RESUMEN

We have previously reported a multipurpose silicone elastomer vaginal ring providing sustained release of dapivirine (an antiretroviral) and levonorgestrel (a progestin) for HIV prevention and hormonal contraception. During initial development, issues arose due to reaction between the ethynyl group in the levonorgestrel molecule and the hydride-functionalised polydimethylsiloxane components in the silicone elastomer formulation. This unwanted reaction occurred both during and to a lesser extent after ring manufacture, impacting the curing process, the mechanical properties of the ring, and the in vitro release of levonorgestrel. Recently, we reported custom silicone elastomer grades that minimise this reaction. In this follow-on study, we describe the manufacture, in vitro drug release, mechanical, and pharmaceutical stability testing of ring formulations prepared from a custom silicone elastomer and containing 200 mg dapivirine and 80, 160, 240 or 320 mg levonorgestrel. The rings showed mechanical properties similar to marketed ring products, sustained in vitro release of both drugs over 30 days in quantities deemed clinically relevant, offered acceptable assay values, and provided good product stability over 15 weeks at 40 °C and 75% relative humidity.

RESUMEN

Vaginal rings releasing antiretrovirals - either alone or in combination with contraceptive progestins - are being developed for prevention of human immunodeficiency virus (HIV) transmission via vaginal sex. Following Phase I trials, significant discolouration was observed on the surface of investigational silicone elastomer antiretroviral-contraceptive matrix-type vaginal rings containing either 25 mg dapivirine or 200 mg dapivirine plus levonorgestrel. In this study, potential causes of the discolouration have been assessed in vitro using simulated vaginal and menstrual fluids (SVF and SMF, respectively) to model in vivo exposure. The fluid compositions also included hydrogen peroxide (H2O2), hydrogen peroxide plus a copper intrauterine device (IUD), or synthetic dyes (representing personal care and household cleaning products). No discolouration was observed for rings exposed to SVF + hydrogen peroxide (with or without an IUD). However, the SVF + dye compositions showed significant ring discolouration, with staining patterns similar to those observed with rings that had been exposed to highly-coloured personal care and household cleaning products during clinical trial use. Exposure of rings to SMF compositions invariably caused yellow surface discolouration, dark spotting and markings, similar to the staining patterns observed following clinical use. The darker marks on the ring surface were identified as blood debris derived from the SMF. The study indicates that surface discolouration of rings in vivo can be attributed to exposure to menstrual fluid or highly coloured personal care or household cleaning products. Discolouration of the rings was not associated with any specific safety risks for the user, though severe discolouration could potentially impact acceptability and adherence.

RESUMEN

A dapivirine-releasing silicone elastomer vaginal ring for reducing women's risk of HIV acquisition has recently been approved. A next-generation multipurpose vaginal ring releasing dapivirine and levonorgestrel is currently in development, offering hormonal contraception and HIV prevention from a single device. Previously, we reported challenges with incorporating levonorgestrel into rings manufactured from addition-cure silicone elastomers due to an irreversible chemical reaction between the levonorgestrel molecule and the hydride-functionalised crosslinker component of the silicone elastomer formulation, leading to low drug content assay, cure inhibition, and reduced ring mechanical properties (which may account for the increased incidence of ring expulsion in vivo). Here, we report on the development and testing of various custom silicone elastomer materials specifically formulated to circumvent these issues. After extensive testing of the custom silicones and subsequent manufacture and testing (Shore M hardness, pot life, content assay, oscillatory rheology, mechanical testing) of rings containing both dapivirine and levonorgestrel, a lead candidate formulation was selected that was amenable to practical ring manufacture via injection molding, exhibited no substantial levonorgestrel binding, and offered suitable mechanical properties.

