal

events in the sample (depending on the sensitivity of the instrument) and to identify the

temperatures at which these events occur. But it does not directly reveal the cause of a

thermal event. The exact nature of the thermal transitions has to be determined with

complementary methods such as microscopic observations, X-ray diffraction or

spectroscopic techniques to distinguish, for example, between melting, polymorphic

transitions, loss of water from hydrates or decomposition of the substance. Previous

experimentations have also shown that these observations occur irrespective of the heating

and cooling rates used for the DSC experiments.



C . P O W D E R X R A Y D I F F R A C T I O N

Such a plot (29 Vs % Intensity) can be considered a fingerprint of the crystal structure and

to differentiate with different crystallographic status of the system. One peak will be

exhibited for all repeating planes with the same spacing. Itz powder was crystalline in

nature (Fig. 23) and showing characteristic peaks at 14.1°, 17.8°, 20.6°, 23.2°, 25.7° and

26.5°. Overlapping images of different bulk lipids are shown in Fig. 55 . Prepared SLNs

were not having characteristic peaks of Itz except some diminished peaks around 21° and

23°. Thus corroborating the results with DSC we may say that Itz is present in amorphous

state in SLN.

Table 32: Data obtained by differential scanning calorimetry. AH and Peak were obtained by direct software analysis while Peak width and Crystallinity Index are derivative, obtained manually.

Peak(°C)

Peakwidth

Samples AH (j/g) Crystallinity Index (% )

Stearic acid 352.163 65.048 6 .2 210 0

Compritol 888 243.249 70.954 4.3210 0

Compritol E ATO 245.127 70.235 4.4010 0

Stearic acid + Compritol 888 (1:1) 211.475 68.352 5.9210 0

Stearic acid + Compritol E ATO (1:1) 192.384 69.043 6 .1 0 10 0

Compritol 888 + Compritol E ATO (1:1) 167.253 70.624 3.9610 0

Itz 105.063 170.408 5.8110 0

SLN (Compritol E ATO) 102.421 66.219 14.26 41.78

SLN (Compritol 8 8 8 ) 45.232 69.246 15.42 18.59

SLN (Compritol E ATO + Compritol 8 8 8 )

56.768 68.983 14.47 33.94

SLN (Stearic acid + Compritol 8 8 8 ) 62.422 69.069 10.75 29.51

SLN (Stearic acid + Compritol E ATO) 81.452 70.196 11.50 42.33


Production of nanoparticles also reduces the degree of crystallinity (Radomska-

Soukharev, A., 2007). The reduction in crystallinity is due to the partial formation of

lower energy lipid modifications. In addition, surfactants distributed to the melted lipid

phase during the production process can also distort crystallization. Broad hump around

19° is present in all the SLNs which may be the shifted peak of lipids as seen in Fig. 55.

Thus we see that lipids in all the cases have attained their small ranged lattice pattern.

Minutely considering the diffractogram all five images of SLN was divided into three

groups. Diffractogram of SLN of SA + CT 888 was matching with SA + CT E ATO,

diffractogram of CT 888 was similar with CT E ATO while diffractogram of CT 888 + CT

E ATO was distinct from others. Thus we see that with different compositions of lipids,

different lattice pattern was developed.

D . S U R F A C E M O R P H O L O G Y S T U D I E S ( T E M )

Result of TEM investigation is shown in Figure 54. Shape of the lipidic nanoparticles is

appearing round and homogenous. Particle size in the image is ranging from 196 to 395

nm. More precise information on particle size and size distribution are given in section

Table 31.

E . % E N T R A P M E N T E F F I C I E N C Y

The outcome of % EE was in accordance with the results obtained from XRD and DSC

studies i.e. as we break the crystalline lattice of solid by developing a mixture, we could

accommodate more amount of drug. Highest d values were noted with mixtures of SA: CT

E ATO and SA: CT 888 which translated into maximum entrapment of drug in the lattice.

Enthalpy (AH), which is an indirect estimation of

crystallinity of the material, was lowest in case of these two mixtures. Thus, we may

funnel out a conclusion that by above given method we may develop substantial

imperfections in a carrier which results into higher drug loading.

Considering the different parameters T 10 was selected as

an optimized SLN.




4 . D E T E R M I N A T I O N O F R E S I D U A L S O L V E N T C O N T E N T

Dichloromethane was found to be absent in formulations. Recommended limit of ICH

Q3C guideline (ICH, 1997) in case of Dichloromethane is 6 mg/day. And the residual

amount limit in USP XXIII is 6 mg/day.

Figure 54: TEM micrograph of SLN.

Figure 55: Overlaid XRD patterns of Itz and SLNs



5 . L I P I D S T A B I L I T Y B Y G C - M S S T U D Y

After keeping the SLN at room temperature for 3 months, lipid stability was

performed. Data obtained are given in Table 33. There is no major decomposition in any

case. This could be attributed to the fact that surfactant concentration was low and

incubation time was small.

Table 33: The percentage of the lipid content in SLN formulations (initially and after 3 months) of the incubation at 25 °C.

F o r m u l a t i o n s

T 1T 2T 3T 4T 5T 6T 7T 8T 9

T 1 0T 1 1T 1 2

L i p i d c o n t e n t ( i n i t i a l l y ) i n %

99.32

100.0

98.16

97.3

96.42

98.44

100.0

96.7

98.6

99.2

100.0

100.0

L i p i d c o n t e n t ( a f t e r 3 m o n t h s ) i n %

98.6

99.3

96.43

96.7

96.2

97.32

98.7

95.2

96.4

97.5

98.5

97.6

6 . C H A R A C T E R I Z A T I O N O F G E L

A . M E A S U R E M E N T O F S O L - G E L T R A N S I T I O N T E M P E R A T U R E ( T SOL-GEL)

The gelation temperature is defined as the point where the elasticity modulus is half way

between the values for the solution and for the gel. Carbopol has been reported to interact

strongly with low molecular weight drugs and causes precipitation (Blanco-Fuente et al.,

2002). However, the physicochemical status of drug or SLN may not affect gelling

temperature significantly. The drug was entrapped inside the lipid core and hence not

available for any interaction. Different gelation temperatures are given in Table 34. With

increase of fraction of P 407, gelation temperature was falling down. Even at higher

concentration it may happen at 4° C. Gelation phenomenon is a result of body centered

cubic packing of spherical micelles. Temperature plays an important role in the micelle



formation through temperature dependent hydration of the ethylene oxide units. Water is a

good solvent for PEO as well as PPO chains of polymer at low temperatures. However, at

higher temperature the solubility of PPO is reduced and micelle formation occurs. At low

temperatures in aqueous solutions, a hydration layer surrounds P 407 molecules. However,

when the temperature is raised, the hydrophilic chains of the copolymer become

desolvated as a result of the breakage of the hydrogen bonds that had been established

between the solvent and these chains. This phenomenon favors hydrophobic interactions

among the polyoxypropylene domains, and leads to gel formation. Aqueous solutions of

poloxamers are stable in the presence of acids, alkalis, and ions (Escobar-Chavez et al.,

2006) so it may also be expected to form gel in vaginal milieu.

Table 34: Gelation temperature of different compositions of gel.

C o d e

G 1

G 2

G 3

G 4

C o m p o n e n t s ( % w / v )

0.2% CP 934, 15% P 407 (Placebo)

Itz-SLN (1% w/v), 0.2% CP 934, 15% P 407

Itz-SLN (1% w/v), 0.2% CP 934, 18% P 407

Itz-SLN (1% w/v), 0.2% CP 934, 20% P 407

G e l l i n g t e m p e r a t u r e ( ° C )

41

40

38

35

At low pH (about pH 3) the carbopol chains exist in a spiral

coiled form, exhibiting a relatively low viscosity. With the progression of pH, the

carboxyl groups of the Carbopol become ionized, causing an increased repulsion of

negative charges. It leads the molecular structure to unwind and a gradual rise in viscosity.

