Pre-clinical development of a combination microbicide vaginal ring containing dapivirine and darunavir

Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the preclinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir.

Methods: Macaque rings containing 25 mg dapivirine, 300 mg darunavir and 100 mg dapivirine, and 300 mg darunavir were manufactured and characterised by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28-day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values calculated.

Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present, and the release medium. In macaques, serum concentrations of both microbicides were maintained between 101–102 pg/mL. Vaginal fluid levels ranged between 103–104 ng/g and 104–105 ng/g for dapivirine and darunavir, respectively. Tissue concentrations ranges for each drug were: vagina (1.8×103–3.8×103 ng/g) > cervix (9.4×101–3.9×102 ng/g) > uterus (0–108 ng/g) > rectum (0–40 ng/g). Measured IC50 values were > 2 ng/mL for both compounds.

Conclusions: Based on these results, and in light of recent clinical progress of the 25mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate.

Development of a Temperature-Recording Vaginal Ring for Monitoring User Adherence

Background: Vaginal ring devices are being actively developed for controlled delivery of HIV microbicides and as multi-purpose prevention technology (MPT) products combining hormonal contraception with prevention of HIV and other sexually transmitted diseases. Presently, there is no reliable method for monitoring user adherence in HIV vaginal ring trials; previous acceptability studies have included some type of participant self-reporting mechanism, which have often been unreliable. More objective, quantitative and accurate methods for assessing adherence are needed.
Methods: A silicone elastomer vaginal ring containing an encapsulated miniature temperature recording device has been developed that can capture and store real-time temperature data during the period of designated use. Devices were tested in both simulated vaginal environments and following vaginal placement in cynomolgus macaques. Various use protocols and data sampling rates were tested to simulate typical patient usage scenarios. Results: The temperature logging devices accurately recorded vaginal temperature in macaques, clearly showing the regular diurnal temperature cycle. When environmental temperature and vaginal temperature was significantly different, the device was able to accurately pinpoint the insertion and removal times. Based on the data collected it was possible to infer removal periods as short as 5 min when the external environmental temperature was 25 °C. Accuracy increased with data sampling rate. Conclusions: This work provides proof-of-concept for monitoring adherence using a vaginal ring device containing an encapsulated temperature logger. The addition of one or more active agents into the ring body is not anticipated to affect the temperature monitoring function. A clinical study to compare self- reported user adherence data with that obtained by the device would be highly informative.

A Temperature-Monitoring Vaginal Ring for Measuring Adherence

Background

Product adherence is a pivotal issue in the development of effective vaginal microbicides to reduce sexual transmission of HIV. To date, the six Phase III studies of vaginal gel products have relied primarily on self-reporting of adherence. Accurate and reliable methods for monitoring user adherence to microbicide-releasing vaginal rings have yet to be established.

Methods

A silicone elastomer vaginal ring prototype containing an embedded, miniature temperature logger has been developed and tested in vitro and in cynomolgus macaques for its potential to continuously monitor environmental temperature and accurately determine episodes of ring insertion and removal.

Results

In vitro studies demonstrated that DST nano-T temperature loggers encapsulated in medical grade silicone elastomer were able to accurately and continuously measure environmental temperature. The devices responded quickly to temperature changes despite being embedded in different thickness of silicone elastomer. Prototype vaginal rings measured higher temperatures compared with a subcutaneously implanted device, showed high sensitivity to diurnal fluctuations in vaginal temperature, and accurately detected periods of ring removal when tested in macaques.

Conclusions

Vaginal rings containing embedded temperature loggers may be useful in the assessment of product adherence in late-stage clinical trials.

A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/ swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9 min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6 ng/L of original sample. Intra- and inter-day precision analysis showed relative
standard deviations (RSD) of 1.2–9.6% and 1.3–12.0% respectively. The method was applied to the analysis of aquatic samples (n = 206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n = 22), cylindrospermopsin (n = 25), microcystin-RR (n = 17), microcystin-LR (n = 12), microcystin-LY (n = 1), microcystin-LF (n = 1) and nodularin (n = 5). For microcystins, the levels detected ranged from 0.001 to 1.51 mg/L, with two samples showing combined levels above the guideline set by the WHO of 1 mg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.