Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine

Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively. The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.     

Stereoselective uptake of the GABA-transaminase inhibitors gamma-vinyl gaba and gamma-acetylenic GABA into neurons and astrocytes

The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomersR- andS-GVG andR- andS-GAG it could be shown that in both cell types only theS-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system forS-GVG (K _m 78.2±20.3 M;V _max 0.71±0.06 nmol · min^–1 · mg^–1 cell protein). In case ofS-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes.S-GVG andS-GAG were found to be weak inhibitors of GABA uptake suggesting thatS-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake ofS-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T toS-GVG.     

Increased gamma-aminobutyric acid (GABA), homocarnosine and beta-alanine in cerebrospinal fluid of patients treated with gamma-vinyl GABA (4-amino-hex-5-enoic acid)

γ-Vinyl GABA, an enzyme-activated irreversible inhibitor of GABA transaminase (GABA-T), was administered orally to 15 patients with various neurological conditions at daily doses of 0.5, 1, 2 or 6 g/day for 3 days. CSF samples were obtained by lumbar puncture before treatment and within 24 hours after the last dose and the CSF concentrations of free GABA, total GABA, homocarnosine, β-alanine and γ-vinyl GABA determined by ion-exchange chromatography with fluorometric detection. γ-Vinyl GABA treatment produced dose-dependent increases in free GABA, conjugated GABA (defined as total minus free GABA), homocarnosine and β-alanine. The concentrations of CSF γ-vinyl GABA also depended on the dose administered. These results indicate that γ-vinyl GABA enters the CNS after oral administration and alters GABA metabolism by inhibition of GABA-T and suggest that such treatment may achieve therapeutic benefit in conditions where such neurochemical alterations are desirable.     

Placental transfer of vigabatrin (gamma-vinyl GABA) and its effect on concentration of amino acids in the embryo of TO mice

BACKGROUND: The mechanism of the teratogenicity of vigabatrin (VGB) is unknown. The objectives of this study were to determine the placental transfer of VGB and to evaluate the effect of VGB on maternal, placental, and fetal concentrations of amino acids. METHODS: A single dose of 400 mg/kg VGB in physiological saline was administered intraperitoneally to a group of Theiler outbred (TO) mice on gestational day (GD) 10. The controls received a proportionate volume of saline. Maternal blood samples, embryos, and placentas were collected at 3.5, 6.0, and 9.0 hr after treatment and their total amino acid concentrations determined in an ion-exchange amino acid analyzer. RESULTS: At 3.5 hr, there was a decrease in concentrations of some amino acids in the blood, placenta, and embryos of VGB-treated mice, but the decrease in methionine was most marked. gamma-aminobutyric acid (GABA) was significantly higher in the VGB group in both the embryos and the placentas at 3.5 hr but at 6.0 and 9.0 hr the differences were not significant. Vigabatrin levels were higher in the placenta than in the embryo at 3.5 hr, but at 6.0 hr there was an overlap of the VGB peak with that of tryptophan with very much lower levels than at 3.5 hr. At 9.0 hr, there was no vigabatrin peak in either the placenta or the embryo. CONCLUSIONS: Maternal exposure to VGB results in peak levels of the drug after 3.5 hr in the placenta and embryo. Methionine concentration is most severely affected in VGB-treated mothers, placentas, and fetuses. We speculate that this deficiency could be a possible mechanism for the teratogenic effects of vigabatrin.     

Differential effects of methylphenidate and cocaine on GABA transmission in sensory thalamic nuclei

Methylphenidate (MPH) is widely used to treat children and adolescents diagnosed with attention deficit/hyperactivity disorder. Although MPH shares mechanistic similarities to cocaine, its effects on GABAergic transmission in sensory thalamic nuclei are unknown. Our objective was to compare cocaine and MPH effects on GABAergic projections between thalamic reticular and ventrobasal (VB) nuclei. Mice (P18‐30) were subjected to binge‐like cocaine and MPH acute and sub‐chronic administrations. Cocaine and MPH enhanced hyperlocomotion, although sub‐chronic cocaine‐mediated effects were stronger than MPH effects. Cocaine and MPH sub‐chronic administration altered paired‐pulse and spontaneous GABAergic input differently. The effects of cocaine on evoked paired‐pulse GABA‐mediated currents changed from depression to facilitation with the duration of the protocols used, while MPH induced a constant increase throughout the administration protocols. Thalamic reticular nucleus GAD67 and VB Ca_V3.1 protein levels were measured using western blot to better understand their link to increased GABA release. Both proteins were increased by sub‐chronic administration of cocaine. MPH showed effects on GABAergic transmission that seems less disruptive than cocaine. Unique effects of cocaine on postsynaptic VB calcium currents might explain deleterious cocaine effects on sensory thalamic nuclei. These results suggest that cocaine and MPH produced distinct presynaptic alterations on GABAergic transmission.     

