Selective and reliable determination of obacunone in rat plasma using solid-phase extraction by liquid chromatography tandem mass spectrometry: Application to a pharmacokinetic study.

This study aimed to develop a highly selective, sensitive and fast liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the determination of obacunone in rat plasma. Sample preparation was accomplished by a simple solid-phase extraction procedure. Chromatographic separation was carried out on an ACQUITY BEH C18 column using acetonitrile/methanol (1:1, v/v) and 0.1% formic acid in water as mobile phase at a flow rate of 0.4 mL/min. Quantification was performed with multiple reactions monitoring in positive ion mode with the precursor-to-product ion transitions at m/z 455.2 > 161.1 for obacunone and m/z 515.2 > 161.1 for nomilin (internal standard). The assay was demonstrated to be linear over the concentration range of 0.1-1,000 ng/mL with correlation coefficient >0.999 (r > 0.999). The intra- and inter-day accuracy ranged from -8.33 to 10.40%, while the precision was <10.41%. The mean extraction recovery was >75.32%, and the assay was free of matrix effect. The validated LC-MS/MS method was successfully applied to the pharmacokinetic study of obacunone in rats after oral and intravenous administrations. The oral bioavailability of obacunone was 13.59%.

A Rapid, Selective and Sensitive UPLC-MS/MS Method for Quantification of Nomilin in Rat Plasma and Its Application in a Pharmacokinetic Study.

Nomilin is a potential anticancer agent. In this study, a rapid, sensitive, and simple ultra-performance liquid chromatography with tandem mass spectrometry methodology was established and validated to quantify nomilin in rat plasma. Plasma samples were prepared through liquid-liquid extraction using ethyl acetate. Chromatographic separation was performed using an Acquity HSS T3 column. Acetonitrile and water containing 0.1% (v/v) formic acid were used as mobile phases at a flow rate of 0.3 mL/min. Nomilin and quercetin (internal standard) were detected and quantified via a triple quadrupole tandem mass spectrometer in the positive ion mode with multiple reaction monitoring. Tandem mass spectrometry detection was performed by monitoring the fragmentations of m/z 515.3 → m/z 161.0 and m/z 303.2 → m/z 153.1 of nomilin and quercetin, respectively. Good linearity (R(2) > 0.996) was observed in the concentration range of 1 ng/mL to 500 ng/mL with a lower limit of quantification of 1 ng/mL for nomilin. The average extraction recoveries of nomilin and quercetin were > 82.3% and 82.0%, respectively. Intra- and interday precisions were less than 15% and accuracy ranged from 85.0% to 90.1%. Indeed, the proposed method was successfully applied to analyze the pharmacokinetics of nomilin after 3 and 50 mg/kg nomilin were administered to rats via intravenous and oral routes, respectively.

Metabolism, Excretion and Pharmacokinetics of MLN3897, a CCR1 Antagonist, in Humans.

UNLABELLED: MLN3897 is a small molecule antagonist of the C-C chemokine receptor-1. Since preclinical studies showed that the molecule was metabolized into two halves, the metabolism, excretion, and pharmacokinetics of MLN3897 were investigated in humans using MLN3897 14C-radiolabeled either on the chlorophenyl (CP) or the tricyclic (TC) half of MLN3897 after an oral dose. OBJECTIVE: To evaluate the mass balance, metabolism and pharmacokinetics of MLN3897 in two cohorts of six randomized healthy subjects. METHOD: After receiving informed consent, subjects were dosed after an overnight fast of 10-hours followed by at least 4- hours after dosing on day-1. Each cohort received a single 29 mg oral dose of either the CP or the TC as an oral solution in water. Serial blood samples, urine and feces were collected over a 10-day period post-dose. RESULTS: For both radiolabeled moieties, 55-59% of the dose was recovered in feces and 32% recovered in urine. MLN3897 was metabolized extensively in humans, with minor amounts of intact MLN3897 detected in the urine and feces. N-oxidation of the tricyclic moiety (M28) and N-dealkylation of the piperidinyl moiety were the primary metabolic pathways leading to further formation of the carboxylic acid metabolite (M19) and the (4-(4-chlorophenyl)-3,3- dimethylpiperidin-4-ol) metabolite (M40). Oxidative metabolites M11, M19, M28, M44 were present at >10% of the total circulating radioactivity and also at >25% of MLN3897 exposure. Metabolites resulting from the chlorophenyl-labeled moiety (M40) had significantly more systemic exposure compared to the tricyclic-labeled moiety (M19).

