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Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC–MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods – one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan – using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

The disposition of a novel kynurenine monooxygenase inhibitor, CHDI-340246, was investigated in vitro and in animals.In vitro, there was minimal metabolic turnover of CHDI-340246 in all species. The protein binding was higher in human plasma (99.7%) relative to other species.In all species, blood clearance was low ( 60%) in all species.In rats, [14C]CHDI-340246 showed wide distribution of radioactivity in all tissues except brain and testes. In rats, the parent drug was the major circulating moiety with minor amounts of a sulphate conjugate of an O-dealkylated metabolite. The elimination occurred via the urinary route and to a lesser extent by biliary route, but mostly as metabolites. In cynomolgus monkeys, the parent drug predominated in plasma with only trace amounts of metabolites detected.Acyl glucuronide conjugate of CHDI-340246 was not detected in plasma of rats or monkeys.Overall, the ADME profile of CHDI-340246 was favourable in rats and monkeys for potential evaluation of KMO inhibition in humans.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aβ- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting. ©

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs).

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood–brain barrier and converted via the kynurenine pathway to N-formyl- l -kynurenine, which is further degraded to l -kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC–MS/MS method to quantify l -kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy- l -kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098–100 ng/ml for 3HK, 9.8–20,000 ng/ml for KYN, 0.49–1000 ng/ml for KYNA and AA. The method was linear (r > 0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%).

AMPA receptors (AMPARs) have been demonstrated to be an important therapeutic CNS target. A series of substituted benzotriazinone and benzopyrimidinone derivatives were prepared with the aim to improve in vivo activity over the previously reported bis-benzoxazinone based AMPAKINE® series from our laboratory. These compounds were shown to be potent, positive allosteric AMPAR modulators that have better in vivo activity and improved metabolic stability over the analogous benzoxazinone derivatives.

The purpose of these studies was to determine the time course for onset of effects of hypoglycemia on insulin gene transcription in vivo. Using insulin infusions, we found that insulin-induced hypoglycemia decreased levels of precursors for insulin mRNA, reflecting changes in new mRNA synthesis, to new steady-state values within 100 min. These changes were followed by declines in processed insulin mRNA. An alternate infusion technique was developed to lower plasma glucose levels from a constant level of 120–130 to 50–60 mg/dl in

AMPA receptors (AMPARs) are an important therapeutic target in the CNS. A series of substituted benzobistriazinone, benzobispyrimidinone and related derivatives have been prepared with high potency and selectivity for the allosteric binding site of AMPARs. Further improvements have been made to previously reported series of positive AMPAR modulators and these compounds exhibit excellent in vivo activity and improved in vivo metabolic stability with up to 100% oral bioavailability in rat.

A limited number of reports of estrogen levels in prepubertal and early pubertal boys have been published because previous estrogen assays have lacked adequate sensitivity to quantitate circulating estrogen concentrations. Development of a new ultrasensitive assay has permitted measurement of estrogen levels in 23 normally growing boys progressing through puberty. Concentrations were measured at approximately 4-month intervals over a 5- to 8-yr period. The levels increased with maturation in all and correlated directly with chronological age, bone age, weight, height, pubertal stage, and testosterone and insulin-like growth factor-I levels. Of these factors, the level of testosterone had the greatest influence on the estrogen concentration. The time from peak growth velocity also significantly correlated with estrogen level. The estrogen level correlated positively with growth velocity before the time of peak growth velocity and negatively after peak growth velocity. The estrogen levels first increased significantly an average of 3 yr after pubertal onset and reached a peak by 5 yr after pubertal onset. Peak growth velocity was attained an average of 3 yr after pubertal onset. The greatest increase in the rate of rise of the estrogen level was an 11-fold rise during the year in which puberty began. The next most significant increase was a 4.8-fold rise 3 yr after pubertal onset. With respect to pubertal stage, the greatest absolute change occurred from stage 4 to stage 5 and the greatest fold change occurred from stage 1 to stage 2. The estrogen level did not significantly correlate with the 24-h GH level. In conclusion, circulating estrogen levels are very low in all boys prepubertally and rise steadily during adolescent development. The estrogen level is closely related to testosterone concentration and to the time of peak growth velocity. These findings are consistent with the hypothesis that estrogen at low levels augments skeletal growth and maturation in boys (as well as girls). They are also consistent with the hypothesis that continued exposure to estrogen leads to epiphyseal fusion. Further studies are required to define the separate and combined roles of estrogen, GH, and testosterone, as well as other factors, on growth and sexual development at puberty.