The reuse of hemodialyzers: an assessment of safety and potential savings.

OBJECTIVE: To evaluate the safety and potential cost savings of hemodialyzer reuse. DATA SOURCES: All English and French articles published from 1960 to 1991 related to hemodialyzer reuse (retrieved through an Index Medicus and MEDLINE search [corrected]), the indexes of eight North American journals from 1960 onward, conference proceedings, association guidelines, and US and Canadian laws and regulations. RESULTS: For health care personnel the reuse of hemodialyzers did not entail any increased risk of infection or exposure to toxic substances if proper control measures were taken. For patients there was no evidence to suggest any excess risk of complications or death as long as precise and appropriate procedures are observed. The "first-use syndrome" can be prevented and should no longer be considered as a reason to favour reuse. A cost-minimization analysis indicated that five uses might save up to $3629 per patient yearly. Thus, the adoption of a policy of reuse in Canada for all eligible patients undergoing long-term hemodialysis could result in direct savings of about $5.8 to $8.9 million per year. CONCLUSION: The health risks associated with hemodialyzer reuse can be reduced to acceptable levels through the rigorous observance of proper quality-assurance and quality-control measures and the use of automated reconditioning equipment. Such a policy could achieve modest savings for the health care system. A decision to reuse should be formally adopted by the institution and accompanied by a precise definition of the standards of quality assurance and control.     

ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy

The availability of ChimeriVax vaccine technology for delivery of flavivirus protective antigens at the time West Nile (WN) virus was first detected in North America in 1999 contributed to the rapid development of the vaccine candidate against WN virus described here. ChimeriVax-Japanese encephalitis (JE), the first live- attenuated vaccine developed with this technology has successfully undergone phase I and II clinical trials. The ChimeriVax technology utilizes yellow fever virus (YF) 17D vaccine strain capsid and nonstructural genes to deliver the envelope gene of other flaviviruses as live-attenuated chimeric viruses. Amino acid sequence homology between the envelope protein (E) of JE and WN viruses facilitated targeting attenuating mutation sites to develop the WN vaccine. Here we discuss preclinical studies with the ChimeriVax-WN virus in mice and macaques. ChimeriVax-WN virus vaccine is less neurovirulent than the commercial YF 17D vaccine in mice and nonhuman primates. Attenuation of the virus is determined by the chimeric nature of the construct containing attenuating mutations in the YF 17D virus backbone and three point mutations introduced to alter residues 107, 316, and 440 in the WN virus E protein gene. The safety, immunogenicity, and efficacy of the ChimeriVax-WN(02) vaccine in the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.     

Ambiguities in the preclinical quality assessment of microparticulate vaccines

Vaccination techniques do not always stimulate immunity because of the inappropriate mobilization of immune responses, and the frequency of vaccinations required is impractical in many developing countries. Such limitations have spurred the development of new vaccine-delivery approaches. Microparticles made of poly(lactide-co-glycolide) can induce adaptive immunity after a single administration of a vaccine. However, the preclinical assessment of such vaccines is not standardized, making it difficult to compare pharmaceutical with immunological data. The relevance of and the ambiguity in the assessment of microparticulate vaccines with respect to the current knowledge on immunity are discussed, in addition to the application of this knowledge to rational vaccine design.     

Diabetic cardiomyopathy: the preclinical phase.

Left ventricular function was assessed by measuring sytolic time intervals in insulin-requiring diabetics with and without significant microangiopathy. The results were compared with those in normal controls. Significant microangiopathy was defined as proteinuria over 3 g/24 h or proliferative retinopathy. Left ventricular function was also assessed one and a half years later by echocardiography in four patients with microangiopathy. Patients with angina, previous myocardial infarction, hypertension, and alcoholism were excluded. All had normal electrocardiograms and chest radiographs. Diabetics with microangiopathy had impaired left ventricular function, whereas those with uncomplicated diabetes had normal function. This finding supports the existence of a specific diabetic cardiomyopathy due to microangiopathy rather than the metabolic defect. The association of microangiopathy and impaired left ventricular function may explain the high immediate mortality and the high incidence of cardiogenic shock and congestive heart failure after myocardial infarction in diabetics.     

