Modulation of Multidrug Resistance Protein 1-mediated Transport Processes by the Antiviral Drug Ritonavir in Cultured Primary Astrocytes.

The Multidrug Resistance Protein 1 (Mrp1) is an ATP-dependent efflux transporter and a major facilitator of drug resistance in mammalian cells during cancer and HIV therapy. In brain, Mrp1-mediated GSH export from astrocytes is the first step in the supply of GSH precursors to neurons. To reveal potential mechanisms underlying the drug-induced modulation of Mrp1-mediated transport processes, we investigated the effects of the antiviral drug ritonavir on cultured rat primary astrocytes. Ritonavir strongly stimulated the Mrp1-mediated export of glutathione (GSH) by decreasing the Km value from 200 nmol/mg to 28 nmol/mg. In contrast, ritonavir decreased the export of the other Mrp1 substrates glutathione disulfide (GSSG) and bimane-glutathione. To give explanation for these apparently contradictory observations, we performed in silico docking analysis and molecular dynamics simulations using a homology model of rat Mrp1 to predict the binding modes of ritonavir, GSH and GSSG to Mrp1. The results suggest that ritonavir binds to the hydrophilic part of the bipartite binding site of Mrp1 and thereby differently affects the binding and transport of the Mrp1 substrates. These new insights into the modulation of Mrp1-mediated export processes by ritonavir provide a new model to better understand GSH-dependent detoxification processes in brain cells.

Interactions of anti-COVID-19 drug candidates with hepatic transporters may cause liver toxicity and affect pharmacokinetics.

Transporters in the human liver play a major role in the clearance of endo- and xenobiotics. Apical (canalicular) transporters extrude compounds to the bile, while basolateral hepatocyte transporters promote the uptake of, or expel, various compounds from/into the venous blood stream. In the present work we have examined the in vitro interactions of some key repurposed drugs advocated to treat COVID-19 (lopinavir, ritonavir, ivermectin, remdesivir and favipiravir), with the key drug transporters of hepatocytes. These transporters included ABCB11/BSEP, ABCC2/MRP2, and SLC47A1/MATE1 in the canalicular membrane, as well as ABCC3/MRP3, ABCC4/MRP4, SLC22A1/OCT1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, and SLC10A1/NTCP, residing in the basolateral membrane. Lopinavir and ritonavir in low micromolar concentrations inhibited BSEP and MATE1 exporters, as well as OATP1B1/1B3 uptake transporters. Ritonavir had a similar inhibitory pattern, also inhibiting OCT1. Remdesivir strongly inhibited MRP4, OATP1B1/1B3, MATE1 and OCT1. Favipiravir had no significant effect on any of these transporters. Since both general drug metabolism and drug-induced liver toxicity are strongly dependent on the functioning of these transporters, the various interactions reported here may have important clinical relevance in the drug treatment of this viral disease and the existing co-morbidities.

Network analysis and molecular mapping for SARS-CoV-2 to reveal drug targets and repurposing of clinically developed drugs.

Novel coronavirus (SARS-CoV-2), turned out to be a global pandemic with unstoppable morbidity and mortality rate. However, till date there is no effective treatment found against SARS-CoV-2. We report on the major in-depth molecular and docking analysis by using antiretroviral (Lopinavir and ritonavir), antimalarial (Hydroxychloroquine), antibiotics (Azithromycin), and dietary supplements (Vitamin C and E) to provide new insight into drug repurposing molecular events involved in SARS-CoV-2. We constructed three drug-target-pathways-disease networks to predict the targets and drugs interactions as well as important pathways involved in SARS-CoV-2. The results suggested that by using the combination of Lopinavir, Ritonavir along with Hydroxychloroquine and Vitamin C may turned out to be the effective line of treatment for SARS-CoV-2 as it shows the involvement of PARP-1, MAPK-8, EGFR, PRKCB, PTGS-2, and BCL-2. Gene ontology biological process analysis further confirmed multiple viral infection-related processes (P < 0.001), including viral life cycle, modulation by virus, C-C chemokine receptor activity, and platelet activation. KEGG pathway analysis involves multiple pathways (P < 0.05), including FoxO, GnRH, ErbB, Neurotrophin, Toll-like receptor, IL-17, TNF, Insulin, HIF-1, JAK-STAT, Estrogen, NF-kappa, Chemokine, VEGF, and Thyroid hormone signaling pathway in SARS-CoV-2. Docking study was carried out to predict the molecular mechanism Thus, the potential drug combinations could reduce viral infectivity, viral replication, and abnormal host inflammatory responses and may be useful for multi-target drugs against SARS-CoV-2.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.

