Protease Inhibitors Ritonavir Research Articles

Myeloma Exhibits Dependence on Atypical Glucose Transporters: Targeting MCL-1 Through GLUT4 Inhibition

Abstract 1850Multiple Myeloma (MM) is an incurable heterogeneous B cell malignancy characterized by hyperdiploidy and/or aberrant chromosomal translocations warranting strategies that can target diverse molecular subtypes. One such unifying feature in MM entails an increased avidity for glucose that forms the basis for clinical imaging of myeloma by 18fluoro-deoxyglucose positron emission tomography. We are however not yet able to target abnormal glucose utilization for therapy. Given that glucose entry is a key rate-limiting step in glycolysis we performed an unbiased multi-cell line gene-expression profiling study combined with functional knock-down experiments to determine critical glucose transporters facilitating glucose entry in myeloma. These studies revealed a critical dependence of myeloma on the insulin-responsive glucose transporter GLUT4, and GLUTs 8 and 11. Our data demonstrate that myeloma cells exhibit a significant reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption and viability. The functional significance of these transporters was further bolstered by detection of similar patterns of expression in myeloma patient samples. We also tested the therapeutic utility of targeting plasma-membrane localized GLUT4 in MM cell lines and patient samples with the FDA approved HIV protease inhibitor ritonavir that has an off-target inhibitory effect on GLUT4, providing proof of principle that GLUTs can be targeted for therapeutic benefit.In this study we have investigated the mechanism by which GLUT4 suppression elicits cell death in myeloma. GLUT4 contributes to a significant fraction of glucose entry in myeloma while GLUTs 8 and 11 appear to have a significant impact on myeloma cell viability relating to alternative enigmatic subcellular functions not reliant on glucose entry per se. To further elucidate the mechanisms associated with cell death upon glucose deprivation in response to GLUT4 suppression, we examined expression levels of key apoptotic effectors in three myeloma cell lines expressing GLUT 1, 4, 8 or 11 shRNAs. Suppression of GLUT4 appears to selectively engage an apoptotic cascade involving suppression of MCL-1 and BCL-xL in addition to an increase in pro-apoptotic BAX expression. We do not detect up-regulation of PUMA or NOXA that have previously been demonstrated to play a role in glucose-deprivation induced cell death in other cell types. The suppression of MCL-1 is associated with an upstream activation of GSK-3. Expression of an MCL-1 ubiquitination-resistant mutant prevents reduction of MCL-1 protein upon GLUT4 knockdown, and reverses the toxicity elicited by GLUT4 suppression. In addition we do not detect the appearance of the cleaved pro-apoptotic fragment of MCL-1. These observations suggest glucose-deprivation induced cell death in response to GLUT4 reduction is mediated by ubiquitination and proteasomal degradation of MCL-1.In sum we have discovered a novel myeloma specific method to target glucose entry and elicit apoptosis via suppression of MCL-1 expression. MCL-1 is a key effector of chemo-resistance in MM with levels of expression correlating to disease severity. Therefore strategies that can target MCL-1 will have significant utility in treatment of this fatal malignancy. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and potentially other glucose-driven cancers. Disclosures:No relevant conflicts of interest to declare.

Timing of the Components of the HIV Life Cycle in Productively Infected CD4 + T Cells in a Population of HIV-Infected Individuals

We estimate the time required for HIV to complete separate stages of its infection cycle in productively infected CD4+ T cells in vivo by comparing initial delays after administration of single antiretroviral drugs until HIV RNA reduction in peripheral blood. Data were obtained from monotherapy studies of eight antiretroviral drugs from all currently licensed HIV drug classes: CCR5 blockers (maraviroc), fusion inhibitors (enfuvirtide), nucleoside and nonnucleoside reverse transcriptase inhibitors (abacavir, tenofovir, and rilpivirine), integrase inhibitors (raltegravir), and protease inhibitors (ritonavir and nelfinavir). We find that HIV requires an average of 52 h between export of virions in one generation to export in the next, with most of this (33 h) taken up by reverse transcription. Reverse transcription in vivo was three times longer than in vitro and began soon after virion fusion, as we determined no difference in mean times for commencement of reverse transcription and virion fusion as calculated by timing of the effects for tenofovir and maraviroc. Approximately 7 h is required between HIV integration and virion production. First-phase HIV RNA decay (half-life of 17 h over all drugs) seemed to slow as the stage being inhibited by the drug was further from viral production. The mean estimated half-life of plasma virions was 5 min, significantly shorter than previous estimates.

