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.

A Combination Vaginal Ring Releasing Dapivirine and Darunavir

Background: Combination microbicide vaginal rings, containing two or more antiretrovirals targeting different steps in the HIV replicative process, may be more effective than single microbicide products at preventing sexual transmission of HIV. Here, we report the preclinical development, including in vitro release and macaque pharmacokinetics, of matrix-type silicone elastomer rings containing dapivirine (DPV; an experimental non-nucleoside reverse transcriptase inhibitor) and darunavir (DRV; a marketed protease inhibitor). Methods: Macaque rings containing 25 mg DPV, 300 mg DRV and 100 mg DPV, and 300 mg DRV 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: DRV caused a concentration-dependent reduction in the DPV 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 DPV and DRV, respectively. Vaginal tissue concentrations decreased in rank order: 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 (HIV-1 BaL) determined from macaque vaginal fluid samples were < 2 ng/mL for both compounds. Conclusions: Based on these results, and in light of the ongoing clinical progress of the 25mg DPV ring, a combination vaginal ring containing DPV and DRV is a viable second-generation HIV microbicide candidate.

Darunavir: A Critical Review of Its Properties, Use and Drug Interactions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desenvolvimento de métodos analíticos qualitativos para a análise de darunavir comprimidos

Darunavir is a protease inhibitor used in the treatment of HIV infection. It is a pillar of the drug cocktail for patients diagnosed with the virus. Quality control in the pharmaceutical industry, to verify the content of active substance and study the physicochemical characteristics of the drug, is essential to ensure final product quality. Until now, standardized methods for the analysis of darunavir have not been available in official compendia. This justifies new research, to develop and validate analytical methods, as well as physicochemical and pharmaceutical analysis for this drug, both as a raw material and a finished product. Thus, in this study, (a) the average weight of darunavir tablets and (b) the melting point of the pure drug were determined, and the following analytical techniques were performed: (c) thin-layer chromatography, (d) ultraviolet spectroscopy, (e) infrared spectroscopy and (f) high performance liquid chromatography. By developing the above techniques, it is possible to make a qualitative assessment of the quality of darunavir tablets.

Estabilidade, polimorfismo, dissolução e correlação in vivo-in vitro de Darunavir comprimidos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stability-indicating thin-layer chromatographic method for determination of darunavir in complex darunavir-beta-cyclodextrin in the presence of its degradation products

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A Critical Review of Properties of Darunavir and Analytical Methods for Its Determination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise quimico-farmacêutica de Darunavir etanolato em comprimidos e de seu complexo de inclusão

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characteristics, complexation and analytical methods of darunavir

What is it? Darunavir is a protease inhibitor used in the treatment of HIV infection. It is an important drug of therapy cocktail for patients infected with the virus. On the market there are darunavir ethanolate tablets of 75, 150, 300, 400, 600 and 800mg, because this is the most stable form. It is commercialized by Janssen-Cilag with the name PrezistaTM. Why we started? This drug has low water solubility and poor bioavailability, therefore requires administration in doses relatively high to the success of the therapeutic effect. The complexation of drugs by using cyclodextrin is welcome in this respect to improve the solubility and hence increase the dissolution rate of poorly soluble drugs. A monograph about this compound has not been described, thus it is an extremely important quality control of darunavir to demonstrate its effectiveness and safety. What we did? Some existing analytical techniques have been discussed in this manuscript, focusing on bioanalytical and pharmaceutical quality control applications. What we found? This review showed the published analytical methods reported for the determination of darunavir and discuss about its characteristics and complexation with cyclodextrin.

Development and validation of first derivative spectrophotometric method for quantification of darunavir in tablets

Aims: Darunavir is widely used in HIV/AIDS therapy. It is a HIV protease inhibitor that has excellent efficacy against the virus. The aim of this study is to develop and validate an analytical method fast and free of interferences for determination of darunavir ethanolate as raw material and tablet dosage form. Methodology: As the formulation excipients show high interference in darunavir determination by a direct UV absorption measurement a derivative spectrophotometry was applied. A selective, easy and fast method was achieved employing simple and cheap instrumentation by using first-order derivative spectrophotometry. Results: The first-derivation of spectrum of the drug measured between 200 and 400 nm allowed identification of the analyte and showed absence of placebo interference. The assay was based on the absorbance at 276nm. The linear concentration range was established from 11 to 21 μg/mL. The intra-day and inter-day precision expressed as RSD was 0.06% and 3.75% respectively with mean recovery of 99.84%. Conclusion: The proposed analytical method is able to quantify darunavir as raw material and tablets and can be used routinely by any laboratory applying a spectrophotometer with a derivative accessory. The great difference of the method proposed here is that it proves to be free of placebo interferences as well as simple, fast and low cost.

