Marcus H. Henderson

Vaginal distribution of Replens and K-Y Jelly using three imaging techniques

BACKGROUND Determination of vaginal distribution is important to the development of potential vaginal microbicidal or spermicidal products. STUDY DESIGN This was a descriptive study of three imaging techniques with a randomized crossover assignment of two gels and activity status within each technique. METHOD Each of three sites utilized one technique. Three nulligravid women and three parous women were to be enrolled at each site. We studied the effects of time, ambulation, parity and body mass index on vaginal spreading of two commonly used gels, K-Y Jelly and Replens. Imaging by magnetic resonance imaging and gamma scintigraphy was performed at 5, 20, 35 and 50 min after insertion of 3.5 mL of gel. Imaging with a fiberoptic probe was performed at 5 and 20 min after insertion. RESULTS Initial application of the gel resulted in approximately two thirds of maximum coverage possible, both in linear extent along the vaginal axis and in surface area covered. Over the next 45 min, spreading increased to about three quarters of the maximum possible. Ambulation generally increased linear spreading and the proportions of women with gel at the introitus and os. Effects of parity and body mass index (BMI) were similar on most measures of gel spreading, with nulligravid women tending toward greater spread than parous women and women of high BMI usually showing somewhat greater spread than women of normal weight. Differences between the two gels were not seen when all conditions of application were considered together. CONCLUSION In vivo imaging of gel distribution demonstrated that ambulation, parity and BMI affect vaginal gel spreading. The three imaging techniques have advantages and disadvantages and provide complementary information for microbicide development.

Optical instrument for measurement of vaginal coating thickness by drug delivery formulations

An optical device has been developed for imaging the human vaginal epithelial surfaces, and quantitatively measuring distributions of coating thickness of drug delivery formulations—such as gels—applied for prophylaxis, contraception or therapy. The device consists of a rigid endoscope contained within a 27-mm-diam hollow, polished-transparent polycarbonate tube (150mm long) with a hemispherical cap. Illumination is from a xenon arc. The device is inserted into, and remains stationary within the vagina. A custom gearing mechanism moves the endoscope relative to the tube, so that it views epithelial surfaces immediately apposing its outer surface (i.e., 150mm long by 360° azimuthal angle). Thus, with the tube fixed relative to the vagina, the endoscope sites local regions at distinct and measurable locations that span the vaginal epithelium. The returning light path is split between a video camera and photomultiplier. Excitation and emission filters in the light path enable measurement of fluorescence of t...

Quantitative Analysis of Microbicide Concentrations in Fluids, Gels and Tissues Using Confocal Raman Spectroscopy

Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data. These results demonstrate that confocal Raman spectroscopy holds promise as a tool for practical, minimally invasive, label-free measurement of microbicide drug concentrations in fluids, gels and tissues.

Transport Theory for HIV Diffusion through In Vivo Distributions of Topical Microbicide Gels

Topical microbicide products are being developed for the prevention of sexually transmitted infections. These include vaginally-applied gels that deliver anti-HIV molecules. Gels may also provide partial barriers that slow virion diffusion from semen to vulnerable epithelium, increasing the time during which anti-HIV molecules can act. To explore the barrier function of microbicide gels, we developed a deterministic mathematical model for HIV diffusion through realistic gel distributions. We applied the model to experimental data for in vivo coating distributions of two vaginal gels in women. Time required for a threshold number of virions to reach the tissue surface was used as a metric for comparing different scenarios. Results delineated how time to threshold increased with increasing gel layer thickness and with decreasing diffusion coefficient. We note that for gel layers with average thickness > approximately 100 microm, the fractional area coated, rather than the gel layer thickness, was the primary determinant of time to threshold. For gel layers < approximately 100 microm, time to threshold was brief, regardless of fractional area coated. Application of the model to vaginal coating data showed little difference in time to threshold between the two gels tested. However, the protocol after gel application (i.e., with or without simulated coitus) had a much more significant effect. This study suggests that gel distribution in layers of thickness >100 microm and fractional area coated >0.8 is critical in determining the ability of the gel to serve as a barrier to HIV diffusion.

Measuring macrodiffusion coefficients in microbicide hydrogels via postphotoactivation scanning.

