Antiretroviral eluting intravaginal rings to prevent the sexual transmission of HIV

Female-controlled preventative technologies are being developed to decrease HIV sexual transmission rates in resource poor regions of the world where the pandemic is most prevalent. Intravaginal rings (IVRs) comprise extended duration vaginal drug delivery vehicles which may provide sustained release of antiretrovirals at local inhibitory concentrations to prevent initial HIV infection during coitus. Few IVR formulations have been researched for HIV prophylaxis although numerous antiretrovirals are excellent candidates. Poor progress is due in part to limitations of conventional IVR technology in delivering antiretrovirals with diverse physiochemical properties and dosing requirements. Furthermore, there is limited understanding of drug vaginal pharmacokinetics and potential toxicological effects on the local environment. Consequently, in this dissertation several candidate antiretrovirals were formulated in new IVR platforms and animal models were developed to characterize the IVR formulations in vivo. In the first part of the dissertation, polyurethane IVRs were designed that delivered the potent and dual-acting pyrimidinedione congeners for up to one month. The pyrimidinediones attained concentrations throughout the nonhuman primate vaginal tract that were expected to be inhibitory against HIV, with no observed detrimental effects to the vaginal environment. In the second part of the dissertation, an IVR was developed to simultaneously deliver dapivirine and tenofovir which possess contrasting hydrophilicity and differing mechanisms of action against HIV. A two segment ring design was utilized which independently optimized tenofovir and dapivirine release by using compositionally different polyurethanes. In the final two parts of the dissertation, a hydrophilic polyurethane reservoir IVR was engineered and tested in a new sheep model, whereby tenofovir vaginal concentrations from the IVR were similar to the clinically effective tenofovir vaginal gel but for 90 day duration. The tunable IVR platform allowed for achievement of desired drug release rates and ring mechanical stiffness which were time-independent. No major toxicological effects were observed in sheep, and extensive IVR in vitro characterization was performed to ensure that a chemically and physically stabilized product was achieved. The work reported herein describes the design and characterization of antiretroviral-eluting intravaginal rings which each hold promise as preventative technologies to prevent the sexual transmission of HIV.

ANTIRETROVIRAL ELUTING INTRAVAGINAL RINGS TO PREVENT THE SEXUAL TRANSMISSION OF HIV by Todd Joseph Johnson A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Bioengineering The University of Utah December 2012 Copyright © Todd Joseph Johnson 2012 All Rights Reserved The Universit y of Utah Graduat e School STATEMENT OF DISSERTATION APPROVAL The dissertation of Todd Joseph Johnson has been approved by the following supervisory committee members: Patrick F. Kiser , Chair 07/30/2012 Date Approved Robert W. Hitchcock , Member 07/30/2012 Date Approved Vladimir Hlady , Member 07/30/2012 Date Approved Michael S. Kay , Member 07/30/2012 Date Approved Steven E. Kern , Member 07/30/2012 Date Approved and by Patrick A. Tresco , Chair of the Department of Bioengineering and by Charles A. Wight, Dean of The Graduate School. ABSTRACT Female-controlled preventative technologies are being developed to decrease HIV sexual transmission rates in resource poor regions of the world where the pandemic is most prevalent. Intravaginal rings (IVRs) comprise extended duration vaginal drug delivery vehicles which may provide sustained release of antiretrovirals at local inhibitory concentrations to prevent initial HIV infection during coitus. Few IVR formulations have been researched for HIV prophylaxis although numerous antiretrovirals are excellent candidates. Poor progress is due in part to limitations of conventional IVR technology in delivering antiretrovirals with diverse physiochemical properties and dosing requirements. Furthermore, there is limited understanding of drug vaginal pharmacokinetics and potential toxicological effects on the local environment. Consequently, in this dissertation several candidate antiretrovirals were formulated in new IVR platforms and animal models were developed to characterize the IVR formulations in vivo. In the first part of the dissertation, polyurethane IVRs were designed that delivered the potent and dual-acting pyrimidinedione congeners for up to one month. The pyrimidinediones attained concentrations throughout the nonhuman primate vaginal tract that were expected to be inhibitory against HIV, with no observed detrimental effects to the vaginal environment. In the second part of the dissertation, an IVR was developed to iv simultaneously deliver dapivirine and tenofovir which possess contrasting hydrophilicity and differing mechanisms of action against HIV. A two segment ring design was utilized which independently optimized tenofovir and dapivirine release by using compositionally different polyurethanes. In the final two parts of the dissertation, a hydrophilic polyurethane reservoir IVR was engineered and tested in a new sheep model, whereby tenofovir vaginal concentrations from the IVR were similar to the clinically effective tenofovir vaginal gel but for 90 day duration. The tunable IVR platform allowed for achievement of desired drug release rates and ring mechanical stiffness which were time-independent. No major toxicological effects were observed in sheep, and extensive IVR in vitro characterization was performed to ensure that a chemically and physically stabilized product was achieved. The work reported herein describes the design and characterization of antiretroviral-eluting intravaginal rings which each hold promise as preventative technologies to prevent the sexual transmission of HIV. To my best friend and companion, Dorthyann TABLE OF CONTENTS ABSTRACT .....................................................................................................................iii LIST OF TABLES ...........................................................................................................viii NOMENCLATURE ........................................................................................................ix ACKNOWLEDGEMENTS .............................................................................................xii CHAPTER 1 INTRODUCTION ...............................................................................................1 1.1 Vaginal Drug Delivery ...................................................................................2 1.2 HIV and Microbicides....................................................................................2 1.3 Intravaginal Rings as Microbicides ...............................................................5 1.4 IVR Manufacturing ........................................................................................7 1.5 IVR Design and Classification.......................................................................9 1.6 Acceptability and Adherence .........................................................................12 1.7 Microbicide IVR Limitations and Unknowns................................................15 1.8 Conclusions ....................................................................................................21 1.9 Dissertation Chapter Overview ......................................................................22 1.10 References ....................................................................................................26 2 SAFE AND SUSTAINED VAGINAL DELIVERY OF PYRIMIDINEDIONE HIV-1 INHIBITORS FROM POLYURETHANE INTRAVAGINAL RINGS..................................................................................................................38 2.1 Abstract ..........................................................................................................39 2.2 Introduction ....................................................................................................39 2.3 Materials and Methods ...................................................................................40 2.4 Results ............................................................................................................42 2.5 Discussion ......................................................................................................44 2.6 Acknowledgements ........................................................................................46 2.7 References ......................................................................................................46 vii 3 SEGMENTED POLYURETHANE INTRAVAGINAL RINGS FOR THE SUSTAINED COMBINED DELIVERY OF ANTIRETROVIRAL AGENTS DAPIVIRINE AND TENOFOVIR ......................................................................48 3.1 Abstract ..........................................................................................................49 3.2 Introduction ....................................................................................................49 3.3 Materials and Methods ...................................................................................50 3.4 Results and Discussion ..................................................................................52 3.5 Conclusion .....................................................................................................56 3.6 Acknowledgements ........................................................................................57 3.7 References ......................................................................................................57 4 A NINETY DAY TENOFOVIR RESERVOIR INTRAVAGINAL RING FOR MUCOSAL HIV PROPHYLAXIS ............................................................59 4.1 Abstract ..........................................................................................................59 4.2 Introduction ....................................................................................................60 4.3 Materials and Methods ...................................................................................63 4.4 Results ............................................................................................................72 4.5 Discussion ......................................................................................................76 4.6 Acknowledgements ........................................................................................87 4.7 References ......................................................................................................89 5 ENGINEERING OF A NEW INTRAVAGINAL RING TECHNOLOGY FOR HIV PROPHYLAXIS .................................................................................109 5.1 Abstract ..........................................................................................................109 5.2 Introduction ....................................................................................................110 5.3 Materials and Methods ...................................................................................114 5.4 Results ............................................................................................................123 5.5 Discussion ......................................................................................................129 5.6 Acknowledgements ........................................................................................141 5.7 References ......................................................................................................142 6 CONCLUSIONS AND FUTURE RECOMMENDATIONS ..............................158 6.1 Chapter Conclusions ......................................................................................159 6.2 Challenges and Future Directions ..................................................................162 6.3 References ......................................................................................................170 LIST OF TABLES Table Page 2.1 Antiviral activity of PYD1 and PYD, unformulated and after in vitro release ..........40 2.2 Chemical stability of formulated PYD1 and PYD2 stressed at 40°C ........................42 2.3 Ectocervical and introitus PYD vaginal tissue levels for each individual biopsy specimen .........................................................................................................45 3.1 Antiretroviral agents tenofovir and dapivirine ...........................................................50 4.1 Sheep safety study design ..........................................................................................96 4.2 Mean (standard deviation) pharmacokinetic parameters for the TFV IVR and TFV gel ......................................................................................................................97 4.3 Study 2 TFV vaginal fluid, vaginal tissue, and plasma concentrations from the TFV IVR ..............................................................................................................98 5.1 IVR formulation matrix .............................................................................................146 5.2 HPU-B1 molecular weight as a function of 40°C IVR storage time .........................147 5.3 The bulk swelling and enthalpic water peak areas of the hydrated HPU-B1 IVRs as a function of 40°C storage time .............................................................................148 5.4 The enthalpic water peak areas of the hydrated and equilibrated HPU IVRs ...........149 NOMENCLATURE HIV Human immunodeficiency virus STI Sexually transmitted infection RT Reverse transcriptase RTI Reverse transcriptase inhibitor NtRTI Nucleotide analogue reverse transcriptase inhibitor NNRTI Nonnucleoside reverse transcriptase inhibitor HRT Hormone replacement therapy IVR Intravaginal ring API Active pharmaceutical ingredient ARV Antiretroviral HME Hot melt extrusion PYD Pyrimidinedione PYD1 IQP-0528 pyrimidinedione PYD2 IQP-0532 pyrimidinedione TFV Tenofovir DPV Dapivirine PU Polyurethane NWS-PU Nonwater swellable polyurethane x WS-PU Water swellable polyurethane HPU Hydrophilic polyurethane HPU-35 HP-60D-35 Tecophilic polyurethane HPU-60 HP-60D-60 Tecophilic polyurethane HPU-B1 75/25 HP-60D-60/HP-93A-100 Tecophilic polyurethane HPU-B2 50/50 HP-60D-60/HP-93A-100 Tecophilic polyurethane HPU-B3 25/75 HP-60D-60/HP-93A-100 Tecophilic polyurethane EVA Polyethylene-co-vinyl acetate EVA-R NuvaRing PTMO Polytetramethylene oxide H12MDI 4,4'-dicyclohexylmethane diisocyanate PEO Polyethylene oxide HPLC High-performance liquid chromatography LC/MS Liquid chromatography-tandem mass spectrometry DSC Differential scanning calorimetry TGA Thermogravimetric analysis WAXS Wide angle X-ray scattering SAXS Small angle X-ray scattering FTIR Fourier transform infrared spectroscopy LOQ Limit of quantification LLOQ Lower limit of quantification PK Pharmacokinetic xi AUC Area under the curve Tmax Time of maximum observed concentration Cmax Maximum observed concentration C log P Calculated log partition coefficient EC50 50% effective concentration IC50 50% inhibitory concentration CPE Cytopathic effect SFU Syncytium-forming unit AZT Zidovudine XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide H&E Haematoxylin and eosin IFN Interferon IL Interleukin TNF Tumor necrosis factor MIP Macrophage inflammatory protein G-CSF Granulocyte colony-stimulating factor DAPI 4',6-diamidino-2-phenylindole PBS Phosphate-buffered saline DMA Dimethylacetamide DCM Dichloromethane THF Tetrahydrofuran ACKNOWLEDGEMENTS I am truly grateful for all of the guidance and support from the many people who have enabled this achievement to be realized. First, I would