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Antimicrob Agents Chemother 1998 Oct;42(10):2637-44
Agouron Pharmaceuticals, Inc., San Diego, California 92121, USA.
[Medline record in process]
Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.
PMID: 9756769, UI: 98443459
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Antimicrob Agents Chemother 1998 Sep;42(9):2153-9
Departments of Medicine and Pharmacology Albany Medical College, Albany, New York 12208, USA. GLDrusano@AOL.com
The use of combinations of anti-human immunodeficiency virus (anti-HIV) agents targeted to different molecular targets will most likely result in increased viral suppression and may also delay or prevent the emergence of resistant HIV strains. The purpose of the present study was to develop information on the in vitro anti-HIV activities of combinations of the reverse transcriptase inhibitor 1592U89 and the protease inhibitor 141W94 to help guide the choice of dosages in clinical trials. Triplicate in vitro dose-response matrices were prepared with MT-2 cells infected with HIV type 1 (HIV-1) strain IIIB. In order to account for the effects of protein binding, tissue culture medium with 10% fetal bovine serum was supplemented with the human serum proteins alpha1 acid glycoprotein (1 mg/ml) and albumin (40 mg/ml). The three-dimensional drug interaction surface for 1592U89 and 141W94 was constructed with the program MacSynergy II. As analyzed relative to a Bliss Independence null reference model, this combination was synergistic, with volumes of synergy exceeding 100 (99% confidence). Analysis of the data set with a fully parametric form of an equation for the quantitation of drug interaction developed by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev. 47:331-385, 1995) resulted in an interaction term statistically significantly greater than 0.0, indicating true synergy. Both methods concur that this combination is significantly synergistic. These data, with favorable findings from phase I/II trials for each drug alone, suggest that the combination of 1592U89 plus 141W94 should be further evaluated in clinical trials.
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Drugs 1998 Jun;55(6):837-42; discussion 843-4
Adis International Limited, Auckland, New Zealand. demail@adis.co.nz
Amprenavir is a viral protease inhibitor with specificity for the HIV protease enzyme. The resistance profile of amprenavir appears to differ from that of other protease inhibitors such as saquinavir and indinavir. Twelve hours after single-dose administration of amprenavir 1200mg to HIV-infected individuals, the mean plasma concentration of the drug was more than 10-fold greater than the 50% inhibitory concentration for HIV-1IIIB in peripheral blood lymphocytes. In a small nonblind study, amprenavir monotherapy increased CD4+ cell count and decreased viral load in 37 patients with HIV infection and no previous exposure to protease inhibitor therapy. Combination therapy comprising amprenavir and other antiretroviral agents (abacavir, zidovudine, lamivudine, indinavir, saquinavir or nelfinavir) decreased viral load and increased CD4+ cell counts in patients with HIV infection. Antiviral efficacy was maintained during up to 24 weeks' follow-up. Available data suggest that rash, headache and diarrhoea or loose stools are the most frequent adverse events associated with amprenavir therapy.
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Adv Exp Med Biol 1998;436:75-83
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139, USA.
VX-478 (141W94), a potent inhibitor of HIV protease, is in late stage clinical trials for the treatment of HIV infection and AIDS. Resistant viruses were raised in vitro by passage of HIV-1IIIB in the presence of increasing concentrations of VX-478 and the related hydroxyethylamino sulfonamide inhibitor VB-11,328. By direct PCR analysis of selected viruses, a number of mutations were identified (L10F, M46I, I47V, I50V and I84V) in the protease gene. These mutations were introduced into recombinant HIV-1 protease and the mutant enzymes assayed against a panel of inhibitors of diverse chemical structure. For VX-478, significant increases in IC90 and Ki were observed for virus or protease, respectively, containing I50V single mutation or an M46I/I47V/I50V triple mutation. The mutant proteases were also characterized for their kinetic competence to process substrates representing cleavage sites of gag-pol viral polypeptide. The kinetic data were interpreted with the aid of molecular modeling to understand the effect of mutations on inhibitor binding and processing of the gag-pol polypeptide to generate infective virions.
J Pharm Sci 1998 Mar;87(3):306-10
Department of Pharmaceutics, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut 06877, USA.