B . I N V I T R O D R U G R E L E A S E K I N E T I C S

The release behavior (Figure 56) could be helpful in predicting the gel’s performance in

the vaginal milieu. Highest release (72.4% in 20 hrs) was achieved by G2, followed by G3

(66.6% in 20 hrs) and then G4 (62.2% in 20 hrs). A faster release of Itz expected in vivo

because of the presence of salts and proteins. Error bars have been removed from the

figure to make the data more understandable. All the three formulations were showing a

sustained release profile. One unexpected phenomena observed in the formulations were

an initial burst release, which was not expected considering the presence of drug in solid

lipidic core and then dispersed into gel matrix. Since we had allowed stirring the final



formulation for hours, some amount of drug must have oozed out from the lipidic core and

dispersed in gel matrix. So, during release study, this fraction of drug was responsible for

burst release. With progression of time, all the formulations achieved a sustained release

profile. Concentration of polymer was also found to have role in release profile. With

increasing polymer (P 407) concentration (15-18 %w/w), drug release was found to be

decreasing. More concentrated gels were reported to dissolve at a slower rate than less

concentrated ones because of the decreased water diffusion through the gel. Dissolution of

polymer is actually a controlling factor in drug release but it is not the only factor also,

because the surface is eroding at a constant rate. It is actually a two step process. Other

than burst release, the drug molecule first oozes out from the solid lipid core then diffuse

through gel matrix.

Figure 56: In vitro drug release profile of Gels (G2, G3 and G4). Bar graphs from the figure have been omitted considering the clarity of the results.

Hence considering the gelation temperature and In vitro drug

release, G4 was chosen for further evaluation.

7 . I N V I T R O C E L L T O X I C I T Y S T U D I E S O F G E L ( G 4 )Fig. 57 shows the cellular viability of HeLa-S3 cells, which was investigated over various

concentrations of Itz gel in PBS. Different concentrations of G4 did not show any

cytotoxicity except at a concentration of 1000 ^g/ml, where cell viability decreased by

about 7%. Thus, we may conclude that neither Itz molecule nor excipients used were

having any cytotoxic effect on vaginal tissues.



Figure 57: Percentage viability of HeLa cells against various concentrations of G4 and

placebo.

8 . P R E C L I N I C A L E V A L U A T I O N O F G E L ( G 4 )A . B I O A D H E S I O N A N D I R R I T A T I O N T E S T S

Intensity of dye in animals containing optimized gel (G 4) was much higher than control. It

shows the bioadhesive potential of the formulation. The basic components of the mucus

layers are mucin glycoproteins with oligosaccaride side chains. Sialic acids are located at

the terminal ends of the oligosaccharide chains. Since the pKa of sialic acid is 2.6, the

mucin network carries a substantial negative charge at physiological vaginal pH of pH 4 -

5. This charged portion interacts with the ionic components of the gel and results into

bioadhesion.

Treated (A) Control (B)Figure 58: Morphology of vaginal tissues after application of mucoadhesive thermo sensitive gels. The vaginal tissues of the treated rats (A) and control (B) were isolated, fixed in 10% neutral carbonated-buffered formaldehyde, embedded in paraffin, and cut into slices. After hematoxylin-eosin staining, the slices were observed under a light microscope (X100).



Optimized gel (G 4) was also found without any sign of toxicity. On a 0 - 4 scale basis, it

was given the score zero, considering the negligible signs of irritation. Even after

pathological examinations, it was found to be safe (Figure 58a & 58b).

B . I n v iv o a n t i f u n g a l s t u d i e s

A single dose of developed gel brought the microbial count to a negligible level after 21

days (Figure 59). But the marketed formulation was not sufficient to minimize the

infection after a single dose. Hence, a sustained release property was also observed in

vivo. Kinetics of infection was little different in initial days. Marketed formulation was

able to cut down the infection at a higher pace. Reason behind may be the drug release

characteristics. SLN was not having a burst release property as the Itz molecules was

supposed to be present in the core of particle.

Figure 59: Outcome of vaginal infection by C. albicans in oophorectomized, Estradiol-treated rats after first infections with 108 C. albicans cells. Each curve represents the mean of the fungal CFU of six rats.

9 . A C C E L E R A T E D S T A B I L I T Y S T U D I E S F O R D E T E R M I N A T I O N O F S H E L F L I F E

After exposure to variable heat conditions and humidity, no changes in physical

appearance, pH and HPTLC chromatograms were observed, indicating the physical and

chemical stability of the sample. Little decrease in viscosity at higher temperature (60°C)



was observed but it regained the viscosity after keeping at room temperature. Furthermore

the gels passed the antimicrobial tests after storage in accelerated conditions for 90 days.

Data obtained from the degradation kinetics were further

subjected to fit in Arrhenius plot (Fig. 60 and 61). The degradation rate constant (at 25°C)

was found to be 3.696 x 10-4. Amount of drug remained after 90 days at 60°C was about

89%. Furthermore, shelf life predicted for the formulation (G 4) was 0.78 years.

T i m e i n d a y s

Figure 60: Degradation plots of optimized formulation at different accelerated conditions.

Figure 61: Arrhenius plot for the degradation of optimized formulation (G4).




An analysis and understanding of the available treatment stratagems for various vaginal

afflictions led us to devising a model formulation based on strategies to overcome the

physiological challenges offered by the target site. The purported formulation bearing

SLNs loaded with Itz offers to assimilate itself in the vaginal milieu with prolonged muco-

retention, high tolerability and enhanced antifungal efficacy. Some new techniques of

excipients selection have been also introduced in this study.



4e. Formulation development of Novel vaginal dosage form using

Herbal component, Tea tree oil (Melaleuca alternifolia) - a

thermosensitive gel incorporating nanoemulsion


4 e . D E V E L O P M E N T O F T H E R M O - S E N S I T I V E M U C O A D H E S I V E G E L

C O N T A I N I N G S Y N E R G I S T I C N A N O E M U L S I O N

The dual loading of Itz and tea tree oil in a single formulation seems promising as it would

elaborate the microbial coverage. Despite being low solubility of Itz in Tea tree oil, a

homogenous, transparent and stable solution of both was created by co-solvency using

chloroform. Complete removal of chloroform was authenticated by GC-MS and the oil

solution was used in the development of nanoemulsion which was further translated into a

thermo-sensitive and mucoadhesive gel.

4 e . 1 D E V E L O P M E N T A N D O P T I M I Z A T I O N O F N E

4 e . 1 . 1 S C R E E N I N G O F A N O P T I M U M R A T I O F O R O I L m ix

Variable and little excess quantity of Itz was dissolved in a fixed quantity of CHCl3 and

then a constant composition (30% CHCl3: TTO 70% ) was made in volumetric flask. All

the flasks were left at room temperature (25±1°C) with continuous shaking for three days.

Total volume was kept constant with addition of TTO if there is any decrease in volume

due to evaporation of CHCl3 . Saturation solubility of the drug in the oil phase was

determined using our previously reported analytical method (Mirza et al., 2012).

