Material and Methods

Vaginal mucoadhesive tablets of clotrimazole for vaginal candidiasis were prepared. Tablets mainly contain cysteamine thiomer which was thiol derivative of natural polysaccharide Xyloglucan. Along with thiomer HPMC and lactose were used. Tablets were optimized for HPMC and thiomer concentration. In vitro antifungal activity of drug was carried out on formulation and marketed formulation.

Results and Conclusion

It was seen formulation has similar release and better mucoadhesion than marketed tablet. Permeation study shows that xyloglucan thiomer enhances permeation of formulation by forming covalent bonding. Formulation containing thiomer has better antifungal activity.

Abstract

Vaginal candidiasis is a common infection affecting most women in reproductive age. The topical treatment is preferred as infection resides in mucosal layer and it ensures patient compliance. An important factor in topical delivery is retention of dosage form in vagina, mucoadhesive drug delivery can overcome this limitation. The purpose of this research was to develop vaginal drug delivery system of clotrimazole which, after vaginal administration should have the ability to prolong residence time with desired In vitro release profile. Mucoadhesive clotrimazole tablets were prepared using thiomeric derivative xyloglucan derived from tamarind seeds. A Simplex Centroid design was employed to optimize the quantities of thiomer, HPMC K100M and lactose, in order to get high mucoadhesion and desired drug release. In vitro antifungal activity of formulation based on zone of inhibition showed better performance of thiomer tablet over marketed mucoadhesive formulation.

Keywords: Clotrimazole; Vaginal Tablet; Thiomer; Simplex Centroid Design

Abbreviations

CLO: Clotrimazole; XG: xyloglucan; TKP : Tamrind seed kernel;
SDA : sabouraud Dextrose Agar; DMF: Di Methyl Formamide;
MIC: Minimum Inhibitory Concentration

Introduction

Infectious vaginitis accounts for 90% of all cases in women of reproductive age [1,2] commonly called candida vaginitis. Candidiasis is a fungal infection developed in mucosa that usually causes a watery, white, cottage cheese-like vaginal discharge [3].

The discharge is irritating to the vagina and the surrounding skin. Conventional treatment includes both combined treatment of oral and vaginal administration. Vaginal administration has following advantages like bypassing first pass metabolism, local drug delivery, low toxicity [4] For many years imidazole derivatives are used as drug of choice for treating infection. Clotrimazole is BCS class II drug which has prominent antifungal action Clotrimazole works to kill individual Candida or fungal cells by altering the permeability of the fungal cell wall. It binds to phospholipids in the cell membrane and inhibits the biosynthesis of ergosterol and other sterols required for cell membrane production. This leads to the cell’s death via loss of intracellular elements [5]. Clotrimazole for local treatment is available in various formulations like tablets, creams, gels. However, a major problem with these formulation is less residence time [6]. There is little literature regarding formulation attempt to improve effectiveness of vaginal formulations by use of cyclodextrins, mucoadhesive polymers etc. [6].

Xyloglucan is polysaccharide made from tamarind seed kernel (TKP) [7,8,9,10] modification of xyloglucan using various thiol residues is reported [11,12,13] thiolation enhances mucoadhesion by means of covalent bonding with mucin and enhances transmucosal penetration by causing reversible and efficient opening of the tight junctions of the mucosal epithelium Thiomers possess CYP 34 enzyme inhibition properties have been reported to exert an inhibitory effect on efflux pumps [14]. In addition, it possesses antibacterial and antifungal activity and due to its good mucoadhesive properties resulted from the cationic behavior [12].

The aim of this work was to design mucoadhesive vaginal tablets of clotrimazole by using xyloglucan cysteamine thiomers a matrix in order to improve drug residence time compared to commercially available mucoadhesive tablet. The prepared tablets were optimized by using mixture design to possess desired values of mucoadhesion, release and T50. In vitro antifungal activity was performed to assess effectiveness of formulation.

Material

Clotrimazole was gift from Medly pharma. Xyloglucan was kind gift from Encore Natural Polymers Pvt. Ltd, Ahmadabad and HPMC K 100M was purchased from Sigma Aldrich. Sabauraud dextrose broth and agar were procured from oxide. Solvents used were of analytical grade. All other chemicals were procured from local sources and were of analytical grade.

