Formulation Design and Evaluation of Clindamycin Microspheres in aBioadhesive Gel for Local Therapy of Vaginal Candidiasis

INTRODUCTION

Vaginal candidiasis is a most fungal disease and also one of the most common vaginal infections in women, in the fertile period. Many psychological and emotional stress related problems are associated with vaginitis such as reduced immunity, prolonged antibiotic therapy, use of contraceptives, malnutrition, pregnancy, obesity, diabetes etc.¹ The infectious vaginitis is of 3 types: candidiasis, trichomoniasis and bacterial vaginosis.^2,3 Vaginitis is defined as a spectrum of conditions that cause vaginal and sometimes vulvar symptoms, such as itching, burning, irritation, odour, and vaginal discharge.⁴

Vaginal route of drug delivery is considered as a preferred route for the delivery of drugs within or through the vaginal mucosa for local or systemic pharmacological action. Creams, tablets, gels, suppositories, ointments, etc. are commonly used as vaginal drug delivery systems.⁵ Bioadhesive vaginal delivery systems have several advantages when compared to conventional dosage forms, so they are preferred for this route. The formulations are readily localized in the region of application thus improving the bioavailability of drugs.⁶

Clindamycin is an antibiotic useful for the treatment of several bacterial infections. It is also a second antimicrobial agent for the treatment of bacterial vaginitis. Chemically, clindamycin is Methyl 7-chloro-6,7,8-trideoxy-6-{[(4R)-1-methyl4-propyl-L-propyl]amino}-1-thio-L-threo-αDgalacto-octopyranoside.7 Clindamycin may be bacteriostatic or bactericidal in action. It acts by inhibiting protein synthesis at the level of the bacterial ribosome. The antibiotic binds preferentially to the 50S ribosomal subunit and affects the process of peptide chain initiation. The conventional formulations of vaginal drug delivery systems (VDDS) are associated with poor retention due to the self- cleansing action of vaginal tract, leading to poor compliance. To overcome this problem, bioadhesive polymers are added to VDDS and formulated. Clindamycin loaded microsphere in a bioadhesive gel for the local therapy of vaginal candidiasis to provide enhanced therapeutic effect.

Microspheres are defined by a monolithic spherical structure with the drug or therapeutic agent distributed throughout the matrix either as a molecular dispersion or as a dispersion of particles having a size range of 1-1000 µm.^8,9 The spherical shape of microspheres also in-creases the surface area which increases the bioavailability of dosage form.⁹ It provides con-stant and prolonged therapeutic effect and reduced GI toxic effects and dosing frequency. Therefore patient compliance is increased. The morphology of microsphere allows a controllable variability in degradation and drug release.¹⁰

Shekhar K et al. worked on the development of Cefotaxime sodium microcapsules for the treatment of microbial disease. From the study, it shows that the release ratios were very slow in as we go on increasing the polymer ratio, resulted in sustained release of the drug and showed more effective and better patience compliance than conventional drug delivery systems.¹¹ Ahmed F et al. developed acid buffering bioadhesive vaginal (ABBV) gel for the treatment of mixed vaginal infections. The ex vivo retention study showed that the bioadhesive polymers hold the gel for 12-13h inside the vaginal tube. The in vivo antimicrobial study indicated that the ABBV gel had better antimicrobial action than the commercial intra vaginal drug delivery systems.¹² Akhani Jolly R et al. worked on development and evaluation of Nystatin loaded thermo-sensitive mucoadhesive in situ gel for vaginal drug delivery using thermo-sensitive polymers. The results suggested that the formulation prepared using the combination of Pluronic F127 (15%w/v), Pluronic F68 (15%w/v) and HPMC K4M (1%w/v) showed good bioadhesive strength, mucoadhesion force with improved antifungal activity and sustained pattern of drug release.¹³ The aim of present work is to formulate and evaluate Clindamycin Microspheres in a bioadhesive gel for local therapy of Vaginal Candidiasis and to analyse the data statistically.

