Psychosine, a marker of Krabbe phenotype and treatment effect.

Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.

Antibacterial synergism: a proposal for chemotherapeutic potentiation between trimethoprim and sulfamethoxazole.

Sulfamethoxazole and other sulfa drugs are moderately potent inhibitors of Escherichia coli dihydrofolate reductase. They also significantly potentiate the inhibition of this enzyme by trimethoprim. The molecular basis for inhibition potentiation is the simultaneous binding of trimethoprim and sulfa by the enzyme. This potentiation may explain the synergism observed when these drugs are used in antibacterial chemotherapy.

Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate.

A central eight-stranded beta-pleated sheet is the main feature of the polypeptide backbone folding in dihydrofolate reductase. The innermost four strands and two bridging helices are geometrically similar to but are connected in a different way from those in the dinucleotide binding domains found in nicotinamide-adenine dinucleotide-linked dehydrogenases. Methotrexate is bound in a 15-angstrom-deep cavity with the pteridine ring buried in a primarily hydrophobic pocket, although a strong interaction occurs between the side chain of aspartic acid 27 and N(1), N(8), and the 2-amino group of methotrexate.

The α2,3-selective potentiator of GABAA receptors, KRM-II-81, reduces nociceptive-associated behaviors induced by formalin and spinal nerve ligation in rats.

Clinical evidence indicates that positive allosteric modulators (PAMs) of GABAA receptors have analgesic benefit in addition to efficacy in anxiety disorders. However, the utility of GABAA receptor PAMs as analgesics is compromised by the central nervous system side effects of non-selective potentiators. A selective potentiator of GABAA receptors associated with α2/3 subunits, KRM-II-81(5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole), has demonstrated anxiolytic, anticonvulsant, and antinociceptive effects in rodents with reduced motoric side effects. The present study evaluated the potential of KRM-II-81 as a novel analgesic. Oral administration of KRM-II-81 attenuated formalin-induced flinching; in contrast, diazepam was not active. KRM-II-81 attenuated nociceptive-associated behaviors engendered by chronic spinal nerve ligation (L5/L6). Diazepam decreased locomotion of rats at the dose tested in the formalin assay (10 mg/kg) whereas KRM-II-81 produced small decreases that were not dose-dependent (10-100 mg/kg). Plasma and brain levels of KRM-II-81 were used to demonstrate selectivity for α2/3- over α1-associated GABAA receptors and to define the degree of engagement of these receptors. Plasma and brain concentrations of KRM-II-81 were positively-associated with analgesic efficacy. GABA currents from isolated rat dorsal-root ganglion cultures were potentiated by KRM-II-81 with an ED50 of 32 nM. Measures of respiratory depression were reduced by alprazolam whereas KRM-II-81 was either inactive or produced effects with lower potency and efficacy. These findings add to the growing body of data supporting the idea that α2/3-selective GABAA receptor PAMs will have efficacy and tolerability as pain medications including those for neuropathic pain. Given their predicted anxiolytic effects, α2/3-selective GABAA receptor PAMs offer an additional inroad into the management of pain.

An antidepressant-related pharmacological signature for positive allosteric modulators of α2/3-containing GABAA receptors.

Data from transgenic animals and novel pharmacological agents has realigned scientific scrutiny on the therapeutic potential of positive allosteric modulators (PAMs) of α2/3-containing GABAA receptors. Evidence for analgesic, anticonvulsant, and anxiolytic activity of α2/3-selective PAMs has been presented along with the clinical potential for a milder motor-impacting profile compared to non-selective GABAA receptor PAMs. A new series of α2/3-selective PAMs was recently introduced which has anxiolytic and anticonvulsant activity in rodent models. These molecules also produce efficacy against pain in multiple animal models. Additionally, co-morbid states of depression are prevalent among patients with pain and patients with anxiety. Compounds were shown to be selective for α2 and α3 constructs over α1 (except KRM-II-82), α4, α5, and α6 proteins in electrophysiological assays in transfected HEK-293T cells. Utilizing the forced-swim assay in mice that detects conventional and novel antidepressant drugs, we demonstrate for the first time that α2/3-selective PAMs are active in the forced-swim assay at anxiolytic-producing doses. In contrast, activity in a related model, the tail-suspension test, was not observed. Diazepam was not active in the forced-swim assay when given alone but produced an antidepressant-like effect in mice when given in conjunction with the α1-preferring antagonist, ß-CCT, that attenuated the motor-impairing effects of diazepam. We conclude that these α2/3-selective PAMs deserve further scrutiny for their potential treatment of major depressive disorder. If effective, such a mechanism could add a beneficial antidepressant component to the anxiolytic, analgesic, and anticonvulsant spectrum of effects of these compounds.