Hydrogels can function as biocompatible, engineered systems to provide controlled release of drugs in vivo. Whether they are employed as gel implants, tissue engineered scaffolds, or delivery systems for transdermal or mucosal delivery, hydrogels often function by controlling the diffusion of particles into or out of their polymer matrices. Biologically relevant predictions of hydrogel functionality in vivo (e.g., pharmacokinetic profiles) require accurate measurements of particle transport. However, determinations of diffusion coefficients in hydrogels are highly influenced by the length scales over which measurements are made. Because of heterogeneity within hydrogel macromolecular structures, local microdiffusion coefficients deviate from ones measured over longer length scales, especially as diffusing particle size increases. Therefore, in vitro measurement of diffusion coefficients should be performed on length scales similar to those of the in vivo transport processes of interest. Several techniques (e.g., FRAP, single particle tracking, scanning microphotolysis, multiple image photography) have been widely utilized to measure particle diffusion within hydrogels, but most provide measurements on molecular or cellular length scales (<30 μm). Other methods (e.g., diffusion chambers, semi-infinite slabs) obtain bulk diffusion measurements over centimeter length scales. While these scales are relevant to many biological processes, they do not capture diffusion on the intermediate range of the order of hundreds of micrometers. This intermediate length scale is prevalent in many biomedical applications and is especially relevant to the HIV/ AIDS prevention field. Here, hydrogels are being used to deliver antiviral microbicidal compounds directly to potential sites of infection as well as to inhibit HIV transport from semen to epithelium. These hydrogels create layers 50–500 μm thick within the lower human female reproductive tract. Understanding the rates at which antiviral agents, as well as HIV itself, migrate through the gel layers is essential to understanding their role in prophylaxis against HIV transmission. This understanding of particle transport within hydrogels can be extended to other drug delivery applications as well. To address this experimental gap, we have created and evaluated a novel method to measure macroscale particle diffusion in hydrogels. Using UV photolysis of caged fluorescent compounds, we generated a region of uniformly high fluorescence intensity with distinct linear margins between regions of very low intensity. The optical creation of a sharp concentration gradient overcomes the limitations of mechanically forming interfaces that are inherent in previous semi-infinite slab configurations and allows diffusion to be studied over intermediate length scales. The sealed chamber and lack of translational movement eliminate convective flow and enable diffusion measurements over long times and, therefore, extended distances. A high-resolution fluorescent scanner quantifies concentration profiles resulting from 1-D diffusion of fluorescent particles outward from the photoactivated linear region. These profiles are imaged over time. A custom MATLAB software program determines a unique solution for the diffusion coefficient by numerically fitting all concentration profiles to the partial differential equation for diffusion. The method has produced accurate, robust quantification of diffusion coefficients over a range of length scales and within a range of liquid or semisolid media. Within the HIV/AIDS prevention field, the method will be applicable to quantify both drug and viral transport within drug delivery vehicles. Moreover, the technique has broad applicability to any processes that involve diffusion through polymeric materials over comparable length scales. This paper presents a description of the method, provides validation measurements, and applies the new technique to measure the diffusion of a small fluorescein molecule (322 Da) and a larger macromolecule (10 kD dextran) in placebo gels used in previous and ongoing microbicide clinical trials.

Label-free measurement of microbicidal gel thickness using low- coherence interferometry

Spectral-domain low-coherence interferometry (LCI) was used to measure the thickness of microbicidal gels applied to a cylindrical calibration test socket. Microbicides are topical formulations containing active ingredients targeted to inhibit specific pathogens that are currently under development for application to the epithelial lining of the lower female reproductive tract to combat sexually transmitted infections such as HIV. Understanding the deployment and drug delivery of these formulations is vital to maximizing their effectiveness. Previously, in vivo measurements of microbicidal formulation thickness were assessed using fluorescence measurements of fluorescein-labeled gels via an optical endoscope-based device. Here we present an LCI-based device that measures the thickness of a formulation without the use of any exogenous agents by analyzing the interference pattern generated between the reflections from the front and back surface of the sample. Results are presented that validate the effectiveness and performance of the LCI measurement in a clinically relevant system as compared to an existing fluorescence-based method. The impact of the new LCI-based design on in vivo measurements is discussed.

Co-localized confocal Raman spectroscopy and optical coherence tomography (CRS-OCT) for depth-resolved analyte detection in tissue.

We report the development of a combined confocal Raman spectroscopy (CRS) and optical coherence tomography (OCT) instrument (CRS-OCT) capable of measuring analytes in targeted biological tissues with sub-100-micron spatial resolution. The OCT subsystem was used to measure depth-resolved tissue morphology and guide the acquisition of chemically-specific Raman spectra. To demonstrate its utility, the instrument was used to accurately measure depth-resolved, physiologically-relevant concentrations of Tenofovir, a microbicide drug used to prevent the sexual transmission of HIV, in ex vivo tissue samples.

Portable colposcopy in low-resource settings.

Screening for cervical dysplasia is an important public health effort worldwide. In unscreened populations, the incidence of cervical cancer ranges between 2 and 4% of the adult female population, whereas less than 0.1% of the screened population of Caucasian women has cervical cancer in the United States. In developing countries, cervical cytology is difficult to implement successfully because of the cost, cultural constraints, limited access to pathology services, etc. Bypassing cytology and going directly to colposcopy has been successfully implemented as a screening strategy for dysplasia in low resource settings. In this article we describe the development and utilization of a portable binocular colposcope that does not require electricity.

Applications of biomedical engineering in reproductive biomedicine: sensing and drug delivery to the lower female reproductive tract

Current intravaginal products for contraception and prophylaxis against sexually transmitted diseases lack the efficacy that is both needed and technically possible. A multidisciplinary effort-that integrates gynecology, pathology, microbiology, and reproductive biology with engineering and materials science-can provide critical and missing information to the process of design and development of better products. Several engineering sub-disciplines can contribute to this effort, including electrical, mechanical and chemical engineering. Specific contributions will pertain to the polymeric delivery vehicles for drug delivery, the active ingredients of those vehicles, and to methods of testing, both in vitro and in vivo.

Design and validation of a multiplexed low coherence interferometry instrument for in vivo clinical measurement of microbicide gel thickness distribution

We present a multiplexed, Fourier-domain low coherence interferometry (mLCI) instrument for in vivo measurement of intravaginal microbicide gel coating thickness distribution over the surface of the vaginal epithelium. The mLCI instrument uses multiple delivery fibers to acquire depth resolved reflection profiles across large scanned tissue areas. Here mLCI has been adapted into an endoscopic system with a custom imaging module for simultaneous, co-registered measurements with fluorimetric scans of the same surface. The resolution, optical signal-to-noise, and cross-talk of the mLCI instrument are characterized to evaluate performance. Validation measurements of gel thickness are made using a calibration socket. Initial results from a clinical study are presented to show the in vivo capability of the dual-modality system for assessing the distribution of microbicide gel vehicles in the lower human female reproductive tract.