To combat infection and inhibit viral replication of HIV in the brain, antiretroviral agents must cross the blood-brain barrier (BBB). An in vitro BBB model consisting of bovine brain microvessel endothelial cells grown on porous filters was used to study and compare the transport of nevirapine, a potent and selective nonnucleoside reverse transcriptase inhibitor, with other HIV antiretroviral agents currently in use for the treatment of HIV infection. These included nucleoside reverse transcriptase inhibitors (didanosine, stavudine, zalcitabine, zidovudine), a nonnucleoside reverse transcriptase (delaviridine), and protease inhibitors (indinavir, saquinavir, VX-478). Nevirapine was the most permeable antiretroviral agent studied in the BBB model. The order of in vitro BBB permeability was nevirapine >> VX-478 > didanosine, stavudine, zalcitabine, zidovudine > indinavir > saquinavir. There was an apparent bell-shaped relationship between in vitro BBB permeability and octanol/phosphate-buffered saline distribution coefficient (D) where all lipophilic (log D > 2.5) as well as hydrophilic (log D < -0.5) antiretrovirals were less permeable than nevirapine (log D = 1.8). There were no significant effects on the in vitro BBB permeability of nevirapine in combination with other antiretroviral agents. Saquinavir was the only drug shown to have an affinity for the P-glycoprotein efflux pump, which may have contributed to its very low permeability. The apparent ability of nevirapine to readily permeate the BBB and enter the brain, where it may inhibit replication of HIV, potentially increases its therapeutic value.
J Infect Dis 1997 May;175(5):1063-70
CIBA-GEIGY Ltd., Pharmaceutical Research, Basel, Switzerland.
Protein binding can impair the potency of human immunodeficiency virus (HIV) protease inhibitors. Therefore, the activity of a novel compound, CGP 61755, was studied in the absence or presence of alpha1-acid glycoprotein (alpha1AGP). In MT-2 cells, the activity loss was 4-fold (EC90 without alpha1AGP, 29 nM vs. 122 nM with alpha1AGP). In primary lymphocytes, the loss was 8-fold (EC90, 45 nM vs. 364 nM). In identical experiments, the activity loss in MT-2 cells and lymphocytes was 2- and 3-fold, respectively, for indinavir, 11- and 10-fold for saquinavir, and 11- and 48-fold for ritonavir. For SC-52151, a 17-fold loss was seen in MT-2 cells, whereas no EC90 with alpha1AGP was reached in lymphocytes. This study demonstrates that the impact of alpha1AGP on in vitro activity varies greatly among different HIV protease inhibitors. The magnitude of such differences is greater in human lymphocytes than in a standard cell line.
Clin Pharmacokinet 1997 Mar;32(3):194-209
Department of Pharmacology and Therapeutics, University of Liverpool, England.
Since its introduction in 1987, zidovudine monotherapy has been the treatment of choice for patients with HIV infection. Unfortunately it has been established that the beneficial effects of zidovudine are not sustained due to the development of resistant viral strains. This has led to the strategy of combination therapy, and in 1995 treatment with zidovudine plus didanosine, or zidovudine plus zalcitabine, was demonstrated to be more effective than zidovudine monotherapy in preventing disease progression and reducing mortality in patients with HIV disease. Recent work demonstrates an even greater antiviral effect from triple therapy with 2 nucleosides, zidovudine plus zalcitabine with the addition of saquinavir, a new protease inhibitor drug. The HIV protease enzyme is responsible for the post-translational processing of gag and gag-pol polyprotein precursors, and its inhibition by drugs such as saquinavir, ritonavir, indinavir and VX-478 results in the production of non-infectious virions. As resistance may also develop to the protease inhibitors they may be used in combination, and future strategies may well include quadruple therapy with 2 nucleoside analogues plus 2 protease inhibitors. Administration of protease inhibitors alone or in combination with other drugs does raise a number of important pharmacokinetic issues for patients with HIV disease. Some protease inhibitors (e.g. saquinavir) have kinetic profiles characterised by reduced absorption and a high first pass effect, resulting in poor bioavailability which may be improved by administrating with food. Physiological factors including achlorhydria, malabsorption and hepatic dysfunction may influence the bioavailability of protease inhibitors in HIV disease. Protease inhibitors are very highly bound to plasma proteins (> 98%), predominantly to alpha 1-acid glycoprotein. This