Oral coadministration of elacridar and ritonavir enhances brain accumulation and oral availability of the novel ALK/ROS1 inhibitor lorlatinib.

Lorlatinib, a novel generation oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor with high membrane and blood-brain barrier permeability, recently received accelerated approval for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), and its further clinical development is ongoing. We previously found that the efflux transporter P-glycoprotein (MDR1/ABCB1) restricts lorlatinib brain accumulation and that the drug-metabolizing enzyme cytochrome P450-3A (CYP3A) limits its oral availability. Using genetically modified mouse models, we investigated the impact of targeted pharmacological inhibitors on lorlatinib pharmacokinetics and bioavailability. Upon oral administration of lorlatinib, the plasma AUC0-8h in CYP3A4-humanized mice was ∼1.8-fold lower than in wild-type and Cyp3a-/- mice. Oral coadministration of the CYP3A inhibitor ritonavir caused reversion to the AUC0-8h levels seen in wild-type and Cyp3a-/- mice, without altering the relative tissue distribution of lorlatinib. Moreover, simultaneous pharmacological inhibition of P-glycoprotein and CYP3A4 with oral elacridar and ritonavir in CYP3A4-humanized mice profoundly increased lorlatinib brain concentrations, but not its oral availability or other relative tissue distribution. Oral lorlatinib pharmacokinetics was not significantly affected by absence of the multispecific Oatp1a/1b drug uptake transporters. The absolute oral bioavailability of lorlatinib over 8 h in wild-type, Cyp3a-/-, and CYP3A4-humanized mice was 81.6%, 72.9%, and 58.5%, respectively. Lorlatinib thus has good oral bioavailability, which is markedly restricted by human CYP3A4 but not by mouse Cyp3a. Pharmacological inhibition of CYP3A4 reversed these effects, and simultaneous P-gp inhibition with elacridar boosted absolute brain levels of lorlatinib by 16-fold without obvious toxicity. These insights may help to optimize the clinical application of lorlatinib.

Nelfinavir and Ritonavir Kill Bladder Cancer Cells Synergistically by Inducing Endoplasmic Reticulum Stress.

The human immunodeficiency virus (HIV) protease inhibitor nelfinavir acts against malignancies by inducing endoplasmic reticulum (ER) stress. The HIV protease inhibitor ritonavir, on the other hand, not only induces ER stress but also inhibits P-glycoprotein's pump activity and thereby enhances the effects of its substrate drugs. We therefore postulated that ritonavir in combination with nelfinavir would kill bladder cancer cells effectively by inducing ER stress cooperatively and also enhancing nelfinavir's effect. Nelfinavir was shown to be a P-glycoprotein substrate, and the combination of nelfinavir and ritonavir inhibited bladder cancer cell growth synergistically. It also suppressed colony formation significantly. The combination significantly increased the number of cells in the sub-G1 fraction and also the number of annexin V+ cells, confirming robust apoptosis induction. The combination induced ER stress synergistically, as evidenced by the increased expression of glucose-regulated protein 78, ER-resident protein 44, and endoplasmic oxidoreductin-1-like protein. It also increased the expression of the mammalian target of rapamycin (mTOR) inhibitor AMP-activated protein kinase and caused dephosphorylation of S6 ribosomal protein, demonstrating that the combination also inhibited the mTOR pathway. We also found that the combination enhanced histone acetylation synergistically by decreasing the expression of HDACs 1, 3, and 6.

Influence of Ethanol on Darunavir Hepatic Clearance and Intracellular PK/PD in HIV-Infected Monocytes, and CYP3A4-Darunavir Interactions Using Inhibition and in Silico Binding Studies.