RESUMEN

Epidemiological findings have discussed recurrent and persistent vulvovaginal candidiasis to be a major manifestation of HIV infected women. Conversely, women with vulvovaginal candidiasis have higher risk of acquiring HIV transmitted during intercourse. Common treatments for such conditions include combined antiretroviral and antifungal therapy. Drug-Drug interaction is a major problem encountered due to common CYP450 metabolic pathway of azoles and antiretroviral drugs. Ebselen (EB), lipophilic, organo-selenium compound has demonstrated promising anti-HIV and anti-fungal activity. The aim of current research was to develop and characterize a rapidly soluble and non-cytotoxic vaginal film of ebselen which could serve dual purpose of treating vulvovaginal candidiasis and pre-exposure prophylactic (PrEP) against HIV. Ebselen/cyclodextrin polymer/Soluplus® (1:10:10) ternary complex (EßpolySol) showed 200 fold enhancement in aqueous solubility and no degradation of EB in thermogravimetry analysis. EßpolySol film with tensile strength of 33.12 ± 1.98 N/cm2 disintegrated within 30 sec, presented instant drug release with no apparent precipitation in simulated vaginal fluid. EßpolySol film showed compatibility with HEC-1A monolayer and HeLa cells compared to VCF®. EßpolySol film showed MIC of 20 µM against Candida species and IC50 of 0.71 µM against HIV.

Asunto(s)

Candidiasis Vulvovaginal , Infecciones por VIH , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Azoles , Candidiasis Vulvovaginal/tratamiento farmacológico , Candidiasis Vulvovaginal/prevención & control , Celulosa , Ciclodextrinas , Femenino , Infecciones por VIH/tratamiento farmacológico , Células HeLa , Humanos , Isoindoles , Compuestos de Organoselenio , Polietilenglicoles , Polímeros , Polivinilos

RESUMEN

Here we report the first use of an additive manufacturing (AM) technique based on high pressure material jetting of molten thermoplastic for the fabrication of dapivirine (DPV) loaded vaginal rings (VRs). The VRs are compared to those produced conventionally using injection molding (IM). VRs (outer diameter 54.0 mm, cross-sectional diameter 4.0 mm) were manufactured by either injection molding or Arburg Plastic Freeforming (APF) - a proprietary droplet deposition modelling (DDM) process, using medical grade thermoplastic polyurethanes (TPUs) loaded with 10% w/w DPV. This unique DDM process was used to produce rings of 100, 50 and 10% matrix infill density. DDM printed VRs with 10% density (57-62 mg drug load) exhibited up to seven-fold increase in DPV release compared to injection molded rings containing 190-194 mg DPV. This work has shown that DDM using the APF technique can be used to manufacture drug delivery devices of varying geometries, densities and surface areas to give precise levels of control over the drug release kinetics. This work presents a new opportunity to increase the release of poorly water-soluble compounds or to achieve desired dosing levels using lower drug loadings than those required using conventional thermoplastic processing techniques.

Asunto(s)

Fármacos Anti-VIH/química , Dispositivos Anticonceptivos Femeninos , Sistemas de Liberación de Medicamentos/instrumentación , Diseño de Equipo , Poliuretanos/química , Pirimidinas/química , Liberación de Fármacos

RESUMEN

With a dapivirine-releasing vaginal ring having successfully completed late-stage clinical testing for HIV prevention and currently undergoing regulatory review, there is now growing interest in next-generation multipurpose prevention technologies that seek to combine antiretroviral and contraceptive drugs within a single product. Here, we focus on ongoing efforts to develop a silicone elastomer vaginal ring releasing both dapivirine and levonorgestrel. Specifically, we evaluate various strategies aimed at both better understanding and reducing the tendency of levonorgestrel to bind with the elastomer, including: (i) formulation and post-manufacturing strategies aimed at reducing the extent of levonorgestrel reaction with addition-cure silicone elastomers; (ii) evaluation of a simple silicone system to model the complex elastomer; (iii) use of model compounds representing the enone and ethinyl moieties of levonorgestrel to probe the mode of addition of levonorgestrel to addition-cure silicone elastomers; and (iv) solution and solid-state 13C NMR analysis to probe the structural features of the levonorgestrel-silicone system. The results demonstrate that both the enone and ethinyl groups within levonorgestrel undergo hydrosilylation reactions with the hydrosiloxane groups in the silicone elastomer leading to covalent binding. The results also highlight potential strategies for further optimising the dapivirine + levonorgestrel silicone vaginal ring formulation to ensure that the levonorgestrel is available for release.