like to acknowledge my advisor, Dr. Patrick Kiser, for his strong scientific training - specifically how to think critically, retain skepticism, strive for excellence, and "tell the story". Although graduate school has taught me there will always be more to learn, I feel that I have grown immensely as a scientist under his guidance. I am also appreciative of the support from the members of our lab group who provided assistance and insight along the way. I would like to thank my committee members Drs. Vladimir Hlady, Steven Kern, Michael Kay, and Robert Hitchcock. Their constructive criticism and advice has been invaluable. I am also grateful to collaborators Jim Smith at the CDC and Meredith Clark and David Friend at CONRAD for their involvement and assistance in the many aspects of my work. Finally, I would like to thank my family and friends for their unwavering support and encouragement. I am blessed to have an amazing new wife, Dorthyann Isackson, who has journeyed with me in also receiving her PhD in Bioengineering. I will forever be grateful to my parents for instilling in me the value of hard work and a good education. CHAPTER 1 INTRODUCTION This chapter includes excerpts from the manuscript: STATE OF THE ART IN INTRAVAGINAL RING TECHNOLOGY FOR TOPICAL PROPHYLAXIS OF HIV INFECTION Patrick F. Kiser, Todd J. Johnson, Justin T. Clark Aids Reviews 2012;14(1):62-77 Reproduced with permission from Permanyer Publishing 2 1.1 Vaginal Drug Delivery Although the medical device and combination drug-device fields are often perceived as relatively new, intravaginal insertion of devices and therapeutics have existed for centuries with the primary objective of preventing conception (68). Intravaginal drug dosing, while less commonly used compared to oral or transdermal routes of administration, can offer certain advantages. Clearly, women-specific local indications such as vaginal infections (vaginitis) are excellent candidates for vaginal drug delivery. Less known, however, is that many small molecules including hormonal compounds demonstrate high vaginal absorption and thus can achieve effective systemic concentrations for indications including contraception and hormone replacement therapy (HRT) for post-menopausal women. Vaginal drug delivery is also advantageous as it is non-invasive, may be self-administered, avoids first-pass hepatic drug metabolism, often demonstrates high bioavailability with low fluctuation in systemic concentrations (i.e., peaks and troughs), and when intended for local delivery often shows less systemic side effects since a significantly lower dose achieves effectiveness (2, 101). Potential drawbacks of intravaginal drug delivery will be discussed in greater detail below and vary with the type of dosage form, but may include interference with coitus, cultural taboos associated with touching of the reproductive anatomy, and undesirable product attributes such as excess vaginal wetness or leakage. 1.2 HIV and Microbicides Virtually all currently marketed vaginal drug products are intended to treat or prevent unwanted pregnancy, menopausal-related symptoms, and vaginal inflammation 3 due to infection (vaginitis) (100). However, recently there has been considerable effort to expand indications including uterine fibroids and cancer, diabetes, and prevention of diseases via vaccine or chemoprophylactic delivery - primarily sexually transmitted infections including the Human Immunodeficiency Virus (HIV) (3, 8, 28, 34, 40). Although HIV infects both men and women, a strong rationale exists for developing HIV prophylactic intravaginal dosage forms; 22 of the world's 33 million HIV-infected individuals reside in sub-Saharan Africa, where HIV incidence on average is 1.3 times higher in women than men and an even higher disparity is observed with young adults aged 15-24 (82). The startlingly high HIV incidence in sub-Saharan African young women - up to 20% in some regions (82) - is poorly understood and likely complicated by many intertwined biological and social factors (16, 53, 65). Vaccine development has been the focal point of efforts to prevent the spread of HIV since it was discovered in the early 1980s, yet to-date only one clinical trial has shown any significant protective effect against HIV (31). To protect this high risk, sizeable female population against HIV sexual transmission in the absence of an efficacious vaccine, women-controlled prophylactic vaginal products termed microbicides have been under development for approximately two decades (73). Thus far, vaginal gel dosage forms have been the only microbicides clinically evaluated for HIV prophylactic effectiveness, in part due to their formulation simplicity and low production cost. The first microbicide clinical trial was conducted in 2002 with a Nonoxynol-9 gel, a nonionic surfactant which has been used for years as a contraceptive spermicide and was more recently found to inactivate HIV by disrupting its envelope. Although in vitro antiviral results were promising, limited safety data was generated prior 4 to efficacy studies in women, where Nonoxynol-9 exposure increased the rate of HIV infection in women who frequently applied the product (84). Similarly, the surfactant C31G, composed of alkyl dimethyl glycine and alkyl dimethyl amine oxide, did not prevent HIV infection and may have increased the incidence of adverse events in women (25, 63). The broad consensus from these early microbicide clinical trials was that these surfactants possess relatively low potency against HIV and harm the protective vaginal mucosa due to their nonspecific mechanism of action, allowing passage of virus to underlying immune cells and/or recruiting susceptible immune cells via an inflammatory response. Polyanions were also evaluated for prophylactic HIV effectiveness, but fared no better with cellulose sulfate demonstrating more HIV infections in the active versus placebo arm (83). Carrageenan-based gels PRO2000 and Carraguard showed no harm but were not effective (50, 75). In the Carraguard trial, gel applicators were examined post-use for evidence of user adherence, and it was discovered that women were not using the gel as instructed and were over-reporting adherence which was disturbingly low at 42% on average. More recently, the CAPRISA 004 trial evaluated the reverse transcriptase inhibitor (RTI) tenofovir formulated at 1 wt% in a vaginal gel (the prodrug form of tenofovir is already taken orally as part of highly active antiretroviral therapy for HIV-infected individuals). Although adherence to the gel dosage regimen was still low (less than 75% on average), the product demonstrated 39% overall effectiveness to become the first clinically effective microbicide and validating RTIs for microbicide applications (1). However, the follow-on VOICE trial tested the same tenofovir gel formulation and found no effectiveness, albeit with a different dosing regimen (87). Altogether, two major conclusions have been made from the microbicide trials performed to-date: First, 5 targeting specific steps in the HIV replication cycle with highly active and target-specific compounds will improve effectiveness over nonspecific and low activity compounds due to improved safety and specificity. Second, delivering these compounds from dosage forms that provide controlled, sustained inhibitory vaginal concentrations and potentially increased user adherence will improve effectiveness over vaginal gels which have demonstrated short duration drug pharmacokinetics and poor user adherence (32, 87). 1.3 Intravaginal Rings as Microbicides Due to the relative infancy of the microbicide field, microbicide formulations have typically leveraged many of the older vaginal drug delivery platforms developed for contraception, HRT, and vaginal infections. To date, intravaginal drug delivery vehicles primarily comprise gels and rings although newer and/or less utilized dosage forms include capsules, creams, foams, films, pessaries, sponges, suppositories, diaphragms, tablets, and tampons (100). Excluding intravaginal rings (IVRs), all of the above dosage forms may need to be frequently applied due to their relatively short therapeutically effective duration (hours to days), often require dosing before or after sex (coitally dependent), and typically provide dynamic vaginal pharmacokinetics. In contrast, IVRs are capable of weeks to months duration and can provide controlled drug release kinetics to more consistently maintain desired vaginal drug concentrations. Furthermore, IVRs possess favorable user attributes such as the potential for discreet use without interference or detection during sex, allowance for sexual spontaneity due to their long duration, and avoidance of product messiness or leakiness. Although not proven, microbicide IVRs are expected to improve both vaginal pharmacokinetics and user adherence over the 6 commonly used gel dosage form and are increasingly desired by the HIV prevention field in the search for a highly effective HIV prophylactic product (29, 86, 87). Torus shaped IVRs are prepared from "rubbery" polymers that deform elastically when pinched into an oval or figure-eight shape. The use of intravaginal elastomeric ring-shaped devices to deliver active pharmaceutical ingredients (APIs) is now over four decades old (24, 38, 47, 51, 101). It is not widely known that the contraceptive IVR was one of the first controlled release devices studied in the 1970s at the dawn of modern drug delivery technology (6). These revolutionary drug delivery devices were the first to take advantage of the new mechanistic understanding of controlled drug release from solid implants (9-12, 72). When the IVR is placed in the vaginal lumen, the drug concentration initially is homogeneous throughout the IVR, but immediately upon contact with vaginal tissue a spatial concentration gradient ensues. The drug present on the ring surface (at the polymer/tissue interface) is the first to diffuse from the IVR into the contacting tissue, transiting through a thin conducting layer of vaginal fluid or directly into tissue (Figure 1.1). The rate of drug release is interdependent on a number of factors including the solubility of the drug in the elastomer, the diffusion coefficient of the drug in the elastomer, the solubility of the drug in vaginal fluid, the volume of the vaginal fluid, the partition coefficient of the drug between the IVR and the vaginal fluid and tissue, the rate of diffusion and elimination of the drug through the vaginal tissue, and the rate of anterior to posterior advection of the vaginal fluid. To date, the contribution of each effect to vaginal pharmacokinetics and biodistribution is poorly understood. 7 1.4 IVR Manufacturing Four of the five commercial IVRs available to women are based on the silicone technology with indications for contraception (Progering® and Fertiring®) and HRT (Femring® and Estring®) (6, 20). Silicone IVR manufacturing is strictly limited to reaction injection molding wherein silicone elastomer base, cross-linking catalyst, and drug are mixed and subsequently injected under high pressure into a mold where the silicone addition-cure or condensation-cure reaction occurs under elevated temperature (48). The most recent commercially available IVR, marketed in 2002, is the dual hormone contraceptive IVR NuvaRing® (88-91), which was one of the first marketed thermoplastic hot melt extruded combination drug-device products. Polymers employed for microbicide IVR applications similarly include thermoset silicones (46, 49, 100), thermoplastic ethylene-co-vinyl acetate (EVA) (43), and more recently a variety of thermoplastic polyurethanes (14, 33, 39). Hot melt extrusion (HME) (30) along with injection molding (103) are two common thermoplastic processing methods that can be used to fabricate IVRs. HME is a continuous process whereby a screw or screws forces a molten material through an opening in an enclosed barrel under elevated temperature and pressure. Depending on the application, twin-screw extruders may be used to facilitate mixing of additives (i.e., excipients or API) or a single screw extruder may be used to provide more precise pressure and tighter control of shaped-product dimensions. Although HME has been used for decades in the plastics and medical device industries, its potential has only recently begun to be realized in the pharmaceutical industry (23). Over the last decade the number of patents, publications, and marketed pharmaceutical products has exponentially increased (67). There are several advantages of HME for 8 pharmaceutical and combination drug-device products including simultaneous API/carrier compounding and shaping, increased API bioavailability due to formation of a solid state solution (molecularly dissolved API), solvent-free processing to negate use of volatile and/or toxic solvents, and potentially reduced manufacturing costs since it is a continuous process in contrast to conventional pharmaceutical batch-processing (18). Furthermore, due in part to its extensive development in the plastics and medical device industries, HME is a highly controlled and monitored process that enables quality by design and process analytical technology - concepts strongly favored by the Food and Drug Administration (67). HME is an extremely versatile process and a variety of shaped and multilayered products may be made depending on equipment and accessory configuration. For example, the reservoir IVR NuvaRing® is made via coaxial extrusion which involves simultaneously using two extruders to feed a drug-loaded EVA core and an EVA rate-controlling membrane into a crosshead, resulting in a continuously extruded layered reservoir type product. Two significant drawbacks of HME for pharmaceutical applications are API chemical and physical stability; APIs that are thermally labile may be poor HME candidates although steps can be taken to minimize the extent of thermal degradation. Also, APIs that are formulated in the polymeric carrier above their maximum solubility at storage temperature can achieve API supersaturation during the elevated processing temperatures, resulting in a thermodynamically physically unstable system where API may bulk- or surface-crystallize during storage (7, 14, 90). Therefore, chemical and physical API stability must be considered in the potential development of a HME drug product. 