Immunodeficiency Virus Protease Inhibitor Ritonavir Research Articles

Functionalized graphene oxide tablets for sample preparation of drugs in biological fluids: Extraction of ritonavir, a HIV protease inhibitor, from human saliva and plasma using LC–MS/MS

In this work, graphene oxide based tablets (GO-Tabs) were prepared by applying functionalized GO as a thin layer on a polyethylene substrate. The graphene oxide was functionalized with amine groups (-NH2 ) by Poly (ethylene glycol) bis(3-aminopropyl) terminated (GO-NH2 -PEG-NH2 ). The functionalized GO-Tabs were used for extraction of ritonavir in human saliva samples. The analysis of ritonavir in plasma and saliva samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gradient LC system with MS/MS in the positive ion mode (ESI+) was used. The transitions m/z 721→269.0 and m/z 614→421 were used for the ritonavir and the internal standard indinavir, respectively. In this study, the determination of the HIV protease inhibitor ritonavir in human saliva samples was carried out using functionalized GO-Tab and LC-MS/MS and the method was validated. Standard calibration curve for plasma and saliva samples was constructed from 5.0 to 2000 nmol L-1 . The limit of detection was 0.1 nmol L-1 and the limit of quantification was 5.0 nmol L-1 in both plasma and saliva matrices. The intra- and inter-assay precision values were found to be between 1.5 and 5.8%, and the accuracy values ranged from 88.0% to 108% utilizing saliva and plasma samples. The extraction recovery was over 80%, and the presented functionalized GO-Tabs could be re-used for more than ten extractions without deterioration in recovery.

MP18-15 RITONAVIR AND OPROZOMIB INHIBIT RENAL CANCER GROWTH BY INDUCING ENDOPLASMIC RETICULUM STRESS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (MP18)1 Apr 2020MP18-15 RITONAVIR AND OPROZOMIB INHIBIT RENAL CANCER GROWTH BY INDUCING ENDOPLASMIC RETICULUM STRESS Takako Asano*, Kazuki Okubo, and Akinori Sato Takako Asano*Takako Asano* More articles by this author , Kazuki OkuboKazuki Okubo More articles by this author , and Akinori SatoAkinori Sato More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000843.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. The human immunodeficiency virus protease inhibitor ritonavir inhibits molecular chaperones and increases the amount of unfolded proteins in cells. We postulated that combining ritonavir with the novel proteasome inhibitor oprozomib would kill renal cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby inducing ER stress triggering apoptosis. METHODS: The viability and clonogenicity of renal cancer cells (769-P, Caki-1, Caki-2) treated with ritonavir (20–40 μM) and/or oprozomib (50–200 nM) were assessed by MTS assay and colony formation assay. Flow cytometry was used to detect and quantify apoptosis. The induction of ER stress and the expression of acetylated histone, ubiquitinated proteins, AMP-activated protein kinase (AMPK), S6 ribosomal protein (S6), and histone deacetylases (HDACs) were assessed using western blot analysis. Combination indexes were calculated using the Chou-Talalay method. RESULTS: The combination of ritonavir and oprozomib inhibited renal cancer growth synergistically (combination indexes < 1) and suppressed colony formation significantly (p < 0.05). The combination caused renal cancer cell apoptosis synergistically: either 40 μM ritonavir or 50 nM oprozomib alone induced only moderate apoptosis, but in combination they induced drastic apoptosis (up to 97.6% annexin V-positive cells). Mechanistically, the combination caused ubiquitinated unfolded proteins to accumulate and thereby induced ER stress synergistically. This ER stress induction was essential to the combination’s cytotoxic action because inhibition of unfolded protein accumulation by the protein synthesis inhibitor cycloheximide markedly attenuated the combination-induced apoptosis. Furthermore, the combination-induced ER stress caused dephosphorylation of S6 by increasing the expression of mammalian target of rapamycin (mTOR) inhibitor AMPK, showing that the combination also inhibited the mTOR pathway. Interestingly, we also found that the combination increased histone acetylation synergistically by decreasing the expression of HDACs 1, 3, and 6. CONCLUSIONS: The combination of ritonavir and oprozomib inhibits renal cancer growth by synergistically inducing ER stress. Histone acetylation is also an important mechanism of its cytotoxicity. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e241-e241 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takako Asano* More articles by this author Kazuki Okubo More articles by this author Akinori Sato More articles by this author Expand All Advertisement PDF downloadLoading ...

Ritonavir Interacts With Belinostat to Cause Endoplasmic Reticulum Stress and Histone Acetylation in Renal Cancer Cells.