Cushing's syndrome and adrenal axis suppression in a patient treated with ritonavir and corticosteroid eye drops

Cushing's syndrome and adrenal axis suppression in a patient treated with ritonavir and corticosteroid eye drops

Management of HIV infection in treatment-naive patients: a review of the most current recommendations.

The most current guidelines issued by the Department of Health and Human Services (DHHS) on the management of human immunodeficiency virus (HIV) infection in treatment-naive patients are reviewed. Treatment guidelines are updated frequently because of the emergence of data demonstrating the risks and benefits of antiretroviral therapy. The DHHS guidelines strongly recommend initiating therapy in patients with certain conditions regardless of CD4 cell count and in patients with CD4 cell counts of <350 cells/mm(3). Although supporting data are less definitive, treatment is also recommended for patients with CD4 cell counts of 350-500 cells/mm(3). Treatment for patients with CD4 cell counts of >500 cells/mm(3) is controversial. Although cumulative observational data and biological evidence support treatment at higher CD4 cell counts, randomized controlled trial data to support this are not available, and the risk of antiretroviral toxicities, resistance, non-adherence, and cost should be considered in individual patients. The preferred regimens have been consolidated to four options, including a dual-nucleoside reverse transcriptase inhibitor backbone (tenofovir plus emtricitabine) with a nonnucleoside reverse transcriptase inhibitor (efavirenz), a ritonavir-boosted protease inhibitor (atazanavir plus ritonavir or darunavir plus ritonavir), or an integrase strand-transfer inhibitor (raltegravir). Regimens are classified as alternative or acceptable when they have potential safety or efficacy concerns, have higher pill burdens, or require more-frequent administration compared with preferred regimens. The DHHS 2011 guidelines advocate earlier antiretroviral therapy initiation than recommended in recent years, and preferred regimens have been refined to maximize efficacy, safety, and quality of life for treatment-naive HIV-infected patients.

Ritonavir inhibits the two main prasugrel bioactivation pathways in vitro: a potential drug–drug interaction in HIV patients

Prasugrel is an antiplatelet prodrug used in patients with acute coronary syndrome. Prasugrel is mainly bioactivated by cytochromes P450 3A4/5 and CYP2B6. HIV patients are at risk of cardiovascular disease, and the protease inhibitor ritonavir is a potent inhibitor of these 2 CYPs. The aim of this in vitro study was to determine the impact of ritonavir in prasugrel metabolism. Human liver microsomes (HLMs) and recombinant microsomes were used to identify the enzymes responsible for the bioactivation of prasugrel. Prasugrel concentrations of 5 to 200 μM were used for Km determination. Inhibition by ritonavir was characterized using HLMs at concentrations of 0.1 to 30 μM. Prasugrel active metabolite determination was performed with a validated liquid chromatography-mass spectrometry method. Using recombinant microsomes, prasugrel biotransformation was mainly performed by CYP2B6, CYP2D6, CYP2C19, CYP3A4, and CYP3A5. With specific inhibitors of CYP3A, CYP2B6, CYP2D6, CYP2C9, and CYP2C19, active metabolite production was decreased by 38% ± 15% with 4-(4-chlorobenzyl)pyridine (CYP2B6 inhibitor) and by 45 ± 16% with ketoconazole (CYP3A inhibitor). The Km value for prasugrel metabolism in HLMs was determined to be 92.5 μM. Ritonavir at 0.1 to 30 μM was shown to be a potent dose-dependent inhibitor of prasugrel. In this in-vitro study, we found a potent inhibition of prasugrel bioactivation by ritonavir compared to the specific inhibitors of CYP3A and CYP2B6 due to the simultaneous inhibition of CYP2B6 and CYP3A by ritonavir. This finding suggests a potential significant drug-drug interaction between these two drugs.

