Protease Inhibitors Ritonavir Research Articles

Effects of lopinavir–ritonavir combined therapy during the rat pregnancy: Morphological and biochemical aspects

Objective To evaluate the biochemical and morphological effects in rats subjected to three different dose associations of the protease inhibitors lopinavir and ritonavir administered throughout the entire period of pregnancy. Study design The animals were treated throughout pregnancy with daily oral doses of lopinavir + ritonavir starting at the day one of pregnancy, and were divided into four groups: E1, 13.3 + 3.3 mg/kg; E2, 39.9 + 9.9 mg/kg; E3, 119.7 + 29.9 mg/kg and C, control (drug vehicle, propyleneglycol). The animals were then sacrificed and maternal blood and fetal and maternal organ samples were taken for morphological and biochemical analysis. Results No major changes were identified in the group treated with the lowest dose as compared with the control. In the group E2, we found hepatocytes with signs of atrophy, eosinophilic cytoplasm, picnotic nuclei and vasodilatation. The proximal convoluted tubules of maternal kidneys showed eosinophilic areas and hyperchromatic nuclei, as well as signs of vasodilation. In the group treated with the highest dose (group E3), in the maternal kidneys and livers, the morphological changes were similar to those found in E2, although more prominent. Regarding the fetal organs, the single abnormality observed was some liver vasodilation in the group E3 (highest dose). The treatment with lopinavir + ritonavir caused discrete, yet significant, alterations of aspartate aminotransferase activity, blood urea nitrogen and creatinine plasma levels. Conclusions Our results showed that the administration of a combination of lopinavir plus ritonavir to pregnant rats can cause morphological as well as functional changes in maternal and fetal liver and kidneys and, in higher than therapeutic doses, might be toxic to those animals.

Nelfinavir induces liposarcoma apoptosis and cell cycle arrest by upregulating sterol regulatory element binding protein-1

"HIV protease-induced lipodystrophy syndrome" is associated with the use of HIV protease inhibitors for treatment of HIV infection. In-vitro studies suggest that alteration of sterol regulatory element binding protein-1 levels underlie its pathogenesis. We postulated that HIV protease inhibitors may represent a novel class of antiliposarcoma agents. SW872, FU-DDLS-1 and LiSa-2 liposarcoma, and HT1080 and 293 nonliposarcoma cell lines were treated with HIV protease inhibitors (nelfinavir, ritonavir, saquinavir, indinavir and amprenavir), and clonogenic assays were performed. Nelfinavir exhibited the most potent inhibition of clonogenicity, and further assays for proliferation, cell cycle and apoptosis were performed with nelfinavir. Immunoblots were performed for sterol regulatory element binding protein-1, proapoptotic and cell cycle-related protein expression after nelfinavir treatment. Finally, a sterol regulatory element binding protein-1-inducible SW872 cell line was developed to examine the phenotype resulting from upregulated sterol regulatory element binding protein-1. Nelfinavir selectively inhibited clonogenicity and proliferation, and induced G1 cell cycle block and induced apoptosis in a dose-dependent manner in SW872 and LiSa-2 cells, whereas it had minimal or no effect on these parameters in FU-DDLS-1 or nonliposarcoma cells. Nelfinavir induced significant sterol regulatory element binding protein-1 expression in a dose-dependent and time-dependent fashion in sensitive SW872 and LiSa-2 cells, modestly in HT1080 cells, but not in nelfinavir-insensitive FU-DDLS-1 and 293 cells without inducing adipocytic differentiation. Forced expression of sterol regulatory element binding protein-1 in inducible-SW872 cells led to the induction of proapoptotic and antiproliferative proteins, and consequent reduction of cellular proliferation. Our data indicate that nelfinavir represents a novel class of antiliposarcoma agent that acts by selectively upregulating sterol regulatory element binding protein-1 expression in liposarcomas.

