Virus Protease Inhibitors Research Articles

Telaprevir combination therapy induced a sustained virologic response in nonresponsive chronic HCV infection

ACP Journal Club17 August 2010Telaprevir combination therapy induced a sustained virologic response in nonresponsive chronic HCV infectionJuerg Reichen, MDJuerg Reichen, MDUniversity Clinic of Visceral Surgery and Medicine, Berne, Switzerland (J.R.)Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-153-4-201008170-02007 SectionsAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail Source CitationMcHutchison JG, Manns MP, Muir AJ, et al. Telaprevir for previously treated chronic HCV infection. N Engl J Med. 2010;362:1292-303. https://pubmed.ncbi.nlm.nih.gov/20375406Clinical Impact RatingsGastroenterology: Infectious Disease: References1 Kuntzen T, Timm J, Berical A, et al. Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naïve patients. Hepatology. 2008;48:1769-78. [PMID: 19026009] Google Scholar2 Hézode C, Forestier N, Dusheiko G, et al. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med. 2009;360:1839-50. [PMID: 19403903] Google Scholar Author, Article, and Disclosure InformationAffiliations: University Clinic of Visceral Surgery and Medicine, Berne, Switzerland (J.R.)This article was published at Annals.org on 3 August 2010. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetails Metrics 17 August 2010Volume 153, Issue 4Page: JC2-7KeywordsAdverse eventsAnemiaChronic hepatitis CDrugsHepatitis AHepatitis CInfectious diseasesPatientsRashes ePublished: 17 August 2010 Issue Published: 17 August 2010 Copyright & PermissionsCopyright © 2010 by American College of Physicians. All Rights Reserved.PDF downloadLoading ...

Micellar electrokinetic chromatography method development for determination of impurities in Ritonavir

Ritonavir is a synthetic peptidomimetic human immunodeficiency virus (HIV) protease inhibitor employed in the treatment of AIDS since 1996. Synthetic precursors are potential impurities in the final product. In the present work a micellar electrokinetic chromatography (MEKC) method for the separation of Ritonavir from three available synthetic precursors was developed. The optimized separation is performed in a background electrolyte composed of sodium tetraborate (pH 9.6; 15 mM) containing sodium dodecylsulfate (30 mM) and acetonitrile (18%, v/v). Mass spectrometry was used to confirm the identity of the tested substances. Good repeatability was observed for migration time (RSD about 0.4%) and peak area (RSD about 0.8%). The limits of detection (LOD) obtained allow the determination of two of the impurities at levels as low as 0.005% m/m, and one at a level of 0.3% m/m.

Viral respiratory tract infections and asthma: The course ahead

Inquiries into the relationships between viral respiratory tract illnesses and the inception and exacerbation of asthma are being facilitated by recent advances in research approaches and technology. In this article we identify important knowledge gaps and future research questions, and we discuss how new investigational tools, including improved respiratory tract virus detection techniques, will permit current and future researchers to define these relationships and the host, virus, developmental, and environmental mechanisms that regulate them. A better understanding of these processes should facilitate the development of improved strategies for the prevention and treatment of virus-induced wheezing illnesses and asthma exacerbations and, possibly, the ultimate goal of discovering effective approaches for the primary prevention of asthma.

In situ formation of nanoparticles upon dispersion of melt extrudate formulations in aqueous medium assessed by asymmetrical flow field-flow fractionation