The histone deacetylase (HDAC) inhibitor belinostat increases the amount of unfolded proteins in cells by promoting the acetylation of heat shock protein 90 (HSP90), thereby disrupting its chaperone function. The human immunodeficiency virus protease inhibitor ritonavir, on the other hand, not only increases unfolded proteins by suppressing HSP90 but also acts as a proteasome inhibitor. We thought that belinostat and ritonavir together would induce endoplasmic reticulum (ER) stress and kill renal cancer cells effectively. The combination of belinostat and ritonavir induced drastic apoptosis and inhibited the growth of renal cancer cells synergistically. Mechanistically, the combination caused ER stress (evidenced by the increased expression of the ER stress markers) and also enhanced histone acetylation by decreasing the expression of HDACs. To our knowledge, this is the first study that showed a beneficial combined effect of belinostat and ritonavir in renal cancer cells, providing a framework for testing the combination in renal cancer patients.

The human immunodeficiency virus protease inhibitor ritonavir is potentially active against urological malignancies.

The human immunodeficiency virus protease inhibitor ritonavir has recently been shown to have antineoplastic activity, and its use in urological malignancies is under investigation with an eye toward drug repositioning. Ritonavir is thought to exert its antineoplastic activity by inhibiting multiple signaling pathways, including the Akt and nuclear factor-kappaB pathways. It can increase the amount of unfolded proteins in the cell by inhibiting both the proteasome and heat shock protein 90. Combinations of ritonavir with agents that increase the amount of unfolded proteins, such as proteasome inhibitors, histone deacetylase inhibitors, or heat shock protein 90 inhibitors, therefore, induce endoplasmic reticulum stress cooperatively and thereby kill cancer cells effectively. Ritonavir is also a potent cytochrome P450 3A4 and P-glycoprotein inhibitor, increasing the intracellular concentration of combined drugs by inhibiting their degradation and efflux from cancer cells and thereby enhancing their antineoplastic activity. Furthermore, riotnavir’s antineoplastic activity includes modulation of immune system activity. Therapies using ritonavir are thus an attractive new approach to cancer treatment and, due to their novel mechanisms of action, are expected to be effective against malignancies that are refractory to current treatment strategies. Further investigations using ritonavir are expected to find new uses for clinically available drugs in the treatment of urological malignancies as well as many other types of cancer.

Ritonavir-boosted indinavir but not lopinavir inhibits erythrocytic stage Plasmodium knowlesi malaria in rhesus macaques.

The inhibitive activities of the human immunodeficiency virus protease inhibitors ritonavir (RTV) boosted indinavir (IDV) and RTV boosted lopinavir (LPV) for erythrocytic stage malaria were evaluated in rhesus macaques. The IDV/RTV regimen effectively inhibits the replication of Plasmodium knowlesi with clinically relevant doses, whereas the LPV/RTV regimen did not show activity against plasmodium infection.

Investigating and targeting chronic lymphocytic leukemia metabolism with the human immunodeficiency virus protease inhibitor ritonavir and metformin

Chronic lymphocytic leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity. Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal. Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with human immunodeficiency (HIV) protease inhibitor ritonavir, which inhibits GLUT4, elicits toxicity similar to that elicited upon glucose deprivation. CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for the treatment of diabetes in patients with HIV, demonstrating the tolerance to this combination in humans. Our studies strongly substantiate further investigation of Food and Drug Administration approved ritonavir and metformin for CLL.

Ritonavir acts synergistically with panobinostat to enhance histone acetylation and inhibit renal cancer growth.

There is currently no curative treatment for advanced renal cancer. Enhancing histone acetylation is a promising epigenetic-based therapy for cancer; however, in solid tumors, the efficacy of histone deacetylase (HDAC) inhibitors alone is limited. The human immunodeficiency virus protease inhibitor ritonavir is also a CYP3A4 inhibitor and we hypothesized that combining ritonavir with the HDAC inhibitor panobinostat, one of the substrates of CYP3A4, may effectively eliminate renal cancer cells by enhancing the activity of panobinostat. The combination of ritonavir and panobinostat synergistically inhibited cancer cell growth and cancer cell colony formation, while only slightly inhibiting the growth of renal proximal tubule epithelial cells. This combination significantly induced apoptosis in cancer cells and this apoptosis was considered to be caspase-dependent, since the pan-caspase inhibitor Z-VAD-FMK reduced the number of Annexin V-positive cells. In murine subcutaneous xenograft models using Caki-1 cells, a 10-day treatment with the combination of ritonavir and panobinostat significantly inhibited tumor growth. Panobinostat alone increased histone acetylation in a dose-dependent manner and the co-administration of ritonavir synergistically enhanced this acetylation. Furthermore, this combination inhibited the expression of HDACs, which may also play a role in the enhancement of histone acetylation. Thus, the present study may provide a basis for testing the combination of ritonavir and panobinostat for patients with advanced renal cancer.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

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.