may influence their antiviral activity in vitro and may also predispose to plasma protein displacement interactions. Such interactions are usually only of clinical relevance if the metabolism of the displaced drug is also inhibited. This is precisely the situation likely to pertain to the protease inhibitors, as ritonavir may displace other protease inhibitor drugs, such as saquinavir, from plasma proteins and inhibit their metabolism. Protease inhibitors are extensively metabolised by the cytochrome P450 (CYP) enzymes present in the liver and small intestine. In vitro studies suggest that the most influential CYP isoenzyme involved in the metabolism of the protease inhibitors is CYP3A, with the isoforms CYP2C9 and CYP2D6 also contributing. Ritonavir has an elimination half-life (t1/2 beta) of 3 hours, indinavir 2 hours and saquinavir between 7 and 12 hours. Renal elimination is not significant, with less than 5% of ritonavir and saquinavir excreted in the unchanged form. As patients with HIV disease are likely to be taking multiple prolonged drug regimens this may lead to drug interactions as a result of enzyme induction or inhibition. Recognised enzyme inducers of CYP3A, which are likely to be prescribed for patients with HIV disease, include rifampicin (rifampin) [treatment of pulmonary tuberculosis], rifabutin (treatment and prophylaxis of Mycobacterium avium complex), phenobarbital (phenobarbitone), phenytoin and carbamazepine (treatment of seizures secondary to cerebral toxoplasmosis or cerebral lymphoma). These drugs may reduce the plasma concentrations of the protease inhibitors and reduce their antiviral efficacy. If coadministered drugs are substrates for a common CYP enzyme, the elimination of one or both drugs may be impaired. Drugs which are metabolised by CYP3A and are likely to be used in the treatment of patients with HIV disease include the azole antifungals, macrolide antibiotics and dapsone; therefore, protease inhibitors may interact with these drugs.
Antimicrob Agents Chemother 1997 Mar;41(3):654-60
Department of Infectious Diseases Research, Abbott Laboratories, Illinois 60064, USA. Dale.J.Kempf@abbott.com
Coadministration with the human immunodeficiency virus (HIV) protease inhibitor ritonavir was investigated as a method for enhancing the levels of other peptidomimetic HIV protease inhibitors in plasma. In rat and human liver microsomes, ritonavir potently inhibited the cytochrome P450 (CYP)-mediated metabolism of saquinavir, indinavir, nelfinavir, and VX-478. The structural features of ritonavir responsible for CYP binding and inhibition were examined. Coadministration of other protease inhibitors with ritonavir in rats and dogs produced elevated and sustained plasma drug levels 8 to 12 h after a single dose. Drug exposure in rats was elevated by 8- to 46-fold. A > 50-fold enhancement of the concentrations of saquinavir in plasma was observed in humans following a single codose of ritonavir (600 mg) and saquinavir (200 mg). These results indicate that ritonavir can favorably alter the pharmacokinetic profiles of other protease inhibitors. Combination regimens of ritonavir and other protease inhibitors may thus play a role in the treatment of HIV infection. Because of potentially substantial drug level increases, however, such combinations require further investigation to establish safe regimens for clinical use.
J Biol Chem 1996 Dec 27;271(52):33231-5
Department of Virology, Glaxo Wellcome, Stevenage SG1 2NY, United Kingdom.
Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V >> L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0. 54 s-1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.
Other Formats: Links: OBJECTIVE: To highlight recent developments in the field of antiretroviral therapy and viral load monitoring. METHODS: Review of studies detailing the efficacy of the antiretroviral agents and combinations furthest along in clinical development and the application of plasma HIV RNA quantification as a disease marker. RESULTS: Developments in the field of antiretroviral therapy have led to substantial advances in the approach to management of HIV-infected persons. These include the end of the zidovudine (ZDV) monotherapy era; the demonstration of a survival benefit conferred by antiretroviral therapy in patients with CD4 counts of 200-500x10(6)/l; the further development of newer nucleoside analog combinations (e.g., ZDV-lamivudine, stavudine-didanosine, stavudine-lamivudine, ZDV-1592U89) and the non-nucleoside reverse transcriptase inhibitor class of compounds; and, perhaps most importantly, the advent of the