9 1.5 IVR Design and Classification IVRs are generally categorized as either matrix or reservoir type and offer different advantages. A matrix IVR contains drug which is homogenously dissolved or dispersed throughout the entirety of the elastomeric polymer, whereas a reservoir IVR typically utilizes a drug loaded polymeric core surrounded by a thin layer of polymer of the same or different composition (termed a rate-controlling membrane) to impede and thus regulate drug diffusion (66) (Figure 1.2). Matrix IVRs, owing to their simpler design and construction, are often cheaper to fabricate yet typically provide time-dependent release kinetics proportional to the square root of time with less flexibility in modulating the release kinetics (46). Conversely, reservoir IVRs are generally more costly to fabricate but offer near time-independent drug release kinetics and easier modulation of the release rate (91). The literature provides general equations describing drug release from matrix- and reservoir-type drug delivery devices (Equation 1.1 and 1.2, respectively) based on the Higuchi equation (26): 􀜳􃌍 􀵌􄰒 􁈾􃸒􁈺􃨃􀍴􇐇􀜣􂌍 􀵆􄘇 􀜥􂔒􁈻􃬇􀜥􂔇􀜦􂘇􀝐􅀒􁈿􃼋􀬴􃐁􀇤􎐋􀬹􃤠 􀜳􃌍 􀵌􄰇 􀜦􂘇􀜥􂔇􀝐􅀀 􀂊􈨠 where Q is the cumulative amount of drug released per unit surface area, A is the drug loading per unit volume, C is the solubility of drug in the polymer per unit volume, D is the diffusion coefficient in the polymer, t is time, and h is the rate controlling membrane thickness (for reservoir devices, C and D refer to the solubility and diffusion coefficient Equation (1.1) Equation (1.2) 10 in the outer polymer). An additional consideration beyond the desired drug release profile is the drug release rate; reservoir IVRs of a given polymer will typically demonstrate decreased release rates at early time compared to matrix IVRs due to the rate-controlling membrane which impedes drug diffusion (56). Therefore, selection of a matrix-type or reservoir-type IVR design will depend on the clinical needs and which drug release specifications are most critical to achieve. For example, delivery of contraceptive hormones requires low but consistent daily release rates (i.e., micrograms per day) due to their relatively narrow therapeutic window and high activity; therefore reservoir IVRs are more suitable than matrix IVRs in this instance. An interesting exception to IVR classification exists when matrix IVRs demonstrate time-independent drug release. In the case termed partition controlled release, the drug demonstrates elevated solubility in the polymer and significantly lower solubility in the vaginal fluid and furthermore is formulated at high weight percent in the polymer with a fairly large polymer diffusion coefficient (9). If these conditions are satisfied, time-independent release has been observed since dissolution into the vaginal fluid is the rate limiting step, in contrast to the more typical matrix controlled release discussed above where diffusion through the polymer to the IVR surface is the rate limiting step (69). Although the primary function of an IVR is to deliver therapeutics to the vaginal tract, IVRs must also possess certain dimensions and mechanical properties in order to be inserted and retained in the vaginal tract - both from an ease-of-use and device functionality perspective. Vaginal rings should exhibit an elastic nature since the IVR must be compressed to be inserted, yet have sufficient recoil force once in place so as to be retained in the vaginal tract. A force balance thus exists between the elastic recoil of 11 the IVR and the musculature of the vaginal wall, with ring dimensions and material elastic modulus determining the final conformation and retention of the IVR. Marketed vaginal ring outer diameters and cross-sectional diameters range from 54 to 58 and 4 to 9 mm, respectively (2). The four marketed silicone rings (Estring®, Femring®, Progering®, and Fertiring®) possess a significantly lower elastic modulus than the marketed EVA ring (NuvaRing®). As a result, the silicone rings encompass the larger dimensions to increase ring rigidity whereas the EVA ring is the smallest with a 54 by 4 mm outer diameter and cross-sectional diameter, respectively. To arrive at these finalized ring dimensions during product development, several clinical studies evaluated silicone or EVA rings of various cross-sectional diameters and elastic moduli in women who used them for extended durations up to a year, and user acceptability, functionality, and/or effects to the vaginal epithelium were monitored. It was discovered that rings with relatively lower cross-sectional diameters (resulting in a mechanically softer ring) had a higher rate of vaginal expulsion or ring movement due to their lower elastic recoil force (41, 70). However, if the ring elastic recoil force was too great, there was some evidence that users had greater difficulty inserting the ring and that the ring may have caused trauma to the vaginal epithelium (96). Logically there exists an optimum range of IVR stiffness keeping in mind that there will be a wide range of vaginal shapes and sizes among the female user population (60-62). In one particularly large clinical study, four rings of wide-ranging stiffness were evaluated and all were observed to provide no vaginal trauma compared to the naïve arm (27). Since there is no quantitative model to determine the ideal mechanical properties of a vaginal ring, early microbicide IVR development may benefit by demonstrating similar ring stiffness to the above mentioned marketed products 12 which have exhibited high user acceptability and safety in large user populations. Also, a mechanical model relating the force required for the point compression of thin elastic rings may be useful for IVR research to better understand the influence of variables such as ring dimensions and elastic modulus on ring stiffness (Equation 1.3) (37). 􀜨􂠍 􀵌􄰀 􀀃􀌇 􀟨􎠋􀬶􃘇􀜧􂜇􀝎􄸋􀯢􎈋 􀬸􃠒 􁈺􃨇􀟨􎠋􀬶􃘍 􀵆􄘃 􀍺􇨒􁈻􃬒􁈺􃨇􀜴􃐍 􀵆􄘇 􀝎􄸋􀯢􎈒􁈻􃬋􀬷􃜇 􀜻􃬠 where F is force, E is the elastic modulus, ro is the cross-sectional outer radius, R is the ring outer radius, and Y is the reduction in ring outer diameter. The fourth-power force dependence yet third-power volume dependence on the cross-sectional ring diameter (assuming a fixed ring outer diameter) allows for flexibility when designing an IVR. Also, IVR compression force is linearly related to the elastic modulus of the IVR matrix, which can be affected by the class or grade of polymer used and by incorporation of drugs or excipients. Although this mechanical model ignores polymer viscoelasticity and assumes linear stress-strain behavior, which is invalid for high ring deformations such as during ring insertion where polymer, the model can still be a useful tool for ring design, especially when considering small ring deformations such as those seen in a magnetic resonance imaging assessment of NuvaRing® retention in the female vaginal tract (5). 1.6 Acceptability and Adherence In the context of microbicides, the terms "acceptability" and "adherence" are often misused interchangeably and therefore should be defined and distinguished. Acceptability refers to the extent to which a product is desirable to the target user population while Equation (1.3) 13 adherence refers to the extent to which users obey specified dosing instructions. Although acceptability and adherence will often correlate, the specific relationship between the two has not been well defined in the context of microbicides. Clearly, however, an efficacious microbicide product must be desirable to women (i.e., possess high acceptability) or the product will not be used (i.e., demonstrate low adherence) and its real world effectiveness will be limited (52). Few microbicide IVR acceptability studies have been performed but it is hypothesized that microbicide IVR acceptability will be similar to contraceptive IVRs, which have been extensively evaluated. In a multinational clinical trial, 66% of the women initially preferred oral contraceptives but after NuvaRing® use 81% preferred the vaginal ring (57). The change in contraceptive preference was mainly attributed to ease of use and not having to remember to take a pill each day, and overall acceptability of the IVR was approximately 96% (71). However, an important consideration is that IVR acceptability in developed countries - where NuvaRing® has been well studied - may be different than in developing countries where most microbicide IVRs will be used and where women's perceptions and cultural practices will be diverse. A study in Brazil concluded that women found IVRs to be more acceptable than other microbicide dosage forms such as gels (35), with long duration and spontaneity being offered as the primary reason. Another relevant study for the microbicide field examined African women sex workers' acceptability of IVRs for HIV prevention (76). Most women favored an IVR over a gel due to its long duration and covert use - although women were concerned that men might detect the ring. Women's opinions are likely coupled to men's opinion in the many patriarchal societies in the developing world; therefore, men's concerns and opinions on IVRs should not be discounted. In the study, men's primary concerns were 14 feeling the ring during intercourse, yet overall thought it more acceptable than a gel that may give undesired vaginal lubrication. Since both sexes were concerned about the possibility of male contact with the ring during coitus, the study suggests that IVR dimensions should be minimized to decrease the probability of this event. Most recently, an extensive acceptability study was performed where 157 young African women inserted a placebo silicone ring for 12 weeks (86). In general, women found the IVR favorable and were attracted by its potential for discreetness, noninterference with coitus, and continuous use capability which could allow for spontaneity or protection against HIV in the case of rape. 67% of female participants considered it important for their male partner to not detect the ring during intercourse, and 63% and 13% feared that their partner may become angry or physically abusive, respectively, if the ring was discovered. It is worth noting that both African acceptability studies evaluated silicone IVRs with roughly twice the diameter of the contraceptive NuvaRing®. Although the majority of men did not feel the ring during intercourse, minimizing IVR dimensions will likely increase women's confidence in the discreet capability of the IVR and decrease the probability of undesired male contact with the ring during coitus. An excellent review article by Heise et al. discusses microbicide prevention trials and considerations when interpreting success of user-controlled methods (36). The authors define efficacy as an improvement in outcome under ideal conditions (perfect use) whereas effectiveness is the improvement in outcome achieved in practice (imperfect or inconsistent use). The microbicide field is concerned with adherence in clinical trials, especially following the Carraguard vaginal gel clinical trial where women adhered to the dosing regimen only 42% of the time (75). This result brought the 15 realization that product efficacy is meaningless for prospective microbicide products demonstrating low user adherence, as the overall effectiveness is so low. In the CAPRISA 004 trial with the TFV 1% gel, user adherence positively correlated with HIV prophylaxis. The hope is that a long-lasting, coitally-independent IVR will increase adherence and therefore effectiveness, shortening the gap between efficacy and effectiveness. We warn that there are little data supporting this hope. In fact, the 21 day NuvaRing® is only as good at preventing unplanned pregnancy as the once daily oral contraceptive (6), and only a 9-12% increase in adherence is achieved for various pharmaceutical products when going from daily to weekly frequency with an overall trend of intermittent dosing having higher adherence than frequent dosing (42, 58). However, the messy, nondiscreet, and burdensome gel dosage form may not be a fair comparison to the oral dosage form which generally demonstrates good user adherence. Little data exists on nonuser reported once per month administration, but methods to accurately measure microbicide adherence are being developed (79, 81). 1.7 Microbicide IVR Limitations and Unknowns As discussed previously, an advantage of the EVA polymer technology over the silicone polymer technology is the ability for continuous and controlled manufacturing. Equally important, however, is the ability to more easily modulate ring stiffness (since silicone possesses a relatively narrow elastic modulus range) so that IVR cross-sectional diameter can be reduced to potentially increase user acceptability. Although NuvaRing® development has advanced IVR technology, both silicone and EVA IVR platforms have only demonstrated success to deliver hydrophobic small molecules at micrograms/day 16 levels. Numerous microbicide IVRs have been developed using these platforms to deliver hydrophobic small molecule antiretrovirals and one product is advancing for clinical efficacy evaluation (55). Clearly, however, the vaginal drug delivery field would benefit from IVR platforms which 1) can provide controlled and sustained delivery of doses higher than micrograms/day and 2) can deliver APIs besides hydrophobic small molecules. Unfortunately, many hydrophilic APIs are minimally soluble in the silicone and EVA polymers and therefore often cannot be delivered by simple diffusion at desired release rates for sustained duration. The hydrophilic small molecule tenofovir is a key example of current IVR technology inadequacy; Tenofovir was recently proven effective against HIV when formulated in a vaginal gel yet lacks a suitable IVR delivery system due to insufficient release rates from conventional EVA and silicone polymers (77). In addition to hydrophilic small molecules, there are a diverse set of macromolecular and biopharmaceutical APIs (including nucleic acids, peptides, and proteins) (97-99) that may be potential topical microbicides. However, biologics will require unconventional IVR designs as they often cannot withstand conventional IVR processing temperatures required for injection molding and hot melt extrusion, and/or have limited diffusivity in elastomeric polymers due to their size and limited polymer solubility (98). Therefore, designing IVRs for long-term delivery of drugs with wide ranging physicochemical properties and desired release kinetics is challenging and will require new and innovative technology. The primary objective of microbicide IVRs is to deliver antiretrovirals locally to prevent initial infection of susceptible immune cells in the vaginal tract, and therefore knowledge of spatiotemporal vaginal drug concentrations is deemed crucial to device 17 success. Since all of the currently marketed IVRs target systemic hormonal delivery, there is very limited knowledge about the vaginal biodistribution and pharmacokinetics of drugs that are delivered from IVRs. Prior to initiation of the dissertation work reported herein, there were no peer-reviewed in vivo studies in women or large animals to examine safety, vaginal biodistribution and pharmacokinetics, or potential efficacy of microbicide IVR formulations. To evaluate some of these critical unknowns prior to testing in women, several animal models offering unique advantages and disadvantages are now being utilized by the field (92, 94). A comparison of vaginal mucosal models is presented in literature (17, 78). The cheapest yet least relevant animal models for HIV prophylaxis are rodents and rabbits. Their vaginal anatomy and physiology are quite different from women, but clearly are a step forward from in vitro studies and may offer a quick, low cost option for screening potential formulations prior to testing in more relevant animal models. The rabbit vaginal irritation model is widely used for preliminary microbicide safety studies as it is currently a required preclinical test by the Food and Drug Administration (21, 22), and more recently pilot pharmacokinetic studies have been performed with rabbits (13, 15). A major drawback of testing microbicide IVRs in rabbits has been the need to suture devices in place to avoid expulsion from the vaginal tract. The invasive suturing procedure causes tissue trauma at the implant site and may likely impact safety and pharmacokinetic outcomes. To improve the relevancy and usefulness of this model, a shape memory polyurethane fixture was recently invented in our laboratory which allows for nonsurgical device insertion and retention of the test article in the vagina. 