Vincent Tong

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This studys objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.

Discovery of a Highly Potent, Selective, and Bioavailable Soluble Epoxide Hydrolase Inhibitor with Excellent Ex Vivo Target Engagement

4-Substituted piperidine-derived trisubstituted ureas are reported as highly potent and selective inhibitors for sEH. The SAR outlines approaches to improve activity against sEH and reduce ion channel and CYP liability. With minimal off-target activity and a good PK profile, the benchmark 2d exhibited remarkable in vitro and ex vivo target engagement. The eutomer entA-2d also elicited vasodilation effect in rat mesenteric artery.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Kinetics of Valproic Acid Glucuronidation: Evidence for in Vivo Autoactivation

Sigmoidal or autoactivation kinetics has been observed in vitro for both cytochrome P450- and UDP-glucuronosyltransferase-catalyzed enzymatic reactions. However, the in vivo relevance of sigmoidal kinetics has never been clearly demonstrated. In the current study we investigate the kinetics of valproic acid glucuronide (VPAG) formation both in vivo in adult sheep and in vitro in sheep liver microsomes (pool of 10). After a 100 mg/kg i.v. bolus dose of valproic acid (VPA) to adult sheep (n = 5), the majority of the dose was recovered in urine as VPAG (∼79%). Eadie-Hofstee plots of the VPAG formation rate (calculated from urinary excretion rate data for VPAG) were characteristic of autoactivation kinetics and provided estimates of the apparent maximum velocity of an enzymatic reaction (Vmaxapp), the substrate concentration resulting in 50% of Vmaxapp (S50app), and Hill coefficient (n) of 2.10 ± 0.75 μmol/min/kg, 117 ± 56 μM, and 1.34 ± 0.14, respectively. Comparable estimates of Vmaxapp (2.63 ± 0.33 μmol/min/kg), S50app (118 ± 53 μM), and n (2.06 ± 0.47) describing overall VPA elimination from plasma were obtained by fitting VPA unbound plasma concentration-time data to a two-compartment model with elimination described by the Hill equation. Consistent with our in vivo observations, Eadie-Hofstee plots of VPAG formation in sheep liver microsomes were characteristic of autoactivation kinetics. To our knowledge, these data provide the first clear demonstration that autoactivation kinetics observed in vitro in liver preparations can translate to the in vivo situation at least under certain experimental conditions and confirm its relevance.

Determination of an arylether antiarrhythmic and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates using liquid chromatography-tandem mass spectrometry

A method was developed and validated for the quantification of (+/-)-trans-[2-morpholino-1-(1-naphthalene-ethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5-3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Atypical Kinetics of Valproic Acid Glucuronidation In vitro and In vivo in Humans

Atypical kinetics has been observed in vitro for both cytochrome P450 and UDP-glucuronosyltransferase catalyzed enzymatic reactions. The in vitro and in vivo kinetics of valproic acid- glucuronide (VPA-glucuronide) formation was investigated in humans. VPA-glucuronide formation kinetics was investigated in vitro using human cryoperserved hepatocytes (pool of 10). Estimates of Vmax app (39.5 ± 3.3 pmol/min/106 cells), S50 app (224 ± 34 µM) and n (2.34 ± 0.28) was obtained by fitting VPA-glucuronide formation rate vs VPA incubation concentration data to the Hill equation. In vitro EadieHofstee plots were “hooked” and characteristic of atypical sigmoidal/autoactivation kinetics. In vivo Eadie-Hofstee plots of VPA-glucuronide formation rate (calculated from urinary excretion rate data for VPA-glucuronide) were constructed using data from four human subjects given a 1000 mg VPA oral dose [1]. The positive slope of linear regression lines for these plots was consistent with in vivo atypical sigmoidal/autoactivation kinetics. In summary, these data build upon our previous observations in sheep and provide the first demonstration that VPA-glucuronide formation exhibits atypical kinetics in vitro in human hepatocytes. Available in vivo data is consistent with in vitro results suggesting that VPA-glucuronidation exhibits atypical sigmoidal/autoactivation kinetics in vivo in humans.