RESUMEN
We report the in vitro assessment of pharmacotoxicity for the high-affinity GHB receptor ligand, NCS-382, using neuronal stem cells derived from mice with a targeted deletion of the aldehyde dehydrogenase 5a1 gene (succinic semialdehyde dehydrogenase(SSADH)-deficient mice). These animals represent a phenocopy of the human disorder of GABA metabolism, SSADH deficiency, that metabolically features accumulation of both GABA and the GABA-analog γ-hydroxybutyric acid in conjunction with a nonspecific neurological phenotype. We demonstrate for the first time using MDCK cells that NCS-382 is actively transported and capable of inhibiting GHB transport. Following these in vitro assays with in vivo studies in aldh5a1-/- mice, we found the ratio of brain/liver GHB to be unaffected by chronic NCS-382 administration (300mg/kg; 7 consecutive days). Employing a variety of cellular parameters (reactive oxygen and superoxide species, ATP production and decay, mitochondrial and lysosomal number, cellular viability and necrosis), we demonstrate that up to 1mM NCS-382 shows minimal evidence of pharmacotoxicity. As well, studies at the molecular level indicate that the effects of NCS-382 at 0.5mM are minimally toxic as evaluated using gene expression assay. The cumulative data provides increasing confidence that NCS-382 could eventually be considered in the therapeutic armament for heritable SSADH deficiency.
Asunto(s)
Benzocicloheptenos/metabolismo , Benzocicloheptenos/toxicidad , Errores Innatos del Metabolismo de los Aminoácidos , Animales , Anticonvulsivantes/metabolismo , Anticonvulsivantes/toxicidad , Biomarcadores , Supervivencia Celular , Discapacidades del Desarrollo , Células Epiteliales , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Humanos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Células-Madre Neurales/metabolismo , Neuronas , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular , Succionato-Semialdehído Deshidrogenasa/deficiencia , Superóxidos/metabolismoRESUMEN
HYPOTHESIS: An enzymatic assay for quantification of γ-hydroxybutyric acid (GHB) in biofluids can be employed for targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) in selected populations. RATIONALE: We used a two-tiered study approach, in which the first study (proof of concept) examined 7 urine samples derived from patients with SSADHD and 5 controls, and the second study (feasibility study) examined a broader sample population of patients and controls, including plasma. OBJECTIVE: Split samples of urine and plasma (anonymized) were evaluated by enzymatic assay, gas chromatography alone (proof of concept) and gas chromatography-mass spectrometry, and the results compared. METHOD: Multiple detection methods have been developed to detect GHB. We evaluated an enzymatic assay which employs recombinant GHB dehydrogenase coupled to NADH production, the latter quantified on a Cobas Integra 400 Plus. Results: In our proof of concept study, we analyzed 12 urine samples (5 controls, 7 SSADHD), and in the feasibility study we evaluated 33 urine samples (23 controls, 10 SSADHD) and 31 plasma samples (14 controls, 17 SSADHD). The enzymatic assay carried out on a routine clinical chemistry analyzer was robust, revealing excellent agreement with instrumental methods in urine (GC-FID: r = 0.997, p ≤ 0.001; GC-MS: r = 0.99, p ≤ 0.001); however, the assay slightly over-estimated GHB levels in plasma, especially those in which GHB levels were low. Conversely, correlations for the enzymatic assay with comparator methods for higher plasma GHB levels were excellent (GC-MS; r = 0.993, p ≤ 0.001). CONCLUSION: We have evaluated the capacity of this enzymatic assay to identify patients with SSADHD via quantitation of GHB. The data suggests that the enzymatic assay may be a suitable screening method to detect SSADHD in selected populations using urine. In addition, the assay can be used in basic research the elucidate the mechanism of the underlying disease or monitor GHB- levels for the evaluation of drug candidates. SYNOPSIS: An enzymatic assay for GHB in biofluids was evaluated as a screening method for SSADHD and found to be reliable in urine, but in need of refinement for application to plasma.
