Richard Allen Cone - US grants

Rat cauda epidiidymal (CE) sperm are stored immotile in the epididymis and become vigorously motile when ther are ejaculated. We found that these sperm are mechanically immobilized in the cauda epididymis by "immobilin", a large mucin-like glycoprotein that makes the epedidymal fluid highly viscoelastic. During ejaculation, immobilin is both diluted and degraded, greatly reducing the biscoelasticity of the fluid and thereby initiating sperm motility. In this grant application we propse to continue our research on the initiation of sperm motility. Our major aims are: 1) To fully characterize the rheological properties of CE fluid using an oscillating ball microrheometer. 2) To carry out a thorough biochemical analysis of immobilin, including the characterization of its carbohydrate side chains and the disulfide bonds and other crosslinks that help to produce high viscoelasticity. 3) To determine the cellular changes that occur in a sperm cell as it first begins to swim by using selective extracellular electrodes to measure changes in sperm cell properties such an membrane potential, cytoplasmic calcium activity, and respiration rate, and by using other investigate the "probing" mechanism by which a sperm cell detects the change in external viscoelasicity that allows it to initiate motility. For example, we will analyze motility initiation using high speed and high resolution video tapes to determine how the sperm probes its enviroment, and how the probing mechanism responds to different rheological properties of the surrounding fluid. 5) Finally, we will investigate how the mechanisms of motility initiation in other animals compares to the mechanism in rat. Our motivation for pursuing this research is to contribute to the development of better baarrier methods of contraception.

The overall aim of this project is to characterize the mechanisms by which a rhodopsin molecule excites the visual receptor. In the present proposal there are three major objectives: (1) Recent work in this project has demonstrated that low concentrations of oxidizing agents can rapidly close the light-regulated cation channels ("PL") in isolated rod outer segments (ROS), and that exceedingly low concentrations of reducing agents, as well as membrane impermeant sulfhydryl binding reagents, can rapidly open cation channels ("PSH") in the isolated ROS. With the evidence available to date, the SH-sensitive permeability, PSH, is indistinguishable from the light-regulated permeability, PL, so perhaps both represent the same cation channel. Thus, the first aim of this project is to isolate and characterize the molecules in the ROS plasma membrane that contain these highly sensitive SH groups. The binding of less than 10,000 SH reagents/ROS opens PSH. This is such a small #/ROS that in addition to radioactive labels, two new labeling techniques will be tried: colloidal gold particles (individually visible in EM), and/or fluorescently labeled methacrylate microspheres (individually visible in fluorescence LM) will be functionalized with SH seeking reagents and then reacted with isolated ROS to determine rates of reaction, total number/ROS, and spatial distribution of these SH sites. Patch-clamp methods will be used to attempt to purify, identify, and characterize the channels responsible for PL and PSH. (2) In addition to studying these SH-containing molecules, attempts will be made to identify other molecular sites that are highly susceptible to oxidation upon isolation of the ROS. It seems likely that some of the degradative diseases of photoreceptors may result from diminished protection or replacement of these "most labile" sites. The rates of both lipid peroxidation and SH oxidation will be measured in isolated ROS exposed to various concentrations of oxidizers and compared with the rates at which the oxidizers close the PL channels. (3) As in previous years of this project, the role of diffusion in setting the timecourse of visual excitation will continue to be investigated. The effects of experimentally inducing changes in the viscosities of the disc membrane and the cytoplasm will be evaluated by monitoring both the receptor potential and the concurrent light-scattering signals. Fluorescence photobleaching and recovery techniques will be used to quantitate the changes induced in the viscosities.

