Comparative disposition and metabolism of paraherquamide in sheep, gerbils, and dogs.

The disposition and metabolism of paraherquamide (PHQ), a potent and broad-spectrum anthelminthic, were examined in sheep, dogs, and gerbils. The metabolism of PHQ in these species was extensive and marked by significant species differences both in vitro and in vivo. In sheep and gerbils, PHQ metabolism occurs mainly at the pyrrolidine moiety, generating several metabolites that, for the most part, retained nematodicidal activity in vitro. In dogs, the dioxepene group was also extensively metabolized, ultimately resulting in formation of a catechol and loss of pharmacological activity. After oral administration of [3H]PHQ to intact sheep, gerbils, and dogs, the majority of the administered radioactivity was recovered in feces. Intact PHQ accounted for 0% (dogs) to approximately 30% (sheep and gerbils) of drug-related material in feces. A detailed investigation of the composition of the intestinal content of sheep indicated that a significant amount of the dose was still present in the rumen 24 h after dose and that PHQ underwent significant dehydration in the cecum. The oral pharmacokinetic parameters of PHQ in sheep and dogs suggest that its absorption is rapid in both species but that its apparent elimination rate is significantly higher in the dog (t(1/2) approximately 1.5 h) than it is in sheep (t(1/2) approximately 8.5 h). The short elimination half-life and the absence of PHQ or other active components in the dog gastrointestinal tract provide a potential explanation of the lack of efficacy of PHQ in this species.

Characterization of two cyclic metabolites of sitagliptin.

Two novel metabolites of the dipeptidyl peptidase inhibitor sitagliptin (MK-0431, (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)-butan-2-amine), were identified after purification from dog urine. The metabolites (referred to as M2 and M5) were characterized by hydrogen/deuterium exchange tandem mass spectrometry and NMR spectroscopy nuclear Overhauser effect experiments as the cis and trans stereoisomers formed by cyclization of the primary amino group with the alpha carbon of the piperazine ring, following oxidative desaturation.

Metabolism and disposition of a potent and selective GABA-Aalpha2/3 receptor agonist in healthy male volunteers.

[14C]7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine ([14C]-TPA023; 99 microCi/dose) was administered to five young, healthy, fasted male subjects as a single oral dose (3.0 mg) in solution (propylene glycol/water, 10:90 v/v). The parent compound was rapidly absorbed (plasma Tmax approximately 2 h), exhibited an apparent terminal half-life of 6.7 h, and accounted for approximately 53% of the total radioactivity in plasma. After 7 days of collection, the mean total recovery of radioactivity in the excreta was 82.6%, with 53.2% and 29.4% in urine and feces, respectively. Radiochromatographic analysis of the excreta revealed that TPA023 was metabolized extensively, and only trace amounts of unchanged parent were recovered. Radiochromatograms of urine and feces showed that TPA023 underwent metabolism via three pathways (t-butyl hydroxylation, N-deethylation, and direct N-glucuronidation). The products of t-butyl hydroxylation and N-deethylation, together with their corresponding secondary metabolites, accounted for the majority of the radioactivity in the excreta. In addition, approximately 10.3% of the dose was recovered in urine as the triazolo-pyridazine N1-glucuronide of TPA023. The t-butyl hydroxy and N-desethyl metabolites of TPA023, the TPA023 N1-glucuronide, and the triazolo-pyridazine N1-glucuronide of N-desethyl TPA023 were present in plasma. In healthy male subjects, therefore, TPA023 is well absorbed and is metabolized extensively (t-butyl hydroxylation and N-deethylation > glucuronidation), and the metabolites are excreted in urine and feces.

Replacement of Phe6, Phe7, and Phe11 of D-Trp8-somatostatin-14 with L-pyrazinylalanine. Predicted and observed effects on binding affinities at hSST2 and hSST4. An unexpected effect of the chirality of Trp8 on NMR spectra in methanol.

