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1.
J Inherit Metab Dis ; 41(4): 699-708, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29460030

RESUMO

We present an update to the status of research on succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD), a rare disorder of GABA metabolism. This is an unusual disorder featuring the accumulation of both GABA and its neuromodulatory analog, gamma-hydroxybutyric acid (GHB), and recent studies have advanced the potential clinical application of NCS-382, a putative GHB receptor antagonist. Animal studies have provided proof-of-concept that enzyme replacement therapy could represent a long-term therapeutic option. The characterization of neuronal stem cells (NSCs) derived from aldehyde dehydrogenase 5a1-/- (aldh5a1-/-) mice, the murine model of SSADHD, has highlighted NSC utility as an in vitro system in which to study therapeutics and associated toxicological properties. Gene expression analyses have revealed that transcripts encoding GABAA receptors are down-regulated and may remain largely immature in aldh5a1-/- brain, characterized by excitatory as opposed to inhibitory outputs, the latter being the expected action in the mature central nervous system. This indicates that agents altering chloride channel activity may be therapeutically relevant in SSADHD. The most recent therapeutic prospects include mTOR (mechanistic target of rapamycin) inhibitors, drugs that have received attention with the elucidation of the effects of elevated GABA on autophagy. The outlook for novel therapeutic trials in SSADHD continues to improve.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Benzocicloeptenos/uso terapêutico , Deficiências do Desenvolvimento/tratamento farmacológico , Terapia de Reposição de Enzimas , Antagonistas GABAérgicos/uso terapêutico , Succinato-Semialdeído Desidrogenase/deficiência , Serina-Treonina Quinases TOR/antagonistas & inibidores , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Deficiências do Desenvolvimento/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Succinato-Semialdeído Desidrogenase/metabolismo
2.
J Inherit Metab Dis ; 39(6): 877-886, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27518770

RESUMO

Recent studies have identified a role for supraphysiological gamma-aminobutyric acid (GABA) in the regulation of mechanistic target of rapamycin (mTOR), a protein kinase with pleiotropic roles in cellular development and homeostasis, including integration of growth factors and nutrient sensing and synaptic input in neurons (Lakhani et al. 2014; Vogel et al. 2015). Aldehyde dehydrogenase 5a1-deficient (aldh5a1 -/- ) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA that disrupts mitophagy and increases mitochondria number with enhanced oxidant stress. Treatment with the mTOR inhibitor, rapamycin, significantly attenuates these GABA-related anomalies. We extend those studies through characterization of additional rapamycin analog (rapalog) agents including temsirolimus, dual mTOR inhibitors [Torin 1 and 2 (Tor 1/ Tor 2), Ku-0063794, and XL-765], as well as mTOR-independent autophagy inducers [trehalose, tat-Beclin 1, tacrolimus (FK-506), and NF-449) in aldh5a1 -/- mice. Rapamycin, Tor 1, and Tor 2 rescued these mice from premature lethality associated with status epilepticus. XL-765 extended lifespan significantly and induced weight gain in aldh5a1 -/- mice; untreated aldh5a1 -/- mice failed to increase body mass. Expression profiling of animals rescued with Tor 1/Tor 2 and XL-765 revealed multiple instances of pharmacological compensation and/or correction of GABAergic and glutamatergic receptors, GABA/glutamate transporters, and GABA/glutamate-associated proteins, with Tor 2 and XL-765 showing optimal outcomes. Our studies lay the groundwork for further evaluation of mTOR inhibitors in aldh5a1 -/- mice, with therapeutic ramifications for heritable disorders of GABA and glutamate neurotransmission.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Deficiências do Desenvolvimento/tratamento farmacológico , Ácido Glutâmico/metabolismo , Nascimento Prematuro/mortalidade , Succinato-Semialdeído Desidrogenase/deficiência , Succinato-Semialdeído Desidrogenase/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Deficiências do Desenvolvimento/metabolismo , Modelos Animais de Doenças , Camundongos , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pirimidinas/farmacologia , Quinoxalinas/farmacologia , Sirolimo/farmacologia , Sulfonamidas/farmacologia , Transmissão Sináptica/efeitos dos fármacos
3.
J Pharmacol Exp Ther ; 352(3): 462-70, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25512370