This Program Project will study entry paths used by STD pathogens, develop and test human monoclonal antibodies as microbicides for blocking pathogen entry, and characterize how antibodies are deployed and function in preventing infectious entry of pathogens in the female reproductive tract. Project 1. Mucosal entry paths used by STD pathogens will investigate entry paths that topical microbicides must blocks: (a) Cell vectors, such as HIV-infected leukocytes, may penetrate genital epithelial and carry HIV directly to target cells in the lymph nodes. (b) Microtrauma that occurs during consensual intercourse, increasing the risk that pathogens will contact target cells. (c) Upper tract exposure. Uterine peristalis causes uptake of vaginal fluids that may expose the upper reproductive tract to STD pathogens. Project 2. Blocking STD pathogen entry with mucosal antibodies will develop monoclonal human antibodies as highly potent and specific microbicides. Pharmacokinetics of a "plantibody" against HSV-2 (a human monoclonal produced in corn for large scale, inexpensive production) will be determined in mouse and rabbit vaginas. New human monoclonals will be developed against HPV, and against leukocyte cell vectors for HIV. Protective efficacy will be tested in vitro and in SCID mouse models. Using mouse monoclonals to identify adhesins and host receptors, human monoclonals against chlamydia and gonorrhea will be generated and tested in mice. Monoclonals will also be tested for their ability to markedly reduce a commensal bacterium in the mouse vagina. Project 3. Uptake of Ig by vaginal epithelial cells will investigate how cervico-vaginal epithelial cells (in tissue culture and in vivo_ deploy Ig in the vaginal epithelium, and the effects of physiological modulators (hormones, cytokines) on Ig uptake, storage and release. Human monoclonals identified in Project 2 will be applied to explants of human genital tissues and immortalized epithelial cell lines, to determine whether monoclonals that have been taken up by epithelial cells are effective for blocking HIV, HSV-2, and chlamydia infections, and for blocking adhesions and transepithelial migration of leukocytes.

[unreadable] DESCRIPTION (provided by applicant): The "Duet" is a cervical barrier device that, unlike a diaphragm, delivers gel on both its upper and lower surfaces to protect the vagina as well as the cervix. This application proposes 3 related projects: [unreadable] [unreadable] 1) "BufferGel-Duet", a single-use product for protection against STIs, HIV/AIDS, BV, and pregnancy. (a) Complete development of manufacturing steps to produce Duet for clinical efficacy trials. (b) Complete development of packaging, stability, and shelf-life testing of BufferGel-Duet. (c) Seek FDA permission via ReProtect's BufferGel IND to pursue clinical trials of BufferGel-Duet for preventing STIs (chlamydia, gonorrhea, trichomoniasis, and HIV), BV, and for contraception. [unreadable] [unreadable] 2) Reusable Duet, a low-cost, and maximally protective barrier device for use in low resource areas. Submit IDE to FDA for Duet as an OTC cervical barrier device for use with microbicides. [unreadable] [unreadable] 3) Membrane permeant acids: Determine whether lactic and acetic acid can safely increase microbicide efficacy. Lactobacilli acidify the vagina to pH~4 by secreting lactic and acetic acids. At this pH these acids can rapidly inactivate pathogens by permeating cell and bacterial membranes, but these permeant acids can also be toxic to mucosal surfaces. We hypothesize that adding lactic and acetic acids at physiological concentrations to microbicides formulated at pH~4 will speed acid-inactivation of pathogens without causing significant toxicity. To test this hypothesis: (a) Measure in vitro the speed at which lactic acid and acetic acid at vaginal pH (~4) inactivate sexually transmitted pathogens, BV-associated microbes, and sperm, (b) Add physiological concentrations of these acids to BufferGel (which uses an impermeant buffering gel to reinforce vaginal acidity) and measure the extent to which they increase its protective efficacy in animal models for herpes, chlamydia, and trichomonas. (c) Test whether adding these acids to BufferGel increases vaginal toxicity, or susceptibility to herpes or chlamydia, by using sensitive tests in mouse and rabbit models. [unreadable] [unreadable] RELEVANCE: The aim is to maximize protective efficacy of microbicides that women can use for protection against STIs and HIV. The products will also prevent pregnancy and help prevent BV. BufferGel is now in clinical trials for contraception (used with a diaphragm) and HIV prevention (used alone). "Duet" covers the cervix for maximum protection of this susceptible site for infections, and delivers microbicide gel to protect both the cervix and vagina. The reusable Duet will be low in cost and useful for low-resource areas. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The "Duet" is a cervical barrier device that, unlike a diaphragm, delivers gel on both its upper and lower surfaces to protect the vagina as well as the cervix. This application proposes 3 related projects: [unreadable] [unreadable] 1) "BufferGel-Duet", a single-use product for protection against STIs, HIV/AIDS, BV, and pregnancy. (a) Complete development of manufacturing steps to produce Duet for clinical efficacy trials. (b) Complete development of packaging, stability, and shelf-life testing of BufferGel-Duet. (c) Seek FDA permission via ReProtect's BufferGel IND to pursue clinical trials of BufferGel-Duet for preventing STIs (chlamydia, gonorrhea, trichomoniasis, and HIV), BV, and for contraception. [unreadable] [unreadable] 2) Reusable Duet, a low-cost, and maximally protective barrier device for use in low resource areas. Submit IDE to FDA for Duet as an OTC cervical barrier device for use with microbicides. [unreadable] [unreadable] 3) Membrane permeant acids: Determine whether lactic and acetic acid can safely increase microbicide efficacy. Lactobacilli acidify the vagina to pH~4 by secreting lactic and acetic acids. At this pH these acids can rapidly inactivate pathogens by permeating cell and bacterial membranes, but these permeant acids can also be toxic to mucosal surfaces. We hypothesize that adding lactic and acetic acids at physiological concentrations to microbicides formulated at pH~4 will speed acid-inactivation of pathogens without causing significant toxicity. To test this hypothesis: (a) Measure in vitro the speed at which lactic acid and acetic acid at vaginal pH (~4) inactivate sexually transmitted pathogens, BV-associated microbes, and sperm, (b) Add physiological concentrations of these acids to BufferGel (which uses an impermeant buffering gel to reinforce vaginal acidity) and measure the extent to which they increase its protective efficacy in animal models for herpes, chlamydia, and trichomonas. (c) Test whether adding these acids to BufferGel increases vaginal toxicity, or susceptibility to herpes or chlamydia, by using sensitive tests in mouse and rabbit models. [unreadable] [unreadable] RELEVANCE: The aim is to maximize protective efficacy of microbicides that women can use for protection against STIs and HIV. The products will also prevent pregnancy and help prevent BV. BufferGel is now in clinical trials for contraception (used with a diaphragm) and HIV prevention (used alone). "Duet" covers the cervix for maximum protection of this susceptible site for infections, and delivers microbicide gel to protect both the cervix and vagina. The reusable Duet will be low in cost and useful for low-resource areas. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The "Duet" is a cervical barrier device that, unlike a diaphragm, delivers gel on both its upper and lower surfaces to protect the vagina as well as the cervix. This application proposes 3 related projects: [unreadable] [unreadable] 1) "BufferGel-Duet", a single-use product for protection against STIs, HIV/AIDS, BV, and pregnancy. (a) Complete development of manufacturing steps to produce Duet for clinical efficacy trials. (b) Complete development of packaging, stability, and shelf-life testing of BufferGel-Duet. (c) Seek FDA permission via ReProtect's BufferGel IND to pursue clinical trials of BufferGel-Duet for preventing STIs (chlamydia, gonorrhea, trichomoniasis, and HIV), BV, and for contraception. [unreadable] [unreadable] 2) Reusable Duet, a low-cost, and maximally protective barrier device for use in low resource areas. Submit IDE to FDA for Duet as an OTC cervical barrier device for use with microbicides. [unreadable] [unreadable] 3) Membrane permeant acids: Determine whether lactic and acetic acid can safely increase microbicide efficacy. Lactobacilli acidify the vagina to pH~4 by secreting lactic and acetic acids. At this pH these acids can rapidly inactivate pathogens by permeating cell and bacterial membranes, but these permeant acids can also be toxic to mucosal surfaces. We hypothesize that adding lactic and acetic acids at physiological concentrations to microbicides formulated at pH~4 will speed acid-inactivation of pathogens without causing significant toxicity. To test this hypothesis: (a) Measure in vitro the speed at which lactic acid and acetic acid at vaginal pH (~4) inactivate sexually transmitted pathogens, BV-associated microbes, and sperm, (b) Add physiological concentrations of these acids to BufferGel (which uses an impermeant buffering gel to reinforce vaginal acidity) and measure the extent to which they increase its protective efficacy in animal models for herpes, chlamydia, and trichomonas. (c) Test whether adding these acids to BufferGel increases vaginal toxicity, or susceptibility to herpes or chlamydia, by using sensitive tests in mouse and rabbit models. [unreadable] [unreadable] RELEVANCE: The aim is to maximize protective efficacy of microbicides that women can use for protection against STIs and HIV. The products will also prevent pregnancy and help prevent BV. BufferGel is now in clinical trials for contraception (used with a diaphragm) and HIV prevention (used alone). "Duet" covers the cervix for maximum protection of this susceptible site for infections, and delivers microbicide gel to protect both the cervix and vagina. The reusable Duet will be low in cost and useful for low-resource areas. