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Toxicol Appl Pharmacol 1997 Sep;146(1):40-52
Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Oral administration of the HIV protease inhibitor L-689,502 caused cholestasis and hepatocyte injury in rats and dogs. These changes occurred rapidly, with elevations in serum transaminase observed as early as 6 hr after oral dosing in dogs. The acute phase of this hepatotoxic response was characterized in more detail in rats. Following intravenous administration, bile flow was decreased in a dose-dependent manner with greater than 90% decrease in less than 30 min at a dose of 5 mg/kg. The decrease in bile flow was associated with a decrease in erythritol clearance. The decrease in bile flow was not due to disruption of biliary tight junctions. Sucrose clearance was not increased and biliary bile acid concentrations in treated animals were not different from controls. Unlike control animals, bile flow was not stimulated by infusion of the bile acid tauroursodeoxycholic acid in animals treated with L-689,502. These cholestatic effects may be due, in part, to direct hepatocyte injury. Histological examination of perfusion-fixed livers 30 min after L-689,502 administration revealed periportal changes including hepatocyte vacuolation and occasional single cell necrosis. On a subcellular level, the nucleus and mitochondria were intact in less-severely affected cells. However, extensive vacuolation with multilamellar inclusions was pronounced in these cells. In addition, canalicular ectasia was also observed which was consistent with the cholestatic changes that were seen. In summary, L-689,502 is a potent, rapid acting hepatotoxin in dogs and rats. The mechanism by which this agent induces cholestasis is novel compared to other well-characterized cholestatic agents such as alpha-naphtylisothiocyanate and ethinyl estradiol. Copyright 1997 Academic Press.
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Antiviral Res 1997 Mar;34(1):1-15
Department of Microbiology, School of Dentistry, University of the Pacific, San Francisco, CA 94115, USA.
Macrophages are recognized as a major reservoir of HIV-1 in infected individuals. We examined the effect of an inhibitor of the viral protease, L-689,502, on virus production by monocyte-derived macrophages infected with HIV-1BaL. Continuous treatment with L-689,502 drastically inhibited virus production in a dose-dependent manner in the range of 10-200 nM, in some cases by more than 1000-fold, compared to untreated cells. Since liposomes can be targeted to macrophages in vivo, we examined whether the inhibitor was effective following delivery in liposomes. The inhibitor encapsulated in multilamellar liposomes was more effective than the free drug in inhibiting virus production in macrophages, throughout the concentration range studied. The EC90 of the liposomal inhibitor was 2.9- to 4.5-fold lower than that of the free compound. L-689,502 encapsulated in sterically stabilized liposomes with prolonged circulation time inhibited virus production at a level comparable to the free inhibitor. When macrophages were infected and treated for only a limited time, L-689,502 in multilamellar liposomes was the most effective of the three treatments. In chronically infected H9 cells treated continuously, the free inhibitor was more effective than the liposome-encapsulated drug, but virus production was reduced only to 40-60% of controls. In contrast, treatment of acutely infected H9 cells with either free or encapsulated L-689,502 inhibited virus production by up to three orders of magnitude. Our results indicate that liposomes may be useful for the delivery of HIV protease inhibitors with low aqueous solubility and low oral bio-availability, and for the targeting of these drugs to lymph nodes.
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Virology 1996 May 1;219(1):307-13
Department of Medicine, University of Alabama at Birmingham 35294, USA.
In addition to Gag, Pol, and Env, primate lentiviruses encode other virion-associated proteins, including Vpr, Vpx, and Vif. Vpr- and Vpx-staphylococcal nuclease and chloramphenicol acetyltransferase fusion proteins incorporate into human immunodeficiency virus (HIV) virions and retain enzyme activity when expressed in trans with HIV proviruses (Wu et al., J. Virol. 69, 3389, 1995). To explore whether the viral protease (PR) could be expressed as a proteolytically active fusion protein, the HIV PR coding region was fused in-frame with the HIV-2 vpx and HIV-1 vpr genes. Using a vaccinia virus-T7 expression system, the Vpx-PR fusion protein was expressed and formed homodimers. Coexpression with Pr55Gag demonstrated that Vpx-PR possessed Gag-specific proteolytic activity and inhibited the production of Gag virus-like particles. Trans-expression of a PR-Vpr fusion protein with HIV-1 provirus caused a profound reduction in viral protein expression and virion production. Importantly, the PR-Vpr fusion protein caused a similar level of inhibition and intracellular cleavage of Pr55Gag precursor protein when coexpressed with protease defective HIV-1 provirus. The inhibitory effect of PR-Vpr expression on virion production was markedly greater than that of PR alone. These results indicate that Vpr arguments the intracellular proteolytic activity of PR when expressed as a fusion protein and thus may be relevant for the expression of PR in intracellular immunization strategies against HIV infection. Moreover, the ability to express and package enzymatically active PR-Vpr fusion protein, independent of Gag/Pol, may provide a novel means to study enzyme function.