The HIV protease inhibitor ritonavir and the proteasome inhibitor bortezomib induce synergistic cytotoxicity on soft tissue sarcoma cells in vitro

9566 Background: HIV-protease inhibitors like Ritonavir produces regressions in HIV-associated Kaposi‘s sarcoma, reportedly due to proteasome inhibition. We have here assessed the effect of Ritonavir and the proteasome inhibitor Bortezomib on the viability and proteasome activity of the soft tissue sarcoma cell lines RD (rhabdomyosarcoma) and A-673 (ewing‘s sarcoma) in vitro. Methods: Cytotoxixity was assessed by MTS-test. To directly dissect proteasome activity in viable sarcoma cells, we used a recently developed cell-permeable affinity label that for the first time allows to assess the individual activity profile of the different catalytically active proteasomal subunits in living cells (Berkers et al., Nature Methods, May 2005). Results: Both types of cells were resistant towards Bortezomib and Ritonavir monotherapy at therapeutic concentrations (20 nM for Bortezomib, 20μM for Ritonavir). Ritonavir induced cytotoxicity in sarcoma cell lines with an IC50 of 40μM. The combination of clinically meaningful concentrations of Ritonavir (20μM) with Bortezomib (10–20 nM) induced robust synergistic cytotoxicity in both sarcoma cell lines. Using affinity labelling of proteasome activity, we directly demonstrate that a) Botezomib abrogates beta5, but also beta1-proteosomal activity in sarcoma cells and b) that Ritonavir at 20μM does not affect proteasom activity. Conclusions: We here present the first analysis of active proteasomal subunits in living human sarcoma cell lines. Ritonavir and Velcade induce synergistic cytotoxicity at clinically meaningful concentrations in vitro, which is not mediated through a direct effect of Ritonavir on proteasome activity. Based on our data, the synergistic combination of Ritonavir and Bortezomib might warrant further clinical testing in soft tissue sarcoma in vivo. No significant financial relationships to disclose.

Evolution on distributive lattices

We consider the directed evolution of a population after an intervention that has significantly altered the underlying fitness landscape. We model the space of genotypes as a distributive lattice; the fitness landscape is a real-valued function on that lattice. The risk of escape from intervention, i.e., the probability that the population develops an escape mutant before extinction, is encoded in the risk polynomial. Tools from algebraic combinatorics are applied to compute the risk polynomial in terms of the fitness landscape. In an application to the development of drug resistance in HIV, we study the risk of viral escape from treatment with the protease inhibitors ritonavir and indinavir.

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. ER-positive estradiol-dependent lines (IC50, 12-24 micromol/L) and ER-negative (IC50, 45 micromol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-alpha levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 +/- 8 micromol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K(D), 7.8 micromol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90alpha short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

HIV-1 protease inhibitor ritonavir potentiates the effect of 1,25-dihydroxyvitamin D 3 to induce growth arrest and differentiation of human myeloid leukemia cells via down-regulation of CYP24

HIV-1 protease inhibitor, ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explored the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] to its inactive form 1,24,25,(OH) 3. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH) 2D 3 induced expression of CYP24, and pre-incubation of these cells with RTV decreased this transcripts, resulting in increased intracellular levels of 1,25(OH) 2D 3 and potentiation of the ability of 1,25(OH) 2D 3 to induce growth arrest and differentiation of these cells. Taken together, inhibition of CYP24 might open a new paradigm for therapy using Vitamin D compounds.

HIV protease inhibitor ritonavir inhibits cholesterol efflux from human macrophage derived foam cells

HIV protease inhibitor ritonavir inhibits cholesterol efflux from human macrophage derived foam cells

Differential Inhibition of Cytochrome P450 3A4, 3A5 and 3A7 by Five Human Immunodeficiency Virus (HIV) Protease Inhibitors in vitro