Bioisosteres of ethyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo [1,5-a][1,4]diazepine-3-carboxylate (HZ-166) as novel alpha 2,3 selective potentiators of GABAA receptors: Improved bioavailability enhances anticonvulsant efficacy.

HZ-166 has previously been characterized as an α2,3-selective GABAA receptor modulator with anticonvulsant, anxiolytic, and anti-nociceptive properties but reduced motor effects. We discovered a series of ester bioisosteres with reduced metabolic liabilities, leading to improved efficacy as anxiolytic-like compounds in rats. In the present study, we evaluated the anticonvulsant effects KRM-II-81 across several rodent models. In some models we also evaluated key structural analogs. KRM-II-81 suppressed hyper-excitation in a network of cultured cortical neurons without affecting the basal neuronal activity. KRM-II-81 was active against electroshock-induced convulsions in mice, pentylenetetrazole (PTZ)-induced convulsions in rats, elevations in PTZ-seizure thresholds, and amygdala-kindled seizures in rats with efficacies greater than that of diazepam. KRM-II-81 was also active in the 6 Hz seizure model in mice. Structural analogs of KRM-II-81 but not the ester, HZ-166, were active in all models in which they were evaluated. We further evaluated KRM-II-81 in human cortical epileptic tissue where it was found to significantly-attenuate picrotoxin- and AP-4-induced increases in firing rate across an electrode array. These molecules generally had a wider margin of separation in potencies to produce anticonvulsant effects vs. motor impairment on an inverted screen test than did diazepam. Ester bioisosters of HZ-166 are thus presented as novel agents for the potential treatment of epilepsy acting via selective positive allosteric amplification of GABAA signaling through α2/α3-containing GABA receptors. The in vivo data from the present study can serve as a guide to dosing parameters that predict engagement of central GABAA receptors.

Further evaluation of the potential anxiolytic activity of imidazo[1,5-a][1,4]diazepin agents selective for α2/3-containing GABAA receptors.

Positive allosteric modulators of GABAA receptors transduce a host of beneficial effects including anxiolytic actions. We have recently shown that bioavailability and anxiolytic-like activity can be improved by eliminating the ester functionality in imidazo[1,5-a][1,4]diazepines. In the present series of experiments, we further substantiate the value of heterocyle replacement of the ester for potential treatment of anxiety. None of three esters was active in a Vogel conflict test in rats that detects anxiolytic drugs like diazepam. Compounds 7 and 8, ester bioisosters, were selective for alpha 2 and 3 over alpha 1-containing GABAA receptors but also had modest efficacy at GABAA alpha 5-containing receptors. Compound 7 was efficacious and potent in this anxiolytic-detecting assay without affecting non-punished responding. The efficacies of the esters and of compound 7 were predicted from their efficacies as anticonvulsants against the GABAA antagonist pentylenetetrazole (PTZ). In contrast, the related structural analog, compound 8, did not produce anxiolytic-like effects in rats despite anticonvulsant efficacy. These data thus support the following conclusions: 1) ancillary pharmacological actions of compound 8 might be responsible for its lack of anxiolytic-like efficacy despite its efficacy as an anticonvulsant 2) esters of imidazo[1,5-a][1,4]diazepines do not demonstrate anxiolytic-like effects in rats due to their low bioavailability and 3) replacement of the ester function with suitable heterocycles markedly improves bioavailability and engenders molecules with the opportunity to have potent and efficacious effects in vivo that correspond to human anxiolytic actions.

Regional differences in fiber tractography predict neurodevelopmental outcomes in neonates with infantile Krabbe disease.