MATERIALS AND METHODS

Clindamycin (Yarrow Chem Products, Mumbai); Eudragit RS 100, Eudragit RL 100 (Evonic Deggusa PVT LTD, Mumbai); Magnesium Stearate, n- hexane, Acetone (Loba chemie, PVT LTD, Mumbai); and Liquid Paraffin (Nice chemicals PVT LTD, Kerala). All other chemicals used were of analytical grade.

PREFORMULATION STUDY

Solubility analysis

A saturated solution of the drug was prepared in respective solvents and shaken under ambient conditions for 24 h. At the end of 24 h, 1 ml of the solution was pipette out and the concentration (mg/ml) was determined by using UV Spectrophotometer.

Determination of λmax

A solution of Clindamycin containing a concentration of 10 µg/ ml was prepared in me-thanol and UV spectrum was taken using Shimadzu (UV 1800) double beam spectrometer and scanned between 200-400 nm. The maxima obtained in the graph were considered as λmax for the drug Clindamycin.

Sample preparation and analysis by FTIR14

IR spectra of drug and excipients were taken using FTIR spectrophotometer (Jasco 4100 type A). The drug and polymer were mixed physically in 1:1 ratio and mixtures were stored in an oven at 400 C and 75 % relative humidity in closed containers for one month. FTIR spectrum of samples was taken by using the KBr pellet method. Pellets of samples and KBr were prepared using hydraulic press and scanned over the wave number range 4000-600 cm-1 at the ambient temperature.

Sample preparation and analysis by DSC15

The samples were prepared by a physical mixture of drug and excipients (1:1) using a clean dried glass mortar and pestle. Samples 5-10 mg were accurately weighed and hermetically sealed in aluminium pans. Thermograms were obtained using Shimadzu (DSC 60) instrument, heating at a constant rate of 10 ml/min, over a temperature range of 40-6000 C. To maintain on inert atmosphere, nitrogen gas was purged at a rate of 10 ml/ min.

Development of Analytical method

Preparation of the Simulated Vaginal Fluid¹⁶

The pH of the mixture was adjusted to 4.2 using 0.1 m HCL

Standard stock solution

Accurately weighed the quantity of 100 mg Clindamycin was dissolved in 5ml methanol using 100 ml volumetric flask and volume was made up to 100 ml with 4.2 pH SVF to produce 1000 µg/ml of solution.

Sub stock solution

5 ml of solution was pipette out from the stock solution in 100 ml volumetric flask and the volume was made up with 4.2 SVF. This gives the concentration of 50µg/ml. 0.6, 1.0, 1, 4, 1.8, 2.2, 2.6 and 3 ml of stock solution were pipette out in 10 ml volumetric flask and the volume was made up with PH 4.2 SVF to give the concentration of 3, 5, 7,9,11 and 15µg/ml of the solution of Clindamycin. The absorbance was measured in the UV visible spectrophotometer (Shimadzu -1800) at 207nm using 4.2 pH SVF as blank. 

Preparation of microspheres by Solvent Evaporation Method¹⁷

Clindamycin microspheres were prepared by a solvent evaporation method using Eudragit RS-100 and Eudragit RL-100 with different drug/ polymer ratios. Weighed quantity of polymer was dissolved in 30 ml of acetone with stirring. 1g of drug and 100 mg magnesium stearate dispersed in the polymer solution. The resultant milky white dispersion was poured into a vessel containing a mixture of 270 ml liquid paraffin and 30 ml of n-hexane. Then stirred for 5h using a homogenizer fitted with a four-blade “butterfly” propeller with a diameter of 50 mm, stirring was continued for 3h at 1000 rpm or until the acetone was completely evaporated. Following removal of acetone, the resultant microspheres were harvested by vacuum filtration after which they were washed four times with 25 ml of n-hexane and dried at room temperature for 24h.

Preparation of Gel

Accurately weighed quantity of 1g carbapol 934P was dispersed in distilled water by continuous stirring for 15-20 min with the help of glass rod in which 10 g of glycerol was previously added. Mixture was stirred until thickening occurred and neutralized by drop wise addition of 50% w/w triethanolamine. Mixing was continued until a transparent gel appeared.