18 Amongst large animal models, the sheep model has only recently gained traction and greater use for microbicide safety and pharmacokinetic evaluation. The sheep vaginal anatomy and stratified squamous epithelium are similar to humans and thus offer two significant improvements over small animal models. Due to their anatomical similarity to women, human-sized IVRs may be tested in sheep although significant deviations include different native vaginal microflora and a vaginal fluid pH of approximately 8 as opposed to 4.5 in women (78, 95). The pig animal model, particularly the minipig species, has remarkably similar anatomy and physiology to women and also shows promise for microbicide safety and pharmacokinetic testing (85). Pigs display a similar acidic pH to women although their microflora is generally dissimilar (19, 44, 54). Depending on the species utilized, a human-sized IVR could potentially be tested in pigs. Although the preceding animal models each have unique advantages and may be appropriate for certain stages and objectives during microbicide IVR development, nonhuman primates are considered the most relevant animals for HIV prophylaxis research since they exhibit the most similar anatomy and physiology to humans (80, 94). One particular advantage of pigtail and rhesus macaques for microbicide testing is that their vaginal microflora is most similar to women, with the presence of lactic acid secreting lactobacillus which maintains an acidic vaginal pH to provide an innate defense against pathogens (59, 102). As a result, the potential negative effects of microbicide formulations on endogenous microflora, in addition to the vaginal epithelium, can be evaluated in the pigtail and rhesus models. Another potential advantage of the pigtail and rhesus models is the capability to perform efficacy studies of microbicide formulations using the related simian immunodeficiency and simian-human immunodeficiency 19 viruses, although the ability of these viral challenge studies to accurately predict HIV infection and prophylaxis in women is uncertain and the subject of debate (80). In deciding between rhesus and pigtail macaque models for IVR evaluation, several aspects must be considered. Readers are pointed towards comprehensive reviews of microbicide animal models, which includes rhesus and pigtail macaques (93, 94), but two major differences are highlighted: First, pigtail macaques cycle monthly (akin to women) whereas rhesus are seasonal breeders (64). Therefore, rhesus macaques may require progesterone administration to synchronize their menstrual cycle. Unfortunately, this treatment thins the vaginal epithelium and thereby increases API as well as virus permeability (45, 93). Secondly, the supply of pigtail macaques is quite limited and therefore large studies and/or cost may necessitate the use of rhesus macaques. Altogether, both species still cost significantly more to purchase and house compared to the other animal models discussed, thereby potentially limiting experimental design and statistical analyses. Another drawback of the macaque model is scaling issues related to their smaller vaginal anatomy compared to women, complicating the correlation of pharmacokinetics between macaques and women and preventing the testing of human-sized vaginal rings. The Centers for Disease Control and Prevention recently evaluated various size rings in both rhesus and pigtail macaques and found that 25 mm outer diameter IVRs fit well with no mucosal inflammation or lesions (64). If ring outer diameter is reduced from the human-sized 55 mm to 25 mm yet the same cross-sectional diameter is maintained, ring mass and surface area is reduced by approximately 60%. Since the drug release rate is proportional to the change in ring surface area (26), the reduction in ring size also 20 corresponds to a 60% decrease in release rate. The average woman weighs approximately 10 times that of a macaque (59, 74), therefore simply scaling drug release by body mass (weight/weight) results in an approximately 6-fold higher overall exposure of drug in the macaque than in women. Similarly, an approximation of vaginal volume, given mean length and diameter measurements (4, 59), also results in a 6-fold difference between macaques and women. Therefore, these issues should be considered when designing macaque studies and performing pharmacokinetic extrapolations from macaques to women. Animal models for microbicide IVR evaluation are only now being developed. To date, there are many unknowns related to microbicide IVR in vivo performance, particularly vaginal biodistribution and pharmacokinetics, formulation effects on the vaginal epithelium and endogenous microflora, and the required vaginal concentrations for potential effectiveness against HIV. Many unanswered questions exist, such as how long after ring insertion does it take for vaginal fluid and tissue drug concentrations to reach protective levels? Specifically, what are the spatiotemporal drug concentrations proximal to the ring at the endocervix as well as near the introitus which is centimeters away from ring placement? How long after IVR removal are protective concentrations maintained? Drug release kinetics from IVRs have primarily been tested in vitro using sink conditions wherein the concentration of the drug in the release media is never allowed to exceed one-tenth its maximum solubility. Whether these in vitro conditions are predictive of in vivo performance is unclear, especially for hydrophobic antiretrovirals which display poor aqueous solubility and may thus achieve saturated concentrations in the vaginal fluid. The selection of an appropriate animal model should depend on the 21 main study objectives and questions asked, and several factors should be considered including cost and study size, anatomy and physiology, ring size and scaling, and whether or not efficacy studies are deemed important. 1.8 Conclusions In summary, this work is motivated by an overall desire to develop prophylactic methods and products to prevent HIV transmission. Specifically, the focus of this dissertation work is to develop long-lasting intravaginal ring technologies which can deliver proven antiretrovirals to the vaginal tract to prevent the male-to-female sexual transmission of HIV. Intravaginal rings offer several advantages over other candidate vaginal dosage forms to locally deliver antiretrovirals; First, their extended duration capability can minimize final product cost through amortization. Second, their long-lasting, non-messy, coitally independent, and discreet potential may help increase user adherence. Finally, their inherent ability to provide long-duration, controlled drug release may enable IVRs to better attain and maintain effective prophylactic vaginal concentrations in contrast to less controlled and short-duration dosage forms. Although intravaginal rings hold promise for microbicide applications, there has been little design innovation since their initial development decades ago for contraceptive and hormone replacement indications. As a result, IVR delivery of therapeutics which are not highly active hydrophobic small molecules is generally suboptimal. Furthermore, as intravaginal ring technologies have historically aimed for systemic delivery of therapeutics, there is little understanding of drug vaginal biodistribution and pharmacokinetics from IVRs, or of IVR effects on the vaginal environment pertaining to 22 safety. Thus, the primary focus of this dissertation work is to 1) design new intravaginal rings capable of delivering a variety of antiretrovirals and 2) develop new in vitro tests and in vivo models to increase IVR performance understanding and thereby provide new IVR platforms and products to prevent the sexual transmission of HIV. 1.9 Dissertation Chapter Overview Each following chapter is a separately published manuscript or is pending publication. An overview of each chapter and its publication information is provided below. 1.9.1 Chapter 2 overview In Chapter 2, we sought to design and test in vivo a polyurethane intravaginal ring to deliver the promising small molecule antiretroviral agent congeners IQP-0528 and IQP-0532. In particular, this dose-finding and safety study aimed to determine 1) whether loading-dependent drug release is observed in vivo as is observed in vitro, 2) what IVR drug loading would achieve vaginal concentrations deemed to be sufficient to prevent HIV infection, and 3) whether the formulations cause any harm or irritation to the vaginal mucosa and endogenous microflora. The two congeners were evaluated at various loadings in pigtail macaques and compared to placebo and naïve control arms. The primary study output was vaginal pharmacokinetics and safety. This was the first peer-reviewed publication reporting drug safety and pharmacokinetics of a microbicide IVR in nonhuman primates. 23 Publication: Johnson T. J., Srinivasan P., Albright T. H., Watson-Buckheit K., Rabe L., Martin A., Pau C.P., Hendry R.M., Otten R., McNicholl J., Buckheit R., Smith J., Kiser P.F. Safe and Sustained Vaginal Delivery of Pyrimidinedione HIV-1 Inhibitors from Polyurethane Intravaginal Rings. Antimicrobial Agents and Chemotherapy, 2012. 56(3): p. 1291-1299. 1.9.2 Chapter 3 overview In Chapter 3, we sought to develop a combination IVR to simultaneously deliver the two promising antiretroviral molecules dapivirine and tenofovir which possess different mechanisms of HIV inhibition and contrasting hydrophilicity. Combination microbicides are under development to reduce the probability of drug-resistant virus which could arise from single agent HIV prophylaxis. The delivery of tenofovir from conventional hydrophobic polymer IVRs has proven inadequate owing to its hydrophilicity and low polymer miscibility. Our main aims were to 1) design and characterize a new IVR platform to improve tenofovir release rates over conventional IVRs, and 2) provide sustained release of both dapivirine and tenofovir for up to 30 days. This was the first peer-reviewed publication of an advanced multi-segment IVR platform to deliver multiple antiretrovirals, and the first report of using hydrophilic polyurethanes for IVR applications. Publication: Johnson T.J., Gupta K.M., Fabian J., Albright T.H., Kiser P.F. Segmented Polyurethane Intravaginal Rings for the Sustained Combined Delivery of Antiretroviral Agents Dapivirine and Tenofovir. European Journal of Pharmaceutical Sciences, 2010. 39: p. 203-212. 24 1.9.3 Chapter 4 overview In Chapter 4, we sought to design and evaluate in a sheep animal model a tenofovir (TFV) reservoir IVR which could avoid the time-dependent TFV release kinetics and ring mechanical stiffness observed with the matrix IVR developed in Chapter 3. Our main aims were to develop a ring that could 1) attain similar vaginal TFV concentrations as the clinically effective TFV 1% gel but for 90 day duration, 2) show no harmful vaginal effects, and 3) achieve time-independent ring mechanical stiffness similar to commercially available IVRs. This was the first publication to demonstrate similar or higher TFV vaginal concentrations from an IVR compared to the TFV 1% gel, and furthermore in a time-independent fashion for 90 days with no significant toxicological effects. Publication: Johnson T.J., Clark M.R., Albright T.H., Nebeker J.S., Tuitupou A.L., Clark J.T., Fabian J., McCabe R.T., Chandra, N., Doncel, G.F., Friend D.R., Kiser P.F. A Ninety Day Tenofovir Reservoir Intravaginal Ring for Mucosal HIV Prophylaxis. Antimicrobial Agents and Chemotherapy. Under Review. 1.9.4. Chapter 5 overview In Chapter 5, we sought to further design and characterize the TFV IVR prototype reported in Chapter 4. Our main aims were to 1) design and test IVRs of various composition and dimension to arrive at an optimized design and better understand the design space, and 2) determine the underlying mechanism behind drug release rate changes as a function of product storage time in order to achieve a thermodynamically stable formulation. This manuscript reported the engineering of an unconventional 25 reservoir IVR platform which could be used to deliver a spectrum of therapeutics and is the first publication to demonstrate and explain the impact of polyurethane microphase separation kinetics on drug flux. This was also the first IVR platform capable of delivering daily milligram quantifies of an API for greater than 30 days, and moreover while maintaining time-independent ring mechanical stiffness. Publication: Johnson T.J., Nebeker J.S., Tuitupou A.L., Smith E.M., Clark J.T., Fabian J., McCabe R.T., Clark M.R., Friend D.R., Kiser P.F. Engineering of a New Intravaginal Ring Technology for HIV Prophylaxis. Pharmaceutical Research. Manuscript in preparation. 1.9.5. Chapter 6 overview In Chapter 6, brief conclusions of the dissertation work will be drawn and recommendations will be made regarding future research in the microbicide IVR field. 26 1.10 References 1. Abdool Karim, Q., S. S. Abdool Karim, J. A. Frohlich, A. C. Grobler, C. Baxter, L. E. Mansoor, A. B. Kharsany, S. Sibeko, K. P. Mlisana, Z. Omar, T. N. Gengiah, S. Maarschalk, N. Arulappan, M. Mlotshwa, L. Morris, and D. Taylor. 2010. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 329:1168- 1174. 2. Alexander, N. J., E. Baker, M. Kaptein, U. Karck, L. Miller, and E. Zampaglione. 2004. Why consider vaginal drug administration? Fertil Steril 82:1-12. 3. Baloglu, E., Z. A. Senyigit, S. Y. Karavana, and A. Bernkop-Schnurch. 2009. Strategies to prolong the intravaginal residence time of drug delivery systems. J Pharm Pharm Sci 12:312-336. 4. Barnhart, K. T., A. Izquierdo, E. S. Pretorius, D. M. Shera, M. Shabbout, and A. Shaunik. 2006. Baseline dimensions of the human vagina. Hum Reprod 21:1618-1622. 5. Barnhart, K. T., K. Timbers, E. S. Pretorius, K. Lin, and A. Shaunik. 2005. In vivo assessment of nuvaring placement. Contraception 72:196-199. 6. Brache, V., and A. Faundes. 2010. Contraceptive vaginal rings: a review. Contraception 82:418-427. 7. Bruce, C. D., K. A. Fegely, A. R. Rajabi-Siahboomi, and J. W. McGinity. Aqueous film coating to reduce recrystallization of guaifenesin from hot-melt extruded acrylic matrices. Drug Dev Ind Pharm 36:218-226. 8. Cefalu, W. T. 2001. Novel routes of insulin delivery for patients with type 1 or type 2 diabetes. Ann Med 33:579-586. 9. Chien, Y. W., and H. J. Lambert. 1974. Controlled drug release from polymeric delivery devices. II: differentiation between partition-controlled and matrix-controlled drug release mechanisms. J Pharm Sci 63:515-519. 10. Chien, Y. W., H. J. Lambert, and D. E. Grant. 1974. Controlled drug release from polymeric devices. I: technique for rapid in vitro release studies. J Pharm Sci 63:365-369. 11. Chien, Y. W., and E. P. Lau. 1976. Controlled drug release from polymeric delivery devices IV: in vitro--in vivo correlation of subcutaneous release of norgestomet from hydrophilic implants. J Pharm Sci 65:488-492. 27 12. Chien, Y. W., S. E. Mares, J. Berg, S. Huber, H. J. Lambert, and K. F. King. 1975. Controlled drug release from polymeric delivery devices. III: in vitro-in vivo correlation for intravaginal release of ethynodiol diacetate from silicone devices in rabbits. J Pharm Sci 64:1776-1781. 13. Clark, M. R., and D. R. Friend. 2012. Pharmacokinetics and topical vaginal effects of two tenofovir gels in rabbits. AIDS Res Hum Retroviruses. http://online.liebertpub.com/doi/abs/10.1089/ aid.2011.0328?journalCode=aid. 14. Clark, M. R., T. J. Johnson, R. T. McCabe, J. T. Clark, A. Tuitupou, H. Elgendy, D. R. Friend, and P. F. Kiser. 2012. A hot-melt extruded intravaginal ring for the sustained delivery of the antiretroviral microbicide UC781. J Pharm Sci 101:576-587. 15. Clark, M. R., P. F. Kiser, A. Loxley, C. McConville, R. K. Malcolm, and D. R. Friend. 2011. Pharmacokinetics of UC781-loaded intravaginal ring segments in rabbits: a comparison of polymer matrices. Drug Deliv Transl Res 1:238-246. 16. Corbin, J., and L. T. Bonde. 2012. Intersections of context and HIV/AIDS in sub-saharan africa: what can we learn from feminist theory? Perspect Public Health 132:8-9. 17. Costin, G. E., H. A. Raabe, R. Priston, E. Evans, and R. D. Curren. 2011. Vaginal irritation models: the current status of available alternative and in vitro tests. Altern Lab Anim 39:317-337. 18. Crowley, M. M., F. Zhang, M. A. Repka, S. Thumma, S. B. Upadhye, S. K. Battu, J. W. McGinity, and C. Martin. 2007. Pharmaceutical applications of hot-melt extrusion: part I. Drug Dev Ind Pharm 33:909-926. 19. D'cruz, O. J., D. Erbeck, and F. M. Uckun. 