Methods

Preparation of Xyloglucan Thiomer

Being reported as detailed paper separately by method reported in literature. Briefly TKP was dispersed in cold water and xyloglucan was precipitated using ethanol as per method described in literature to remove proteins and fibres [15].

Synthesis of Xyloglucan-Cysteamine Conjugates (Thiomer)

Part A: Oxidation of Xyloglucan [16]

The XG was oxidized by using Sodium periodate. The reaction was continued for 6 h in the dark at 250C. and was quenched by the addition of ethylene glycol.The Oxidized Xyloglucan was purified by dialysis (Hi Media molecular cut off 11500 Da) against distilled water for 12 h and the product formed was precipitated in ethanol and vacuum dried at room temperature.

Part B: Thiolation of Thiomer

Cysteamine derivative (Thiomer) [16]

The preparation of thiolated xyloglucan was a 2 step reaction. Firstly, to 1g of aldehydic xyloglucan, 40 ml of distilled water was added, followed by addition of 0.5g cysteamine. The pH was adjusted to 5 with 5M HCl. The mixture was adjusted to 50 ml with distilled water followed by incubation for 3 hours under stirring at room temperature. After this, 4g of sodium cyanoborohydride was added to the solution and stirred for 72 hours at room temperature. For the isolation of polymer and elimination of un reacted product, the reaction mixture was dialyzed 6 times in tubing (molecular weight cut-off 12 KDa: Dialysis membrane 150; Hi Media, Mumbai, India) at 10⁰C in dark. The reaction mixture was dialyzed 1 time against distilled water; 1 time against 0.2mM HCl and then 2 times against the same medium but holding 1% NaCl to quench ionic interactions between sulfhydryl compound and cationic polymer. It was then dialyzed 2 times against 0.2mM HCl followed by lyophilization (-78⁰C, 0.002 mbar, Labcono Freezone 2.5 Lyophilizer, USA) and stored at 4⁰C for further use.

Analytical Method [17]

Solutions of clotrimazole ranging from 10-50 μg/ml in methanol were prepared. The absorbance of these solutions was measured at 260nm against a reagent blank (methanol) using UV/Visible Double Beam Spectrophotometer (Lab India 3000+) standard curve was constructed by plotting absorbance versus concentration in μg/ml

Drug Excipient Compatibility

Drug excipient interaction study was carried out by FTIR spectroscopy (Shimadzu, 8400-S) using KBr press palate technique. Clotrimazole was combined with cysteamine thiomer, and HPMC K100M (1:1 ratio) and stored at 40ºC for two weeks. IR spectra of clotrimazole and mixtures were recorded and studied

Preparations of Vaginal Tablets [18]

Tablets were prepared by using wet granulation technique. All the contents like HPMC, lactose, magnesium stearate, talc, thiomer were accurately weighed and granulated using 10% PVP in ethanol as binder solution. dough is formed. Wet mass was passed through sieve no 18 granules were oven dried at 40ºC for 30 min. These granules were compressed on 12-station rotary tablet compression machine (Rimek, Karnavati, India) using an 8-mm standard concave die punch set.

Optimization

Optimization was carried out using Simplex Mixture experimental design. Design-Expert (Version 10.4.0; Stat-Ease Inc., Minneapolis, Minnesota, USA) was used for mathematical modeling and assessment of the responses.

Then the optimum level of these variables was determined by Simplex Mixture design including center point which is the most accepted response surface methodology (RSM). The simplex centroid design for a 3-component system (A, B, and C) is represented by an equilateral triangle in a 2-dimentional space Fig. 3. The amount of cysteamine thiomer (A) HPMC K100M (B, and lactose C were selected as independent variables as shown in Table 2. clotrimazole was mixed with required quantity of polymer HPMC K100M (A), cysteamine Thiomer (B), and lactose (C) and mixed for 5 min, quantity of talc and magnesium stearate was kept invariant in all batches showed in Table 1.

The percent release values of drug after 24 h; T50 and Mucoadhesive force were selected as dependent variables. The levels of the three factors were selected on the basis of the preliminary studies carried out before implanting the experimental design. According to the Table 3 runs were examined.

