A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques.

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbit and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a TAF dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. The level of inflammation in the primates was markedly lower in the placebo group than in the active-implant group. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve a sustained target dose, we observed an unacceptable inflammatory response locally at the implant tissue interface.

A Dose Ranging Pharmacokinetic Evaluation of IQP-0528 Released from Intravaginal Rings in Non-Human Primates.

PURPOSE: Design of intravaginal rings (IVRs) for delivery of antiretrovirals is often guided by in vitro release under sink conditions, based on the assumption that in vivo release will follow a similar release profile. METHODS: We conducted a dose-ranging study in the female reproductive tract of pigtail macaques using matrix IVRs containing IQP-0528, a poorly soluble but highly potent antiretroviral drug with an IC90 of 146 ng/mL. These IVRs consisted of drug-loaded segments, 15.6% IQP-0528 in Tecoflex 85A, comprising either all, half, or a quarter of the entire ring. RESULTS: In vitro release under sink conditions demonstrates loading-proportional release, with a cumulative 30-day release of 48.5 ± 2.2 mg for our 100% loaded ring, 24.8 ± .36 mg from our 50% loaded ring, and 13.99 ± 1.58 mg from our 25% loaded ring. In vivo, while drug concentration in vaginal fluid is well in excess of IQP-0528's EC90, we find no statistical difference between the different ring loadings in either swab drug levels or drug released from our rings. CONCLUSIONS: We show that in vitro release may not accurately reflect in vivo release, particularly for poorly soluble drugs. All tested loadings of our IVRs are capable of delivering IQP-0528 well in excess of the IC90.

Intravaginal flux controlled pump for sustained release of macromolecules.

PURPOSE: To design a flux controlled pump (FCP) capable of 30-day, controlled release of macromolecules to the vaginal mucosa. METHODS: The FCP is composed of a single chamber fabricated from a rigid thermoplastic with orifices and encloses a pellet of water-swellable polymer containing the drug substance. We performed testing both in vitro and in rabbits. To ensure vaginal retention in the rabbit, we designed and attached an oval shape-memory polyether urethane retainer to the FCP allowing for long-term intravaginal evaluation of a solid dosage form without invasive surgical implantation. RESULTS: The orifices and swelling properties of the polymer pellet control water entry for polymer hydration and expansion, and subsequent extrusion of the drug-containing gel from the orifice. A FCP device containing a pellet composed of hydroxypropyl cellulose compounded with a model macromolecule, achieved controlled in vitro release for 30 days with an average release rate of 24 ± 2 µg/day (mean ± SD) and range of 16 to 42 µg/day. We observed a slightly lower average release rate in vivo of 20 ± 0.6 µg/day (mean ± SD). CONCLUSIONS: The size of the orifice and nature of the swelling polymer controls the hydration rate and thereby macromolecule release rate and duration from this FCP.

A 90-day tenofovir reservoir intravaginal ring for mucosal HIV prophylaxis.

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 various hydrophilicities was filled with a high-density TFV/glycerol/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 an approximately 10-mg/day release for 90 days with mechanical stiffness similar to that of the 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 10(4) ng/g, 10(6) ng/g, and 10(1) ng/ml, respectively, near or exceeding the highest observed concentrations in a TFV 1% gel control group. TFV vaginal fluid concentrations were approximately 1,000-fold greater than levels shown to provide significant protection in women using the TFV 1% gel. There were no toxicological findings following placebo and TFV IVR treatment for 28 or 90 days, although slight to moderate increases in inflammatory infiltrates in the vaginal epithelia were observed in these animals compared to naïve animals. In summary, the controlled release of TFV from this reservoir IVR provided elevated sheep vaginal concentrations for 90 days to merit its further evaluation as an HIV prophylactic.

Safe and sustained vaginal delivery of pyrimidinedione HIV-1 inhibitors from polyurethane intravaginal rings.

The potent antiretroviral pyrimidinediones IQP-0528 (PYD1) and IQP-0532 (PYD2) were formulated in polyurethane intravaginal rings (IVRs) as prophylactic drug delivery systems to prevent the sexual transmission of HIV-1. To aid in the selection of a pyrimidinedione candidate and the optimal loading of the drug in the IVR delivery system, four pyrimidinedione IVR formulations (PYD1 at 0.5 wt% [PYD1(0.5 wt%)], PYD1(1 wt%), PYD2(4 wt%), and PYD2(14 wt%)) were evaluated in pigtail macaques over 28 days for safety and pyrimidinedione vaginal biodistribution. Kinetic analysis of vaginal proinflammatory cytokines, native microflora, and drug levels suggested that all formulations were safe, but only the high-loaded PYD2(14 wt%) IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 µg/ml to vaginal fluid and 1 µg/g to vaginal tissue, a level over 1,000 times the in vitro 50% effective concentration. The in vitro release of PYD1 and PYD2 under nonsink conditions correlated well with in vivo release, both in amount and in kinetic profile, and therefore may serve as a more biologically relevant means of evaluating release in vitro than typically employed sink conditions. Lastly, the pyrimidinediones in the IVR formulation were chemically stable after 90 days of storage at elevated temperature, and the potent nanomolar-level antiviral activity of both molecules was retained after in vitro release. Altogether, these results point to the successful IVR formulation and vaginal biodistribution of the pyrimidinediones and demonstrate the usefulness of the pigtail macaque model in evaluating and screening antiretroviral IVR formulations prior to preclinical and clinical evaluation.