After determining the saturation solubility of Itz (at room

temperature) in oil phase, variable amounts of Itz (< saturation solubility at room

temperature) were added in different containers (sequentially adding 30% CHCl3 and TTO

70%; v/v) and left for incubation (at 40°C for 2 h with intermittent shaking). Thus,

allowing the CHCl3 in the Oilmix phase to evaporate. After the incubation period small

quantity was centrifuged (Tomy MX-305) at 5000 rpm for 15 minutes while the remaining

quantities were left undisturbed to observe any precipitation. Consequently, the maximum

quantity of Itz that remained solubilized after complete evaporation of chloroform from

the Oilmix was determined (also the solution should be homogenous after sufficient

incubation period). Complete removal of Chloroform was authenticated by GC-MS

system [Agilent 7890A series (Germany)] equipped with split-split less injector and CTC-

PAL auto sampler attached to an apolar HP-5MS capillary column (30 cm x 0.25 mm i.d.

and 0.25 ^m film thickness) and fitted to a mass detector. Carrier gas flow rate (Helium)

was 1ml/min, split: split less ratio 1:100, injector temperature was 70°C, detector



temperature 250°C, while column temperature was kept at 60°C for 2 min followed by

linear programming from 70 to 230 °C (at rate of 5°C/min), and then kept isothermally at

230 °C for 2 min. Transfer line was heated at 280 °C. Split ratio was kept at 1:100. Mass

spectra were acquired in EI mode (70 eV); in m/z range 30-400. The amount of sample

was injected through head space. The residual solvent of the formulation were identified

by comparison of their mass spectra to those from Wiley 275 and NIST/NBS libraries,

using different search engines.

4 e . 1 . 2 C Y T O T O X I C P O T E N T I A L O F I T Z , T T O A N D O I L m ix

The cytotoxicity of Itz, TTO and Oilmix were evaluated by 3-[4-5-dimethylthiazol-2-4]-2,

5- diphenyltetrazolium bromide (MTT) assay using HeLa-S3 cell lines. HeLa-S3 cell lines

obtained from National Center for Cell Science (NCCS, Pune) and grown under 5% CO2

in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2.0 mML-1 glutamine,

and 1.5 mg/mL NaHCO3. Exponentially growing HeLa epithelial cells are seeded into 96-

well plate containing RPMI 1640 medium at a density of 106 cells/well. The cells are

allowed to grow for 24 h at 37°C prior exposure to samples. On the day of treatment,

RPMI 1640 medium is replaced with fresh medium. Test samples were placed on top of

the cells and allowed to incubate for 24 h at 37°C. After incubation, cells were washed

with phosphate-buffered saline (PBS) to remove the Oilmix, and 100 ^l of fresh medium

with 10 ^l of MTT solution (5 mg/mL in 0.1 M PBS, pH 7.2) was added to each well.

Cells containing only medium and MTT were considered as negative controls (without

formulation treated). Plates were then incubated for 4 h at 37°C in a CO2 incubator. After

incubation, MTT reaction medium was discarded and cells were washed with PBS. Then,

100 ^l DMSO was added in each well to dissolve blue formazan crystals, and optical

density was measured at 570 nm with a 96-well multiscanner ELISA reader with DMSO

serving as blank. The percent viability was calculated by the following formula:


OD of test sample% Cell viability = ;--------— X 100

OD or control sample



4 e . 1 . 3 . S E L E C T I O N O F Sm ix

4 e . 1 . 3 . A . S c r e e n i n g o f S u r f a c t a n t

A kit (Greth and Wilson, 1961) containing surfactants of different HLB values were used

to develop a series of emulsions with a constant composition of Oilmix and aqueous phase,

its stability was also studied after 3 days. Required HLB for the phase was deduced from

the inference obtained (Table 35).

Table 35: Selection of HLB for Oilmix composition. Stability of the emulsion was observed for 3 days at room temperature. Results obtained are indicative of required HLB for the development of a stable emulsion of proposed Oilmix.

K i t N o . K i t c o n s t i t u e n t H L B v a l u e O b s e r v a t i o n a f t e r 3 d a y s

8% Span 80 + 92% Span 85 Substantial phase separation

88% Span 80 + 12% Span 85

83% Span 80 + 17% Tween 85

65% Span 80 + 35% Tween 80

46% Span 80 + 54% Tween 80 10

28% Span 80 + 72% Tween 80 12 Little phase separation

9% Tween 20 + 91% Tween 80 14 No phase separation

60% Tween 20 + 40% Tween 80 16 No phase separation

4 e . 1 . 3 . B . S c r e e n i n g o f C o - s u r f a c t a n t s

16% w/v of different aqueous co-surfactants solutions was made (Azeem et al., 2009). To

the 2.5 ml of this solution, 5 ^L of Oilmix was added, mixed, vortexed and observed for

transmittance by UV spectroscopy at 600 nm. If there is not a sudden drop in

transmittance, 5 ^L of Oilmix was added repeatedly to reach a point where transmittance

falls sharply. Volume of Oilmix consumed was noted for each surfactant and was compared

with each other.


2

4 tt

6 tt

8 tt

tt

4 e . 1 . 4 C O N S T R U C T I O N O F P S E U D O T E R N A R Y P H A S E D I A G R A M

For the determination of existence zone of NE, pseudo ternary phase diagrams were

constructed using aqueous titration method. Surfactants and co-surfactants (Smix) in each

group were mixed in different volume ratios (1:0, 1:1, 1:2, 1:3, 2:1, 3:1, 4:1) and the stock

of 100 mL were prepared.

For each phase diagram, oil and specific Smix were mixed

thoroughly in different volume ratios from 1:9 to 9:1 in different glass vials. Nine different

combinations of oil and Smix, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 were made so that

maximum ratios were covered for the study to delineate the boundaries of phases precisely

formed in the diagram. Visual observations were made for transparency and viscosity of

o/w NEs. The physical state of the NE was marked on pseudo-three-component phase

diagrams with one apex representing aqueous phase, second Oilmix and third Smix. These

observations were made for each Smix ratio in the group separately and phase diagrams

were constructed separately. From each phase diagram, different compositions were

selected and subjected to thermodynamic stability tests. The unstable or metastable

formulations were discarded.

4 e . 1 . 5 T H E R M O D Y N A M I C S T A B I L I T Y S T U D I E S

Several formulations with minimum percentage of Smix from different phase diagrams

were taken for following studies.

A . H e a t i n g - c o o l i n g c y c l e

Formulations were subjected to different temperature 4 °C and 45 °C and storage of

formulations at each temperature was not less than 48 h. Formulation were exposed for six

cycles and then examined for stability at these temperatures.

B . C e n t r i f u g a t i o n t e s t

Formulations were subjected to centrifugation at 3500 rpm for 30 min and observed for

phase separation.

C . F r e e z e - t h a w c y c l e

Three freeze-thaw cycles between -21 °C and +25 °C with formulation were performed

and formulations were stored at each temperature for not less than 48 h.



4 e . 1 . 6 E V A L U A T I O N O F S T A B L E N E

A . G l o b u l e S i z e A n a l y s i s

Globule size analysis was carried according to the method described in section 4d.4.1. All

measurements were done in triplicate using disposable polystyrene cuvettes (Malvern

Instruments, UK). Size of the NE was also measured for several weeks to check any

increase in size.

B . R e f r a c t i v e i n d e xRefractive index of drug-loaded formulations was determined using an Abbe's type

refractrometer (Precision Standard Testing Equipment Corporation, India).

C . p H M e a s u r e m e n t s

The pH of the formulations was measured by a pH meter (Mettler Toledo MP 220,

Greifensee, Switzerland) in triplicate at 25°C.

