RESUMEN
OBJECTIVE: Glycine encephalopathy, also known as nonketotic hyperglycinemia (NKH), is an inherited neurometabolic disorder with variable clinical course and severity, ranging from infantile epileptic encephalopathy to psychiatric disorders. A precise phenotypic characterization and an evaluation of predictive approaches are needed. METHODS: Longitudinal clinical and biochemical data of 25 individuals with NKH from the patient registry of the International Working Group on Neurotransmitter Related Disorders were studied with in silico analyses, pathogenicity scores, and molecular modeling of GLDC and AMT variants. RESULTS: Symptom onset (p < 0.01) and diagnosis occur earlier in life in severe NKH (p < 0.01). Presenting symptoms affect the age at diagnosis. Psychiatric problems occur predominantly in attenuated NKH. Onset age ≥ 3 months (66% specificity, 100% sensitivity, area under the curve [AUC] = 0.87) and cerebrospinal fluid (CSF)/plasma glycine ratio ≤ 0.09 (57% specificity, 100% sensitivity, AUC = 0.88) are sensitive indicators for attenuated NKH, whereas CSF glycine concentration ≥ 116.5µmol/l (100% specificity, 93% sensitivity, AUC = 0.97) and CSF/plasma glycine ratio ≥ 0.15 (100% specificity, 64% sensitivity, AUC = 0.88) are specific for severe forms. A ratio threshold of 0.128 discriminates the overlapping range. We present 10 new GLDC variants. Two mild variants resulted in attenuated, whereas 2 severe variants or 1 mild and 1 severe variant led to severe phenotype. Based on clinical, biochemical, and genetic parameters, we propose a severity prediction model. INTERPRETATION: This study widens the phenotypic spectrum of attenuated NKH and expands the number of pathogenic variants. The multiparametric approach provides a promising tool to predict disease severity, helping to improve clinical management strategies. ANN NEUROL 2022;92:292-303.
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Hiperglicinemia no Cetósica , Glicina/líquido cefalorraquídeo , Glicina/genética , Humanos , Hiperglicinemia no Cetósica/diagnóstico , Hiperglicinemia no Cetósica/genética , Hiperglicinemia no Cetósica/patología , Mutación , FenotipoRESUMEN
The glycine cleavage system H protein (GCSH) is an integral part of the glycine cleavage system with its additional involvement in the synthesis and transport of lipoic acid. We hypothesize that pathogenic variants in GCSH can cause variant nonketotic hyperglycinemia (NKH), a heterogeneous group of disorders with findings resembling a combination of severe NKH (elevated levels of glycine in plasma and CSF, progressive lethargy, seizures, severe hypotonia, no developmental progress, early death) and mitochondriopathies (lactic acidosis, leukoencephalopathy and Leigh-like lesions on MRI). We herein report three individuals from two unrelated Indian families with clinical, biochemical, and radiological findings of variant NKH, harboring a biallelic start loss variant, c.1A > G in GCSH.
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Proteína H del Complejo de la Glicina Descarboxilasa/genética , Hiperglicinemia no Cetósica/genética , Preescolar , Femenino , Glicina/sangre , Glicina/líquido cefalorraquídeo , Humanos , Hiperglicinemia no Cetósica/etiología , Masculino , Mutación , LinajeRESUMEN
The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30-60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.