Activity and safety of synthetic lectins based on benzoboroxole-functionalized polymers for inhibition of HIV entry.

Lectins derived from plant and microbial sources constitute a vital class of entry inhibitors that target the oligomannose residues on the HIV envelope gp120. Despite their potency and specificity, success of lectin-based entry inhibitors may be impeded by high manufacturing costs, formulation and potential mitogenicity. Therefore, there exists a gap in the HIV microbicides pipeline that underscores the need for mass producible, synthetic, broad-spectrum, and biocomptabile inhibitors of HIV entry. Here, we present the development of a polymeric synthetic lectin, based on benzoboroxole (BzB), which exhibits weak affinity (∼25 M(-1)) for nonreducing sugars, similar to those found on the HIV envelope. High molecular weight BzB-functionalized polymers demonstrated antiviral activity that increased with an increase in ligand density and molecular weight of the polymer construct, revealing that polyvalency improves activity. Polymers showed significant increase in activity from 25 to 75 mol % BzB functionalization with EC(50) of 15 µM and 15 nM, respectively. A further increase in mole functionalization to 90% resulted in an increase of the EC(50) (59 ± 5 nM). An increase in molecular weight of the polymer at 50 mol % BzB functionalization showed a gradual but significant increase in antiviral activity, with the highest activity seen with the 382 kDa polymer (EC(50) of 1.1 ± 0.5 nM in CEM cells and 11 ± 3 nM in TZM-bl cells). Supplementing the polymer backbone with 10 mol % sulfonic acid not only increased the aqueous solubility of the polymers by at least 50-fold but also demonstrated a synergistic increase in anti-HIV activity (4.0 ± 1.5 nM in TZM-bl cells), possibly due to electrostatic interactions between the negatively charged polymer backbone and the positively charged V3-loop in the gp120. The benzoboroxole-sulfonic acid copolymers showed no decrease in activity in the presence of a seminal concentration of fructose (p > 0.05). Additionally, the copolymers exhibit minimal, if any, effect on the cellular viability, barrier properties, or cytokine levels in human reconstructed ectocervical tissue after 3 days of repeated exposure and did not show pronounced activity against a variety of other RNA and DNA viruses.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

Controlling the hydration rate of a hydrophilic matrix in the core of an intravaginal ring determines antiretroviral release.

Intravaginal ring technology is generally limited to releasing low molecular weight species that can diffuse through the ring elastomer. To increase the diversity of drugs that can be delivered from intravaginal rings, we designed an IVR that contains a drug matrix encapsulated in the core of the IVR whereby the mechanism of drug release is uncoupled from the interaction of the drug with the ring elastomer. We call the device a flux controlled pump, and it is comprised of compressed pellets of a mixture of drug and hydroxypropyl cellulose within the hollow core of the ring. The pump orifice size and chemistry of the polymer pellets control the rate of hydration and diffusion of the drug-containing hydroxypropyl cellulose gel from the device. A mechanistic model describing the hydration and diffusion of the hydroxypropyl cellulose matrix is presented. Good agreement between the quantitative model predictions and the experimental studies of drug release was obtained. We achieved controlled release rates of multiple antiretrovirals ranging from µg/d to mg/d by altering the orifice design, drug loading, and mass of pellets loaded in the device. This device could provide an adaptable platform for the vaginal drug delivery of many molecules.

Engineering a segmented dual-reservoir polyurethane intravaginal ring for simultaneous prevention of HIV transmission and unwanted pregnancy.