protease inhibitor era. Trials of ritonavir and saquinavir have proven that clinical benefit can be conferred by protease inhibitors, and three-drug combination regimens, such as indinavir-ZDV-lamivudine, have shown the potential for degrees of viral suppression not previously seen. Newer protease inhibitors, such as nelfinavir and VX-478/GW141W94, hold promise for further advances. The concurrent development of assays to quantitatively measure plasma HIV RNA has provided laboratory tools to improve our understanding of disease pathogenesis, to assess the in vivo potency of treatment regimens and to characterize the risk of disease progression. CONCLUSIONS: Recent progress in HIV disease pathogenesis, antiretroviral therapy and viral load monitoring indicates the interdependence of these factors. The current optimism in the field is warranted but complex challenges must be met if the fulfilment of this hope is to be realized by the world community. Publication Types: Review Review, tutorial MeSH Terms: Adult Anti-HIV Agents/therapeutic use* Clinical Trials Drug Therapy, Combination Human HIV Infections/drug therapy* Viral Load* Substances: Anti-HIV Agents PMID: 8970706, UI: 97125634 Other Formats: Links: Order this document AIDS 1996 Nov;10 Suppl 1:S15-9 Rational approaches to resistance: using saquinavir. Boucher C Department of Virology, Eykman-Winkler Institute, University Hospital Utrecht, The Netherlands. AIM: To review drug-resistance patterns of HIV protease inhibitors, with particular reference to saquinavir, and how resistance and cross-resistance patterns may influence disease management. RESISTANCE TO SAQUINAVIR: Resistance to saquinavir in vitro and in vivo is associated with mutations L90M and G48V in HIV protease. L90M is the predominant mutation in vivo. Clinically, G48V is uncommon and the double mutation rare. This pattern of mutation differs from those seen with other protease inhibitors. CROSS-RESISTANCE: Long-term treatment with saquinavir in most cases does not induce a significant decrease in sensitivity to saquinavir itself or other protease inhibitors. Where significant resistance to saquinavir does develop (i.e. fourfold increase in the median inhibitory concentration), there are observed instances of cross-resistance. Preliminary phenotypic studies of patients on combination therapy with saquinavir (plus zalcitabine and/or zidovudine) for 1 year indicate that > 80% should subsequently respond to indinavir, ritonavir or VX-478. CONCLUSIONS: Resistance to saquinavir develops slowly and in a minority of patients on long-term therapy. Reduced susceptibility to saquinavir is associated with mutations different from those associated with other HIV protease inhibitors. Saquinavir appears to be a good first-choice protease inhibitor for combination therapy with HIV reverse transcriptase inhibitors as it should provide prolonged antiretroviral activity without limiting subsequent therapeutic options. Publication Types: Review Review, tutorial MeSH Terms: Anti-HIV Agents/therapeutic use* Anti-HIV Agents/administration & dosage Drug Resistance, Microbial Drug Therapy, Combination Genotype Human HIV/genetics HIV/enzymology HIV/drug effects HIV Infections/virology HIV Infections/drug therapy* HIV Protease/genetics HIV Protease Inhibitors/therapeutic use* HIV Protease Inhibitors/administration & dosage Point Mutation Saquinavir/therapeutic use* Saquinavir/administration & dosage Substances: Saquinavir HIV Protease Inhibitors Anti-HIV Agents HIV Protease PMID: 8970671, UI: 97125599 Other Formats: Links: Order this document Drugs 1996 Oct;52(4):541-6; discussion 547-8 Ritonavir. Lea AP, Faulds D Adis International Limited, Auckland, New Zealand. Ritonavir is a protease inhibitor with an HIV-1 resistance profile similar to that of indinavir, but different from that of saquinavir. Ritonavir has good oral bioavailability, and may increase the bioavailability of other protease inhibitors including saquinavir, nelfinavir, indinavir and VX-478. Clinically significant drug interactions have been predicted between ritonavir and a range of medications. In patients with HIV-1 infection, ritonavir markedly reduced viral load within 2 weeks of treatment onset and also increased CD4+ cell counts. In a large placebo-controlled trial in patients with advanced HIV infection, the addition of ritonavir to existing therapy reduced the risk of mortality by 43% and clinical progression by 56% after 6.1 months. Triple therapy with ritonavir plus zidovudine, in combination with lamivudine or zalcitabine, reduced HIV viraemia to below detectable levels in most patients with