Table 1: Trial runs of simplex centroid design for optimization of mucoadhesive clotrimazole tablet

 

Batch
code

clotrimazole
(mg)

A
(thiomer) (mg)

B
(HPMC K100 M)
(mg)

C
(Lactose)
(mg)

Talc
(mg)

Mg Stearate
(mg)

Total
(mg)

F1

100

40

80

40

3

3

306

F2

100

80

80

40

3

3

306

F3

100

40

120

40

3

3

306

F4

100

66.66

66.66

66.66

3

3

306

F5

100

40

40

80

3

3

306

F6

100

80

40

80

3

3

306

F7

100

120

40

120

3

3

306

Table 2: variables and levels

 

Independent variables

 

Symbol

Levels

0

1

 

X1 = Cystamine thiomer

X2 = HPMC K100M

X3 = Lactose

 

A

B

C

 

40

40

4

 

120

120

120

Dependent variables

Units

Constraint

Y1 = T50

Y2=Release aft. 24 h

Y3=Mucoadhesive Force

h

%

g

Minimize

Maximize

Maximize

Table 3: Formulation for trial runs of simplex centroid mixture design

Formulation batches

Drug

Thiomer

HPMC

Lactose

Mag stearate

Talc

F1

100mg

66.66

66.66

66.66

3

3

F2

100mg

40

120

40

3

3

F3

100mg

40

80

80

3

3

F4

100mg

40

40

120

3

3

F5

100mg

80

40

80

3

3

F6

100mg

80

80

40

3

3

F7

100mg

120

40

40

3

3

Evaluations of Tablets

Pre and Post Compression Properties

Pre-compression properties such as angle of repose, bulk density and tapped density, hausner’s ratio, and carr’s index were measured by methods reported in literature [19].

Formulated matrix tablets were evaluated for Thickness using Vernier caliper, Weight variation, Hardness (Monsanto hardness tester), Friability (Roche friabilator) Average drug content [19].

Measurement of Swelling Index [20]

The extent of swelling was measured in terms of percentage weight gain by the tablet. The swelling behavior of all formulations was studied. Each formulation tablet was weighed individually (W1) and placed separately in Petri dishes containing 25 ml of 0.1 N HCl. At regular intervals of 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 hrs. The tablet was withdrawn, soaked using filter paper without pressing. The swollen tablet was re-weighed (W2) and the swelling index of each tablet were calculating using the equation:


W2 W1 W1 *100 MathType@MTEF@5@5@+= feaagGart1ev2aqatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaaeaaaaaaaaa8 qacaWGxbGaaGOmaiabgkHiTmaalaaapaqaa8qacaWGxbGaaGymaaWd aeaapeGaam4vaiaaigdaaaGaaiOkaiaaigdacaaIWaGaaGimaaaa@3EF4@

In vitro Drug Release [21]

Drug release from tablets was studied out using USP dissolution rate test apparatus-II. The dissolution study for different formulations were conducted at 37 ± 0.5ºC and 20 rpm in 500 ml acetate buffer pH 4.6, and tested for drug release up to 24h. Five ml of sample was withdrawn at specific time interval and filtered through Whatman filter paper (Pore size 0.22 μm). Same amount of fresh dissolution media was replaced to maintain sink conditions, and absorbance was measured using Double beam UV-Spectrophotometer (LABINDIA® 3000+) at 260 nm and concentration of clotrimazole was calculated. The release studies were conducted in triplicate and the mean values were plotted verses time similarly dissolution of marketed mucoadhesive clotrimazole tablet was performed. And profile of F1 and marketed tablets were compared with help of similarity factor F2.

Ex vivo Mucoadhesive Force of Formulated Tablets

Mucoadhesive force was evaluated using a Texture Analyzer (CT3 Texture Analyzer, Brookfield Engineering Labs, Inc., Model Texture Pro CT V1.4 Build 17). Fresh sheep vaginal mucosa was obtained from a local slaughter house and was used within 2 h of slaughtering. The mucosal membrane was washed with distilled water and then with acetate buffer pH 4.6, subsequently it was carefully attached to a 10-mm cylindrical probe (TA 3/100 probe) using a double-sided adhesive tape. The upper platform was moved downward manually near to the tablet surface and then the mucosa was brought toward the polymer sample at a constant speed of 1mm/s until a predetermined compressive force of 0.5 N was applied with holding time 60 s with load cell 1000gm. The probe was then removed with return speed of 1 mm/s to a distance of 15 mm and maximum detachment force (N) was determined for each sample with data rate 15 points/sec. For each new sample, a different mucosa sample was used.