Incorporation of microspheres in a gel

Microsphere containing Clindamycin incorporated into the 1 % w/w carbopol 934P gel. Mixing well by using an electrical mixer at 25 rpm for 2 min to get Clindamycin micro-sphere incorporated gel.

EVALUATION OF MICROSPHERES

Appearance

The general appearance of microsphere was identified visually. It includes size, shape, and colour.

Particle size

Particle size analysis carried by optical microscopic method. A minute quantity of microspheres was dispersed in glycerine and then spread on clean glass slide and average size of 100 microspheres was determined in each batch.

Percentage yield

Practical yield was calculated as the weight of Clindamycin microspheres recovered from each batch in relation to the sum of starting material. The percentage yield of prepared Clindamycin microsphere was determined by using the formula:

% yield = Actual weight of product/ Total weight of excipients and drug X 100

Determination of percentage drug entrapment efficiency (PDE)¹⁸

The percentage drug entrapment of each batch was calculated as per the following formula: % PDE= Practical drug content/ Theoretical drug content X 100

Theoretical drug content was determined by assuming that the entire CD present in of the polymer solution used, gets entrapped in CD microspheres. And no loss occurs at any stage of preparation of CD microspheres and the percent drug loading was calculated by using the formula:

% loading = Weight of drug/Weight of Microspheres X 100

Practical drug content was analysed by using the following procedure, Weighed amount of CD microspheres equivalent to 100 mg of CD was dissolved in 100 ml of pH 4.2 SVF. This solution was kept overnight for the complete dissolution of the CD in SFV. This solution was filtered and further diluted to make a conc. of 10μg/ml solution. The absorbance of the solu-tion was measured at 207nm using UV Visible spectrophotometer against SVF as blank and calculated for the percentage of drug present in the sample.

Scanning Electron Microscopy (SEM)¹⁹

It is used to determine particle size distribution, surface topography, texture and to ex-amine the morphology of the fractured of sectioned surface. SEM studies were carried out by using the JEOL JSM 6380 LA (Japan). Dried Clindamycin microspheres were place on an electron microscope brass stub and coated with an iron sputter. Picture of Clindamycin microspheres were taken by random scanning of the stub.

In vitro dissolution studies of Microspheres

Clindamycin microspheres containing 100 mg equivalent weight of CD were placed in the basket of the dissolution vessel containing 900 ml of pH 4.2 SVF as dissolution medium at 50 rpm. The dissolution media were maintained at the temperature of 37±0.50 C. 2 ml of the dissolution media was withdrawn at predetermined time intervals and same amount of fresh dissolution media was replaced. The withdrawn samples were filtered through Whatmann filter paper and the absorbance was measured using UV visible spectrophotometer at 207 nm. Dissolution profile was analysed by plotting drug release versus time plot.

EVALUATION OF GEL

Physical appearance

Clindamycin incorporated gel were visually inspected for colour, clarity, homogeneity presence of particles and fibres.

Determination of pH²⁰

The pH of Clindamycin microsphere incorporated carbopol gels were determined by using digital pH meter. One gram of gel was dissolved in 25 ml water and the electrode was then dipped into gel formulation for 30 min until constant reading obtained.

Drug content analysis

Accurately weighed gel equivalent to 10 mg of drug was suspended in 25ml of SVF. The volume was made up to 100 ml. After proper dilution absorbance was measured using UV 207 nm.

Drug content uniformity

To ensure the homogeneity of drug content in the formulation of gel, five tubes were sampled from the different locations in the mixer and assayed for the drug content.

Extrudability study²¹

The prepared gel was based upon the quantity of percentage gel extruded from the tube on application of certain load. The formulation under study was filled in a clean, lacquered aluminium collapsible one ounce tube with nasal tip 5mm opening. It was then placed in between two glass slides and was clamped and extrudability was determined by weighing the amount of gels extruded through the tip when a constant load of 1 kg was placed on the slides and gels extruded was collected and weighed.

Spreadability²²

Spreadability of Clindamycin incorporated gels were determined 48 h after preparation, by measuring two 20x20 cm glass plates after 1 min. The mass of the upper plate was stan-dardized at 125 g. The spreadability was calculated by using the formula

S= m.l/t,

Where ‘S’ is spreadability,

‘m’ is the weight tied to the upper slide,

‘l’ is the length of the glass slide, and t is the time taken.