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): We have developed mucus-penetrating nanoparticles (MPP) suitable for sustained delivery of small-molecule microbicides (Lai et al, Proceedings of the National Academy of Sciences 2007; 104(5):1482-7). Conventional particles (CP) are mucoadhesive and stick to the outer layers of mucus that are shed most rapidly out of the vagina. By densely coating MPP with low molecular weight polyethylene glycol (PEG) we found that unexpectedly large MPP 100-500 nm in diameter can be engineered to rapidly penetrate human cervicovaginal (CV) mucus and thereby reach the unstirred layer of mucus adhering to the epithelial surface. These MPP will likely significantly increase vaginal residence time and improve epithelial microbicide distribution. The aim of this R21/R33 project is to develop MPP for the sustained delivery of small-molecule microbicides to increase their protective efficacy, acceptability, and user reliability. 'User failure' is the primary failure mode of barrier methods, and microbicides are likely to be used more reliably if applied daily on a coitally-dissociated basis. Another failure mode well-documented in animal models is inadequate microbicide distribution - the infectious inoculum reaches surfaces unprotected by the microbicide. MPP can provide a once-a-day, coitally-dissociated method that is likely to achieve complete and essentially uniform epithelial distribution. MPP will not likely provide the month-long delivery of a vaginal ring, but MPP have advantages that are not immediately apparent: 1) The vaginal epithelium is highly permeable to small water-soluble molecules - thus uniform epithelial distribution can best be achieved by uniform sustained delivery of small water soluble microbicides directly to the entire epithelial surface, not just to the vicinity of a vaginal ring. 2) Uterine peristalsis exposes the upper reproductive tract to vaginally deposited pathogens, and reliable protection of the upper tract is more likely to be achieved by MPP that can transport, and then locally deliver, small water-soluble molecules to the epithelia surfaces of the upper tract. In the R21 phase we propose to develop acyclovir-loaded MPP to evaluate in our mouse HSV models for efficacy, duration, vaginal distribution, and toxicity. The MPP will be composed biodegradable copolymers that we have shown are capable of sustained delivery of a wide range of bioactive molecules. The key milestone for R21 will be to develop acyclovir-MPP that provide at least one day of protection in the mouse. In the R33 phase, we will use the knowledge gained from the R21 phase to speed the development of an anti- HIV-MPP for sustained release of the best anti-HIV microbicide candidate then available (Fall, 2010), with tenofovir being a likely choice. The R33 anti-HIV-MPP will be optimized for drug delivery based on R21 results, and be tested for toxicity in mouse models and for efficacy in the Hu-BLT-SCID mouse/HIV model by Dr. Victor Garcia at UT Southwestern. World-wide, there is a great need for methods women can use to protect against AIDS and other sexually transmitted diseases. Several small-molecule vaginal microbicides are being developed that block HIV from infecting and/or replicating in target cells. The aim of this project is to enhance the protective efficacy of these small-molecule microbicides by developing mucus-penetrating nanoparticles that will improve coverage of susceptible tissues to increase reliability of protection, and to increase duration of protection so that the microbicides can be applied regularly, on a daily basis, and not require coitally-related applications. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mucus secretions from the female reproductive tract are extremely difficult to penetrate even by virus-sized objects. Why then do these barriers sometimes fail to protect against sexually transmitted pathogens? Our pilot data suggests some pathogens are too large to penetrate mucus mesh-spacing (pore size), and mucus also traps some pathogens like fly-paper before they can reach target cells. In contrast, abnormal mucus may not be so protective. About 1 in 3 women have bacterial vaginosis (BV), and they are at >2-6-fold increased risk of infection by HIV and other viral, bacterial, and protozoan pathogens. BV is a polymicrobial overgrowth of vaginal flora that decimates healthy lactobacilli, transforms