Immaturities in NB hematopoiesis include defects in both myelopoiesis and thrombopoiesis. 25% of babies admitted to the NICU experience thrombocytopenia(Castle et al, J Peds 108:1986), and it is a risk factor for neonatal morbidity and mortality (Andrews et al, J Peds 110:1987). TPO and rhG-CSF have been shown to be potent stimulators of thrombopoiesis and myelopoiesis, respectively. (Blood 86:(10), 1995) The present study investigates the effects of TPO alone and in combination with rhG-CSF in modulating NB rat hematopoiesis. NB Sprague Dawley rats (24-36 hrs, n=16/tx) were given SC TPO (10 μg/kg, (provided by Genentech), rhG-CSF (100μg/kg) (provided by Amgen), TPO + rhG-CSF (same doses) or PBS/HSA × 14 days. CBC and PLT were obtained on alternate days from d 0-20. Random animals (n=3/tx) were sacrificed on d 14 for bone marrow CFU-GM, MEG and proliferation studies. TPO alone or + rhG-CSF had no effect on HCT or RBC. However, significant increases in ANC (/mm3) were seen with TPO alone vs PBS/HSA beginning on d 2 (1386 ± 101.3 vs 1057.0 ± 68.6, p< 0.05), d 8 (1032.8 ± 96.3 vs 509.0 ± 62.7, p< 0.05), d 14 (1490.4 ± 233.6 vs 835.2 ± 72.3, p< 0.05). A significant increase in ANC (/mm3)was also seen with TPO + rhG-CSF vs rhG-CSF alone beginning on d 4 (2486.0 ± 256.1 vs 1714.4 ± 342.8, p< 0.01), d 8 (2207.2 ± 218.2 vs 1476.0 ± 179.1, p< 0.001), d 14(2124.0 ± 150.2 vs 1175.2 ± 169.2, p< 0.01). Significant increases in PLT (K/mm3) were seen with TPO alone d 4-18 with no effect seen with rhG-CSF alone. However, a significant abrogation of PLT was seen with TPO + rhG-CSF vs TPO alone beginning on d 8 (1674.4 ± 235.5 vs 2188 ± 148.1, p < 0.05), d 14 (2466.0 ± 330.8 vs 2980.8± 100.9, p < 0.05). Significant increases were seen in CFU-Meg and GM with TPO alone & TPO+ rhG-CSF vs C (colonies/2×105c) (MEG: 23.3 ± 1.0, 20.33 ± 3.8 vs 6.0 ± 1.0, p< 0.05) (GM: 144.0 ± 4.51, 106.7 ± 15.4 vs 84.3 ± 6.5, p< 0.01). Megakaryocyte proliferation was increased in TPO alone & TPO + rhG-CSF vs PBS/HSA (%C) (250.1 ± 44.3 & 246.8±21.1 vs 100, p< 0.05). These data suggests that TPO induces myeloid and megakaryocytic progenitor cells, and increases neutrophil and platelet production in the NB rat. However, the addition of rhG-CSF abrogates the TPO effect on platelet production. Further studies are required to determine the clinical implications of the combination of TPO + rhG-CSF in the treatment of neonatal thrombocytopenia and neutropenia.

A LONGITUDINAL ASSESSMENT OF ESTROGEN LEVELS DURING NORMAL PUBERTY IN BOYS AS DETERMINED BY AN ULTRA-SENSITIVE BIOASSAY. † 532A