An alanine scan performed in the 1970s suggested that Phe(6) and Phe(11) are required for the binding of somatostatin (SRIF-14). Molecular modeling studies and replacement of Phe(6) and Phe(11) with a cystine bridge affording ligands with the retention of high biological activity, however, led to the alternate conclusion that Phe(6) and Phe(11) stabilize the bioactive conformation of SRIF-14. Subsequent studies revealed that Phe(11) shields Phe(6) in a "herringbone" arrangement. More recently, a report from this laboratory demonstrated that Spartan 3-21G MO calculations can be invaluable in explaining SARs in medicinal chemistry. For example, the ability of benzene and indole rings to bind the Trp(8) binding pocket for SRIF-14 and the inability of pyrazine to do so was explained through differences in electrostatic potentials. To investigate the role of Phe(6) and Phe(11) more fully, we report here the synthesis of two analogues of D-Trp(8)-SRIF in which Phe(6) and Phe(11) were replaced by the pryazinylalanine congeners, respectively. The NMR spectra in D(2)O and the K(i)s fully support the proposition that Phe(11) stabilizes the bioactive conformation through pi-bonding or aromatic edge-to-face interaction and that pyrazinylalanine(6) can be shielded by Phe(11). The data also show unexpectedly that Phe(6), via the pi-bond, interacts with the receptor, consistent with the original interpretation of the alanine scan. Heretofore it had only been known that Lys(9) interacts with an aspartate anion of the receptor. These conclusions are supported by recent studies of Lewis et al. on the effects on K(i)s of Ala(6)-SRIF-14-amide at the five receptor subtargets. We also found that pyrazinylalanine(7)-D-Trp(8)-SRIF-14 does not bind, suggesting a repulsive interaction with the receptor. Taken together, our results not only validate predictions based on Spartan 3-21G MO analysis but also provide valuable information about the nature of the receptor interaction at the molecular level. Finally, the chirality of Trp(8) was unexpectedly found to have a striking effect on NMR spectra in methanol, especially at lower temperatures.

In vitro and in vivo metabolism of a potent and selective integrin alpha v beta 3 antagonist in rats, dogs, and monkeys.

Compound A (3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyrindin-2-yl)propyl]-imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)-propionic acid), a potent and selective antagonist of integrin alpha(v)beta(3) receptor, is under development for treatment of osteoporosis. This study describes metabolism and excretion of A in vivo in rats, dogs, and monkeys, and metabolism of A in vitro in primary hepatocytes from rats, dogs, monkeys, and humans. In all three animal species studied, A was primarily excreted as unchanged drug and, to a lesser degree, as phase I and phase II metabolites. Major biotransformation pathways of A included glucuronidation/glucosylation on the carboxylic group to form acyl-linked glucuronides/glucosides; and oxidation on the tetrahydronaphthyridine moiety to generate a carbinolamine and its further metabolized products. Minor pathways involved O-demethylation and hydroxylations on the alkyl chain. Only in rats, a glutathione adduct of A was also observed, and its formation is proposed to be via an iminium intermediate on the tetrahydronaphthyridine ring. Similar metabolic pathways were observed in the incubates of hepatocytes from the corresponding animals as well as from humans. CYP 3A and 2D subfamilies were capable of metabolizing A to its oxidative products. Overall, these in vitro and in vivo findings should provide useful insight on possible biotransformation pathways of A in humans.

Identification of UGT2B9*2 and UGT2B33 isolated from female rhesus monkey liver.

Two UDP-glucuronosyltransferases (UGT2B9(*)2 and UGT2B33) have been isolated from female rhesus monkey liver. Microsomal preparations of the cell lines expressing the UGTs catalyzed the glucuronidation of the general substrate 7-hydroxy-4-(trifluoromethyl)coumarin in addition to selected estrogens (beta-estradiol and estriol) and opioids (morphine, naloxone, and naltrexone). UGT2B9(*)2 displayed highest efficiency for beta-estradiol-17-glucuronide production and did not catalyze the glucuronidation of naltrexone. UGT2B33 displayed highest efficiency for estriol and did not catalyze the glucuronidation of beta-estradiol. UGT2B9(*)2 was found also to catalyze the glucuronidation of 4-hydroxyestrone, 16-epiestriol, and hyodeoxycholic acid, while UGT2B33 was capable of conjugating 4-hydroxyestrone, androsterone, diclofenac, and hyodeoxycholic acid. Three glucocorticoids (cortisone, cortisol, and corticosterone) were not substrates for glucuronidation by liver or kidney microsomes or any expressed UGTs. Our current data suggest the use of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation, and estriol-17-glucuronidation to assay UGT1A01, UGT2B9(*)2, and UGT2B33 activity in rhesus liver microsomes, respectively.

