Sustained release of microbicides by newly engineered vaginal rings.

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.

Development of vaginal rings for sustained release of nonhormonal contraceptives and anti-HIV agents.

OBJECTIVES: Construction of vaginal rings to deliver nonhormonal contraceptives and 3'-azido-3'-deoxythymidine (AZT) as an anti-HIV agent and determination of their daily release and efficacy in vitro. MATERIALS AND METHODS: Intravaginal rings of 0.5-0.7 cm rim and 5-5.5 cm in diameter were cast in the molds. The rings were composed of biosoluble acacia gum or nonbiodegradable hydrogel of 2-hydroxyethyl methacrylate (HEMA) and sodium methacrylate (SMA) [P(HEMA-co-SMA)]. The rings were impregnated with nonhormonal contraceptives such as ferrous gluconate to cause spermiostasis, l-ascorbic acid to increase the viscosity of the cervical mucus, and pharmalytes of pH 4-5 or AZT. RESULTS: The daily release rate of nonhormonal contraceptives as well as AZT from the rings was efficacious in vitro. Cumulative effect of nonhormonal contraceptives showed complete spermiostasis within 30 s up to 10 and 28 days, respectively. Daily release of AZT from both rings was also likely to be efficacious to inhibit HIV proliferation in vitro for 10 and 28 days, respectively. CONCLUSION: The data indicate that the rings described here can be developed for intravaginal delivery of nonhormonal contraceptives and/or anti-HIV agents.

Boc-lysinated-betulonic acid: a potent, anti-prostate cancer agent.

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.

Expression of a recombinant bifunctional protein from a chimera of human lutropin receptor and human chorionic gonadotropin beta-subunit.

Human lutropin (hLH) and human chorionic gonadotropin (hCG) are structurally and functionally similar and play important roles in reproduction via a common gonadal receptor (LH-R). However, hormone specific hCG-beta subunit contains 24 additional amino acid carboxyl terminal peptide (CTP), which produce specific antibodies to hCG-beta with little cross-reaction with LH. A chimeric protein containing both hLH-R and hCG-beta would provide a unique bifunctional antigen for immunocontraception. In this study is described the synthesis of a chimeric DNA construct of full-length of hLH-receptor and hCG-beta and its expression in Sf9 cells to produce a bifunctional protein. Recombinant protein was recognized by antibodies to LH-R as well as anti-hCG-beta in Western Blots, thus indicating the preservation of immunological epitopes for both hLH-R and hCG-beta in the chimera. Specific ligand binding of recombinant hLH-R component was demonstrated by the displacement of bound labeled hCG at increasing concentrations of unlabeled hCG, indicating that, the presence of hCG-beta component of the chimera did not interfere with the binding of hCG to LH-R. hCG-beta was also present in the recombinant chimeric protein as shown by a specific hCG-beta chemiluminescence assay. Treatment of transfected Sf9 cells with hCG induced dose-dependent increase in the stimulation of intracellular cAMP production, which showed that the ligand binding had functional activity. These results demonstrate that the chimeric DNA construct of hLHR-hCG-beta expressed a bifunctional protein containing both hLH-R and hCG-beta activities, which could provide a unique potential antigen for immunocontraception in vertebrates.

A hypothetical mathematical construct explaining the mechanism of biological amplification in an experimental model utilizing picoTesla (PT) electromagnetic fields.

We seek to answer the conundrum: What is the fundamental mechanism by which very weak, low frequency Electromagnetic fields influence biosystems? In considering the hydrophobicity of intramembranous protein (IMP) H-bonds which cross the phospholipid bilayer of plasma membranes, and the necessity for photonic recycling in cell surface interactions after dissipation of energetic states, present models lack structure and thermodynamic properties to maintain (DeltaE) sufficient energy sources necessary for amplifications by factors of 10(12). Even though one accepts that the ligand-receptor association alters the conformation of extracellular, extruding portions of IMP's at the cell surface, and that this change can be transmitted to the cytoplasm by the transmembranous helical segments by nonlinear vibrations of proteins with generation of soliton waves, one is still unable to account for repair and balanced function. Indeed, responses of critical molecules to certain magnetic field signals may include enhanced vibrational amplitudes, increased quanta of thermal energies and order inducing interactions. We may accept that microtrabecular reticulum-receptor is associated with actin filaments and ATP molecules which contribute to the activation of the cyclase enzyme system through piezoelectricity. Magnetic fields will pass through the membrane which sharply attenuates the electric field component of an EM field, due to its high impedance. Furthermore, EM oscillations are converted to mechanical vibrations; i.e., photon-phonon transduction, to induce molecular vibrations of frequencies specifically responsible for bioamplifications of weak triggers at the membrane surface, as well as GAP junctions. The hydrogen bonds of considerable importance are those in proteins (10(12)Hz) and DNA (10(11)Hz) and may be viewed as centers of EM radiation emission in the range from the mm microwaves to the far IR. However, classical electrodynamical theory does not yield a model for biomolecular resonant responses which are integrated over time and account for the connection between the phonon field and photons. Jacobson Resonance does supply an initial physical mechanism, as equivalencies in energy to that of Zeeman Resonance (i.e., zero-order magnetic resonance) and cyclotron resonance may be derived from the DeBroglie wave particle equation. For the first time, we view the introduction of Relativity Theory to biology in the expression, mc(2)=BvLq, where m is the mass of a particle in the 'box' or 'string' (molecule in a biosystem), c is the velocity of electromagnetic field in space, independent of its inertial frame of reference, B is the magnetic flux density,v is the velocity of the carrier or 'string' (a one or two dimensional 'box') in which the particle exists, L is its dimension (length) and q represents a unit charge q=1C, by defining electromotive force as energy per unit charge. Equivalencies suggest that qvBL is one of the fundamental expressions of energy of a charged wave-particle in magnetic fields, just as Zeeman and cyclotron resonance energy expressions, gbetaB and qhB/2pim, and is applicable to all charged particles (molecules in biological systems). There may exist spontaneous, independent and incessant interactions of magnetic vector B and particles in biosystems which exert Lorentz forces. Lorentz forces may be transmitted from EM field to gravitational field as a gravity wave which return to the phonon field as microgravitational fluctuations to therein produce quantum vibrational states that increase quanta of thermal energies integrated over time. This may account for the differential of 10(12) between photonic energy of ELF waves and the Boltzman energy kT. Recent data from in vivo controlled studies are included as empirical support for the various hypotheses presented.

Effect of immunization with bovine luteinizing hormone receptor on ovarian function in cats.

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.