David D. Christ

Potent, Orally Bioavailable Delta Opioid Receptor Agonists for the Treatment of Pain : Discovery of N,N-Diethyl-4-(5-hydroxyspiro-[chromene-2,4'-piperidine]-4-yl)benzamide (ADL5859)

Selective delta opioid receptor agonists are promising potential therapeutic agents for the treatment of various types of pain conditions. A spirocyclic derivative was identified as a promising hit through screening. Subsequent lead optimization identified compound 20 (ADL5859) as a potent, selective, and orally bioavailable delta agonist. Compound 20 was selected as a clinical candidate for the treatment of pain.

Potent and selective aggrecanase inhibitors containing cyclic P1 substituents

Anti-succinate hydroxamates with cyclic P1 motifs were synthesized as aggrecanase inhibitors. The N-methanesulfonyl piperidine 23 and the N-trifluoroacetyl azetidine 26 were the most potent aggrecanase inhibitors both having an IC(50)=3nM while maintaining >100-fold selectivity over MMP-1, -2, and -9. The cyclic moieties were also capable of altering in vivo metabolism, hence delivering low clearance compounds in both rat and dog studies as shown for compound 14.

The chimpanzee (Pan troglodytes) as a pharmacokinetic model for selection of drug candidates: Model characterization and application

The chimpanzee (CHP) was evaluated as a pharmacokinetic model for humans (HUMs) using propranolol, verapamil, theophylline, and 12 proprietary compounds. Species differences were observed in the systemic clearance of theophylline (∼5-fold higher in CHPs), a low clearance compound, and the bioavailability of propranolol and verapamil (lower in CHPs), both high clearance compounds. The systemic clearance of propranolol (∼1.53 l/h/kg) suggested that the hepatic blood flow in CHPs is comparable to that in humans. No substantial differences were observed in the in vitro protein binding. A preliminary attempt was made to characterize cytochrome P450 (P450) activities in CHP and HUM liver microsomes. Testosterone 6β-hydroxylation and tolbutamide methylhydroxylation activities were comparable in CHP and HUM liver microsomes. In contrast, dextromethorphan O-demethylation and phenacetin O-deethylation activities were ∼10-fold higher (per mg protein) in CHP liver microsomes. Intrinsic clearance estimates in CHP liver microsomes were higher for propranolol (∼10-fold) and theophylline (∼5-fold) and similar for verapamil. Of the 12 proprietary compounds, 3 had oral clearances that differed in the two species by more than 3-fold, an acceptable range for biological variability. Most of the observed differences are consistent with species differences in P450 enzyme activity. Oral clearances of proprietary compounds in HUMs were significantly correlated to those from CHPs (r = 0.68; p = 0.015), but not to estimates from rat, dog, and monkey. In summary, the chimpanzee serves as a valuable surrogate model for human pharmacokinetics, especially when species differences in P450 enzyme activity are considered.

Pharmacokinetics and Pharmacodynamics of DPC 333 ((2R)-2-((3R)-3-Amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)), a Potent and Selective Inhibitor of Tumor Necrosis Factor α-Converting Enzyme in Rodents, Dogs, Chimpanzees, and Humans

DPC 333 ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) is a potent and selective inhibitor of tumor necrosis factor (TNF)-α-converting enzyme (TACE). It significantly inhibits lipopolysaccharide-induced soluble TNF-α production in blood from rodents, chimpanzee, and human, with IC50 values ranging from 17 to 100 nM. In rodent models of endotoxemia, DPC 333 inhibited the production of TNF-α in a dose-dependent manner, with an oral ED50 ranging from 1.1 to 6.1 mg/kg. Oral dosing of DPC 333 at 5.5 mg/kg daily for 2 weeks in a rat collagen antibody-induced arthritis model suppressed the maximal response by approximately 50%. DPC 333 was distributed widely to tissues including the synovium, the site of action for antiarthritic drugs. Pharmacokinetic and pharmacodynamic studies in chimpanzee revealed a systemic clearance of 0.4 l/h/kg, a Vss of 0.6 l/kg, an oral bioavailability of 17%, and an ex vivo IC50 for the suppression of TNF-α production of 55 nM (n = 1). In a phase I clinical trial with male volunteers after single escalating doses of oral DPC 333, the terminal half-life was between 3 and 6 h and the ex vivo IC50 for suppressing TNF-α production was 113 nM. Measurement of the suppression of TNF-α production ex vivo may serve as a good biomarker in evaluating the therapeutic efficacy of TACE inhibitors. Overall, the pharmacological profiles of DPC 333 support the notion that suppression of TNF-α with TACE inhibitors like DPC 333 may provide a novel approach in the treatment of various inflammatory diseases including rheumatoid arthritis, via control of excessive TNF-α production.

α,β-Cyclic-β-benzamido hydroxamic acids: Novel oxaspiro[4.4]nonane templates for the discovery of potent, selective, orally bioavailable inhibitors of tumor necrosis factor-α converting enzyme (TACE)

Two novel oxaspiro[4.4]nonane beta-benzamido hydroxamic scaffolds have been synthesized in enantio- and diasteriomerically pure form. These templates proved to be exceptional platforms that have led to the discovery of potent inhibitors of TACE that are active in a cellular assay measuring suppression of LPS-induced TNF-alpha. Furthermore, these inhibitors are selective against related MMPs, demonstrate permeability in a Caco-2 assay, and display good oral bioavailability.