BACKGROUND: Krabbe disease is a fatal neurodegenerative disease caused by rapid demyelination of the central and peripheral nervous systems. The only available treatment, unrelated umbilical cord blood transplantation, is effective only if performed before clinical symptoms appear. Phenotypic expressions of disease-causing mutations vary widely, but genotype-phenotype relationships are unclear. Therefore, we evaluated diffusion tensor imaging (DTI) tractography with volumetric analysis as a biomarker of early white matter changes and functional disability in presymptomatic infants. METHODS: We obtained DTI and structural scans of newborns with early-infantile Krabbe disease (n = 9) diagnosed by family history or newborn screening. We compared white matter fiber tract properties to those of normal controls (n = 336) and assessed the ability of tract-based properties to predict longitudinal development in four functional domains (cognitive, fine motor, gross motor, adaptive behavior) after treatment with unrelated umbilical cord blood transplantation. We also assessed the relationship between the standard evaluation (modified Loes score) and DTI results, and the volumetric differences between the Krabbe subjects and normal controls. FINDINGS: Reductions in fractional anisotropy were significant in the corticospinal tract in the Krabbe patients compared to controls, which strongly correlated with motor and cognitive outcomes after transplantation. Significant regional differences were observed in the splenium and uncinate fasciculus in Krabbe patients and these differences correlated only with cognitive outcomes. Regional brain volumes of Krabbe patients were slightly larger than controls. Loes scores did not correlate with DTI results. INTERPRETATION: Neonatal microstructural abnormalities correlate with neurodevelopmental treatment outcomes in patients treated for infantile Krabbe disease. DTI with quantitative tractography is an excellent biomarker for evaluating infants with Krabbe disease identified through newborn screening.

CTL lysis: there is a hyperbolic relation of killing rate to exocytosable granzyme A for highly cytotoxic murine cytotoxic T lymphocytes.

The lysis of susceptible targets by efficient cytotoxic T lymphocytes (CTL) increases both with time and with the ratio of CTL to target. Simple methods for calculating a killing rate constant from the time dependence of killing and for calculating the relation of the killing rate constant to the concentration of exocytosable granzyme A are given. Application of these methods to the killing of target cells by the highly efficient mouse CTL AR1 is presented. AR1 needed granzyme A for efficient killing. AR1 contained sufficient exocytosable granzyme A to kill at about 80% of the rate possible at infinite exocytosable granzyme A.

Quantitative structure-activity relationships for the inhibition of Escherichia coli dihydrofolate reductase by 5-(substituted benzyl)-2,4-diaminopyrimidines.

Quantitative structure-activity relationships for the inhibition of Escherichia coli (MB 1428) dihydrofolate reductase (DHFR) by 61 5-(substituted benzyl)-2,4-diaminopyrimidines are reported and analyzed. The 61 compounds include 17 congeners whose activities have not been previously reported, five of which have a 5'-substituent larger than a methoxy group. The correlation equations indicated that the molar refractivity (MR) values of the 5'-substituent, just as with the 3'- and 4'-substituents, contributed maximally at the value of 0.79 with no increment of binding for compounds with MR larger than 0.79 (which corresponds to a 5'-methoxy substitution). This experimental result is in agreement with the crystal structure of the Escherichia coli DHFR-trimethoprim complex, which shows a reasonably large trimethoprim-binding site. The inhibition of E. coli (MB 1428) DHFR by nine of the 17 new benzylpyrimidines is at lower concentrations than for trimethoprim. However, all 17 are much less potent than trimethoprim in inhibition of growth of E. coli (1515).

On the optimization of hydrophobic and hydrophilic substituent interactions of 2,4-diamino-5-(substituted-benzyl)pyrimidines with dihydrofolate reductase.