acute, and some patients with advanced HIV infection in 2 small trials. Early results suggest combination therapy with ritonavir and saquinavir increases CD4+ cell counts and decreases HIV RNA levels in patients with previously untreated HIV infection. Publication Types: Review Review, tutorial MeSH Terms: Administration, Oral Anti-HIV Agents/therapeutic use Anti-HIV Agents/pharmacokinetics Anti-HIV Agents/pharmacology* Biological Availability Comparative Study Cytochrome P-450/metabolism Cytochrome P-450/genetics CD4-Positive T-Lymphocytes/drug effects* CD4-Positive T-Lymphocytes/cytology Dose-Response Relationship, Drug Double-Blind Method Drug Therapy, Combination Drug Tolerance Human HIV Infections/mortality HIV Infections/genetics HIV Infections/drug therapy* HIV Protease Inhibitors/therapeutic use HIV Protease Inhibitors/pharmacokinetics HIV Protease Inhibitors/pharmacology* Lamivudine/therapeutic use Lamivudine/pharmacology Lamivudine/administration & dosage Leukocyte Count/drug effects Randomized Controlled Trials Ritonavir/therapeutic use Ritonavir/pharmacokinetics Ritonavir/pharmacology* Ritonavir/metabolism RNA, Messenger/metabolism Zalcitabine/therapeutic use Zalcitabine/pharmacology Zalcitabine/administration & dosage Zidovudine/therapeutic use Zidovudine/pharmacology Zidovudine/administration & dosage Substances: Cytochrome P-450 Zalcitabine Zidovudine Lamivudine RNA, Messenger Ritonavir HIV Protease Inhibitors Anti-HIV Agents PMID: 8891466, UI: 97046545 Other Formats: Links: Order this document J Biol Chem 1996 Jul 26;271(30):17979-85 Kinetic characterization of human immunodeficiency virus type-1 protease-resistant variants. Pazhanisamy S, Stuver CM, Cullinan AB, Margolin N, Rao BG, Livingston DJ Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139, USA. Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding. MeSH Terms: Amino Acid Sequence Binding Sites Comparative Study Hydrolysis HIV Protease/genetics* HIV Protease/drug effects HIV Protease Inhibitors/pharmacology HIV-1/genetics* HIV-1/enzymology* Isoquinolines/pharmacology Kinetics Models, Molecular Molecular Sequence Data Mutation* Oligopeptides/metabolism Pyridines/pharmacology Quinolines/pharmacology Selection (Genetics) Substrate Specificity Sulfonamides/pharmacology Variation (Genetics) Substances: VX 478 Indinavir Saquinavir Sulfonamides Quinolines Pyridines Oligopeptides Isoquinolines HIV Protease Inhibitors HIV Protease PMID: 8663409, UI: 96279343 Other Formats: Links: Order this document Rapid Commun Mass Spectrom 1996;10(9):1019-26 In vitro metabolism of a potent HIV-protease inhibitor (141W94) using rat, monkey and human liver S9. Singh R, Chang SY, Taylor LC Glaxo Wellcome Inc., Research Triangle Park, NC 27709, USA. Compound 141W94 (Vertex VX478) (3S)-tetrahydro-3-furyl N-[((S,2R)-3-(4-amino-N-isobutylbenzenesulfonamido)-1-benzyl- 2-hydroxypropyl] carbamate, is a potent HIV-protease inhibitor and is currently undergoing clinical trials. The purpose of this study was the rapid identification of the phase I and II in vitro metabolite of 141W94 using mass spectrometry. Four different sources of liver S9 fractions were used for studying comparative in vitro metabolism of 141W94. They were obtained from Arochlor-induced rat, normal (untreated) rat, cynomolgus monkey and human livers. Selected incubations were supplemented with uridine diphosphate glucuronic acid and the reduced form of glutathione. The predominant species seen in the incubation mixture was the parent compound 141W94. Metabolites arising from ring opening to form the diol and carboxylic acid and oxidation of the tetrahydrofurran ring (formation of dihydrofuran) were identified. In addition, of the two monohydroxylated products identified, one resulted from hydroxylation on the aniline ring and the other from hydroxylation at the benzylic position. Two different glucuronides were also observed. Comparing the three species, very little metabolism was seen in the normal (non-induced) rat. The metabolic profile and extent of metabolism with induced rat, monkey and human S9 was similar. Induced rat S9 incubation showed the formation of two unique metabolites that were not seen in non-induced rat, monkey and human S9 fractions. They were the monohydroxylated glucuronide and a carbamate cleavage product. The metabolites were identified using mass spectrometry based on their molecular masses and fragmentation patterns. MeSH Terms: Animal Aroclors/pharmacology Chromatography, High Pressure Liquid Comparative Study Human HIV Protease Inhibitors/metabolism* In Vitro Liver/metabolism* Liver/cytology Macaca fascicularis Rats