Ultrasonography in Patients with Suspected Acute Appendicitis.

BACKGROUND: To evaluate the usefulness and limitations of graded compression ultrasonography in the diagnosis of clinically equivocal cases of suspected acute appendicitis at the setting of mid zonal military hospital of India. METHODS: A prospective study, graded compression ultrasonography with self localization was carried out with 3.5 MHz convex, 5 MHz convex and 7.5 MHz linear transducers (Wipro GE) in 69 clinically equivocal suspected cases of acute appendicitis. With maximal compression the anteroposterior diameter of appendix was measured from outer to outer wall. The main criterion for diagnosing appendicitis was demonstration of a non compressible appendix with anteroposterior dimension of 7mm or more. RESULT: Sonologically 36 (52%) cases were diagnosed as appendicitis. Anteroposterior outer diameter of inflamed appendices ranged from 7mm to 21mm (mean 10.5mm). 30 (83%) of 36 patients could accurately self localize the point of maximum tenderness. There were 01 false positive and 04 false negative cases. Sensitivity and specificity were 89.7% and 96.6% respectively. Positive and negative predictive values were 97.2% and 87.8% respectively. Alternative diagnoses were offered in 33 (47.8%) cases. Amongst these 33 cases, 14(42.4%) had abdominal pain of unknown origin. Gynaecologic, urologic and gastrointestinal aetiologies were established in 10(30.3%), 07(21.2%) and 02(6%) cases respectively. CONCLUSION: Graded compression ultrasonography superadded with self localization is an accurate means of diagnosing/excluding appendicitis in clinically equivocal cases of acute appendicitis and it is of great value in establishing alternative diagnoses.

Testing paradigm for prediction of development-limiting barriers and human drug toxicity.

The financial investment grows exponentially as a new chemical entity advances through each stage of discovery and development. The opportunity exists for the modern toxicologist to significantly impact expenditures by the early prediction of potential toxicity/side effect barriers to development by aggressive evaluation of development-limiting liabilities early in drug discovery. Improved efficiency in pharmaceutical research and development lies both in leveraging "best in class" technology and integration with pharmacologic activities during hit-to-lead and early lead optimization stages. To meet this challenge, a discovery assay by stage (DABS) paradigm should be adopted. The DABS clearly delineates to discovery project teams the timing and type of assay required for advancement of compounds to each subsequent level of discovery and development. An integrative core pathology function unifying Drug Safety Evaluation, Molecular Technologies and Clinical Research groups that effectively spans all phases of drug discovery and development is encouraged to drive the DABS. The ultimate goal of such improved efficiency being the accurate prediction of toxicity and side effects that would occur in development before commitment of the large prerequisite resource. Good justification of this approach is that every reduction of development attrition by 10% results in an estimated increase in net present value by $100 million.

Whole-body autoradiography in drug discovery.

In the drug discovery process the pharmacokinetic screening, drug stability studies, evaluation of metabolites, CYP involvement, enzyme induction and inhibition, and excretion studies play a major role. The use of more sensitive and novel detection systems have made the discovery process less cumbersome than in previous years. In particular, the use of whole-body autoradiography (WBA) for tissue distribution, which was once considered an impractical tool, owing to the long turn around time (4-10 weeks), is coming to the forefront for rapidly resolving issues encountered in discovery. In today's research environment early lead compounds can be radio-labeled and whole-body sections imaged quickly (3-5 days) using new techniques, which has made (14)C- and (3)H-WBA a viable tool. The technique has been used in vivo in species from mice to monkeys, and ex vivo and/or in vitro in larger animals and humans. WBA has considerable merit in identifying "pharmacodeficient" compounds and providing insight on mechanistic questions. WBA data can provide information related to tissue pharmacokinetics, routes of elimination, CYP or Pgp mediated drug-drug interactions, tissue distribution, site specific drug localization and retention, metabolism, clearance, compound solubility issues, routes of administration, penetration into specific targets (e.g., tumors), tissue binding (e.g., melanin), and interspecies kinetics. Thus, WBA is quickly becoming part of the battery of studies conducted during the lead optimization process to select optimal drug candidates. Examples of the use of the WBA tool in early discovery are reviewed.

Tumorigenicity of racemic and optically pure bay region diol epoxides and other derivatives of the nitrogen heterocycle dibenz[a,h]acridine on mouse skin.