In recent years melt extrudates (e.g. Meltrex ®) have proven to be a promising formulation tool for poorly water-soluble and poorly bioavailable drugs. During the hot-melt extrusion process solid dispersions are formed. For several of these formulations improved bioavailabilities have been reported; the mechanism behind, however is still not very well understood. The aim of this study was to investigate whether solid dispersions prepared by melt extrusion upon dispersion in aqueous medium form particles and/or supramolecular assemblies. The formulation investigated here contained the human immunodeficiency virus (HIV) protease inhibitors lopinavir and ritonavir, polyvinylpyrrolidone–vinyl acetate copolymer (Kollidon ® VA64), sorbitan monolaurate (Span ® 20) and hydrophilic fumed silica (Aerosil ® 200). The aqueous dispersions originating from both, API-containing and placebo formulation were investigated using photon correlation spectroscopy (PCS) and asymmetrical flow field-flow fractionation (AsFlFFF) with subsequent online multi-angle light-scattering (MALS) particle size analysis. The content of both APIs in the AsFlFFF-fractions was quantified using high performance liquid chromatography–mass spectrometry. PCS indicated sub-micron particles. AsFlFFF revealed the co-existence of up to three different types of colloidal to nanoparticulate assemblies in the aqueous dispersions. Even though a complete resolution of the composition of the sub-fractions could not be achieved, the following types could be clearly distinguished: The first fraction eluting from AsFlFFF, appears to be colloidal polymer. Only marginal amounts of the APIs were found associated with the polymer. Secondly, API-rich nanoparticles eluted. Thirdly, nanoparticulate assemblies assigned to sorbitan monolaurate and/or hydrophilic fumed silica were identified. A limited amount of drug was found associated with this fraction. Using AsFlFFF–MALS the size of particles in fractions could be determined. From this experience AsFlFFF is regarded as promising technique for investigation of particles/structures originating during dispersion of melt extrudates in aqueous medium in terms of size and type of nanoparticles and their API-content.

Binary selectivity for HCV NS3/4A protease versus host proteases via irreversible inactivation at a non‐catalytic cysteine

Viral protease inhibitors typically achieve potency via interactions with the catalytic apparatus, but as a result are often poorly selective. Viral protease inhibitors must also block emerging mutants. We have developed a fundamentally different approach to the design of covalent inhibitors by targeting non‐catalytic cysteine residues unique to viral targets but absent in host proteases, enabling extreme, “binary” selectivity. An estimated 170 million people worldwide are infected with hepatitis C virus (HCV). HCV NS3/4A protease (HCVP) is a key target for HCV therapy. We have identified a class of irreversible HCVP inhibitors that bond covalently to the non‐catalytic Cys159 in HCVP. Cys159 is conserved across all HCV genotypes but is absent in host proteases. MALDI‐TOF MS and in vitro assays show that the lead inhibitor bonds to and inhibits HCVP across multiple genotypes and drug‐resistant variants. In replicon assays covalent silencing leads to potent inhibition of HCVP and prolonged duration of action (>24 hrs) after brief exposure to inhibitor. Covalent silencing yields potent inhibition of HCVP in vivo, in a new mouse model. Inhibitors with this profile may profoundly improve hepatitis C patient treatment across all HCV genotypes. This design strategy is also readily applicable to a wide range of therapeutically important proteases. This work was supported by Avila Therapeutics.

Induction of P-glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells

Extended treatment with human immunodeficiency virus (HIV) protease inhibitors (HPIs) is standard in HIV/AIDS therapy. While these drugs have helped decrease the overall incidence of AIDS defining illnesses, the relative prevalence of HIV/AIDS dementia has increased. HPIs may cause induction of blood-brain barrier (BBB) drug transporters (P-glycoprotein; P-gp) and thereby limit entry of HPIs into brain tissue, increasing the probability that the brain could become an HIV sanctuary site. Using bovine brain microvessel endothelial cells (BMEC) as an in-vitro model of the BBB, the potential for the HIV protease inhibitor ritonavir to cause induction of P-gp activity and expression was examined. BMEC were isolated from fresh cow brain by enzymatic digest and density centrifugation. Primary culture BMEC were co-incubated with ritonavir or vehicle control for 120 h. Quantitative drug accumulation of rhodamine 123 (Rh123) and fluorescence microscopy were used as measures of P-gp activity. P-gp expression was assessed using quantitative Western blotting. Ritonavir decreased Rh123 cell accumulation and increased P-gp immunoreactive protein in a concentration-dependent manner. Fluorescent microscopy mirrored Rh123 quantitative studies. In BMEC pretreated with 30 microM ritonavir, Rh123 accumulation was decreased 40% and immunoreactive P-gp protein increased 2-fold. Collectively, a strong correlation between decreased Rh123 BMEC accumulation and increased P-gp immunoreactive protein was observed (Spearman r2 = 0.77, P < 0.0001). Thus extended exposure of BMEC to ritonavir caused a concentration-dependent increase in P-gp activity and expression. Similar findings may occur at the clinical level with prolonged HIV protease inhibitor use, giving insight into the central nervous system as an HIV sanctuary site and eventual development of HIV dementia.