Asunto(s)

Fármacos Anti-VIH/química , Anticonceptivos Femeninos/química , Dispositivos Anticonceptivos Femeninos , Levonorgestrel/química , Pirimidinas/química , Elastómeros de Silicona/química

RESUMEN

OBJECTIVES: The primary aim was to investigate post-use ring weight as a potential measure of cumulative adherence to a progesterone-releasing vaginal ring. STUDY DESIGN: We weighed and quantified residual progesterone in 115 vaginal rings following 90-day use by participants in an acceptability trial conducted in Nigeria, Senegal and Kenya. The primary objective was to correlate residual progesterone content with post-use ring weight. Secondary objectives included correlating ring weight with putative duration of ring use, and, where participants used two rings consecutively in the study, correlating residual content between these paired rings. RESULTS: Mean ring weight and progesterone content of used rings was 8.62±0.24 g and 1245±245 mg respectively, versus 9.37±0.02 and 2058±21 mg for control rings. Most used rings (90.4%) had residual progesterone levels less than 85% of the nominal loading. Linear regression showed a strong positive linear trend between residual progesterone content and post-use ring weight for all rings (r2=0.82). Duration of ring use was inversely associated (p=.00020) with ring weight. CONCLUSIONS: Post-use ring weight is highly correlated with residual progesterone content, a benchmark objective cumulative measure of adherence, and thus potentially useful as a surrogate objective measure of cumulative adherence to a progesterone-releasing vaginal ring. IMPLICATION STATEMENT: For vaginal rings containing a high initial drug loading and releasing a relatively large fraction of the initial loading during clinical use, post-use ring weight may offer a simple and inexpensive alternative to residual content testing for accurate monitoring of user adherence.

Asunto(s)

Anticonceptivos Femeninos/análisis , Dispositivos Anticonceptivos Femeninos , Cooperación del Paciente , Progesterona/análisis , Ensayos Clínicos como Asunto , Anticonceptivos Femeninos/administración & dosificación , Femenino , Humanos , Kenia , Modelos Lineales , Nigeria , Progesterona/administración & dosificación , Senegal

RESUMEN

In two recent Phase III clinical trials, use of a 25 mg dapivirine vaginal ring significantly reduced HIV acquisition rates. Post hoc analysis from one of the trials indicated higher rates of protection among women over the age of 21 years when compared to younger women, most likely due to reduced adherence in the latter group. There is currently no information available on how release of dapivirine from the ring is affected by either its intermittent removal from the vagina or women's cleaning of the ring before re-insertion. Here, in vitro drug stability and product performance characteristics of the dapivirine ring were assessed under simulated conditions of real-world use. The impact of systematic deviations from the 28-day continuous use protocol upon in vitro release performance, was investigated. Also, the effect of ring exposure to a range of common household chemicals - including bath salts, bleach, detergent and personal lubricants - was examined through measurement of dapivirine content and stability. Dapivirine in vitro release under intermittent schedules was similar to that obtained under the normal continuous schedule ignoring the periods of interruption. Ring exposure to various household chemicals had no discernible impact on dapivirine assay value, degradation or stability.

Asunto(s)

Fármacos Anti-VIH/química , Dispositivos Anticonceptivos Femeninos , Pirimidinas/química , Blanqueadores , Liberación de Fármacos , Estabilidad de Medicamentos , Productos Domésticos , Lubricantes

RESUMEN

The past fifteen years have witnessed a resurgence of interest in vaginal ring technologies for drug delivery applications, mostly driven by the impetus for development of vaginally-administered antiretroviral microbicides to help reduce the high acquisition rates for human immunodeficiency virus (HIV) among Sub-Saharan African women. Currently, the lead candidate microbicide is a 28-day silicone elastomer vaginal ring releasing dapivirine (Ring-004), an experimental non-nucleoside reverse transcriptase inhibitor. The ring was tested in two pivotal Phase III clinical studies in 2016 and is currently undergoing review by the European Medicines Agency. Recently, we described a new type of silicone elastomer vaginal ring offering sustained release of the protein molecule 5P12-RANTES, a potent experimental chemokine analogue that potently blocks the HIV CCR5 coreceptor. Building on our previous work, here we report the preclinical development of a new combination vaginal ring that offers sustained release of both 5P12-RANTES and dapivirine, in which the 5P12-RANTES is incorporated into an exposed core within the ring body and the dapivirine in the sheath. In this way, in vitro release of dapivirine matches closely that for Ring-004. Also, we report the pharmacokinetic testing of this combination ring formulation in sheep, where vaginal concentrations of both drugs are maintained over 28 days at levels potentially useful for preventing HIV infection in women.

