Browsing by Author "Katz, David F"

This dissertation focuses on enhancing our understanding of microbicide transport mechanisms from gel delivery vehicles into vaginal mucosal tissues through the development and application of novel transport assays. We demonstrated the capabilities of Raman spectroscopy to measure four leading microbicide drugs, Tenofovir, IQP-0528, MIV 150, and Dapivirine, in fluids, microbicide gels, and biological tissues. Strong linear dependences of spectral Raman intensities on drug concentrations suggested that Raman spectrometry could be used in practice for quantitative analysis of drug concentrations in biologically-relevant matrices. Concentration measurements in fluids and tissues demonstrated good correlation with gold standard LC-MS/MS data.

Next, we investigated the accuracy of confocal Raman measurements of uniform and non-uniform (linear) concentration distributions of Vitamin K in a tissue phantom. A chemometric method was used to perform the quantitative analyses of concentration distributions and to correct for signal attenuation due to elastic scattering that increased with depth into the tissue. The measured concentration distributions demonstrated an expected trend of the linear profile up to a depth of 180 to 200 μm, thereby validating the confocal Raman approach for concentration profiling in scattering matrices.

We developed a label-free assay using Raman spectroscopy to quantify the drug diffusion coefficient within a gel. The concentration distributions of Tenofovir in gel over time and space were measured and the concentration profiles were fitted to PDE diffusion models to obtain diffusion coefficients. The gel was tested undiluted, and in 10-50% serial dilutions with vaginal fluid simulant, to capture the range of conditions that likely occur in vivo. Tenofovir exhibited diffusion-like behavior that could be fitted to the diffusion model reasonably well (R2: 0.86 – 0.99). The dilutions with vaginal fluid simulant increased diffusion coefficients of Tenofovir in the test gel by up to 50%.

Finally, we developed a real-time transport assay using a combined CRS-OCT instrument constructed by our team to study and distinguish the microbicide transport mechanisms between epithelial and stromal layers of vaginal tissue. Measurements were corrected by taking into account the effects of out-of-focus light contributions from the Tenofovir overlying gel layer. Data were translated to fundamental transport properties, the diffusion and partition coefficients. The diffusion coefficient in stroma was found to be 2 to 15 times larger than that in epithelium, suggesting that epithelium could present a diffusion barrier to Tenofovir transport in vaginal tissue.

The series of transport assays developed here can provide useful information of microbicide transport mechanisms. The transport parameters can be used as salient measures to investigate the drug delivery performance of different drugs and gel vehicles. The parameters are important inputs to the deterministic transport models that predict the gels’ dosing regimen in PK studies. Collectively, this thesis will contribute to a better understanding of anti-HIV drug delivery and pharmacokinetics, and be of significant help in creation of products for prevention of HIV/AIDS.

Our lab focuses on developing and optimizing drug delivery systems for applications in women’s health. In this field, development of drugs and drug delivery systems is hindered by a heavy reliance on empirically derived data, usually obtained from non-standardized, highly variable in vitro and in vivo animal experiments. Further, without a mechanistic understanding of the various phenomena progressing during drug delivery, experiments tend to explore complex parameter spaces blindly and randomly. Deterministic mathematical models can improve the efficiency of this process by informing rational drug and product design. In this work, we were interested in two applications: 1. drug delivery of topically applied anti-HIV microbicides to the female reproductive tract; and 2. Localized intratumoral injections of ethanol-ethyl cellulose mixtures for treatment of cervical lesions. Development of topically applied anti-HIV microbicides to prevent sexual HIV transmission is inefficient, with in vitro and in vivo tests having limited applicability to real product use. This issue is exacerbated by the dependence of drug performance on adherence and drug-administration conditions, which are not tested until clinical trials. Further, the lack of a standardized pharmacodynamic (PD) metric that is dependent on the heterogenous dynamics of viral transport and infection makes it difficult to identify the most promising drug candidates. Here we develop a deterministic mathematical model that incorporates drug pharmacokinetics (PK) and viral transport and dynamics to estimate the probability of infection (POI) as a PD metric that can be computed for a variety of anti-HIV drugs in development. The model reveals key mechanistic insights into the spatiotemporally dependent dynamics of infection in the vaginal mucosa, including susceptibility to infection at different phases in the menstrual cycle. Further, it and can be used as a platform to test novel drugs under several conditions, such as the timing of drug administration relative to the time of HIV exposure. Localized injections of ablative agents, immunotherapeutics and chemotherapeutics have potential for increased therapeutic efficacy against tumors and reduced systemic effects. However, injection outcomes thus far have been largely unsatisfactory, due to unintended leakage of the active pharmaceutical ingredients (APIs) to non-target tissues. Adding a gelling or precipitating agent to the injection can help ameliorate this limitation, by acting to contain the API within the target tissue. One such example is injection of ethanol-ethyl cellulose mixtures. Due to the insolubility of ethyl cellulose in water, this polymer phase-separates in the aqueous tumor environment, forming a fibrous gel that helps contain ethanol, the current ablating agent (and chemotherapeutic drugs in the future), within the boundaries of the tumor. Our collaborators have shown that this strategy can be an effective low-cost treatment strategy for superficial solid tumors, with cervical cancer and cervical dysplasia, and liver cancer, being promising targets. Here we present a mathematical model that enables characterization of the injection process. Our model uses Cahn Hilliard theory to model the phase separation of a precipitating or gelling agent during injection into poroelastic tissue. This theory is linked to the soft mechanics of tissue deformation during the injection, and to mass transport theory for the API. The model predicts key elements of the injection process, including the pressure field, the soft tissue displacement field, the phase constitution of the precipitating or gelling agent in the tissue, and the concentration distribution of the API in the tissue. The model enables us to explore relationships between these elements and fundamental injection and tissue parameters. This can inform design of optimized injection protocols. Select model predictions include that larger injection volumes do not significantly affect cavity volumes but do lead to faster transport of the API to target tumor tissue. However, although higher flow rates lead to larger cavities – in the absence of tissue fracture, and when injected volume is held constant – they also lead to slower delivery of the API into the target tumor tissue. This is due to the shorter injection times. Importantly, concentration distributions of the API are not sensitive to the speeds of precipitation of the precipitating agents or to diffusion coefficients of the API in the dense (gelled) phase of the injectate material. The model presented here enables first-pass exploration of injection parameter space for select tissue types (properties). This can aid in optimization of localized therapeutic injections in a range of applications.