The effects of five HIV protease inhibitors (amprenavir, indinavir, nelfinavir, ritonavir and saquinavir) on cytochrome P450 (CYP) 3A4, 3A5 and 3A7 activities were studied in vitro using testosterone 6beta-hydroxylation in recombinant CYP3A4, CYP3A5 and CYP3A7 enzymes. The protease inhibitors showed differential inhibitory effects on the three CYP3A forms. Ritonavir and saquinavir were non-selective and preferential inhibitors of CYP3A4 and CYP3A5 (K(i) 0.03 microM and 0.6-0.8 microM for ritonavir and saquinavir, respectively), and weaker inhibitors of CYP3A7 (K(i) 0.6 microM and 1.8 microM, respectively). Nelfinavir was a potent and non-selective inhibitor of all three CYP3A forms (K(i) 0.3-0.4 microM). Amprenavir and indinavir preferentially inhibited CYP3A4 (K(i) 0.1 microM and 0.2 microM, respectively), with weaker inhibitory effects on CYP3A5 (K(i) 0.5 microM and 2.2 microM, respectively) and CYP3A7 (K(i) 2.1 microM and 10.6 microM, respectively). In conclusion, significant differences exist in the inhibitory potency of protease inhibitors for different CYP3A forms. Ritonavir, nelfinavir, saquinavir and amprenavir seem to be prone to drug-drug interactions by inhibiting both CYP3A4 and CYP3A5. Especially nelfinavir and ritonavir also have a potential to inhibit foetal CYP3A7-mediated drug metabolism and some endogenous pathways that may be crucial to normal foetal development, while indinavir has the lowest potential to inhibit CYP3A5 and CYP3A7.

Melt Extrusion Can Bring New Benefits to HIV Therapy

The incorporation of poorly soluble drugs into convenient oral dosage forms is one of the biggest challenges encountered in drug formulation. Melt extrusion, the process of converting a thermoplastic raw material into a product of uniform shape and density by forcing it through a die, offers one solution. Ingredients are mixed, compressed, and plasticized as they pass through a proprietary extruder, resulting in a solid dispersion or solid suspension in which the active molecules are uniformly spread through a polymeric matrix.Melt extrusion has been used in a wide range of medical applications, from stents to ophthalmic implants. Application of melt extrusion to the manufacturing of tablets is relatively new, complex, and is technically challenging. To date, only four drugs have been successfully manufactured using melt extrusion technology; however, many other pharmaceutical formulations may benefit from melt extrusion technology.Kaletra®, containing the anti-HIV protease inhibitors lopinavir and ritonavir, is the first co-formulated pharmaceutical compound to be successfully tableted using a proprietary melt extrusion process. Melt extrusion appears to have overcome the poor solubility and negligible oral bioavailability of previous experimental solid formulations of lopinavir/ritonavir. Importantly for patients, compared with the capsule formulation of Kaletra®, melt extrusion tablets require fewer doses to be taken each day, do not need to be refrigerated, and do not need to be taken with food.The melt extrusion tablet formulation of Kaletra® represents a significant step towards a simpler, more convenient dosage form for these protease inhibitors for many patients with HIV.

Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose (600 mg twice daily), ritonavir also inhibits CYP2D6. The effect of low-dose ritonavir on CYP2D6 is unknown and was investigated in this study. This was a 1-arm, 2-period, fixed-order study in 13 healthy male volunteers who were extensive metabolizers of CYP2D6. The first period examined baseline CYP2D6 activity by evaluating the pharmacokinetics of a single dose of desipramine and by metabolic phenotyping with dextromethorphan. During the second period, participants took ritonavir, 100 mg twice daily, for 2 weeks, followed by repeat assessment of desipramine pharmacokinetics and the dextromethorphan metabolic phenotype in the presence of ritonavir. Low-dose ritonavir (100 mg twice daily) significantly increased the exposure to single-dose desipramine, as reflected in a geometric mean ratio (with ritonavir/without ritonavir) of 1.26 (95% confidence interval, 1.13-1.40) for the desipramine area under the concentration versus time curve from time 0 to infinity (P < .001). Coadministration of low-dose ritonavir did not significantly affect the dextromethorphan/dextrorphan urinary metabolic ratio and did not convert any extensive metabolizer to a poor metabolizer. Low-dose ritonavir (100 mg twice daily) exerts a modest inhibitory effect on the activity of CYP2D6 in extensive metabolizers, as assessed with desipramine as the index substrate. This effect was not apparent with the dextromethorphan/dextrorphan metabolic ratio as an indicator for CYP2D6 activity. It is expected that the effect of low-dose ritonavir on CYP2D6 will not require standard dose reductions for CYP2D6 substrates.