The Human Immunodeficiency Virus Protease Inhibitor Ritonavir Inhibits Lung Cancer Cells, in Part, by Inhibition of Survivin

Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity. RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis. Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 μM). Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

Extreme bradycardia due to multiple drug–drug interactions in a patient with HIV post‐exposure prophylaxis containing lopinavir–ritonavir

Extreme bradycardia due to multiple drug–drug interactions in a patient with HIV post‐exposure prophylaxis containing lopinavir–ritonavir

Effect of protease inhibitors (indinavir and ritonavir) on the pharmacokinetics of gliclazide in rabbits

The objective of this study was to investigate the effect of protease inhibitors (indinavir and ritonavir) on the pharmacokinetics of gliclazide in rabbits and to evaluate the mechanism of interaction of the combination. Studies in rabbits were conducted with oral doses of gliclazide, selected protease inhibitor, and their combination with a 1-week washout period between each treatment (single dose followed by multiple dose treatment). Blood samples were collected at regular time intervals by marginal ear vein puncture and serum gliclazide levels were analyzed by high-pressure liquid chromatography. Pharmacokinetic analysis was performed by noncompartmental analysis using WinNonlin Software. In combination, ritonavir significantly increased serum gliclazide levels and altered the pharmacokinetic parameters of gliclazide in rabbits while indinavir had no significant effect. The percentage increase of serum gliclazide level was 22.34% and 27.78% following single-dose and multiple-dose treatment of ritonavir, respectively. The interaction of ritonavir with gliclazide is pharmacokinetic at a metabolic level (by CYP3A4 inhibition) in normal rabbits, while the interaction of indinavir with gliclazide is pharmacodynamic, which needs dose adjustment, and care should be taken when these

Nordihydroguaiaretic acid (NDGA) inhibits ritonavir-induced endothelial dysfunction in porcine pulmonary arteries

SummaryBackgroundHIV infection and treatment with highly active antiretroviral therapy (HAART) including HIV protease inhibitor ritonavir (RTV) have been associated with endothelial dysfunction and cardiovascular disease including pulmonary arterial hypertension. The objective of this study was to determine if nordihydroguaiaretic acid (NDGA), a natural herbal antioxidant found in the creosote bush Larrea tridentate, can protect vascular tissues against RTV-induced vascular injury.Material/MethodsFresh porcine pulmonary artery (PA) rings were treated with a clinically relevant concentration of RTV (15 μmol/L) with or without NDGA for 24 hours, and then subjected to myograph analysis for vasomotor reactivity. Expression of endothelial nitric oxide synthase (eNOS) in both treated PA rings and human pulmonary artery endothelial cells (HPAECs) was analyzed by real-time PCR and immunohistochemistry. Oxidative stress levels were analyzed with the lucigenin-enhanced chemiluminescence and glutathione assay.ResultsIn response to bradykinin at 10−10 mol/L, RTV-treated PA rings showed a 39% reduction in endothelium-dependent vasorelaxation compared with the control vessels (P<0.05); when co-cultured with NDGA (1.75 or 3.50 μmol/L), the relaxation increased by 25% and 48%, respectively. RTV also decreased the maximal contraction and endothelium-independent vasorelaxation in RTV-treated vessels, while NDGA improved these vasomotor responses. In addition, treatment of RTV significantly decreased eNOS mRNA levels in both porcine PAs and HPAECs, and reduced eNOS immunoreactivity in porcine PAs, while NDGA significantly inhibited this effect of RTV. Furthermore, NDGA significantly blocked RTV-induced increase of superoxide anion in the PA rings and inhibited RTV-induced decrease of glutathione in HPAECs.ConclusionsNDGA effectively inhibits the detrimental effects of HIV protease inhibitor RTV on vasomotor functions in porcine PAs. NDGA also blocks RTV-induced decrease of eNOS expression and increase of oxidative stress in both porcine PAs and HPAECs. This study may provide valuable information for the development of effective strategies for the prevention and treatment of HAART-associated cardiovascular complications.