2005. A study of the potential of the pig as a model for the vaginal irritancy of benzalkonium chloride in comparison to the nonirritant microbicide PHI-443 and the spermicide vanadocene dithiocarbamate. Toxicol Pathol 33:465-476. 20. Dezarnaulds, G., and I. S. Fraser. 2003. Vaginal ring delivery of hormone replacement therapy--a review. Expert Opin Pharmacother 4:201-212. 21. Doncel, G. F., N. Chandra, and R. N. Fichorova. 2004. Preclinical assessment of the proinflammatory potential of microbicide candidates. J Acquir Immune Defic Syndr 37:S174-S180. 22. Doncel, G. F., and M. R. Clark. 2010. Preclinical evaluation of anti-HIV microbicide products: new models and biomarkers. Antiviral Res 88 Suppl 1:S10-18. 28 23. Douroumis, D. (ed.). 2012. Hot-melt extrusion: pharmaceutical applications. Wiley, West Sussex. 24. Duncan, G. W. 1970. Medicated devices and methods. US patent 3,545,439. 25. Feldblum, P. J., A. Adeiga, R. Bakare, S. Wevill, A. Lendvay, F. Obadaki, M. O. Olayemi, L. Wang, K. Nanda, and W. Rountree. 2008. SAVVY vaginal gel (C31G) for prevention of HIV infection: a randomized controlled trial in nigeria. PLoS One 3:e1474. 26. Fetherston, S. M., R. K. Malcolm, and A. D. Woolfson. 2010. Controlled-release vaginal ring drug-delivery systems: a key strategy for the development of effective HIV microbicides. Ther Deliv 1:785-802. 27. Fraser, I. S., M. Lacarra, D. R. Mishell Jr, F. Alvarez, V. Brache, P. Lähteenmäki, K. Elomaa, E. Weisberg, and H. A. Nash. 2000. Vaginal epithelial surface appearances in women using vaginal rings for contraception. Contraception 61:131-138. 28. Friend, D. R. 2012. Drug delivery in multiple indication (multipurpose) prevention technologies: systems to prevent HIV-1 transmission and unintended pregnancies or HSV-2 transmission. Expert Opin Drug Deliv 9:417-427. 29. Friend, D. R. 2011. Intravaginal rings: controlled release systems for contraception and prevention of transmission of sexually transmitted infections. Drug Deliv Transl Res 1:185-193. 30. Ghebre-Selassie, I., and C. Martin (ed.). 2007. Pharmaceutical extrusion technology. Informa Healthcare, New York. 31. Girard, M. P., and S. A. Plotkin. 2012. HIV vaccine development at the turn of the 21st century. Curr Opin HIV AIDS 7:4-9. 32. Grant, R. M., D. Hamer, T. Hope, R. Johnston, J. Lange, M. M. Lederman, J. Lieberman, C. J. Miller, J. P. Moore, D. E. Mosier, D. D. Richman, R. T. Schooley, M. S. Springer, R. S. Veazey, and M. A. Wainberg. 2008. Whither or wither microbicides? Science 321:532-534. 33. Gupta, K. M., S. M. Pearce, A. E. Poursaid, H. A. Aliyar, P. A. Tresco, M. A. Mitchnik, and P. F. Kiser. 2008. Polyurethane intravaginal ring for controlled delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1. J Pharm Sci 97:4228-4239. 34. Gupta, S., R. Gabrani, J. Ali, and S. Dang. 2011. Exploring novel approaches to vaginal drug delivery. Recent Pat Drug Deliv Formul 5:82-94. 29 35. Hardy, E., E. M. Hebling, M. H. Sousa, A. F. Almeida, and E. Amaral. 2007. Delivery of microbicides to the vagina: difficulties reported with the use of three devices, adherence to use and preferences. Contraception 76:126-131. 36. Heise, L. L., C. Watts, A. Foss, J. Trussell, P. Vickerman, R. Hayes, and S. McCormack. 2011. Apples and oranges? Interpreting success in HIV prevention trials. Contraception 83:10-15. 37. Huston, R., and H. Josephs. 2009. Practical stress analysis in engineering design, 3rd ed. CRC Press, Boca Raton. 38. Johansson, E. D. B., and R. Sitruk-Ware. 2004. New delivery systems in contraception: vaginal rings. Am J Obstet Gynecol 190:S54-S59. 39. Kaur, M., K. M. Gupta, A. E. Poursaid, P. Karra, A. Mahalingam, H. A. Aliyar, and P. F. Kiser. 2011. Engineering a degradable polyurethane intravaginal ring for sustained delivery of dapivirine. Drug Deliv Transl Res 1:15. 40. Keskar, V., P. S. Mohanty, E. J. Gemeinhart, and R. A. Gemeinhart. 2006. Cervical cancer treatment with a locally insertable controlled release delivery system. J Control Release 115:280-288. 41. Koetsawang, S., G. Ji, U. Krishna, A. Cuadros, G. I. Dhall, R. Wyss, J. Rodriquez la Puenta, A. T. Andrade, T. Khan, E. S. Kononova, J. P. Lawson, U. Parekh, M. Elstein, V. Hingorani, N. Wang, Z. Yao, B. M. Landgren, R. Boukhris, L. Lo, and S. Boccard. 1990. Microdose intravaginal levonorgestrel contraception: a multicentre clinical trial. II: expulsions and removals. Contraception 41:125-141. 42. Kruk, M. E., and N. Schwalbe. 2006. The relation between intermittent dosing and adherence: preliminary insights. Clin Ther 28:1989-1995. 43. Loxley, A., M. Mitchnick, O. Okoh, J. McConnell, L. Goldman, C. Morgan, M. Clark, and D. R. Friend. 2011. Ethylene vinyl acetate intravaginal rings for the simultaneous delivery of the antiretroviral UC781 and contraceptive levonorgestrel. Drug Deliv Transl Res 1:247-255. 44. Maes, D., M. Verdonck, and A. De Kruif. 1999. Vaginal microecology and vulval discharge in swine. Old Herborn University Seminar Monograph 12:39-50. 45. Malcolm, K., D. Lowry, L. Green, R. Shattock, M. Mitchnick, L. Geer, P. Klaase, J. Moore, and R. Veazey. 2010. Pre-treatment with depo-provera modifies the pharmacokinetics of CMPD167 in rhesus macaques following vaginal ring administration. Presented at the Microbicides 2010. Pittsburgh, PA. 30 46. Malcolm, K., D. Woolfson, J. Russell, P. Tallon, L. McAuley, and D. Craig. 2003. Influence of silicone elastomer solubility and diffusivity on the in vitro release of drugs from intravaginal rings. J Control Release 90:217-225. 47. Malcolm, R. K. 2003. The intravaginal ring. Drugs and the Pharmaceutical Sciences 126:775-790. 48. Malcolm, R. K., K.-L. Edwards, P. Kiser, J. Romano, and T. J. Smith. 2010. Advances in microbicide vaginal rings. Antiviral Res 88:S30-S39. 49. Malcolm, R. K., A. D. Woolfson, C. F. Toner, R. J. Morrow, and S. D. McCullagh. 2005. Long-term, controlled release of the HIV microbicide TMC120 from silicone elastomer vaginal rings. J Antimicrob Chemother 56:954- 956. 50. McCormack, S., G. Ramjee, A. Kamali, H. Rees, A. M. Crook, M. Gafos, U. Jentsch, R. Pool, M. Chisembele, S. Kapiga, R. Mutemwa, A. Vallely, T. Palanee, Y. Sookrajh, C. J. Lacey, J. Darbyshire, H. Grosskurth, A. Profy, A. Nunn, R. Hayes, and J. Weber. 2010. PRO2000 vaginal gel for prevention of HIV-1 infection (microbicides development programme 301): a phase 3, randomised, double-blind, parallel-group trial. Lancet 376:1329-1337. 51. Mishell, D. R., Jr., M. Talas, A. F. Parlow, and D. L. Moyer. 1970. Contraception by means of a silastic vaginal ring impregnated with medroxyprogesterone acetate. Am J Obstet Gynecol 107:100-107. 52. Morrow, K. M., and M. S. Ruiz. 2008. Assessing microbicide acceptability: a comprehensive and integrated approach. AIDS Behav 12:272-283. 53. Napierala Mavedzenge, S., R. Olson, A. M. Doyle, J. Changalucha, and D. A. Ross. 2011. The epidemiology of HIV among young people in sub-saharan africa: know your local epidemic and its implications for prevention. J Adolesc Health 49:559-567. 54. Ndesendo, V. M. K., V. Pillay, Y. E. Choonara, L. C. Du Toit, L. C. R. Meyer, E. Buchmann, P. Kumar, and R. A. Khan. 2011. In vivo evaluation of the release of zidovudine and polystyrene sulfonate from a dual intravaginal bioadhesive polymeric device in the pig model. J Pharm Sci 100:1416-1435. 55. Nel, A. 2012. Safety and efficacy trial of a dapivirine vaginal matrix ring in healthy HIV-negative women. http://www.clinicaltrials.gov/ct2/show/NCT01539226. 56. Nel, A., S. Smythe, K. Young, K. Malcolm, C. McCoy, Z. Rosenberg, and J. Romano. 2009. Safety and pharmacokinetics of dapivirine delivery from matrix 31 and reservoir intravaginal rings to HIV-negative women. J Acquir Immune Defic Syndr 51:416-423. 57. Novak, A., C. de la Loge, L. Abetz, and E. A. van der Meulen. 2003. The combined contraceptive vaginal ring, nuvaring: an international study of user acceptability. Contraception 67:187-194. 58. Osterberg, L., and T. Blaschke. 2005. Adherence to medication. N Engl J Med 353:487-497. 59. Patton, D. L., Y. C. Sweeney, C. C. Tsai, and S. L. Hillier. 2004. Macaca fascicularis vs. macaca nemestrina as a model for topical microbicide safety studies. J Med Primatol 33:105-108. 60. Pendergrass, P. B., M. W. Belovicz, and C. A. Reeves. 2003. Surface area of the human vagina as measured from vinyl polysiloxane casts. Gynecol Obstet Invest 55:110-113. 61. Pendergrass, P. B., C. A. Reeves, M. W. Belovicz, D. J. Molter, and J. H. White. 2000. Comparison of vaginal shapes in afro-american, caucasian and hispanic women as seen with vinyl polysiloxane casting. Gynecol Obstet Invest 50:54-59. 62. Pendergrass, P. B., C. A. Reeves, M. W. Belovicz, D. J. Molter, and J. H. White. 1996. The shape and dimensions of the human vagina as seen in three-dimensional vinyl polysiloxane casts. Gynecol Obstet Invest 42:178-182. 63. Peterson, L., K. Nanda, B. K. Opoku, W. K. Ampofo, M. Owusu-Amoako, A. Y. Boakye, W. Rountree, A. Troxler, R. Dominik, R. Roddy, and L. Dorflinger. 2007. SAVVY (C31G) gel for prevention of HIV infection in women: a phase 3, double-blind, randomized, placebo-controlled trial in ghana. PLoS One 2:e1312. 64. Promadej-Lanier, N., J. M. Smith, P. Srinivasan, C. F. McCoy, S. Butera, A. D. Woolfson, R. K. Malcolm, and R. A. Otten. 2009. Development and evaluation of a vaginal ring device for sustained delivery of HIV microbicides to non-human primates. J Med Primatol 38:263-271. 65. Quinn, T. C., and J. Overbaugh. 2005. HIV/AIDS in women: an expanding epidemic. Science 308:1582-1583. 66. Rathbone, M. J., J. Hadgraft, and M. S. Roberts (ed.). 2008. Modified-release drug delivery technology, 2nd ed. Informa Healthcare, New York. 32 67. Repka, M. A., S. Shah, J. Lu, S. Maddineni, J. Morott, K. Patwardhan, and N. N. Mohammed. 2012. Melt extrusion: process to product. Expert Opin Drug Deliv 9:105-125. 68. Riddle, J. M., and J. K. Evans. 1994. Contraception and abortion from the ancient world to the renaissance. History: Reviews of New Books 22:138-138. 69. Roseman, T. J., and S. H. Yalkowsky. 1974. Letter: influence of solute properties on release of p-aminobenzoic acid esters from silicone rubber: theoretical considerations. J Pharm Sci 63:1639-1641. 70. Roumen, F. J. M. E., and T. O. M. Dieben. 1999. Clinical acceptability of an ethylene-vinyl-acetate nonmedicated vaginal ring. Contraception 59:59-62. 71. Sarkar, N. 2005. The combined contraceptive vaginal device (nuvaring): a comprehensive review. Eur J Contracept Reprod Health Care 10:73-78. 72. Segal, S. J. 1971. Beyond the laboratory: recent research advances in fertility regulation. Fam Plann Perspect 3:17-21. 73. Shattock, R. J., and Z. Rosenberg. 2012. Microbicides: topical prevention against HIV. Cold Spring Harb Perspect Med 2:a007385. 74. Shedlock, D. J., G. Silvestri, and D. B. Weiner. 2009. Monkeying around with HIV vaccines: using rhesus macaques to define 'gatekeepers' for clinical trials. Nat Rev Immunol 9:717-728. 75. Skoler-Karpoff, S., G. Ramjee, K. Ahmed, L. Altini, M. G. Plagianos, B. Friedland, S. Govender, A. De Kock, N. Cassim, T. Palanee, G. Dozier, R. Maguire, and P. Lahteenmaki. 2008. Efficacy of carraguard for prevention of HIV infection in women in south africa: a randomised, double-blind, placebo-controlled trial. Lancet 372:1977-1987. 76. Smith, D. J., S. Wakasiaka, T. D. Hoang, J. J. Bwayo, C. Del Rio, and F. H. Priddy. 2008. An evaluation of intravaginal rings as a potential HIV prevention device in urban kenya: behaviors and attitudes that might influence uptake within a high-risk population. J Womens Health 17:1025-1034. 77. Sparks, M. H., K.-L. Edwards, K. Malcolm, P. F. Kiser, T. J. Johnson, and A. Loxley. 2009. Drug release characteristics of dapivirine and tenofovir from vaginal rings consisting of ethylene vinyl acetate, silicone or polyurethane polymers: options for HIV prevention. Presented at the AAPS Annual Meeting and Exposition. Los Angeles, CA. 33 78. Squier, C. A., M. J. Mantz, P. M. Schlievert, and C. C. Davis. 2008. Porcine vagina ex vivo as a model for studying permeability and pathogenesis in mucosa. J Pharm Sci 97:9-21. 79. Stirratt, M. J., and C. M. Gordon. 2008. Adherence to biomedical HIV prevention methods: considerations drawn from HIV treatment adherence research. Curr HIV/AIDS Rep 5:186-192. 80. Thomas, C. 2009. Roadblocks in HIV research: five questions. Nat Med 15:855- 859. 81. Tolley, E. E., P. F. Harrison, E. Goetghebeur, K. Morrow, R. Pool, D. Taylor, S. N. Tillman, and A. van der Straten. 2009. Adherence and its measurement in phase 2/3 microbicide trials. AIDS Behavior 14:1124-1136. 82. UNAIDS. 2010. Report on the global AIDS epidemic. Joint United Nations Programme on HIV/AIDS (UNAIDS). 83. Van Damme, L., R. Govinden, F. M. Mirembe, F. Guedou, S. Solomon, M. L. Becker, B. S. Pradeep, A. K. Krishnan, M. Alary, B. Pande, G. Ramjee, J. Deese, T. Crucitti, and D. Taylor. 2008. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med 359:463-472. 84. Van Damme, L., G. Ramjee, M. Alary, B. Vuylsteke, V. Chandeying, H. Rees, P. Sirivongrangson, L. Mukenge-Tshibaka, V. Ettiegne-Traore, C. Uaheowitchai, S. S. Karim, B. Masse, J. Perriens, and M. Laga. 2002. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 360:971-977. 85. van der Laan, J. W., J. Brightwell, P. McAnulty, J. Ratky, and C. Stark. 2010. Regulatory acceptability of the minipig in the development of pharmaceuticals, chemicals and other products. J Pharmacol Toxicol Methods 62:184-195. 86. van der Straten, A., E. Montgomery, H. Cheng, L. Wegner, G. Masenga, C. von Mollendorf, L. Bekker, S. Ganesh, K. Young, J. Romano, A. Nel, and C. Woodsong. 2012. High acceptability of a vaginal ring intended as a microbicide delivery method for HIV prevention in african women. AIDS Behav 1:1-12. 87. van der Straten, A., L. Van Damme, J. E. Haberer, and D. R. Bangsberg. 2012. Unraveling the divergent results of pre-exposure prophylaxis trials for HIV prevention. AIDS 26:F13-19. 34 88. van Laarhoven, H., J. Veurink, M. A. Kruft, and H. Vromans. 2004. Influence of spinline stress on release properties of a coaxial controlled release device based on EVA polymers. Pharm Res 21:1811-1817. 89. van Laarhoven, J. A. 2005. Physical-chemical aspects of a coaxial sustained release device based on poly-EVA. University of Utrecht dissertation. 90. van Laarhoven, J. A., M. A. Kruft, and H. Vromans. 2002. Effect of supersaturation and crystallization phenomena on the release properties of a controlled release device based on EVA copolymer. J Control Release 82:309- 317. 91. van Laarhoven, J. A., M. A. Kruft, and H. Vromans. 2002. In vitro release properties of etonogestrel and ethinyl estradiol from a contraceptive vaginal ring. Int J Pharm 232:163-173. 92. Van Rompay, K. K. 2010. Evaluation of antiretrovirals in animal models of HIV infection. Antiviral Res 85:159-175. 93. Veazey, R. S. 2008. Microbicide safety/efficacy studies in animals: macaques and small animal models. Curr Opin HIV AIDS 3:567-573. 94. Veazey, R. S., R. J. Shattock, P. J. Klasse, and J. P. Moore. 2012. Animal models for microbicide studies. Curr HIV Res 10:79-87. 95. Vincent, K. L., N. Bourne, B. A. Bell, G. Vargas, A. Tan, D. Cowan, L. R. Stanberry, S. L. Rosenthal, and M. Motamedi. 2009. High resolution imaging of epithelial injury in the sheep cervicovaginal tract: a promising model for testing safety of candidate microbicides. Sex Transm Dis 36:312-318. 96. Weisberg, E., I. S. Fraser, J. Baker, D. Archer, B. M. Landgren, S. Killick, P. Soutter, T. Krause, and C. d'Arcangues. 2000. A randomized comparison of the effects on vaginal and cervical epithelium of a placebo vaginal ring with non-use of a ring. Contraception 62:83-89. 97. Welch, B. D., J. N. Francis, J. S. Redman, S. Paul, M. T. Weinstock, J. D. Reeves, Y. S. Lie, F. G. Whitby, D. M. Eckert, C. P. Hill, M. J. Root, and M. S. Kay. 2010. Design of a potent d-peptide HIV-1 entry inhibitor with a strong barrier to resistance. J Virol 84:11235-11244. 