A Phase I, Single-Ascending-Dose Study in Healthy Subjects to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of DS-2969b, a Novel GyrB Inhibitor.

DS-2969b is a novel GyrB inhibitor in development for the treatment of Clostridium difficile infection. The aim of this study was to assess the safety, tolerability, pharmacokinetics, and effects on normal gastrointestinal microbiota groups of single daily oral ascending doses of DS-2969b in healthy subjects. The study enrolled 6 sequential ascending dose cohorts (6 mg, 20 mg, 60 mg, 200 mg, 400 mg, and 600 mg). In each cohort, 6 subjects were administered DS-2969b and 2 subjects were administered matching placebo. DS-2969b was safe and well tolerated at all dose levels examined. All adverse events related to DS-2969b were mild to moderate in severity and predominantly related to the gastrointestinal tract. DS-2969a (free form of DS-2969b) plasma concentrations increased with increasing doses; however, both the maximum serum concentration and area under the plasma concentration-time curve generally increased less than dose proportionally. DS-2969a was predominantly eliminated in the urine, with feces as a minor route of elimination. While the overall proportion of DS-2969a eliminated in the feces was low, target fecal levels sufficient for C. difficile eradication were achieved within 24 hours of administration with doses of 60 mg or higher. In exploratory analyses, DS-2969b appeared to reduce bacterial counts in 8 of the 25 groups of normal intestinal microbiota examined, suggesting that DS-2969b has only a mild effect on intestinal microbiota. Data from this study support and encourage further development of DS-2969b as a novel treatment for C. difficile infection.

LC-MS/MS determination of tideglusib, a novel GSK-3ß inhibitor in mice plasma and its application to a pharmacokinetic study in mice.

A sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of tideglusib in mice plasma using warfarin as an internal standard (I.S.) as per regulatory guidelines. Sample preparation was accomplished through liquid-liquid extraction process. Chromatographic separation was performed on Atlantis dC18 column using mobile phase A (acetonitrile) and B (5mM ammonium acetate in water) in a flow-gradient mode. Elution of tideglusib and the I.S. occurred at ∼2.06 and 1.29min, respectively. The total chromatographic run time was 3.2min. A linear response function was established in the concentration range of 20.2-1008ng/mL. The intra- and inter-day accuracy and precision were in the range of 4.61-12.6 and 6.04-11.8%, respectively. This novel method has been applied to a pharmacokinetic study in mice.

Preclinical abuse liability assessment of ABT-126, an agonist at the α7 nicotinic acetylcholine receptor (nAChR).

ABT-126 is a nicotinic acetylcholine receptor (nAChR) agonist that is selective for the α7 subtype of the receptor. nAChRs are thought to play a role in a variety of neurocognitive processes and have been a pharmacologic target for disorders with cognitive impairment, including schizophrenia and Alzheimer's disease. As part of the preclinical safety package for ABT-126, its potential for abuse was assessed. While the involvement of the α4ß2 subtype of the nicotinic receptor in the addictive properties of nicotine has been demonstrated, the role of the α7 receptor has been studied much less extensively. A number of preclinical assays of abuse potential including open-field, drug discrimination and self-administration were employed in male rats. ABT-126 had modest effects on locomotor activity in the open-field assay. In nicotine and d-amphetamine drug discrimination assays, ABT-126 administration failed to produce appreciable d-amphetamine-like or nicotine-like responding, suggesting that its interoceptive effects are distinct from those of these drugs of abuse. In rats trained to self-administer cocaine, substitution with ABT-126 was similar to substitution with saline, indicating that it lacks reinforcing effects. No evidence of physical dependence was noted following subchronic administration. Overall, these data suggest that ABT-126 has a low potential for abuse. Together with other literature on this drug class, it appears that drugs that selectively activate α7 nAChRs are not likely to result in abuse or dependence.

Late-stage optimization of a tercyclic class of S1P3-sparing, S1P1 receptor agonists.

Poor solubility and cationic amphiphilic drug-likeness were liabilities identified for a lead series of S1P3-sparing, S1P1 agonists originally developed from a high-throughput screening campaign. This work describes the subsequent optimization of these leads by balancing potency, selectivity, solubility and overall molecular charge. Focused SAR studies revealed favorable structural modifications that, when combined, produced compounds with overall balanced profiles. The low brain exposure observed in rat suggests that these compounds would be best suited for the potential treatment of peripheral autoimmune disorders.

Utilizing physiologically based pharmacokinetic modeling to inform formulation and clinical development for a compound with pH-dependent solubility.