PURPOSE: Although the prevalence of alcohol consumption is higher in HIV+ people than general public, limited information is available on how alcohol affects the metabolism and bioavailability of darunavir (DRV). METHODS: DRV was quantified by using LC-MS/MS method. All in vitro experiments were performed using human liver microsomes and HIV-infected monocytic cells. CYP3A4 and DRV/Ritonavir (RTV) docking was performed using GOLD suite 5.8. RESULTS: Ethanol (20 mM) significantly decreased apparent half-life and increased degradation rate constant of RTV-boosted DRV but not for DRV alone. Similarly, ethanol exposure increased hepatic intrinsic clearance for RTV-boosted DRV with no significant influence on DRV alone. Ethanol showed a limited influence on intracellular total DRV exposure in the presence of RTV without altering maximum concentration (Cmax) values in HIV-infected monocytic cells. Ethanol alone elevated HIV replication but this effect was nullified with the addition of DRV or DRV + RTV. Additionally, inhibitory potency of DRV was significantly reduced in the presence of ethanol. Our docking results projected that ethanol increases the average distance between DRV and CYP3A4 heme, and alter the orientation of DRV-CYP3A4 binding. CONCLUSIONS: Collectively these findings suggest that DRV metabolism is primarily influenced by ethanol in the liver, but has minor effect in HIV-residing monocytes.

Potential Drug Interactions Mediated by Renal Organic Anion Transporter OATP4C1.

Organic anion-transporting polypeptide 4C1 (OATP4C1) is an organic anion transporter expressed in the basolateral membrane of the renal proximal tubules. It plays a major role in the urinary excretion of both exogenous drugs and endogenous compounds. Our previous studies have indicated the importance of OATP4C1 in pathologic and physiologic conditions; however, the majority of its pharmacologic characteristics remained unclear. Therefore, to provide essential information for clinical drug therapy decisions and drug development, we clarified drug interactions mediated by OATP4C1. To elucidate potential drug interactions via OATP4C1, we screened 53 representative drugs commonly used in clinical settings. Next, we evaluated the IC50 values of drugs that inhibited OATP4C1 by more than 50%. To apply our results to clinical settings, we calculated the drug-drug interaction (DDI) indices. The screening analysis using an OATP4C1-expressing cell system demonstrated that 22 out of 53 therapeutic drugs inhibited OATP4C1-mediated triiodothyronine transport. In particular, OATP4C1-mediated transport was strongly inhibited by 10 drugs. The IC50 values of 10 drugs-nicardipine, spironolactone, fluvastatin, crizotinib, levofloxacin, clarithromycin, ritonavir, saquinavir, quinidine, and verapamil-obtained in this study were 51, 53, 41, 24, 420, 200, 8.5, 4.3, 100, and 110 µM, respectively. The IC50 values of these drugs were higher than the plasma concentrations obtained in clinical practice. However, ritonavir showed the highest DDI index (1.9) for OATP4C1, suggesting that it may strongly influence this transporter and thus cause drug interactions seen in clinical settings. Our finding gives new insight into the role of OATP4C1 in clinical DDIs.

Mechanisms and Predictions of Drug-Drug Interactions of the Hepatitis C Virus Three Direct-Acting Antiviral Regimen: Paritaprevir/Ritonavir, Ombitasvir, and Dasabuvir.

To assess drug-drug interaction (DDI) potential for the three direct-acting antiviral (3D) regimen of ombitasvir, dasabuvir, and paritaprevir, in vitro studies profiled drug-metabolizing enzyme and transporter interactions. Using mechanistic static and dynamic models, DDI potential was predicted for CYP3A, CYP2C8, UDP-glucuronosyltransferase (UGT) 1A1, organic anion-transporting polypeptide (OATP) 1B1/1B3, breast cancer resistance protein (BCRP), and P-glycoprotein (P-gp). Perpetrator static model DDI predictions for metabolizing enzymes were within 2-fold of the clinical observations, but additional physiologically based pharmacokinetic modeling was necessary to achieve the same for drug transporters. When perpetrator interactions were assessed, ritonavir was responsible for the strong increase in exposure of sensitive CYP3A substrates, whereas paritaprevir (an OATP1B1/1B3 inhibitor) greatly increased the exposure of sensitive OATP1B1/1B3 substrates. The 3D regimen drugs are UGT1A1 inhibitors and are predicted to moderately increase plasma exposure of sensitive UGT1A1 substrates. Paritaprevir, ritonavir, and dasabuvir are BCRP inhibitors. Victim DDI predictions were qualitatively in line with the clinical observations. Plasma exposures of the 3D regimen were reduced by strong CYP3A inducers (paritaprevir and ritonavir; major CYP3A substrates) but were not affected by strong CYP3A4 inhibitors, since ritonavir (a CYP3A inhibitor) is already present in the regimen. Strong CYP2C8 inhibitors increased plasma exposure of dasabuvir (a major CYP2C8 substrate), OATP1B1/1B3 inhibitors increased plasma exposure of paritaprevir (an OATP1B1/1B3 substrate), and P-gp or BCRP inhibitors (all compounds are substrates of P-gp and/or BCRP) increased plasma exposure of the 3D regimen. Overall, the comprehensive mechanistic assessment of compound disposition along with mechanistic and PBPK approaches to predict victim and perpetrator DDI liability may enable better clinical management of nonstudied drug combinations with the 3D regimen.