vaginal mucus to a watery secretion, and partially eliminates the acidity of the vagina. We have developed mucus penetrating particles (MPP) that reveal the mucus mesh-spacing in fresh human samples. We can thus identify pathogens that are slowed in ex vivo genital mucus fluids by steric occlusion (pathogen size > mesh-spacing) and/or trapped by adhesion. Our pilot observations indicate native (acidic) cervicovaginal mucus from women with healthy vaginal flora adhesively traps HPV, HIV, and HSV. But, if partially neutralized with NaOH to mimic neutralizing actions of BV, HIV and HSV can penetrate this mucus barrier. Our overall hypothesis is that mucus secretions from women with healthy vaginal flora are highly protective against pathogen penetration, but that this protection is greatly diminished in women with BV. To test this, Aim 1 will extend our pilot observations and include all mucus secretions through which STD pathogens are usually transmitted: endocervical mucus, healthy cervicovaginal mucus, BV secretions, and semen. We will use multiple particle tracking and MPP to characterize the meshspacing and protective viscoelastic properties of these genital secretions at the nano- to micro- length scales experienced by pathogens. Aim 2 will identify the secretions that can block or retard penetration by major STD pathogens (HIV, HSV, HPV, N. gonorrhoeae and C. trachomatis), and secretions that are rapidly penetrated. We will also determine the exact viscosity and elasticity experienced by each pathogen in the various human secretions. This will provide important quantitation of the extent of impediment each pathogen experiences. Taken together, the results from Aims 1 & 2 will also distinguish pathogens blocked sterically, and those trapped adhesively, in each secretion. Aim 3 will investigate the roles of lactic acid, and lactate anions, in causing STD pathogens to be trapped by mucus. Our pilot results show lactic acid specifically alters the surfaces of HIV and HSV, and this may help explain how they are trapped by healthy mucus. This project will likely clarify how abnormal mucus secretions caused by BV increase susceptibility to penetration of a broad range of viral, bacterial, and protozoal pathogens. The results may also support the creation of new methods that use lactic acid to help prevent BV and enhance the protective effects of mucus secretions

RFA-AI-10-011 Microbicide Innovation Program (MIP VI) (R21/R33) Mucus penetrating particles for rectal microbicide delivery Project Summary For reliable protection against STD transmission, rectal microbicides must be formulated in a way that will deliver the active agent to all the surfaces that are susceptible to infection. These include the entire rectum as well as a large fraction of the colon (due to peristaltic stirring of colonic contents). Colorectal surfaces are columnar epithelia that are mechanically and osmotically fragile, and are highly susceptible to STD transmission. Although continuous mucus secretion by these susceptible surfaces helps protect against trauma and pathogens, this continuously secreted mucus also poses a significant barrier against effective delivery of microbicides to the epithelial surface. Recently we developed novel mucus penetrating nanoparticles (MPP) that can overcome this barrier and provide sustained, well-distributed delivery of drugs to mucosal surfaces. Our hypothesis is that MPP will significantly increase the protective efficacy of rectal microbicides by achieving more uniform and complete colorectal distribution, sustained drug activity, and thus longer duration and more complete protection compared to drug delivered in gels (free drug) or drug delivered in conventional nanoparticles, CP, that adhere to mucus and fail to penetrate mucus barriers. In the R21 phase, we will determine optimal MPP properties for penetration of mouse colorectal mucus, and we will characterize the uniformity of MPP distribution and retention times in the mouse colorectum compared to CP and free drug. We will then prepare drug-loaded biodegradable and biocompatible MPP that provide sustained release of antiviral drugs (valacyclovir for HSV and UC-781 for HIV). We will deliver these MPP in both a rectal enema format and a rectal lubricant gel format since both formats are frequently used for enhancing rectal intercourse. Moreover, an enema may deliver MPP to large regions of the colon unlikely to be reached by a gel. The key milestone for the R21 phase will be development of valacyclovir-MPP and UC-781- MPP that provide more complete and persistent coverage of the rectal epithelial surface, with minimal toxicity, compared to CP formulations or free drug. In the R33 phase, we will extensively test these MPP formulations for safety and protective efficacy in our mouse/HSV rectal model and in the hu-BLT-SCID mouse/HIV model (via a subcontract with Dr. J. Victor Garcia-Martinez at UNC).