Asunto(s)

Fármacos Anti-VIH/administración & dosificación , Quimiocinas CC/administración & dosificación , Dispositivos Anticonceptivos Femeninos , Infecciones por VIH/prevención & control , Pirimidinas/administración & dosificación , Animales , Fármacos Anti-VIH/farmacocinética , Quimiocinas CC/farmacocinética , Preparaciones de Acción Retardada/administración & dosificación , Combinación de Medicamentos , Femenino , Pirimidinas/farmacocinética , Ovinos , Vagina/metabolismo

RESUMEN

With almost 2 million new HIV infections worldwide each year, the prevention of HIV infection is critical for stopping the pandemic. The only approved form of pre-exposure prophylaxis is a costly daily pill, and it is recognized that several options will be needed to provide protection to the various affected communities around the world. In particular, many at-risk people would benefit from a prevention method that is simple to use and does not require medical intervention or a strict daily regimen. We show that silk fibroin protein can be formulated into insertable discs that encapsulate either an antibody (IgG) or the potent HIV inhibitor 5P12-RANTES. Several formulations were studied, including silk layering, water vapor annealing and methanol treatment to stabilize the protein cargo and impact the release kinetics over weeks. In the case of IgG, high concentrations were released over a short time using methanol treatment, with more sustained results with the use of water vapor annealing and layering during device fabrication. For 5P12-RANTES, sustained release was obtained for 31 days using water vapor annealing. Further, we show that the released inhibitor 5P12-RANTES was functional both in vitro and in ex vivo colorectal tissue. This work shows that silk fibroin discs can be developed into formidable tools to prevent HIV infection.

Asunto(s)

Quimiocinas CC/administración & dosificación , Fibroínas/administración & dosificación , Infecciones por VIH/prevención & control , Inmunoglobulina G/administración & dosificación , Línea Celular , Quimiocinas CC/química , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Liberación de Fármacos , Fibroínas/química , Humanos , Inmunoglobulina G/química , Profilaxis Pre-Exposición

RESUMEN

Antiretroviral-releasing vaginal rings are at the forefront of ongoing efforts to develop microbicide-based strategies for prevention of heterosexual transmission of the human immunodeficiency virus (HIV). However, traditional ring designs are generally only useful for vaginal administration of relatively potent, lipophilic, and small molecular weight drug molecules that have sufficient permeability in the non-biodegradable silicone elastomer or thermoplastic polymers. Here, we report a novel, easy-to-manufacture 'exposed-core' vaginal ring that provides sustained release of the protein microbicide candidate 5P12-RANTES, an experimental chemokine analogue that potently blocks the HIV CCR5 coreceptor. In vitro release, mechanical, and stability testing demonstrated the utility and practicality of this novel ring design. In a sheep pharmacokinetic model, a ring containing two »-length excipient-modified silicone elastomer cores - each containing lyophilised 5P12-RANTES and exposed to the external environment by two large windows - provided sustained concentrations of 5P12-RANTES in vaginal fluid and vaginal tissue between 10 and 10,000 ng/g over 28days, at least 50 and up to 50,000 times the reported in vitro IC50 value.

Asunto(s)

Antagonistas de los Receptores CCR5/administración & dosificación , Quimiocinas CC/administración & dosificación , Dispositivos Anticonceptivos Femeninos , Sistemas de Liberación de Medicamentos , Administración Intravaginal , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Antagonistas de los Receptores CCR5/farmacocinética , Quimiocinas CC/farmacocinética , Preparaciones de Acción Retardada , Femenino , Infecciones por VIH/prevención & control , Humanos , Concentración 50 Inhibidora , Ovinos