ARRY-403 is a glucokinase activator developed for the treatment of diabetes. Less than dose-proportional exposure was observed during single ascending dose studies with ARRY-403. A physiologically based pharmacokinetic (PBPK) model for ARRY-403 was developed through integration of in vitro physicochemical data with precipitation time estimations based on results from the single ascending dose studies; PBPK modeling indicated that the primary cause of the less than dose-proportional exposure was dose-limited absorption because of pH-dependent solubility. The impact of dose, particle size, and fasted or fed state on ARRY-403 exposure was examined through sensitivity analyses and used to refine the PBPK model. On the basis of the marked pH-dependent solubility of ARRY-403, the refined PBPK model was used to simulate the effects of acid-reducing agents (ARAs) on ARRY-403 exposure, as these agents are widely available and could be coadministered with ARRY-403. The simulations indicated that a clinical study with an ARA was warranted; in a clinical study, famotidine had a marked effect on ARRY-403 exposure. This approach, based on the "predict, learn, and confirm" paradigm, demonstrates the utility of integrating physicochemical properties, in vitro experiments, and clinical results using PBPK to inform formulation development and to guide clinical study design.

A novel GSK-3beta inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo.

Amyloid deposits, neurofibrillary tangles, and neuronal cell death in selectively vulnerable brain regions are the chief hallmarks in Alzheimer's (AD) brains. Glycogen synthase kinase-3 (GSK-3) is one of the key kinases required for AD-type abnormal hyperphosphorylation of tau, which is believed to be a critical event in neurofibrillary tangle formation. GSK-3 has also been recently implicated in amyloid precursor protein (APP) processing/Abeta production, apoptotic cell death, and learning and memory. Thus, GSK-3 inhibition represents a very attractive drug target in AD and other neurodegenerative disorders. To investigate whether GSK-3 inhibition can reduce amyloid and tau pathologies, neuronal cell death and memory deficits in vivo, double transgenic mice coexpressing human mutant APP and tau were treated with a novel non-ATP competitive GSK-3beta inhibitor, NP12. Treatment with this thiadiazolidinone compound resulted in lower levels of tau phosphorylation, decreased amyloid deposition and plaque-associated astrocytic proliferation, protection of neurons in the entorhinal cortex and CA1 hippocampal subfield against cell death, and prevention of memory deficits in this transgenic mouse model. These results show that this novel GSK-3 inhibitor has a dual impact on amyloid and tau alterations and, perhaps even more important, on neuronal survival in vivo further suggesting that GSK-3 is a relevant therapeutic target in AD.

Pharmacokinetic studies of a novel 1,2,4-thiadiazole derivative, inhibitor of Factor XIIIa, in the rabbit by a validated HPLC method.

Activated Factor XIII (FXIIIa) stabilizes fibrin clot by covalent cross-linking of fibrin strands in the fibrin, making it resistant to physiological and pharmacologically induced fibrinolysis. Inhibition of Factor XIIIa offers a novel approach to treatment of thrombosis. Selected derivatives of 1,2,4-thiadiazoles, presently in discovery and development, may offer new treatment strategies as inhibitors of Factor XIIIa. In order to evaluate its pharmacokinetic (PK) profile and to facilitate the selection of drug candidates for drug discovery and development process, we developed and validated a simple and selective reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection for the determination of N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]-2-nitrobenzensulfonamide (5624) in rabbit plasma. The plasma protein precipitation and sample preparation was achieved by using acetonitrile, followed by organic phase evaporation to dryness and the residue reconstitution in the mobile phase. The 5624 recovery from the plasma was about 90%. Chromatography was performed on a C18 column using a gradient of acetonitrile in water as a mobile phase. A chemically related compound, N-[6-(imidazo[1,2-d][1,2,4]thiadiazol-3-ylamino)hexyl]naphthalene-1-sulfonamide (5422), was used as an internal standard. Limit of detection (LOD), based on signal to noise ratio>3, was 0.2 microM (on-column amount of about 7 ng), while limit of quantification (LOQ), based on signal to noise ratio>10, was 0.5 microM (on-column amount of about 20 ng). The plasma samples for the PK study were collected at defined time points during and after 5624 slow intravenous infusion (25 mg/kg) to male White New Zealand rabbits and analyzed by RP-HPLC method. The PK parameters, such as half-life, volume of distribution, total clearance, elimination rate constant etc., were determined. The PK profile of 5624 offered insights in the design and development of additional new compounds, derivatives of 1,2,4-thiadiazole, with desired PK properties.