Ritonavir Inhibits NF-AT Activation Through Effects on the PI-3 Kinase/Akt Pathway

The HIV protease inhibitor ritonavir has been reported to have activities unrelated to inhibition of HIV protease, including anti-tumor activity in vivo and in vitro, induction of lipodystrophy in vivo, inhibition of the 20S proteasome, and inhibition of NFkB activation. Here we show that ritonavir also inhibits activation of NF-AT by PMA plus ionomycin and by the HHV-8 vGPCR. Inhibition of NF-AT activation occurs through the PI-3 kinase/Akt/GSK3 pathway, since ritonavir treatment leads to decreased Akt phosphorylation and a resultant decrease in GSK-3 phosphorylation. Treatment with ritonavir also inhibits the expression of NF-AT-dependent pro-inflammatory factors. Inhibition of multiple signaling pathways may help to explain the anti-tumor and other effects of ritonavir that are unrelated to its anti-retroviral activity. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Spinal epidural lipomatosis: a manifestation of HAART-associated lipodystrophy

Spinal epidural lipomatosis: a manifestation of HAART-associated lipodystrophy

Acquired Bortezomib (Velcade®)-Resistance Is Accompanied by Downmodulation of Proteasome Activity and Upregulation of Tripeptidyl Peptidase II (TPPII) and a Ubiquitin-Specific Protease.

Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.

Ritonavir Impairs Lipoprotein Lipase–Mediated Lipolysis and Decreases Uptake of Fatty Acids in Adipose Tissue

The use of the HIV protease inhibitor ritonavir (RTV) is frequently associated with hypertriglyceridemia and lipodystrophy. The aim of our study was to determine the mechanism underlying the observed hypertriglyceridemia. Feeding female APOE*3-Leiden transgenic mice a Western-type diet supplemented with RTV (35 mg/kg per day) for 2 weeks resulted in a 2-fold increase in fasting plasma triglyceride (TG) levels, which was specific for very low-density lipoprotein (VLDL). RTV did not change the hepatic VLDL-TG production. Instead, RTV did increase the postprandial TG response to an oral fat load (area under the curve, 25.5+/-12.1 versus 13.8+/-6.8 mmol/L per hour in controls; P<0.05). Likewise, RTV hampered the plasma clearance of intravenously injected glycerol tri[3H]oleate-labeled VLDL-like emulsion particles (half time, 19.3+/-10.5 versus 5.0+/-1.3 minutes in controls; P<0.05) associated with a decrease of 44% in plasma lipoprotein lipase activity. Accordingly, RTV decreased the uptake of TG-derived fatty acids (FAs) into adipose tissue, as well as the uptake of albumin-bound FA. We conclude that RTV causes hypertriglyceridemia via decreased lipoprotein lipase-mediated clearance of VLDL-TG. In addition, RTV specifically impairs the uptake of FA in adipose tissue, which may contribute to the lipodystrophy that is frequently observed in HIV-infected subjects on antiretroviral therapy.

Simultaneous determination of HIV protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in human plasma by high-performance liquid chromatography-tandem mass spectrometry

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially 'toxic' drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).