RESUMEN

Many novel vaginal/rectal microbicide formulations failed clinically due to safety concerns, indicating the need for the early investigation of lead microbicide formulations. In this study, the preclinical safety of an HIV-1 gp120 and mannose responsive microbicide delivery system (MRP) is evaluated in C57BL/6 mice. MRP was engineered through the layer-by-layer coating of calcium carbonate (CaCO3) with Canavalia ensiformis lectin (Con A) and glycogen. MRP mean particle diameter and zeta potential were 857.8 ± 93.1 nm and 2.37 ± 4.12 mV, respectively. Tenofovir (TFV) encapsulation and loading efficiencies in MRP were 70.1% and 16.3% w/w, respectively. When exposed to HIV-1 rgp120 (25 µg/mL), MRP released a significant amount of TFV (∼5-fold higher) in vaginal and seminal fluid mixture compared to the control (pre-exposure) level (∼59 µg/mL) in vaginal fluid alone. Unlike the positive control treated groups (e.g., nonoxynol-9), no significant histological damages and CD45+ cells infiltration were observed in the vaginal and major reproductive organ epithelial layers. This was probably due to MRP biocompatibility and its isosmolality (304.33 ± 0.58 mOsm/kg). Furthermore, compared to negative controls, there was no statistically significant increase in pro-inflammatory cytokines such as IL1α, Ilß, IL7, IP10, and TNFα. Collectively, these data suggest that MRP is a relatively safe nanotemplate for HIV-1 gp120 stimuli responsive vaginal microbicide delivery system.

Asunto(s)

Antiinfecciosos/uso terapéutico , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Administración Intravaginal , Animales , Carbonato de Calcio/metabolismo , Quimiocinas/metabolismo , Dispersión Dinámica de Luz , Femenino , Infecciones por VIH/tratamiento farmacológico , Inmunohistoquímica , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-7/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Concentración Osmolar , Tenofovir/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismo , Vagina/virología

RESUMEN

This work investigates the impact of vaginal ring size and drug loading on the in vitro release, safety, ease of fit, and pharmacokinetics in cynomolgus macaques of matrix-type silicone elastomer vaginal rings containing a combination of the non-nucleoside reverse transcriptase inhibitor dapivirine and the protease inhibitor darunavir. Drug-free and drug-loaded vaginal rings having three different geometries were manufactured by reaction injection molding. In vitro drug release was assessed using both a solvent/water mixture and a vaginal fluid simulant. Macaques fitted with drug-free vaginal rings for 28 days were assessed by colposcopy, cytological evaluation of cervico-vaginal lavage and histological evaluation of tissue after ring removal. The 20 × 4.5 mm combination ring, deemed most appropriate for vaginal fit and comfort in the macaques, was evaluated for pharmacokinetics over 28 days. Substantial differences were observed in the in vitro release profiles between the three ring sizes. However, these differences were not manifest in vivo, where measured drug concentrations after 20 × 4.5 mm ring use were not significantly different from those reported previously with a 25 × 6 mm ring. These results suggest that ring placement and fit is an important species-specific study parameter that should be optimised prior to pharmacokinetic testing.

Asunto(s)

Fármacos Anti-VIH/farmacocinética , Dispositivos Anticonceptivos Femeninos , Darunavir/farmacocinética , Pirimidinas/farmacocinética , Animales , Combinación de Medicamentos , Liberación de Fármacos , Femenino , Macaca fascicularis

RESUMEN

A silicone elastomer vaginal ring providing sustained release over 28 days of the anti-retroviral microbicide dapivirine has recently completed phase III clinical testing and showed moderate protection against HIV acquisition. In support of the product licensure program, we report the impact of dapivirine packing polymorphism on the thermal and solubility characteristics of dapivirine and on the in vitro performance of the 25 mg dapivirine ring product. This is the first time that polymorphism has been reported for a drug-releasing vaginal ring product. Thermal, particle size, powder X-ray diffraction, and thermodynamic solubility analyses of dapivirine polymorphic forms I and IV, both of which are persistent at room temperature and with form I being the thermodynamically stable form, were conducted for both micronized and non-micronized materials. No significant differences in solubility between DPV forms I and IV were observed in media commonly used for in vitro release testing. Matrix-type silicone elastomer vaginal rings were manufactured and the impact of dapivirine polymorphism on key in vitro parameters (compression and tensile behavior; content assay; in vitro release; residual content assay) was investigated. The data demonstrate that dapivirine packing polymorphism has no significant impact on in vitro performance of the 25 mg dapivirine vaginal ring.