Glucocorticoid receptor modulators informed by crystallography lead to a new rationale for receptor selectivity, function, and implications for structure-based design.

The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands. Compound 10, the archetype of a new family of dibenzoxepane and dibenzosuberane sulfonamides, is a potent anti-inflammatory agent with selectivity for the GR versus other steroid receptors and a differentiated gene expression profile versus clinical glucocorticoids (lower GR transactivation with comparable transrepression). A stereospecific synthesis of this chiral molecule provides the unique topology needed for biological activity and structural biology. In vivo activity of 10 in acute and chronic models of inflammation is equivalent to prednisolone. The crystal structure of compound 10 within the GR ligand binding domain (LBD) unveils a novel binding conformation distinct from the classic model adopted by cognate ligands. The overall conformation of the GR LBD/10 complex provides a new basis for binding, selectivity, and anti-inflammatory activity and a path for further insights into structure-based ligand design.

Rational design of phosphoinositide 3-kinase α inhibitors that exhibit selectivity over the phosphoinositide 3-kinase ß isoform.

Of the four class I phosphoinositide 3-kinase (PI3K) isoforms, PI3Kα has justly received the most attention for its potential in cancer therapy. Herein we report our successful approaches to achieve PI3Kα vs PI3Kß selectivity for two chemical series. In the thienopyrimidine series of inhibitors, we propose that select ligands achieve selectivity derived from a hydrogen bonding interaction with Arg770 of PI3Kα that is not attained with the corresponding Lys777 of PI3Kß. In the benzoxepin series of inhibitors, the selectivity observed can be rationalized by the difference in electrostatic potential between the two isoforms in a given region rather than any specific interaction.

Kinetics of human serum butyrylcholinesterase inhibition by a novel experimental Alzheimer therapeutic, dihydrobenzodioxepine cymserine.

Cholinergic loss is the single most replicated neurotransmitter deficiency in Alzheimer's disease (AD) and has led to the use of acetylcholinesterase inhibitors (AChE-Is) and unselective cholinesterase inhibitors (ChE-Is) as the mainstay of treatment. AChE-Is and ChE-Is, however, induce dose-limiting adverse effects. Recent studies indicate that selective butyrylcholinesterase inhibitors (BuChE-Is) elevate acetylcholine (ACh) in brain, augment long-term potentiation, and improve cognitive performance in rodents without the classic adverse actions of AChE-Is and ChE-Is. BuChE-Is thereby represent a new strategy to ameliorate AD, particularly since AChE activity is depleted in AD brain, in line with ACh levels, whereas BuChE activity is elevated. Our studies have focused on the design and development of cymserine analogues to induce selective time-dependent brain BuChE inhibition, and on the application of innovative and quantitative enzyme kinetic analyses to aid selection of drug candidates. The quantitative interaction of the novel inhibitor, dihydrobenzodioxepine cymserine (DHBDC), with human BuChE was characterized. DHBDC demonstrated potent concentration-dependent binding with BuChE. The IC(50) and specific new kinetic constants, such as K(T50), P(PC), K(T1/2) and R(I), were determined at dual substrate concentrations of 0.10 and 0.60 mM butyrylthiocholine and reaction times, and are likely attainable in humans. Other classical kinetic parameters such as K(ia), K(ma), V(ma) and V(mi) were also determined. In synopsis, DHBDC proved to be a highly potent competitive inhibitor of human BuChE in comparison to its structural analogue, cymserine, and represents an interesting drug candidate for AD.

Studies on the novel antiallergic agent HSR-609: its penetration into the central nervous system in mice and guinea pigs and its selectivity for the histamine H1-receptor.