CD-1 female mice were initiated with a single topical application of 500 nmol dibenz[a,h]acridine (DB[a,h]Acr), its racemic trans-1,2-, 3,4-, 8,9- and 10,11-dihydrodiols, racemic DB[a,h]Acr 3,4-diol 1,2-epoxide-1 and -2 or racemic DB[a,h]Acr 10,11-diol 8,9-epoxide-1 and -2, where the benzylic hydroxyl group is either cis (isomer 1) or trans (isomer 2) to the epoxide oxygen. The mice were subsequently treated twice weekly with 12-O-tetradecanoylphorbol 13-acetate for 25 weeks. High tumorigenicity was observed only for DB[a,h]Acr, its 10,11-dihydrodiol and DB[a,h]Acr 10,11-diol 8,9-epoxide-2 (3.3, 1.2 and 1.6 tumors/mouse, respectively). The tumor-initiating activity of a 50 nmol dose of DB[a,h]Acr and the optically active (+)- and (-)-enantiomers of DB[a,h]Acr 10,11-dihydrodiol and of the optically active DB[a,h]Acr 10,11-diol 8,9-epoxide-1 and -2 were also studied. Only DB[a,h]Acr, (-)-DB[a,h]Acr (10R,11R)-dihydrodiol and the bay region (+)-(8R,9S,10S,11R)-diol epoxide-2 were highly active (1.6, 1.7 and 2.4 tumors/mouse, respectively). These results are consistent with previous studies which showed that the corresponding bay region RSSR diol epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[c]phenanthrene as well as the aza-polycyclic dibenz[c,h]acridine are the most tumorigenic isomers.

Metabolites of caspofungin acetate, a potent antifungal agent, in human plasma and urine.

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [(3)H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine gamma-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding gamma-hydroxy acid, N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetyl-M2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation.

In vitro and in vivo studies on the metabolism of tirofiban.

Tirofiban hydrochloride [L-tyrosine-N-(butylsulfonyl)-O-[4-(4-piperidinebutyl)] monohydrochloride, is a potent and specific fibrinogen receptor antagonist. Radiolabeled tirofiban was synthesized with either (3)H-label incorporated into the phenyl ring of the tyrosinyl residue or (14)C-label in the butane sulfonyl moiety. Neither human liver microsomes nor liver slices metabolized [(14)C]tirofiban. However, male rat liver microsomes converted a limited amount of the substrate to a more polar metabolite (I) and a relatively less polar metabolite (II). The formation of I was sex dependent and resulted from an O-dealkylation reaction catalyzed by CYP3A2. Metabolite II was identified as a 2-piperidone analog of tirofiban. There was no evidence for Phase II biotransformation of tirofiban by microsomes fortified with uridine-5'-diphospho-alpha-D-glucuronic acid. After a 1 mg/kg i.v. dose of [(14)C]tirofiban, recoveries of radioactivity in rat urine and bile were 23 and 73%, respectively. Metabolite I and unchanged tirofiban represented 70 and 30% of the urinary radioactivity, respectively. Tirofiban represented >90% of the biliary radioactivity. At least three minor biliary metabolites represented the remainder of the radioactivity. One of them was identified as I. Another was identified as II. When dogs received 1 mg/kg i.v. of [(3)H]tirofiban, most of the radioactivity was recovered in the feces as unchanged tirofiban. The plasma half-life of tirofiban was short in both rats and dogs, and tirofiban was not concentrated in tissues other than those of the vasculature and excretory organs.

Nonlinear pharmacokinetics of efavirenz (DMP-266), a potent HIV-1 reverse transcriptase inhibitor, in rats and monkeys.