Drug Permeation Studies [15]

Drug permeation studies were done on clotrimazole tablet using Franz diffusion cellto check the permeation enhancement. the membrane used was sheep vaginal mucosa Simulated vaginal fluid was chosen as buffer medium. Aliquots of 1 ml were taken. Equal volume of medium was replaced to maintain sink conditions the samples were analyzed spectrophotometrically.

Antifungal Activity

It was done using 2 methods namely:
I. Ditch plate method
II. Cup plate method

Ditch Plate Method [22]

Considering poor solubility of CLO in various solvents “ditch plate method” was used to check efficacy of drug.

Ditch was made in plate containing SDA. in that ditch suspension of drug in sterile media was poured. These plates were streaked with micro organism. Plates were incubated for 24-48 hrs in incubator.

Cup Plate Method

Preparation of Test Sample

Mucoadhesive antifungal tablet of CLO is taken. It is suspended into100 ml conical flask containing simulated vaginal fluid. The flasks were kept in orbital shaker for 24 hrs. Samples were withdrawn after 1 h and 24 h. The withdrawn samples were suitably diluted with 10 % DMF solution and used to determine MIC.

Preparation of Std Solution

Same method as above described was used except instead of tablet std CLO was taken

Preparation of Media

1. Sabouraud Dextrose broth and agar DIFCO/OXOID was prepared according to standard procedure. Then media was sterilized by autoclaving at 121ºC for 30 min at 15 psi.

1. Fungi strain: Candida albicans ATCC 10231

Inoculum Preparation

1. Organism were sub cultured from fresh slants on SDA aseptically. Then they were incubated at 25ºC for 24 h.

2. Inoculum was prepared by picking some colonies of fresh culture from fresh Petri plate.

3. Colonies were suspended in 5 ml saline and matched with Mcfarland standard 0.5 [23].

Test Procedure

All procedures were done aseptically. Pour plate technique was carried out on which wells were bored using sterile T- Borer. Standard and test solutions were added in wells plates were kept at 25ºC for 24-48 hrs.

After incubation period plates were checked for zone of inhibition. Inhibitory action of solvents was checked by well method to check if itself has any antifungal action.

Results and Discussion

Characteristic features that prompted selection of xyloglucan for thiomerization are:[14,24]

1. Xyloglucan exhibit mucoadhesive property in native state.
2. Xyloglucan is useful as a sustained release polymer.
3. It is stable over wider pH and temperature ranges.
4. It can be oxidized to undergo ring opening generating reactive aldehyde group.
5. Xyloglucan is biocompatible and biodegradable.

The initial reaction of aldehydic xyloglucan with the 1⁰C amine led to formation of carbinol amine 3 which forms imine on dehydration. The imine was protonated under weakly acidic conditions to form an iminium ion 4. The iminium moiety was reduced by sodium cyanoborohydride to form thiolated xyloglucan (thiomer). The reaction was performed at pH 5 in order to generate large number of free thiol moieties.

Analytical Method

Clotrimazole followed linearity between concentration range of 10-50 μg/ml the equation of line was Y =0.01X+0.2618 and the R2 was 0.992 for methanol

For acetate buffer the equation of line was 0.0248x + 0.0274 and R2 was 0.9742

Drugexcipient Compatibility

Fig 1 depicts the IR spectra depicts no interaction between drug and excipients as the exclusive drug peak 3446 cm-1 (N-H stretch amidic group), 3361 cm-1 (-CH stretch aliphatic group), 742.9 cm-1 (-C-Cl stretch) were recognized with shift wave numbers. Absence of new peak and shift in existing peaks confirmed the lack of interaction between the drug and excipient.

Preparation of Tablets

Simplex mixture designs are especially useful in cases where the relative proportion of ingredients have effect on the performance properties of the formulation. In present case the mucoadhesive polymers HPMC K100M, xyloglucan cysteamine thiomer and diluent lactose are capable of regulating the mucoadhesion and drug release according to the relative proportion in which they are incorporated in formulation. The design allows estimation of the parameters of the quadratic model and requires fewer runs in case of three variables.