The Effect of the HIV Protease Inhibitor Ritonavir on Proliferation, Differentiation, Lipogenesis, Gene Expression and Apoptosis of Human Preadipocytes and Adipocytes

HIV patients in highly active antiretroviral therapy (HAART) develop lipodystrophy and insulin resistance. Protease inhibitors have been shown to alter adipocyte metabolism in murine cell lines. In this study, biological effects of the HIV protease inhibitor, ritonavir, were investigated on human SGBS preadipocytes and adipocytes. Ritonavir dose-dependently impaired preadipocyte proliferation and adipogenic differentiation. Gene expression analysis measured by real-time PCR, showed no effect of ritonavir (up to 20 microM) on expression of mRNA of PPARgamma2 and SREBP1c, but suppressed adiponectin mRNA while increasing IL-6 mRNA expression. In human adipocytes, ritonavir at therapeutic concentrations inhibited insulin-stimulated lipogenesis, reduced GLUT4 mRNA, fatty acid synthase and adiponectin expression, while increasing IL-6 mRNA expression. Finally, long-term treatment (72 and 120 h) of SGBS adipocytes but not preadipocytes with ritonavir induced apoptosis in up to 15% of the cells. All together, these data show effects of ritonavir on human preadipocytes and adipocytes aiming at reducing adipose tissue mass and increasing insulin resistance. These in vitro findings may partly explain the clinical findings in patients under HAART. Furthermore, SGBS cells may serve as a useful tool in further investigation of the mechanism of protease inhibitor action in human adipocytes.

HIV Protease Inhibitors: Suppression of Insulin Secretion by Inhibition of Voltage-Dependent K+Currents and Anion Currents

We have shown before that the human immunodeficiency virus (HIV) protease inhibitors ritonavir and nelfinavir, but not indinavir, suppress insulin secretion from mouse pancreatic B-cells via reduction of the cytosolic free calcium concentration ([Ca(2+)](c)). This was not because of an effect on ATP-dependent K(+) channels (K(ATP) channels) or L-type Ca(2+) channels. The study was intended to elucidate the mechanisms by which distinct HIV protease inhibitors decrease [Ca(2+)](c) and thus evoke their adverse side effect on insulin release. Membrane potential and whole-cell currents were measured with the patch-clamp technique, and [Ca(2+)](c) was determined with a fluorescence dye. Ritonavir and nelfinavir both inhibited the same component(s) of voltage-dependent K(+) currents with a concomitant change in action potential wave form, whereas indinavir was ineffective. Comparison with other blockers of voltage-dependent K(+) currents revealed that suppression of distinct noninactivating current component(s) altered action potential wave form and decreased [Ca(2+)](c) similar to ritonavir and nelfinavir, whereas blockage of inactivating component(s) was without effect. Complete inhibition of voltage-dependent K(+) currents by 80 mM TEA(+) drastically increased [Ca(2+)](c), demonstrating that voltage-dependent K(+) channels are not the sole target of ritonavir and nelfinavir. Accordingly, the Ca(2+)-lowering effect of ritonavir was preserved in the presence of 80 mM TEA(+). This effect was mimicked by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Consequentially, ritonavir and nelfinavir inhibited a DIDS-sensitive anion current in B-cells. We suggest that ritonavir and nelfinavir decrease insulin secretion by inhibition of voltage-dependent K(+) channels and anion channels, which are essential to provide counterion currents for Ca(2+) influx across the plasma membrane.

Effects of 5 HIV Protease Inhibitors on Vasomotor Function and Superoxide Anion Production in Porcine Coronary Arteries

HIV protease inhibitors (PIs) have been implicated to cause cardiovascular complications. Previous studies demonstrated that the PI ritonavir (RTV) caused endothelial dysfunction in porcine arteries. This study investigated and compared the effects of 5 commonly used PIs on vasomotor function, endothelial nitric oxide synthase (eNOS) expression, and oxidative stress in porcine coronary arteries. Vessel rings were incubated with 15 microM of RTV, amprenavir (APV), saquinavir (SQV), indinavir (IDV), or nelfinavir (NFV) for 24 hours. Vasomotor function was studied using a myograph system. The contractility of the rings was significantly reduced for RTV and SQV. In response to bradykinin at 10(-5) M, the endothelium-dependent relaxation was significantly reduced for RTV, APV, and SQV. The eNOS mRNA levels were significantly reduced for RTV, APV, and SQV. Furthermore, the superoxide anion (O(2)(-)) levels of the vessels were significantly increased for RTV and APV. It was found that nitric oxide production was decreased, whereas the level of nitrotyrosine proteins was increased in RTV-treated vessels. Furthermore, antioxidant seleno-L-methionine (SeMet) reversed RTV-induced O(2)(-) production and vasomotor dysfunction. Thus, the HIV PIs RTV, APV, and SQV at 15 microM have more potent in vitro effects on vasomotor dysfunction, eNOS downregulation, and O(2)(-) production than IDV and NFV. The antioxidant SeMet can block these adverse effects of RTV. The results suggest that antioxidant therapy may have applications for controlling PI-associated cardiovascular complications.