Efavirenz (EFV, Sustiva, Stocrin, DMP-266, L-743,726) is a potent and selective non-nucleoside inhibitor of HIV-1 reverse transcriptase. Pharmacokinetics of EFV was studied in rats and monkeys, the safety assessment species. In rats, after 2 and 5 mg/kg i.v. administrations, the mean CLp, Vdss, and T1/2 were 67 ml/min/kg, 5.0 liters/kg, and 1 h, respectively. EFV was metabolized completely, and the products were excreted almost exclusively via bile. At the higher dose of 15 mg/kg, the CLp was reduced by 36%, implying saturation of metabolism processes. A similar phenomenon occurred in monkeys, where the CLp declined by 60% as the i.v. dose was increased from 5 to 15 mg/kg. After oral dosing, the bioavailability of EFV in rats (10 mg/kg) and monkeys (2 mg/kg) was 16% and 42%, respectively. Higher doses in both species led to disproportionate increases in the AUC and higher Tmax values, suggesting saturation of metabolism and/or prolongation of absorption. The delay in Tmax was more pronounced in monkeys where the plasma concentrations reached plateaus and were sustained for 4 to 20 h. In rats, the prolongation of absorption was due to delayed gastric emptying as demonstrated by >10-fold slower transit of [14C]polyethylene glycol through the stomach of EFV-pretreated animals. The delayed gastric emptying in monkeys also was observed when the animals dosed at 160 mg/kg exhibited emesis, 8 h postdose, which was found to contain a substantial portion of the dose. These results demonstrated that in rats and monkeys, both delayed gastric emptying and saturation of metabolic processes played significant roles in the nonlinear pharmacokinetics of EFV.

Metabolic profiles of montelukast sodium (Singulair), a potent cysteinyl leukotriene1 receptor antagonist, in human plasma and bile.

Montelukast sodium [1-([(1(R)-(3-(2-(7-chloro-2-quinolinyl)-(E)- ethenyl)phenyl)-3-(2-(1-hydroxy-1-methylethyl)phenyl)propyl)thio]methyl)cyclopropylacetic acid sodium salt] (MK-476, Singulair) is a potent and selective antagonist of the cysteinyl leukotriene (Cys-LT1) receptor and is under investigation for the treatment of bronchial asthma. To assess the metabolism and excretion of montelukast, six healthy subjects received single oral doses of 102 mg of [14C]montelukast, and the urine and feces were collected. Most of the radioactivity was recovered in feces, with

Hepatic microsomal metabolism of montelukast, a potent leukotriene D4 receptor antagonist, in humans.

Montelukast (L-706,631, MK-0476, SINGULAIR), a potent and selective leukotriene D4 (CysLT1) receptor antagonist, is currently under development for the treatment of asthma. In vitro studies were conducted using human liver microsomes to evaluate: 1) the difference in the metabolic kinetics of montelukast between adult and pediatric subjects; 2) the relative contribution of flavin-containing monooxygenase and cytochrome P450 (P450) to the sulfoxidation; and 3) the P450 isoforms responsible for montelukast oxidation. No statistically significant difference was observed in the in vitro kinetics for acyl glucuronidation and oxidative metabolism between the two age groups. Results from studies on heat inactivation of flavin-containing monooxygenase and immunochemical inhibition by an anti-rat NADPH P450 reductase antibody on montelukast oxidation indicated that all oxidative metabolism of montelukast-including diastereomeric sulfoxidations, as well as 21- and methyl-hydroxylations-are catalyzed exclusively by P450. Five in vitro approaches have been used to identify the P450 isoforms responsible for the human liver microsomal oxidation of montelukast. The experimental results consistently indicated that CYP3A4 catalyzes sulfoxidation and 21-hydroxylation, whereas CYP2C9 selectively mediates methyl-hydroxylation.

Disposition of indinavir, a potent HIV-1 protease inhibitor, after an oral dose in humans.

Indinavir, N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-tertiary- butylaminocarbonyl-4-(3-pyridylmethyl)piperazino]-4(S)- hydroxy-2(R)-phenylmethylpentanamide (L-735,524,MK-639, ayl-4- Crixivan), is a potent and specific inhibitor of the HIV-1(3 protease for the treatment of AIDS. Disposition of [14C]indinavir was investigated in six healthy subjects after single oral administration of 400 mg. AUC, Cmax, and Tmax values for indinavir were 492 microM x min, 4.7 microM, and 50 min, respectively. The AUC value for the total radioactivity in plasma was 1.9 times higher than that of indinavir, indicating the presence of metabolites. The major excretory route was through feces, and the minor through urine. Mean recovery of radioactivity in the feces was 83.4%. In the urine, mean recoveries of the total radioactivity and unchanged indinavir were 18.7% and 11.0% of the dose, respectively. HPLC radioactivity and LC-MS/MS analyses of urine showed the presence of indinavir and low levels of quaternary pyridine N-glucuronide (M1), 2',3'-trans-dihydroxyindanylpyridine N-oxide (M2), 2',3'-trans-dihydroxyindan (M3) and pyridine N-oxide (M4a) analogs, and despyridylmethyl analogs of M3 (M5) and indinavir (M6). M5 and M6 were the major metabolites in urine. The metabolic profile in plasma was similar to that in urine. Quantitatively, the metabolites in feces accounted for >47% of the dose, which along with the urinary excretion of approximately 19%, suggested that the absorption of the drug was appreciable. In the feces, radioactivity was predominantly due to M3, M5, M6, and the parent compound. Thus, in urine and feces, the prominent metabolic pathways were oxidations and oxidative N-dealkylations. Excretion of the quaternary N-glucuronide metabolite in the urine, which is a minor metabolite in human, was specific to primates.