Evaluation of Tablets

Powder blends for 7 trial runs were prepared by using different amounts of thiomer, HPMC K100M and Lactose. These blends were assessed for micromeritic properties. Bulk density of powder blends was in range of 0.371-0.412g/ml which indicates good compressibility, angle of repose; 25.24⁰C - 30.02⁰C Hausner’s ratio 1.11 - 1.24 and Carr’s index found in range, , 9.81 % - 14.48% respectively which points to good flow behavior (Table 4).

Evaluation of tablets for different post compression parameters such as weight variation, hardness, thickness and friability was observed to be within the accepted limits (Table 4) [25]. The tablet weight varied between 306.3-306.7 mg which is within ± 7.5% allowance, hardness of tablets of all batches in range between 5.5 to 6.5 Kg/cm2. Maximum friability

Figure 1: IR spectra of A) clotrimazole B) physical mixture
Table 4: Micromeritics properties Table 5: Model F, P value and R2

   1

Bulk density (g/ml)

0.37±0.20

0.33±0.12

0.31±0.12

0.34±0.11

0.41±0.30

0.32±0.12

0.35±0.15

2

Tapped density (g/ml)

0.43±0.13

0.39±0.33

0.43±0.24

0.38±0.17

0.47±0.14

0.40±0.27

0.43±0.05

3

Angle of repose

25.24±0.14

27.32±0.18

30.02±0.11

29.11±0.13

28.04±0.12

29.31±0.17

27.08±0.07

4

Carr’s index

11.1±0.14

12.08±0.05

14.06±0.14

13.21±0.17

9.81±0.11

12.64±0.25

14.48±0.34

5

Hausner’s ratio

1.11 ± 0.12

1.14 ± 0.17

1.21 ± 0.02

1.17 ± 0.14

1.24 ± 0.08

1.17 ± 0.13

1.13 ± 0.11

6

Weight variation (±5%)

306.7±2.69

306.2±3.29

306.2±4.61

306.9±3.21

306.7±3.09

306.7±2.34

306.5±3.68

7

Hardness(Kg/Cm2)

6.02 ±0.11

5.71 ±0.07

6.26±0.21

6.30 ±0.14

6.12 ±0.15

5.56±0.24

5.6±0.16

8

Friability (%)

0.61

0.54

0.42

0.58

0.43

0.68

0.41

9

Average drug content

98.9

99.2

98.6

98.4

97.8

98.2

99.5

observed was 0.61% that is less than recommended 1%. In all batches drug content was found to be in the range of 97-99.5 %.

Swelling Index

Fig 2 shows there is slow and gradual increase in swelling index with time from 1 to 8 h and concentration of HPMC K100M, as thiomer does not swell instantaneously upon contact with water. It shows that tablets have good swelling power. Swelling of F2 batch is highest because it contains a higher amount of HPMC K100M. If swelling is more then it increases the path length required for water to travel inside the core of tablet, which helpful to sustain the release.

In vitro Drug Release

Fig.3 shows that effect of HPMC K100M, thiomer and lactose on drug release of Clotrimazole. Drug release decreased as the concentration of thiomer in tablet increased. Further, addition of HPMC K100M in the tablet also decreased the drug release and extended over a period of 12 h. addition of lactose in the tablet increased the drug release.

The dissolution study was carried out for all 7 formulations (F1- F7); the data showed T50 in the range of 15-17 h; release after 24 h was in the range of 62-95%. The release of Clotrimazole from all batches was variable and was in the range of 62-95%.The optimization of the formula using DOE technique was carried out using Design Expert 10.4 software. The concentration of thiomer, HPMC K100M and Lactose was optimized to achieve T50; release after 24 h. The software provided following equations for both the response parameters.

Figure 2: Swelling index of all batches
Figure 3: In vitro release of formulation batch F1-F7

Equation: T50=0.0893*A+0.10182*B-0.051818*C+1.44*AB- 1.14*BC-1.18*AC……………. (1)

Equations 1 and 2 shows the effect of variables on T50; andrelease after 12 h both the equations were statistically significant and followed the quadratic order. The T50values showed a increased with increasing values of HPMC K 100M and thiomer and the coefficients of both terms indicates a opposite magnitude of effect. But, as concentration of lactose increased there is decreased in T50and coefficient of both HPMC K 100M and lactose showed slighlty increased in T50.The T50 values showed a negative effect on both thiomer and lactose. The combined effect of both terms showed decreased with increasing values of thiomer and lactose. There was a mild interaction which shows and increase in T50 values.