RESUMEN
Gamma-hydroxybutyrate (GHB) is a drug of abuse, an approved therapeutic for narcolepsy, an agent employed for facilitation of sexual assault, as well as a biomarker of succinic semialdehyde dehydrogenase deficiency (SSADHD). Our laboratory seeks to identify surrogate biomarkers in SSADHD that can shed light on the developmental course of this neurometabolic disease. Since GHB may be quantified in hair as a potential surrogate to identify victims of drug-related assault, we have opted to examine its level in SSADHD. We quantified GHB in hair derived from ten patients with SSADHD, and documented a significant negative age correlation. These findings are consistent with recent results in patient biological fluids, including plasma and red blood cells. These findings may provide additional insight into the developmental course of SSADHD (Jansen et al., J Inherit Metab Dis 39:795-800, 2016).
RESUMEN
γ-Hydroxybutyric acid (GHB), a minor metabolite of the inhibitory neurotransmitter GABA, can accumulate to significant concentrations in the heritable disorder of GABA degradation, succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD). Moreover, GHB may be employed in therapeutic settings (treatment of narcolepsy), as well as instances of illicit activity, including acquaintance sexual assault and the induction of euphoria. High-affinity binding sites for GHB in the brain have been identified, although the absolute identity of these receptors remains unclear. Pharmacological antagonism of GHB binding may have multiple instances of therapeutic relevance. The high affinity GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid) has not been piloted in humans. To address the potential clinical utility of NCS-382, we have piloted initial studies of its toxicology in HepG2 and primary hepatocyte cells. At high dose (0.5mM), NCS-382 showed no capacity for inhibition of microsomal CYPs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and minimal potential for activation of xenobiotic nuclear receptors. Additional cellular integrity and functional assays (viability, oxidative stress, apoptosis, ATP production) revealed little evidence for cytotoxicity, and a low degree of dysregulation of >370 genes actively engaged in the mediation of cellular toxicity. In vitro testing indicates a low probability of cellular toxicity associated with NCS-382.
Asunto(s)
Benzocicloheptenos/farmacología , Hidroxibutiratos/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismoRESUMEN
Vigabatrin (VGB; γ-vinylGABA) is a unique antiepileptic directly elevating CNS GABA via inactivation of the GABA metabolic enzyme GABA-transaminase. VGB is effective in treating infantile spasms, a rare seizure disorder associated with significant morbidity. The potential for unexplained bilateral constriction of the visual field associated with VGB intervention can severely limit its temporal utility. Removal of this potential adverse effect with adjuvant intervention(s) would represent a significant advance in epilepsy therapeutics.
Asunto(s)
Anticonvulsivantes/efectos adversos , Autofagia/efectos de los fármacos , Serina-Treonina Quinasas TOR/efectos de los fármacos , Vigabatrin/efectos adversos , Trastornos de la Visión/inducido químicamente , Potenciales Evocados Visuales , Humanos , Lactante , Recién Nacido , Transducción de Señal , Espasmos Infantiles , Trastornos de la Visión/fisiopatología , Ácido gamma-Aminobutírico/biosíntesisRESUMEN
Aldehyde dehydrogenase 5a1-deficient (aldh5a1-/-) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1-/- mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1-/- mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1-/- mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1-/- mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Discapacidades del Desarrollo/tratamiento farmacológico , Discapacidades del Desarrollo/metabolismo , Eliminación de Gen , Succionato-Semialdehído Deshidrogenasa/deficiencia , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Discapacidades del Desarrollo/genética , Modelos Animales de Enfermedad , Femenino , Glutatión/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Naftiridinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Succionato-Semialdehído Deshidrogenasa/genética , Succionato-Semialdehído Deshidrogenasa/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5-41 years; median 8). Both metabolites negatively correlated with age (P < 0.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10 years. Declining plasma GABA achieved this approximate steady state at 30-40 years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/sangre , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Biomarcadores/sangre , Discapacidades del Desarrollo/sangre , Discapacidades del Desarrollo/metabolismo , Succionato-Semialdehído Deshidrogenasa/deficiencia , Ácido gamma-Aminobutírico/metabolismo , Adolescente , Adulto , Niño , Preescolar , Epilepsia/sangre , Epilepsia/metabolismo , Femenino , Humanos , Hidroxibutiratos/metabolismo , Masculino , Succionato-Semialdehído Deshidrogenasa/sangre , Succionato-Semialdehído Deshidrogenasa/metabolismo , Transmisión Sináptica/fisiología , Adulto JovenRESUMEN