Species differences in the pharmacokinetics and metabolism of indinavir, a potent human immunodeficiency virus protease inhibitor.

Indinavir, a potent and specific inhibitor of human immunodeficiency virus protease, is undergoing clinical investigation for the treatment of acquired immunodeficiency syndrome. The studies described herein were designed to characterize the absorption, distribution, metabolism, and excretion of the drug in rats, dogs, and monkeys. Indinavir exhibited marked species differences in elimination kinetics. The plasma clearance was in the rank order: rat (107 ml/min/kg) > monkey (36 ml/min/kg) > dog (16 ml/min/kg). Significant differences in the bioavailability of indinavir also were observed. When given orally as a solution in 0.05 M citric acid, the bioavailability varied significantly from 72% in the dog to 19% in the monkey, and 24% in the rat. These differences in bioavailability were attributed mainly to species differences in the magnitude of hepatic first-pass metabolism. The distribution of indinavir was studied only in rats, both intravenously and orally. Intravenously, indinavir was distributed widely throughout the body. Brain uptake studies showed that indinavir penetrated the blood-brain barrier, but that the penetration was limited. After oral administration, indinavir was distributed rapidly into and out of the lymphatic system. The rapid lymph transfer is of clinical relevance, because a primary clinical hallmark of acquired immunodeficiency syndrome is the depletion of CD4 lymphocytes. Biliary and urinary recovery studies revealed that metabolism was the major route of indinavir elimination in all species, and N-dealkylation, N-oxidation, and hydroxylation seemed to be the major pathways. Although limited to qualitative aspects, the metabolite profile obtained from in vitro microsomal studies generally reflected the in vivo oxidative metabolism of indinavir in all species studies. Results from the chemical and immunochemical inhibition studies indicated the possible involvement of isoforms of the CYP3A subfamily in the oxidative metabolism of indinavir in rats, dogs, and monkeys. This is consistent with our previous studies, which have shown that CYP3A4 is the isoform responsible for the oxidative metabolism of indinavir in human liver microsomes. Furthermore, the in vivo oxidative metabolism of indinavir in rats, dogs, and monkeys was qualitatively similar to that in humans. The high degree of similarity in the metabolite profiles of drug metabolism between animals and humans validates the use of these animal models for toxicity studies of indinavir. Attempts were made to quantitatively extrapolate in vitro metabolic data to in vivo metabolism. With the application of the well-stirred and parallel-tube models, the hepatic clearance and hepatic extraction ratio were calculated using the in vitro Vmax/Km values. In rats, the predicted hepatic clearance (31 ml/ min/kg) and hepatic extraction ratio (0.47) agreed well with the observed in vivo hepatic clearance (43 ml/min/kg) and hepatic extraction ratio (0.68). In addition, the hepatic clearance of indinavir was predicted reasonably well in dogs and monkeys. Based on the in vitro intrinsic clearance of human liver microsomes, a small but significant hepatic first-pass metabolism (ca. 25%) is expected in humans.

In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4.