RESUMO

Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of nonalcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindividual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 µCi/kg) was administered intravenously, and plasma and bile (0-150 or 0-240 minutes), liver and kidney, and cumulative urine were analyzed for morphine and M3G. The antinociceptive response to M6G (5 mg/kg) was assessed (0-12 hours) after direct intraperitoneal administration since rats do not produce M6G. NASH caused a net decrease in morphine concentrations in the bile and plasma and a net increase in the M3G/morphine plasma area under the concentration-time curve ratio, consistent with upregulation of UDP-glucuronosyltransferase Ugt2b1. Despite increased systemic exposure to M3G, NASH resulted in decreased biliary excretion and hepatic accumulation of M3G. This shift toward systemic retention is consistent with the mislocalization of canalicular Mrp2 and increased expression of sinusoidal Mrp3 in NASH and may correlate to increased antinociception by M6G. Increased metabolism and altered transporter regulation in NASH provide a mechanistic basis for interindividual variability in morphine disposition that may lead to opioid-related toxicity.


Assuntos
Derivados da Morfina/sangue , Morfina/administração & dosagem , Morfina/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Animais , Infusões Intravenosas , Masculino , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia
4.
J Pharmacol Exp Ther ; 351(3): 576-84, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25253884

RESUMO

Dietary substances, including herbal products and citrus juices, can perpetrate interactions with conventional medications. Regulatory guidances for dietary substance-drug interaction assessment are lacking. This deficiency is due in part to challenges unique to dietary substances, a lack of requisite human-derived data, and limited jurisdiction. An in vitro-in vivo extrapolation (IVIVE) approach to help address some of these hurdles was evaluated using the exemplar dietary substance grapefruit juice (GFJ), the candidate marker constituent 6',7'-dihydroxybergamottin (DHB), and the purported victim drug loperamide. First, the GFJ-loperamide interaction was assessed in 16 healthy volunteers. Loperamide (16 mg) was administered with 240 ml of water or GFJ; plasma was collected from 0 to 72 hours. Relative to water, GFJ increased the geometric mean loperamide area under the plasma concentration-time curve (AUC) significantly (1.7-fold). Second, the mechanism-based inhibition kinetics for DHB were recovered using human intestinal microsomes and the index CYP3A4 reaction, loperamide N-desmethylation (KI [concentration needed to achieve one-half kinact], 5.0 ± 0.9 µM; kinact [maximum inactivation rate constant], 0.38 ± 0.02 minute(-1)). These parameters were incorporated into a mechanistic static model, which predicted a 1.6-fold increase in loperamide AUC. Third, the successful IVIVE prompted further application to 15 previously reported GFJ-drug interaction studies selected according to predefined criteria. Twelve of the interactions were predicted to within the 25% predefined criterion. Results suggest that DHB could be used to predict the CYP3A4-mediated effect of GFJ. This time- and cost-effective IVIVE approach could be applied to other dietary substance-drug interactions to help prioritize new and existing drugs for more advanced (dynamic) modeling and simulation and clinical assessment.


Assuntos
Bebidas , Citrus paradisi , Citocromo P-450 CYP3A/metabolismo , Interações Alimento-Droga/fisiologia , Loperamida/sangue , Adulto , Biomarcadores/sangue , Estudos Cross-Over , Feminino , Previsões , Humanos , Loperamida/administração & dosagem , Masculino , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Pessoa de Meia-Idade , Estudos Prospectivos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia , Adulto Jovem
5.
Drug Metab Dispos ; 41(2): 518-28, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23223498

