Protease Inhibitors Ritonavir Research Articles

Evolution of Human Immunodeficiency Virus Type 1 Protease Genotypes and Phenotypes In Vivo under Selective Pressure of the Protease Inhibitor Ritonavir

We examined the population dynamics of human immunodeficiency virus type 1 pro variants during the evolution of resistance to the protease inhibitor ritonavir (RTV) in vivo. pro variants were followed in subjects who had added RTV to their previously failed reverse transcriptase inhibitor therapy using a heteroduplex tracking assay designed to detect common resistance-associated mutations. In most cases the initial variant appeared rapidly within 2 to 3 months followed by one or more subsequent population turnovers. Some of the subsequent transitions between variants were rapid, and some were prolonged with the coexistence of multiple variants. In several cases variants without resistance mutations persisted despite the emergence of new variants with an increasing number of resistance-associated mutations. Based on the rate of turnover of pro variants in the RTV-treated subjects we estimated that the mean fitness of newly emerging variants was increased 1.2-fold (range, 1.02 to 1.8) relative to their predecessors. A subset of pro genes was introduced into infectious molecular clones. The corresponding viruses displayed impaired replication capacity and reduced susceptibility to RTV. A subset of these clones also showed increased susceptibility to two nonnucleoside reverse transcriptase inhibitors and the protease inhibitor saquinavir. Finally, a significant correlation between the reduced replication capacity and reduced processing at the gag NC-p1 processing site was noted. Our results reveal a complexity of patterns in the evolution of resistance to a protease inhibitor. In addition, these results suggest that selection for resistance to one protease inhibitor can have pleiotropic effects that can affect fitness and susceptibility to other drugs.

The HIV Protease Inhibitors Saquinavir, Ritonavir, and Nelfinavir Induce Apoptosis and Decrease Barrier Function in Human Intestinal Epithelial Cells

Diarrhoea is a frequent adverse effect of HIV protease inhibitors (PIs) which may be due to intestinal barrier disruption. We investigated whether tight junction dysregulation, apoptosis or necrosis are responsible for this epithelial damage. Saquinavir, nelfinavir, and ritonavir were added to the mucosal or serosal side of HT-29/B6 colon cell monolayers. Transepithelial resistance was monitored for 72 h to assess epithelial barrier function. Apoptosis and necrosis were investigated by light and electron microscopy and quantified by nucleosome ELISA and LDH measurement, respectively. Tight junction components were analysed by Western blots of occludin and zonula occludens. Apoptosis induction in normal human intestinal epithelium was examined by measurement of poly(ADP-ribose) polymerase (PARP) cleavage in Western blots of mucosal tissue explants cultured with PIs for 24 h. HIV PIs decreased transepithelial resistance by more than 44% in HT-29/B6 monolayers. Histology revealed massive apoptotic body formation but no evidence for necrosis after PI treatment. Correspondingly, LDH release was lower than 0.2%/h of total LDH, independent of PI treatment, and nucleosomes were increased up to 22-fold after drug treatment versus control. Occludin and zonula occludens-1 expression in the membrane were not diminished. PARP cleavage increased in normal human intestinal tissue treated with PIs. PI-induced barrier disruption in intestinal epithelial cells is not due to necrosis or tight junction alterations, but to induction of massive apoptosis which may lead to leak-flux diarrhoea in vivo. Our findings suggest that induction of apoptosis by PIs could have potential for antitumour therapy.

HIV Protease Inhibitors Activate the Unfolded Protein Response in Macrophages: Implication for Atherosclerosis and Cardiovascular Disease

Human immunodeficiency virus (HIV) protease inhibitors have been successfully used in highly active antiretroviral therapy for HIV-1 infection. Treatment of patients infected with HIV with HIV protease inhibitors is unfortunately associated with a number of clinically significant metabolic abnormalities and an increased risk of premature atherosclerosis and myocardial infarction. However, the cellular/molecular mechanisms of the HIV protease inhibitor-induced lipid dysregulation and atherosclerosis remain elusive. Macrophages are the most prominent cell type present in atherosclerotic lesions and play essential roles in both early lesion development and late lesion complications. In this study, we demonstrate that three different HIV protease inhibitors (ritonavir, indinavir, and atazanavir) induce endoplasmic reticulum stress and activate the unfolded protein response in mouse macrophages. Furthermore, at therapeutic concentrations (5-15 microM), these HIV protease inhibitors were found to increase the levels of transcriptionally active sterol regulatory element binding proteins, decrease endogenous cholesterol esterification, cause the accumulation of free cholesterol in intracellular membranes, deplete endoplasmic reticulum calcium stores, activate caspase-12, and increase apoptosis in macrophages. These findings provide possible cellular mechanisms by which HIV protease inhibitors promote atherosclerosis and cardiovascular disease in HIV-1 infected patients treated with HIV protease inhibitors.