A novel lipid hydroperoxide-derived modification to arginine.

The guanidine group present in the amino acid arginine was found to react with the lipid hydroperoxide-derived bifunctional electrophile, 4-oxo-2-nonenal. The reaction between N(alpha)-tert-butoxycarbony-l-arginine and 4-oxo-2-nonenal resulted in the formation of an adduct (adduct A) that subsequently dehydrated on heating to adduct B. Liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy were used to assign the structure of adduct B as (N(delta),N(omega)(')-etheno-2'-heptanon-2' '-one)-N(alpha)-t-Boc-arginine. The reaction proceeded from initial reaction of the primary N(omega)-amino group at the C-1 aldehyde of 4-oxo-2-nonenal. Subsequently, an intramolecular Michael addition of a secondary N(delta)-amino group occurring at C-3 resulted in formation of the cyclic carbinolamine adduct A. Dehydration and rearrangement of the exocyclic imine resulted in the formation of adduct B, which contained a stable imidazole ring. The tetra peptide LRDE reacted with 4-oxo-2-nonenal primarily at arginine rather than at the amino terminus. This suggests that arginine-containing proteins can react with lipid hydroperoxide-derived 4-oxo-2-nonenal to form a novel imidazole modification.

Metabolism of rofecoxib in vitro using human liver subcellular fractions.

The metabolism of rofecoxib, a potent and selective inhibitor of cyclooxygenase-2, was examined in vitro using human liver subcellular fractions. The biotransformation of rofecoxib was highly dependent on the subcellular fraction and the redox system used. In liver microsomal incubations, NADPH-dependent oxidation of rofecoxib to 5-hydroxyrofecoxib predominated, whereas NADPH-dependent reduction of rofecoxib to the 3,4-dihydrohydroxy acid metabolites predominated in cytosolic incubations. In incubations with S9 fractions, metabolites resulting from both oxidative and reductive pathways were observed. In contrast to microsomes, the oxidation of rofecoxib to 5-hydroxyrofecoxib by S9 fractions followed two pathways, one NADPH-dependent and one NAD+-dependent (non-cytochrome P450), with the latter accounting for about 40% of total activity. The 5-hydroxyrofecoxib thus formed was found to undergo NADPH-dependent reduction ("back reduction") to rofecoxib in incubations with liver cytosolic fractions. In incubations with dialyzed liver cytosol, net hydration of rofecoxib to form 3,4-dihydro-5-hydroxyrofecoxib was observed, whereas the 3,4-dihydrohydroxy acid derivatives were formed when NADPH was present. Although 3,4-dihydro-5-hydroxyrofecoxib could be reduced to the 3,4-dihydrohydroxy acid by cytosol in the presence of NADPH, the former species does not appear to serve as an intermediate in the overall reductive pathway of rofecoxib metabolism. In incubations of greater than 2 h with S9 fractions, net reductive metabolism predominated over oxidative metabolism. These in vitro results are consistent with previous findings on the metabolism of rofecoxib in vivo in human and provide a valuable insight into mechanistic aspects of the complex metabolism of this drug.

Characterization of 2'-deoxycytidine adducts derived from 4-oxo-2-nonenal, a novel lipid peroxidation product.

Analysis of the reaction between 2'-deoxycytidine and 4-oxo-2-nonenal by LC/MS revealed the presence of three major products (adducts A(1), A(2), and B; [M + H](+) = 364). Adducts A(1) and A(2) were isomeric, and each dehydrated to form adduct B. The structure of adduct B was shown by LC/MS and NMR spectroscopy to be an etheno-2'-deoxycytidine adduct 1' '-[1-(2'-deoxy-beta-d-erythro-pentofuranosyl)-1H-imidazo[2,1-c]pyrimidin-2-oxo-4-yl]heptane-2' '-one. A time course experiment performed at 65 degrees C (pH 5-8) showed that the transformation of both A(1) and A(2) was pH-dependent. In acidic conditions, adducts A(1) and A(2) dehydrated primarily to adduct B. In contrast, in basic conditions, adducts A(1) and A(2) hydrolyzed primarily to dCyd. The data are consistent with adducts A(1) and A(2) being substituted ethano adducts that dehydrate to adduct B, a substituted 3,N(4)-etheno-2'-deoxycytidine adduct.

