Simulated Vaginal Fluid Research Articles

Dilution of Microbicide Gels With Vaginal Fluid and Semen Simulants: Effect on Rheological Properties and Coating Flow

Microbicides are agents applied topically to the vagina to prevent HIV transmission. Microbicide products formulated as semi-solid dosage forms or "gels" coat vulnerable tissue to deliver active ingredients. Effective microbicide delivery vehicles must have appropriate rheological properties to ensure appropriate deployment in vivo. Microbicide products become diluted by fluids in the vagina after application; dilution affects vehicle rheological properties and mechanics of vaginal distribution, thus affecting efficacy. To simulate the changes that might occur after application, this study analyzed the effects of small dilutions (10-30%) with vaginal fluid and semen simulants on three semi-solid vaginal formulations: a cellulose lubricant (KY Jelly), a polyacrylic acid moisturizer (Replens), and a carrageenan prototype microbicide (Carraguard). Rheological behavior was characterized using cone-and-plate rheometry. Data were fitted to either the power-law, Carreau, or Herschel-Bulkley model. Rheological parameters from these fits were input to models of coating flow due squeezing, and the simulated area coated output from these models was used to compare the responses of the different formulations to the two diluents for varying degrees of dilution. There were differences in the responses of the three materials to dilution. Even small dilutions altered the rank order of vaginal coating rates compared to the undiluted formulations.

Biophysical Analysis of Prototype Microbicidal Gels

The objective of this study was to evaluate the distribution and retention (deployment) of four prototype vehicles for delivery of prophylactic microbicides against vaginal HIV transmission. Study gels were created with different molecular compositions, producing different biophysical properties governing vaginal deployment. The study employed three techniques: direct rheological measurement of gel properties, direct observation of gel surface coating erosion, and dissolution by a vaginal fluid simulant, and mathematical modeling of gel squeezing flow processes. Results suggest significant differences in extent of vaginal coating after gel application and in erosion of these gel layers due to contact with ambient vaginal fluid and shearing. The relationships between gel rheological properties, coating flow and erosion of coating were not always anticipated from differences in gel molecular composition.

Temperature and pH Sensitive Hydrogels: An Approach Towards Smart Semen-Triggered Vaginal Microbicidal Vehicles

Microbicides are drug delivery systems (DDSs) for the prevention of sexual transmission of HIV and other STDs. A topically applied vaginal microbicidal gel should provide uniform coating of vaginal tissue, retention of this gel layer prior to intercourse, and controlled release kinetics of antivirals to inactivate the viral load potentially introduced during sexual activity. Here, we describe the microbicide-oriented characterization of a DDS made with a dual pH sensitive and thermosensitive smart polymer gel composed of a random terpolymer of N-isopropyl acrylamide, butyl methacrylate, and acrylic acid. The system was engineered to coat vaginal tissue with a stable gel layer and to release entrapped model agents in a burst release profile in response to the presence of the infecting agent: semen. The gel rheology, layer erosion properties, model drug release kinetics, and cytocompatibility of the terpolymer system were studied. Negligible erosion of the gel in the presence of vaginal fluid simulant suggests prolonged retention. Burst release of molecular and macromolecular model compounds was observed when the system's pH changed from the vaginal pH to the pH of semen, and cytotoxicity studies showed that the terpolymer is equally cytocompatible as a commonly used polymeric vaginal carrier.

Erosion of microbicide formulation coating layers: Effects of contact and shearing with vaginal fluid or semen

An effective vaginal microbicide formulation must distribute and maintain an epithelial coating layer. The post-application durability of this coating is significantly affected by the vaginal environment. A new in vitro assay quantified coating layer erosion after contact and shear with simulated vaginal fluid or semen. Coating layer persistence and viscosity of both fluid and gel layers were assessed versus time. Five vaginal formulations were studied. In all gels, there was an overall trend of rapid (∼30 min) and significant viscosity loss. Although there were differences across gels and between simulants, greater erosion occurred after contact with the low-pH vaginal fluid simulant (>50% viscosity decrease), as compared to an alkaline semen simulant. These in vitro results suggest significant differences in vivo of vaginal coating retention by the test gels. This new assay can be diversified to create a spectrum of biologically relevant conditions which collectively simulate the natural history of vaginal formulation residence.