Interaction between HIV protease inhibitors (PIs) and hepatic transporters in sandwich cultured human hepatocytes: implication for PI-based DDIs.

Although HIV protease inhibitors (PIs) produce profound metabolic interactions through inactivation/inhibition of CYP3A enzymes, their role as victims of transporter-based drug-drug interactions (DDIs) is less well understood. Therefore, this study investigated if the PIs, nelfinavir (NFV), ritonavir (RTV), lopinavir (LPV) or amprenavir (APV) were transported into sandwich-cultured human hepatocytes (SCHH), and whether OATPs contributed to this transport. The findings showed that, except for (3) H-APV, no significant decrease in the total hepatocyte accumulation of the (3) H-PIs was detected in the presence of the corresponding unlabeled PI, indicating that the uptake of the other PIs was not mediated. Further, hepatocyte biliary efflux studies using (3) H-APV and unlabeled APV confirmed this decrease to be due to inhibition of sinusoidal influx transporter(s) and not the canalicular efflux transporters. Moreover, this sinusoidal transport of APV was not OATP-mediated. The results indicate that the hepatic uptake of NFV, RTV or LPV was primarily mediated by passive diffusion. The hepatic uptake of APV was mediated by an unidentified sinusoidal transporter(s). Therefore, NFV, RTV or LPV will not be victims of DDIs involving inhibition of hepatic influx transporters; however, the disposition of APV may be affected if its sinusoidal transport is inhibited.

Efflux transporters- and cytochrome P-450-mediated interactions between drugs of abuse and antiretrovirals.

Multidrug regimens and corresponding drug interactions cause many adverse reactions and treatment failures. Drug efflux transporters: P-gp, MRP, BCRP in conjunction with metabolizing enzymes (CYPs) are major factors in such interactions. Most effective combination antiretrovirals (ARV) therapy includes a PI or a NNRTI or two NRTI. Coadministration of such ARV may induce efflux transporters and/or CYP3A4 resulting in sub-therapeutic blood levels and therapeutic failure due to reduced absorption and/or increased metabolism. A similar prognosis is true for ARV-compounds and drugs of abuse combinations. Morphine and nicotine enhance CYP3A4 and MDR1 expression in vitro. A 2.5 fold rise of cortisol metabolite was evident in smokers relative to nonsmokers. Altered functions of efflux transporters and CYPs in response to ARV and drugs of abuse may result in altered drug absorption and metabolism. Appropriate in vitro models can be employed to predict such interactions. Influence of genetic polymorphism, SNP and inter-individual variation in drug response has been discussed. Complexity underlying the relationship between efflux transporters and CYP makes it difficult to predict the outcome of HAART as such, particularly when HIV patients taking drugs of abuse do not adhere to HAART regimens. HIV(+) pregnant women on HAART medications, indulging in drugs of abuse, may develop higher viral load due to such interactions and lead to increase in mother to child transmission of HIV. A multidisciplinary approach with clear understanding of mechanism of interactions may allow proper selection of regimens so that desired therapeutic outcome of HAART can be reached without any side effects.

Analyses of nanoformulated antiretroviral drug charge, size, shape and content for uptake, drug release and antiviral activities in human monocyte-derived macrophages.

Long-term antiretroviral therapy (ART) for human immunodeficiency virus type one (HIV-1) infection shows limitations in pharmacokinetics and biodistribution while inducing metabolic and cytotoxic aberrations. In turn, ART commonly requires complex dosing schedules and leads to the emergence of viral resistance and treatment failures. We posit that the development of nanoformulated ART could preclude such limitations and affect improved clinical outcomes. To this end, we wet-milled 20 nanoparticle formulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz, collectively referred to as "nanoART," then assessed their performance using a range of physicochemical and biological tests. These tests were based on cell-nanoparticle interactions using monocyte-derived macrophages and their abilities to uptake and release nanoformulated drugs and affect viral replication. We demonstrate that physical characteristics such as particle size, surfactant coating, surface charge, and most importantly shape are predictors of cell uptake and antiretroviral efficacy. These studies bring this line of research a step closer to developing nanoART that can be used in the clinic to affect the course of HIV-1 infection.