RESUMO

DB868 [2,5-bis [5-(N-methoxyamidino)-2-pyridyl] furan], a prodrug of the diamidine DB829 [2,5-bis(5-amidino-2-pyridyl) furan], has demonstrated efficacy in murine models of human African trypanosomiasis. A cross-species evaluation of prodrug bioconversion to the active drug is required to predict the disposition of prodrug, metabolites, and active drug in humans. The phase I biotransformation of DB868 was elucidated using liver microsomes and sandwich-cultured hepatocytes from humans and rats. All systems produced four NADPH-dependent metabolites via O-demethylation (M1, M2) and N-dehydroxylation (M3, M4). Compartmental kinetic modeling of the DB868 metabolic pathway suggested an unusual N-demethoxylation reaction that was supported experimentally. A unienzyme Michaelis-Menten model described the kinetics of M1 formation by human liver microsomes (HLMs) (K(m), 11 µM; V(max), 340 pmol/min/mg), whereas a two-enzyme model described the kinetics of M1 formation by rat liver microsomes (RLMs) (K(m1), 0.5 µM; V(max1), 12 pmol/min/mg; K(m2), 27 µM; V(max2), 70 pmol/min/mg). Human recombinant CYP1A2, CYP3A4, and CYP4F2, rat recombinant Cyp1a2 and Cyp2d2, and rat purified Cyp4f1 catalyzed M1 formation. M2 formation by HLMs exhibited allosteric kinetics (S(50), 18 µM; V(max), 180 pmol/mg), whereas M2 formation by RLMs was negligible. Recombinant CYP1A2/Cyp1a2 catalyzed M2 formation. DB829 was detected in trace amounts in HLMs at the end of the 180-min incubation and was detected readily in sandwich-cultured hepatocytes from both species throughout the 24-h incubation. These studies demonstrated that DB868 biotransformation to DB829 is conserved between humans and rats. An improved understanding of species differences in the kinetics of DB829 formation would facilitate preclinical development of a promising antitrypanosomal prodrug.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Modelos Biológicos , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologia , Animais , Biotransformação , Células Cultivadas , Remoção de Radical Alquila , Feminino , Hepatócitos/enzimologia , Humanos , Hidroxilação , Isoenzimas , Cinética , Masculino , Metilação , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Oxirredução , Pró-Fármacos/química , Ratos , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Tripanossomicidas/química
6.
Neurochem Int ; 125: 151-162, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30822440

RESUMO

The anticonvulsant vigabatrin (VGB; SabrilR) irreversibly inhibits GABA transaminase to increase neural GABA, yet its mechanism of retinal toxicity remains unclear. VGB is suggested to alter several amino acids, including homocarnosine, ß-alanine, ornithine, glycine, taurine, and 2-aminoadipic acid (AADA), the latter a homologue of glutamic acid. Here, we evaluate the effect of VGB on amino acid concentrations in mice, employing a continuous VGB infusion (subcutaneously implanted osmotic minipumps), dose-escalation paradigm (35-140 mg/kg/d, 12 days), and amino acid quantitation in eye, visual and prefrontal cortex, total brain, liver and plasma. We hypothesized that continuous VGB dosing would reveal numerous hitherto undescribed amino acid disturbances. Consistent amino acid elevations across tissues included GABA, ß-alanine, carnosine, ornithine and AADA, as well as neuroactive aspartic and glutamic acids, serine and glycine. Maximal increase of AADA in eye occurred at 35 mg/kg/d (41 ±â€¯2 nmol/g (n = 21, vehicle) to 60 ±â€¯8.5 (n = 8)), and at 70 mg/kg/d for brain (97 ±â€¯6 (n = 21) to 145 ±â€¯6 (n = 6)), visual cortex (128 ±â€¯6 to 215 ±â€¯19) and prefrontal cortex (124 ±â€¯11 to 200 ±â€¯13; mean ±â€¯SEM; p < 0.05), the first demonstration of tissue AADA accumulation with VGB in mammal. VGB effects on basic amino acids, including guanidino-species, suggested the capacity of VGB to alter urea cycle function and nitrogen disposal. The known toxicity of AADA in retinal glial cells highlights new avenues for assessing VGB retinal toxicity and other off-target effects.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Aminoácidos/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Vigabatrina/farmacologia , 4-Aminobutirato Transaminase/antagonistas & inibidores , Aminoácidos/sangue , Aminoácidos/genética , Animais , Anticonvulsivantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Retina/efeitos dos fármacos , Retina/metabolismo
7.
Pharmacol Res Perspect ; 7(1): e00456, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30631446