D . T r a n s m i s s i o n E l e c t r o n M i c r o s c o p y ( T E M )

Morphology and structure of the NE were studied using Morgagni 268D electron

microscope (Fei Company, Netherlands) operating at 70 kV. Combination of bright field

imaging at increasing magnification and of diffraction modes was used to reveal the form

and size of the NE. In order to perform TEM observations, a drop of the NE was suitably

diluted with water and applied on a carbon-coated grid, then treated with a drop of 2%

phosphotungstic acid and left for 30 s. The coated grid was dried and then taken on a slide

and covered with a cover slip and observed under the microscope.

E . V i s c o s i t y m e a s u r e m e n t

The viscosity of the NEs were determined by using Brookfield R/S plus rheometer

(Brookfield Engineering, Middleboro, MA) using a C50-1 spindle in triplicate at 25°C.

4 e . 2 D E V E L O P M E N T A N D O P T I M I Z A T I O N O F G E L

Literature reports suggests that patients are known to better tolerate gels than inserts or

ointments while using vaginal dosage forms (Edsman et al., 1998). Hence it was

envisaged to have a gel as a carrier for the purported NE. Polymers used for the

development of gel were CP 934 and Poloxamer 407. Carbopols are widely used gelling

agents in case of vaginal drug delivery and also shown to have mucoadhesion properties



(Wang and Lee, 2002). Poloxamer 407 (Pluronic® F127) a thermo-sensitive polymer

which shows excellent water solubility, good drug release characteristics, low toxicity and

irritation, and has also shown compatibility with other excipients (Zaki et al., 2007).

Thermo sensitive gels were prepared according to the cold

method (Choi et al., 1998). Briefly, Carbopol 934 (0.3% w/v) was slowly added to citrate

phosphate buffer (0.1 M, pH 4.0) at 4° C with gentle mixing for 3 hours and allowed to

hydrate. Then it was centrifuged at 3000 rpm for 20 min to remove the air bubbles. Care

was taken to allow the minimum air bubbles level during handling. Pluronic® F 127

(15%, 18% & 20%, w/v) was then added to CP 934 solution and allowed to dissolve

overnight at 4° C. Optimized NEs were converted into final formulations in such a way

that total strength of Itz in gel remains 0.5% w/v.

Conventional gels containing dispersed Itz (0.5% and 1% w/v) were

also prepared using method described earlier. Smix in equal strength was used to disperse

the Itz into the polymer solution. Similarly, another gel containing 1% w/v of TTO was

also developed. The gels developed by using Itz were not clear. These gels were used in

permeation and in vivo studies for comparative evaluations.

A . M e a s u r e m e n t o f s o l - g e l t r a n s i t i o n t e m p e r a t u r e ( T sol- gel)

In a 20 ml transparent vial containing a magnetic bar, 5 ml gel was placed. Whole

assembly was put in a water bath. It was heated at a rate of 2°C/min with constant stirring

(150 rpm). When magnetic bar stopped moving due to gelation, the temperature displayed

on the thermistor was noted as the gelation temperature.

B . V i s c o s i t y m e a s u r e m e n t

As described in section 4e.1.6E the temperature of the laboratory was maintained at 37°C

and sufficient time was given to the formulation to be converting into gel.

C . E v a l u a t i o n o f g e l m u c o a d h e s i o n

Mucoadhesion of the gels were determined using methods described earlier from our

laboratory (Chopra et al., 2007). In brief, mucoadhesion was determined by measuring the

force required to detach the formulation (maximum detachment force, MDF) from

Cellophane membrane treated with SVF using a software- controlled penetrometer, TA-

XT2 Texture Analyzer (Stable Micro Systems, UK) with a 5 kg load cell. Gels were

placed between two cellophane membranes hydrated with SVF and attached horizontally



to upper and lower probes. The pre-test speed was set up at 1 mm/s, the test speed at 0.5

mm/s, and the penetration depth at 5 mm with an acquisition rate of 100 points/s. A

downward force of 10 g was applied for 3 min to ensure intimate contact between the

membrane and the sample. The probe was then moved upwards at a constant speed of 0.5

mm/s and the force required to detach the membrane (upper probe) from the surface of

each formulation was determined as the peak value in the resultant force-time plot. The

study was carried out at body temperature (37° C) in three replicates for each sample. The

work of adhesion per unit area (wA^^s), was characterized by the work executed on the

matrices when the two contact phases i.e. the cellophane membrane (a ) and the

mucoadhesive gels (fi), formed an interface of unit area which were subsequently

separated reversibly to form unit areas of each of the aS- and fi5-interfaces. This

relationship is mathematically described Eq. (1).

Further, volume of vaginal fluid is about 0.75 ml (Owen, Dunmire et al.

1999) and volume of applied liquid dosage form is about 1-3 ml. Hence to mimic the

conditions of vaginal milieu, gels were diluted 1:1 with SVF (previously maintained at

37°C) and tested the influence of dilution on MDF.

D . v iv o d r u g p e r m e a t i o n s t u d y








was filled with SVF and stirred with a magnetic rotor at a speed of 100 rpm in hot air oven

maintaining temperature at 37±1°C. The whole media was replaced with a fresh one after

every 30 min to stabilize it. After running the six cycles of stabilization, 1 mL of sample

(G2, G3, G4 and a conventional gel containing 0.5% w/v Itz) was placed into donor

compartment. Also 0.75 ml of SVF was added into donor compartment to mimic the



conditions of vaginal milieu. The receptor compartment was having 20 ml phosphate

buffer (pH 4.5) solution. The samples were withdrawn at regular interval (0.5, 1, 2, 3, 4, 5,

6, 7, 8, 9, 10, 12, 14, 16 and 24 h), filtered through 0.45 ^m membrane filter and analyzed

for drug content by HPTLC (Mirza et al., 2012). The cumulative amount of drug (Itz)

permeated through the membrane (^g/cm2) was plotted as a function of time (t) for each

formulation. The permeability parameters were determined according to the methods

reported in literature.

4 e . 3 . I N V I V O A N T I F U N G A L S T U D I E S O F O P T I M I Z E D N E G E L

A rat infection model was used to study the final formulation in vivo (De Bernardis et al.,

1997) after getting approval from Institutional animal ethical committee of Jamia

Hamdard (proposal number 742). Eighteen female wistar rats were divided in groups of

three for the application of optimized gel (G 4), conventional Itz-gel (1% w/v),

conventional TTO-gel (1% w/v). Control was not taken in the study (due to animal

constraints) since in another study in our lab, a placebo gel with nearly same polymer

compositions was found to produce insignificant decline in infection kinetics. Six days

prior to inoculation of infection, all animals were maintained under pseudo estrus by


albicans (108 cells per ml of saline) were inoculated. Then after, 1 g of each gel was

applied. During specified time intervals 1 ^L of the fluid from vaginal cavity was

withdrawn, diluted and spread on Sabouraud agar containing chloramphenicol (50 mg/ml).

It was then incubated at 32°C in the BOD incubator (LHC-78-Labhospmake, India) for 48

hrs and CFUs were counted. A rat was considered infected when at least 1 CFU was

present in the vaginal sample (i.e., a count of >103 CFU/ml). A graph was plotted between

time (days) and no. of CFU present in vaginal fluid.

4 e . 4 B I O A D H E S I O N A N D I R R I T A T I O N T E S T S

In vivo bioadhesion studies were carried out after taking permission from Institutional

Animal Ethical Committee of Jamia Hamdard (proposal number 712). Experimentation

was carried out according to the earlier reported method (Chopra et al., 2007). Briefly, 0.5

g of the Optimized gel (G 4) was mixed with 0.4% trypan blue dye. Resulting dark blue

colored formulation was inserted into the rat vaginal with a 2 mL plastic syringe (without



needle). For each group three rats were taken. After 24 h of administration, the rats were

sacrificed and the retention of bioadhesive formulation at the administration site was

visualized by dye intensity. For control group simulated vaginal fluid (pH 4.5) was taken.

