Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy.

(19)F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of (19)F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the (3)H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with (19)F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.

Incidence of cancer after a first episode of idiopathic venous thromboembolism treated with 3 months or 1 year of oral anticoagulation.

BACKGROUND: A prolonged treatment with oral anticoagulants has been claimed to reduce the incidence of newly diagnosed cancer in the long-term follow-up of patients with venous thromboembolism. OBJECTIVES: In a multicenter prospective study we assessed the incidence of newly diagnosed clinically overt cancer in patients with a first episode of idiopathic venous thromboembolism (VTE) treated with oral anticoagulants for 3 months or 1 year. PATIENTS AND METHODS: Consecutive patients with an idiopathic venous thromboembolism who had completed 3 months of oral anticoagulant therapy without having a recurrence, bleeding or newly diagnosed cancer were randomized to discontinue oral anticoagulant therapy or to continue it for nine additional months. Idiopathic venous thromboembolism was defined as thrombosis occurring in the absence of known cancer, known thrombophilia, or temporary risk factors for venous thromboembolism. All patients were followed up for at least 1 year after randomization. RESULTS: A total of 429 patients, 265 patients with DVT and 164 with PE, were followed up for an average of 43.7 months after randomization. A newly diagnosed cancer occurred in 32 patients (7.5%), 13 (6.2%) of the 210 patients treated for 3 months and 19 (8.7%) of the 219 patients treated for 1 year (RR = 0.71, 95% confidence interval 0.36-1.41). CONCLUSIONS: The incidence of newly diagnosed clinically overt cancer is not reduced in patients with idiopathic venous thromboembolism treated with 1-year anticoagulant treatment compared with patients treated for 3 months.

A new method for chemoselective conjugation of unprotected peptides to dauno- and doxorubicin.

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

Sensitivity and specificity of denaturing high-pressure liquid chromatography for unknown protein C gene mutations.

Screening methods for unknown DNA sequence variations are laborious, expensive, and relatively insensitive. To evaluate the sensitivity and specificity of denaturing high-pressure liquid chromatography (DHPLC) screening for unknown protein C gene (PROC) mutations, we studied 31 PROC-deficient patients. Eleven amplimers containing 4 kb of the PROC gene and spanning all exons, splice junctions, and the putative promoter and 3'-untranslated regions were amplified by PCR for each patient. Each amplimer (n = 341) was sequenced with a fluorescence-based method, and screened by DHPLC. Sequencing identified 10 unique mutations and three polymorphisms. Combining all mutations and polymorphisms, 227 amplimers were homozygous wildtype, and 63 and 51 were heterozygous and homozygous mutant, respectively. DHPLC screening correctly identified all amplimers (100% sensitivity and specificity). DHPLC is a rapid, automated, sensitive and specific screening method for unknown mutations within the PROC gene, and may be a useful screening method for unknown mutations within other genes.

Building effective prophylaxis of deep vein thrombosis in the outpatient setting.

Perioperative prophylaxis of venous thromboembolism (VTE) usually coincides with the duration of hospital stay, typically lasting between 5 and 14 days. However, mounting evidence indicates that the risk of postoperative VTE persists for at least five weeks after orthopaedic surgery and six weeks following general surgery, suggesting that prolonged prophylaxis may reduce the burden of thromboembolic complications. As interest in extending prophylaxis grows, however, economic pressures and patient preferences are contributing to ever shorter hospital stays, giving rise to an exploration of administering prolonged prophylaxis to ambulatory patients at home. Effective outpatient prophylaxis for VTE must be safe, simple to use and convenient for patients to maximize compliance. Low-molecular-weight heparins (LMWHs) are at least as safe and effective as standard low-dose unfractionated heparin (UFH) in preventing thromboembolic events, and can be administered subcutaneously at a fixed daily dose without monitoring. Clinical studies in patients with total hip replacement have demonstrated that LMWHs, administered at home once daily for up to four weeks following hospital discharge, are safe and well-tolerated and significantly reduce the incidence of post-discharge deep vein thrombosis. Due to the high costs of treating thromboembolic complications and post-thrombotic syndrome, appropriately targeted prolonged thromboprophylaxis may be cost-effective.