1H-NMR studies on a potent and selective antagonist at human melanocortin receptor 4 (hMC-4R).

Melanocortin receptor 4 (MC-4R) is involved in the regulation of energy balance and body weight, and recognizes alpha-, beta-, and gamma-melanocyte stimulating hormones (alpha-, beta-, gamma-MSH). In the search for compounds that regulate food intake and body weight, two synthetic lactam-derivative ligands of alpha-MSH were discovered, MTII and SHU9119. MTII is an agonist and reduces food intake in rats, whereas SHU9119 is an antagonist, and increases food intake and body weight in rats. MTII and SHU9119 are nonselective compounds to MC-4R. To enhance the potency and selectivity at the human MC-4R (hMC-4R), we recently synthesized analogs of SHU9119 (M. A. Bednarek, T. MacNeil, R. N. Kalyani, R. Tang, Van der L. H. T. Ploeg, and D. H. Weinberg, Journal of Medicinal Chemistry, 2001, Vol. 44, pp. 401-409), wherein compound 1 was the most selective for hMC-4R. Replacement of D-Nal by L-Nal in compound 1 made compound 2 weakly active. Comparison of the structures by NMR and molecular modeling of compounds 1 and 2 vs SHU9119 and MTII indicated that, even though they existed as an average of several conformations in solution, there were distinctions in their structures. The gamma-methylene protons of Arg in compound 1 were nonequivalent and shielded probably by the aromatic ring of Nal. The NHi-NHi+1 NOE cross peaks and the temperature coefficients of the amide protons around the "essential core" Nal/Phe7-Arg8-Trp9, required for high affinity and high selectivity at hMC-4R, were indicative of a possible turn structure for these compounds but with differences in their NOE strengths and temperature coefficient values. Molecular modeling of these compounds based on their NMR data showed that the essential core appeared as a "V" shape with two different orientations, one for compound 1 and some of the conformers of SHU9119 and MTII, and the other for compound 2 and some other conformers of SHU9119 and MTII. The remaining conformers of SHU9119 and MTII, which did not map to compound 1 or 2, suggested that they were outside of the hMC-4R binding envelop. These observations may lead to conjectures as to why compound 1 is highly active and selective toward hMC-4R.

Metabolic activation of a pyrazinone-containing thrombin inhibitor. Evidence for novel biotransformation involving pyrazinone ring oxidation, rearrangement, and covalent binding to proteins.

Compound I, (2-[3-[(2,2-difluoro-2(2-pyridyl)ethyl)amino]-6-methyl-2-oxohydropyrazinyl]-N-[(3-fluoro(2-pyridyl))methyl]acetamide, is a potent competitive inhibitor of thrombin that reacts stoichiometrically with the protease. Compounds of this class possess therapeutic potential as anticoagulation agents. During the metabolic characterization of compound I, evidence was obtained for extensive metabolic activation of the pyrazinone ring system. Following administration of (14)C-labeled I to rats, significant levels of irreversibly bound radioactivity to proteins were detected in rat plasma and liver. LC/MS/MS analysis of metabolites formed in rat and human liver microsomes fortified with glutathione (GSH) revealed the presence of two structurally distinct GSH adducts. It is proposed that the first of these GSH conjugates derives from a two electron oxidation of the 6-methyl-2-oxo-3-aminopyrazinone moiety to afford an electrophilic imine-methide intermediate, while the second is formed by addition of GSH to an epoxide formed by P-450-mediated oxidation of the double bond at the 5-6 position of the pyrazinone ring. The addition of GSH to the proposed epoxide facilitates opening of the pyrazinone ring and a rearrangement to afford a stable, rearranged imidazole-containing metabolite. Elucidation of the metabolic activation pathways of I provides structural guidance for the design of thrombin inhibitors with decreased potential for the generation of chemically reactive intermediates.

Structure-function relationships of rat liver CYP3A9 to its human liver orthologs: site-directed active site mutagenesis to a progesterone dihydroxylase.