Rats have simple, cuboidal or columnar epithelium which is highly

sensitive to mucosal irritation when compared to human vagina. Adult wistar rats (3

months old; 150-180 g) were used for the in vivo toxicity studies. Tap water and food

pellets were available ad libitum throughout the study. Room temperature was maintained

at 25±2°C with relative humidity 50±5%. Strength of each group was taken three. The rats

were treated once daily with 0.5 g of gel for 14 days. Prior to every administration, rats

were evaluated for vaginal or vulval irritation, discharge or bleeding from vagina. After

the completion of 14 days, study period, they were scored for possible erythema. The

mean erythemal scores were recorded (ranging from 0 to 4) depending on the degree of

erythema as follows: no erythema = 0, slight erythema (barely perceptible-light pink) = 1,

moderate erythema (dark pink) = 2, moderate to severe erythema (light red) = 3, and

severe erythema (extreme redness) = 4. Then, they were sacrificed and transverse section

of the vaginal tissue was examined by experienced pathologist for the severity of epithelial

loss and atrophy. Control group was given same volume of SVF.

R E S U L T S A N D D I S C U S S I O N

The present work embarked on preparing a nano emulsion (NE) of Itz (Itz) using Tea tree

oil (TTO). It offered a great formulation challenge to us despite being highly lipophilic,

the solubility of Itz in most of the oil was limited to <100 ^g/mL (the only exceptions

being medium chain triglycerides, in which it showed a solubility of 140.79±7.23 ^g/mL).

The solubility of Itz in TTO was found to be 1.3±0.2 mg/ml. The purpose of combining

TTO and Itz was based on two main reasons. First, TTO (obtained from the leaves of the

Australian native tea tree, Melaleuca alternifolia) has a well established broad spectrum

antimicrobial activity (Mondello et al., 2006) and has been indicated in vaginal affliction

of both fungal and bacterial origin. Itz on the other hand, is a potent and broad spectrum

antifungal agent (Saag and Dismukes, 1988) primarily used to treat vaginal candidiasis.

Thus a novel formulation comprising the two antimicrobials is expected to work

synergistically with an exceptional broad spectrum of antimicrobial action. Additionally,



t h e u s e o f t h e s e c o m p o n e n t s i s e x p e c t e d t o y i e l d a h i g h e r h y d r o p h o b i c i t y o f o i l p h a s e a s

t h e h y d r o p h o b i c i t y o f T T O i s l o w ( s o l u b l e i n m e t h a n o l , e t h a n o l a n d o t h e r s o l v e n t s w i t h

s i m i l a r d i p o l e m o m e n t s ) . I t i s g e n e r a l l y o b s e r v e d t h a t a d i s p e r s e d p h a s e i n N E m u s t b e

e s s e n t i a l l y i n s o l u b l e i n t h e c o n t i n u o u s p h a s e s o a s t o d i m i n i s h t h e r a t e o f O s t w a l d r i p e n i n g

d e s p i t e t h e v e r y h i g h L a p l a c e p r e s s u r e s . A l t h o u g h t h e s u p p r e s s i o n o f O s t w a l d r i p e n i n g c a n

b e a c h i e v e d b y o t h e r m e a n s ( M a s o n e t a l . , 2 0 0 6 ) a l s o , b u t c h o o s i n g a v e r y i n s o l u b l e l i q u i d

f o r t h e d i s p e r s e d p h a s e i s t h e e a s i e s t m e t h o d . I t z ( l o g P ~ 6 . 2 ) h a s a n a p p r e c i a b l y h i g h

h y d r o p h o b i c i t y w h e r e a s T T O e l i c i t s l o w . S o , i t i s p r o p o s e d t h a t p r e p a r a t i o n o f a

h o m o g e n o u s a n d t r a n s p a r e n t s o l u t i o n o f b o t h b y s o m e m e a n s i s e x p e c t e d t o d e l i v e r a h i g h

h y d r o p h o p h o b i c o i l p h a s e w h i c h m a y b e f u r t h e r u s e d f o r p r e p a r i n g a n a n o e m u l s i o n .

S i n c e , I t z i s f r e e l y s o l u b l e i n d i c h l o r o m e t h a n e a n d c h l o r o f o r m , a

c o n c e p t w a s h y p o t h e s i z e d t o u s e c h l o r o f o r m a s a c o - s o l v e n t a n d a t r a n s p a r e n t a n d s t a b l e

s o l u t i o n o f T T O + I t z a s a n o i l p h a s e f o r p r e p a r i n g a n o / w N E . T h e d e v e l o p e d a n d s t a b l e

N E w a s t h e n c o n v e r t e d i n t o t h e r m o s e n s i t i v e g e l s u s i n g C a r b o p o l 9 3 4 ( C P 9 3 4 ) a n d

P l u r o n i c ® F 1 2 7 a n d c h a r a c t e r i z e d f o r S o l - g e l t r a n s i t i o n t e m p e r a t u r e , m u c o a d h e s i o n ,

v i s c o s i t y a n d i n v i t r o p e r m e a t i o n s t u d i e s . T h e o p t i m i z e d g e l w a s t h e n c a r r i e d f o r w a r d f o r

i n v i v o s t u d i e s ( f u n g a l c l e a r a n c e k i n e t i c s ) , b i o a d h e s i o n a n d i r r i t a t i o n t e s t s .

T h e i d e a o f u s i n g c h l o r o f o r m a s a c o - s o l v e n t t o b r i n g t h e d r u g i n s o l u t i o n

s t a t e a n d t h e n s o l u b i l i z i n g t h e d r u g s o l u t i o n i n t o o i l p h a s e o r i g i n a t e d f r o m o u r p r e v i o u s

r e p o r t s ( M i r z a e t a l . , 2 0 1 2 ) . O u r f i n d i n g s s u g g e s t e d t h a t t h e h i g h l y c r y s t a l l i n e n a t u r e o f I t z

i s a m a j o r i m p e d i m e n t f o r i t s o i l s o l u b i l i t y . O n c e , i t i s b r o u g h t i n t o a m o l e c u l a r l y

d i s p e r s e d f o r m i t b e c o m e s e a s y t o g e t h i g h a n d s t a b l e s o l u b i l i t y c o n s i d e r i n g i t s i n t r i n s i c

l i p o p h i l i c p r o p e r t i e s . T h i s O i l mix i s u s e d a s a n o i l p h a s e f o r t h e d e v e l o p m e n t o f N E . T h e

s u c c e s s o f s u c h N E w i l l d e p e n d o n t h e c h o i c e o f c o m p o s i t i o n o f o i l p h a s e ( a m o u n t o f I t z ,

T T O a n d C H C l 3) w h i c h w i l l y i e l d a s t a b l e f o r m u l a t i o n ( a s C H C l 3 i s v o l a t i l e i n n a t u r e ) .

S u g g e s t i v e l y a % o f I t z c h o s e n f o r t h e d e v e l o p m e n t o f N E i s t h e o n e w h i c h r e m a i n s

s o l u b i l i s e d i n T T O a f t e r t h e c o m p l e t e e v a p o r a t i o n o f C H C l s ( u s e d a s a c o - s o l v e n t ) .




1 D E V E L O P M E N T A N D O P T I M I Z A T I O N O F N E

1 .1 S C R E E N I N G O F A N O P T I M U M R A T I O F O R O I L m ix A N D D R U G C O N T E N T

Variable and little excess quantity of Itz was dissolved in a fixed quantity of CHCl3 and

then a constant composition (30% CHCl3: TTO 70% ) was made in volumetric flask. All

the flasks were left at room temperature (25±1°C) with continuous shaking for three days.