Development of a Dog Microdialysis Model for Determining Synovial Fluid Pharmacokinetics of Anti-Arthritis Compounds Exemplified by Methotrexate

AbstractPurpose. The purpose of this study was to develop and validate an animal model of drug disposition in synovial fluid (SF) by comparing microdialysis with arthrocentesis using the anti-arthritic drug methotrexate (MTX). Methods. Microdialysis probes were calibrated in vitro with the no net flux method using dog synovial fluid. The probes were implanted surgically into the stifle joint space of four dogs and were dialyzed overnight using a portable microinfusion pump. The membrane integrity of the probes was monitored by retrodialysis using an internal standard. After an intravenous bolus of 2.5 mg/kg of MTX, unbound concentrations in synovial fluid, as well as total plasma concentrations, were measured by liquid chromatography tandam mass spectrometer (LC/MS/MS) in samples collected from 0 to 48 h postdose. Results. The probe membrane remained intact at least 48 h after implantation. The mean probe recovery and unbound fraction of MTX in SF were 46.8% and 44.8%, respectively. The unbound fraction of MTX was 44% in synovial fluid. MTX penetrated into the joint space rapidly, with maximal concentrations of 6.6 μM reached at approximately 1 h postdose. The unbound MTX area under the curve in SF was approximately 40% of the total area under the curve in plasma. These data agree well with the previous data obtained for MTX using arthrocentesis. Conclusion. In contrast with arthrocentesis, microdialysis enables the collection of multiple serial SF samples from individual animals with minimal trauma and potential blood contamination. This animal model should prove valuable for studying the disposition of new anti-arthritis compounds or biomarkers in SF.

Pharmacokinetic-pharmacodynamic Relations of Losartan and Exp3174 in a Porcine Animal Model

The pharmacokinetics of losartan and EXP3174, an active metabolite of losartan, were evaluated in the anesthetized pig after both a single intravenous dose (3 mg/kg) and during constant intravenous infusion. The pharmacodynamic activities of losartan and EXP3174 were determined during constant intravenous infusion as the degree of inhibition of angiotensin II-induced increase in the diastolic pressure. The systemic plasma clearance of losartan was 22.1 +/- 4.4 ml/min/kg (mean +/- SEM) and had an apparent volume of distribution at steady state of 0.56 +/- 0.16 L/kg after a 3-mg/kg intravenous dose. The elimination half-life of losartan was 40 +/- 6 min. Less than 2% of the intravenous losartan doses was estimated to be present as unconjugated EXP3174. The plasma clearance of EXP3174 was approximately 50% that of losartan, 11.8 +/- 1.5 ml/min/kg, and had a smaller steady-state apparent volume of distribution, 0.18 +/- 0.04 L/kg. The elimination half-life for EXP3174 was slightly longer than that of losartan (52 min). The time course of the pharmacodynamic effects of losartan and EXP3174 closely followed their respective plasma concentrations. The apparent dissociation constant of EXP3174 to the angiotensin II receptor was estimated, based on the total plasma concentrations, to be approximately 5 times lower than that for losartan.

16 – Nonpeptide Angiotensin II Receptor Antagonist: Losartan

Publisher Summary Losartan is the prototype of a new class of orally active, nonpeptide angiotensin II (AII) receptor antagonists. Losartan inhibits specifically the renin-angiotensin system (RAS) without the agonist effects of the peptide receptor antagonists or the bradykinin (BK) potentiating effects of the angiotensin-converting enzyme (ACE) inhibitors. The BK potentiation effects of ACE inhibitors, however, have given rise to the mechanistic issues. Losartan, by contrast, is selective for the AII AT 1 receptor subtype, which mediates all of the known biological effects of AII. Because of its specificity and selectivity of action, losartan is being used to define the functional role of AII in many physiological or pathophysiological settings. This chapter discusses the methods and strategies by which the prototype nonpeptide AII receptor antagonist losartan was (1) synthesized, (2) characterized in vitro , (3) characterized in vivo , (4) analyzed in blood or urine, and (5) evaluated in man. At present, these methods/strategies are being applied to the discovery of a new class of inhibitors of the renin-angiotensin system.

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor

1. The methylester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog givenradiolabelled roxifibanshowedlimited oralabsorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiraland chiralcentres of the isoxazolinering. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Glucuronidation in the chimpanzee (Pan troglodytes): Studies with acetaminophen, oestradiol and morphine

The chimpanzee has recently been characterized as a surrogate for oxidative drug metabolism in humans and as a pharmacokinetic model for the selection of drug candidates. In the current study, the glucuronidation of acetaminophen, morphine and oestradiol was evaluated in the chimpanzee to extend the characterization of this important animal model. Following oral administration of acetaminophen (600 mg) to chimpanzees (n = 2), pharmacokinetics were comparable with previously reported human values, namely mean oral clearance 0.91 vs. 0.62 ± 0.05 l h−1 kg−1, apparent volume of distribution 2.29 vs. 1.65 ± 0.25 l kg−1, and half-life 1.86 vs. 1.89 ± 0.27 h, for chimpanzee vs. human, respectively. Urinary excretions (percentage of dose) of acetaminophen, acetaminophen glucuronide and acetaminophen sulfate were also similar between chimpanzees and humans, namely 2.3 vs. 5.0, 63.1 vs. 54.7, and 25.0 vs. 32.3%, respectively. Acetaminophen, oestradiol and morphine glucuronide formation kinetics were investigated using chimpanzee (n = 2) and pooled human liver microsomes (n = 10). and (or ) for acetaminophen glucuronide, morphine 3- and 6-glucuronide, and oestradiol 3- and 17-glucuronide formation were comparable in both species. Eadie–Hofstee plots of oestradiol 3-glucuronide formation in chimpanzee microsomes were characteristic of autoactivation kinetics. Western immunoblot analysis of chimpanzee liver microsomes revealed a single immunoreactive band when probed with anti-human UGT1A1, anti-human UGT1A6, and anti-human UGT2B7. Taken collectively, these data demonstrate similar glucuronidation characteristics in chimpanzees and humans.