Metabolic and neurologic consequences of chronic lopinavir/ritonavir administration to C57BL/6 mice.

It is well established that HIV antiretroviral drugs, particularly protease inhibitors, frequently elicit a metabolic syndrome that may include hyperlipidemia, lipodystrophy, and insulin resistance. Metabolic dysfunction in non-HIV-infected subjects has been repeatedly associated with cognitive impairment in epidemiological and experimental studies, but it is not yet understood if antiretroviral therapy-induced metabolic syndrome might contribute to HIV-associated neurologic decline. To determine if protease inhibitor-induced metabolic dysfunction in mice is accompanied by adverse neurologic effects, C57BL/6 mice were given combined lopinavir/ritonavir (50/12.5-200/50 mg/kg) daily for 3 weeks. Data show that lopinavir/ritonavir administration caused significant metabolic derangement, including alterations in body weight and fat mass, as well as dose-dependent patterns of hyperlipidemia, hypoadiponectinemia, hypoleptinemia, and hyperinsulinemia. Evaluation of neurologic function revealed that even the lowest dose of lopinavir/ritonavir caused significant cognitive impairment assessed in multi-unit T-maze, but did not affect motor functions assessed as rotarod performance. Collectively, our results indicate that repeated lopinavir/ritonavir administration produces cognitive as well as metabolic impairments, and suggest that the development of selective aspects of metabolic syndrome in HIV patients could contribute to HIV-associated neurocognitive disorders.

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.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids.

HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.

Nucleoside reverse transcriptase inhibitors prevent HIV protease inhibitor-induced atherosclerosis by ubiquitination and degradation of protein kinase C.

HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Calpha (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.

Interaction of anti-HIV protease inhibitors with the multidrug transporter P-glycoprotein (P-gp) in human cultured cells.

The anti-HIV protease inhibitors represent a new class of agents for treatment of HIV infection. Saquinavir, ritonavir, indinavir, and nelfinavir are the first drugs approved in this class and significantly reduce HIV RNA copy number with minimal adverse effects. They are all substrates of cytochrome P450 3A4, and are incompletely bioavailable. The drug transporting protein, P-glycoprotein (P-gp), which is highly expressed in the intestinal mucosa, could be responsible for the low oral bioavailability of these and other drugs which are substrates for this transporter. To determine whether these protease inhibitors are modulators of P-gp, we studied them in cell lines which do and do not express P-gp. Saquinavir, ritonavir and nelfinavir significantly inhibited the efflux of [3H]paclitaxel and [3H]vinblastine in P-gp-positive cells, resulting in an increase in intracellular accumulation of these drugs. However, similar concentrations of indinavir did not affect the accumulation of these anticancer agents. In photoaffinity labeling studies, saquinavir and ritonavir displaced [3H]azidopine, a substrate for P-gp, in a dose-dependent manner. These data suggest that saquinavir, ritonavir, and nelfinavir are inhibitors and possibly substrates of P-gp. Because saquinavir has a low bioavailability, its interaction with P-gp may be involved in limiting its absorption.

Active apical secretory efflux of the HIV protease inhibitors saquinavir and ritonavir in Caco-2 cell monolayers.

PURPOSE: To investigate in vitro the mechanisms involved in the gastro-intestinal absorption of the HIV protease inhibitor, saquinavir mesylate (Invirase), whose oral bioavailability is low, variable, and significantly increased by co-administration with ritonavir, also an HIV protease inhibitor but with higher oral bioavailability. METHODS: Confluent epithelial layers of human Caco-2 cells mimicking the intestinal barrier. RESULTS: Both saquinavir and ritonavir showed polarized transport through Caco-2 cell monolayers in the basolateral to apical direction (secretory pathway), exceeding apical to basolateral transport (absorptive pathway) by factors of 50-70 and 15-25, respectively. Active efflux was temperature dependent, saturable and inhibited by verapamil and cyclosporin A. Saquinavir and ritonavir decreased each other's secretory permeability and hence elevated their net transport by the absorptive pathway. CONCLUSIONS: Saquinavir and ritonavir are both substrates for an efflux mechanism in the gut, most likely P-glycoprotein, which acts as a counter-transporter for both drugs. Together with sensitivity to gutwall metabolism by cytochrome P-450 3A, this may partially account for the low and variable oral bioavailability of saquinavir in clinical studies and for its increased bioavailability after co-administration with ritonavir.