98. Whaley, K. J., J. Hanes, R. Shattock, R. A. Cone, and D. R. Friend. 2010. Novel approaches to vaginal delivery and safety of microbicides: biopharmaceuticals, nanoparticles, and vaccines. Antiviral Res 88 Suppl 1:S55- 66. 35 99. Woodrow, K. A., Y. Cu, C. J. Booth, J. K. Saucier-Sawyer, M. J. Wood, and W. Mark Saltzman. 2009. Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA. Nature Materials 8:526-533. 100. Woolfson, A. D. 2006. Potential use of vaginal rings for prevention of heterosexual transmission of HIV: a controlled-release strategy for HIV microbicides. Am J Drug Deliv 4:7-20. 101. Woolfson, A. D., R. K. Malcolm, and R. Gallagher. 2000. Drug delivery by the intravaginal route. Crit Rev Ther Drug 17:509-555. 102. Yu, R. R., A. T. Cheng, L. A. Lagenaur, W. Huang, D. E. Weiss, J. Treece, B. E. Sanders-Beer, D. H. Hamer, P. P. Lee, Q. Xu, and Y. Liu. 2009. A chinese rhesus macaque (macaca mulatta) model for vaginal lactobacillus colonization and live microbicide development. J Med Primatol 38:125-136. 103. Zema, L., G. Loreti, A. Melocchi, A. Maroni, and A. Gazzaniga. 2012. Injection molding and its application to drug delivery. J Control Release 159:324- 331. 36 Figure 1.1. Diagram of in vivo drug release and transport from an IVR. A. Diffusive transport of drug from an intravaginal ring directly into vaginal tissue or first into a thin conducting layer of vaginal fluid and then into vaginal tissue, with transport into the blood being the ultimate sink. B. Advective transport of the drug in vaginal fluid from the anterior vagina near the IVR (ectocervix) to the posterior vagina (introitus). 37 Figure 1.2. Comparison of matrix and reservoir IVR designs and their corresponding drug release kinetics. CHAPTER 2 SAFE AND SUSTAINED VAGINAL DELIVERY OF PYRIMIDINEDIONE HIV-1 INHIBITORS FROM POLYURETHANE INTRAVAGINAL RINGS Todd J. Johnson, Priya Srinivasan, Theodore H. Albright, Karen Watson-Buckheit, Lorna Rabe, Amy Martin, Chou-Pong Pau, R. Michael Hendry, Ron Otten, Janet McNicholl, Robert Buckheit, Jr., James Smith, Patrick F. Kiser Antimicrobial Agents and Chemotherapy 2012;56(3):1291-1299 Reprinted with permission from American Society for Microbiology 39 40 41 42 43 44 45 46 47 CHAPTER 3 SEGMENTED POLYURETHANE INTRAVAGINAL RINGS FOR THE SUSTAINED COMBINED DELIVERY OF ANTIRETROVIRAL AGENTS DAPIVIRINE AND TENOFOVIR Todd J. Johnson, Kavita M. Gupta, Judit Fabian, Theodore H. Albright, Patrick F. Kiser European Journal of Pharmaceutical Sciences 2010;39:203-212 Reprinted with permission from Elsevier License Number: 2918341215351 49 50 51 52 53 54 55 56 57 58 CHAPTER 4 A NINETY DAY TENOFOVIR RESERVOIR INTRAVAGINAL RING FOR MUCOSAL HIV PROPHYLAXIS 4.1 Abstract A vaginal gel containing the antiretroviral tenofovir (TFV) recently demonstrated 39% protection against HIV infection in women. We designed and evaluated a novel reservoir TFV intravaginal ring (IVR) to potentially improve product effectiveness by providing a more controlled and sustained vaginal dose to maintain cervicovaginal concentrations. Polyurethane tubing of varying hydrophilicity was filled with a high density 65/33/2 weight percent TFV/glycerin/water semisolid paste and then end-sealed to create IVRs. In vitro, TFV release increased with polyurethane hydrophilicity, with 35 weight percent water swelling polyurethane IVRs achieving approximately 10 mg/day release for 90 days with similar mechanical stiffness to commercially available NuvaRing®. This design was evaluated in two 90 day in vivo sheep studies for TFV pharmacokinetics and safety. Overall, TFV vaginal tissue, vaginal fluid, and plasma levels were relatively time-independent over the 90 day duration at approximately 104 ng/g, 106 ng/g, and 101 ng/mL, respectively, near or exceeding peak concentrations in a TFV 1% gel control group. TFV vaginal fluid concentrations were approximately 1000- fold greater than levels shown to provide significant protection in women with the TFV 60 1% gel. There were no toxicological findings following placebo or TFV IVR treatment for 28 or 90 days, although modest increases in inflammatory infiltrates in the vaginal epithelia were considered to be TFV- and/or ring-related and warrant additional safety and efficacy assessment. In summary, this reservoir IVR provided controlled yet significant daily release of TFV to maintain elevated sheep vaginal concentrations for 90 days and merit further evaluation as a HIV prophylactic. 4.2 Introduction Recent progress in antiretroviral HIV prevention research has advanced the field from concept toward medical practice (46). The CAPRISA 004 study demonstrated that a vaginal gel containing the reverse transcriptase inhibitor tenofovir (TFV) was partially effective in preventing HIV transmission in women (1), with significant protection observed in women who maintained preventative TFV concentrations of at least 1000 ng/mL in vaginal fluid (23). However, the overall effectiveness (39%) likely was reduced by poor user adherence to the inconvenient "before-and-after-sex" dosing regimen. The correlation of adherence and TFV vaginal fluid concentrations to protection was a key finding (23, 24, 45), indicating the need for vaginal drug delivery systems that attain and maintain elevated user adherence and vaginal drug concentrations. More recently, the VOICE trial tested the same TFV 1% gel formulation as CAPRISA 004 but with a once daily dosage regimen, and failed to show any effectiveness in women. Here as well, low adherence may have contributed to the gel's inability to prevent HIV transmission (54). As a result, we (6, 21) and others (4, 35, 36, 44, 49) aim to develop 61 TFV drug delivery systems to provide sustained protective vaginal tissue concentrations and potentially increase user adherence. The micromolar anti-HIV activity of TFV motivated selection of the high dose in the CAPRISA 004 trial (up to two 40 mg doses within 24 hours). Inter- and intra-user TFV vaginal fluid and tissue concentrations were likely dependent on several poorly understood factors and processes, such as adherence, time between gel applications, vaginal product clearance, menstrual cycle phase, vaginal fluid volume and composition, and frequency of intercourse (i.e., more sex acts over a given time would result in higher vaginal TFV administration since the dosing regimen was coitally dependent). Moreover, episodic dosage forms like gels are intrinsically short acting; the TFV 1% gel formulation attains peak vaginal tissue concentrations in women two hours post-vaginal application and diminishes rapidly thereafter (45). Finally, the anatomical site and kinetics of HIV transmission itself are poorly understood (19). Therefore, a drug delivery system that maintains elevated yet controlled and consistent TFV cervicovaginal tract drug concentrations over a duration longer than HIV transmission and throughout multiple episodic HIV exposures has the potential to increase efficacy over the dynamic drug levels provided by vaginal gels. The silicone intravaginal ring (IVR), invented in 1970 (8, 34), was designed to elute hormones for a 30 day duration and provide sustained drug levels in the range of 10 to 100 μg/day. Since then there has been little innovation in IVR technology. In fact, current IVR technology is inadequate to meet the high topical dose requirements of TFV. Groups have claimed successful TFV formulation and delivery from silicone- and ethylene vinyl acetate-based IVRs (4, 35, 36, 44, 49), yet the in vitro and in vivo daily 62 delivery rates reported were micrograms, rather than milligrams, as is required for HIV prophylaxis with TFV. The TFV release from silicone and ethylene vinyl acetate polymers is therapeutically insignificant primarily due to TFV's hydrophilicity and resultant low solubility in these elastomeric polymers commonly used for IVRs. Although IVRs have a higher per-unit cost compared to gels, for a global health application they offer the advantage of distributing the per-unit cost over many days, weeks, or months (15). Therefore, from an economic perspective a long-lasting and low manufacturing-cost IVR could be more affordable than a frequently applied gel since hundreds of gel units would be needed per year compared to several rings. However, developing a long-duration IVR to deliver milligrams-per-day of TFV for at least 90 days requires a design capable of delivering approximately 1 gram of TFV from a 3 - 6 gram IVR. With such a high weight fraction of drug incorporated in the IVR, maintaining both time-independent TFV release and ring mechanical stiffness for a 90 day duration presents a significant design challenge. We recently reported TFV delivery from hydrophilic polyether urethane (HPU) matrix IVRs formulated with up to 20 weight percent (wt%) TFV (6, 21). Although TFV release rates were greatly improved compared to hydrophobic polymer IVRs, matrix IVRs inherently demonstrate decreased drug release rates with time. Matrix IVRs with a high fraction of undissolved drug similarly display time-dependent decreases in mechanical stiffness as the drug is released. Therefore, we developed and evaluated in vitro and in vivo the first reservoir IVR using a water-absorbable polyurethane as a rate controlling membrane capable of delivering 10 to 30 mg of TFV daily for up to 90 days. Herein, we report the ring's in vitro/in vivo TFV release, mechanics, and vaginal safety and TFV pharmacokinetics in a sheep model. 63 4.3 Materials and Methods 4.3.1 Materials Hydrophilic aliphatic polyether urethane (HPU) Tecophilic® HP-60D-20, HP- 60D-35, and HP-60D-60 grades were purchased from Lubrizol Advanced Materials (Wickliffe, OH), with equilibrium aqueous mass absorption of approximately 20, 35, and 60 wt% and shore hardnesses of 43D, 42D, and 41D, respectively. A custom-synthesized 35 wt% swelling HPU with 78A shore hardness was provided by DSM Biomedical (Berkeley, CA). Tenofovir monohydrate was supplied by Gilead Sciences (Foster City, CA). TFV 1% gel was supplied by Patheon Pharmaceuticals (Cincinnati, OH). USP grade glycerol and water were purchased from Spectrum Chemical (New Brunswick, NJ). Starch 1500® USP grade partially pregelatinized maize starch was provided at no cost by Colorcon (Harleysville, PA). Unless noted, all solvents and reagents were ACS grade. 4.3.2 Ring fabrication HPU resins were dried overnight in a compressed air micro dryer (Dri-Air, East Windsor, CT) to less than 0.05 wt% water content as determined using Karl Fisher titration (Mettler-Toledo, Columbus, OH). The dried pellets were fed into a 3/4-inch single screw hot-melt extruder attached to an advanced torque rheometer drive (C.W. Brabender, South Hackensack, NJ) with a tubing crosshead (Guill Tool, West Warwick, RI). The extruder heating zones (1 - 3) were set at 150, 160, 170°C and the tubing crosshead tip and die temperatures were 150°C and 130°C, respectively. Upon leaving the crosshead, the extrudate was drawn down using a CPC2-12 combination puller/cutter (Conair, Cranberry Township, PA) to create a final tubing product with 0.7 mm wall 64 thickness and 5.5 mm cross-sectional diameter. The extruded tubes were cut to 171 mm in length, weighed, and the tubing lumens were filled with either 100% TFV powder or a 65/33/2 wt% TFV/glycerol/water mixture, resulting in 1.6 g of TFV loaded into each IVR lumen. The TFV/glycerol/water semisolid was mixed using a Hobart mixer with "B" beater attachment (Troy, OH) and back-filled into the tubing lumen using a high pressure hydraulic filling system (Dymax, Torrington, CT). TFV powder was manually filled into the tubing lumen. The tubing ends were sealed using an induction welder equipped with a stainless steel reverse bonding die, and the resultant plugged ends were welded together using a stainless steel split die induction welder (PlasticWeld Systems, Inc., Newfane, NY) to create an IVR. A custom machined 12 cavity aluminum mold was used to shape anneal the IVRs in a circular conformation and minimize tubing lumen kinking. IVRs were placed in the mold which was first heated via water circulation to 65°C for 15 minutes followed by 5 minutes cooling at 10°C. 4.3.3 In vitro TFV release and IVR mechanical testing In vitro release and ring mechanical compression testing were performed as previously described (6, 21). Briefly, IVRs were immersed in 50 mL of 25 mM sodium acetate buffer (pH 4.2) at 37°C and 80 rpm. Release media was periodically collected for TFV concentration measurement by high-performance liquid chromatography (HPLC) and replaced every 24 hours to maintain sink conditions. Simultaneously, IVRs were periodically subjected to mechanical compression testing. Briefly, a custom machined probe attached to an Instron 3342 uniaxial mechanical testing system was used to compress the IVR 25% of its initial 55 mm diameter at a rate of 1 mm/sec and record the 65 result required force. The force value corresponding to 10% ring compression was compared across all samples. 4.3.4 Drug content analysis for in vitro studies TFV in vitro release sample concentrations were determined by HPLC using a method previously described (6). Briefly, 2 μL of sample was injected on an Agilent 1200 series HPLC with diode array detector and Phenomenex Luna C18 5 μm, 150 x 4.6 mm column. A 15 minute gradient method consisted of 100% mobile phase A (potassium phosphate buffer, pH 6.0) switching to 100% mobile phase B (acetonitrile/ potassium phosphate buffer, pH 6.0) upon run completion. The flow rate was 1.5 mL/minute with a typical TFV retention time of 7 min with TFV detection at 260 nm. Cumulative percent release was calculated by numerically integrating between collection time points using the trapezoidal rule and subsequently dividing by the original amount of TFV in the IVR. 4.3.5 Sheep pharmacokinetic study (study 1) The 78A shore hardness HPU IVR (35 wt% swelling) and the clinically tested TFV 1% gel were comparatively evaluated for pharmacokinetics in 1 - 2 year old Dorset Crossbred sheep. The study was conducted at MPI Research (Mattawan, MI), which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The study protocol was reviewed and approved according to the standard procedures of the performing laboratory's Institutional Animal Care and Use Committee. The sheep were housed indoors, and fluorescent lighting was provided for approximately 12 hours per day. On occasion, the 66 dark cycle was interrupted due to study-related activities. Reproductive cycles were not synchronized, and the study was performed September through December. Eight animals in treatment Group 1 received a TFV IVR that was inserted in the posterior vagina on day 0 and retained in the vaginal tract for 90 days. The exterior of the vagina was cleaned with chlorhexadine solution and the IVR was inserted as aseptically as practical using a gloved hand. Following insertion, a speculum was used to confirm correct placement of the ring and the animals were examined daily for evidence that the ring was still in place. If the vaginal ring was expelled or otherwise needed to be replaced, a new ring was inserted and the old ring was recovered for residual drug content analysis (if found). Seven animals in treatment Group 2 received once daily vaginal administration of 4 mL of the TFV 1% gel for 28 days (40 mg/day). Each gel dose was administered using a pre-filled, single-dose polypropylene applicator (HTI Plastics, Lincoln, NE) similar to that used in the clinical gel administration regimen. Vaginal tissue, vaginal fluid, and blood plasma periodically were collected for drug content analysis throughout the dosage duration and up to three days following the last gel dose or IVR removal, as depicted in Figure 4.1. Blood samples (approximately 1 mL) were collected from all animals via the jugular vein, placed in tubes containing K3EDTA anticoagulant, and centrifuged under refrigeration to isolate the blood plasma. At all specified time points, two Weck-Cel® (distributed by Beaver-Visitec International, Inc., Waltham, MA) swabs and biopsies were collected per animal for TFV concentration determination in vaginal fluid and tissue, respectively, one proximal to the ring (posterior vagina, 5-7 cm from the introitus) and one distal to the ring (2-4 cm from the introitus). Weck-Cel swabs were pre-weighed, inserted and allowed to absorb fluid for approximately 1 minute and subsequently were 67 removed and re-weighed to determine fluid mass uptake. A Kevorkian-Younge biopsy forceps (Miltex, York, PA) and speculum were used to acquire approximately 50 mg vaginal tissue biopsies. Local and general anesthetics and analgesics were given on the day of biopsy collection and a chlorhexidine solution was used to clean the exterior vagina. Vaginal tissue biopsy location (left or right side) was alternated with timepoint to minimize tissue trauma and encourage healing. If bleeding was observed after biopsy collection, direct pressure with gauze or gauze with thrombin was applied to encourage clotting. Upon completion of the 90 day time point, IVRs were removed and analyzed for remaining drug content as described below. At study termination on days 93 (Group 1, 72 hours post-IVR removal) and 31 (Group 2, 72 hours post-last gel dose), surviving animals were euthanized by an intravenous overdose of sodium pentobarbital solution followed by exsanguination via severing the femoral or axillary vessels, and vaginal tissues were collected for final drug content determination. All samples (blood plasma, Weck-Cel swabs, and tissues) were snap frozen and stored at -70°C until bioanalysis was performed, as described below. 