L-754,394, a furanopyridine derivative, is an experimental anti-HIV agent which has been shown to be an unusually potent and selective inhibitor of cytochrome P450 3A enzymes in a number of mammalian species. In the present studies, L-754,394 was demonstrated to undergo NADPH-dependent metabolic activation in hepatic microsomal preparations from rats, dogs, rhesus monkeys, and humans to electrophilic intermediates which became bound covalently to cellular proteins. The extent of binding was species-dependent, the highest levels being observed with liver microsomes from rhesus monkeys. Inclusion in incubation media of the nucleophilic trapping agents glutathione, cysteine, or methoxyamine led to a modest (15-25%) decrease in the covalent binding, while trichloropropylene oxide, an inhibitor of epoxide hydrolase, had no effect. When L-754,394 was incubated with monkey liver microsomes, the corresponding dihydrofurandiol was identified as a metabolite by liquid chromatography-tandem mass spectrometry. In contrast, when incubations were carried out in the presence of methoxyamine, the O-methyloxime derivative of the ring-opened dihydrodiol tautomer was formed, while inclusion of glutathione or N-acetylcysteine led to the formation of S-linked conjugates of a putative furan epoxide. Collectively, these results are taken to indicate that L-754,394 undergoes cytochrome P450-dependent oxidation of the fused furan ring system, leading to the formation of chemically-reactive intermediates. One or more of these electrophilic species may be responsible for the autocatalytic destruction of cytochrome P450 enzymes which accompanies L-754,394 metabolism in vitro and in vivo.

Role of cytochrome P450 3A4 in human metabolism of MK-639, a potent human immunodeficiency virus protease inhibitor.

MK-639 (L-735,524) is a potent human immunodeficiency virus protease inhibitor under investigation in the treatment of acquired immunodeficiency syndrome. Five in vitro approaches have been used to identify the cytochrome P450 isoform(s) responsible for the human microsomal oxidative metabolism of MK-639. These approaches are: 1) chemical inhibition; 2) immunochemical inhibition; 3) metabolism by cDNA-expressed human cytochrome P450 enzymes; 4) a correlation analysis; and 5) competitive inhibition of marker activities. Ketoconazole and troleandomycin, both selective inhibitors for cytochrome P450 3A4 (CYP3A4), markedly inhibited the formation of all oxidative metabolites of MK-639; whereas other inhibitors (furafylline, sulfaphenazole, quinidine, S-mephenytoin, and diethyldithiocarbamate) had little effect on MK-639 metabolism. This suggested the involvement of CYP3A4 in MK-639 metabolism. Consistent with this, an anti-rat CYP3A1 rabbit polyclonal antibody, which shows a cross-reactive inhibition of CYP3A4-dependent testosterone 6beta-hydroxylation in human liver microsomes, completely inhibited MK-639 metabolism. Human recombinant CYP3A4 showed a high metabolic activity to form all MK-639 metabolites found in native human liver microsomes. In addition, the formation of individual MK-639 metabolites correlated well with each other and with testosterone 6beta-hydroxylation in 12 different human liver microsomes, whereas no correlation was observed between MK-639 metabolite formation and bufuralol 1'-hydroxylation (or tolbutamide methyl hydroxylation). Furthermore, MK-639 strongly inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner. Kinetic analysis showed that MK-639 is a very potent competitive inhibitor for testosterone 6beta-hydroxylation, with a Ki value of approximately 0.5 mu M. Collectively, these results consistently indicate that CYP3A4 is the isoform responsible for the oxidative metabolism of MK-639 in human liver microsomes.

Species differences in the metabolism of a potent HIV-1 reverse transcriptase inhibitor L-738,372. In vivo and in vitro studies in rats, dogs, monkeys, and human.

In vivo and in vitro metabolism of 6-chloro-4(S)-cyclopropyl-3,4-dihydro-4-((2-pyridyl) ethynyl)quinazolin-2(1H)-one (L-738,372), a potent human immunodeficiency virus-type 1 reverse transcriptase inhibitor, has been investigated in rats, dogs, and monkeys. Following 0.9 mg/kg iv and 9 mg/kg po doses, systemic blood clearance (CLB) and bioavailability (F) of L-738,372 were species-dependent and inversely related (CLB = 48, 15, and 3 ml/min/kg; F = 6, 62 and 94%, in dogs, rats, and monkeys, respectively). Incubation of L-738,372 with rat liver slices and liver microsomes from all species studied led to the formation of two hydroxylated metabolites, M1 and M2. Kinetic studies of the microsomal metabolism of L-738,372 indicated that M1 was formed by a much higher affinity, but lower capacity enzyme(s) than that which catalyzed M2 formation in rats, dogs, and monkeys. The total intrinsic clearance of metabolite formation (CL(int) total = CL(int) M1 + CL(int) M2) was highest in dogs, followed by rats and monkeys. In dogs, CL(int) total was caused almost exclusively by CL(int) M1. Extrapolation of the CL(int) total values to the hepatic clearances (19, 8.4, and 0.9ml/min/kg in dogs, rats, and monkeys, respectively) showed a similar rank order to the CLB observed in vivo. Good agreement between these in vivo and in vitro results suggests that the species differences in hepatic first-pass metabolism, and not the intrinsic absorption, contributed significantly to the observed differences in F.(ABSTRACT TRUNCATED AT 250 WORDS)