Curcumin Blocks HIV Protease Inhibitor Ritonavir-Induced Vascular Dysfunction in Porcine Coronary Arteries

HIV protease inhibitor ritonavir (RTV) is associated with many cardiovascular complications and causes vascular dysfunction through oxidative stress. In the present study, we determined the effects of RTV and curcumin (a pigment derived from turmeric) on porcine coronary arteries. Artery rings were incubated with 15 microM concentration of RTV and curcumin (5 or 10 microM) for 24 hours. Vasomotor function was studied with a myograph tension system. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels were studied using real-time polymerase chain reaction, Western blot, and immunohistochemistry. Nitric oxide was measured using Griess assay. Superoxide anion levels were determined by lucigenin enhanced chemiluminescence. RTV considerably reduced vessel contraction by 71%, endothelium-dependent relaxation by 59%, and endothelium-independent relaxation by 52%, as compared with controls. Curcumin effectively blocked RTV-induced vasomotor dysfunction. RTV-treated arteries showed substantial reductions of eNOS mRNA by 77%, eNOS protein by 72%, and nitric oxide release by 37% as compared with controls. The RTV plus curcumin-treated vessels showed substantial increases of eNOS and nitric oxide levels as compared with the RTV-treated vessels. Finally, there was a 47% increase of superoxide anion production in the RTV-treated vessels as compared with controls. Again, curcumin effectively reversed this effect of RTV. HIV protease inhibitor RTV impairs vasomotor functions, reduces eNOS expression and nitric oxide release, and increases oxidative stress in porcine coronary arteries. Curcumin effectively blocks these detrimental effects of RTV.

Switch from Ritonavir to Indinavir in Combination Therapy for HIV‐1–Infected Children

Protease inhibitors are an effective component of combination antiretroviral treatment for children infected with human immunodeficiency virus 1 (HIV-1), but tolerance or toxicity issues sometimes require an alternative therapeutic strategy. HIV-1-infected children aged 2-17 years received combination therapy with either stavudine plus ritonavir or with zidovudine, lamivudine, and ritonavir as part of a randomized clinical trial. Twenty-one months after the start of the trial, ritonavir in capsule formulation became unavailable. The treatment regimen for 25 children was switched from ritonavir capsules to indinavir capsules (500 mg/m(2) every 8 h). The other study drugs remained unchanged. A matched-pairs analysis was performed to compare the results for these 25 children with the results for 25 matched children whose treatment regimen continued to include ritonavir (in liquid formulation). There were no significant differences in the percentage of children with an HIV-1 RNA load of <or=200 copies/mL between the group receiving indinavir and the matched group receiving ritonavir (52% vs. 68%, at the start of indinavir treatment; 42% vs. 52%, at week 12; and 50% vs. 56%, at week 24). Similarly, there were no significant differences between the treatment groups with respect to median CD4 cell counts over time. Toxicities observed in the children treated with indinavir were most often flank pain or headache (16%), renal dysfunction (16%), hematuria (12%), and skin rash (12%) and were consistent with toxicities reported elsewhere. There was no evidence that the switch from ritonavir to indinavir therapy altered the pharmacokinetics of indinavir as a result of residual cytochrome P450 induction or inhibition caused by ritonavir. The switch from one protease inhibitor (ritonavir) to another (indinavir) as a component of combination antiretroviral treatment in this patient population was a practical therapeutic strategy.