4.3.6 TFV measurement in plasma, vaginal fluid, and vaginal tissue TFV was extracted from vaginal tissue, vaginal fluid, and plasma and quantified by LC/MS/MS similar to methods described elsewhere (7, 36). Briefly, for TFV plasma quantification, 100 μL of standard, quality control sample or study sample was mixed with 50 μL of a working internal standard TFV-d6 solution at 50 ng/mL. A 500 μL aliquot of 0.5% formic acid in water was added to all plate wells, and the samples were transferred to a pre-conditioned Oasis MCX solid-phase extraction plate. The plate was 68 then washed with 800 μL of 0.5% formic acid in water and 800 μL of methanol, followed by sample elution with 600 μL of 5:95 ammonium hydroxide:methanol. The eluent was evaporated to dryness, and the residue was reconstituted with 200 μL of 0.1% ammonium hydroxide in water. The samples were vortexed and an aliquot of each sample was injected onto an LC-MS/MS system for analysis. Similarly, TFV was measured in tissue by mixing 200 μL of standard, quality control sample or study sample with 50 μL of a working internal standard solution containing TFV-d6 at 50 ng/mL. The samples were vortexed and centrifuged, and an aliquot of each sample was injected onto an LC-MS/MS system for analysis. Lastly, TFV levels in swabs were processed by mixing 50 μL of standard, quality control sample or study sample with 50 μL of a working internal standard TFV-d6 solution (200 ng/mL) in a tube containing a clean spear or study sample. A 950 μL aliquot of 50:50 methanol:water was added to the standards and quality control samples, whereas a 1000 μL aliquot of 50:50 methanol:water was added to the study samples. The samples were vortexed and centrifuged, and an aliquot of the supernatant was injected onto an LC-MS/MS system for analysis. The chromatography varied with sample type: plasma samples were analyzed using a Phenomenex Synergi Polar-RP column, 75 mm x 2 mm (4 μm particle size) with a gradient flow of 0.1% acetic acid in water, 0.1% acetic acid in acetonitrile, and 0.2% ammonium hydroxide in water at a flow rate of 300 to 500 mL/minute. Swabs were analyzed using the same column but with an isocratic flow of water/acetonitrile/acetic acid/ammonium hydroxide (930:70:5:1) (v/v/v/v) at a flow rate of 200 μL/minute. Tissue samples were analyzed using a BioBasic AX column, 50 x 3.0 mm (5 μm particle size) with a gradient flow of acetonitrile:10 mM ammonium acetate in water at pH 6 (30:70) 69 and acetonitrile:1 mM ammonium acetate in water pH 10.5 (30:70) at a flow rate of 400 to 1000 mL/minute. The analyte and internal standards were detected using a Sciex API 5000 triple quadrupole LC-MS/MS system equipped with an ESI (TurboIonSpray®) ionization source operated in the positive and negative ion mode. Multiple-reaction-monitoring mode transitions of the respective ions were used to monitor TFV and TFV-d6 and may have been slightly modified to optimize system performance. For TFV, m/z 288 to 176 was monitored with a retention time of 1.15 - 3.67 min. For TFV-d6, m/z 294 to 182 was monitored with a retention time of 1.15 - 3.65 min. The lower limits of quantification (LOQ) for TFV in plasma, Weck-Cel, and vaginal tissue were 1.00 ng/mL, 5.00 ng/spear, and 20.0 ng/g, respectively. 4.3.7 TFV pharmacokinetic analysis Individual pharmacokinetic parameters were determined by non-compartmental methods using WinNonlin® Phoenix software (Pharsight Corporation, Sunnyvale, CA). Area under the curve (AUC) estimates were determined using the linear trapezoidal rule with the linear interpolation calculation method. Values below the respective LOQs were treated as "0" for the analysis. Nominal sample collection time was used for the analysis. Pharmacokinetic parameters were defined as follows: Tmax: Time of maximum observed concentration; Cmax: Maximum observed concentration occurring at Tmax; and AUC0-90 days: Area under the curve from the time of dosing to 90 days (IVR group only). 70 4.3.8 Quantification of IVR residual TFV content TFV was extracted out of the recovered IVRs to determine the total amount of TFV released. Each IVR was cut into approximately 1 cm segments that were placed together in a 50 mL volumetric flask, and 100 mM pH 7.4 phosphate buffer was added to dissolve the entirety of the residual TFV. After removing the polymer segments, an aliquot of the resulting solution was diluted volumetrically and analyzed for TFV content by HPLC as previously described (21). Original semisolid material used for IVR manufacture was kept and extracted in the same manner to calculate percent recovery based on IVR fill mass. 4.3.9 Sheep toxicology study (study 2) The local tolerance and systemic toxicity of the 78A shore hardness HPU TFV IVR (35 wt% swelling) was assessed in female Welsh Mule sheep (age: 49 months; weight at start of study was 59.5 - 83.5 kg) over a 1 month (28 day) or 3 month (90 day) period, in comparison with a placebo IVR or sham control (i.e., underwent restraint and sham insertion, but no ring was inserted). The placebo IVR consisted of 60/38/2 wt% starch/glycerol/water in the tubing lumen but otherwise the IVR was manufactured using identical HPU tubing, processes, and equipment as the TFV IVR. The study was conducted at Huntingdon Life Sciences (Huntingdon Life Sciences, United Kingdom), which is fully accredited by AAALAC International. The study design, outlined in Table 4.1, was based on the current International Conference on Harmonisation (ICH) Harmonised Tripartite Guidance on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (20), and the 71 study protocol was reviewed and approved according to the standard procedures of the performing laboratory's Ethical Review Process Committee. Physical examinations were conducted on all animals pre-treatment. The animals were observed at least twice daily throughout the study for any clinical abnormalities or signs of reaction to treatment. More frequent observations were made on the day of dosing. Body weights were recorded weekly and food consumption was recorded on a daily basis. Hematology, blood chemistry, and urinalysis were conducted on all animals pre-treatment and pre-termination (day 28 or day 90). Upon termination of the study, all animals were sacrificed and gross necropsy was conducted. Most major body organs were isolated and weighed. Tissues were then fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at approximately 4 - 5 μm thickness and stained with haematoxylin and eosin (H&E) for histological examination by light microscopy. In addition to the standard analyses described above, H&E stained slides of the posterior vaginal tissue (i.e., proximal to IVR location) were used for vaginal irritation scoring in accordance with the rabbit vaginal irritation method of Eckstein et al. (9). Approximately 10 areas were analyzed using a Nikon 600 microscope and individual scores from 0 - 4 were assigned depending on the extent of congestion, edema, leukocyte infiltration and epithelial damage. The individual scores were combined to yield a total group score (0 - 16) that in rabbits correlates with human irritation potential. Similar to the sheep pharmacokinetic study, TFV IVRs were retrieved from the animals prior to termination at 28 and 90 days and analyzed for remaining drug content as described above. In addition, blood plasma, vaginal fluid (Weck-Cel swabs), and vaginal 72 tissue was collected from the TFV IVR treated animals prior to termination and analyzed for TFV content using methods described above. 4.3.10 Statistical analysis Data were analyzed using one-way and two-way ANOVA and unpaired two-tailed Student's t-test. A p-value less than 0.05 was considered statistically significant. 4.4 Results 4.4.1 In vitro characterization and optimization An elastomeric HPU tubular IVR design was utilized as it maximized the weight ratio of drug to total device, could be filled with solid or liquefied drug, and provided sufficient time-independent mechanical stiffness and drug release. Previous research beyond the scope of this publication determined that HPU IVRs of 5.5 mm outer cross-sectional diameter, 0.7 mm wall thickness, and 55 mm outer diameter (Figure 4.2) would provide ring mechanical properties, lumen volume, and drug release kinetics suitable for the proposed TFV IVR (see Chapter 5). The in vitro TFV release rate was evaluated using various core compositions and equilibrium percent swelling HPU IVRs. IVRs containing only TFV powder in the lumen showed low yet steadily increasing TFV release rates with time which did not reach steady-state by 28 days (Figure 4.3A). When the highly water miscible and HPU-permeable compound glycerol was added into the tubing lumen, the lumen was rapidly hydrated and attained the equilibrium TFV release rate after only a 1 day transient state. The TFV release rate increased with polymer equilibrium swelling for 20, 35, and 60 wt% swelling Tecophilic HPUs. The 20 wt% 73 swelling IVRs did not achieve the desired 10 mg/day minimum release rate and were stopped after 28 days, whereas the 35 and 60 wt% swelling IVRs achieved approximately 17 and 25 mg/day steady-state release rates, respectively. However, TFV was depleted from the 60 wt% swelling IVRs before the target 90 day duration. The TFV percent cumulative release profile was linear until greater than 90% of the initial drug load was depleted from the IVRs (Figure 4.3B). The softer shore hardness 35 wt% swelling HPU IVR released 10 mg/day of TFV for 90 days (Figure 4.4A) and demonstrated mechanical stiffness similar to NuvaRing (Figure 4.4B). Furthermore, the HPU IVR stiffness did not change considerably with time when comparing the dry state, hydrated state, and after a majority of the TFV was released (i.e., 0, 1, and 90 days). Ten percent ring compression values were compared as this is approximately the in vivo compression of NuvaRing, as determined by Magnetic Resonance Imaging (3). 4.4.2 TFV pharmacokinetics in sheep (study 1) The HPU TFV IVRs were evaluated in a sheep model to compare the TFV pharmacokinetics to the clinically tested TFV 1% gel. Throughout the 90 day duration one animal expelled multiple rings, one animal expelled one ring, and two animals required a replacement ring due to a ring manufacturing defect and a procedural error during pinch biopsy collection which damaged the ring. However, pharmacokinetic data from these sheep did not differ from sheep that retained a single IVR for the full 90 day duration and thus no data were excluded. One sheep in the TFV gel group expired on day 15, with complications from anesthesia being determined as the cause of death (non-treatment related). The calculated time-averaged in vivo TFV release rate from devices 74 that were retained for 90 days in vivo was 17.0 ± 1.1 mg/day (mean ± standard deviation, N = 5) as determined by residual TFV extraction, approximately 70% higher than the 10 mg/day in vitro release rate. TFV vaginal fluid concentrations from the IVR group attained their steady-state levels of approximately 106 ng/g by day 1, which were maintained for the remainder of the 90 day duration (Figure 4.5A). Mean TFV concentrations from IVR insertion through 90 days were 3.5-fold higher proximal to the ring than distal. Comparatively, steady-state mean TFV vaginal fluid concentrations from the IVR group (approximately 106 ng/g) were similar to mean TFV vaginal fluid concentrations at 8 hours post-gel dose (Figure 4.5B). Mean TFV concentrations from the gel group were 1.4-fold higher proximal than distal. Up to 3 days beyond IVR removal (Figure 4.6) and after the last gel dose, vaginal fluid concentrations for both groups were approximately 104 ng/g. Eight hours post-IVR insertion, TFV vaginal tissue concentrations were approximately 102 ng/g (Figure 4.7A). TFV tissue concentrations at day 14 (the next biopsy time point) through day 90 were approximately 104 ng/g, with concentrations trending slightly higher with time. For the full duration of IVR residence in the vaginal tract (0 to 90 days), mean TFV tissue concentrations were similar proximal and distal to the ring. Mean TFV tissue concentrations were 104 ng/g 8 hours after the 15th gel dose, similar to day 14 TFV concentrations with the IVR group (Figure 4.7B). Eight hours after the 15th dose and 24 hours after the 28th gel dose, mean TFV levels decreased to approximately 103 and 102 ng/g, respectively. TFV tissue concentrations 3 days after IVR removal and 3 days after the last gel administration were significantly lower, with several biopsies from both groups below the LOQ. Across all gel group time points, mean TFV 75 tissue concentrations were 3.1-fold higher proximal than distal. TFV-diphosphate concentration determination in vaginal tissue was also attempted but was undetectable or highly variable in both IVR and gel groups and thus was excluded from further analysis (data not shown). Mean TFV plasma concentrations at day 7 through day 90 from the IVR group were steady at approximately 15 ng/mL, with the exception of day 14 where mean concentrations were 28 ng/mL (Figure 4.8A). In contrast, the gel group achieved similar TFV concentrations at 2 hours but rapidly decayed thereafter (Figure 4.8B). Also in the gel group, mean post-dose TFV plasma concentrations decreased with increased dose (mean TFV concentrations from the 1st dose > 15th dose > 28th dose). TFV was undetectable in plasma at 24 hours post-IVR removal or gel dose. TFV pharmacokinetic parameters for vaginal fluid, vaginal tissue, and plasma (Table 4.2) were consistent with the data presented above. 4.4.3 Toxicological evaluation A second sheep study was performed to evaluate the systemic toxicity and local irritation potential of TFV IVRs compared to placebo IVRs and a sham control. IVRs were well tolerated and retained, with only one IVR expulsion noted on the day of scheduled removal (day 90). No treatment-related toxicological findings were observed regarding body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, and gross pathology. As determined by histopathology, the degree or incidence of leukocytic infiltration in the vagina at 1 and 3 months increased with ring presence although no epithelial disruption was observed (TFV or placebo, Figure 4.9). 