Plasma kinetics and efficacy of oral megazol treatment in Trypanosoma brucei brucei-infected sheep.

Experimentally infected sheep have been previously developed as an animal model of trypanosomosis. We used this model to test the efficacy of megazol on eleven Trypanosoma brucei brucei-infected sheep. When parasites were found in blood on day 11 post-infection, megazol was orally administered at a single dose of 40 or 80mg/kg. After a transient aparasitaemic period, all animals except two relapsed starting at day 2 post-treatment, which were considerated as cured on day 150 post-treatment and showed no relapse after a follow-up period of 270 days. In order to understand the high failure of megazol treatment to cure animals, a kinetic study was carried out. Plasma concentrations of megazol determined, by reverse-phase high-performance liquid chromatography at 8h post-treatment in these animals, were lowered, suggesting slow megazol absorption, except in cured animals. However, megazol plasma profiles in uninfected sheep after a single oral dose of megazol showed a fast megazol lowered absorption associated with a short plasma half-life of drug. Inter-individual variation of megazol pharmacokinetic properties was also observed. These findings suggested that the high failure rates of megazol treatment were related to poor drug availability after oral administration in sheep. In conclusion, megazol could cure sheep with T. b. brucei infection but oral administration was not an effective route.

Liquid chromatography-mass spectrometry assay of a thiadiazole derivative in mice: application to pharmacokinetic studies.

Modern atmospheric pressure ionization (API) ion-trap mass spectrometry in connection with fast chromatographic separations using a short narrow-bore C8 column was developed to determine 5-phenyl-3-thioureido-1,2,4-thiadiazole (301029), a novel virus inhibitor in serum. Both 301029 and an internal standard (I.S.) were separated from serum samples by acetonitrile deproteinization and extraction without time-consuming reconstitution. The chromatographic separation was achieved on a C8 reversed-phase narrow-bore column using acetonitrile-water-acetic acid (90:10:0.01, v/v/v) as a mobile phase. The mass spectrometric analysis was performed by atmospheric pressure chemical ionization (APCI) mode with positive ion detection. Single ion monitoring (SIM) scan mode of m/z 237 and 158 was used to quantitatively determine 301029 and I.S., respectively. The low limit of quantitation was 25 ng/ml. The assay exhibited a linear range of 25-2500 ng/ml. Recovery from serum proved to be 100-113%. The precision (C.V.) and accuracy (RE) of the method were 2-12% and 94-112%, respectively. The present method was applied to determine the pharmacokinetic parameters of 301029 following oral administration of the agent to mice at 5 g/kg. The results revealed that the elimination half-life of 301029 was 413 min and the area under serum concentration-time curve was 354 microg/ml/min.

Pharmacokinetics, metabolism and excretion of megazol, a new potent trypanocidal drug in animals.

The pharmacokinetics of megazol (CAS 19622-55-0) was investigated after intraperitoneal and oral administration of the drug (80 mg/kg) to mice. The plasma levels were significantly higher after oral administration of drug than after intraperitoneal route (33.8 micrograms/ml compared with 19.0 micrograms/ml for Cmax, 158714 micrograms.h/l compared with 96057 micrograms.h/l for AUC). When suramin (CAS 145-63-1) was administered 24 h before oral administration of megazol, megazol absorption was accelerated (2 h compared with 4 h for Tmax) but the amount absorbed was lower (19.9 micrograms/ml compared with 33.8 micrograms/ml for Cmax and 95547 micrograms.h/l vs 158714 micrograms.h/l for AUC). In the infected mice previously treated with suramin, all estimated pharmacokinetic parameters of plasma megazol were significantly modified, in particularly an increase in the apparent volume of distribution (5.6 l/kg compared with 0.9 l/kg) with a prolongation of the elimination half-life (3 h compared with 0.7 h) of megazol. Excretion of the total radioactivity of megazol was also evaluated after oral administration of 3H-megazol to rats. Total radioactivity was eliminated predominantly via the urinary route (80%) vs. 10.5% in the faeces, 9.5% remaining in the body 8 days after dosing. When unlabelled megazol was orally administered to rats with absence or presence of suramin, megazol recovered in urine and faeces 72 h dosing was: 55.7%/2% vs 20.6%/1.6%, respectively. In the urine, unchanged megazol was present as characterized by LC-MS/MS as well as 4 unknown metabolites. This study indicates that suramin significantly affects the pharmacokinetics of megazol and its elimination.

Sensitive method for the determination of KC 12291 in rat plasma and urine by high-performance liquid chromatography.

