Intravenous Infusion of the ß3-Adrenergic Receptor Antagonist APD418 Improves Left Ventricular Systolic Function in Dogs With Systolic Heart Failure.

BACKGROUND: Unlike ß1- and ß2-adrenergic receptors (ARs), ß3-AR stimulation inhibits cardiac contractility and relaxation. In the failing left ventricular (LV) myocardium, ß3-ARs are upregulated, and can be maladaptive in the setting of decompensation by contributing to LV dysfunction. This study examined the effects of intravenous infusions of the ß3-AR antagonist APD418 on cardiovascular function and safety in dogs with systolic heart failure (HF). METHODS AND RESULTS: Three separate studies were performed in 21 dogs with coronary microembolization-induced HF (LV ejection fraction [LVEF] of approximately 35%). Studies 1 and 2 (n = 7 dogs each) were APD418 dose escalation studies (dosing range, 0.35-15.00 mg/kg/h) designed to identify an effective dose of APD418 to be used in study 3. Study 3, the sustained efficacy study, (n = 7 dogs) was a 6-hour constant intravenous infusion of APD418 at a dose of 4.224 mg/kg (0.70 mg/kg/h) measuring key hemodynamic endpoints (e.g., EF, cardiac output, the time velocity integral of the mitral inflow velocity waveform representing early filling to time-velocity integral representing left atrial contraction [Ei/Ai]). Studies 1 and 2 showed a dose-dependent increase of LVEF and Ei/Ai, the latter being an index of LV diastolic function. In study 3, infusion of APD418 over 6 hours increased LVEF from 31 ± 1% to 38 ± 1% (P < .05) and increased Ei/Ai from 3.4 ± 0.4 to 4.9 ± 0.5 (P < .05). Vehicle had no effect on the LVEF or Ei/Ai. In study 3, APD418 had no significant effects on the HR or the systemic blood pressure. CONCLUSIONS: Intravenous infusions of APD418 in dogs with systolic HF elicit significant positive inotropic and lusitropic effects. These findings support the development of APD418 for the in-hospital treatment of patients with an acute exacerbation of chronic HF.

Safety, Pharmacokinetics, and Efficacy of Olorinab, a Peripherally Acting, Highly Selective, Full Agonist of the Cannabinoid Receptor 2, in a Phase 2a Study of Patients With Chronic Abdominal Pain Associated With Crohn's Disease.

Background: This randomized, open-label phase 2a study investigated the safety/tolerability, pharmacokinetics, and efficacy of olorinab-a highly selective, peripherally acting, full agonist of the cannabinoid receptor 2-in patients with Crohn's disease (CD) experiencing abdominal pain. Methods: Eligible subjects 18-80 years of age with quiescent to mildly active CD were randomized to receive olorinab 25 or 100 mg three times daily for 8 weeks. The primary objective was to assess safety/tolerability. Results: Fourteen subjects received olorinab 25 mg (N = 6) or 100 mg (N = 8). Ten subjects [4 (67%) in the 25-mg group and 6 (75%) in the 100-mg group] reported a total of 34 treatment-emergent adverse events (TEAEs; 32 grade 1/2, not serious events; 2 grade 3, serious, not treatment-related events). No dose reductions or discontinuations due to TEAEs or deaths were reported. Dose-proportional increases in olorinab exposure from 25 to 100 mg were observed, with minimal accumulation at both doses. At week 8, the mean (SD) change from baseline in average abdominal pain score at peak olorinab plasma concentrations was -4.61 (1.77) in the 25-mg group (P = 0.0043) and -4.57 (2.17) in the 100-mg group (P = 0.0036). The change from baseline at week 8 in the mean (SD) number of pain-free days per week was +1.60 (2.61) in the 25-mg group and +2.33 (3.62) in the 100-mg group. No subject required pain medication on study. Conclusions: Patients with quiescent to mildly active CD receiving olorinab experienced mild-to-moderate adverse events and an improvement in abdominal pain scores in this study.

Rapid identification of a novel phosphodiesterase 7B tracer for receptor occupancy studies using LC─MS/MS.

Phosphodiesterase 7B (PDE7B) inhibition has been considered as a therapeutic target for the treatment of several neurological disorders. Currently, there are no radio-labeled tracers available to determine receptor occupancy (RO) of this target. Developing such a tracer could greatly facilitate the identification of viable PDE7B inhibitors. In the current study, a liquid chromatography tandem mass spectrometry (LC─MS/MS) method was utilized to evaluate the brain distribution of unlabeled tracer candidates following intravenous micro-dosing. This novel approach resulted in an accelerated identification of a potential novel RO tracer for PDE7B. The identified molecule, Compound 30, showed reasonable target-tissue specificity (striatum/cerebellum ratio of 2.2) and suitable uptake (0.25% of the injected dose/g brain tissue) as demonstrated in rats dosed with the unlabeled compound. Compound 30 was subsequently labeled with tritium (3H). In vitro characterization of 3H-Compound 30 demonstrated that this compound possessed a high target affinity with a subnanomolar Kd (0.8 nM) and a Bmax of 58 fmol/mg of protein using rat brain homogenate. Intravenous microdosing of 3H-Compound 30 showed preferential binding in the rat striatum, consistent with the mRNA distribution of PDE7B. In vitro displacement study with other structurally distinct PDE7B target-specific inhibitors using rat brain homogenate indicated that 3H-Compound 30 is an ideal tracer for Ki analysis. This is the first report of a preclinical tracer for PDE7B. With further characterization, Compound 30 may ultimately show the appropriate properties required to be further developed as a PDE7B PET ligand for clinical studies.