Ritonavir-Based Highly Active Antiretroviral Therapy in Human Immunodeficiency Virus Type 1-Infected Infants Younger Than 24 Months of Age

Few data are available regarding clinical outcomes or dosing requirements for the protease inhibitor ritonavir in human immunodeficiency virus (HIV)-infected children younger than under 24 months of age. This prospective, multicenter phase I/II open label treatment trial used ritonavir, zidovudine and lamivudine to treat protease inhibitor-naive, HIV-infected infants between the ages of 4 weeks and 24 months. Two sequential dosing cohorts were treated with 350 or 450 mg/m(2) ritonavir every 12 hours; this report includes results of pharmacokinetics, safety, tolerability and efficacy through 104 weeks of follow-up of all subjects. Fifty HIV-infected children were treated. By week 16, 36 had achieved HIV-1 RNA <400 copies/mL (72% intent-to-treat, 84% as-treated analysis); by week 104, 18 maintained durable viral suppression (36% intent-to-treat, 46% as-treated). Poor medication adherence by caregiver report contributed to virologic failure. Few subjects experienced treatment-limiting toxicity: emesis or ritonavir refusal in 6 (12%); and severe but reversible anemia or elevated serum hepatic transaminases in 1 (4%) each. Apparent oral clearance was higher and the median predose concentrations were substantially lower than those found in adults. Median z scores for weight and height for age/gender were below normal at baseline but improved by week 104. A combination regimen of ritonavir, zidovudine and lamivudine was generally safe and produced sustained viral suppression in more than one-third of infants who initiated therapy before 2 years of age. Improved palatability of liquid preparations of protease inhibitors, supporting infrastructure and behavioral approaches to improve medication adherence with antiretrovirals will likely be necessary to further improve efficacy.

HIV protease inhibitor ritonavir increases endothelial monolayer permeability

HIV protease inhibitors (PIs) are often associated with metabolic and cardiovascular complications although they are effective anti-HIV drugs. In this study, we determined whether HIV PI ritonavir could increase endothelial permeability, one of the important mechanisms of vascular lesion formation. Human dermal microvascular endothelial cells (HMECs) treated with ritonavir showed a significant increase of endothelial permeability in a dose- and time-dependent manner assayed with a transwell system. Ritonavir significantly reduced the mRNA levels of tight junction proteins zonula occluden-1, occludin, and claudin-1 by 40–60% as compared to controls ( P < 0.05) by real-time PCR analysis. Protein levels of these tight junction molecules were also substantially reduced in the ritonavir-treated cells. In addition, HMECs treated with ritonavir (7.5, 15, and 30 μM) showed a substantial increase of superoxide anion production by 10%, 32%, and 65%, respectively, as compared to controls. Antioxidants (EGCG and SeMet) effectively reduced ritonavir-induced endothelial permeability. Furthermore, ritonavir activated ERK1/2 (phosphorylation), but not P38 and JNK. Specific ERK1/2 inhibitor, PD89059, significantly abolished ritonavir-induced endothelial permeability by 92%. Thus, HIV PI ritonavir increases endothelial permeability, decreases levels of tight junction proteins, and increases superoxide anion production. ERK1/2 activation is involved in the signal transduction pathway of ritonavir-induced endothelial permeability.