Characterisation and in vitro evaluation of bioadhesive liposome gels for local therapy of vaginitis

The purpose of this study was to design and evaluate a new vaginal delivery system for the local treatment of vaginitis. Liposomes containing two commonly applied drugs in the treatment of vaginal infections, namely clotrimazole and metronidazole, were prepared by the proliposome and the polyol dilution methods. Both types of liposomes were characterised and compared for particle size, polydispersity, entrapment efficiency, and tested for in vitro stability in media that mimic human vaginal conditions (buffer, pH 4.5, and vaginal fluid simulant). To achieve application viscosity and to further improve their stability, liposomes containing drugs were incorporated in a bioadhesive gel made of Carbopol ® 974P NF resin. In vitro release studies have demonstrated that even after 24 h of incubation in vaginal fluid simulant (at 37 °C) more than 30% of the originally entrapped clotrimazole (or 50% of metronidazole) was still retained in the gel. Storage stability studies have proved the ability of Carbopol ® 974P NF gel to preserve original size distributions of incorporated liposomes. All the performed experiments confirm the applicability of bioadhesive liposome gels as a novel delivery system for local therapy of vaginal infections.

Development and in vitro evaluation of a liposomal vaginal delivery system for acyclovir

Design of a liposome delivery system for vaginal administration of acyclovir, able to provide sustained release and improved bioavailability of the encapsulated drug for the local treatment of genital herpes was investigated. Acyclovir was encapsulated in liposomes prepared by the polyol dilution method, whereby various phospholipid compositions were used: egg phosphatidylcholin (PC)/egg phosphatidylglycerol (PG) 9 : 1, egg phosphatidylcholine (PC) and egg phosphatidycholine (PC)/stearylamine (SA) 9 : 3. All liposome preparations were characterized and compared for particle size, polydispersity, encapsulation efficiency and tested for in vitro stability in different media chosen to simulate human vaginal conditions: buffer, pH 4.5 (corresponding to normal human vaginal pH), vaginal fluid simulant (medium developed so as to mimic the fluid produced in the vagina) with or without mucin. To be closer to in vivo application of liposomes and to achieve further improvement of their stability, liposomes were incorporated in a vehicle suitable for vaginal self-administration. Bioadhesive hydrogel made from Carbopol 974P NF resin with adequate pH value and desirable viscosity was chosen as a vehicle for liposomes containing acyclovir. In vitro release studies of liposomes incorporated in the hydrogel proved their applicability as a novel vaginal delivery system with localized and sustained release of encapsulated acyclovir. Even after 24 h of incubation in vaginal fluid simulant more than 35% of the originally encapsulated drug was retained in the hydrogel.

Preparation and Evaluation of Proliposomes Containing Clotrimazole

Clotrimazole (CT)-containing proliposomes were prepared by penetrating an ethanol solution of CT and Egg phosphatidylcholine (PC) into microporous sorbitol particles, followed by vacuum evaporation of the solvent. As a result, CT proliposomes with free-flowing flowability were obtained. On contact with water, the proliposomes were rapidly converted into a liposomal dispersion, in which a certain amount of CT was entrapped by the liposomes. The result in scanning electronic micrograph confirmed the formation of liposomes structures from proliposomes, and the particles revealed round or ellipse. The ratio of drug to total lipid, ratio of PC to cholesterol and ratio of lipid to sorbitol affected the entrapment efficiency (EE%). The EE% of optimized formulation (CT 10 mg, 0.1 g total lipid, PC/CH ratio is 60 : 40 and 1 g sorbitol) in this investigation was 96.2+/-1.5%. The proliposomes system can provide sustaining release in simulated vaginal fluid at 37+/-1 degrees C for 24 h. In-vivo performance of blank proliposomes, a physical mixture of sorbitol and drug, clotrimazole proliposomes and commercial ointment formulation were evaluated using antifungal activity test. At 7 d post-dose, the c.f.u. of C. albicans decreased in proliposomes-treated groups than ointment and the physical mixture (t-Student, p<0.05). The results indicated that CT-containing vaginal proliposomes prolonged drug release and may increase amount of drug retention into the mucosa to result in more antifungal efficacy. In addition, CT-proliposomes did not affect the morphology of vaginal tissues. Therefore, the dosage form might be further developed for safe, convenient, and effective treatment of vaginal candidasis with reduced dosing interval.

