Microbicide Product

Octylglycerol is a low molecular weight compound that has been shown to have in vitro activity against STD’s including HIV without negatively effecting Lactobacillus crispatus. In order to successfully deliver the active agent octylglycerol, it must be formulated into a stable, safe, and effective dosage form. A liposomal cream formulation was chosen to prolong the residence time of the octylglycerol inside the vagina, and to provide means of targeted delivery to the infectious agents. Lipid to monoglyceride ratio was optimized to maximize in-vitro efficacy while sparing innate micro flora. This formulation was assessed for anti-HIV , HSV, and N. gonorrhoeae activity in vitro. In all cases the active formulation had effectiveness whereas the placebo exhibited no activity. Physical stability of the formulation was assessed by monitoring pH, viscosity, particle size, weight, odor, and appearance. Acceptable physical and chemical stability of the formulation was found for all evaluations made. Studies were conducted to identify the potential for hydrolysis of lipid in this formulation. No hydrolytic degradation products were found when the formulation was evaluated at a number of pH values. These studies showed the chemical and physical stability of the liposomal formulation, as well as the confirmation of its maintained efficacy against HIV, HSV-1, HSV-2, and N. gonorrhoeae. Therefore, a rectal safety assessment study was initiated using the Pigtail Macaque model.

Introduction

Approximately 12 million individuals in the United States are infected with Sexually Transmitted Diseases (STDs) each year. Since current prevention strategies have been only partially successful, the use of topical microbicides offer a new approach to the prevention of STDs, since the product can be self-administered by women before sexual intercourse. An ideal microbicide would prevent STDs caused by bacterial pathogens such Chlamydia trachomatis and Neisseria gonorrhoeae, as well as those caused by HIV, HPV, HSV and the parasite Trichomonas vaginalis. In addition, the formulation should be safe to the vaginal epithelium and rectum, have a pH close to that of the vagina, cause no disruption to the normal vaginal flora, and present rheological stability. One potential active ingredient for such formulations was found in human breast milk. Human breast milk contains numerous antimicrobial compounds, some of which are lipid based. Previous studies showed that milk lipids could inactivate a number of bacteria and enveloped virus, including HIV. Although the exact mechanism is not clear, it has been suggested that the fatty acids and their monoesters are incorporated into the lipid membrane, causing destabilization of the lipid bilayer. Some of these antimicrobial lipids were modified to increase their stability and aqueous solubility while maintaining their antimicrobial activity. Preliminary studies have showed that the synthesized lipid octylglycerol (1-O-octyl-sn-glycerol) showed 100% killing of C. trachomatis serovar D and F (106 inclusion-forming units) at a concentration of 15mM after 30min of contact. In addition, studies have shown no vaginal irritation in the rabbit model by lipid concentrations as high as 120mM.

Liposomes can be utilized as a novel drug delivery system. They can be formulated to prolong the residence time of the active agent within the targeted site of action. The formulation of octylglycerol utilizing this technology, offers a promising and novel microbicide product.

Objectives

Viscosity Measurements: Viscosity was monitored to ensure physical stability. Changes in viscosity would indicate destabilization of the product. These evaluations were conducted on a Brookfield DVIII+ cone/plate viscometer, spindle CP41, at 30rpm.

pH Measurements: pH measurements were made using an AccuFet solid state probe calibrated at pH 4 and 7.

Vial Weight: Vial weights were monitored to assess product dehydration. Product was packaged in sealed glass vials.

In vitro release: A 15mm Franz-cell system was set up at 37 ° C, using a synthetic membrane (Spectropor 1). The receptor phase used was PBS pH7.4. Samples were removed every 30 minute for the first 2 hours and then every hour for a total period of 6h. The samples were analyzed by gas chromatographic (GC) method developed for the quantitation of octylglycerol. In this method the lipid is extracted from an aqueous medium using a chloroform:aqueous medium at a 1:4 ratio. The chloroform layer is then evaporated dry and 100uL of BSTFA added. The GC column used was a 0.25 mm diameter DB17 from J&W Scientific. Injection temperature was set a 250°C. The temperature program for the column starts at 100°C and increases at a rate of 10°C/min up to a temperature of 250°C. The temperature is kept at 250°C for 7 minutes.

Microbiological evaluations: Bacteria were cultured on an appropriate medium overnight at 37°C in air plus 5% CO2. Isolated colonies were suspended in saline containing 0.5mM ACES buffer pH 7.0 to a density of 0.5 Mcfarland units, diluted 1:10 in the same buffer and 10 µl of cells added to 90 µl the test solutions and incubated as above for 30 min. Samples were taken (25 µl) and place on the appropriate medium and spread using an inoculation loop. Samples containing 10 or more colonies after over night incubation were considered positive. Liposomes were diluted 1:10 in ACES buffered saline mixed thoroughly and assayed for killing as described above.

HSV evaluation: Between 105 and 106 tissue culture infective doses of HSV-1 or HSV-2 were mixed with the liposomes and incubated for 1hour at 37 degrees in the presence of 1% fetal bovine serum. After incubation , the infectivity of each mixture was titrated by the serial dilution endpoint method. Dilutions (10-fold) were inoculated into mono-layers of Vero cells. The plates were examined for 3-5 days and examined daily for cytopathic effect.

HIV evaluation: 2 x 105 PHA-stimulated PBMC were platted per well on a 96 well plate and centrifuged at 800 rpm for 5 minutes to pellet the cells at the bottom of the well. Plates were held in the incubator until ready to be inoculated with the virus. 100mL of HIV (50,000 p24 equivalent) was incubated for 30 minutes at 25°C with equal volume of test material. Centrifuged at 10,000rpm for 15 min, filtered and centrifuged at 22,000 rpm for 1h. Pelleted viruses were then resuspended in 200mL of the RPMI medium containing 20% FCS and 5% natural IL2 and inoculated onto PBMC in the 96 well plate. Following 5 days of incubation at 37°C, production of HIV was monitored by measuring HIVp24 using the antigen capture assay (Dupont).

Lipid Stability evaluation: Phosphatidylcholine can be hydrolyzed into stearic acid and lyso-phosphatidylcholine. The integrity of the phosphatidylcholine in the liposomal octylglycerol formulation was evaluated at the 4 weeks time point using a normal phase HPLC assay.

Safety test in monkeys: Product safety was assessed in the pigtailed macaque model. Product was administered rectally daily for three days and effects examined 30 minutes and 24 hours after each application. pH and rectal lavage evaluations were made.

Liposome Product Formulation: Liposome formulations containing various lipid to octylglycerol ratios were formulated (data not shown). Based upon in-vitro test results the final octylglycerol content was chosen as 1%.

Results

See our Octyglycerol Study!