RESUMO

Vigabatrin (VGB; (S)-(+)/(R)-(-) 4-aminohex-5-enoic acid), an antiepileptic irreversibly inactivating GABA transaminase (GABA-T), manifests use-limiting ocular toxicity. Hypothesizing that the active S enantiomer of VGB would preferentially accumulate in eye and visual cortex (VC) as one potential mechanism for ocular toxicity, we infused racemic VGB into mice via subcutaneous minipump at 35, 70, and 140 mg/kg/d (n = 6-8 animals/dose) for 12 days. VGB enantiomers, total GABA and ß-alanine (BALA), 4-guanidinobutyrate (4-GBA), and creatine were quantified by mass spectrometry in eye, brain, liver, prefrontal cortex (PFC), and VC. Plasma VGB concentrations increased linearly by dose (3 ± 0.76 (35 mg/kg/d); 15.1 ± 1.4 (70 mg/kg/d); 34.6 ± 3.2 µmol/L (140 mg/kg/d); mean ± SEM) with an S/R ratio of 0.74 ± 0.02 (n = 14). Steady state S/R ratios (35, 70 mg/kg/d doses) were highest in eye (5.5 ± 0.2; P < 0.0001), followed by VC (3.9 ± 0.4), PFC (3.6 ± 0.3), liver (2.9 ± 0.1), and brain (1.5 ± 0.1; n = 13-14 each). Total VGB content of eye exceeded that of brain, PFC and VC at all doses. High-dose VGB diminished endogenous metabolite production, especially in PFC and VC. GABA significantly increased in all tissues (all doses) except brain; BALA increases were confined to liver and VC; and 4-GBA was prominently increased in brain, PFC and VC (and eye at high dose). Linear correlations between enantiomers and GABA were observed in all tissues, but only in PFC/VC for BALA, 4-GBA, and creatine. Preferential accumulation of the VGB S isomer in eye and VC may provide new insight into VGB ocular toxicity.


Assuntos
Anticonvulsivantes/farmacocinética , Vigabatrina/farmacocinética , Transtornos da Visão/prevenção & controle , 4-Aminobutirato Transaminase/antagonistas & inibidores , Animais , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/química , Avaliação Pré-Clínica de Medicamentos , Olho/efeitos dos fármacos , Olho/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Estereoisomerismo , Distribuição Tecidual , Vigabatrina/efeitos adversos , Vigabatrina/química , Transtornos da Visão/induzido quimicamente , Córtex Visual/efeitos dos fármacos , Córtex Visual/metabolismo , Campos Visuais/efeitos dos fármacos
8.
Lab Chip ; 19(19): 3152-3161, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31469131

RESUMO

The lung is a complex organ; it is both the initial barrier for inhaled agents and the site of metabolism and therapeutic effect for a subset of systemically administered drugs. Comprised of more than 40 cell types that are responsible for various important functions, the lung's complexity contributes to the subsequent challenges in developing complex in vitro co-culture models (also called microphysiological systems (MPS), complex in vitro models or organs-on-a-chip). Although there are multiple considerations and limitations in the development and qualification of such in vitro systems, MPS exhibit great promise in the fields of pharmacology and toxicology. Successful development and implementation of MPS models may enable mechanistic bridging between non-clinical species and humans, and increase clinical relevance of safety endpoints, while decreasing overall animal use. This article summarizes, from a biopharmaceutical industry perspective, essential elements for the development and qualification of lung MPS models. Its purpose is to guide MPS developers and manufacturers to expedite MPS utilization for safety assessment in the biopharmaceutical industry.