We studied the pharmacological characteristics of HSR-609 (3-[4-(8-fluoro-5,11-dihydrobenz[b]oxepino[4,3-b]pyridin-11- ylidene)- piperidino]propionic acid dihydrate), a novel amphoteric antiallergic agent, on the central nervous system (CNS). Its selectivity for the histamine H1-receptor and its ability to penetrate into the CNS were compared with those of typical antiallergic agents and the nonamphoteric basic compound PY-608 (8-fluoro-5,11-dihydro-11-(1-methyl-4-piperidylidene)benz[b]oxe pino- [4,3-b]pyridine), which has a chemical structure similar to that of HSR-609. In the in vitro study, HSR-609 had a high affinity for H1-receptors in the guinea pig cerebral cortex in comparison to affinities for muscarinic and serotonin 5-HT2-receptors in the rat cerebral cortex, while the selectivity of PY-608 for the H1-receptor was low. The inhibitory effects of these antiallergic agents on histamine-induced increase of vascular permeability in mice (ED50) were compared with the displacement of [3H]mepyramine binding to H1-receptors in mouse brain ex vivo (ID50). The ID50/ED50 ratio of HSR-609 was much larger than those of cyproheptadine, ketotifen and PY-608 and larger than those of terfenadine and cetirizine. HSR-609 was found to display selective displacement of the [3H]mepyramine binding to H1-receptors for lung vs cerebral cortex as found with terfenadine in guinea pigs ex vivo. These findings suggest that HSR-609 has high selectivity for the H1-receptor and poor ability to penetrate into the CNS in mice and guinea pigs due to its amphoteric chemical structure.

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization.

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzox adi azole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid liquid extraction following the derivatization. The conjugate was analyzed by ion-pair-HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.

[Effects of a new antiarrhythmic drug, KW-3407, on canine ventricular arrhythmia models].

The antiarrhythmic and direct cardiovascular effects of the new antiarrhythmic agent KW-3407, 5-[[2-(diethylamino)ethyl]amino]-7-methoxy-5,11- dihydro[1]benzoxepino[3,4-b]pyridine 1.5 fumarate, were examined. To evaluate antiarrhythmic effects, two-stage coronary ligation-, digitalis- and adrenaline-induced spontaneously occurring arrhythmias were used. KW-3407, 20 mg/kg/10 min, suppressed these three arrhythmia models, similar to flecainide, mexiletine and phenytoin. The antiarrhythmic plasma concentrations, IC50, of KW-3407 for 24-hr and 48-hr coronary ligation-, digitalis- and adrenaline-induced arrhythmias were 18.1, 14.4, 18.3 and 21.4 micrograms/ml, respectively; and these values were similar to one another. In the canine blood perfused atrioventricular (AV) node, sinoatrial node and papillary muscle preparations, KW-3407 decreased the sinoatrial rate and contractile force, and increased the coronary blood flow and AV conduction times, but these effects were weaker than those of disopyramide and flecainide and were short-lived. These results indicate that KW-3407 can be expected to become a clinically useful antiarrhythmic drug.

Tolerance and pharmacokinetics of a new antiulcer compound in man after single oral doses.

5-[(2-Diethylamino)ethyl]amino-5,11-dihydro[1]benzoxepino[3,4- b]pyridine trihydrochloride (KW-5805) is a new antiulcer compound, which was effective in animal studies by increasing gastric mucus and stimulating defensive factors such as glucosamine-synthesizing enzymes rather than by inhibiting aggressive factors. In this placebo-controlled, double-blind study the tolerability and pharmacokinetics of KW-5805 have been evaluated first time in man. Single oral doses of 2.5 to 320 mg were administered to 3 healthy young male subjects per dose level. One additional subject received placebo at each dose level. Plasma and urine samples were collected up to 24 h after administration and analysed gaschromatographically respectively by a high performance liquid chromatography. The mean maximum concentrations in plasma of KW-5805 occurred between 1.17 and 3.33 h after administration, independent of the dose. The half-lives of elimination varied between 6.63 and 11.9 h. 13.4-23.7, 9.6-13.4 and 6.5-11.0% of the administered dose were recovered in the urine after 24 h as unchanged substance, as monodeethylated (M-1) and as hydroxylated (M-3) metabolite, respectively. KW-5805 was not associated with any clinically significant effect on vital signs, ECG or laboratory investigations. Subjectively and objectively the substance was well tolerated in the dose range administered.