The impact of glutamine supplementation on the symptoms of ataxia-telangiectasia: a preclinical assessment

Background

Our previous studies of Alzheimer’s disease (AD) suggested that glutamine broadly improves cellular readiness to respond to stress and acts as a neuroprotectant both in vitro and in AD mouse models. We now expand our studies to a second neurodegenerative disease, ataxia-telangiectasia (A-T). Unlike AD, where clinically significant cognitive decline does not typically occur before age 65, A-T symptoms appear in early childhood and are caused exclusively by mutations in the ATM (A-T mutated) gene.

Results

Genetically ATM-deficient mice and wild type littermates were maintained with or without 4 % glutamine in their drinking water for several weeks. In ATM mutants, glutamine supplementation restored serum glutamine and glucose levels and reduced body weight loss. Lost neurophysiological function assessed through the magnitude of hippocampal long term potentiation was significantly restored. Glutamine supplemented mice also showed reduced thymus pathology and, remarkably, a full one-third extension of lifespan. In vitro assays revealed that ATM-deficient cells are more sensitive to glutamine deprivation, while supra-molar glutamine (8 mM) partially rescued the reduction of BDNF expression and HDAC4 nuclear translocation of genetically mutant Atm^?/? neurons. Analysis of microarray data suggested that glutamine metabolism is significantly altered in human A-T brains as well.

Conclusion

Glutamine is a powerful part of an organism’s internal environment. Changes in its concentrations can have a huge impact on the function of all organ systems, especially the brain. Glutamine supplementation thus bears consideration as a therapeutic strategy for the treatment of human A-T and perhaps other neurodegenerative diseases.

     

Glutamate and GABA: a painful combination.

Regulation of release of inhibitory neurotransmitter is a key element of plasticity in dorsal horn function. In this issue of Neuron, Kerchner et al. report that neurotransmitter release from inhibitory dorsal horn neurons is affected by activation of presynaptic kainate-type glutamate receptors.     

Epidemiological designs for vaccine safety assessment: methods and pitfalls

Three commonly used designs for vaccine safety assessment post licensure are cohort, case-control and self-controlled case series. These methods are often used with routine health databases and immunisation registries. This paper considers the issues that may arise when designing an epidemiological study, such as understanding the vaccine safety question, case definition and finding, limitations of data sources, uncontrolled confounding, and pitfalls that apply to the individual designs. The example of MMR and autism, where all three designs have been used, is presented to help consider these issues.     

Combination of dipeptidylpeptidase IV inhibitor and low dose thiazolidinedione: preclinical efficacy and safety in db/db mice

Thiazolidinediones (TZDs) are currently the most efficacious class of oral antidiabetics. However, they carry the burden of weight gain and haemodilution, which may lead to cardiovascular complications. The present study was designed to ascertain whether a combination of dipeptidyl peptidase IV (DPP IV) inhibitor with low dose of a thiazolidinedione absolves TZD associated weight gain and oedema without compromising its efficacy. In this study, we examined the efficacy and safety of lower dose (1 mg/kg/day) of rosiglitazone, a thiazolidinedione, in combination with 5 mg/kg/day dose of LAF-237 (vildagliptin), a known DPP IV inhibitor, in aged db/db mice after 14 days of treatment and compared the combination with therapeutic dose (10 mg/kg) of rosiglitazone. The combination therapy showed similar efficacy as that of 10 mg/kg/day rosiglitazone in lowering random blood glucose (53.8%, p<0.001 and 54.3%, p<0.001 respectively), AUC ((0-120) min) during oral glucose tolerance test (OGTT) (38.6 %, p<0.01; 38.3%, p<0.01 respectively) and triglyceride levels (63.9% and 61% respectively; p<0.01). Plasma active glucagon like peptide-1 (GLP-1) and insulin levels were found to be elevated significantly (p<0.01 and p<0.05 respectively) in both LAF-237 and combination treated groups following oral glucose load. LAF-237 alone had no effect on random glucose and glucose excursion during OGTT in severely diabetic db/db mice. Interestingly, the combination treatment showed no significant increase in body weight as compared to the robust weight gain by therapeutic dose of rosiglitazone. Rosiglitazone at 10 mg/kg/day showed significant reduction (p<0.05) in haematocrit, RBC count, haemoglobin pointing towards haemodilution associated with increased mRNA expression of Na(+), K(+)-ATPase-alpha and epithelial sodium channel gamma (ENaCgamma) in kidney. The combination therapy escaped these adverse effects. The results suggest that combination of DPP IV inhibitor with low dose of thiazolidinedione can interact synergistically to represent a therapeutic advantage for the clinical treatment of type 2 diabetes without the adverse effects of haemodilution and weight gain associated with thiazolidinediones.     