Anti-HIV-1 activity of the neurokinin-1 receptor antagonist aprepitant and synergistic interactions with other antiretrovirals

Neurokinin-1 receptor (NK1R) antagonists interfere with binding of neuropeptide substance P to NK1R and exhibit novel anti-HIV-1 activities. Since NK1R antagonists effectively penetrate the blood-brain barrier to reduce the inflammatory response within the brain, we wished to evaluate their potential as anti-HIV-1 candidates for targeting HIV-1 infections of the central nervous system. A series of small molecule agents were evaluated for anti-NK1R and anti-HIV-1 activity using peripheral blood mononuclear cells (PBMCs). The most promising of these, aprepitant (Emend, Merck and Co. Inc.), was investigated for potential synergies with other antiretroviral drugs. Anti-NK1R activity was tested by measuring intracellular calcium increase triggered by substance P. Anti-HIV-1 activity was evaluated by measuring p24 antigen in culture supernatants of PBMC following exposure to HIV. The concentration of drug which produced 50% reduction in intracellular calcium levels or viral production in 7-day PBMC cultures was determined. The combined effect of aprepitant with each of the major classes of anti-HIV-1 drugs was evaluated in synergy studies. Aprepitant had the highest anti-HIV-1 activity of the NK1R antagonists examined and was equally active against all major HIV-1 subtypes. Aprepitant acted synergistically with protease inhibitors (ritonavir and saquinavir), but not with nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, or viral entry inhibitors. The ability of aprepitant to penetrate the blood-brain barrier, its safety record as an FDA-approved drug for reducing nausea and vomiting in chemotherapy, and synergistic activity with other anti-HIV-1 drugs make it a promising candidate for treatment of HIV infection.

Targeting the PI3K/AKT/Survivin Pathway with Ritonavir In Therapy-Resistant Mantle Cell Lymphoma

AbstractAbstract 2855Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma accounting for about 6% of non-Hodgkin's lymphoma cases in the US. While multiple therapy regimens are available to treat MCL, patients ultimately relapse within 3–4 years from therapy-resistant MCL, making MCL carry the worst prognosis of all non-Hodgkin's B cell lymphomas. To improve therapies for patients with MCL, we must understand the biological causes for relapse and therapy-resistance in MCL and develop mechanisms to target the unique properties in relapsing MCL. Recently, we have isolated and produced therapy-resistant MCL cell lines by inoculating NOD-SCID mice with a human MCL cell line, Granta-519 (GP), and subsequently treating with CHOP chemotherapy regimen plus bortezomib. When mice relapsed following therapy, tumor cells were isolated from the kidneys and livers, and cultured to produce stable cell lines, named GRK and GRL, respectively. GRK and GRL cells exhibited increased proliferative and therapy-resistant properties in vitro, and GRL showed increased aggressiveness in vivo also, as compared with GP. Using quantitative PCR (qRT-PCR) to assess the activation of pathways relating to lymphoma progression, the PI3K/Akt/survivin pathway was found to be differentially activated in GRL and GRK compared to GP, including a 6.5 fold increase in survivin in GRL. Therefore, we inhibited survivin in GRL using the FDA-approved protease inhibitor ritonavir (Abbott Laboratories), as recent studies suggest this compound is able to downregulate survivin in lymphoblastoid B cells (Dewan, et al. Int J Cancer 2009; 124[3]: 622–629). When GRL cells were incubated with ritonavir plus vincristine or doxorubicin, MTT assays showed significant inhibition of cell proliferation/survival (p < 0.05) compared to the effects of ritonavir alone. Also, flow cytometric analysis of Annexin V demonstrated dose-dependent induction of apoptosis upon ritonavir treatment in GRL. In addition, qRT-PCR analysis demonstrated a decrease in mRNA expression of the pro-survival genes cyclin D2, Rel A, survivin, and BCL2 in GP and GRL cells after ritonavir treatment. Together, these studies demonstrate the potentials of utilizing ritonavir in a combined treatment regimen designed to target therapy-resistant MCL. As ritonavir is already an FDA-approved drug, these studies hold promise for expediting future in vivo and clinical studies that may eliminate therapy-resistant cells responsible for relapse in MCL patients.(This research was supported by the Lymphoma Research Foundation, New York, NY). Disclosures:No relevant conflicts of interest to declare.