Roles and Mechanisms of Human Immunodeficiency Virus Protease Inhibitor Ritonavir and Other Anti-Human Immunodeficiency Virus Drugs in Endothelial Dysfunction of Porcine Pulmonary Arteries and Human Pulmonary Artery Endothelial Cells

The objective of this study was to determine the effects of highly active antiretroviral therapy (HAART) drugs on pulmonary endothelial function. Porcine pulmonary arteries or human pulmonary arterial endothelial cells (HPAECs) were incubated with eight HAART drugs [ritonavir, indinavir, lopinavir, zidovudine (AZT), abacavir, stavudine, didanosine (ddI), and lamivudine] individually or in combination [three HAART drugs (3-plex; indinavir, stavudine, and ddI)] at their clinical plasma concentrations for 24 hours. Endothelium-dependent vasorelaxation in response to bradykinin was reduced significantly by the ritonavir in a concentration-dependent manner. Five other HAART drugs (indinavir, lamivudine, abacavir, AZT, and ddI) and the 3-plex significantly also impaired endothelium-dependent vasorelaxation in response to bradykinin. Five HAART drugs (ritonavir, indinavir, lamivudine, abacavir, and AZT) significantly decreased endothelial nitric oxide synthase (eNOS) expression and increased superoxide anion levels in both vessels and HPAECs. Furthermore, both ritonavir and AZT substantially activated ERK2 in HPAECs. Additionally, the antioxidants ginsenoside Rb1 and ginkgolide A effectively reversed HAART drug-induced vasomotor dysfunction and eNOS down-regulation. Inhibition of ERK1/2 also partially blocked ritonavir- and AZT-induced down-regulation of eNOS and vasomotor dysfunction. Thus, HAART drugs significantly impair endothelial functions of porcine pulmonary arteries and HPAECs, which may be mediated by eNOS down-regulation, oxidative stress, and ERK1/2 activation. These findings suggest that HAART drugs may contribute to the high incidence of pulmonary artery hypertension in human immunodeficiency virus-infected patients.

WNT/β-Catenin Signaling Is Involved in Regulation of Osteoclast Differentiation by Human Immunodeficiency Virus Protease Inhibitor Ritonavir: Relationship to Human Immunodeficiency Virus-Linked Bone Mineral Loss

Untreated human immunodeficiency virus (HIV) infection is accompanied by reduced bone mineral density, which appears to be exacerbated by certain HIV protease inhibitors (PIs). The mechanisms leading to this apparent paradox, however, remain unclear. We have previously shown that, the HIV envelope glycoprotein gp120 used at levels similar those in plasmas of untreated HIV(+) patients, induced expression of the osteoclast (OC) differentiation factor RANKL in CD4+ T cells. In addition, the HIV PI ritonavir abrogated the interferon-gamma-mediated degradation of the RANKL nuclear adapter protein TRAF6, a physiological block to RANKL activity. Here, using oligonucleotide microarrays and quantitative polymerase chain reaction, we explored potential upstream mechanisms for these effects. Ritonavir, but not the HIV PIs indinavir or nelfinavir, up-regulated the production of transcripts for OC growth factors and the non-canonical Wnt Proteins 5B and 7B as well as activated promoters of nuclear factor-kappaB signaling, but suppressed genes involved in canonical Wnt signaling. Similarly, ritonavir blocked the cytoplasmic to nuclear translocation of beta-catenin, the molecular node of the Wnt signaling pathway, in association with enhanced beta-catenin ubiquitination. Exposure of OC precursors to LiCl, an inhibitor of the canonical Wnt antagonist GSK-3beta, suppressed OC differentiation, as did adenovirus-mediated overexpression of beta-catenin. These data identify, for the first time, a biologically relevant role for Wnt signaling via beta-catenin in isolated OC precursors and the modulation of Wnt signaling by ritonavir. The reversal of these ritonavir-mediated changes by interferon-gamma provides a model for possible intervention in this metabolic complication of HIV therapy.