Altered Adipokine Response in Murine 3T3-F442A Adipocytes Treated with Protease Inhibitors and Nucleoside Reverse Transcriptase Inhibitors

Abnormal levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and adiponectin have been observed in HIV patients with lipodystrophy. However, because these patients have long drug and disease histories, it is unclear which drugs are responsible for these abnormalities. We have therefore investigated the effects of individual antiretrovirals on adipokine expression and adipogenesis using a murine cell line in vitro. Differentiating murine 3T3-F442A adipocytes were incubated with 20 microM nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine or stavudine, or protease inhibitors (PIs) indinavir, ritonavir, saquinavir or nelfinavir, in the presence and absence of rosiglitazone (10 microM). Adipogenesis was assessed using glycerol-3-phosphate dehydrogenase activity, while expression of TNF-alpha, IL-6 and adiponectin at protein and mRNA levels was assessed by ELISA and quantitative real-time PCR, respectively. Nelfinavir, ritonavir and saquinavir inhibited adipogenesis and up-regulated the expression of TNF-alpha and IL-6, but this effect was not seen with indinavir, zidovudine and stavudine. Adiponectin expression was significantly reduced in both NRTI- and PI-treated cells, although the most profound reductions were found with ritonavir and saquinavir. Co-incubation with rosiglitazone led to a partial attenuation of the change in TNF-alpha, IL-6 and adiponectin secretion. Our data suggest that the PIs nelfinavir, ritonavir and saquinavir have potent effects in inhibiting adipocyte differentiation whilst up-regulating TNF-alpha and IL-6 mRNA levels and decreasing adiponectin levels. These changes were partially attenuated by rosiglitazone. Taken together, the data show that antiretrovirals have complex effects on adipocyte function, which may be mediated by an altered adipokine response.

Pronounced inhibition of both intestinal and hepatic cytochrome P450 3A4 activity by lopinavir/ritonavir in HIV infected patients

Background/Aims The HIV protease inhibitors ritonavir and lopinavir both are substrates, inhibitors, and (weak) inducers of cytochrome P450 3A4 (CYP3A4) in humans. The present study was conducted to quantify the changes of intestinal and hepatic CYP3A4 activity caused by combined therapeutic administration of the two drugs in HIV infected patients. Methods In this open, two-period trial, midazolam (MID, 1.5 mg orally followed by 1.0 mg i.v. 4h later) was administered to 30 treatment-naïve HIV positive patients before and at least 14 days after the start of an antiretroviral regimen containing 400 mg lopinavir / 100 mg ritonavir (Kaletra®) bid. Plasma concentrations of MID and 1'-OH-midazolam (OHM) were analyzed up to 12h after the oral dose using LC-MS/MS (limit of quantification, LOQ, 0.2 ng/mL for both analytes). Results Hepatic CYP3A4 activity, measured as hepatic MID clearance, was reduced from 71.9 l/h (CV, 34 %) to 25.5 l/h (CV, 69 %, given are geometric means (geometric CVs)). Intestinal CYP3A4 activity, measured as intestinal MID extraction, was reduced from 0.43 (CV, 31 %) to 0.09 (CV, 60 %). OHM formation fell below LOQ in all patients when Kaletra® was taken in, and the apparent half-life increased from 1.2h (CV, 23 %) to 2.6h (CV, 49 %). Conclusions Both intestinal and hepatic CYP3A4 are pronouncedly inhibited by ritonavir / lopinavir suggesting that drugs metabolized by CYP3A4 should be dosed very cautiously in these patients. Clinical Pharmacology & Therapeutics (2005) 77, P19–P19; doi: 10.1016/j.clpt.2004.11.073

Ginsenosides block HIV protease inhibitor ritonavir-induced vascular dysfunction of porcine coronary arteries

Human immunodeficiency virus (HIV) protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. Ginsenosides have been shown to have potential benefits on the cardiovascular system through diverse mechanisms, including antioxidative property. The objective of this study was to determine whether ginsenosides could prevent coronary arteries from RTV-induced dysfunction. Porcine coronary artery rings were incubated with RTV and ginsenosides Rb1, Rc, and Re for 24 h. Vasomotor function was recorded by a myograph tension system. In response to the thromboxane A(2) analog U-46619, the contraction of the vessel rings was significantly reduced. When cocultured with Rb1, Rc, and Re, the contractility significantly increased. In response to bradykinin at 10(-5) M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls (P < 0.05). When cocultured with Rb1, Rc, and Re, the relaxation significantly increased 100%, 90%, and 134%, respectively, compared with the RTV-alone groups (P > 0.05). In response to sodium nitroprusside, RTV significantly reduced vasorelaxation. In addition, the endothelial nitric oxide synthase (eNOS) mRNA levels were significantly reduced by 78% for RTV group (P < 0.05) by real-time PCR analysis. The eNOS protein levels measured by Western blot analysis and nitrite concentrations measured by Griess assay were also decreased, whereas O(2)(-) production by lucigenin-enhanced chemiluminescence was significantly increased in the RTV-treated group. These effects of RTV were effectively blocked by ginsenosides. Thus HIV protease inhibitor RTV significantly impaired the vasomotor function of porcine coronary arteries. This effect may be mediated by the downregulation of eNOS and overproduction of O(2)(-). These results suggest that ginsenosides can effectively block RTV-induced vascular dysfunction.