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.

The Bile Acid Sensor FXR Protects against Dyslipidemia and Aortic Plaques Development Induced by the HIV Protease Inhibitor Ritonavir in Mice

BackgroundAlthough human immunodeficiency virus (HIV)–related morbidity and mortality rates in patients treated with a combination of high active antiretroviral therapy (HAART) have declined, significant metabolic/vascular adverse effects associated with the long term use of HIV protease inhibitors (PIs) have emerged as a significant side effect. Here we illustrate that targeting the bile acid sensor farnesoid X receptor (FXR) protects against dyslipidemia and vascular injury induced HIV-PIs in rodents.Methodology/Principal FindingsAdministration of the HIV PI ritonavir to wild type mice increased plasma triacylglycerols and cholesterol levels and this effect was exacerbated by dosing ritonavir to mice harbouring a disrupted FXR. Dyslipidemia induced by ritonavir associated with a shift in the liver expression of signature genes, Sterol Regulatory Element-Binding Protein (SREBP)-1 and fatty acid synthase. Treating wild type mice with the FXR agonist (chenodeoxycholic acid, CDCA) protected against development of dyslipidemia induced by ritonavir. Administration of ritonavir to ApoE−/− mice, a strain that develop spontaneously atherosclerosis, increased the extent of aortic plaques without worsening the dyslipidemia. Treating these mice with CDCA reduced the extent of aortic plaques by 70% without changing plasma lipoproteins or the liver expression of signature genes. A beneficial effect on aortic plaques was also obtained by treating ApoE−/− mice with gemfibrozil, a PPARα agonist. FXR activation counter-regulated induction of expression/activity of CD36 caused by HIV-PIs in circulating monocytes and aortic plaques. In macrophages cell lines, CDCA attenuated CD36 induction and uptake of acetylated LDL caused by ritonavir. Natural and synthetic FXR ligands reduced the nuclear translocation of SREBP1c caused by ritonavir.Conclusions/SignificanceActivation of the bile acid sensor FXR protects against dyslipidemia and atherosclerotic caused by ritonavir, a widely used HIV PI. From a mechanistic stand point it appears that besides reducing the liver expression of genes involved in fatty acid synthesis, FXR activation counter-regulates the expression/activity of CD36 on monocytes. FXR ligands might hold promise in the treatment dyslipidemia induced by ritonavir.

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.

Combination of Suberoylanilide Hydroxamic Acid and Ritonavir is Effective Against Renal Cancer Cells

To investigate the combined effect of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) and the protease inhibitor ritonavir on renal cancer cells. Renal cancer cells (769P, 786O, A498, ACHN, Caki-1) and renal proximal tubule epithelial cells were treated with SAHA (0-5 μM) with or without ritonavir (0-50 μM). Cell viability, clonogenecity, and changes in cell cycle were evaluated. The expression of acetylated histone, retinoblastoma protein (Rb), phosphorylated Rb, histone deacetylases, X-linked inhibitor of apoptosis, survivin, and active caspase 3 was assessed using Western blot analysis. SAHA induced histone acetylation and Rb dephosphorylation and inhibited cell growth in a time- and dose-dependent manner. SAHA and ritonavir combined inhibited cell proliferation effectively and promoted histone acetylation and Rb dephosphorylation but only slightly affected renal proximal tubule epithelial cell survival. The combination induced the accumulation of the sub-G(1) fraction, decreased the expression of X-linked inhibitor of apoptosis and survivin, and increased active caspase 3, thus inducing apoptosis. It also inhibited the expression of histone deacetylases. Combination therapy using SAHA and ritonavir inhibited the proliferation of renal cancer cells effectively, perhaps by inhibiting both histone deacetylase function and expression. It might be a useful new regimen for treating renal cancer.