Preparation, in Vitro and in Vivo Evaluation of Liposomal/Niosomal Gel Delivery Systems for Clotrimazole

Clotrimazole, which is an imidazole derivative antifungal agent, was widely used for the treatment of mycotic infections of the genitourinary tract. To develop alternative formulation for the vaginal administration of clotrimazole to provide sustained and controlled release of appropriate drug for local vaginal therapy, liposomes/niosomes were evaluated as delivery vehicles. To optimize the preparation of liposomes/niosomes with regard to size and entrapment efficiency, multilamellar liposomes/niosomes containing drug were prepared by lipid hydration method. The prepared liposomes/niosomes were incorporated into 2% carbopol gel, and the systems were evaluated for drug stability in phosphate-buffered saline (pH 7.4) and simulated vaginal fluid at 37 ± 1°C. Further, the vesicle gel system was evaluated by antifungal activity and tolerability on tissue level in rat.

Effect of temperature and pH on contraceptive gel viscosity

The rheological properties of four commercially available spermicidal gels (two polyacrylic acid derivatives and two carboxymethylcellulose based) and their dilutions with a vaginal fluid simulant (pH 4.2) and a semen simulant (pH 7.7) were measured at 25°C and 37°C over a biologically relevant range of shear rates. All four gels were shear thinning with temperature-dependent rheological properties. The two types of gels responded differently to dilution. The rheological properties of the polyacrylic acid derivative gels were strongly dependent on the type of diluent used. Their viscosities after dilution with the semen simulant were 100 times greater than after comparable dilutions with the vaginal fluid simulant, this effect being due primarily to the higher pH. The cellulose gels did not exhibit such an effect. These results suggest that the polyacrylic acid and cellulose gels interact differently with the vaginal environment in vivo. Such differences could lead to differences in the extent and durability of epithelial coating.

Characterization of a female controlled drug delivery system for microbicides

In this study, the feasibility of a carbopol 934P-HPMC-based gel formulation as a vaginal drug delivery system was evaluated. A vaginal fluid simulant (VFS) was utilized to simulate human vaginal mucus. The viscosity of the carbopol-HPMC gel system and VFS was examined using a cone and plate viscometer. The surface tension of VFS was measured using a capillary rising method. Differential scanning calorimetry (DSC) was employed to investigate the effect of carbopol gel on the conformational changes of rat vaginal membrane. The viscosity of carbopol gel increased as the pH of the system increased from 4.0 to 6.0. The viscosity of HPMC gel remained the same irrespective of pH of the system. The viscosity of the carbopol gel significantly increased, when HPMC (0.5–1.5%) was added to the system. The optimal viscosity value (1.5∼2.0 poises) was achieved at 1.0∼1.5% carbopol (pH 4.0) with the presence of 1.0∼1.5% HPMC. There were mucin concentration dependent changes in viscosity and surface tension of VFS. The results of DSC analysis of rat vaginal membrane showed that the profiles of thermal stability for both carbopol-treated and the untreated control at the temperature ranging from 40 to 90°C were almost identical. The thermal denaturation temperatures (Td) of the carbopol-treated membrane and the untreated control were not significantly different from each other. The combination of carbopol and HPMC seems to be an ideal formulation as a vaginal delivery system. Mucin played an important role in the regulation of viscosity and surface tension of VFS. DSC study demonstrated that carbopol gel showed good biocompatibility and did not cause any conformational changes in rat vaginal membrane.