Preconcentration of iron (III), cobalt (II) and copper (II) nitroso-R complexes on tetradecyldimethylbenzylammonium iodide-naphthalene adsorbent.

Iron, cobalt and copper form coloured water soluble anionic complexes with disodium 1-nitroso-2-naphthol-3-6-disulphonate (nitroso R-salt). The anionic complex is retained quantitatively as a water insoluble neutral ion associated complex (M-nitroso R-TDBA) on tetradecyldimethylbenzylammonium iodide on naphthalene (TDBA(+)I(-)-naphthalene) packed column in the pH range of: Fe(III): 3.1-6.5, Co: 3.4-8.5 and Cu 5.9-8.0 when their solutions are passed individually over this adsorbent at a flow rate of 0.5-5.0 ml/min. The solid mass consisting of an ion associated metal complex along with naphthalene is dissolved out of the column with 5 ml dimethylformamide/chloroform and metals are determined spectrophotometrically. The absorbance is measured at 710 nm for iron, 425 nm for cobalt and 480 nm for copper. Beers law is obeyed in the concentration range 9.2-82 mug of iron, 425 nm for cobalt cobalt and 3.0-62 mug of copper in 5 ml of final DMF/CHCl(3) solution. The molar absorptivities are calculated to be Fe: 7.58 x 10(3), Co: 1.33 x 10(4) and Cu: 4.92 x 10(4)M(-1)cm(-1). Ten replicate determinations containing 25 mug of iron, 9.96 mug of cobalt and 3.17 mug of copper gave mean absorbances 0.677, 0.450 and 0.490 with relative standard deviations of 0.88, 0.98 and 0.92%, respectively. The interference of large number of metals and anions on the estimations of these metals has been studied. The optimized conditions so developed have been employed for the trace determination of these metals in standard alloys, waste water and fly ash samples.

Metabolism of L-689,502 by rat liver slices to potent HIV-1 protease inhibitors.

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.

Metabolites of L-735,524, a potent HIV-1 protease inhibitor, in human urine.

L-735,524, N-[2(R)-hydroxy-1(S)-indanyl]-5-(2(S)-(1,1- dimethylethylaminocarbonyl)-4-[(pyridin-3-yl)methyl]piperazin++ +-1-yl)-4(S)- hydroxy-2(R)-phenylmethylpentanamide, is a potent and specific inhibitor of the human immunodeficiency virus type 1 protease and is undergoing clinical evaluation. In an initial clinical study, noninfected male volunteers were administered single, 1000 mg oral doses of nonlabeled compound. Urine samples were collected over a period of 48 hr. Metabolic profile of the urine was determined by HPLC-UV comparison with that from a human liver slice incubation of radiolabeled L-735,524. Seven significant metabolites were isolated from pooled human urine, and were characterized by NMR, MS, and/or chromatographic comparisons with authentic standards. The major metabolic pathways were identified as: a) glucuronidation at the pyridine nitrogen to yield a quaternized ammonium conjugate, b) pyridine N-oxidation, c) para-hydroxylation of the phenylmethyl group, d) 3'-hydroxylation of the indan, and e) N-depyridomethylation. A minor product was identified as 2',3'-trans-dihydroxyindan analog. Urinary excretion of L-735,524 and its metabolites represented a minor pathway of elimination. The intact parent compound seemed to be the major component in the urine, whereas the level of each metabolite was relatively low.

Metabolism of 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2 (1H)-one (L-696,229), an HIV-1 reverse transcriptase inhibitor, by rat liver slices and in humans.