Practical Guidelines to Interpret Plasma Concentrations of Antiretroviral Drugs

Several relationships have been reported between antiretroviral drug concentrations and the efficacy of treatment, and toxicity. Therefore, therapeutic drug monitoring (TDM) may be a valuable tool in improving the treatment of HIV-1-infected patients in daily practice. In this regard, several measures of exposure have been studied, e.g. trough and maximum concentrations, concentration ratios and the inhibitory quotient. However, it has not been unambiguously established which pharmacokinetic parameter should be monitored to maintain optimal viral suppression. Each pharmacokinetic parameter has its pros and cons. Many factors can affect the pharmacokinetics of antiretroviral agents, resulting in variability in plasma concentrations between and within patients. Therefore, plasma concentrations should be considered on several occasions. In addition, the interpretation of the drug concentration of a patient should be performed on an individual basis, taking into account the clinical condition of the patient. Important factors herewith are viral load, immunology, occurrence of adverse events, resistance pattern and comedication. In spite of the described constraints, the aim of this review is to provide a practical guide for TDM of antiretroviral agents. This article outlines pharmacokinetic target values for the HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir, and the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine. Detailed advice is provided on how to interpret the results of TDM of these drugs.

Evolution of Human Immunodeficiency Virus Type 1 Protease Genotypes and Phenotypes In Vivo under Selective Pressure of the Protease Inhibitor Ritonavir

Evolution of Human Immunodeficiency Virus Type 1 Protease Genotypes and Phenotypes In Vivo under Selective Pressure of the Protease Inhibitor Ritonavir

The HIV protease inhibitor ritonavir synergizes with butyrate for induction of apoptotic cell death and mediates expression of heme oxygenase‐1 in DLD‐1 colon carcinoma cells

The protease inhibitor ritonavir is an integral part of current antiretroviral therapy targeting human immunodeficiency virus. Recent studies demonstrate that ritonavir induces apoptotic cell death with high efficiency in lymphoblastoid cell lines. Moreover, ritonavir can suppress activation of the transcription factor nuclear factor-kappaB and is an inhibitor of interleukin-1beta and tumor necrosis factor-alpha production in peripheral blood mononuclear cells. Thus, ritonavir appears to have anti-inflammatory properties. In the present study, we investigated in DLD-1 colon carcinoma cell effects of ritonavir on apoptotic cell death and expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme that may be critically involved in the modulation of colonic inflammation. Compared to unstimulated control, ritonavir resulted in a moderate increase in the rate of apoptotic cell death as observed after 20 h of incubation. Notably, ritonavir potently synergized with the short-chain fatty acid butyrate for induction of caspase-3-dependent apoptosis in DLD-1 cells. Ritonavir enhanced mRNA and protein expression of HO-1 in DLD-1 cells. Ritonavir-induced HO-1 protein was suppressed by SB203580 or SB202190 and preceded by immediate upregulation of cellular c-Fos and c-Jun protein levels. This process was associated with induction of activator protein-1 as detected by electrophoretic mobility shift analysis. The present data suggest that ritonavir has the potential to curb colon carcinogenesis by reducing cell growth via mechanisms that include apoptosis and by simultaneously modulating colonic inflammation via induction of anti-inflammatory HO-1.

HIV-1 Protease Inhibitor Ritonavir Potentiates the Effect of 1,25-Dihydroxyvitamin D3 to Induce Growth Arrest and Differentiation of Human Myeloid Leukemia Cells Via Inhibition of CYP24.

Ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explores the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its inactive form 1,24,25,(OH)3. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)2D3 (10−7 M, 1h) induced expression of CYP24 by 80-fold, and pre-incubation of these cells with RTV (2X10−5 M, 3h) decreased 1,25(OH)2D3-stumulated expression of CYP24 transcripts by 30 %, resulting in increased levels of 1,25(OH)2D3 in culture media (50.7 vs 82.4 nmol/L). Importantly, pre-incubation of HL-60 cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) also increased intracellular levels of 1,25(OH)2D3 (1.20 vs 2.46 nmol/L) and potentiated the ability of 1,25(OH)2D3 to induce growth arrest of HL-60 cells. Clonogenic assay showed that either RTV (5X10−6 M) or 1,25(OH)2D3 (10−8 M) alone inhibited colonal growth of HL-60 cells by either a mean 30 ± 12 (±SD) % and 40 ± 12 %, respectively; when both were combined, colony formation was inhibited by a mean 80 ± 13 % (p<0.01). Measurement of CD14 expression, NBT reduction, and non-specific esterase staining showed that RTV augmented the prodifferentiative effects of 1,25(OH)2D3 in HL-60 cell. For example, 1,25(OH)2D3 (10−8 M, 2 days) induced approximately 48 % of HL-60 cells to become CD14 antigen positive. RTV (2X10−5 M, 48h) alone did not stimulate expression of CD14 antigen; when 1,25(OH)2D3 was combined with RTV, approximately 65 % of cells expressed CD14. Furthermore, androgen-independent DU145 human prostate cancer cells, that are resistant to growth inhibition by 1,25(OH)2D3, expressed 80-fold higher levels of CYP24 transcripts compared to HL-60 cells, and intracellular levels of 1,25(OH)2D3 were not detectable (<0.3 nmol/L) after exposure of these cells to 1,25(OH)2D3 (10−7 M, 24 h). Of note, pre-incubation of these cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) increased intracellular levels of 1,25(OH)2D3 to 0.94 nmol/L and sensitized DU145 cells to growth inhibition mediated by 1,25(OH)2D3 as measured by MTT assay. Taken together, inhibition of CYP24 might open a new paradigm for therapy using vitamin D compounds.

Antiretrovirals as Antimalarial Agents

Recent studies have indicated that antiretroviral protease inhibitors may affect outcome in malarial disease. We have investigated the antimalarial activities of 6 commonly used antiretroviral agents. Our data indicate that, in addition to the previously published effects on cytoadherence and phagocytosis, the human immunodeficiency virus (HIV)-1 protease inhibitors saquinavir, ritonavir, and indinavir directly inhibit the growth of Plasmodium falciparum in vitro at clinically relevant concentrations. These findings are particularly important in light of both the high rate of malaria and HIV-1 coinfection in sub-Saharan Africa and the effort to employ highly active antiretroviral therapy in these regions.

CADA, a novel CD4-targeted HIV inhibitor, is synergistic with various anti-HIV drugs in vitro

To evaluate the anti-HIV-1 activity of the cyclotriazadisulfonamide CADA against primary isolates in vitro and the combination of CADA with approved anti-HIV drugs for potential synergy. Peripheral blood mononuclear cells (PBMC) were treated with CADA and infected with 16 different clinical isolates. After 8 days of infection, the median inhibitory concentration (IC50) was calculated from the p24 viral antigen content in the supernatant. MT-4 cells were infected with HIV-1NL4.3 and then cultured with CADA or other antiretroviral drugs (i.e., several reverse transcriptase, protease and entry inhibitors), alone and in combination. After 4 days, IC50 was determined for the various drugs in replicate assays. Analysis of combined effects was performed using the median effect principle (CalcuSyn; Biosoft). The entry inhibitor CADA exerted a potent and consistent anti-HIV-1 activity against a wide range of R5, R5/X4 and X4 primary isolates in PBMC. From the two-drug studies, combination indices showed synergy between CADA and reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, zalcitabine, didanosine, abacavir, tenofovir, nevirapine, delavirdine and efavirenz), and protease inhibitors (lopinavir, saquinavir, indinavir, nelfinavir, amprenavir and ritonavir). In addition, the combination of CADA with the gp41 fusion inhibitor T-20 (enfuvirtide), the CXCR4 antagonist AMD3100 and the gp120-specific interacting plant lectins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) also resulted in a synergistic inhibition. Compounds that can specifically downmodulate the CD4 receptor in PBMC have broad-spectrum anti-HIV activity against primary isolates and act synergistically when used in conjunction with currently available antiretroviral drugs. They deserve further study as potential candidate anti-HIV drugs.