Total volume was kept constant with addition of TTO if there is any decrease in volume

due to evaporation of CHCls. Saturation solubility of the drug in the oil phase was

determined using our previously reported analytical method (Mirza et al.2012).

After determining the saturation solubility (at room

temperature) Itz in oil phase, variable amounts of Itz (< saturation solubility at room

temperature) were added in different containers (sequentially adding 30% CHCl3 and TTO

70%; v/v) and left for incubation (at 40°C for 2 h with intermittent shaking). Thus,

allowing the CHCl3 in the Oilmix phase to evaporate. After the incubation period small

quantity was centrifuged (Tomy MX-305) at 5000 rpm for 15 minutes while the remaining

quantities were left undisturbed to observe any precipitation. Consequently, the maximum

quantity of Itz that remained solubilized after complete evaporation of chloroform from

the Oilmix was determined (also the solution should be homogenous after sufficient

incubation period). Complete removal of Chloroform was authenticated by GC-MS

system [Agilent 7890A series (Germany)] by the method described earlier.

Table 36: Determination of a homogenous, transparent and stable Oilmix. Quantities of Itz was determined that remained solubilized after complete removal of chloroform. It was solely due to inherent lipophilic property of Itz when its crystallinity is broken down with the use of co-solvent.

O i l mix c o m p o s i t i o nT r a n s p a r e n c y o f s o l n

( a f t e r 2 h r s a t 4 0 ° CC H C l 3 d e t e c t e d

i n G C - M SI t z s o l u b i l i t y

( m g / m l )(TTO 70%: 30% CHCl3) Clear Not detected 5.0 ± 0.1

(TTO 70%: 30% CHCl3) Clear Not detected 6.0 ± 0.2

(TTO 70%: 30% CHCl3)Precipitation after longer

timeNot detected 7.3 ± 0.1

(TTO 70%: 30% CHCl3)Precipitation after some

timesNot detected 8.5 ± 0.09

(TTO 70%: 30% CHCl3) Non-transparent Not detected 9.6 ± 0.6



To be on a safer side, Itz concentration (5 mg/ml) was taken

for stock preparation. A stock of 10 ml was prepared and allowed the chloroform to

evaporate and then again making the volume up to 10 ml with TTO. This stock (Oilmix)

was used in aqueous titration for the development of pseudo ternary phase diagram.

Representative chromatogram of GC-MS study is given in Fig. 62.

From the library search it was evident that there was not any dreg of chloroform in the

Oilmix.

Figure 62: GC-MS chromatogram of Oilmix after the incubation period. It was supposed that after the given incubation period and intermittent shaking would let the chloroform evaporate completely, which has been authenticated by the chromatogram obtained.

1 . 2 C Y T O T O X I C P O T E N T I A L O F I T Z , T T O A N D O I L m ix

The cytotoxicity data of all the three components in a 72 hrs study are given in Table 37 .

The results obtained were interpreted by Graph pad prism 5. A representative graph

between Log concentration of TTO and percent inhibitions, obtained by statistical analysis

is also given in Fig 63.

Table 37: Results of MTT assays of Itz, TTO and Oilmix. None of the components (TTO, Itz

T i m e i n t e r v a l s S u b s t a n c e s I C 50 i n ^ g / m l R 2

24 Hrs

Itz 40.52 0.9856

TTO 37.55 0.987

Oilmix 39.48 0.9872

48 Hrs

Itz 32.16 0.9821

TTO 35.16 0.9894

Oilmix 32.34 0.9827

72 Hrs

Itz 22.25 0.9913

TTO 31.98 0.9849

Oilmix 33.51 0.9796



R values obtained in all the cases were > 0.982. In case of Itz, a significant decrease in

IC50 was observed as time passes. In case of TTO, IC50 was decreasing but not very

sharply. Whereas in case of Oil phase, a decrease in the IC50 was observed up to 48 hr

studies but then it increased very slightly.

Figure 63: A representative graph of percentage cell growth inhibition Vs Log concentration of TTO against HeLa-S3 cells.

1 . 3 S E L E C T I O N O F S m ix

As per general observation, required hydrophilic lipophilic balance (HLB) value to form

o/w NE should be greater than 10. In our particular Oilmix it was found to be 14. Hence

Tween 20 was chosen as a surfactant. Ionic surfactants were excluded from the study

because of their pH susceptibility, higher CMC values and irritant nature. Care must be

given to pH susceptible moieties when we intended to develop a formulation for vaginal

uses. Substantial variations in pH in healthy and diseased conditioned have been observed

and also there is a defined pH gradient in the cavity (lowest near the anterior fornix and

highest near the cervix). Further, o/w NEs based on nonionic surfactants are likely to offer

better in vivo stability (Kawakami et al., 2002). The right blend of low and high HLB

surfactants leads to the formation of a stable NE formulation (Craig et al., 1995). As

transient negative interfacial tension and fluid interfacial film is rarely achieved by the use

of a single surfactant, usually necessitating the addition of a co-surfactant. Also the

presence of a co-surfactant decreases the bending stress of the interface and allows the

interfacial film sufficient flexibility to take up different curvatures required to form micro

/ NE over a wide range of composition (Kawakami et al., 2002). Hence in our study



Labrasol was taken as Co-surfactants from the inferences obtained from Table 42.

Labrasol had the potential of solubilizing > 200% TTO than other co-surfactants taken

(Solutol, Transcutol and Labrafil). Transparency and elegancy is an important

characteristic of NE, hence transmittance was taken as an important parameter for the

selection of co-surfactant. On the other hand, as per general observation value of

interfacial tension (yow) should be equal to droplet size for a simple dispersed system (o/w

emulsion) to be formed (as inferred from Laplace’s equation, Ap = 2 yow/R). So, in case of

NE the Smix should be sufficient enough and the value of yow should be much lower than

conventional emulsions considering the size of droplet.

Table 38: % Transmittance analysis obtained for different Co-surfactant against variable quantities of Oilmix. Volume of Oilmix given in the table is cumulative quantities. End point is fixed to a point when there is a sudden drop in transmittance

S . N oT o t a l v o l u m e o f

O i l mix ( ^ L )

% T r a n s m i t t a n c e w i t h d i f f e r e n t c o - s u r f a c t a n t s

L a b r a s o l S o l u t o l T r a n s c u t o l L a b r a f i l

1. 0 65.4% 64.2% 65.6% 63.7%

2. 5 63.9% 61.3% 66.4% 61.9%

3. 10 62.4% 0.4% 63.1% 60.3%

4. 15 62.1% 9% 7%

5. 20 61.3%

6. 25 60.6%

7. 30 58.7%

8. 35 0.8%

1 . 4 C O N S T R U C T I O N O F P S E U D O T E R N A R Y P H A S E D I A G R A M

The existence of NE formation zone can be illustrated with the help of the pseudo ternary

phase diagram. During titration formulations were carefully observed so that the

metastable systems were not selected, although the free energy required to form a NE is

very low and the formation is thermodynamically spontaneous (Craig et al., 1995). Effect

of surfactant and co-surfactant mass ratio on NE formation was evaluated for the further

optimization. A noticeable change in NE area was obtained with increasing ratio of co



surfactant (Labrasol). A larger NE area was observed when Smix was used in 1:1

composition but it decreased for 1:2 ratio and remained nearly same for other higher ratios

like 1:3 and 1:4 (Fig. 64). While in case of increasing ratio of surfactant, NE region

increased as we move from 1:1 to 2:1 ratio and then decreased (for 3:1 and 4:1). Thus we

see that the areas of one phase NE zones are dependent on surfactant composition which

may be attributed to differences in the packing of surfactant and co-surfactant at the o/w

interface. Also the o/w NE region was found towards the water-rich apex of the phase

diagram. Thus we see that a proper ratio of Smix is important to obtain the wide NE region.