In-vitro and in-vivo pharmacokinetic interactions of amprenavir, an HIV protease inhibitor, with other current HIV protease inhibitors in rats.

The drug interactions between a new human immune deficiency virus (HIV) protease inhibitor, amprenavir, and four other protease inhibitors which are presently used have been characterized by in-vitro metabolic studies using rat liver microsomal fractions and in-vivo oral administration studies. The metabolic clearance rates (Vmax/Km) of amprenavir, saquinavir, indinavir and nelfinavir in rat liver microsomes were 50.67+/- 3.77, 170.88 +/- 15.34, 73.01 +/- 2.76 and 126.06 +/- 6.23 microLmin(-1) (mg protein)(-1), respectively, and the degree of metabolicclearance was in the order of saquinavir > nelfinavir > indinavir > amprenavir > ritonavir. The inhibition constants (Ki) of ritonavir for amprenavir, indinavir, nelfinavir and saquinavir were 2.29, 0.95, 1.01 and 1.64 microM, respectively, and that of indinavir for amprenavir was 0.67, indicating that amprenavir metabolism in rat liver microsomes was strongly inhibited by indinavir. The Ki values of amprenavir for indinavir, nelfinavir and saquinavir were 7.41, 2.13 and 16.11 microM, respectively, and those of nelfinavirand saquinavirforamprenavirwere 9.15 and 34.57 microM, respectively. The area under the concentration vs time curve (AUC) of amprenavir after oral co-administration with saquinavir, indinavir, nelfinavir or ritonavir (20 mg kg(-1) for each oral dose in rats) was increased by 1.6-, 2.0-, 1.2- and 9.1-fold, respectively. The AUC values of saquinavir, indinavir and nelfinavir by co-administration with amprenavir showed about 7.3-, 1.3-, and 7.9-fold increase, respectively. These observations suggested that the oral bioavailability of amprenavir was not so affected by co-administration with saquinavir, nelfinavir or indinavir, compared with ritonavir, whereas amprenavir markedly affected the oral bioavailability of saquinavir and nelfinavir. In addition, the in-vivo effects after co-administration of two kinds of HIV protease inhibitors cannot always be predicted from in-vitro data, suggesting the presence of other interaction processes besides metabolism in the liver. However, these results provide useful information for the treatment of AIDS patients when they receive a combination therapy with two kinds of HIV protease inhibitor.

Pharmacokinetic Interactions between HIV-1 Protease Inhibitors in Rats: Study on Combinations of Two Kinds of HIV-1 Protease Inhibitors

The drug interactions between four human immune deficiency virus (HIV-1) protease inhibitors have been characterized by in-vitro metabolic studies using rat liver microsomal fractions and in-vivo oral administration. In this study, a new HPLC analytical method developed by us was used for the simultaneous determination of saquinavir and nelfinavir in rat plasma and microsomes. The metabolic clearance rates (Vmax/Km) of saquinavir, nelfinavir, and indinavir were 170.9 +/- 10.9, 126.1 +/- 4-4, and 73.0 +/- 2.0 microL min(-1) (mg protein)(-1), respectively. Ritonavir was the strongest inhibitor with inhibition constants (Ki) of 1.64 microM for saquinavir, 0.95 microM for indinavir, and 1.01 microM for nelfinavir. Nelfinavir was the second strongest inhibitor with Ki's of 2.35 microM for saquinavir and 2.14 microM for indinavir. Indinavir was the third strongest inhibitor with Ki's of 2.76 microM for nelfinavir and 3.55 microM for saquinavir. Saquinavir was the weakest inhibitor for the other three HIV- 1 protease inhibitors. After oral co-administration in combination with another HIV-1 protease inhibitor, the AUCs of saquinavir, indinavir, and nelfinavir were significantly increased compared with mono-treatment. The AUCs of saquinavir were increased about 10.1-, 3.1- and 45.9-fold in the presence of indinavir, nelfinavir and ritonavir, respectively. The AUCs of indinavir were increased about 6.8-, 5.9- and 9.4-fold in the presence of nelfinavir, saquinavir and ritonavir, respectively. The AUCs of nelfinavir were increased about 2.2-, 6.6- and 8.5-fold in the presence of indinavir, saquinavir and ritonavir, respectively. The in-vivo effects observed after co-administration of two kinds of HIV-1 protease inhibitor were not always expected from in-vitro data, suggesting the presence of other interaction processes besides metabolism in the liver. These results provide useful information for the treatment of AIDS patients receiving combination therapy with two HIV-1 protease inhibitors.