The HIV/AIDS pandemic and its impact on women prompt the investigation of prevention strategies to interrupt sexual transmission of HIV. Long-acting drug delivery systems that simultaneously protect womenfrom sexual transmission of HIV and unwanted pregnancy could be important tools in combating the pandemic. We describe the design, in silico, in vitro and in vivo evaluation of a dual-reservoir intravaginal ring that delivers the HIV-1 reverse transcriptase inhibitor tenofovir and the contraceptive levonorgestrel for 90 days. Two polyether urethanes with two different hard segment volume fractions were used to make coaxial extruded reservoir segments with a 100 µm thick rate controlling membrane and a diameter of 5.5 mm that contain 1.3 wt% levonorgestrel. A new mechanistic diffusion model accurately described the levonorgestrel burst release in early time points and pseudo-steady state behavior at later time points. As previously described, tenofovir was formulated as a glycerol paste and filled into a hydrophilic polyurethane, hollow tube reservoir that was melt-sealed by induction welding. These tenofovir-eluting segments and 2 cm long coaxially extruded levonorgestrel eluting segments were joined by induction welding to form rings that released an average of 7.5 mg tenofovir and 21 µg levonorgestrel per day in vitro for 90 days. Levonorgestrel segments placed intravaginally in rabbits resulted in sustained, dose-dependent levels of levonorgestrel in plasma and cervical tissue for 90 days. Polyurethane caps placed between segments successfully prevented diffusion of levonorgestrel into the tenofovir-releasing segment during storage.Hydrated rings endured between 152 N and 354 N tensile load before failure during uniaxial extension testing. In summary, this system represents a significant advance in vaginal drug delivery technology, and is the first in a new class of long-acting multipurpose prevention drug delivery systems.

Quantitative evaluation of a hydrophilic matrix intravaginal ring for the sustained delivery of tenofovir.

In vitro testing and quantitative analysis of a matrix, hydrophilic polyether urethane (HPEU) intravaginal ring (IVR) for sustained delivery of the anti-HIV agent tenofovir (TFV) are described. To aid in device design, we employed a pseudo-steady-state diffusion model to describe drug release, as well as an elastic mechanical model for ring compression to predict mechanical properties. TFV-HPEU IVRs of varying sizes and drug loadings were fabricated by hot-melt extrusion and injection molding. In vitro release rates of TFV were measured at 37 °C and pH 4.2 for 30 or 90 days, during which times IVR mechanical properties and swelling kinetics were monitored. Experimental data for drug release and mechanical properties were compared to model predictions. IVRs loaded with 21% TFV (w/w) released greater than 2mg TFV per day for 90 days. The diffusion model predicted 90 day release data by extrapolating forward from the first 7 days of data. Mechanical properties of IVRs were similar to NuvaRing, although the matrix elastic modulus decreased up to three-fold following hydration. This is the first vaginal dosage form to provide sustained delivery of milligram quantities of TFV for 90 days. Drug release and mechanical properties were approximated by analytical models, which may prove useful for the continuing development of IVRs for HIV prevention or other women's health indications.

Enzymatic triggered release of an HIV-1 entry inhibitor from prostate specific antigen degradable microparticles.

This paper describes the design, construction and characterization of the first anti-HIV drug delivery system that is triggered to release its contents in the presence of human semen. Microgel particles were synthesized with a crosslinker containing a peptide substrate for the seminal serine protease prostate specific antigen (PSA) and were loaded with the HIV-1 entry inhibitor sodium poly(styrene-4-sulfonate) (pSS). The particles were composed of N-2-hydroxyproplymethacrylamide and bis-methacrylamide functionalized peptides based on the PSA substrates GISSFYSSK and GISSQYSSK. Exposure to human seminal plasma (HSP) degraded the microgel network and triggered the release of the entrapped antiviral polymer. Particles with the crosslinker composed of the substrate GISSFYSSK showed 17 times faster degradation in seminal plasma than that of the crosslinker composed of GISSQYSSK. The microgel particles containing 1 mol% GISSFYSSK peptide crosslinker showed complete degradation in 30 h in the presence of HSP at 37°C and pSS released from the microgels within 30 min reached a concentration of 10 µg/mL, equivalent to the published IC(90) for pSS. The released pSS inactivated HIV-1 in the presence of HSP. The solid phase synthesis of the crosslinkers, preparation of the particles by inverse microemulsion polymerization, HSP-triggered release of pSS and inactivation of HIV-1 studies are described.

Inhibition of the transport of HIV in vitro using a pH-responsive synthetic mucin-like polymer system.