76 When quantifying the irritation potential in sheep, slight increases in the mean vaginal irritation scores were observed for both placebo and TFV IVRs compared to sham controls (Figure 4.10). Specifically, mean vaginal irritation scores for sham, placebo IVRs, and TFV IVRs at 1 month were 1.8, 2.4 and 3.2, respectively, and at 3 months were 2.2, 3.2 and 3.8, respectively. Differences between the TFV IVR and sham control were found to be statistically different at both time points (p=0.03 and 0.01 for 1 and 3 months, respectively). Upon animal necropsy at 28 and 90 days, IVRs were recovered for drug content analysis and plasma, vaginal fluid, and vaginal tissue were analyzed for TFV concentration. The time-averaged TFV release rate of IVRs retrieved from sheep after 28 and 90 days of treatment was 14.0 ± 2.8 and 16.9 ± 1.6 mg/day, respectively (mean ± SD, N = 5). TFV vaginal tissue, vaginal fluid, and plasma concentrations at 28 and 90 days were generally similar to the pharmacokinetic study (Table 4.3). 4.5 Discussion We designed and tested a new reservoir intravaginal ring for long-duration TFV vaginal delivery. IVR fabrication incorporated materials and manufacturing methods commonly utilized by the pharmaceutical and medical device industries, making the IVR a scalable and cost-effective product for resource-poor regions where the HIV pandemic is most prevalent (51). The 35 wt% swelling HPU released at least 10 mg/day of TFV in vitro for 90 days with time-independent IVR mechanical properties. In sheep receiving IVRs, TFV concentrations in plasma, vaginal fluid, and vaginal tissue were near time-independent for 90 days at levels similar to peak concentrations in the TFV 1% gel group. 77 Lastly, in the sheep safety study, no significant toxicological effects were observed although modest increases in inflammatory infiltrates in the vaginal epithelia were considered to be TFV- and/or ring-related. 4.5.1 In vitro studies The steady-state in vitro TFV release rate increased with HPU equilibrium swelling, thus allowing simple modulation of the release rate by selecting an HPU with appropriate equilibrium swelling. Since TFV is virtually insoluble in the non-swelling polymer phase (6), increased water presence allowed for the water-soluble TFV to dissolve and diffuse through the aqueous phase of the hydrated polymer. The lower durometer 35 wt% swelling HPU released approximately 10 mg/day TFV for 90 days with time-independent IVR mechanical stiffness similar to NuvaRing, which was utilized as a benchmark for mechanical stiffness due to its high user acceptance and extensive safety record in women (43). Glycerol incorporation in the IVR core formulation yielded several benefits over 100% TFV powder including easier lumen filling and increased TFV density to maximize TFV loading. Although TFV comprised approximately 36% of the ring's total mass, the ring mechanical stiffness remained unchanged throughout the 90 day duration even after the majority of the TFV was released. This unique performance attribute was due to the semisolid core whose presence or absence did not significantly impact the overall ring elasticity, in contrast to the solid core TFV matrix IVR that was initially quite stiff but softened upon hydration and as TFV was released (6). 78 Glycerol also acted as an osmotic agent to rapidly draw water into the TFV-loaded core and nearly eliminated the time to steady-state TFV release. Following initial HPU hydration, we hypothesized that the low amount of water at the inner surface of the HPU lumen mixed with the high concentration of glycerol present, resulting in a large inward osmotic driving force since glycerol and water are infinitely miscible. Following lumen hydration, TFV quickly saturated the aqueous solution to establish a fixed TFV concentration gradient across the tubing wall and allow TFV release by membrane-controlled steady-state diffusion. In the absence of a highly water-miscible molecule in the tubing lumen, TFV steady-state release was not achieved after several weeks since TFV alone is a relatively poor osmotic agent (~1% w/v solubility in the acidic aqueous media utilized) (6). The excess of undissolved and highly mobile TFV in the tubing lumen allowed for true time-independent TFV release until virtually the entire load was depleted, thus minimizing drug waste. Conventional solid core polymeric reservoir IVRs, such as NuvaRing, create environmental and cost concerns since up to 85% of the initial drug load remains in the IVR at the end their planned durations (60). Another advantage of the HPU reservoir IVR design is its tunability, where HPU equilibrium swelling, cross-sectional diameter, wall thickness, and shore hardness (elastic modulus) may be independently varied to achieve the desired TFV loading, TFV release rate, and ring stiffness. Commonly utilized silicone IVRs typically demonstrate a lower elastic modulus than thermoplastic elastomers and therefore require a wider cross-sectional diameter and/or the addition of an inert filler excipient to increase ring stiffness (12). As increasing IVR dimensions may negatively impact user acceptability (48), we 79 minimized the HPU IVR cross-sectional diameter while still ensuring that the target TFV release rate and duration was achieved. When accounting for the overall societal costs associated with HIV infection including healthcare and inability to work, TFV 1% gel is expected to be quite cost effective by South African standards at a projected cost of approximately $5/month (58, 61). The 90-day reservoir TFV IVR, with an estimated per-unit manufacturing cost of less than $1 (personal communication and estimate from ProMed Pharma LLC, Plymouth, MN), should therefore cost significantly less per month than the gel. Despite its unconventional design, the reported IVR combines existing pharmaceutical and medical device materials, techniques, and equipment to minimize associated manufacturing costs and ensure affordability in resource-poor countries. From a supply chain and distribution perspective, a year's supply of gel would require significant transportation efforts both for the supplier and the end user as compared to 4 rings. In addition to lower transportation costs and end-user convenience, the IVR would also minimize waste management issues associated with the number of gel applicators and packaging required for frequent use. 4.5.2 In vivo pharmacokinetics In the CAPRISA 004 clinical trial evaluating the TFV 1% gel, women with vaginal fluid concentrations greater than 103 ng/mL were significantly more protected against HIV infection than women with concentrations less than 103 ng/mL (23). In the sheep pharmacokinetic study described in this report, the TFV IVR attained near steady-state vaginal fluid concentrations at day 1 (106 ng/g or ng/mL), which were 80 approximately 1000 times higher than the clinically protective concentration, and this level was maintained for the remainder of the 90 day duration. Vaginal dosing of the TFV 1% gel both in a controlled clinical pharmacokinetic study previously reported by Schwartz et al. (45) and in our sheep gel group demonstrated peak 106 ng/g TFV vaginal fluid concentrations several hours post-dose which were equivalent to the observed steady-state TFV IVR concentrations. Although TFV tissue concentrations were not measured in the CAPRISA 004 trial, the clinical pharmacokinetic study by Schwartz et al. reported approximately 105 ng/g peak vaginal tissue concentrations at 2 hours post-dose which plateaued at around 104 ng/g thereafter (45). Our sheep gel group attained approximately 103 - 104 ng/g tissue levels 8 hours post-dose, but were lower at 24 hours post-dose. TFV tissue concentrations at 8 hours post-IVR insertion were comparatively lower than 8 hours post-gel dose. However, mean TFV tissue concentrations of approximately 104 - 105 ng/g were observed at the next sampling time point (day 14) through the remaining dosing duration (day 90) which were similar to Cmax gel tissue concentrations. Since tissue biopsy time points between 8 hours and 14 days were not collected, it is not known whether similarly high tissue concentrations were achieved earlier than 14 days with the TFV IVR. However, since vaginal fluid and plasma concentrations were near steady-state by days 1 and 7, respectively, it seems probable that near steady-state tissue levels were reached within the first several days as vaginal tissue acts as the intermediary compartment for drug between vaginal fluid and the systemic circulation (62). The elevated sheep tissue TFV concentrations reported herein from the TFV HPU IVR were approximately 1000 times 81 higher than sheep tissue TFV concentrations from an alternative TFV IVR design (35), reflecting the nearly 1000-fold greater TFV in vitro release rate from the HPU IVR. The mean TFV concentrations in vaginal fluid, vaginal tissue, and plasma in the IVR group were similar to or greater than peak concentrations attained in the gel group. This is an interesting result considering that the time-averaged daily TFV release from the IVR, as determined by residual device extraction, was less than one-half of the daily TFV gel dose. The percent of dosed TFV which is absorbed following TFV 1% vaginal gel administration has not been reported, but it is likely that a significant fraction is not absorbed because the gel leaks out of the vaginal tract (2, 33). The gel more rapidly achieved high TFV vaginal and systemic concentrations and therefore may be advantageous for coitally dependent, intermittent use where it is applied just prior to sex (similar to CAPRISA 004 dosing). Conversely, the IVR may be advantageous when women desire a long-lasting, coitally independent, and discreet dosage form. It is generally assumed that antiretrovirals should be well distributed throughout the vaginal tract to maximize HIV prevention. TFV vaginal fluid and tissue concentrations measured proximal and distal to the IVRs' placement were nearly identical over the 90 day period and trended similar to the TFV 1% gel. This finding corroborates previous TFV IVR pharmacokinetic studies in sheep where TFV levels proximal and distal were indistinguishable (36). Conversely, vaginal biodistribution of hydrophobic antiretrovirals delivered from IVRs has been evaluated in women and macaques and both studies have shown a higher drug concentration proximal to the ring than distal (22, 40). Therefore, if uniform vaginal biodistribution proves necessary, 82 hydrophilic antiretrovirals such as TFV may be better suited for IVR delivery than hydrophobic antiretrovirals. Reservoir drug delivery devices typically offer near time-independent release of molecules given sink conditions are satisfied (25, 42). Time-independent TFV release rates were attained by day 2 in vitro, and similarly TFV vaginal fluid concentrations stabilized by day 2 in vivo, suggesting that the device was releasing drug in a zero-order fashion as expected. Following a 3 - 7 day lag time, TFV plasma concentrations in the IVR group were steady for the remaining 90 days with the exception of day 14. It is known that hydrophobic compounds can be more rapidly transported across the epithelium for systemic absorption than hydrophilic compounds (62), yet there has been limited reports of hydrophilic API plasma pharmacokinetics from IVRs, in part due to IVR formulation limitations of such APIs as previously discussed. Also, the reported TFV IVR did not provide an initial burst of drug as do conventional matrix and reservoir IVRs. Nonetheless, a similar plasma concentration-time profile has been observed with the contraceptive NuvaRing whereby plasma concentrations of the hydrophobic small molecules ethinyl estradiol and etonogestrel peaked approximately 1 week after IVR insertion in women (50). A significantly higher release rate was observed in vivo than in vitro, which is atypical for controlled release devices. Of note is that TFV is an acidic compound, with increased aqueous solubility at higher pH. The drug release rate from reservoir IVRs is proportional to the dissolved concentration of drug in the tubing lumen (55). Therefore, the sheep neutral vaginal pH may increase the dissolved TFV concentration in the IVR lumen and thus demonstrate higher TFV flux than in the in vitro pH 4.2 acetate buffer 83 release media. The pH of the healthy human vagina is acidic due to buffering by lactic acid-secreting microflora (62). Therefore, future studies in women will be needed to determine whether the increased in vivo flux in sheep is also observed in women. The 35 wt% swelling HPU TFV IVR demonstrated similar TFV in vivo release rates and TFV vaginal concentrations in both sheep studies, albeit the second study did not include as many time points, as its primary objective was toxicological evaluation. Nonetheless, the studies at separate institutions utilizing two different sheep breeds confirmed the ability of the HPU TFV IVR to reproducibly provide elevated and sustained TFV vaginal concentrations for up to 90 days. 4.5.3 In vivo toxicology An efficacious microbicide product must not compromise vaginal barrier function. Thinning or disruption of the protective epithelium and/or recruitment of susceptible immune cells via an inflammatory response could increase HIV infection, as was observed with early HIV prophylaxis trials using surfactants and anionic polymers (26, 52, 53). Microbicide IVRs are designed to be present in the vagina for a duration of weeks to months, and beyond brief excursions would likely be reinserted or replaced throughout a woman's sexually active life. The potential long-term vaginal effects of an IVR and its corresponding influence on HIV prophylaxis are therefore a concern. Bounds et al. studied an early version of Femring® that had remarkable mechanical stiffness, and it was concluded that the ring contributed to the creation of ulcerative lesions in the vagina (5). Subsequently, several large clinical safety studies have evaluated an array of medicated and non-medicated IVRs composed of silicone and ethylene vinyl acetate 84 polymers comprising various dimensions and ring stiffness (14, 27, 41, 43, 47, 59). None of these studies found a significant ring contribution in creating epithelial lesions and/or altering vaginal microflora when tested for up to 1 year. Furthermore, several marketed IVRs have now recorded thousands of women-years of use with favorable safety records (16, 43). Polyurethane IVRs have not been clinically evaluated to date, although evaluation in pigtail macaque monkeys shows no significant alteration in native microflora or mucosal and proinflammatory cytokines when compared to naïve animals (22) or animals receiving silicone IVRs (personal communication with J. Smith, Centers for Disease Control and Prevention, Atlanta, GA). Care was taken at the onset of product development to utilize materials that possess strong evidence of safety in humans. Medical-grade polyurethanes have demonstrated long-term biocompatibility in many biomedical and drug delivery applications (28). Of the potential water-miscible excipients, glycerol was utilized, as it is generally recognized as safe by the Food and Drug Administration and is one of the most commonly used excipients in vaginal formulations (17) including in the clinically efficacious TFV 1% gel (1). The overall amount of glycerol in the TFV IVR (825 mg) is approximately equivalent to that incorporated in a single dose of the TFV 1% gel (800 mg), which demonstrated no significant adverse events in women (1, 32). Greater than 95% of the glycerol was released from the ring in the first 3 days (data not shown). Over a 90 day duration, the TFV IVR would deliver significantly less glycerol vaginally than the gel which could be dosed up to twice daily. Following 28 or 90 days of treatment with a single IVR, no s