The inhibition constants (Kiapp) were obtained from the action of 68 2,4-diamino-5-(substituted-benzyl)pyrimidines on dihydrofolate reductase from an Escherichia coli strain MB 1428. Subsequently, these results were used to formulate appropriate quantitative structure-activity relationships (QSAR). Once again these equations emphasize the paramount importance of steric/dispersion factors in enhancing antibacterial potency. Hydrophobicity also plays a role, albeit a minor one. Comparisons with the QSAR obtained versus prokaryotic dihydrofolate reductase (DHFR) demonstrate subtle differences in binding behavior between meta and para substituents which may be effectively maximized in the design of more efficacious and selective antibacterial agents. The bacterial and avian QSAR equations can be used to calculate selectivity indices for trimethoprim, tetroxoprim, and two other specially designed 2,4-diamino-5-(substituted-benzyl)pyrimidines.

Renin inhibitors. Syntheses of subnanomolar, competitive, transition-state analogue inhibitors containing a novel analogue of statine.

Analogues of the renin octapeptide substrate were synthesized in which replacement of the scissile dipeptide with (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine, Sta) transformed the substrate sequence into potent, transition-state analogue, competitive inhibitors of renin. Synthesis and incorporation of the cyclohexylalanyl analogue of Sta, (3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), gave the most potent inhibitors of renin yet reported, including N-isovaleryl-L-histidyl-L-prolyl-L-phenylalanyl-L-histidyl-ACHPA-L -leucyl-L- phenylalanyl amide [Iva-His-Pro-Phe-His-ACHPA-Leu-Phe-NH2,3], with renin inhibitions of Ki = 1.6 X 10(-10) M (human kidney renin), IC50 = 1.7 X 10(-10)M (human plasma renin), IC50 = 1.9 X 10(-9)M (dog plasma renin), and IC50 = 2.1 X 10(-8) M (rat plasma renin). This inhibitor 3, containing ACHPA, was 55-76 times more potent vs. human renin than the comparable Sta-containing inhibitor 1 and 17 times more potent vs. dog renin than 1. Inhibitor 3 lowered blood pressure in sodium-deficient dogs, with in vivo potency 19 times that shown by 1, in close agreement with the relative in vitro potencies. Structure-activity results are presented that show the minimal N-terminus for these inhibitors. An ACHPA-containing pentapeptide, N-[(ethyloxy)carbonyl]-L-phenylalanyl-L- histidyl-ACHPA-L-leucyl-L-phenylalanyl amide [Etoc-Phe-His-ACHPA-Leu-Phe-NH2,8], retained subnanomolar inhibitory potency. Molecular modelling studies are described that suggested the design of ACHPA.

Renin inhibitors. Statine-containing tetrapeptides with varied hydrophobic carboxy termini.

A series of statine-containing tetrapeptides, systematically modified at the carboxy terminus with various hydrophobic aromatic groups, is described. These compounds were tested in vitro for their ability to inhibit porcine, human plasma, and purified human kidney renins. These analogues help to define optimal binding aspects in a region of the enzyme that appears to be specific for spatial arrangement of aromatic groups. Replacement of the metabolically labile Phe amide with nonpeptidal groups proved possible while achieving inhibitory potency in the nanomolar range vs. porcine kidney renin. For the compounds 6i, 6m, and 6o, a large discrepancy in potency between the human plasma and the purified human kidney renin assays was observed. This disparity does not appear to be a consequence of a previously proposed plasma binding component.

Crop loss assessment for California: modeling losses with different ozone standard scenarios.

Crop yield losses were estimated for ambient O3 concentrations and for a series of potential O3 air quality standards for California, including the current statewide 1-h oxidant (O3) standard of 0.10 ppm (196 microg m(-3)), 12-h growing season averages, and other models. A model for statewide losses was developed using hourly O3 data for all sites in the State, county crop productivity data, and available O3 concentration-yield loss equations to determine potential yield losses for each crop in each county in California for 1984. Losses were based on comparison to an estimated background filtered air concentration of 0.025 or 0.027 ppm, for 12 or 7 h, respectively. Potential losses due to ambient air in 1984 were estimated at 19% to 25% for dry beans, cotton, grapes, lemons, onions, and oranges. Losses of 5% to 9% were estimated for alfalfa and sweet corn. Losses of 4% or less were estimated for barley, field corn, lettuce, grain sorghum, rice, corn silage, spinach, strawberries, sugar beets, fresh tomatoes, processing tomatoes, and wheat. Implementation of either a modified rollback to meet the current 1 h California O3 standard (0.10 ppm) or a three-month, 12-h growing season average of 0.045 ppm was necessary to produce large reductions in potential crop losses.