Dermatan sulphate in patients with heparin-induced thrombocytopenia.

Patients with thromboembolic diseases who develop heparin-induced thrombocytopenia (HIT) type II require an alternative anticoagulation strategy. Dermatan sulphate (DS) was administered to five patients with thromboembolic diseases who developed HIT type II and showed an in vitro cross-reactivity with low molecular weight heparins. The platelet count and the extension of thrombosis were monitored during DS administration. In four of the five patients the platelet count rapidly increased after heparin was discontinued and DS started. A low platelet count persisted in the single patient with cross-reactivity to DS, up to 4 d after its discontinuation. None of the patients experienced thrombus extension, haemorrhagic side-effects or other adverse events.

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products.

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.

[Pulmonary embolism and cerebral stroke from paradoxical embolism in a young woman]. / Embolia polmonare ed ictus cerebrale da embolia paradossa in una giovane donna.

We describe a case of pulmonary embolism and ischemic stroke due to paradoxical embolism in a healthy young woman taking oral contraceptives to treat an ovarian cyst. It was not possible to identify the site of the thromboembolus. Ultrasound techniques played an important role in identifying the peripheral arterial obstructions and in diagnosing acute pulmonary hypertension. Transesophageal echocardiography provided detailed information on both the morphology and the evolution of the atrial thrombus straddling the foramen ovale within the aneurysmal interatrial septum. The patient was given anticoagulant treatment, initially with heparin and subsequently with warfarin over a period of six months. Repeated ultrasound controls showed no thrombus, regression of the signs of pulmonary hypertension and, lastly unchanged systemic arterial obstruction.

A continuous assay of hepatitis C virus protease based on resonance energy transfer depsipeptide substrates.

Hepatitis C virus (HCV) is the major causative agent of non-A non-B hepatitis, an important health problem with an estimated 50 million people infected worldwide. Among the possible targets for therapeutic intervention, the serine protease contained within the N-terminal region of nonstructural protein 3 (NS3 protease) is so far the best characterized. In vitro characterization of synthetic substrates based on all the natural cleavage sites (as well as a series of analogs) has consistently revealed poor kinetic parameters, making them unsuitable for sensitive high-throughput screening. To overcome these difficulties, we have recently developed depsipeptide substrates incorporating an ester bond between residues P1 and P&prime1. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates showed very high kcat/Km values, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to synthesize internally quenched depsipeptide fluorogenic substrates based on resonance energy transfer between the donor/acceptor couple 5-[(2'-aminoethyl)amino]naphthalene sulfonic acid/4-[[4'-(dimethylamino)phenyl]azo]benzoic acid, and developed a continuous assay for NS3 activity. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 between 1 nM and 250 pM. The suitability of the assay for evaluation of inhibitors was established using as competitor a tridecapeptide corresponding to the natural NS4A/4B cleavage site; this gave an IC50 of 30 microM, well in agreement with the previously found Km value (40 microM).

Synthetic depsipeptide substrates for the assay of human hepatitis C virus protease.

Hepatitis C virus (HCV) is the major etiological agent of both parenterally transmitted and sporadic non-A, non-B hepatitis. The disease is a major health problem with an estimated 50 million people infected worldwide, a high percentage of whom become chronically infected and are at high risk for liver cirrhosis. The serine protease contained within the N-terminal region of the nonstructural protein 3 (NS3 protease) of HCV is considered a promising target for the development of an antiviral therapy. A prime requisite to study in detail the biochemistry of the protease as well as develop inhibitors is the availability of a fast and sensitive in vitro assay of enzyme activity. However, due to their low kcat/Km values, synthetic peptide substrates based on the natural cleavage sites appear unsuitable for this purpose. We show here that appropriate substrates can be obtained by substituting the scissile amide bond with an ester linkage. The resulting depsipeptides show >100-fold improvement in kcat/Km values, up to 13,000 M-1 s-1, enabling detection of activity with subnanomolar NS3 concentrations. The ester substrates are obtained in high yield entirely by solid-phase synthesis using commercially available materials, without the need for any preassembled building blocks.(c) 1996 Academic Press, Inc.