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Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Glicina/líquido cefalorraquídeo , Hiperalgesia/prevención & control , Neuralgia/tratamiento farmacológico , Sarcosina/análogos & derivados , Animales , Hiperalgesia/metabolismo , Hiperalgesia/patología , Masculino , Actividad Motora , Neuralgia/metabolismo , Neuralgia/patología , Ratas , Ratas Wistar , Sarcosina/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patologíaAsunto(s)
Glicina/sangre , Glicina/líquido cefalorraquídeo , Hiperglicinemia no Cetósica/diagnóstico , Hiperglicinemia no Cetósica/genética , Apnea/diagnóstico , Apnea/epidemiología , Cromatografía por Intercambio Iónico/métodos , Coma/diagnóstico , Coma/epidemiología , Consanguinidad , Humanos , Hiperglicinemia no Cetósica/epidemiología , Hiperglicinemia no Cetósica/mortalidad , Incidencia , Nacimiento Vivo/epidemiología , Hipotonía Muscular/diagnóstico , Hipotonía Muscular/epidemiología , Mutación/genética , Fenotipo , Convulsiones/diagnóstico , Convulsiones/epidemiología , Índice de Severidad de la Enfermedad , Túnez/epidemiologíaRESUMEN
Huntington's Disease (HD) is a progressive, fatal neurodegenerative condition. While generally considered for its devastating neurological phenotype, disturbances in other organ systems and metabolic pathways outside the brain have attracted attention for possible relevance to HD pathology, potential as therapeutic targets, or use as biomarkers of progression. In addition, it is not established how metabolic changes in the HD brain correlate to progression across the full spectrum of early to late-stage disease. In this pilot study, we sought to explore the metabolic profile across manifest HD from early to advanced clinical staging through metabolomic analysis by mass spectrometry in plasma and cerebrospinal fluid (CSF). With disease progression, we observed nominally significant increases in plasma arginine, citrulline, and glycine, with decreases in total and D-serine, cholesterol esters, diacylglycerides, triacylglycerides, phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins. In CSF, worsening disease was associated with nominally significant increases in NAD+, arginine, saturated long chain free fatty acids, diacylglycerides, triacylglycerides, and sphingomyelins. Notably, diacylglycerides and triacylglyceride species associated with clinical progression were different between plasma and CSF, suggesting different metabolic preferences for these compartments. Increasing NAD+ levels strongly correlating with disease progression was an unexpected finding. Our data suggest that defects in the urea cycle, glycine, and serine metabolism may be underrecognized in the progression HD pathology, and merit further study for possible therapeutic relevance.
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Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Evaluación de la Discapacidad , Enfermedad de Huntington/sangre , Enfermedad de Huntington/líquido cefalorraquídeo , Metabolómica , Adulto , Arginina/sangre , Arginina/líquido cefalorraquídeo , Creatina/sangre , Creatina/líquido cefalorraquídeo , Estudios Transversales , Femenino , Glicina/sangre , Glicina/líquido cefalorraquídeo , Humanos , Enfermedad de Huntington/metabolismo , Masculino , Persona de Mediana Edad , Proyectos Piloto , Resultado del Tratamiento , Adulto JovenRESUMEN
AIM: The aim of the study is to report on epidemiological, clinical, and biochemical characteristics of nonketotic hyperglycinemia (NKH) in Tunisia. METHODS: Patients diagnosed with NKH in Laboratory of Biochemistry at Rabta hospital (Tunis, Tunisia) between 1999 and 2018 were included. Plasma and cerebrospinal fluid (CSF) free amino acids were assessed by ion exchange chromatography. Diagnosis was based on family history, patient's clinical presentation and course, and increased CSF to plasma glycine ratio. RESULTS: During 20 years, 69 patients were diagnosed with NKH, with 25 patients originating from Kairouan region. Estimated incidences were 1:55,641 in Tunisia and 1:9,684 in Kairouan. Consanguinity was found for 73.9% of the patients and 42% of the families have history of infantile death due to a disease of similar clinical course than the propositus. Clinical symptoms initiated within the first week of life in 75% of the patients and within the first 3 months in 95.7% ones. The phenotype was severe in 76.8% of the patients. Main symptoms were hypotonia, feeding difficulties, coma, apnea, and seizures. Most patients died within few days to months following diagnosis. CSF to plasma glycine ratio was increased in all patients. CSF and plasma glycine levels were negatively correlated with age of disease onset and severity. CONCLUSION: NKH is quite frequent in Tunisia. Kairouan region has the highest NKH incidence rate, worldwide. However, due to lack of confirmatory enzymatic and genetic tests, NKH diagnosis was based on first-line biochemical tests. Characterization of causal mutations is needed for accurate diagnosis and prenatal diagnosis of this devastating life-threatening disease.