Stability study of darunavir ethanolate tablets applying a new stability-indicating HPLC method

Chemical and physical degradation of drugs may result in altered therapeutic efficacy and even toxic effects. Therefore, the aim of this work was to study the stability of darunavir and to develop and validate a liquid chromatography (LC) method to determine darunavir in raw material and tablets in the presence of degradation products. The novel method showed to be linear from 6.0 to 21.0 μg/mL, with high precision (CV < 2%) and accuracy (recuperation of 99.64%). It is simple and reliable, free of placebo interferences. The robustness of the method was evaluated by a factorial design using seven different parameters. Forced degradation study was done under alkaline, acidic, and oxidative stress at ambient temperature and by heating. The LC method was able to quantify and separate darunavir and its degradation products. Darunavir showed to be unstable under alkaline, acid, and oxidative conditions. The novelty of this study is understanding the factors that affect darunavir ethanolate stability in tablets, which is the first step to unravel the path to know the degradation products. The novel stability-indicating method can be used to monitor the drug and the main degradation products in low concentrations in which there is linearity.

Influence of Darunavir: β-cyclodextrin complex on the solubility of Darunavir

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative study over methods developed for quantification of darunavir in tablets by environmental friendly infrared and capillary electrophoretic techniques

Darunavir, a protease inhibitor used in the treatment of HIV infection, presents few methods for its determination in pharmaceuticals. Infrared (IR) spectroscopy offers the possibility of obtaining spectra relatively quickly, providing interesting information, analytically, qualitatively or quantitatively. Capillary electrophoresis (CE) performs separations of high efficiency in shorter time with reagents and samples in small quantity. These two methods are cost-benefitted when we evaluate the green level and the cost of analysis. Faster and cheaper methods without generating organic waste by IR and CE for the quantification of darunavir were developed and validated, focusing socioeconomic impact of analytical decisions. If the cost of acquisition, maintenance, production, analysis and conditioning of drugs and pharmaceuticals is high, consequently the price of this product in the market will be higher and it cannot be accessible to the patient. Treatment failure not only affects the quality of life of patients, but also contributes significantly to the economic burden of the health system. In this context there is a tool called Analysis of the Life Cycle, which comes to make us think in a multidimensional way focusing the whole, the parts and especially the interaction among the parts of a system.

Validation of a stability indicating capillary electrophoresis method for the determination of darunavir in tablets and comparison with the of infrared absorption spectroscopic method

Darunavir (DRV) is a protease inhibitor used in the treatment of HIV infection, which constitutes a keystone in the therapy of patients infected with this virus. There is no monograph described in official compendia. The literature provides few methods of analysis for the determination of DRV in pharmaceuticals which include TLC, IR, UPLC, HPLC, HPLC-MS, HPLC-MS/MS, but there are no reports of the use of capillary electrophoresis (CE) for the determination of this drug. Thus, this research proposed the development and validation of a CE method for the determination of DRV in tablets. The method was completely validated according to the International Conference on Harmonization guidelines, showing linearity, selectivity, precision, accuracy and robustness. The migration was achieved in less than 1 minute using fused-silica uncoated capillary with an id of 50 μm and total length of 21 cm and voltage of +20 kV. The sample injection was performed in the hydrodynamic mode. The method was linear over the concentration range of 50-200 μg mL-1 with correlation coefficient 0.9998 and limits of detection and quantification of 7.29 and 22.09 μg mL-1, respectively. The drug was subjected to acid, base, oxidation and photolysis degradation. Degradation products were found interfering with the assay of DRV, therefore the method can be regarded as stability indicating. The validated method is useful and appropriate for the routine quality control of DRV in tablets.

Complexation between darunavir ethanolate and beta cyclodextrin: experimental and theoretical studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)