Human immunodeficiency virus type 1 (HIV-1) persists in certain CNS cell populations, despite peripheral control of the infection with modern antiretroviral therapy. Infected and/or activated cells release viral proteins, such as trans-activator of transcription (Tat) and various pro-inflammatory factors such as CCL5, creating a positive loop of neuro-inflammation. This serves as the basis for the resulting sublethal and lethal neuropathology that manifests as a spectrum of HIV-mediated CNS impairments, known as HIV-associated neurocognitive disorders (HAND). Opiates, which exist as an interlinked epidemic with HIV-1 infections, exacerbate these neurological effects through direct and indirect mechanisms that disrupt both glial and neuronal function. We hypothesize this is due to converging actions on the CCL5-CCR5 signaling axis by HIV-1 Tat and morphine co-exposure, primarily mediated at the level of the glia, whose consequent activation leads to neuronal damage. We performed repeated measure studies on mixed glia and neuron co-cultures obtained from C57Bl/6J and/or CCR5 knockout mice, treated with Tat and/or morphine for 72 hours. As established in prior studies, morphine worsened Tat-induced neurotoxicity in wild-type co-cultures; substitution of CCR5-null glia eliminated the interactive effects of Tat and morphine, but substitution of CCR5-null neurons did not. Overall, these results suggest that glial CCR5, but not neuronal CCR5, is a convergence point for the interactive effects of Tat and morphine that result in neuron loss. Additional experiments involving treatments with naloxone, a MOR antagonist, or the CCR5 antagonist maraviroc, confirmed each receptor’s role in mediating Tat + morphine toxicity. Quite surprisingly, in co-cultures of wild-type neurons and CCR5-null glia, morphine entirely protected neurons from the neurotoxic effects of Tat. We hypothesize that this effect may reflect an imbalance of neurotrophic factors, particularly BDNF and its neurotoxic precursor proBDNF, whose levels are altered in HIV+ and illicit drug-using patients and may contribute to changes in neuronal signaling and survival exhibited in HAND. Related behavioral tests of anxiety, motor and cognitive function – three areas of neurologic decline seen in HAND – were performed in inducible Tat-transgenic mice that were treated with maraviroc via oral gavage. Tat-mediated impairment was observed in the Barnes Maze, a measure of spatial memory, and was ameliorated by maraviroc. Finally, we assessed the role of CCR5 in mediating Tat and/or morphine effects on psychomotor sensitization and dendritic morphology. With both in vitro and in vivo studies, our findings support the hypothesis that CCR5 plays a central role in driving HIV-1 Tat and/or morphine-mediated neuronal damage.

Introduction. Chlamydia trachomatis (Ct) is an obligate intracellular bacterium, causing a range of diseases in humans. Interactions between chlamydiae and antibiotics have been extensively studied in the past. Hypothesis/Gap statement: Chlamydial interactions with non-antibiotic drugs have received less attention and warrant further investigations. We hypothesized that selected cytokine inhibitors would alter Ct growth characteristics in HeLa cells. Aim. To investigate potential interactions between selected cytokine inhibitors and Ct development in vitro. Methodology. The CCR5 receptor antagonist maraviroc (Mara; clinically used as HIV treatment), the triterpenoid celastrol (Cel; used in traditional Chinese medicine) and the histamine H1 receptor antagonist azelastine (Az; clinically used to treat allergic rhinitis and conjunctivitis) were used in a genital in vitro model of Ct serovar E infecting human adenocarcinoma cells (HeLa). Results. Initial analyses revealed no cytotoxicity of Mara up to 20 µM, Cel up to 1 µM and Az up to 20 µM. Mara exposure (1, 5, 10 and 20 µM) elicited a reduction of chlamydial inclusion numbers, while 10 µM reduced chlamydial infectivity. Cel 1 µM, as well as 10 and 20 µM Az, reduced chlamydial inclusion size, number and infectivity. Morphological immunofluorescence and ultrastructural analysis indicated that exposure to 20 µM Az disrupted chlamydial inclusion structure. Immunofluorescence evaluation of Cel-incubated inclusions showed reduced inclusion sizes whilst Mara incubation had no effect on inclusion morphology. Recovery assays demonstrated incomplete recovery of chlamydial infectivity and formation of structures resembling typical chlamydial inclusions upon Az removal. Conclusion. These observations indicate that distinct mechanisms might be involved in potential interactions of the drugs evaluated herein and highlight the need for continued investigation of the interaction of commonly used drugs with Chlamydia and its host.