CYP3A9 is an estrogen-inducible ortholog of human liver CYP3A4 with 76.5% sequence identity to CYP3A4. Unlike CYP3A4, it is a very poor testosterone 6beta- and 2beta-hydroxylase, but a relatively better catalyst of progesterone monohydroxylation largely at 6beta, 16alpha, and 21 positions with negligible 6beta, 21-dihydroxylation. We reasoned that such differences in substrate catalyses must be due to differences in the active site architecture of each CYP3A enzyme. Indeed, alignment of CYP3A4 substrate recognition sites (SRSs) with the corresponding regions of CYP3A9 sequence revealed that of the 22 fully divergent residues, 4 reside in SRS regions [P107N (SRS-1), M371G (SRS-5), and L479K and G480Q (SRS-6)]. Accordingly, we substituted these and other divergent CYP3A9 SRS residues with the corresponding residues of CYP3A4 and/or CYP3A5. Our findings of the influence of these site-directed mutations of the CYP3A9 active site on its catalysis of testosterone and three other established but structurally different CYP3A substrates (progesterone, imipramine, and carbamazepine) are described. These findings revealed that some mutations (N107P, N107S, V207T, G371M, and Q480G) not only improved the ability of CYP3A9 to hydroxylate testosterone at the 6beta and 2beta positions, but also converted it into a robust progesterone 6beta, 21-dihydroxylase. The latter in the case of CYP3A9N107P was accompanied by a shift from sigmoidal to hyperbolic enzyme-substrate kinetics. In contrast, the catalytic potential of CYP3A9 mutants K206N, K206S, M240V, and K479L/Q480G was either relatively unchanged or negligible to nonexistent. Together these findings attest to the unique substrate-active site fit of each CYP3A enzyme.

Hemin-mediated restoration of allylisopropylacetamide-inactivated CYP2B1: a role for glutathione and GRP94 in the heme-protein assembly.

Administration of the cytochrome P450 (P450) suicide inactivator allylisopropylacetamide (AIA) to phenobarbital (PB)-pretreated rats results in rapid and marked inactivation of several liver endoplasmic reticulum (ER)-bound P450s. A few of these such as CYP2B1, inactivated due to AIA-mediated prosthetic heme N-alkylation, can be structurally and functionally restored nearly completely by exogenous hemin in vivo or in vitro. Such in vitro hemin-mediated reassembly is unsuccessful with purified AIA-inactivated CYP2B1 and, as shown herein, is not very effective even when heme is incubated with just the corresponding liver microsomes that contain the reconstitutable CYP2B1 protein, thereby implicating a requirement for additional factors provided by the intact liver cell homogenates, ER, and/or cytosol. Using various approaches that include high-performance liquid chromatographic fractionation of the liver cytosolic subfraction as well as chemical and immunological probes such as the Hsp90/GRP94-specific inhibitor geldanamycin (GA) and polyclonal anti-GRP94 antibodies, respectively, we now demonstrate that the in vitro hemin-mediated reassembly of heme-stripped microsomal CYP2B1 requires GSH as well as the ER chaperone GRP94, but not the cytosolic chaperone heat shock protein 90. It remains to be determined whether GSH acts directly or indirectly, via a putative ER thiol reductase, to maintain the conserved active site cysteine-thiol (Cys436 in CYP2B1) in a reduced state, competent for heme binding and repair.

Synthesis of 1,19-Aza-1,19-desoxy-avermectin B(1a): The First Avermectin Macrolactam.

The first synthesis of 1,19-aza-1,19-desoxy-avermectin B(1a) (2) is described. This new macrolactam, prepared efficiently from avermectin B(1a) (1a) in seven steps, was designed to form an intramolecular hydrogen bond between the amide carbonyl and the adjacent C7 tertiary hydroxyl via a six-center hydrogen bonding network. The presence of this intramolecular hydrogen bond is anticipated to confer additional conformational rigidity to the 16-membered macrocycle.

A Tethered Porphyrin Dimer with π Overlap of a Single Pyrrole Ring.

The special pair of the bacterial photosystem has been modeled with a porphyrin dimer (the partial structure is shown). As with the natural system, only one pyrrole ring from each monomer subunit participates in π overlap.