Viscosity measurement²³

A. Brookfield (DV-II+) viscometer were used to determine the viscosity in cps of the microsphere incorporated gel. The gel was placed in the sample holder and the suitable spindle selected was lowered perpendicularly in to the sample. The spindle attached to the viscometer and then it was allowed to rotate at a constant optimum speed at room temperature and readings of the viscosity of the formulation were measured after 2 minutes.

In vitro release studies of Gel²⁴

A modified open diffusion cell was used for drug release from the CD-MG. Egg membrane was used as the permeation barrier. The membrane was soaked overnight in SVF before the study. 1g of gel was kept carefully between the donor and receptor compart-ment. The donor compartment as empty and open to the atmosphere but the receptor com-partment contained 25 ml of pH 4.2 SVF as dissolution medium. The dissolution medium were maintained at 37± 50 C and stirred on a magnetic stirrer with a stirring speed of 25 rpm. 1 ml of dissolution media was withdrawn at predetermined time interval (every 1h) and replaced with equal volumes of fresh medium. The absorbance of the samples was analysed by UVSpectrophotometer at 207 nm.

In vitro Antimicrobial Activity²⁵

Antimicrobial activity of CD-MG and placebo gel was evaluated against Candida albicans j10 by using cup plate method.

Cup Plate Method

The composition of Sabouraud’s dextrose agar was taken in a 250 ml of conical flask and was dissolved in 100 ml of distilled water. The pH was adjusted to 5.6. The medium was sterilized in an autoclave at 15lbs for 15 minutes. After completion of sterilization, the medium was kept aside at room temperature. 0.5 ml diluted suspension in Nacl 0.9% were added to 100 ml of medium at 47± 20C and used as inoculated layer. The medium (20ml) was poured in to a sterilized petri dish to give a depth of 3-4 mm, and was assured that the layer of medium is uniform in thickness by placing petri dish on a levelled surface. Petri dish was divided in to two sectors. After solidifying the medium at room temperature, with the help of a sterile cork borer, cups of 6mm diameter were punched and scooped out from the petri dish. Each bore in different sector was loaded with equal quantity of the placebo gel. The petri dish was then incubated for 24 hours at 370 C. After incubation the zone of inhibition was measured.

Stability studies²⁶

Formulated F8.CD-MG incorporated gel was kept at a temperature of 400 C±20 C and Relative Humidity (RH) 75%RH±5%RH for a period of six months. Initial, third month, six months the samples were evaluated for parameters such as colour, pH, extrudability, spreadability, viscosity, drug content and in vitro drug release.

RESULTS AND DISCUSSION

The purpose of the present study was to formulate Clindamycin microspheres incorporated bioadhesive gel. Clindamycin microspheres were prepared by the solvent evaporation method. The polymers Eudragit RS 100 and Eudragit RL 100 were selected for microsphere preparation as they are insoluble in aqueous media but are permeable and have the ability to produce pH-independent drug release profile and have release rate.

They are the copolymers of acrylic and methacrylic acid containing low amounts of quaternary ammonium groups, approximately 5% and 10% for RS and RL respectively. The quaternary ammonium groups in the RS and RL chemical structures play an important role in controlling drug release because they relate to water uptake followed by the swelling of the polymers.

Magnesium stearate was used as a dispersing agent. The use of magnesium stearate as a dispersion agent decreased the interfacial tension between the lipophilic and hydrophilic phases of the emulsion and further simplified the formation of microspheres. As the solvent evaporated, the viscosity of the individual droplets increased, and highly viscous droplets were observed to coalesce a faster rate than they could be separated. Magnesium stearate formed a thin film around the droplets and thereby reduced the extent of coalescence, before hardening of the microspheres, on the collision of the droplets.

Liquid paraffin was selected as a bulk or outer phase since Clindamycin and Eudragit RS/RL are only very slightly soluble in liquid paraffin. Acetone has a dielectric constant 20.7 and was therefore chosen as the dispersed or inner phase since solvents with dielectric constants between 10 and 40 show poor miscibility with liquid paraffin.