Rel ease=0.44515*A+0.2170*B*0.53140*C+5.292*AB- 5.58*BC+3.167*AC………….(2)

The Release after 24 h values is also seen to decrease with increase in HPMC K 100M and thiomers concentration. The combined effect of both showed opposite effect. As the concentration of thiomer and HPMC K 100M increased the released after 24 h also increased. On the other hand, as the concentration of lactose increased drug released. The combined effect of both thiomer and lactose showed slightly positive effect on drug released.

Table 5 shows that Model F, P value and R2 which indicates that there is only a 2.31%, 0.16% and 04.70% chance that an F-value this large could occur due to noise for T50, release after 24 hr and mucoadhesive force respectively and model were significant. PValues for coefficients indicate that all model terms are within significant range.

Ex vivo Mucoadhesive Force

Fig 7 shows Mucoadhesive force was found in the range of 15- 21g (Fig.7). F1 batch showed highest mucoadhesive force because it contains highest amount of thiomer. The mathematical model obtained for mucoadhesive force was

Mucoadhesion=0.098136*A+0.078136*B+0.048*C+5.236*AB+4.6 11*BC-3.882*AC…….(3)

The equation was statistically significant and followed the quadratic order.This implies thiomer is the highest influencing factor for mucoadhesion followed by HPMC, lactose too shows some effect probably due to its ability to absorb water quickly. Interaction was observed between thiomer and HPMC and HPMC and lactose.

Fig 4,5,6 The response surface plots show pictorial view of interaction between variables effects on a particular response. Thiomer and HPMC individually as well as in combination increase T50 whereas coupling with lactose caused a reduction in T50curved surface for T50; release at 24 h was almost complete at high concentration lactose concentration, thiomer had moderate effect compared to HPMC. together due to rheological synergism these effected higher reduction in drug release.

Mucoadhesion was seen to be highest at high concentration of thiomer and was lower at high HPMC & lactose concentration and combination of HPMC and thiomer did not show any increase in percentage of mucoadhesion which may be due to reduction in covalent bonds at lower the thiomer concentration & lack of synergism as adhesion mechanism of both polymers is different concentration.

The desired criteria for T50 13-17 h; and release after 24 h 68- 89% mucoadhesive force 17-23 g were given to design expert for numerical optimization. This provided formulation solution with desirability 0.99. The formulation was recreated and the T50, releases after 24 h values and mucoadhesion were found to match the predicted results thus validating the statistical design (Table 5).

Figure 4: Response surface for effect variables on T50
Figure 5: Response surface for effect variables release at 24 h
Figure 6: Response surface for effect variables mucoadhesion
Figure 7: Mucoadhesion of all formulation batches
Table 5: Model F, P value and R2

Parameter

T50
(Hr)

Release after 24 hr
(%)

Mucoadhesive force
(g)

F-value

1078.43

21978

260.48

P-value

0.0213

0.0019

0.0470

R2

0.998

0.9998

0.9992

Validation of Optimized Model

Table 6 illustrates that list the composition of the optimized formulations, O1, O2, O3 suggested by software on the basis of desirability. Upon comparison of the observed responses with that of the anticipated responses, the predicted error varied between 0.40- 1.93 %. Thus, the low magnitudes of the error as well as the significant values of R2 in the current study indicated a high prognostic ability of response surface methodology.

Similarity factor between release profile of optimized batch and marketed vaginal tablet was 96.57 % hence it can be concluded our formulation has similar release as compared with marketed clotrimazole extended release mucoadhesive vaginal tablet (Fig 8)

Drug Release Mechanism

From the following table 7 it is seen that drug follows Peppas model as the r2 value for the model is maximum. Peppas model clt-THs follow case I type of release behavior. It displays the diffusion of drug from controlled release polymeric system. The drug transport may be attributed with stresses and state transition in hydrophilic glassy polymers leading to its swelling in water and in biological fluids. It is also comprised of polymer disentanglement and erosion.