Short Communication: Antiretroviral Protease Inhibitors Prevent L6 Muscle Cell Fusion by Reducing Calpain Activity

The antiretroviral protease inhibitors indinavir (IDV) and ritonavir (RTV) are used in highly active antiretroviral therapies (HAART). Side effects from long-term HAART therapy include loss of muscle mass. Myoblasts when cultured in media low in growth factors withdraw from the cell cycle, express muscle-specific differentiation inducers and proteins, and fuse to form myotubes. The neutral protease, calpain, is required for myotube formation and RTV decreased calpain activity in vitro. We found lower calpain activity, but not protein, in homogenates of RTV-treated L6 cells than in control cultures. Importantly, L6 and C2C12 myoblasts did not form myotubes when cultured with 10 or 20 microM IDV or RTV. Control and drug-related L6 myoblasts showed identical decreases in proliferating cell nuclear antigens expression indicating proliferation arrest. Similarly, muscle differentiation inducers MyoD and myogenin and their downstream target, myosin heavy chain, were expressed at similar levels in control and drug-treated cells. Thus, whereas muscle differentiation was unaffected by protease inhibitors, calpain activity was reduced and myotube formation prevented. We conclude that RTV and IDV reduced myotube formation by reducing calpain activity. Our data suggest that protease inhibitors included in HAART might be directly involved in muscle wasting by reducing muscle remodeling.

HIV protease inhibitor ritonavir increases monolayer permeability of endothelial cells

Background. Clinical applications of HIV protease inhibitors for treatment of HIV infection have been associated with significant cardiovascular complications. The objective of this study was to determine the effects of HIV protease inhibitor ritonavir (RTV) on endothelial permeability and expression of junctional proteins as well as the effects of several antioxidants as potential therapeutic agents. Methods. Human coronary artery endothelial cells (HCAECs) were cultured with RTV (0, 7.5, 15, and 30 μM) for different times. Monolayer permeability was studied by using a transwell system and a fluorescence-labeled dextran trancer. The mRNA levels of endothelial junctional proteins including zonula occludens-1 (ZO-1), occludin, claudin-1, and VE-cadherin were quantitatively determined by real-time RT-PCR. Three antioxidants, curcumin (CUR), epigallocatechin gallate (EGCG), and seleno-L-methionine (SLM) at concentrations of 5, 10, and 25 μM, were used for blocking the effects of RTV. Results. RTV treatment showed a significant increase of monolayer permeability and a decrease of mRNA levels of four junctional proteins in a dose- and time-dependent fashion ( P < 0.03, n = 3). Three antioxidants showed a potent blocking effect on RVT (15 μM) in a dose-dependent fashion. Effects of RTV at 15 μM with or without one of antioxidants (25 μM) are shown in the table below. Conclusions. HIV protease inhibitor RTV significantly increases monolayer permeability and reduces expression of junctional proteins in HCAECs. Three antioxidants (CUR, EGCG, and SLM) can effectively block RTV-induced endothelial dysfunction and gene alterations. TABLE—ABSTRACT P47 Permeability ZO-1 Occludin Claudin-1 VE-cadherin Control 100% 100% 100% 100% 100% RTV 207% 76% 44% 15% 87% RTV + CUR 117% 71% 101% 43% 72% RTV + EGCG 139% 107% 101% 373% 107% RTV + SLM 138% 100% 101% 96% 100%

Ginsenoside-Rb1 Prevents HIV protease inhibitor ritonavir-induced vasomotor dysfunction