During the production of the microsphere, it was observed that stirrer speed less than 1000 rpm were not sufficient to produce microspheres, and a huge coalesced mass was obtained. This is due in part to inadequate agitation of the media to disperse the inner phase in discreet droplets within the bulk phase. A stirring speed above 1000 rpm, the turbulence caused frothing and adhesion of the microsphere to the container walls and propeller blade surfaces, resulting in high shear and smaller size of the dispersed droplets. Spherical microspheres were obtained at a stirring rate of 1000 rpm; therefore this speed was used during the production of all microspheres.

PREFORMULATION STUDY

Solubility analysis

The solubility of the CD in 10mg/ml of solvent was carried out and it revealed that it is freely soluble in methanol and alcohol.

λmax determination

A solution of the CD containing concentration 10μg/ ml was prepared in methanol and ab-sorbance was taken using UV-Visible spectrophotometer. The λmax for CD was found to be 207 nm.

FOURIER TRANSFORM INFRARED SPECTROSCOPY AND DIFFERENTIAL SCANNING CALORIMETER STUDY

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR studies were carried out to order to investigate the possible interaction be-tween drug and polymers. IR spectrum for pure drug and physical mixture of drug polymers were obtained and analysed for principle peaks at 3199cm1 (OH stret-ching), 3094cm-1 (CH stretching), 1531cm-1 (NO2 asymmetric stretching), 1364cm1 (NO2 symmetric stretching), 1147cm-1 (C-O stretching), 824cm-1(NO2 stretching).

Differential Scanning Calorimetry (DSC) 

DSC curve obtained for pure Clindamycin, Eudragit RL100, and their mixture are shown in the fig.4 and 5 respectively. Pure Clindamycin powder showed a melting endotherm at 88.430 C. A physical mixture of Clindamycin and Eudragit RL100 showed melting endotherm at 93.20 C. These endotherm peaks showed that there is no interaction between the drug and the polymer.

Formulation studies

The popular method for the encapsulation of drugs within water-insoluble polymers is the emulsion solvent evaporation method. This technique offers several advantages and is preferred over other preparation methods such as spray drying, sonication, and homogenization because it requires only mild condition such as ambient temperature and constant stirring. Thus a stable emulsion can be formed without compromising the activity of the drug. Both polymers are soluble in acetone, so acetone was used as a solvent. Liquid paraffin was used as a dispersion medium. Eight formulations were prepared, to investigate the effect of the increasing amount of the polymer and dispersing agent on the microsphere formation, the drugpolymer, and drug dispersing agent ratio was altered while the amount of solvent and stirring speed were kept constant.

Evaluation of Microspheres

Percentage yield and Percentage drug entrapment efficiency

The percentage yield for CD microspheres were 80.2%, 80.30%, 77.31%, 91.12%, 81.92%, 92.91%, 82.06%, and 92.03% for formulation F1, F2, F3, F4, F5, F6, F7, F8 respectively (Table 4). Entrapment efficiency increases with an increase in polymer concentration. Entrapment efficiency was found to be in the range of 65.12% to 82.10%. The results obtained are given in the (table 4). A maximum of 82.10% drug entrapment efficiency was obtained for the formulation F6 which contain 1:3 drug-polymer ratios.

Mean particle size

Particle size was highly influenced by the stirring speed and the polymer concentration. As shown in (Table 5) it was observed that as the polymer concentration increased, the particle size also increased; so the mean particle size for the formulation F6 (458±0.35), F3 (420±1.66) was higher when compared to all other formulations. It was observed that when the speed of the stirrer was below 1000 rpm, there was no formation of the spherical microsphere. This could be due to inadequate agitation to disperse the inner phase in the total mass. Therefore stirring speed was maintained at 1000 rpm.

Actual drug loading and theoretical drug loading

In vitro drug release studies 

The drug release from batches F1-F8 (Fig 8) indicates that as the polymer concentration is increased the release rate decreased. The formulation F8 containing 1:0.25:1.75 con-centration of drug polymer shows maximum of 90.06% drug release at 12 h compared to other formulations. The release rate for other formulations was found to be 64.18% (F1), 62.52% (F2), 54.28% (F3), 79.16% (F4), 72.38% (F5), 69.07% (F6), 84.18% (F7), and 90.06% (F8).