Drug Permeation Studies

(Fig 9) Permeation studies demonstrate an increased permeation from optimized clotrimazole tablet over the dispersion containing xyloglucan and clotrimazole. This can be attributed to improved transmucosal permeation owing to effect of thiomer

Table 6: Validation of optimized model

Optimized batch

Composition

Predicted results

Experimental results

% Error

 

A
(mg)

B
(mg)

C
(mg)

T50
(h)

Release after 24 h (%)

Mucoadhesive Force (g)

T50

Release after 24 h

Mucoadhesive Force (g)

T50
(h)

Release after 24 h (%)

Mucoadhesive Force (g)

O1

120

40

40

05.3

83

20.5

05.1

82.3

20.3

0.20

0.76

0.5

O2

66.66

66.66

66.66

8.28

75

17.9

9.0

74.19

17.1

0.8

0.9

0.8

O3

40

120

40

11.26

60.2

15.4

12

59.461

14.89

0.8

0.80

0.23

Figure 8: In vitro release of marketed formulation and optimized batch
Table 7: Release model for formulation batches

Release model

F1

F2

F3

F4

F5

F6

F7

Zero order

0.9647

0.9632

0.9644

0.9678

0.9612

0.9678

0.9687

First order

0.957

0.955

0.957

0.956

0.956

0.958

0.9589

Peppas

0.989

0.988

0.986

0.987

0.990

0.9891

0.991

Higuchi

0.9012

0.9054

0.9026

0.9032

0.9058

0.9046

0.9088

Figure 9: Permeation studies
Table 8: Observation of zone of inhibition at various concentration

Sr No

Concentration

Hr

Formulation

Marketed product

Solvents ( 10 %DMF, methanol, propylene glycol, DMSO)

 

 

 

MIC (mm)

MIC (mm)

MIC (mm)

1

0.2µ/well

1 h

13

12

0

2

0.2µ/well

24 h

17

15

 

3

0.4µ/well

1h

19

18

0

4

0.4 µ/well

24 h

25

24

 

Figure 10: MIC of formulation in initial h
Figure 11: Ditch plate method

Antifungal Activity

As drug has poor solubility in water “ditch method” was used to check efficacy. Table 8 shows zone of inhibition at 0.4μ concentration at 1 h and at 24 hr. it can be seen that drug permeation plays important role here. As from formulation there is more release at initial hrs as compared to marketed mucoadhesive formulation. From 24 hr reading we can say that drug retains its efficacy for long period of time. Fig 10,11 shows zone of inhibition.

Conclusion

The mucoadhesive tablets of clotrimazole with thiomer, HPMC K100M and lactose were optimized using simplex centroid design. The Swelling index of the tablet was dependent on HPMC concentration whereas the mucoadhesion was predominantly affected by thiomer. In vitro antifungal activity on optimized formulation shows more effective antifungal action in initial hours as compared to marketed mucoadhesive tablet. Thiomer was found to have improved the antifungal activity.