Tetracycline-Inducible Expression of Individual Secreted Aspartic Proteases in Candida albicans Allows Isoenzyme-Specific Inhibitor Screening

The yeast Candida albicans possesses a gene family that encodes secreted aspartic proteases (Saps), which are important for the virulence of this human fungal pathogen. Inhibitors of the Saps could therefore be used as novel antimycotic agents for the treatment of C. albicans infections. In the present study, we established a bioassay which allows testing of the activity of potential protease inhibitors against specific Sap isoenzymes by their ability to inhibit protease-dependent growth of C. albicans. In a medium containing bovine serum albumin (BSA) as the sole source of nitrogen, C. albicans specifically expresses the Sap2p isoenzyme, which degrades the BSA and thereby enables the fungus to grow. As the other SAP genes are not significantly expressed under these conditions, mutants lacking SAP2 are unable to utilize BSA as a nitrogen source and cannot grow in such a medium. To investigate whether forced expression of SAP genes other than SAP2 would also allow growth on BSA, we constructed a set of strains expressing each of the 10 SAP genes from a tetracycline-inducible promoter in a sap2Delta mutant background. Expression of Sap1p, Sap2p, Sap3p, Sap4p, Sap5p, Sap6p, Sap8p, and a C-terminally truncated, secreted Sap9p restored the growth of the sap2Delta mutant with different efficiencies. This set of strains was then used to test the activities of various aspartic protease inhibitors against specific Sap isoenzymes by monitoring growth on BSA in the presence of the inhibitors. While pepstatin blocked the activity of all of the Saps tested, the human immunodeficiency virus protease inhibitors ritonavir and saquinavir inhibited growth of the strains expressing Sap1p to Sap3p and Sap1p, respectively, but not that of strains expressing other Saps. Therefore, the strain set can be used to test the activity of new protease inhibitors against individual C. albicans Sap isoenzymes by their ability to block the growth of the pathogen.

Human Immunodeficiency Virus Protease Inhibitor Ritonavir Inhibits Cholesterol Efflux from Human Macrophage-Derived Foam Cells

Clinical use of human immunodeficiency virus protease inhibitors such as ritonavir may be associated with cardiovascular disease. The objective of this study was to determine the effects and molecular mechanisms of ritonavir on cholesterol efflux from human macrophage-derived foam cells, which is a critical factor of atherogenesis. Human THP-1 monocytes and peripheral blood mononuclear cells were preincubated with acetylated low-density lipoprotein and [(3)H]cholesterol to form foam cells, which were then treated with apolipoprotein A-I for cholesterol efflux assay. A clinically relevant concentration of ritonavir (15 mumol/L) significantly reduced cholesterol efflux from THP-1 and peripheral blood mononuclear cells to apolipoprotein A-I by 30 and 29%, respectively, as compared with controls. In addition, ritonavir significantly decreased the expression of scavenger receptor B1 and caveolin-1, whereas it significantly increased superoxide anion production and activated extracellular signal-regulated kinase (ERK) 1/2 in macrophages. Mitochondrial membrane potential was significantly reduced, whereas NADPH oxidase subunits were increased in ritonavir-treated macrophages. Consequently, the antioxidant seleno-l-methionine, the specific ERK1/2 inhibitor PD98059, or infection of a recombinant adenovirus encoding the dominant-negative form of ERK2 effectively blocked ritonavir-induced decrease of cholesterol efflux. Therefore, human immunodeficiency virus protease inhibitor ritonavir significantly inhibits cholesterol efflux from macrophages, which may be mediated by mitochondrial dysfunction, oxidative stress, ERK1/2 activation, and down-regulation of scavenger receptor B1 and caveolin-1.

Adiponectin ameliorates dyslipidemia induced by the human immunodeficiency virus protease inhibitor ritonavir in mice.

Although the clinical application of HIV protease inhibitors (PIs) has markedly reduced HIV-related morbidity and mortality, it is now recognized that PI-based therapy often causes serious metabolic disorders, including hyperlipidemia and premature atherosclerosis. The etiology of these adverse effects remains obscure. Here, we demonstrate that deficiency of the fat-derived hormone adiponectin might play a role. The steady-state mRNA levels of the adiponectin gene and secretion of this protein from 3T3-L1 adipocytes are significantly decreased after treatment with several PIs (indinavir, nelfinavir, and ritonavir), with ritonavir having the greatest effect. Intragastric administration of ritonavir into mice decreases plasma concentrations of adiponectin and concurrently increases the plasma levels of triglyceride, free fatty acids, and cholesterol. Adiponectin replacement therapy markedly ameliorates ritonavir-induced elevations of triglyceride and free fatty acids. These beneficial effects of adiponectin are partly due to its ability to decrease ritonavir-induced synthesis of fatty acids and triglyceride, and to increase fatty acid combustion in the liver tissue. In contrast, adiponectin has little effect on ritonavir-induced hypercholesterolemia and hepatic cholesterol synthesis. These results suggest that hypoadiponectinemia is partly responsible for the metabolic disorders induced by HIV PIs, and adiponectin or its agonists might be useful for the treatment of these disorders.