Rats, Sprague-Dawley Species Specificity Spectrophotometry, Ultraviolet Spectrum Analysis, Mass Subcellular Fractions/metabolism Sulfonamides/metabolism* Substances: VX 478 Sulfonamides HIV Protease Inhibitors Aroclors PMID: 8755234, UI: 96298219 Other Formats: Links: Order this document Antiviral Res 1996 Jan;29(1):53-6 In vitro antiviral activity of 141W94 (VX-478) in combination with other antiretroviral agents. St Clair MH, Millard J, Rooney J, Tisdale M, Parry N, Sadler BM, Blum MR, Painter G Division of Virology, Glaxo Wellcome Inc., NC 27709, USA. 141W94 (VX-478) is a novel HIV-1 protease inhibitor with an IC50 of 0.08 microM against HIV-1 (strain IIIB) and a mean IC50 of 0.012 microM against six HIV clinical isolates. 141W94 was synergistic on the basis of isobologram analysis with each of the following reverse transcriptase inhibitors: AZT, 935U83, 524W91, 1592U89 and ddl, 141W94 was also synergistic with saquinavir and additive with either indinavir or ritonavir. Resistance to 141W94 has been reported in vitro passage experiments. The binding of 141W94 to human alpha 1-acid glycoprotein was relatively weak (Kd = 4 microM) and the off-rate for the drug is very fast (> or = 100 s-1). Only a 2-fold reduction of in vitro antiviral activity was observed in the presence of 45% human plasma. No serious drug associated adverse experiences were reported in a Phase I placebo-controlled, single-dose escalation, pharmacokinetic and safety study. The average concentration of 141W94 at 8 and 12 h after single doses of 900 and 1200 mg, respectively, was in excess of 10 times the IC50. As 141W94 is synergistic with a variety of anti-HIV-1 agents and exhibits a unique cross resistance profile compared to other protease inhibitors, 141W94 is considered a good candidate for combination therapy. Publication Types: Clinical trial Clinical trial, phase i MeSH Terms: Drug Resistance, Microbial Drug Synergism Drug Therapy, Combination Human HIV Infections/drug therapy* HIV Protease Inhibitors/therapeutic use* HIV Protease Inhibitors/administration & dosage Isoquinolines/therapeutic use Pyridines/therapeutic use Quinolines/therapeutic use Reverse Transcriptase Inhibitors/therapeutic use Sulfonamides/therapeutic use* Sulfonamides/administration & dosage Thiazoles/therapeutic use Valine/therapeutic use Valine/analogs & derivatives Substances: Valine VX 478 Indinavir Saquinavir Thiazoles Sulfonamides Ritonavir Reverse Transcriptase Inhibitors Quinolines Pyridines Isoquinolines HIV Protease Inhibitors PMID: 8721545, UI: 96283736 Other Formats: Links: Order this document J Infect Dis 1995 Nov;172(5):1238-45 Weak binding of VX-478 to human plasma proteins and implications for anti-human immunodeficiency virus therapy. Livington DJ, Pazhanisamy S, Porter DJ, Partaledis JA, Tung RD, Painter GR Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA. VX-478 is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Ki, 0.6 nM) and of HIV-1 replication in antiviral assays (IC90, 80 nM). The fractional binding of VX-478 to human plasma and to purified plasma proteins was determined by equilibrium dialysis and difference UV spectrophotometry. Binding to alpha 1-acid glycoprotein (89% at 2 microM total drug concentration, Kd of 4 microM) accounts for its fractional binding in plasma (93%). Stopped-flow spectrophotometry methods showed that binding is a reversible two-step process. The measured dissociation rate constant approaches 100 s-1. The antiviral effect of VX-478 was determined in the presence of 45% human plasma, in which the IC90 increased by 1.5-fold compared with control experiments in the presence of 15% fetal bovine serum. The effects of protein binding on the antiviral activity of VX-478 are minor, as expected for a weak drug-protein interaction. Comments: Comment in: J Infect Dis 1996 Jun;173(6):1524-6 MeSH Terms: Acquired Immunodeficiency Syndrome/drug therapy* Animal Antiviral Agents/blood* Blood Blood Proteins/metabolism* Cattle Fetus Human HIV Protease Inhibitors/blood* Kinetics Molecular Structure Orosomucoid/metabolism* Protein Binding Spectrophotometry, Ultraviolet Sulfonamides/blood* Substances: VX 478 Sulfonamides Orosomucoid HIV Protease Inhibitors Blood Proteins Antiviral Agents PMID: 7594659, UI: 96036379 Other Formats: Links: Order this document J Virol 1995 Sep;69(9):5228-35 In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease. Partaledis JA, Yamaguchi K, Tisdale M, Blair EE, Falcione C, Maschera B, Myers RE, Pazhanisamy S, Futer O, Cullinan AB, et al Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211, USA. Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially. MeSH Terms: Amino Acid Sequence Base Sequence Cell Line Comparative Study DNA Primers Human HIV Protease/metabolism* HIV Protease/chemistry HIV Protease Inhibitors/pharmacology* HIV-1/physiology HIV-1/isolation & purification HIV-1/drug effects* Kinetics Microbial Sensitivity Tests Models, Molecular Molecular Sequence Data Molecular Structure Mutagenesis, Site-Directed Point Mutation Polymerase Chain Reaction Protein Conformation Recombinant Proteins/chemistry Recombinant Proteins/biosynthesis Recombinant Proteins/antagonists & inhibitors Structure-Activity Relationship Sulfonamides/pharmacology* T-Lymphocytes Virus Replication/drug effects Substances: VX 478 VB 11328 Sulfonamides Recombinant Proteins HIV Protease Inhibitors DNA Primers HIV Protease PMID: 7636964, UI: 95363927 Other Formats: Links: Order this document Antimicrob Agents Chemother 1995 Aug;39(8):1704-10 Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors. Tisdale M, Myers RE, Maschera B, Parry NR, Oliver NM, Blair ED Wellcome Research Laboratories, Beckenham, Kent, United Kingdom. Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy. MeSH Terms: Amino Acid Sequence Base Sequence Cell Line Drug Resistance, Microbial Human HIV Protease/genetics HIV Protease Inhibitors/pharmacology* HIV-1/genetics HIV-1/enzymology HIV-1/drug effects* Molecular Sequence Data Mutation Polymerase Chain Reaction Reverse Transcriptase Inhibitors/pharmacology Support, Non-U.S. Gov't Zidovudine/pharmacology Substances: Zidovudine Reverse Transcriptase Inhibitors HIV Protease Inhibitors HIV Protease PMID: 7486905, UI: 96100736 the above reports in Macintosh PC UNIX Text HTML format documents on this page through Loansome Doc
AIDS 1996 Nov;10 Suppl 1:S15-9
Department of Virology, Eykman-Winkler Institute, University Hospital Utrecht, The Netherlands.
AIM: To review drug-resistance patterns of HIV protease inhibitors, with particular reference to saquinavir, and how resistance and cross-resistance patterns may influence disease management. RESISTANCE TO SAQUINAVIR: Resistance to saquinavir in vitro and in vivo is associated with mutations L90M and G48V in HIV protease. L90M is the predominant mutation in vivo. Clinically, G48V is uncommon and the double mutation rare. This pattern of mutation differs from those seen with other protease inhibitors. CROSS-RESISTANCE: Long-term treatment with saquinavir in most cases does not induce a significant decrease in sensitivity to saquinavir itself or other protease inhibitors. Where significant resistance to saquinavir does develop (i.e. fourfold increase in the median inhibitory concentration), there are observed instances of cross-resistance. Preliminary phenotypic studies of patients on combination therapy with saquinavir (plus zalcitabine and/or zidovudine) for 1 year indicate that > 80% should subsequently respond to indinavir, ritonavir or VX-478. CONCLUSIONS: Resistance to saquinavir develops slowly and in a minority of patients on long-term therapy. Reduced susceptibility to saquinavir is associated with mutations different from those associated with other HIV protease inhibitors. Saquinavir appears to be a good first-choice protease inhibitor for combination therapy with HIV reverse transcriptase inhibitors as it should provide prolonged antiretroviral activity without limiting subsequent therapeutic options.
Drugs 1996 Oct;52(4):541-6; discussion 547-8
Adis International Limited, Auckland, New Zealand.
Ritonavir is a protease inhibitor with an HIV-1 resistance profile similar to that of indinavir, but different from that of saquinavir. Ritonavir has good oral bioavailability, and may increase the bioavailability of other protease inhibitors including saquinavir, nelfinavir, indinavir and VX-478. Clinically significant drug interactions have been predicted between ritonavir and a range of medications. In patients with HIV-1 infection, ritonavir markedly reduced viral load within 2 weeks of treatment onset and also increased CD4+ cell counts. In a large placebo-controlled trial in patients with advanced HIV infection, the addition of ritonavir to existing therapy reduced the risk of mortality by 43% and clinical progression by 56% after 6.1 months. Triple therapy with ritonavir plus zidovudine, in combination with lamivudine or zalcitabine, reduced HIV viraemia to below detectable levels in most patients with acute, and some patients with advanced HIV infection in 2 small trials. Early results suggest combination therapy with ritonavir and saquinavir increases CD4+ cell counts and decreases HIV RNA levels in patients with previously untreated HIV infection.