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Yes / ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant’s phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance. / X.J. was supported by Medical Research Council (G1001806/1) and Wellcome Trust (097820/Z/11/A) funding and F.F. by a Biotechnology and Biological Sciences Research Council studentship to D.L.R.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxikology Student: Zuzana Matiašková Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Role of selected ABC and SLC transporters in transmembrane permeability of maraviroc: effect on transport in placenta Antiretroviral drug maraviroc is an inhibitor of CCR5-trophic HIV virus and belongs to the group of entry inhibitors. Nowadays, maraviroc is administered as part of combination antiretroviral therapy (cART) primarily in adults, children over the age of two and pregnant women to reduce the risk of transmission of HIV to the fetus. The knowledge of interactions of maraviroc with drug transporters in placenta is crucial for optimizing the therapy during pregnancy, both in terms of efficacy and potential adverse effects. Maraviroc is known substrate of ABCB1 transporter, which plays a protective role to the fetus by its efflux activity in the apical membrane of trophoblast. However, the results of recent study employing dually perfused human placenta suggest involvement of other transport mechanisms in the maraviroc transplacental pharmaocokinetics, especially those operating in the opposite direction to ABCB1. The aim of this study was to evaluate in vitro studies whether, besides ABCB1,...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kateřina Erbenová Supervisor: PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Study of interactions antiretroviral drug maraviroc with drug transporters ABCB1 and ABCG2. Maraviroc is inhibitor of CCR5 HIV virus entry into the cells representing one of the important components of antiretroviral therapy. To optimize the treatment strategies and minimize the therapeutic risks of maraviroc-containing combination antiretroviral therapy, it is important to know the interactions of this drug with other antiretrovirals. In particular, interaction on membrane transporters may affect pharmacokinetics and thereby the tissue concentrations of administered drugs, leading to insufficient efficacy of the therapy or increased toxicity. The aim of this study was to experimentally evaluate interaction of maraviroc with the two most important active drug transporters of the ABC transporter superfamily, ABCB1 (P-glycoprotein) and ABCG2 (BCRP). Using in vitro methods employing cell lines we aimed to fulfil two main goals: (1) to evaluate the inhibitory effect of maraviroc on ABCB1 and ABCG2 transporters and (2) to study if any of these transporters could transfer maraviroc as their substrate. The data...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxikology Student: Zuzana Matiašková Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Role of selected ABC and SLC transporters in transmembrane permeability of maraviroc: effect on transport in placenta Antiretroviral drug maraviroc is an inhibitor of CCR5-trophic HIV virus and belongs to the group of entry inhibitors. Nowadays, maraviroc is administered as part of combination antiretroviral therapy (cART) primarily in adults, children over the age of two and pregnant women to reduce the risk of transmission of HIV to the fetus. The knowledge of interactions of maraviroc with drug transporters in placenta is crucial for optimizing the therapy during pregnancy, both in terms of efficacy and potential adverse effects. Maraviroc is known substrate of ABCB1 transporter, which plays a protective role to the fetus by its efflux activity in the apical membrane of trophoblast. However, the results of recent study employing dually perfused human placenta suggest involvement of other transport mechanisms in the maraviroc transplacental pharmaocokinetics, especially those operating in the opposite direction to ABCB1. The aim of this study was to evaluate in vitro studies whether, besides ABCB1,...