In conjunction with the routine role of delivering the active ingredient, carefully designed drug delivery vehicles can also provide ancillary functions that augment the overall efficacy of the system. Inspired by the ability of the cervicovaginal mucus to impede the movement of HIV virions at acidic pH, we have engineered a pH-responsive synthetic polymer that shows improved barrier properties over the naturally occurring cervicovaginal mucus by inhibiting viral transport at both acidic and neutral pH. The pH-responsive synthetic mucin-like polymer is constructed with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA), each individually copolymerized with a 2-hydroxypropyl methacrylamide (pHPMA) polymer backbone. At pH 4.8, the crosslinked polymers form a transient network with a characteristic relaxation time of 0.9 s and elastic modulus of 11 Pa. On addition of semen, the polymers form a densely crosslinked elastic network with a characteristic relaxation time greater than 60 s and elastic modulus of 1800 Pa. Interactions between the PBA-SHA crosslinked polymers and mucin at acidic pH showed a significant increase in elastic modulus and crosslink lifetime (p < 0.05). A transport assay revealed that migration of HIV and cells was significantly impeded by the polymer network at pH ≥ 4.8 with a diffusion coefficient of 1.60 x 10(-4) µm(2)/s for HIV. Additionally, these crosslinked polymers did not induce symptoms of toxicity or irritation in either human vaginal explants or a mouse model. In summary, the pH-responsive crosslinked polymer system reported here holds promise as a class of microbicide delivery vehicle that could inhibit the transport of virions from semen to the target tissue and, thereby, contribute to the overall activity of the microbicide formulation.

Approaches to improve the stability of the antiviral agent UC781 in aqueous solutions.

In this work, we evaluated the chemical stability profiles of UC781 based solutions to identify excipients that stabilize the microbicidal agent UC781. When different antioxidants were added to UC781 in sulfobutylether-beta-cyclodextrin (SBE-beta-CD) solutions and subjected to a 50 degrees C stability study, it was observed that EDTA was a better stabilizing agent than sodium metabisulfite, glutathione or ascorbic acid. Some antioxidants accelerated the degradation of UC781, suggesting metal-catalyzed degradation of UC781. Furthermore, we observed substantial degradation of UC781 when stored in 1% Tween 80 and 1% DMSO solutions alone or in those with 10mM EDTA. On the other hand, improved stability of UC781 in the presence of 100 and 200mM of EDTA was observed in these solutions. The addition of both EDTA and citric acid in the stock solutions resulted in recovery of more than 60% of UC781 after 12 weeks. Generally, 10% SBE-beta-CD in the presence of EDTA and citric acid stabilized UC781 solutions: the amount of UC781 recovered approaching 95% after 12 weeks of storage at 40 degrees C. We also showed that the desulfuration reaction of the UC781 thioamide involves oxygen by running solution stability studies in deoxygenated media. Improved stability of UC781 in the present study indicates that the incorporation of EDTA, citric acid and SBE-beta-CD and the removal of oxygen in formulations of this drug will aid in increasing the stability of UC781 where solutions of the drug are required.

Segmented polyurethane intravaginal rings for the sustained combined delivery of antiretroviral agents dapivirine and tenofovir.

Dual segment polyurethane intravaginal rings (IVRs) were fabricated to enable sustained release of antiretroviral agents dapivirine and tenofovir to prevent the male to female sexual transmission of the human immunodeficiency virus. Due to the contrasting hydrophilicity of the two drugs, dapivirine and tenofovir were separately formulated into polymers with matching hydrophilicity via solvent casting and hot melt extrusion. The resultant drug loaded rods were then joined together to form dual segment IVRs. Compression testing of the IVRs revealed that they are mechanically comparable to the widely accepted NuvaRing IVR. Physical characterization of the individual IVR segments using wide angle X-ray scattering and differential scanning calorimetry determined that dapivirine and tenofovir are amorphous and crystalline within their polymeric segments, respectively. In vitro release of tenofovir from the dual segment IVR was sustained over 30 days while dapivirine exhibited linear release over the time period. A 90 day accelerated stability study confirmed that dapivirine and tenofovir are stable in the IVR formulation. Altogether, these results suggest that multisegment polyurethane IVRs are an attractive formulation for the sustained vaginal delivery of drugs with contrasting hydrophilicity such as dapivirine and tenofovir.

Polyurethane intravaginal ring for controlled delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1.

Women-controlled methods for prevention of male-to-female sexual transmission of HIV-1 are urgently needed. Providing inhibitory concentrations of HIV-1 reverse transcriptase inhibitors to impede the replication of the virus in the female genital tissue offers a mechanism for prophylaxis of HIV-1. To this end, an intravaginal ring device that can provide long duration delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1, was developed utilizing a medical-grade polyether urethane. Monolithic intravaginal rings were fabricated and sustained release with cumulative flux linear with time was demonstrated under sink conditions for a period of 30 days. The release rate was directly proportional to the amount of drug loaded. Another release study conducted for a week utilizing liposome dispersions as sink conditions, to mimic the partitioning of dapivirine into vaginal tissue, also demonstrated release rates constant with time. These results qualify polyether urethanes for development of intravaginal rings for sustained delivery of microbicidal agents.