Apparent mechanism-based inhibition of human CYP3A in-vitro by lopinavir.

The influence of the viral protease inhibitor lopinavir on the activity of six human cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Column chromatography methodology was developed to separate lopinavir from ritonavir starting from the commercially available lopinavir-ritonavir combination dosage form. Lopinavir produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19 and 2D6. However, lopinavir was an inhibitor of CYP3A. At 250 microM triazolam (the CYP3A index substrate), the mean (+/- s.e., n = 4) IC50 versus triazolam alpha-hydroxylation (where IC50 is the concentration producing a 50% decrement in reaction velocity) was 7.3 (+/- 0.5) microM. Pre-incubation of lopinavir with microsomes prior to addition of triazolam yielded a significantly lower IC50 of 4.1 (+/- 0.5) microM. This is consistent with mechanism-based inhibition of human CYP3A by lopinavir. Although lopinavir is less potent than ritonavir as an inhibitor of CYP3A, lopinavir is nonetheless likely to contribute to net CYP3A inhibition in-vivo during treatment with the lopinavir-ritonavir combination.

Recent Advances in Targeting Viral Proteases for the Discovery of Novel Antivirals

The occurrence of life-threatening viral infections and the establishment of appropriate defense strategies exhibit major challenges to the disease management in our society. The unpredictable character of viral outbreaks will even be enhanced in the future due to human activities such as increasing international travel, deforestation, changes in social conditions, or influences induced by the climate change. The defense against these pathogenic agents requires preparedness, including successful drug design strategies. Viral proteases represent attractive targets for the design of anti-infective lead compounds, as in case that a viral mRNA encoding several types of proteins is recognized as a monocistronic template by the host-cell translation machinery, the presence of protease activities is required for processing the viral polyprotein precursor into structural or non-structural components essential for the formation of new virion particles. In addition, viral proteases can be involved in further processes relevant for viral replication. Numerous efforts have been made to develop potent small-molecule inhibitors of viral proteases, however, until now only a limited number reached the market. In the present contribution, functional aspects of the target proteases, their structural properties, drug design strategies, resulting inhibitors, and resistance management are reviewed and discussed by means of the four essential representative cases of HIV, HCV, SARS coronavirus, and the flaviviruses Dengue and West Nile virus.