A new modality for preventing HIV transmission is emerging in the form of topical microbicides. Some clinical trials have shown some promising results of these methods of protection while other trials have failed to show efficacy. Due to the relatively novel nature of microbicide drug transport, a rigorous, deterministic analysis of that transport can help improve the design of microbicide vehicles and understand results from clinical trials. This type of analysis can aid microbicide product design by helping understand and organize the determinants of drug transport and the potential efficacies of candidate microbicide products.

Microbicide drug transport is modeled as a diffusion process with convection and reaction effects in appropriate compartments. This is applied here to vaginal gels and rings and a rectal enema, all delivering the microbicide drug Tenofovir. Although the focus here is on Tenofovir, the methods established in this dissertation can readily be adapted to other drugs, given knowledge of their physical and chemical properties, such as the diffusion coefficient, partition coefficient, and reaction kinetics. Other dosage forms such as tablets and fiber meshes can also be modeled using the perspective and methods developed here.

The analyses here include convective details of intravaginal flows by both ambient fluid and spreading gels with different rheological properties and applied volumes. These are input to the overall conservation equations for drug mass transport in different compartments. The results are Tenofovir concentration distributions in time and space for a variety of microbicide products and conditions. The Tenofovir concentrations in the vaginal and rectal mucosal stroma are converted, via a coupled reaction equation, to concentrations of Tenofovir diphosphate, which is the active form of the drug that functions as a reverse transcriptase inhibitor against HIV. Key model outputs are related to concentrations measured in experimental pharmacokinetic (PK) studies, e.g. concentrations in biopsies and blood. A new measure of microbicide prophylactic functionality, the Percent Protected, is calculated. This is the time dependent volume of the entire stroma (and thus fraction of host cells therein) in which Tenofovir diphosphate concentrations equal or exceed a target prophylactic value, e.g. an EC50.

Results show the prophylactic potentials of the studied microbicide vehicles against HIV infections. Key design parameters for each are addressed in application of the models. For a vaginal gel, fast spreading at small volume is more effective than slower spreading at high volume. Vaginal rings are shown to be most effective if inserted and retained as close to the fornix as possible. Because of the long half-life of Tenofovir diphosphate, temporary removal of the vaginal ring (after achieving steady state) for up to 24h does not appreciably diminish Percent Protected. However, full steady state (for the entire stromal volume) is not achieved until several days after ring insertion. Delivery of Tenofovir to the rectal mucosa by an enema is dominated by surface area of coated mucosa and whether the interiors of rectal crypts are filled with the enema fluid. For the enema 100% Percent Protected is achieved much more rapidly than for vaginal products, primarily because of the much thinner epithelial layer of the mucosa. For example, 100% Percent Protected can be achieved with a one minute enema application, and 15 minute wait time.

Results of these models have good agreement with experimental pharmacokinetic data, in animals and clinical trials. They also improve upon traditional, empirical PK modeling, and this is illustrated here. Our deterministic approach can inform design of sampling in clinical trials by indicating time periods during which significant changes in drug concentrations occur in different compartments. More fundamentally, the work here helps delineate the determinants of microbicide drug delivery. This information can be the key to improved, rational design of microbicide products and their dosage regimens.

This dissertation explores strategies for reducing HIV-virion transport to mucosal surfaces to prevent HIV infection. Infection requires contact between HIV and an infectable cell, so any means of inhibiting this step could contribute to HIV prevention. Our goals were to quantify the effects of strategies that reduce transport of HIV virions and to evaluate them in the context of HIV prevention. We used fundamental transport theory to design two basic strategies: (1) modifying the effective radius of virions; and (2) modifying the native medium through which virions diffuse. We proposed to implement these strategies using (1) anti-HIV antibodies that would bind and aggregate virions and (2) topically-applied semi-solid gels that coat vaginal epithelial surfaces.