The maximum region of NE was obtained in Smix ratio 2:1 and the

maximum percentage of Oilmix that can be solubilized was only 45.45% wt/wt with

45.45% wt/wt of Smix. Similarly in Smix ratio 1:1, maximum percentage of oil solubilized

was 36.36% wt/wt with 54.55% wt/wt of Smix. A negative free energy of formation is

achieved when large reduction in surface tension is accompanied by significant favorable

entropic changes. In such a case, NE formation is spontaneous and the resulting dispersion

is thermodynamically stable (Lawrence and Rees, 2006).

Figure 64: Pseudoternary phase diagram were using the aqueous titration method, indicating o/w nanoemulsion region of Oilmix, Tween 80 (surfactant), Labrasol (cosurfactant) at different Smix ratios indicated in Group (a) to Group (g). (a) 1:1, (b) 1:2, (c) 1:3, (d) 1:4, (e) 2:1, (f) 3:1, (g) 4:1



1 . 5 T H E R M O D Y N A M I C S T A B I L I T Y S T U D I E S

It is the thermo-stability which differentiates nano or micro emulsions from macro

emulsions which have kinetic instability and eventually lead to phase separation (Shafiq-

un-Nabi et al., 2007). The results obtained from thermodynamic stability studies showed

that the investigated formulations remained macroscopically homogeneous, transparent,

and optically isotropic state without any phase change such as appearance of turbidity or

phase separation. Formulations that survived thermodynamic stability tests were separated

and a composition with minimum surfactant concentration among them was chosen for

further transformation into gels. Thus, the formulation that emerged suitable most was

10% Oilmix, 35% Smix (2:1) and 55% water.

1 . 6 E V A L U A T I O N O F S T A B L E N E

Size analysis was performed to confirm whether the resultant emulsions were indeed NEs.

Average diameter of the optimized NE was found to be 42.13 nm (Fig. 65) with a PDI of

0.116. Zeta potential was found to be -43.62 mV. Emulsifiers act not only as a mechanical

barrier but also through the formation of a surface potential (zeta potential), which can

produce repulsive electrical forces among approaching oil droplets and thus hinders

coalescence. The lower the zeta potential, the greater the net charge of the droplets, and

the more stable the emulsion is (Han et al., 2001).

Figure 65: Globule size analysis of nanoemulsion by zetasizer. A narrow size distribution was (PDI ~ 0.116) quite evident in the figure.

The results were again authenticated by TEM images (Fig. 66). It revealed

that the lipid emulsion droplets were almost spherical in shape, discrete, appearing dark

and has the amorphous core. Some droplet sizes were measured and the droplets are in

nanometer range varying from 40-60 nm. The viscosity of the formulation was found to be



14.03 ± 0.86 cps. Refractive index which indicates the isotropic nature of the formulation

was found to be 1.451 ± 0.012. While on the other hand pH of the formulation was

observed to be 5.53 ± 0.06. Fortunately the pH of the formulation was nearly same as the

pH of the vaginal milieu.

Figure 6 6 : Transmission electron micrograph of optimized nanoemulsion showing the size of

some oil droplets

The selected NE (10% Oilmix, 35% Smix (2:1) and 55% water) was stable

through several weeks, although a little increase in droplet size was noted after 7 weeks

(Fig. 67). Although minimized in our formulation, a well known problem associated with

a dispersed system is Ostwald ripening and coalescence. Following expressions have been

derived from Lifshitz-Slezov and Wagner (LSW) theory (Kabalnov and Shchukin, 1992)

for Ostwald ripening:

Where, Cro is the bulk phase solubility (the solubility of the oil in an infinitely large

droplet), Y is the interfacial tension, Vm is the molar volume of the oil, D is the diffusion

coefficient of the oil in the continuous phase, p is the density of the oil, R is the universal

gas constant, and T is the absolute temperature (°K). If the driving force responsible for

the instability is Ostwald ripening, a linear variation of as a function of time is obtained.



Figure 67: Average particle (n=3) size of the nanoemulsion throughout the stability studies (7 weeks). A minor increase in the average size has been observed. Y-axis has been changed (instead of starting from ‘0 ’) to have a clear picture of the result obtained.

2. DEVELOPMENT AND OPTIMIZATION OF NANOEMULGEL

2. 1 MEASUREMENT OF SOL-GEL TRANSITION TEMPERATURE (T SoL-GEL)

The gelation temperature is defined as the point where the elasticity modulus is half way

between the values for the solution and for the gel. With increasing fraction of P 407,

gelation temperature was found to decreasing (Table 39). For our purpose G4 appeared

suitable most considering the normal physiological body temperature (37°C). Gelation

phenomenon is a result of body centered cubic packing of spherical micelles. Temperature

plays an important role in the micelle formation through temperature dependent hydration

of the ethylene oxide units. Water is a good solvent for PEO as well as PPO chains of the

polymer at low temperatures. However, at higher temperature the solubility of PPO is

reduced and micelle formation occurs. At low temperatures in aqueous solutions, a

hydration layer surrounds P 407 molecules. However, when the temperature is raised, the

hydrophilic chains of the copolymer become desolvated as a result of the breakage of the

hydrogen bonds that had been established between the solvent and these chains. This

phenomenon favors hydrophobic interactions among the polyoxypropylene domains, and

leads to gel formation.



2 . 2 V I S C O S I T Y M E A S U R E M E N T S A N D M U C O A D H E S I O N T E S T S

Results of viscosity measurements are given in Table 39. It was found to vary

proportionally with total polymer content. At low pH (about pH 3-4) the carbopol chains

exist in a spiral coiled form, exhibiting a relatively low viscosity. With the progression of

pH (generally associated with fungal infection in vaginal milieu), the carboxyl groups of

the Carbopol become ionized, causing an increased repulsion of negative charges. It leads

the molecular structure to unwind and a gradual rise in viscosity. For Poloxamer 407,

gelation phenomenon is a result of body centered cubic packing of spherical micelles.

Temperature plays an important role in the micelle formation through temperature

dependent hydration of the ethylene oxide units. Water is a good solvent for PEO as well

as PPO chains of polymer at low temperatures. However, at higher temperature the

solubility of PPO is reduced and micelle formation occurs. At low temperatures in

aqueous solutions, a hydration layer surrounds P 407 molecules. However, when the

temperature is raised, the hydrophilic chains of the copolymer become desolvated as a

result of the breakage of the hydrogen bonds that had been established between the solvent

and these chains. This phenomenon favors hydrophobic interactions among the

polyoxypropylene domains, and leads to gel formation. Aqueous solutions of poloxamers

are stable in the presence of acids, alkalis, and ions (Escobar-Chavez et al., 2006) so it was

also expected to form gel in vaginal milieu.

Table 39: Gel compositions and their different in vitro evaluation parameters (Gelling tempeature, Viscosity, maximum detachement force with and without dilution). Results are given in Mean ± SD (n=3). G1 is a placebo.