Interaction of HIV protease inhibitors with OATP1B1, 1B3, and 2B1

The effects of human immunodeficiency virus (HIV) protease inhibitors (PI) on the accumulation of the fluorescent bile salt analogue cholyl-glycylamido-fluorescein (CGamF) were determined in organic anion transporting polypeptide (OATP)-1B1 and -1B3-expressing Chinese hamster ovary (CHO) cells. In addition, interaction studies in Caco-2 monolayers, known only to express the OATP2B1 isoform, were conducted using the established OATP substrate estrone 3-sulfate (E3S), since no CGamF accumulation was observed in Caco-2 monolayers.CGamF appeared an excellent substrate for the OATP1B subfamily, with net accumulation clearance values of 7.8 and 142 μl min−1 mg−1 protein in OATP1B1 and OATP1B3-transfected cells, respectively. Ki-values reflecting inhibition of CGamF accumulation by HIV PI correlated well between OATP1B1 and OATP1B3-expressing cells. Lopinavir was the most potent inhibitor (Ki = 0.5–1.4 μM) of OATP1B-mediated CGamF accumulation compared with atazanavir, darunavir, ritonavir, and saquinavir (Ki between 1.4 and 3.3 μM).Inhibitory profiles towards OATP2B1-mediated E3S accumulation were different with only indinavir, saquinavir, and ritonavir showing substantial effects.In conclusion, OATP1B3 appears to be a major transport mechanism mediating sodium-independent CGamF accumulation in human liver, and CGamF could be used as a probe substrate for in vitro drug interaction studies. The remarkably potent inhibition of OATP1B1 by lopinavir may explain some clinically relevant drug interactions between lopinavir and OATP1B substrates such as fexofenadine.

Synthesis and NMR assignment of pentacycloundecane precursors of potential pharmaceutical agents

The synthesis and complete NMR elucidation of eight novel pentacycloundecane (PCU) derivatives are reported. These compounds are precursors in the synthesis of PCU-based anti-tuberculosis (TB) agents and potential human immunodeficiency virus (HIV) protease inhibitors. Two-dimensional (2D) NMR techniques were used to assign the NMR spectra for these compounds. Substitution of the cage molecule at (C-8/11) further complicates the assignment, since some of the substituted alkyl chain groups overlap with the cage proton signals. The side chain heteroatoms also introduce a rare through-space deshielding effect to some of the carbon atoms of the cage skeleton. Ring strain in the rigid cage skeleton appears to induce drastic electronic changes in some parts of the cage framework. This observation is more dramatic for the C-4 methylene group of the cage diols and the cage ethers.

A Promising First Step for Combination Oral Therapy Against HCV

Oral hepatitis C virus (HCV) protease inhibitors are expected to be approved in 2011. Adding these agents to standard HCV treatment (peginterferon plus ribavirin) improves the likelihood of sustained virologic response, but the interferon component must be given subcutaneously and has substantial side effects. In the industry-funded INFORM-1 study, researchers explored whether an interferon-free regimen containing two new oral …

Combining metabolic fingerprinting and footprinting to understand the phenotypic response of HPV16 E6 expressing cervical carcinoma cells exposed to the HIV anti-viral drug lopinavir

Recently, it has been reported that the anti-viral drug, lopinavir, which is currently used as a human immunodeficiency virus (HIV) protease inhibitor, could also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In this study, C33A parent control cells and HPV16 E6-transfected cells were exposed to lopinavir at concentrations ranging from 0 to 30 microM. The phenotypic response was assessed by Fourier transform infrared (FT-IR) spectroscopy directly on cells (the metabolic fingerprint) and on the cell growth medium (the metabolic footprint). Multivariate analysis of the data using both principal components analysis (PCA) and canonical variates analysis (PC-CVA) showed trends in scores plots that were related to the concentration of the drug. Inspection of the PC-CVA loadings vector revealed that the effect was not due to the drug alone and that several IR spectral regions including proteins, nucleotides and carbohydrates contributed to the separation in PC-CVA space. Finally, partial least squares regression (PLSR) could be used to predict the concentration of the drug accurately from the metabolic fingerprints and footprints, indicating a dose related phenotypic response. This study shows that the combination of metabolic fingerprinting and footprinting with appropriate chemometric analysis is a valuable approach for studying cellular responses to anti-viral drugs.

Atazanavir plasma concentrations are impaired in HIV-1-infected adults simultaneously taking a methadone oral solution in a once-daily observed therapy setting