We measured diffusion coefficients of HIV virions and HIV-like particles in the presence of antibodies and within semi-solid gels. In experiments with antibodies, we did not observe reductions in the diffusion coefficients. In experiments using particle tracking to measure the diffusion coefficients of virions in vaginal gels, we found that the diffusion coefficients in gels were approximately 10,000 times lower than those in water.

We proceeded to evaluate the potential for semi-solid gels to prevent HIV transmission at mucosal surfaces. From previous experiments in our lab that characterized the topical deployment of vaginal gels in vivo, we know that vaginal gels form an uneven coating on the epithelium with gel layer thicknesses of the order of hundreds of microns. Thus, we determined whether semi-solid gels could function as physical barriers to HIV when deployed as thin, incomplete layers on the epithelium.

We developed an experimental system to test the barrier functioning of thin gel layers. We applied thin gel layers to the porous membrane of a Transwell system, and added a solution of HIV to the top compartment. After incubation, samples were assayed for levels of HIV. We found that thin gel layers reduced levels of HIV in the bottom compartment compared to controls where no gel had been applied: There was a log reduction in levels of HIV in conditions where gel layers of approximately 150 μm thickness had been applied to the membrane after 0-, 4-, and 8-hour incubation. Thus, it appears possible for gel layers of thicknesses found in vivo to function as physical barriers to HIV over biologically-relevant time scales.

We studied how nonuniform deployment of semi-solid gels affects accumulation of virions in tissue using a mathematical model. We used transport theory to develop a model of HIV diffusing from semen, through gel layers where present, to tissue. Our findings suggest that comprehensive coating of over 80% of the tissue surface area and gel layer thicknesses over 100 μm are crucial to the barrier functioning of topical gels. Under these conditions, the level of viral restriction makes a significant contribution to increasing the time required for virions to reach tissue.

Overall, the work presented here applies transport theory in the context of HIV transmission and prevention. Results contribute to theoretical and experimental frameworks that can help understand events in HIV transmission and to design and evaluate new technologies for HIV prevention.

There were 2.5 million people newly infected with HIV in 2007, clearly motivating the need for additional novel prevention methods. In response, topical vaginal antimicrobials, or microbicides, are being developed. These products aim to stop HIV transmission through local, vaginal delivery of antiviral compounds. To succeed, microbicides require a potent active compound within a well-engineered delivery vehicle.

A well-engineered delivery vehicle provides an antiviral compound with the greatest opportunity to interact with HIV and/or infected cells, thereby increasing overall microbicide effectiveness. The theoretical and experimental investigations within this dissertation are concerned with the study of HIV and active compound transport within microbicide delivery vehicles and with the mechanisms by which these transport processes can be affected to maximize viral neutralization. To initially investigate the factors contributing to microbicide effectiveness, a combined pharmacokinetic and pharmacodynamic model of HIV transport and neutralization within a microbicide product was created. Model results suggested that thin (~100µm) layers of microbicide product may protect against HIV infection. Model results also indicated that a specific and engineerable property of delivery vehicles - the ability to restrict viral transport - may increase the overall effectiveness of a microbicide. Two new experimental assays were developed to test the hypothesis that delivery vehicles can slow viral transport. First, a novel methodology was created to measure particle diffusion over length scales relevant to microbicide delivery (50-500µm). Results showed that current vehicles significantly restrict the transport of small molecules and proteins. The second assay was designed to test HIV transport in a biologically relevant, layered (fluid-microbicide-tissue) configuration of a microbicide product in vivo; infectious HIV was placed above a thin layer of a microbicide delivery vehicle. Assay results showed that HIV transport is significantly slowed by two different placebo gels. This experimental confirmation of viral restriction in hydrogels, combined with the theoretical finding that viral restriction increased microbicide effectiveness, strongly motivates the future development of new delivery vehicles that intentionally slow viral transport. These new experimental methodologies can also be used to screen and compare future delivery vehicles to produce optimal microbicide products.

Finally, a two-dimensional, computational finite-element vaginal model was created to evaluate the transport of drugs from an intravaginal ring. This model determined that while IVRs may be effective in the delivery of antiviral compound, their performance is influenced by the flow of vaginal fluid. The analysis also warns about the potential for local toxicity.

Well-engineered delivery vehicles are an essential component to microbicide performance because they maximize the opportunities for active compounds to interact with and neutralize HIV. The studies in this dissertation demonstrate that delivery vehicles have a significant effect on active compound and HIV transport. To create an effective microbicide, vehicle effects on transport processes must be well understood, purposefully engineered, and carefully optimized to ensure maximal interactions between antiviral compounds and virus. Directed engineering of delivery vehicles contribute to the foundation for microbicide success.