Protease inhibitors atazanavir, lopinavir and ritonavir are potent blockers, but poor substrates, of ABC transporters in a broad panel of ABC transporter-overexpressing cell lines

A possible mechanism for HIV therapy failure is the efflux of HIV drugs from viral target cells or certain body compartments by ATP-binding cassette (ABC) transporters, allowing ongoing viral replication. Here, we investigated the interaction between protease inhibitors (PIs) and ABC transporters. To explore the potential blocking capacity of PIs, we exposed cells overexpressing multidrug resistance 1 P-glycoprotein (MDR1 P-gp), multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP) to established cytotoxic substrates with or without one of the PIs atazanavir, lopinavir or ritonavir. Furthermore, to assess whether PIs serve as substrates, cell growth-inhibitory effects of these PIs were evaluated on cells overexpressing 1 of 11 ABC transporters and their parental counterparts. Atazanavir, lopinavir and ritonavir were highly effective in reversing resistance against established substrates in cells overexpressing MDR1 P-gp and MRP1, and, to a lesser extent, BCRP. Concurrently, however, PIs appeared to be relatively poor substrates for ABC transporters. Only a moderate level of resistance to atazanavir was observed in cells overexpressing MRP6 and MRP9 [resistance factor (RF): 2.0-2.6]. Cells overexpressing MDR1 P-gp, MRP3, MRP4 and MRP5 displayed low levels of resistance to atazanavir (RF: 1.3-1.7); MRP7- and MRP9-overexpressing cells to lopinavir (RF: 1.4-1.5); and MRP9-overexpressing cells to ritonavir (RF: 1.4). PIs can act as potent blockers of MDR1 P-gp, MRP1 and BCRP, but they are poor substrates for 11 ABC transporters. Consequently, ABC transporters are unlikely to play a major role in PI failure, but still may contribute to drug-specific adverse events and drug-drug interactions.

Resveratrol protects against protease inhibitor-induced reactive oxygen species production, reticulum stress and lipid raft perturbation

HIV protease inhibitors have been successfully used in highly active antiretroviral therapy of HIV-1 infection, but their benefits are compromised by a number of clinically important adverse side-effects. Several studies showed that protease inhibitors induce sarco/endoplasmic reticulum stress and overproduction of reactive oxygen species (ROS), but the hierarchy of these events was never established in protease inhibitor-treated cells. Our objective was to determine whether ROS production and sarco/endoplasmic reticulum stress were co-induced by protease inhibitors in human primary skeletal myotubes and whether antioxidant treatment with resveratrol could protect against protease inhibitor-induced cellular damages. We analyzed in cultures of primary human skeletal myotubes the effects of four protease inhibitors (atazanavir, lopinavir, ritonavir and saquinavir) on ROS production (by measuring the reduction of nitro blue tetrazolium and by fluorescence microscopy using CM-H2DCFDA), on sarco/endoplasmic reticulum stress induction (by immunofluorescence and electron microscopy) and on the expression and localization at lipid rafts of Caveolin 3 and Flotillin 1, two major components of lipid rafts (by immunoblotting and biochemical experiments). Cells were co-incubated with resveratrol to assess its protective effects. In myotubes, protease inhibitors increased ROS production, altered sarco/endoplasmic reticulum morphology, increased expression of C/EBP homologous protein, a sarco/endoplasmic reticulum stress marker, and decreased expression and localization at lipid rafts of Caveolin 3 and Flotillin 1. Importantly, these deleterious protease inhibitor effects were inhibited by the antioxidant resveratrol. Our findings demonstrate a protective effect of resveratrol against protease inhibitor-induced sarco/endoplasmic reticulum stress in human myotubes.