A sensitive and selective ion-pair reversed-phase HPLC method has been developed for the quantitative determination of KC 12291 and its major metabolite, KC 13194, in rat plasma and urine. An Ultrasphere ODS column constructed by using a mobile phase of 1 mM 1-octanesulfonic acid containing acetonitrile-0.1 M triethylamine phosphate buffer, pH 2.2 (29:71, v/v in plasma and 27:73, v/v in urine), an internal standard and a fluorescent detector (excitation 265 nm, emission 370 nm) were used for the separation and the quantitative determination, respectively. The plasma samples were made alkaline and both compounds were cleaned up by the use of liquid-liquid extraction. The limit of quantification was 10 ng/ml for KC 12291 in plasma and urine and for KC 13194 and 100 ng/ml in plasma, respectively. The assay has been validated with respect to system suitability, accuracy, precision, recovery, stability and ruggedness. All validated parameters were found to be within the necessary limits.

Simple high-performance liquid chromatographic method to analyse megazol in human and rat plasma.

A simple and sensitive high-performance liquid chromatographic method has been developed to measure megazol in human plasma. The method was optimized and validated according to the Washington Concensus Conference on the Validation of Analytical Methods (V.P. Shah et al., Eur. J. Drug Metab. Pharmacokinet., 15 (1991) 249). The criteria of complete validation were specificity, linearity, precision, analytical recovery, dilution and stability. It involved extraction of the plasma with dichloromethane, followed by reversed-phase high-performance liquid chromatography using a Kromasil C8 column and UV detection at 360 nm. The retention times of the internal standard (tinidazol) and megazol were 6.10 and 9.60 min, respectively. The standard curve was linear from 2 ng ml-1 (limit of quantification) to 2000 ng ml-1. The coefficients of variation for all the criteria of validation were less than 6%; 85 to 92% extraction efficiencies were obtained. Megazol was stable during the storage period (one month at -20 degrees C) in plasma and for two months at 25 degrees C in standard solution. The method was tested by measuring the plasma concentration following oral administration to rat and was shown to be suitable for pharmacokinetic studies.

Determination of the muscarinic agent [(3-(3-1-butylthio)-1,2,5-thiadiazol-4-yl)-1-azabicyclo-2.2.2-oct ane], in rat, rabbit, and monkey plasma, using high-performance liquid chromatography in conjunction with tandem mass spectrometry.

A method for determining a selective muscarinic agent, LY297802 (compound I), [(3-(3-1-butylthio)-1,2,5-thiadiazol-4-yl)-1-azabicyclo-2.2.2-octa ne], indicated in the treatment of pain, in rat, rabbit, and monkey plasma is described. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane. The organic fraction was evaporated to dryness and the residue reconstituted with mobile phase. The analytes were detected utilizing HPLC in conjunction with electrospray (ES) tandem mass spectrometry (MS-MS). The limit of quantitation was 0.25 ng/ml, and the response was linear to at least 100 ng/ml.

High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma.

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11-0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasm with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 x 4.6 mm I.D., 5-microns column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than +/- 15%.

Determination of xanomeline and active metabolite, N-desmethylxanomeline, in human plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

We have developed a method for the determination of xanomeline and its pharmacologically active N-desmethyl metabolite. The validated method uses hexane to extract xanomeline and its N-desmethyl metabolite from basified plasma. The hexane extract is dried, reconstituted, and analyzed using a liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometry system. The method was developed to support phase II clinical trials and has proven to be extremely sensitive, fast, and rugged. The method has a limit of quantitation of 75 and 200 pg/ml plasma for xanomeline and the N-desmethyl metabolite, respectively. Sample analysis times were less than 3 min from one injection to the next.

Determination of xanomeline (LY246708 tartrate), an investigational agent for the treatment of Alzheimer's disease, in rat and monkey plasma by capillary gas chromatography with nitrogen-phosphorus detection.

A GC method is described for the determination of xanomeline (LY246708 tartrate) and selected metabolites in rat and monkey plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane. The organic extract was evaporated to dryness and the residue was reconstituted in hexane. The analytes were separated from metabolites and endogenous substances using a DB1701 capillary column. The analytes were detected using nitrogen-phosphorus detection (NPD). The limit of quantitation was determined to be 8 ng/ml, and the response was linear from 8 to 800 ng/ml. The method has been successfully applied to rat and monkey samples pursuant to the development of xanomeline as an agent for the symptomatic treatment of Alzheimer's disease.

Uptake and biodisposition of 2-ylcyanamide-1,3,4-thiadiazole (LY217896) by red blood cells.