Rheological evaluation of thermosensitive and mucoadhesive vaginal gels in physiological conditions

The timely gelation and retention of in situ-gelling vaginal formulations would be fundamental to improve the efficacy of drugs. In this study, various rheological properties of clotrimazole gels were evaluated for predicting their performance in vagina. Two kinds of thermosensitive and mucoadhesive formulations were composed of poloxamer 407 (P407, 15%), polycarbophil (0.2%), and different amounts of P188 (15 vs. 20%). Both formulations were Newtonian at 20 °C but non-Newtonian at 37 °C. Although both liquid formulations gelled below the vaginal temperature, they differed in gelation time and viscoelastic properties in the presence of vaginal fluid simulant. At body temperature, the formulation with 20% of P188 gelled within 35 s but it took two times longer for the other one gelled. Upon dilution with simulated vaginal fluid, the formulation with 20% of P188 retained the rheology of a gel, but the other one lost the viscoelastic properties typical for a gel. Moreover, after dilution with simulated vaginal fluid, the elastic modulus was orders of magnitude higher in the formulations with 20% of P188 relative to the other one. These results indicate that the rheological evaluation at the physiologic conditions needs to be preceded to develop more effective in situ-gelling vaginal formulations.

Assemblies for In Vitro Measurement of Bioadhesive Strength and Retention Characteristics in Simulated Vaginal Environment

ABSTRACTThe vaginal route of administration offers a promising option for local and systemic delivery of drugs. Conventional vaginal formulations are associated with limitations of poor retention, leakage, and messiness, thereby causing inconvenience to users. To overcome these limitations, formulations that adhere to the vaginal mucosa for a sufficient period of time need to be developed. Bioadhesion and retention are desirable characteristics of a vaginal formulation to achieve desired efficacy. These properties can be built in during formulation development by the use of bioadhesive polymers. In the present study, assemblies for in vitro measurement of bioadhesive strength and retention characteristics of vaginal formulations have been developed. A modified simulated vaginal fluid (SVFM) was used to simulate vaginal conditions for bioadhesion studies. Cellophane hydrated with SVFM and isolated sheep vaginal mucosa were used as model membranes. The bioadhesive potential of various polymers and their combinations was evaluated. Among the polymers evaluated, xanthan gum (XG), sodium alginate (SA), Polycarbophil (PC), and their combinations (XG + SA and XG + PC) were found to possess significant bioadhesive strength. In retention experiments, XG, SA, and combinations (XG + SA and XG + PC) were retained in isolated sheep vaginal tissue, while PC exhibited poor retention under experimental conditions. Based on the results of the study conducted, XG, SA, and combinations (XG + SA and XG + PC) have been proposed as potential candidates for developing bioadhesive vaginal drug delivery systems.

Comparison of the rheological properties of Advantage-S and Replens

The rheological properties of Advantage-S and Replens were measured at body (37°C) and room temperature (25°C) over a range of physiologically relevant shear rates. The viscosity of Replens was found to differ from that of Advantage-S, particularly at room temperature. In addition, the two materials differed in their miscibility with a vaginal fluid simulant.

Rheological properties of contraceptive gels

The rheological properties of 4 commercially available contraceptive drug delivery gels and their dilutions with a vaginal fluid simulant were measured. These properties govern the critical functions of spreading and retention of these gels over the vaginal surfaces. Measurements made on Conceptrol, KY Plus, Gynol II, and Advantage-S included stress growth, stress relaxation and residual stress, and the shear rate dependence of viscosity. All gels exhibited non-Newtonian behavior including shear thinning and viscoelasticity. Conceptrol and Gynol II had no residual stress, while both KY Plus and Advantage-S did. The gels differed in their response to dilution with vaginal fluid simulant.