Implicit and explicit tests: evidence for dissociable motor skills in probable Alzheimer's dementia.

Previous authors reported evidence for intact implicit memories (those retrieved without conscious effort) in a serial reaction time task for both Alzheimer's subjects and age-matched controls, although performance on an explicit memory task (requiring conscious effort for retrieval) was poor. The current study assessed latencies on a puzzle-assembly task to assess implicit (procedural) memory for 23 female volunteers. Nine participants suffered from probable Alzheimer's Disease and fourteen did not. Even when subjects had no explicit memory of practicing the task, they demonstrated savings upon relearning. Implications for research on memory dissociations in Alzheimer's Disease are discussed.

Temperament and sensory features of children with autism.

This study sought to characterize temperament traits in a sample of children with autism spectrum disorder (ASD), ages 3-7 years old, and to determine the potential association between temperament and sensory features in ASD. Individual differences in sensory processing may form the basis for aspects of temperament and personality, and aberrations in sensory processing may inform why some temperamental traits are characteristic of specific clinical populations. Nine dimensions of temperament from the Behavioral Style Questionnaire (McDevitt and Carey in Manual for the behavioral style questionnaire, Behavioral-Developmental Initiatives, Scottsdale, AZ, 1996) were compared among groups of children with ASD (n = 54), developmentally delayed (DD; n = 33), and the original normative sample of typically developing children (McDevitt and Carey in J Child Psychol Psychiatr 19(3):245-253, 1978; n = 350) using an ANOVA to determine the extent to which groups differed in their temperament profiles. The hypothesized overlap between three sensory constructs (hyperresponsiveness, hyporesponsiveness, and seeking) and the nine dimensions of temperament was analyzed in children with ASD using regression analyses. The ASD group displayed temperament scores distinct from norms for typically developing children on most dimensions of temperament (activity, rhythmicity, adaptability, approach, distractibility, intensity, persistence, and threshold) but differed from the DD group on only two dimensions (approach and distractibility). Analyses of associations between sensory constructs and temperament dimensions found that sensory hyporesponsiveness was associated with slowness to adapt, low reactivity, and low distractibility; a combination of increased sensory features (across all three patterns) was associated with increased withdrawal and more negative mood. Although most dimensions of temperament distinguished children with ASD as a group, not all dimensions appear equally associated with sensory response patterns. Shared mechanisms underlying sensory responsiveness, temperament, and social withdrawal may be fruitful to explore in future studies.

Correlation of automated volumetric analysis of brain MR imaging with cognitive impairment in a natural history study of mucopolysaccharidosis II.

BACKGROUND AND PURPOSE: Reliable markers for predicting neurologic outcome in patients with MPS II are lacking. The purpose of this study is to explore whether quantitative volumetric measurements of brain MR imaging can be used to differentiate between MPS II patients with and without cognitive impairment. This MR imaging study is the first in MPS II patients to use automated/semi-automated methods to quantify brain volumes in a longitudinal design. MATERIALS AND METHODS: Sixteen male patients with MPS II in a natural history study had annual brain MR imaging and detailed neurodevelopmental assessment over 2 years. Automated and semi-automated methods were used to determine brain volumes. Linear mixed regression models adjusting for age were used to assess the correlation between the volumetric parameters and cognition. RESULTS: Among the 16 MPS II patients, 10 (22 MR imaging studies) had cognitive impairment whereas the other 6 (11 MR imaging studies) had normal cognition. A decreased brain tissue/ICV ratio (-5%; P < .001) and an increased lateral ventricle/ICV ratio (+4%; P = .029) were found in patients with cognitive impairment compared with patients with normal cognition. These changes were apparent in patients as young as 7 years of age in addition to older patients. CONCLUSIONS: Quantitative volumetric measurements of brain MR imaging in MPS II patients can be obtained by using automated and semi-automated segmentation methods. MPS II patients with cognitive impairment have decreased brain tissue volumes, but longer studies with more subjects are required to confirm these results.