Drug Metab Dispos 1995 Feb;23(2):185-9
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
L-689,502, N-[2(R)-hydroxy-1(S)-indanyl]-5(S)-(1,1-dimethylethoxy- carbonyl-amino)-4(S)-hydroxy-6-phenyl-2(R)-(4-[2(R)-(4-morpholinyl) ethoxy]phenyl)methylhexamide, is a potent and specific inhibitor of human immunodeficiency virus-type 1 (HIV-1) protease in vitro. Metabolism of this compound in rat liver slices produced four major and several minor metabolites. The major metabolites were identified as morpholin-2-one, 3'(S)-hydroxyindan and 4'-hydroxyindan analogs, and a 4-O-glucuronic acid conjugate of the parent compound. The metabolites were characterized by Heteronuclear Multiple Quantum Coherence and Nuclear Overhauser Effect techniques in NMR spectroscopy, by MS, and/or comparison with authentic standards. Two of the minor metabolites were similarly characterized as a 2(R)-[4-(2-carboxymethoxy)phenyl]methyl analog and a product with a degraded morpholino ring. The hydroxyindan metabolites were lower in activity than L-689,502, whereas the morpholin-2-one and carboxymethoxyphenyl analogs were approximately 6- and 11-fold more potent as inhibitors of HIV-1 protease, respectively.
Biochemistry 1994 Mar 1;33(8):2004-10
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486.
The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.
J Pharmacol Exp Ther 1992 Oct;263(1):105-11
Merck Research Laboratories, West Point, Pennsylvania.
L-689,502, N-[2(R)-hydroxy-1(S)-indanyl]-5(S)-(1,1-dimethylethoxy- carbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(4-[2-(4- morpholinyl)ethoxy]phenyl)methyl hexanamide, is a potent inhibitor of human immunodeficiency virus-1 protease. The effect of dose on the elimination kinetics of L-689,502 was studied in rats and dogs. After i.v. administration, total plasma clearance of L-689,502 in rats decreased with increasing dose; the clearance decreased from 181 ml/min/kg at 1 mg/kg to 86 ml/min/kg at 20 mg/kg. Similar results were observed in dogs; clearance fell from 29 ml/min/kg at 0.5 mg/kg to 17 ml/min/kg at 10 mg/kg. Bile flow in rats was retarded in a dose-dependent manner after a single i.v. injection of L-689,502. The cholestatic effect was reversible and maximal at 5 mg/kg i.v. Consistent with the cholestatic effect, L-689,502 caused an increase in serum levels of aminotransferase. After i.v. administration of L-689,502 (10 mg/kg), alanine aminotransferase increased from 50 to 370 IU/liter and aspartate aminotransferase from 120 to 700 IU/liter. Moreover, pretreatment of rats with L-689,502 resulted in a significant decrease in the elimination kinetics of antipyrine and diflunisal, as well as of L-689,502 itself. Collectively, these results suggest that the dose-dependent kinetics of L-689,502 in rats and dogs are more likely due to hepatotoxicity caused by the drug than to capacity-limited metabolism.
J Med Chem 1992 May 15;35(10):1685-701
Department of Medicinal Chemistry, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
By tethering of a polar hydrophilic group to the P1 or P1' substituent of a Phe-based hydroxyethylene isostere, the antiviral potency of a series of HIV protease inhibitors was improved. The optimum enhancement of anti-HIV activity was observed with the 4-morpholinylethoxy substituent. The substituent effect is consistent with a model derived from inhibitor docked in the crystal structure of the native enzyme. An X-ray crystal structure of the inhibited enzyme determined to 2.25 A verifies the modeling predictions.
Biochem Biophys Res Commun 1991 Dec 31;181(3):1456-61
Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.
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