The human immundeficiency virus (HIV) protease inhibitor atazanavir is often used in once-daily observed therapy of methadone substituted former opiate drug users. We performed a matched-pairs analysis on 24 patients (12 men/women) taking atazanavir/ritonavir 300/100 mg daily plus reverse transcriptase inhibitors, with (n = 12) or without (n = 12) methadone co-administration. Twenty-four-hour pharmacokinetic profiles of atazanavir/ritonavir were assessed at steady-state and measured by liquid chromatography-tandem mass spectrometry. The geometric mean (GM, t test) minimum and maximum plasma drug concentrations (C(min), C(max)), area under the concentration-time curve (AUC), and total clearance (CL(total)) were compared between the groups of pairs, which were matched for age, sex, weight, and ethnicity. The GM [90% confidence interval (CI)] of the atazanavir C(min), C(max), and AUC of patients taking the methadone oral solution at doses of 20-175 mg/day simultaneously with antiretroviral therapy were impaired compared to patients not taking methadone oral solution: C(min) = 315 (range 197-448) vs. 519 (279-793) ng/mL [GM ratio (GMR) = 0.61, p = 0.229]; C(max) = 1714 (1238-2262) vs. 3190 (2412-4076) ng/mL (GMR = 0.54, p = 0.018); AUC = 21,987 (15,870-29,327) vs. 35,572 (26,211-46,728) ng h/mL (GMR = 0.62, p = 0.074). Methadone dose, which is proportional to the amount of methadone oral solution (10 mg/mL), was significantly correlated to atazanavir C(max) (r (2) = 0.40, p = 0.001) and AUC (r (2) = 0.32, p = 0.006). Ritonavir pharmacokinetics was similar between the groups with C(min), C(max), and AUC GMR of 1.01, 0.80, and 0.96, respectively. The partial decrease in atazanavir plasma concentrations in patients concomitantly taking racemic methadone oral solution in this daily observed therapy setting deserves further attention, and therapeutic drug monitoring should be considered.

The Role of P-Glycoprotein in the Pharmacokinetics and Tissue Distribution of a Hepatitis C Virus Protease Inhibitor

S5, a hepatitis C virus protease inhibitor, displays partially saturable efflux in the Caco-2 system. In addition, the efflux can be reversed by cyclosporine, indicating that S5 may be a human P-glycoprotein (P-gp) substrate. S5 can also activate the ATPase activity in vesicle membranes containing mouse P-gp 1a and 1b, suggesting that S5 may be a substrate for mouse P-gp. The pharmacokinetics and tissue distribution of S5 were evaluated after intravenous and oral administration to wild-type and 1a/1b knockout mice. Plasma and kidney levels of this compound in knockout mice were transiently higher than those in wild-type mice only after oral dosing, indicating effective P-gp efflux occurs in wild-type mice. The levels of S5 in brain samples from knockout mice were higher than those from wild-type mice after both intravenous and oral administration, but much more significantly after intravenous administration. The levels in liver were four time higher in knockout mice than in wild-type mice after oral administration, but were not different between knockout and wild-type mice after intravenous administration. These results suggest that P-gp efflux limits exposure to S5 in the brain and liver, and that the effect is dependent on the route of administration.

Inhibition of oral midazolam clearance by boosting doses of ritonavir, and by 4,4‐dimethyl‐benziso‐(2H)‐selenazine (ALT‐2074), an experimental catalytic mimic of glutathione oxidase

* The viral protease inhibitor ritonavir is known to inhibit clearance of intravenous midazolam. * ALT-2074, a catalytic mimic of glutathione oxidase, inhibits human cytochrome P450 3A (CYP3A) isoforms in vitro. * Short-term administration of low-dose ritonavir increases area under the plasma concentration curve following oral midazolam by a factor of 28. * Therefore ritonavir is an appropriate positive control inhibitor for clinical drug interaction studies involving CYP3A substrates. * Midazolam clearance is weakly inhibited by ALT-2074, consistent with its in vitro profile. We evaluated whether 'boosting' doses of ritonavir can serve as a positive control inhibitor for pharmacokinetic drug-drug interaction studies involving cytochrome P450 3A (CYP3A). The study also determined whether 4,4-dimethyl-benziso-(2H)-selenazine (ALT-2074), an investigational organoselenium compound that acts as a catalytic mimic of glutathione oxidase, inhibits CYP3A metabolism in vivo. Thirteen healthy volunteers received single 3-mg oral doses of midazolam on three occasions: in the control condition, during co-treatment with low-dose ritonavir (three oral doses of 100 mg over 24 h), and during co-treatment with ALT-2074 (three oral doses of 80 mg over 24 h). Ritonavir increased mean (+/-SE) total area under the curve (AUC) for midazolam by a factor of 28.4 +/- 4.2 (P < 0.001), and reduced oral clearance to 4.2 +/- 0.5% of control (P < 0.001). In contrast, ALT-2074 increased midazolam AUC by 1.25 +/- 0.11 (P < 0.05), and reduced oral clearance to 88 +/- 8% of control. Low-dose ritonavir produces extensive CYP3A inhibition exceeding that of ketoconazole (typically 10- to 15-fold midazolam AUC enhancement), and is a suitable positive control index inhibitor for drug-drug interaction studies. ALT-2074 inhibits CYP3A metabolism to a small degree that is of uncertain clinical importance.