280 HIV protease inhibitor ritonavir increases heat sensitivity of renal cancer cells by inhibiting heat-induced NF-kappaB activation

280 HIV protease inhibitor ritonavir increases heat sensitivity of renal cancer cells by inhibiting heat-induced NF-kappaB activation

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.

84 COMBINATION THERAPY WITH 17-ALLYLAMINO-17-DEMETHOXYGELDANAMYCIN AND RITONAVIR INHIBITS EXPRESSION OF HEAT SHOCK FACTOR 1 IN RENAL CANCER CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 201084 COMBINATION THERAPY WITH 17-ALLYLAMINO-17-DEMETHOXYGELDANAMYCIN AND RITONAVIR INHIBITS EXPRESSION OF HEAT SHOCK FACTOR 1 IN RENAL CANCER CELLS Akinori Sato, Takako Asano, Junichi Asakuma, Keiichi Ito, Akio Horiguchi, Makoto Sumitomo, and Tomohiko Asano Akinori SatoAkinori Sato More articles by this author , Takako AsanoTakako Asano More articles by this author , Junichi AsakumaJunichi Asakuma More articles by this author , Keiichi ItoKeiichi Ito More articles by this author , Akio HoriguchiAkio Horiguchi More articles by this author , Makoto SumitomoMakoto Sumitomo More articles by this author , and Tomohiko AsanoTomohiko Asano More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.132AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Phase II clinical trials have shown 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein (HSP) 90, to be only a moderately effective anticancer agent because it generally increases the expression of HSPs. In the present study we combined 17-AAG with the protease inhibitor ritonavir and found that this combination therapy increases the effectiveness of 17-AAG against renal cancer cells by decreasing the expression of heat shock factor (HSF)-1, a transcription factor of HSPs. METHODS Renal cancer cells (769P, A498, ACHN, Caki-1) and renal proximal tubule epithelial cells (RPTEC) were treated with 17-AAG (0-1 μM) alone and in combination with ritonavir (0-50 μM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay, changes in cell cycle were evaluated by flow cytometry, and the expression of HSPs 27, 70, and 90; HSF-1; phosphorylated retinoblastoma protein (Rb); cyclin D1; cyclin-dependent kinase 4 (CDK4); histone deacetylases (HDACs) 1, 2, 3, and 6; acetylated histone; X-linked inhibitor of apoptosis (XIAP); and survivin was assessed by Western blot analysis. Apoptosis was assayed using flow cytometry and detecting active caspase 3 and cleaved poly(ADP-ribose) polymerase (PARP). RESULTS Combinations of 17-AAG and ritonavir inhibited the proliferation of renal cancer cells synergistically in a time- and dose-dependent manner and suppressed colony formation. They increased the sub-G1 fraction to as much as 64.2%, decreased the expression of survivin and XIAP, and increased active caspase 3 and cleaved PARP, thus leading to apoptosis. They inhibited the proliferation of RPTEC less than that of renal cancer cells and caused only small changes in the cell cycle of RPTEC. The combination caused Rb dephosphorylation and suppressed the expression of CDK4; cyclin D1; and HDACs 1, 2, and 6 in all the cancer cell lines and also induced histone acetylation in Caki-1 cells. Expression of HSPs 27, 70, and 90 was increased by 17-AAG alone but reduced by 17-AAG combined with ritonavir. Interestingly, the combination therapy decreased the expression of HSF-1, which may be one of the mechanisms of the combined effect. CONCLUSIONS Combination therapy with 17-AAG and ritonavir might be effective for treating renal cancer because ritonavir decreases the expression of HSF-1 and thereby suppresses the 17-AAG-induced increase of HSPs. Tokorozawa, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e35 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akinori Sato More articles by this author Takako Asano More articles by this author Junichi Asakuma More articles by this author Keiichi Ito More articles by this author Akio Horiguchi More articles by this author Makoto Sumitomo More articles by this author Tomohiko Asano More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...