Scanning electron microscopy

Scanning electron microscopy of the formulation F8 was carried out. It was observed that the microsphere was spherical with a smooth surface (fig 9) indicating that CD was well dispersed inside the carrier. It is also evident that the microspheres exhibited slightly porous surfaces probably due to the high concentration of drug in the microspheres.

EVALUATION OF MICROSPHERE INCORPORATED GEL 

Drug content and uniformity

The drug contents of the prepared gels were found to be 65.18% and 80.10% for F7 CD-MG and F8 CD-MG respectively indicating the applications of the present method for the preparation of novel Clindamycin bioadhesive gel system with high drug content uniformi-ty.

pH measurement

The pH of the gels was found to be 6.6 and 6.5 which is within the limit of the semisolid specifications. The almost neutral pH reflected that the gel will be non- irritant to the vagina.

Spreadability

The spreadability plays an important role in patient compliance and helps in uniform ap-plication of gel to the skin. A good gel takes less time to spread and will have high spreadability.

Extrudability

The extrusion of gel from the tube is important during application and for patient com-pliance. Good extrudability was observed for the prepared gel.

Viscosity

Viscosity is an important parameter for characterizing the gel as it affects the spreadability, extrudability, and release of the drug. The data’s are given in the Table 7.

In vitro drug release studies

The in vitro drug release profile fig 11.indicated that the release from microsphere retarded by incorporating in gel network. The formulation F8 CD-MG containing drug-polymer ratio 1:0.25:1.75 showed 90.12% drug release at 8hr.

Antimicrobial activity

Comparative antimicrobial activity study of selected CD-MG with placebo gel.

The antimicrobial activity of CD-MG and placebo gel was evaluated by cup plate method. The placebo gel did not show any zone of inhibition. The zone of inhibition was observed with the CD-MG formulation containing loaded microspheres. Antimicrobial study with the Sabouraud culture showed that the CD-MG was capable of control the growth of Candida albicans for more than 14h. Zone of inhibition of CD-MG was found to be19mm. Fig 12 shows the Zone of inhibition.

Stability studies 

Stability studies were done according to ICH guidelines. The prepared gel F8.CD MG was packed in aluminium collapsible tube and kept at a temperature of 400 C± 2°C and relative humidity 75%RH±5%RH for a period of 6 months. Initial and third month studies were done for the following parameters viz, pH, drug content, drug content uniformity, extrudability, spreadability, viscosity and in vitro drug release and results were mentioned in Table 8. Stability studies will be continued further up to six months.

CONCLUSION

From the above experimental results, it can be concluded that Preformulation studies like solubility, melting point, and UV analysis of CD were complied with standards. The FTIR spectra revealed that, there was no interaction between polymers and CD. Surface smooth-ness of the CD microspheres was increased by increasing the polymer concentration, which was confirmed by SEM. As the drug to polymer ratio was increased, the mean particle size of CD microspheres was also increased. Entrapment efficiency increase with an increase in polymer concentration. The in vitro release study revealed that the drug release decreases with an increase in the polymer concentration. The melting point of the CD was estimated by DSC and found to comply with the IP standards. The in vitro release study of the CDMG revealed that drug release is increased with theoptimum concentration of drug/ polymer ratio. Antimicrobial study with Sabouraud Culture shows that the F8.±CD-MG was capable to control the growth of Candida albicans for more than 14hr. Stability studies of the formulation were carried out as perICH guide lines. The best formulation F8. CD-MG was subjected to stability studies at 400 C±20 C/75%RH ± 5% RH for 6 months. The physical stability was assessed by the appearance and chemical stability by a change in the drug content and drug release studies for the initial and third month. The results showed that there were no significant changes in the drug content and the in vitro drug release studies. The stability studies will be continued further according to ICH guidelines. From the present study, it may be concluded that the ability of the system to adhere to the vaginal mucosa for an extended period of time as well as to improve the drug availability has great appeal for the convenient treatment of vaginal candidiasis.