Discovered some 35 years ago, succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a rare, autosomal recessively-inherited defect in the second step of the GABA degradative pathway. Some 200 patients have been reported, with broad phenotypic and genotypic heterogeneity. SSADHD represents an unusual neurometabolic disorder in which two neuromodulatory agents, GABA (and the GABA analogue, 4-hydroxybutyrate), accumulate to supraphysiological levels. The unexpected occurrence of epilepsy in several patients is counterintuitive in view of the hyperGABAergic state, in which sedation might be expected. However, the epileptic status of some patients is most likely represented by broader imbalances of GABAergic and glutamatergic neurotransmission. Cumulative research encompassing decades of basic and clinical study of SSADHD reveal a monogenic disease with broad pathophysiological and clinical phenotypes. Numerous metabolic perturbations unmasked in SSADHD include alterations in oxidative stress parameters, dysregulation of autophagy and mitophagy, dysregulation of both inhibitory and excitatory neurotransmitters and gene expression, and unique subsets of SNP alterations of the SSADH gene (so-called ALDH5A1, or aldehyde dehydrogenase 5A1 gene) on the 6p22 chromosomal arm. While seemingly difficult to collate and interpret, these anomalies have continued to open novel pathways for pharmacotherapeutic considerations. Here, we present an update on selected aspects of SSADHD, the ALDH5A1 gene, and future avenues for research on this rare disorder of GABA metabolism.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/fisiopatología , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/fisiopatología , Estudios de Asociación Genética/métodos , Herencia Multifactorial/fisiología , Succionato-Semialdehído Deshidrogenasa/deficiencia , Ácido gamma-Aminobutírico/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Discapacidades del Desarrollo/genética , Humanos , Succionato-Semialdehído Deshidrogenasa/genética , Succionato-Semialdehído Deshidrogenasa/metabolismoRESUMEN
Microwave atomic clocks have been the de facto standards for precision time and frequency metrology over the past 50 years, finding widespread use in basic scientific studies, communications, and navigation. However, with its higher operating frequency, an atomic clock based on an optical transition can be much more stable. We demonstrate an all-optical atomic clock referenced to the 1.064-petahertz transition of a single trapped 199Hg+ ion. A clockwork based on a mode-locked femtosecond laser provides output pulses at a 1-gigahertz rate that are phase-coherently locked to the optical frequency. By comparison to a laser-cooled calcium optical standard, an upper limit for the fractional frequency instability of 7 x 10(-15) is measured in 1 second of averaging-a value substantially better than that of the world's best microwave atomic clocks.
RESUMEN
The frequency comb created by a femtosecond mode-locked laser and a microstructured fiber is used to phase coherently measure the frequencies of both the Hg+ and Ca optical standards with respect to the SI second. We find the transition frequencies to be f(Hg) = 1 064 721 609 899 143(10) Hz and f(Ca) = 455 986 240 494 158(26) Hz, respectively. In addition to the unprecedented precision demonstrated here, this work is the precursor to all-optical atomic clocks based on the Hg+ and Ca standards. Furthermore, when combined with previous measurements, we find no time variations of these atomic frequencies within the uncertainties of the absolute value of( partial differential f(Ca)/ partial differential t)/f(Ca) < or =8 x 10(-14) yr(-1) and the absolute value of(partial differential f(Hg)/ partial differential t)/f(Hg) < or =30 x 10(-14) yr(-1).
RESUMEN
With a fiber-broadened, femtosecond-laser frequency comb, the 76-THz interval between two laser-cooled optical frequency standards was measured with a statistical uncertainty of 2x10(-13) in 5 s , to our knowledge the best short-term instability thus far reported for an optical frequency measurement. One standard is based on the calcium intercombination line at 657 nm, and the other, on the mercury ion electric-quadrupole transition at 282 nm. By linking this measurement to the known Ca frequency, we report a new frequency value for the Hg(+) clock transition with an improvement in accuracy of ~10(5) compared with its best previous measurement.