Asunto(s)

Fármacos Anti-VIH/administración & dosificación , Dispositivos Anticonceptivos Femeninos , Sistemas de Liberación de Medicamentos/instrumentación , Pirimidinas/administración & dosificación , Elastómeros de Silicona/química , Fármacos Anti-VIH/química , Liberación de Fármacos , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Difracción de Polvo , Pirimidinas/química , Solubilidad , Termodinámica , Difracción de Rayos X

RESUMEN

Topical microbicides have the potential to provide effective protection against sexual transmission of HIV. Challenges in developing microbicides include their application in resource-poor settings with high temperatures and a lack of refrigeration, and low user adherence to a rigorous daily regimen. Several protein-based HIV inhibitors show great promise as microbicides, being highly specific and not expected to lead to resistance that would affect the efficacy of current antiretroviral treatments. We show that four potent protein HIV inhibitors, 5P12-RANTES, 5P12-RANTES-L-C37, Grft, and Grft-L-C37 can be formulated into silk fibroin (SF) disks and remain functional for 14 months at 25, 37, and 50 °C. These HIV inhibitor-encapsulated SF disks show excellent inhibition properties in PBMC and in human colorectal and cervical tissue explants, and do not induce inflammatory cytokine secretion. Further, the SF provides a mechanically robust matrix with versatile material formats for this type of application. Finally, a formulation was developed to allow sustained release of functional Grft for 4 weeks at levels sufficient to inhibit HIV transmission. This work establishes the suitability of HIV inhibitor-encapsulated SF disks as topical HIV microbicides that can be further developed to allow easy insertion for extended protection.

RESUMEN

A matrix-type silicone elastomer vaginal ring providing 28-day continuous release of dapivirine (DPV) - a lead candidate human immunodeficiency virus type 1 (HIV-1) microbicide compound - has recently demonstrated moderate levels of protection in two Phase III clinical studies. Here, next-generation matrix and reservoir-type silicone elastomer vaginal rings are reported for the first time offering simultaneous and continuous in vitro release of DPV and the contraceptive progestin levonorgestrel (LNG) over a period of between 60 and 180days. For matrix-type vaginal rings comprising initial drug loadings of 100, 150 or 200mg DPV and 0, 16 or 32mg LNG, Day 1 daily DPV release values were between 4132 and 6113µg while Day 60 values ranged from 284 to 454µg. Daily LNG release ranged from 129 to 684µg on Day 1 and 2-91µg on Day 60. Core-type rings comprising one or two drug-loaded cores provided extended duration of in vitro release out to 180days, and maintained daily drug release rates within much narrower windows (either 75-131µg/day or 37-66µg/day for DPV, and either 96-150µg/day or 37-57µg/day for LNG, depending on core ring configuration and ignoring initial lag release effect for LNG) compared with matrix-type rings. The data support the continued development of these devices as multi-purpose prevention technologies (MPTs) for HIV prevention and long-acting contraception.

Asunto(s)

Anticonceptivos Femeninos/farmacocinética , Dispositivos Anticonceptivos Femeninos , Levonorgestrel/farmacocinética , Pirimidinas/farmacocinética , Anticonceptivos Femeninos/administración & dosificación , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Liberación de Fármacos/efectos de los fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Levonorgestrel/administración & dosificación , Pirimidinas/administración & dosificación

RESUMEN

Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20µg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N.

Asunto(s)

Proteínas Bacterianas/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Proteínas Portadoras/metabolismo , Hidroponía/instrumentación , Nicotiana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Técnicas de Cultivo Celular por Lotes/instrumentación , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Cromatografía por Intercambio Iónico , Hidroponía/métodos , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Espectrometría de Masas en Tándem , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo

RESUMEN

Gels are a drug delivery platform that is being evaluated for application of active pharmaceutical ingredients, termed microbicides, that act topically against vaginal and rectal mucosal infection by sexually transmitted HIV. Despite success in one Phase IIb trial of a vaginal gel delivering tenofovir, problems of user adherence to designed gel application scheduling have compromised results in two other trials. The microbicides field is responding to this dilemma by expanding behavioral analysis of the determinants of adherence while simultaneously improving the pharmacological, biochemical, and biophysical analyses of the determinants of microbicide drug delivery. The intent is to combine results of these two complementary perspectives on microbicide performance and epidemiological success to create an improved product design paradigm. Central to both user sensory perceptions and preferences, key factors that underlie adherence, and to vaginal gel mucosal drug delivery, that underlies anti-HIV efficacy, are gel properties (e.g. rheology) and volume. The specific engineering problem to be solved here is to develop a model for how gel rheology and volume, interacting with loaded drug concentration, govern the transport of the microbicide drug tenofovir into the vaginal mucosa to its stromal layer. These are factors that can be controlled in microbicide gel design. The analysis here builds upon our current understanding of vaginal gel deployment and drug delivery, incorporating key features of the gel's environment, the vaginal canal, fluid production and subsequent gel dilution, and vaginal wall elasticity. These have not previously been included in the modeling of drug delivery. We consider the microbicide drug tenofovir, which is the drug most completely studied for gels: in vitro, in animal studies in vivo, and in human clinical trials with both vaginal or rectal gel application. Our goal is to contribute to improved biophysical and pharmacological understanding of gel functionality, providing a computational tool that can be used in future vaginal microbicide gel design.

RESUMEN

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 µg/g leaf fresh mass (LFM) in transgenic tobacco and 25 µg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.

Asunto(s)

Anticuerpos Neutralizantes/inmunología , VIH/inmunología , Inmunoglobulina A Secretora/inmunología , Proteínas Recombinantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Sitios de Unión/inmunología , Líquidos Corporales/inmunología , Líquidos Corporales/metabolismo , Femenino , Glicosilación , VIH/efectos de los fármacos , VIH/metabolismo , Humanos , Immunoblotting , Inmunoglobulina A Secretora/genética , Inmunoglobulina A Secretora/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/ultraestructura , Plantas Modificadas Genéticamente , Polisacáridos/análisis , Polisacáridos/inmunología , Unión Proteica/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Nicotiana/genética , Nicotiana/metabolismo , Vagina/inmunología , Vagina/metabolismo , Virión/efectos de los fármacos , Virión/inmunología , Virión/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo

RESUMEN

Diaryltriazines (DATAs) constitute a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are being investigated for use as anti-HIV microbicides. The aim of the present study was (1) to assess the biopharmaceutical properties of the DATA series, (2) to select the lead candidate as vaginal microbicide and (3) to develop and evaluate gel formulations of the lead candidate. First, the vaginal tissue permeation potential of the different DATAs was screened by performing permeability and solubility measurements. To obtain a suitable formulation of the lead microbicide candidate, several hydroxyethylcellulose-based gels were assessed for their cellular toxicity, stability and ability to enable UAMC01398 epithelial permeation. Also, attention was given to appropriate preservative selection. Because of its favourable in vitro activity, safety and biopharmaceutical profile, UAMC01398 was chosen as the lead microbicide candidate among the DATA series. Formulating UAMC01398 as a vaginal gel did not affect its anti-HIV activity. Safe and chemically stable gel formulations of UAMC01398 (0.02%) included a non-solubilizing gel and a gel containing sulfobutyl ether-ß-cyclodextrin (SBE-ßCD, 5%) as solubilizing excipient. Inclusion of SBE-ßCD in the gel formulation resulted in enhanced microbicide flux across HEC-1A epithelial cell layers, to an extent that could not be achieved by simply increasing the dose of UAMC01398. The applied rational (pre)formulation approach resulted in the development of aqueous-based gel formulations that are appropriate for further in vivo investigation of the anti-HIV microbicide potential of the novel NNRTI UAMC01398.

Asunto(s)

Antiinfecciosos/farmacocinética , Transmisión de Enfermedad Infecciosa/prevención & control , Infecciones por VIH/prevención & control , Cremas, Espumas y Geles Vaginales/farmacocinética , Antiinfecciosos/química , Antiinfecciosos/toxicidad , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Infecciones por VIH/transmisión , VIH-1/efectos de los fármacos , Humanos , Permeabilidad , Solubilidad , Cremas, Espumas y Geles Vaginales/química , Cremas, Espumas y Geles Vaginales/toxicidad