Healthy subjects were administered single oral doses of 800 mg or 400 mg 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-o ne (L-696,229), a nonnucleoside inhibitor of the human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT). Plasma or urine samples were collected over a period of 48 hr. Pooled plasma (0.5-6 hr) and urine (0-24 hr) samples were analyzed by HPLC-UV and HIV-1 RT inhibition assay using poly rC.dG as a template primer. The parent compound and several common metabolites were detected in both samples. The metabolic profiles were also similar to those obtained from a rat liver slice incubation with [3H]L-696,229. The in vitro metabolites were identified by NMR and MS as 5 alpha-hydroxyethyl- (major), 5,6-dihydrodiol-, 6'-hydroxy-, 6-hydroxymethyl-, and 5-vinyl analogs, and a benzoxazole ring hydrolysis product. Most of the significant metabolites in human plasma and urine were found to be identical to the in vitro metabolites, as established by HPLC-UV and MS. Hydrolysis of the plasma and urine with beta-glucuronidase/sulfatase indicated the presence of significant amounts of conjugates of the parent compound and 5 alpha-hydroxyethyl metabolite. Most of the other primary metabolites were also present in conjugated forms, albeit in small quantities. In addition, two secondary metabolites were isolated and identified from the hydrolyzed urine as 5-acetyl-6'-hydroxy- and 5 alpha-hydroxyethyl-6-hydroxymethyl- analogs.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of L-696,229, an HIV-1 reverse transcriptase inhibitor in rats and humans. Hepatic and extrahepatic metabolism and identification of enzymes involved in the hepatic metabolism.

The metabolism of L-696,229, 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-o ne, a potent human immunodeficiency virus-type 1 reverse transcriptase inhibitor, by rat liver, lung, gut, and kidney microsomes has been studied. L-696,229 was metabolized by rat liver microsomes to several products: the 5 alpha-hydroxyethyl (M1); 5,6-dihydrodiol (M2); 6'-hydroxy (M3); 6-hydroxymethyl (M4); and 5-vinyl (M5) metabolites. For these pathways, liver was the most active metabolizing organ, whereas lung was the major extrahepatic organ in the drug metabolism. In all tissues tested, M1 was the major metabolite. With the exception of M3, gender differences in the hepatic formation of all metabolites were observed. Enzymes responsible for the hepatic metabolism of L-696,229 in rats were also investigated using various enzyme inducers and polyclonal antibodies to rat P-450. Treatment of male rats with dexamethasone (DX) or phenobarbital (PB) caused significant increases in the hepatic formation of the gender-dependent metabolites. Methylcholanthrene (3-MC) greatly enhanced the hepatic formation of M1, M3, and M4. Immunoinhibition studies suggested that CYP2B1/2 and 2E1 were not involved in L-696,229 metabolism, whereas CYP1A was partly responsible for the formation of M1 in untreated rats. CYP3A played an important role in the formation of M1, M2, M4, and M5 in untreated and DX-treated rats. In PB-treated rats, CYP2B1/2 was involved in the increased formation of M1 and M4, whereas CYP3A was partly involved in the enhanced M2 and M4 formation, and primarily responsible for the increased M5 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial and mammalian cell mutagenicity of four optically active bay-region 10,11-diol-8,9-epoxides of the nitrogen heterocycle dibenz[a,h]acridine.

The mutagenic activities of the enantiomers of the diastereomeric pair of bay-region 10,11-diol-8,9-epoxides of dibenz[a,h]acridine (DB[a,h]ACR) were evaluated in histidine-dependent strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells. In strains TA98 and TA100 of S.typhimurium, the (-)-[8S,9R,10R,11S] diol-epoxide was the most mutagenic compound, inducing 1200 and 6900 His+ revertants/nmol respectively. The mutagenic activity of each of the remaining three isomers was essentially independent of the bacterial strain used and had 14-72% of the activity of the [S,R,R,S] isomer. However, in Chinese hamster V79 cells, the (+)-[8R,9S,10S,11R] diol-epoxide was the most mutagenic compound (68 8-azaguanine resistant variants/nmol/10(5) cells), inducing from 2 to 11 times as many mutations as the other three isomers. These results are analogous to previous studies with the bay-region diol-epoxides of other polycyclic hydrocarbons in that the isomer with [R,S,S,R] absolute configuration has had variable activity in the bacterial assays, but has generally been the most active in the mammalian cells. Furthermore, this isomer has almost always been highly tumorigenic in the mouse.