In man, 14C-2-ylcyanamide-1 3 4-thiadiazole (LY217896) accumulates into red blood cells (RBCs) where it is rapidly metabolized. Both in man and ex vivo, within a few hours of administration of 14C-LY217896 at least two intracellular metabolites were detected within the RBCs using HPLC. These metabolites were never detected extracellularly. After 24 h no detectable radioactivity was found in the plasma and all the radioactivity was detected within the cellular fraction. All radioactivity was identified as a single peak within the RBCs, indicating the metabolite(s) to be highly polar compared to LY217896. Parent LY217896 was never detected within the RBCs at any time point, suggesting transport, either by diffusion or a carrier mediated mechanism, was the rate limiting step. Due to the nature of the preparation it was impossible to separately characterize uptake and biotransformation. Nevertheless, uptake/biotransformation was found to be temperature sensitive, sodium independent, and energy dependent. Both niacin and vitamin B6, but not nicotinamide, competitively blocked the uptake and subsequent intracellular metabolism of LY217896.

Determination of xanomeline in human plasma by ion-spray tandem mass spectrometry.

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Oral LY217896 for prevention of experimental influenza A virus infection and illness in humans.

The efficacy and safety of oral LY217896 for prevention of experimental influenza A/Kawasaki/86 (H1N1) virus infection were assessed in susceptible males randomly assigned to receive LY217896 (75 mg) or placebo once daily for 7 days beginning 24 h prior to viral challenge. The rates of virus shedding (100% in both groups), days of viral shedding (3.1 +/- 1.3 for the LY217896 group; 2.8 +/- 1.3 for the placebo group), and titers of virus in nasal washings did not differ between the groups. Mild upper respiratory tract illness (72% in the LY217896 group; 69% in the placebo group) developed in similar proportions of each group. LY217896 was associated with asymptomatic rises in serum uric acid levels and was ineffective in modifying the virologic or clinical course of experimental influenza A (H1N1) virus infection.

Subacute and subchronic toxicology studies of CI-986, a novel anti-inflammatory compound.

CI-986 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)- thione-2-hydroxy-N,N,N-trimethylethanaminium salt) is a novel anti-inflammatory compound classified as a dual inhibitor of cyclooxygenase and 5-lipoxygenase. Studies were undertaken to characterize the preclinical toxicology of the compound. CI-986 was administered to rats for 2 weeks (0, 50, 250, 750, and 1500 mg/kg) or 13 weeks (0, 20, 250, 500, and 1000 mg/kg), dogs for 2 weeks (0, 50, 150, and 500 mg/kg) or 13 weeks (0, 20, 100, and 200 mg/kg), and to monkeys for 2 weeks (0, 50, 250, and 1000 mg/kg). No drug-related deaths resulted. Mild clinical signs of toxicity were noted in rats given doses of 250 mg/kg and above. Drug-related emesis and diarrhea were absent at the low dose in the dog and monkey but increased in incidence and severity at higher doses. Severe clinical signs in monkeys (emesis and diarrhea) necessitated the lowering of the top dose to 500 mg/kg/day (administered b.i.d.) during the second week of the monkey study. Slight decreases (< 23%) in serum protein and/or albumin were noted in all studies at the higher doses. A dose-related increase in alkaline phosphatase was noted in both dog studies, with no other drug-related effect on clinical pathology parameters. A gastric ulcer occurred in one rat administered 500 mg/kg CI-986 for 13 weeks. Gastrointestinal ulcers were not noted at any other dose in rats or at any dose in dogs or monkeys. A dose-related eosinophilia of glandular stomach submucosa was noted in rats after 2 and 13 weeks of drug administration but not in dogs or monkeys. In the 2-week rat study, mean combined sex plasma drug concentrations monitored 2 hr after dose on Day 14 were 0.59, 1.10, 2.64, and 3.43 micrograms/ml for the 50, 250, 750, and 1,500 mg/kg dose groups, respectively. In the 2-week dog studies, maximum plasma drug concentrations on Day 10 or Day 11 were achieved within 2 hr of dose with mean combined sex Cmax values of 0.73, 2.05, and 2.62 micrograms/ml for the 50, 250, and 750 mg/kg groups, respectively. Hepatic microsomal induction characterized by increased microsomal protein, increased microsomal cytochrome P450 content, and increased p-nitroanisole O-demethylation activity was noted in dogs and monkeys but not rats. CI-986 was well tolerated in rats and dogs at the doses employed and in monkeys at doses up to 500 mg/kg (b.i.d.).(ABSTRACT TRUNCATED AT 250 WORDS)