Multimodality imaging in vivo for preclinical assessment of tumor-targeted doxorubicin nanoparticles

This study presents a new multimodal imaging approach that includes high-frequency ultrasound, fluorescence intensity, confocal, and spectral imaging to improve the preclinical evaluation of new therapeutics in vivo. Here we use this approach to assess in vivo the therapeutic efficacy of the novel chemotherapy construct, HerDox during and after treatment. HerDox is comprised of doxorubicin non-covalently assembled in a viral-like particle targeted to HER2+ tumor cells, causing tumor cell death at over 10-fold lower dose compared to the untargeted drug, while sparing the heart. Whereas our initial proof-of-principle studies on HerDox used tumor growth/shrinkage rates as a measure of therapeutic efficacy, here we show that multimodal imaging deployed during and after treatment can supplement traditional modes of tumor monitoring to further characterize the particle in tissues of treated mice. Specifically, we show here that tumor cell apoptosis elicited by HerDox can be monitored in vivo during treatment using high frequency ultrasound imaging, while in situ confocal imaging of excised tumors shows that HerDox indeed penetrated tumor tissue and can be detected at the subcellular level, including in the nucleus, via Dox fluorescence. In addition, ratiometric spectral imaging of the same tumor tissue enables quantitative discrimination of HerDox fluorescence from autofluorescence in situ. In contrast to standard approaches of preclinical assessment, this new method provides multiple/complementary information that may shorten the time required for initial evaluation of in vivo efficacy, thus potentially reducing the time and cost for translating new drug molecules into the clinic.     

Regulatory interrelations between GABA and polyamines. I. Brain GABA levels and polyamine metabolism

Elevation of brain GABA levels by GABA-T inhibition is accompanied by a decrease ofS-adenosylmethionine decarboxylase activity. This is followed by an increase of ornithine decarboxylase activity and a severalfold increase of brain putrescine levels. Spermidine and spermine levels are not significantly affected under these conditions. These unexpected findings support a regulatory interaction between GABA and polyamine metabolism.     

Cell-based therapies: careful safety assessment for minimization of risk

Cell therapy can be numbered among the novel biologic therapeutics that will increase our ability to cure human disease in the years to come. While the general feeling is that in vivo use of ex-vivo expanded cellular products is relatively safe, the attempt at augmenting potency have increased the risk for adverse events. Future efforts are warranted to better characterize cellular products, and collect and analyze clinical trial outcomes through national databases, to identify risks connected to their in vivo use.     

Drug safety assessment in clinical trials: methodological challenges and opportunities

ABSTRACT: Randomized controlled trials are the principal means of establishing the efficacy of drugs. However pre-marketing trials are limited in size and duration and exclude high-risk populations. They have limited statistical power to detect rare but potentially serious adverse events in real-world patients. We summarize the principal methodological challenges in the reporting, analysis and interpretation of safety data in clinical trials using recent examples from systematic reviews. The principle challenges include the lack of an evidentiary gold standard, the limited statistical power of randomized controlled trials and resulting type 2 error, the lack of adequate ascertainment of adverse events and limited generalizability of safety trials that exclude high risk patients. We discuss potential solutions to these challenges. Evaluation of drug safety requires careful examination of data from heterogeneous sources. Meta-analyses of drug safety should include appropriate statistical methods and assess the optimal information size to avoid type 2 errors. They should evaluate outcome reporting biases and missing data to ensure reliable and accurate interpretation of findings. Regulatory and academic partnerships should be fostered to provide an independent and transparent evaluation of drug safety.