Highly sensitive determination of HCV protease inhibitors boceprevir (SCH 503034) and telaprevir (VX 950) in human plasma by LC–MS/MS

The purpose of this study was to develop a specific and highly sensitive method based on fast sample preparation and LC–MS/MS techniques for the determination of the HCV protease inhibitors boceprevir (SCH 503034) and telaprevir (VX 950) in human plasma. Boceprevir, telaprevir and the internal standard dimethylcelecoxib were separated on a Luna C18 column (150 mm × 2.0 mm I.D., 5 μm particle size) under gradient conditions with a mobile phase A consisting of water/ammonia solution (25%) (100:0.05, v/v) and mobile phase B consisting of methanol/ammonia solution (25%) (100:0.05, v/v) and a chromatographic run time of 11 min. The lower limit of quantification (LLOQ) of boceprevir and telaprevir is 0.25 pg on column (25 pg/mL at injection volume of 10 μL). The method possesses a reliable calibration range of 0.025–2.5 ng/mL. Due to the dilution of real life plasma samples by a factor of 10 during the precipitation process the method is suitable to quantify boceprevir and telaprevir at a concentration range of 0.25–25 ng/mL. Variations in accuracy and intraday and interday precision ( n = 6 for each concentration) were <15% over the whole range of calibration. For the first time, a rapid, specific, sensitive, accurate and reproducible LC–MS/MS method in human plasma has been developed and validated. It is suitable to quantify the concentrations of the hepatitis C virus protease inhibitors boceprevir and telaprevir in human plasma.

Contribution and limit of the model of perfused cotyledon to the study of placental transfer of drugs. Example of a protease inhibitor of HIV: Nelfinavir

Objectives The perfused cotyledon model is a very useful method to study placental transfer of drugs. Here we studied placental transfer of the human immunodeficiency virus protease inhibitor nelfinavir using the non-recirculating dual human placental perfusion with a main goal to determining the clearance index of nelfinavir as related to maternal concentrations, and analyze the conditions under which ex vivo and in vivo data can be correlated. Study design Thirteen human cotyledons, obtained after uneventful term pregnancies, were perfused in an open double circuit with nelfinavir (320–4436 μg/l) and a freely diffusing marker antipyrine 20 mg/l, in the presence of an albumin concentration of 2 g/l. Drug concentrations were determined by high-performance liquid chromatography. Results The mean clearance index of nelfinavir was very weak when maternal concentrations were under 500 μg/l (0.03 ± 0.05). For maternal concentrations above 1200 μg/l, the mean fetal transfer rate was 14 ± 3.4%, the mean clearance index was 0.39 ± 0.10 and the fetal concentrations were between 133 and 671 μg/l. There was a good correlation between maternal and fetal concentrations ( r = 0.86; p < 0.001). Conclusions Our study with nelfinavir has achieved a good correlation between ex vivo and in vivo data. Our results also indicate that studies must be conducted under well defined conditions to obtain accurate and comparable data, underlining the fact that the ex vivo perfused cotyledon remains difficult to standardize as a model system.

Novel HIV-1 therapeutics through targeting altered host cell pathways

The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse transcriptase, viral protease or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host's proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be used to better design therapeutics against HIV-1.