C o d e C o m p o n e n t s ( % w / v )

G e l l i n gt e m p e a t u r e

( ° C )

V i s c o s i t y( P a s )

M D F ( g ) ( M e a n ±

S D )

M D F ( g ) a f t e r d i l u t i o n ( M e a n

± S D )

G 10.3% CP 934, 15% P 407 (Placebo)

41 ± 10.903 ± 0.003

25.4 ± 0.6 19.2 ± 0.2

G 2NE (0.5% w/v, Itz), 0.3% CP 934, 15% P 407

41 ± 20.907 ± 0.005

27.1 ± 0.3 21.4 ± 0.5

G 3NE (0.5% w/v, Itz), 0.3% CP 934, 18% P 407

39 ± 2 0.911 ± .002 29.6 ± 0.4 22.7 ± 0.4

G 4NE (0.5% w/v, Itz), 0.3% CP 934, 20% P 407

36 ± 10.913 ±

0 .0 0 131.7 ± 0.3 24.6 ± 0.3


Bioadhesivity or mucoadhesion is essentially defined as the

interfacial force between the polymeric drug delivery system and the mucus layer coating

an epithelium (Peppas and Buri, 1985). It is thus thought to be a result of the presence of

hydrogen bonding groups, strong anionic/cationic charges, high molecular mass, chain

flexibility and surface energy interactions which favor spreading onto the vaginal tissue.

Basic theories such as electronic, adsorption, wetting and diffusion phenomena have been

described and associated with the mechanisms by which bioadhesion occurs (Helfand and

Tagami, 1972) and (Kaelble and Moacanin, 1977). Results of the mucoadhesion were

found to be in the order of G4>G3>G2>G1 (Table 39). Nearly same order was also

observed when the gel was diluted with SVF.

2 . 3 E X V I V O D R U G P E R M E A T I O N S T U D Y

Flux values of all the Gels (G2, G3 and G4) formulations were found in the range from

0.018 ± 0.001 to 0.006 ± 0.0007 mg/cm2/h while for conventional gel it was 0.005 ±

0.0005 mg/cm2/h (Table 40). Values for G3 and G4 were comparable to conventional gel

while, G2 was significantly higher. Here polymer content appears to play a rate

determining role in drug diffusion and permeation. Since polymer content is similar in G4

and conventional gel, the releases were comparable to each other. In spite of similar

polymer content between G4 and conventional gel, a higher flux was obtained with G4.

The reason may be attributed to established permeation capability of TTO and its major

component terpinen-4-ol (Reichling et al., 2006). Another reason that resulted in enhanced

permeation of novel gels may be the nano sized droplets of formulation also with

increased in the interfacial area which influences transportation properties of the drug

(Akhter et al., 2008). It is also assumed that the low interfacial tensions, continuous and

spontaneous fluctuating interfaces of micro-emulsions are supposed to be responsible for

smoother permeation of drug.




Table 40: Ex-vivo permeation profile of different gels. *Mean±SD, n = 3; Jss was calculated from the slope of the linear portion of graph. Kp was calculated by dividing J ss with the concentration of the drug in donor cell (C0). Standard deviation in case of Kp has not been given, considering the small values and bulkiness of the table.

S a m p l e sG 2

G

G 4

Conventional gel

J s s ± S D ( m g / c m 2/ h ) a0.018 ± 0.001

0.008 ± 0.0009

0.006 ± 0.0007

0.005 ± 0.0005

K p

0.00630032

0.00280014

0.00175009

0.00210011

2 . 4 I N V I V O A N T I F U N G A L S T U D I E S O F O P T I M I Z E D N E G E L

The mechanism of action of both the antifungal has been reported to be similar up to some

extent. Itz creates membrane abnormalities by inhibiting cytochrome P450 (inhibiting

ergosterol synthesis) and TTO causes alteration in membranal permeability of candida,

dose dependent respiratory inhibition and inhibition of germ tube formation (Carson et al.,

2006). Respiration of C. albicans is inhibited by approximately 95% after treatment with

1.0% TTO and by approximately 40% after treatment with 0.25% TTO. Similarly,

complete germ tube inhibition has been observed with 0.25 and 0.125% TTO. Thus, a

combination of both (Itz and TTO) is expected to act in a multipronged way.

The results of fungal clearance kinetics have been given in Fig. 68.

The results were according to our expectations regarding synergism. The accelerated

clearance was observed with our synergistic formulation and least clearance was observed

with conventional Itz gel. Comparatively higher clearance of TTO conventional gel may

be due to permeation potential and broader antimicrobial activity with less MIC values of

TTO. Significantly higher CFUs have been observed in Itz gel treated group even after 21

days. Pattern of clearance kinetics graph was almost similar in all the cases which may be

attributed to diffusion characteristics of the drugs through gel matrix.



Figure 6 8 : Fungal clearance kinetics of C. albicans infection in oophorectomized, estradiol- treated rats after infections with 108 C. albicans cells. Each curve represents the mean of six rats. Marked differences in the fungal clearance kinetics of all three formulations (G4, conventional Itz gel and conventional TTO gel) have been observed.

2 . 5 I N V I V O B I O A D H E S I O N A N D I R R I T A T I O N T E S T S

As the gel is applied to the vaginal mucosa mechanical bioadhesion occurred with all

surface voids between the gel and tissue filled and therefore resulted in surface

interlocking. With the gradual hydration, a more unyielding chemically adhesive

interaction progresses between the hydrated polymeric blend and tissue interface either

through ionic or covalent bonding. Weak physical forces like van der Waals and London

forces are involved here in bioadhesion and once the gel diffuses through the surface, it

appears a sintering-like mechanism. The strength of bioadhesion depends on a hybrid

between the dispersive and diffusive phases as well as the contact surface area. The

lifetime of these bonds can vary from microseconds to seconds because, at any time

sufficient energy is (KT, where K is Boltzmann constant and T is the absolute

temperature) available to break these bonds. Hence, during the course of bioadhesion

these physical bonds break several times. The prolong retention of mucoadhesive gel in

the vagina was confirmed by blue staining even after 24 hrs as compared to untreated

animals.


Optimized gel (G 4) was also found without any sign of toxicity. On

a 0 - 4 scale basis, it was given the score zero, considering the negligible signs of

irritation. Even after pathological examinations, it was found to be safe (figure not given).

In a report, The TTO component 1, 8-cineole, which has a reputation as a skin irritant, was

also tested at concentrations up to and including 28% and did not produce any irritant

reactions (Southwell et al., 1997) and the maximum content of 1,8-cineole in tea tree oil

has been reported to be 15% v/v (Council of Standards). So, considering the irritation

potential, we had taken a much smaller amount in our optimized formulation. In another

report, a dosage form containing even up to 10% TTO was non-irritant (Reichling et al.,

2006). In recent medical literatures several reports of contact allergy due to the topical use

of TTO have appeared (Bhushan and Beck, 2006). The reactions have been in response to

100% pure TTO as well as lower concentrations of TTO in various formulations. So, it

should be considered once dispensing the TTO formulation to the patient.

Another point to be considered about the TTO formulation is

developmental toxicity. If the candidiasis is associated with pregnancy care must be taken

in dispensing the dosage form. A study of the embryo foeto-toxicity of a-terpinene, which

is present at approximately 9% in TTO, has demonstrated significant toxicity in a rat

model after oral ingestion (Araujo et al., 1996).

C O N C L U S I O N A N D I N F E R E N C E S D R A W N

Conclusively, it can be stated that the current work harnessed the potential of TTO both as

a formulation ingredient as well as a medicinal agent of natural origin for treating vaginal

afflictions. Moreover its synergism with Itz yields a stable and effective thermosensitive

gel which can be judiciously used in patients with recurrent candidiasis and other related

infections. Local application of the final preparation with sufficient loading of Itz and

goodness of TTO is purported as an effective tool for circumventing an often occurring

affliction in majority of female population. Considering the positive outcomes in different

in vitro and in vivo studies, it may be further explored for clinical applications.



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