Assuntos
Técnicas de Cocultura , Dispositivos Lab-On-A-Chip , Pulmão/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Técnicas de Cocultura/instrumentação , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Técnicas Analíticas Microfluídicas/instrumentação
9.
Pediatr Neurol ; 66: 44-52.e1, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27816307

RESUMO

BACKGROUND: Gamma-vinyl-γ-aminobutyric acid (GABA) (vigabatrin) is an antiepileptic drug and irreversible GABA transaminase inhibitor associated with visual field impairment, which limits its clinical utility. We sought to relate altered visual evoked potentials associated with vigabatrin intake to transcriptional changes in the mechanistic target of rapamycin (mTOR) pathway and GABA receptors to expose further mechanisms of vigabatrin-induced visual field loss. METHODS: Vigabatrin was administered to mice via an osmotic pump for two weeks to increase GABA levels. Visual evoked potentials were examined, eye samples were collected, and gene expression was measured by quantitative reverse transcription-polymerase chain reaction. Similarly, human retinal pigment epithelial cells (ARPE19) were exposed to vigabatrin and treated with mTOR inhibitors for mTOR pathway analysis and to assess alterations in organelle accumulation by microscopy. RESULTS: Dysregulated expression of transcripts in the mTOR pathway, GABAA/B receptors, metabotropic glutamate (Glu) receptors 1/6, and GABA/glutamate transporters in the eye were found in association with visual evoked potential changes during vigabatrin administration. Rrag genes were upregulated in both mouse eye and ARPE19 cells. Immunoblot of whole eye revealed greater than three fold upregulation of a 200 kDa band when immunoblotted for ras-related guanosine triphosphate binding D. Microscopy of ARPE19 cells revealed selective reversal of vigabatrin-induced organelle accumulation by autophagy-inducing drugs, notably Torin 2. Changes in the mTOR pathway gene expression, including Rrag genes, were corrected by Torin 2 in ARPE19 cells. CONCLUSIONS: Our studies, indicating GABA-associated augmentation of RRAG and mTOR signaling, support further preclinical evaluation of mTOR inhibitors as a therapeutic strategy to potentially mitigate vigabatrin-induced ocular toxicity.


Assuntos
Inibidores Enzimáticos/farmacologia , Substâncias Protetoras/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Vigabatrina/toxicidade , Campos Visuais/efeitos dos fármacos , Animais , Linhagem Celular , Potenciais Evocados Visuais/efeitos dos fármacos , Potenciais Evocados Visuais/fisiologia , Olho/efeitos dos fármacos , Olho/patologia , Olho/fisiopatologia , Humanos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Receptores de GABA/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Serina-Treonina Quinases TOR/metabolismo , Campos Visuais/fisiologia
10.
PLoS One ; 12(10): e0186919, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29053743

RESUMO

We explored the utility of neural stem cells (NSCs) as an in vitro model for evaluating preclinical therapeutics in succinic semialdehyde dehydrogenase-deficient (SSADHD) mice. NSCs were obtained from aldh5a1+/+ and aldh5a1-/- mice (aldh5a1 = aldehyde dehydrogenase 5a1 = SSADH). Multiple parameters were evaluated including: (1) production of GHB (γ-hydroxybutyrate), the biochemical hallmark of SSADHD; (2) rescue from cell death with the dual mTOR (mechanistic target of rapamycin) inhibitor, XL-765, an agent previously shown to rescue aldh5a1-/- mice from premature lethality; (3) mitochondrial number, total reactive oxygen species, and mitochondrial superoxide production, all previously documented as abnormal in aldh5a1-/- mice; (4) total ATP levels and ATP consumption; and (5) selected gene expression profiles associated with epilepsy, a prominent feature in both experimental and human SSADHD. Patterns of dysfunction were observed in all of these parameters and mirrored earlier findings in aldh5a1-/- mice. Patterns of dysregulated gene expression between hypothalamus and NSCs centered on ion channels, GABAergic receptors, and inflammation, suggesting novel pathomechanisms as well as a developmental ontogeny for gene expression potentially associated with the murine epileptic phenotype. The NSC model of SSADHD will be valuable in providing a first-tier screen for centrally-acting therapeutics and prioritizing therapeutic concepts of preclinical animal studies applicable to SSADHD.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encéfalo/patologia , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Células-Tronco Neurais/patologia , Succinato-Semialdeído Desidrogenase/deficiência , Trifosfato de Adenosina/metabolismo , Animais , Meios de Cultura , Epilepsia/genética , Técnicas In Vitro , Camundongos , Estresse Oxidativo , Succinato-Semialdeído Desidrogenase/genética
11.
Pharmacol Res Perspect ; 4(6): e00265, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27891231