J Biol Chem 1996 Jul 26;271(30):17979-85
Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.
Rapid Commun Mass Spectrom 1996;10(9):1019-26
Glaxo Wellcome Inc., Research Triangle Park, NC 27709, USA.
Compound 141W94 (Vertex VX478) (3S)-tetrahydro-3-furyl N-[((S,2R)-3-(4-amino-N-isobutylbenzenesulfonamido)-1-benzyl- 2-hydroxypropyl] carbamate, is a potent HIV-protease inhibitor and is currently undergoing clinical trials. The purpose of this study was the rapid identification of the phase I and II in vitro metabolite of 141W94 using mass spectrometry. Four different sources of liver S9 fractions were used for studying comparative in vitro metabolism of 141W94. They were obtained from Arochlor-induced rat, normal (untreated) rat, cynomolgus monkey and human livers. Selected incubations were supplemented with uridine diphosphate glucuronic acid and the reduced form of glutathione. The predominant species seen in the incubation mixture was the parent compound 141W94. Metabolites arising from ring opening to form the diol and carboxylic acid and oxidation of the tetrahydrofurran ring (formation of dihydrofuran) were identified. In addition, of the two monohydroxylated products identified, one resulted from hydroxylation on the aniline ring and the other from hydroxylation at the benzylic position. Two different glucuronides were also observed. Comparing the three species, very little metabolism was seen in the normal (non-induced) rat. The metabolic profile and extent of metabolism with induced rat, monkey and human S9 was similar. Induced rat S9 incubation showed the formation of two unique metabolites that were not seen in non-induced rat, monkey and human S9 fractions. They were the monohydroxylated glucuronide and a carbamate cleavage product. The metabolites were identified using mass spectrometry based on their molecular masses and fragmentation patterns.
Antiviral Res 1996 Jan;29(1):53-6
Division of Virology, Glaxo Wellcome Inc., NC 27709, USA.
141W94 (VX-478) is a novel HIV-1 protease inhibitor with an IC50 of 0.08 microM against HIV-1 (strain IIIB) and a mean IC50 of 0.012 microM against six HIV clinical isolates. 141W94 was synergistic on the basis of isobologram analysis with each of the following reverse transcriptase inhibitors: AZT, 935U83, 524W91, 1592U89 and ddl, 141W94 was also synergistic with saquinavir and additive with either indinavir or ritonavir. Resistance to 141W94 has been reported in vitro passage experiments. The binding of 141W94 to human alpha 1-acid glycoprotein was relatively weak (Kd = 4 microM) and the off-rate for the drug is very fast (> or = 100 s-1). Only a 2-fold reduction of in vitro antiviral activity was observed in the presence of 45% human plasma. No serious drug associated adverse experiences were reported in a Phase I placebo-controlled, single-dose escalation, pharmacokinetic and safety study. The average concentration of 141W94 at 8 and 12 h after single doses of 900 and 1200 mg, respectively, was in excess of 10 times the IC50. As 141W94 is synergistic with a variety of anti-HIV-1 agents and exhibits a unique cross resistance profile compared to other protease inhibitors, 141W94 is considered a good candidate for combination therapy.
J Infect Dis 1995 Nov;172(5):1238-45
Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA.
VX-478 is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Ki, 0.6 nM) and of HIV-1 replication in antiviral assays (IC90, 80 nM). The fractional binding of VX-478 to human plasma and to purified plasma proteins was determined by equilibrium dialysis and difference UV spectrophotometry. Binding to alpha 1-acid glycoprotein (89% at 2 microM total drug concentration, Kd of 4 microM) accounts for its fractional binding in plasma (93%). Stopped-flow spectrophotometry methods showed that binding is a reversible two-step process. The measured dissociation rate constant approaches 100 s-1. The antiviral effect of VX-478 was determined in the presence of 45% human plasma, in which the IC90 increased by 1.5-fold compared with control experiments in the presence of 15% fetal bovine serum. The effects of protein binding on the antiviral activity of VX-478 are minor, as expected for a weak drug-protein interaction.
Comments:
J Virol 1995 Sep;69(9):5228-35
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211, USA.
Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.
Antimicrob Agents Chemother 1995 Aug;39(8):1704-10
Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.
Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.
the above reports in Macintosh PC UNIX Text HTML format documents on this page through Loansome Doc
