ABSTRACT
OBJECTIVE: An effective vaginal microbicide against sexual HIV transmission remains elusive, with requirements for adherence to appropriate application of effective, nontoxic products being a major deterrent. We explored methods to enable sustained release of combinations of antiretroviral microbicides, utilizing intravaginal rings composed of biosoluble Acacia gum or nonbiodegradable hydrogel of 2-hydroxyethyl methacrylate and sodium methacrylate, materials approved for use by the US Food and Drug Administration. DESIGN AND METHODS: The reverse transcriptase inhibitors TMC120, PMPA, 3'-azido-3'-deoxythymidine, and a newly characterized anti-HIV agent, Boc-lysinated betulonic acid, were incorporated into vaginal rings with different combinations. Daily and cumulative release rates of these inhibitors in ring eluates were determined by high-performance liquid chromatography, gas chromatography, or immunoassay. Anti-HIV effects were measured by assessment of p24 Gag antigen in T-cell cultures exposed to HIV-1 isolates. RESULTS: Drug release rates were sustained at concentrations higher than the minimum effective dose for HIV inhibition. The release was maintained for no less than 15 and 28 days from the Acacia gum and 2-hydroxyethyl methacrylate and sodium methacrylate rings, respectively. Boc-lysinated betulonic acid showed more than 90% inhibition of HIV-1 infection in H9 cells, with little toxicity to normal cells. CONCLUSION: The intravaginal rings described here are capable of efficacious drug delivery. Incorporation of several antiretroviral agents, including betulinol derivatives, which act at multiple levels of the HIV life cycle, may provide a synergistic effect to achieve higher efficacy on the inhibition of HIV infection.
Subject(s)
Anti-HIV Agents/administration & dosage , Drug Delivery Systems/instrumentation , HIV Infections/prevention & control , Reverse Transcriptase Inhibitors/administration & dosage , Administration, Intravaginal , Chromatography, Gas , Chromatography, High Pressure Liquid , Contraceptive Devices, Female , Female , Gene Products, gag/metabolism , Gum Arabic/therapeutic use , Humans , Hydrogels/therapeutic use , Immunoassay , Methacrylates/therapeutic use , Treatment OutcomeABSTRACT
Betulonic acid, derived from betulinol, a pentacyclic styrene, has shown a highly specific anti-prostate cancer activity in in vitro cell cultures. However, due to the lack of solubility of betulonic acid in aqueous medium, its potent anti-cancer activity in vivo has not been determined to the fullest extent. The present study describes the chemical synthesis of hydrophilic Boc-lysinated-betulonic acid, which has improved its solubility in an aqueous biocompatible solvent. Evaluation in cytotoxicity assays, Boc-lysinated-betulonic acid dissolved in phosphate-buffered saline (PBS) containing 22% ethanol and 4% human serum albumin, has shown 95.7% inhibition of LNCaP prostate cancer cells in culture after 72 h incubation at a concentration of 100 microM, but with little effect on normally proliferating fibroblast cells. In the in vivo assay, male athymic mice transplanted with human prostate LNCaP xenografts were injected with Boc-lysinated-betulonic acid intraperitoneally at a dose of 30 mg/kg daily for 17 days. The treated mice exhibited 92% inhibition of tumor growth as compared to controls. Histological sections of the tumors showed that Boc-lysinated-betulonic acid arrested mitosis and induced apoptosis, which was confirmed by TUNEL assay, Yo-Pro-1 staining, and the release of cleaved caspase-3 from the ex vivo in tumor culture. These studies, for the first time, demonstrate that a non-toxic hydrophilic lysinated derivative of betulonic acid and its solubility in a biocompatible aqueous medium has enhanced the bioavailability of the drug and has thus unleashed its full anti-prostate cancer activity.
Subject(s)
Antineoplastic Agents/pharmacology , Lysine/chemistry , Oleanolic Acid/analogs & derivatives , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Caspase 3 , Caspases/drug effects , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Hydrophobic and Hydrophilic Interactions , Immunohistochemistry , In Vitro Techniques , Lysine/analogs & derivatives , Male , Mice , Mice, Nude , Mitosis/drug effects , Molecular Conformation , Oleanolic Acid/chemical synthesis , Oleanolic Acid/chemistry , Oleanolic Acid/pharmacology , Prostatic Neoplasms/pathology , Solubility , Stereoisomerism , Structure-Activity Relationship , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: To determine the effect of immunization with bovine luteinizing hormone receptor (LH-R) on ovarian function of cats. ANIMALS: 9 adult female domestic cats. PROCEDURE: 7 cats were immunized with 0.5 mg of LH-R encapsulated in a silastic subdermal implant (3 x 10 mm); 2 served as control cats. Receptors had 80% specific binding to 125I-human chorionic gonadotropin with a binding capacity of 2,682 pM/mg. Cats received booster injections of LH-R. Cats were induced to ovulate with luteinizing hormone (LH) releasing hormone on day 345. Samples of venous blood and vaginal cells were collected through day 395. Observation of estrus behavior continued until day 516. Serum concentrations of estradiol, progesterone, thyroid gland hormones, LH, and LH-R antibody were determined. RESULTS: LH-R antibody was detected in the sera of immunized cats within 21 days after implantation. Detection of LH-R antibody was associated with suppression of serum progesterone to < or = 0.5 ng/mL during the study period, compared with concentrations of 5 to 10 ng/mL in control cats. Immunized cats did not display signs of estrus. Release of LH after administration of LH-releasing hormone indicated an intact hypothalamic-pituitary axis but poor corpus luteum function. Serum estradiol concentrations remained between 30 to 40 pg/mL in immunized and control cats. With the decrease antibody titers, hormone concentrations returned to a pattern consistent with that during fertility. CONCLUSIONS AND CLINICAL RELEVANCE: Active immunization with LH-R suppressed corpus luteum function in cats. The effect was reversible. An LH-R-based antifertility vaccine may have clinical application in other vertebrates.