RESUMO

Gamma-aminobutyric acid (GABA) is an endogenous inhibitory neurotransmitter and precursor of gamma-hydroxybutyric acid (GHB). NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid), a known GHB receptor antagonist, has shown significant efficacy in a murine model of succinic semialdehyde dehydrogenase deficiency (SSADHD), a heritable neurological disorder featuring chronic elevation of GHB that blocks the final step of GABA degradation. NCS-382 exposures and elimination pathways remain unknown; therefore, the goal of the present work was to obtain in vivo pharmacokinetic data in a murine model and to identify the NCS-382 metabolites formed by mouse and human. NCS-382 single-dose mouse pharmacokinetics were established following an intraperitoneal injection (100, 300, and 500 mg/kg body weight) and metabolite identification was conducted using HPLC-MS/MS. Kinetic enzyme assays employed mouse and human liver microsomes. Upon gaining an understanding of the NCS-382 clearance mechanisms, a chemical inhibitor was used to increase NCS-382 brain exposure in a pharmacokinetic/pharmacodynamic study. Two major metabolic pathways of NCS-382 were identified as dehydrogenation and glucuronidation. The Km for the dehydrogenation pathway was determined in mouse (Km = 29.5 ± 10.0 µmol/L) and human (Km = 12.7 ± 4.8 µmol/L) liver microsomes. Comparable parameters for glucuronidation were >100 µmol/L in both species. Inhibition of NCS-382 glucuronidation, in vivo, by diclofenac resulted in increased NCS-382 brain concentrations and protective effects in gamma-butyrolactone-treated mice. These initial evaluations of NCS-382 pharmacokinetics and metabolism inform the development of NCS-382 as a potential therapy for conditions of GHB elevation (including acute intoxication & SSADHD).

12.
Ann Clin Transl Neurol ; 2(6): 699-706, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26125044

RESUMO

Recent findings in mice with targeted deletion of the GABA-metabolic enzyme succinic semialdehyde dehydrogenase revealed a new role for supraphysiological GABA (4-aminobutyric acid) in the activation of the mechanistic target of rapamycin (mTOR) that results in disruption of endogenous mitophagy. Employing biochemical and electron microscopic methodology, we examined the hypothesis that similar outcomes would be observed during intervention with vigabatrin, whose antiepileptic capacity hinges on central nervous system GABA elevation. Vigabatrin intervention was associated with significantly enhanced mitochondrial numbers and areas in normal mice that could be selectively normalized with the rapalog and mechanistic target of rapamycin inhibitor, Torin 1. Moreover, short-term administration of vigabatrin induced apoptosis and enhanced phosphorylation of mechanistic target of rapamycin Ser 2448 in liver. Our results provide new insight into adverse outcomes associated with vigabatrin intervention, and the first evidence that its administration is associated with increased mitochondrial number in central and peripheral tissues that may associate with mechanistic target of rapamycin function and enhanced cell death.

13.
Mol Genet Metab Rep ; 3: 80-87, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26120559

RESUMO

OBJECTIVE: Initial studies on the use of non-physiological amino acids (NPAAs) to block the accretion of Phe in the brain Pahenu2-/- mice revealed that 2-aminoisobutyrate (AIB) and N-methyl-2-aminoisobutyrate (MAIB) were promising lead compounds whose pharmacokinetic parameters warranted investigation. METHODS: Control and Pahenu2-/- mice received intraperitoneal NPAA treatments as test compounds (150, 300 and 500 mg/kg, 1 or 7 days;) followed by collection of sera, liver and brain. LC-MS analysis was developed to quantify both AIB and MAIB in all matrices, and pharmacokinetic parameters for distribution, partitioning, accumulation and MAIB demethylation were determined. RESULTS: MAIB was partially converted to AIB in vivo. AIB and MAIB partitioned similarly from sera to brain and liver, with an approximate 10-fold higher accumulation in liver compared to brain. In comparison to MAIB, AIB accumulated to approximately 3 to 7-fold higher concentration in brain. Analysis of brain and liver revealed a trend toward decreased Phe with increased MAIB sera concentration. CONCLUSIONS: Our data support further pharmacokinetic characterization of MAIB and AIB in preparation for further preclinical safety, toxicity and tolerability studies of both AIB and MAIB.