Introduction. We previously reported that ritonavir (RTV), one of the clinically used HIV protease inhibitors, can cause endothelial injury by increasing oxidative stress. The objective of this study was to determine whether an active ingredient of Panax ginseng, Ginsenoside-Rb1 (Rb1), could prevent coronary arteries from RTV-induced vasomotor dysfunction. Methods. Porcine coronary arteries were cut into 5-mm rings and incubated with RTV (15 μM) or/and Rb1 (1 or 10 mg/L) for 24 h. Vasomotor function was studied with a myograph tension system in response to U46619 (contraction) and bradykinin (relaxation). The mRNA and protein levels of endothelial nitric oxide synthase (eNOS) were determined by real-time PCR and Western blot, respectively. Superoxide anion levels were determined by lucigenin-enhanced chemiluminescence. Results. In response to U46619, the control vessels showed a vessel tension of 168 mN, while RTV-treated vessels had 41 mN, showing a 76% reduction ( P < 0.001, n = 8). The combination of RTV and Rb1 (1 or 10 mg/L) showed a vessel tension of 76 or 90 mN, respectively. In response to bradykinin, the control vessel rings relaxed by 70%, while RTV-treated vessels only relaxed by 29%, which showed a 59% reduction ( P < 0.001). Rb1 (1 or 10 mg/L) plus RTV showed 46 or 58% of vasorelaxation, respectively. The eNOS mRNA expression was decreased by 77% in RTV-treated vessels over controls ( P < 0.05). Western blot and immunohistochemistry also showed a decrease of eNOS protein levels in RTV-treated vessels. Superoxide anion levels in the RTV-treated group was increased by 47% over controls ( P < 0.05). Rb1 treatment (1 and 10 mg/L) significantly inhibited RTV-induced down regulation of eNOS and overproduction of superoxide anion. Conclusions. HIV protease inhibitor RTV significantly decreases vessel contractivity and endothelium-dependent vasorelaxation in porcine coronary arteries. It also decreases eNOS and increases superoxide anion production. Ginsenoside-Rb1 effectively inhibited RTV-induced endothelial dysfunction and oxidative stress in this model.

'MARCHING IN' ON NIH-FUNDED DRUGS

IN 1988, ABBOTT LABORATORIES REceived a National Cooperative Drug Discovery Group for AIDS grant, administered by the National Institute of Allergy & Infectious Diseases at the National Institutes of Health. The $3.5 million, five-year grant supported Abbott scientists in the discovery of the protease inhibitor ritonavir, which was further developed and marketed by Abbott as Norvir. This successful drug, which was introduced in 1996, was thrust into the spotlight when Abbott raised its price in the US. by 400% last December. In many ways, the case of Norvir is a good example of how technology developed with federal funds can be effectively transferred into the commercial sector where it can be developed into a product that benefits countless people. This outcome is, in fact, the goal of the Bayh-Dole Act of 1980—the legislation that governs the transfer of federally funded research from the lab to the marketplace. The Norvir case, however, has ...

209 Enhancing radiation tumor response using protease inhibitor ritonavir in HPV-positive head and neck carcinoma

209 Enhancing radiation tumor response using protease inhibitor ritonavir in HPV-positive head and neck carcinoma

Curcumin prevents HIV protease inhibitor ritonavir-induced vascular injury and superoxide anion production in porcine coronary arteries

Introduction: HIV protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. The objective of this study was to determine whether a dietary antioxidant curcumin could prevent coronary arteries from RTV-induced injury. Methods: Porcine coronary arteries were incubated with RTV and/or curcumin for 24 hours. Vasomotor function was studied with a myograph tension system in response to thromboxane A2 analogue U46619 (contraction), and bradykinin (endothelium-dependent relaxation). Superoxide anion levels were determined by lucigenin enhanced chemiluminescence. Results: In response to U46619, the control vessels showed a 71% reduction (p < 0.001, t-test). The combination of RTV and curcumin showed a vessel tension similar to controls. In response to bradykinin, the control vessel rings relaxed by 70.80%, while RTV treated vessels only relaxed by 29.25%, which showed a 59% reduction as compared to controls (p < 0.001, t-test). Curcumin plus RTV showed 54.20% of vasorelaxation, respectively. Superoxide anion level in the RTV-treated group was increased by 47%, as compared to controls (P < 0.05, t-test). Curcumin treatment significantly inhibited RTV induced superoxide anion production. Conclusions: HIV protease inhibitor RTV significantly decreases smooth muscle cell contractivity and endothelium-dependent vasorelaxation, but increases superoxide anion production in porcine coronary arteries. Curcumin can effectively inhibit RTV-induced vascular injury and oxidative stress.