  1. Ferrer J (2000) Vaginal candidosis: epidemiological and etiological factors. International Journal of Gynecology & Obstetrics 71 (Suppl 1): S21-S27.
  2. Lanchares  JL, Hernandez ML (2000) International Journal of Gynecology & Obstetrics 71(Suppl 1): S29-S35.
  3. Mendling W, Brasch J (2012) Guideline vulvovaginal candidosis (2010) of the German Society for Gynecology and Obstetrics, the Working Group for Infections and Infectimmunology in Gynecology and Obstetrics, the German Society of Dermatology, the Board of German Dermatologists and the German Speaking Mycological Society. Mycoses 55 Suppl 3: 1-13.
  4. Khan AB, Saha C (2014) A Review on Vaginal Drug Delivery System. RGUHS J Pharm Sci 4(4): 142-147.
  5. An imidazole derivative with a broad spectrum of antimycotic activity. It inhibits biosynthesis of the sterol ergostol, an important component of fungal cell membranes. Its action leads to increased membrane permeability and apparent disruption of enzyme systems bound to the membrane.
  6. Baloglu E, Senyigit ZA, Karavana SY, Bernkop-Schnürch A (2009) Strategies to Prolong the Intravaginal Residence Time of Drug Delivery Systems. J Pharm Pharmaceut sci 12(3): 312-336.
  7. Wang A, Wang Z (2011) Pharmaceutical overview of tamarind gum. Int J Macromolecular Sci 1(1): 15-18.
  8. Mishra A, Malhotra AV, (2009) Tamarind Xyloglucan: a polysaccharide with versatile potential. Journal of Material Chemistry 19: 8528-8536
  9. Lillford PJ, Rowlands DW, Lang P, Dentini M, Crescenzi V, et al. (1991) Structure and solution properties of tamarind polysaccharide. Carbohydrates Research 214: 299-314.
  10. Rao PS, Ghosh TP, Krishna S (1946) Extraction and purification of tamarind seed polysaccharide. J Sci Ind Res 4: 705.
  11. Kaur H, Yadav S, AhujaM, DilbaghiN (2012) Synthesis, characterization and evaluation of thiolated tamarind seed polysaccharide as a mucoadhesive polymer. Carbohydrate Polymers 90(4): 1543-1549.
  12. Rahmat D, Sakloetsakun D, Shahnaz G, Perera G, Kaindl R, et al. (2011) A design and synthesis of a movel cationic thiolated polymer. Int J Pharm 411(1-2): 10-17.
  13. Aney J, Maushumi K, Rashmi T (2010) Thiomrs: forms, features an formulations. Journal of Chemical and Pharmaceutical Rsearch 2(6): 316-323.
  14. 14. Iqbal J, Sakloetsakun D, Bernkop-Schnürch A (2011) Thiomers: Inhibition of cytochrome P450 activity. European Journal of Pharmaceutics and Biopharmaceutics 78(3): 361-365.
  15. Madgulkar AR, Bhalekar MR, Asgaonkar KD, Dikpati AA (2016) Synthesis and characterization of novel mucoadhesive derivative of xyloglucan. Carbohydrate polymers 135: 356-362.
  16. Mahajan H. Tyagi V, Patil RR, Dusunge SB (2013) Thiolated xyloglucan: Synthesis, characterization and evaluation as mucoadhesive in situ gelling agent. Carbohydrate polymers 91(2): 618-625.
  17. Silverstein RM, Webster FX, Kiemle DJ (2005) Spectrometric identification of organic compounds. John Wiley and Sons : 150-151.
  18. Ceschel GC, Maffei P, Lombardi Borgia S, Ronchi C, Rossi S (2001) Development of a Mucoadhesive Dosage Form for Vaginal Administration. Drug Development and Industrial Pharmacy 27(6): 541-547.
  19. Lachman L, Liberman HA (2009) The Theory And Practices Of Industrial Pharmacy. Cbs Publishers &Distributers Special Indian Edition 67-71: 296-300.
  20. Bernkop-Schnurch A, Konig V, Leitner V, Krauland A, Brodnik I (2004) Preparation and characterisation of thiolated poly(methacrylic acid)-starch compositions. Eur J Pharm Biopharm. 57(2): 219-224.
  21. Sumathi S, Ray AR (2002) Release behavior of drugs from polysaccharide tablets. J Pharm Sci 5(1): 12-18.
  22. (1996) M22-A2 Quality assurance of commercially prepared microbiological culture media. 2nd ed. Wayne, PA: NCCLS.
  23. McFarland J (1907) Nephelometer: an instrument for media used for estimating the number of  bacteria in suspensions used for calculating the opsonic index and for vaccines. JAMA 14: 1176-1178.
  24. Kumari A, Yadav SK, Yadav SC (2010) Biodegradable polymeric nanoparticles based drug delivery systems. Colloids and Surfaces B Biointerfaces 75(1): 1-18.
  25. (2014) The indian pharmacopoeia Govt of India, Ministry of health and Family welfare. Ghaziabad 1: 177-174.
  26. Grabovac V, Guggi D, Bernkop-Schnürch A (2005) Comparison of the mucoadhesive properties of various polymers. Advanced drug delivery 57(11): 1713-1723.
  27. Rahmat D, Muller C, Barthelmes J, Shahnaz G, Martien R, Bernkop-Schnurch A (2013) Thiolated hydroxyethyl cellulose: design and in vitro evaluation of mucoadhesion and permeation enhancing nanoparticles. Eur J Pharm Biopharm 83(2): 149-155.