14.
J Pharm Biomed Anal ; 98: 260-5, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24951959

RESUMO

Dietary supplements are a multi-billion dollar business, with yearly profit increases. Allegedly safe, these supplements are marketed to a variety of niches, encompassing claims from immune support to weight loss. Six sports nutrition supplements were acquired that were labeled to contain the furanocoumarin(s) bergamottin and/or 6',7'-dihydroxybergamottin (DHB), both of which are potent irreversible inhibitors of the prominent drug metabolizing enzyme cytochrome P450 3A (CYP3A). Both furanocoumarins are typically present in grapefruit juice, which has been shown to inhibit intestinal CYP3A, perpetrating an increase in the systemic exposure of certain concomitant 'victim' drugs. The acquired supplements were analyzed using ultra-performance liquid chromatography coupled to both a photodiode array (PDA) detector and a triple quadrupole mass spectrometer (MS). Contrary to the product labeling, four of the supplements contained no detectable quantities of either furanocoumarin (LOD 0.060µg/capsule), while two of the supplements contained minimal amounts (one contained 12.13 (±0.23) µg bergamottin and 65.51 (±0.64) µg DHB per capsule; the other contained 2.705 (±0.069) µg bergamottin per capsule and no detectable quantities of DHB). A CYP3A inhibition bioassay was used to assess whether the actual content of the furanocoumarins correlated with CYP3A inhibitory activity. Despite the low amounts of bergamottin and DHB, CYP3A inhibition by the supplements was greater than could be accounted for by the two furanocoumarins. The additional activity suggests the presence of other potent or highly abundant CYP3A inhibitors.


Assuntos
Inibidores do Citocromo P-450 CYP3A/química , Suplementos Nutricionais/análise , Furocumarinas/química , Bebidas/análise , Cromatografia Líquida de Alta Pressão/métodos , Citrus/química , Citrus paradisi/química , Espectrometria de Massas/métodos
15.
Toxicol Sci ; 132(1): 53-63, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23288052

RESUMO

Hepatocellular carcinoma (HCC) mostly develops in patients with advanced fibrosis; however, the mechanisms of interaction between a genotoxic insult and fibrogenesis are not well understood. This study tested a hypothesis that fibrosis promotes HCC via a mechanism that involves activation of liver stem cells. First, B6C3F1 mice were administered diethylnitrosamine (DEN; single ip injection of 1mg/kg at 14 days of age). Second, carbon tetrachloride (CCl(4); 0.2ml/kg, 2/week ip starting at 8 weeks of age) was administered for 9 or 14 weeks to develop advanced liver fibrosis. In animals treated with DEN as neonates, presence of liver fibrosis led to more than doubling (to 100%) of the liver tumor incidence as early as 5 months of age. This effect was associated with activation of cells with progenitor features in noncancerous liver tissue, including markers of replicative senescence (p16), oncofetal transformation (Afp, H19, and Bex1), and increased "stemness" (Prom1 and Epcam). In contrast, the dose of DEN used did not modify the extent of liver inflammation, fibrogenesis, oxidative stress, proliferation, or apoptosis induced by subchronic CCl(4) administration. This study demonstrates the potential role of liver stem-like cells in the mechanisms of chemical-induced, fibrosis-promoted HCC. We posit that the combination of genotoxic and fibrogenic insults is a sensible approach to model liver carcinogenesis in experimental animals. These results may contribute to identification of cirrhotic patients predisposed to HCC by analyzing the expression of hepatic progenitor cell markers in the noncancerous liver tissue.


Assuntos
Tetracloreto de Carbono/toxicidade , Dietilnitrosamina/toxicidade , Cirrose Hepática/complicações , Neoplasias Hepáticas Experimentais/etiologia , Animais , Animais Recém-Nascidos , Transformação Celular Neoplásica , Feminino , Imuno-Histoquímica , Cirrose Hepática/induzido quimicamente , Camundongos , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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