In vitro Drug Metabolism Investigation of 7-Ethoxycoumarin in Human, Monkey, Dog and Rat Hepatocytes by High Resolution LC-MS/MS.

BACKGROUND: Recently, it has been an increasing concern on the bioactivation and adverse reactions associated with consumption of herbal and nature products. 7-Ethoxycoumarin is one of coumarin family compounds, but little information is available regarding its potential reactive metabolites. METHOD: 7-ethoxylcoumarin was incubated individually with human, monkey, dog and rat hepatocytes for 2 hr, metabolites were detected, identified and characterized using high resolution liquid chromagraphy - tandem mass spectrometry. RESULTS: Twenty-eight metabolites (M1 - M28) were detected and identified. O-deethylation, glucuronidation, sulfation, oxygenation, oxidative ring-opening, hydrogenation, glutathionation, dehydrogenation, cysteination, glucosidation, methylation, and hydrolysis were observed. At least sixteen metabolites not reported previously, were newly identified. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3 (O-deethylation and glucuronidation), M5 (hydrolysis and mono-oxygenation), M14 (O-deethylation), M16 (hydrolysis), M22 (oxidative ring-opening and oxygenation) and M27 (monooxygenation) exhibited high mass spectrometric responses in human hepatocytes. M3, M5, M8, M13 (mono-oxygenation), M14, M16, M18 (O-deethylation and sulfation), M22 and M27 exhibited high mass spectrometric responses in monkey hepatocytes. M14, M16, M18, M20 (glutathionation and dehydrogenation) and M27 exhibited high mass spectrometric responses in dog hepatocytes. M1 (Odeethylation, mono-oxygenation and glucuronidation), M3, M5, M13, M14, M16, M17 (cysteination), M18, M20, and M22 exhibited high mass spectrometric responses in rat hepatocytes. CONCLUSION: Most of new metabolites via oxidative ring-opening and glutathionation were identified. Species differences in metabolism of 7-ethoxylcoumarin in hepatocytes were observed. The analysis of metabolites suggests that 7-ethoxylcoumarin may undergo 3,4-epoxidation responsible for formation of glutathione and its derived cysteine conjugates, carboxylic acid and its glucuronides, glucosides and sulfate.

A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the determination of endogenous cyclic nucleotides in rat brain.

A robust high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3',5'-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3',5'-cyclic guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure endogenous 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC-MS/MS. The calibration curves for both analytes ranged from 5 to 2000ng/g and showed excellent linearity (r2>0.996). Relative surrogate analyte-to-analyte LC-MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies.

Aminocyclohexylsulfonamides: discovery of metabolically stable alpha(1a/1d)-selective adrenergic receptor antagonists for the treatment of benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS).

Benign prostatic hyperplasia/lower urinary tract symptoms (BPH/LUTS) can be effectively treated by alpha(1) adrenergic receptor antagonists, but these drugs also produce side effects that are related to their subtype non-selective nature. To overcome this limitation, it was hypothesized that an alpha(1a/1d) subtype-selective antagonist would be efficacious while keeping side effects to a minimum. To discover alpha(1a/1d)-selective antagonists and improve metabolic stability of our previously reported compounds, we have designed and synthesized a series of (phenylpiperazinyl)- or (phenylpiperidinyl)-cyclohexylsulfonamides. By incorporating the information obtained from metabolism studies, we were able to discover several compounds that are both alpha(1a/1d) adrenoceptor subtype selective and show increased stability toward human liver microsomal metabolism. The selectivity profile of these compounds provides great improvement over the commercial drug tamsulosin, hence may pave the way to the development of new and efficacious therapeutic agents with reduced side effects.

Disposition of the selective cholesterol absorption inhibitor ezetimibe in healthy male subjects.

Ezetimibe [SCH 58235; 1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone], a selective cholesterol absorption inhibitor, is being developed for the treatment of primary hypercholesterolemia. The absorption, metabolism, and excretion of ezetimibe were characterized in eight healthy male volunteers in this single-center, single-dose, open-label study. Subjects received a single oral 20-mg dose of [14C]ezetimibe (approximately 100 microCi) with 200 ml of noncarbonated water after a 10-h fast. Concentrations of radioactivity and/or ezetimibe (conjugated and unconjugated) were determined in plasma, urine, and fecal samples. Ezetimibe was rapidly absorbed and extensively conjugated following oral administration. The main circulating metabolite in plasma was SCH 60663 [1-O-[4-[trans-(2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl]-beta-D-glucuronic acid], the glucuronide conjugate of ezetimibe. Plasma concentration-time profiles of unconjugated and conjugated drug exhibited multiple peaks, indicating enterohepatic recycling. Approximately 78 and 11% of the administered [14C]ezetimibe dose were excreted in feces and urine, respectively, by 240 h after drug administration. Total recovery of radioactivity averaged 89% of the administered dose. The main excreted metabolite was the glucuronide conjugate of ezetimibe. The primary metabolite in urine (0- to72-h composite) was also the glucuronide conjugate (about 9% of the administered dose). Significant amounts (69% of the dose) of ezetimibe were present in the feces, presumably as a result of SCH 60663 hydrolysis and/or unabsorbed drug. No adverse events were reported in this study. A single 20-mg capsule of [(14)C]ezetimibe was safe and well tolerated after oral administration. The pharmacokinetics of ezetimibe are consistent with extensive glucuronidation and enterohepatic recirculation. The primary metabolic pathway for ezetimibe is by glucuronidation of the 4-hydroxyphenyl group.