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Consanguinidad , Glicina/metabolismo , Hiperglicinemia no Cetósica/diagnóstico , Hiperglicinemia no Cetósica/epidemiología , Hiperglicinemia no Cetósica/fisiopatología , Edad de Inicio , Preescolar , Femenino , Glicina/sangre , Glicina/líquido cefalorraquídeo , Humanos , Lactante , Recién Nacido , Masculino , Fenotipo , Índice de Severidad de la Enfermedad , Túnez/epidemiologíaRESUMEN
Nonketotic hyperglycinemia (NKH) or glycine encephalopathy is an autosomal recessive disorder of glycine metabolism resulting in an excessive accumulation of glycine in all body tissues, including the central nervous system. It is caused by a biochemical defect in the glycine cleavage system and considered as a rare disorder with an estimated prevalence of 1:60,000. The neonatal form presents in the first few days of life with progressive encephalopathy, hypotonia, myoclonic jerks, hiccups, seizures, rapid progression to coma and often death due to central apnea. Surviving infants often have severe developmental delay and refractory seizures. Atypical forms of NKH present with heterogeneous and nonspecific disease course. Classical glycine encephalopathy usually carries a very poor prognosis. We describe two neonates who presented with neonatal encephalopathy, apnea, and progressive lethargy. Increased CSF glycine level along with an elevated CSF to plasma glycine ratio was suggestive of classic NKH. Burst suppression EEG and agenesis of the corpus callosum were supportive findings. Evolution of the EEG patterns and course of the disease are discussed in detail. Transient phases of clinical stabilization and normalized plasma biochemical results may not necessarily reflect the actual encephalopathic process. Serial EEGs are helpful to assess the efficacy of treatment and to modify the therapeutic approach.
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Hiperglicinemia no Cetósica/diagnóstico , Agenesia del Cuerpo Calloso/diagnóstico por imagen , Agenesia del Cuerpo Calloso/etiología , Apnea/etiología , Encéfalo/diagnóstico por imagen , Encefalopatías/etiología , Electroencefalografía , Resultado Fatal , Femenino , Glicina/líquido cefalorraquídeo , Glicina/metabolismo , Humanos , Hiperglicinemia no Cetósica/diagnóstico por imagen , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Convulsiones/etiología , Convulsiones/terapia , Estado Epiléptico/etiologíaRESUMEN
Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.
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Glicina-Deshidrogenasa (Descarboxilante)/deficiencia , Glicina/sangre , Hiperglicinemia no Cetósica/genética , Trastornos Motores/enzimología , Transmisión Sináptica/efectos de los fármacos , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/fisiopatología , Proteína 9 Asociada a CRISPR/metabolismo , Dextrometorfano/administración & dosificación , Dextrometorfano/uso terapéutico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Resultado Fatal , Femenino , Conservantes de Alimentos/uso terapéutico , Glicina/líquido cefalorraquídeo , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Humanos , Hiperglicinemia no Cetósica/diagnóstico , Hiperglicinemia no Cetósica/enzimología , Recién Nacido , Masculino , Persona de Mediana Edad , Trastornos Motores/fisiopatología , Mutación , Fenotipo , Benzoato de Sodio/administración & dosificación , Benzoato de Sodio/uso terapéutico , Resultado del Tratamiento , Pez CebraRESUMEN
No disponible
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Humanos , Masculino , Femenino , Recién Nacido , Lactante , Hiperglicinemia no Cetósica/complicaciones , Hiperglicinemia no Cetósica/diagnóstico , Hiperglicinemia no Cetósica/genética , Glicina/análisis , Glicina/sangre , Glicina/líquido cefalorraquídeo , Cromatografía de Gases , Hormona Adrenocorticotrópica/administración & dosificaciónRESUMEN
BI 425809 is a potent and selective glycine transporter 1 (GlyT1) inhibitor being developed for the treatment of cognitive impairment in Alzheimer disease and schizophrenia. Translational studies evaluated the effects of BI 425809 on glycine levels in rat and human cerebrospinal fluid (CSF). Oral administration of BI 425809 in rats induced a dose-dependent increase of glycine CSF levels from 30% (0.2 mg/kg, not significant) to 78% (2 mg/kg, P < 0.01), relative to vehicle. Similarly, oral administration of BI 425809 in healthy volunteers resulted in a dose-dependent increase in glycine CSF levels at steady state, with a mean 50% increase at doses as low as 10 mg. The peak plasma concentration (Cmax ) of BI 425809 was achieved earlier in plasma than in CSF (tmax 3-5 vs. 5-8 hours, respectively). Generally, BI 425809 was safe and well tolerated. These data provide evidence of functional target engagement of GlyT1 by BI 425809.
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Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Glicina/líquido cefalorraquídeo , Nootrópicos/farmacología , Compuestos Orgánicos/farmacología , Administración Oral , Adulto , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Área Bajo la Curva , Línea Celular , Relación Dosis-Respuesta a Droga , Glicina/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Neuronas , Nootrópicos/farmacocinética , Nootrópicos/uso terapéutico , Compuestos Orgánicos/administración & dosificación , Compuestos Orgánicos/farmacocinética , Cultivo Primario de Células , Ratas , Ratas Wistar , Esquizofrenia/tratamiento farmacológico , Adulto JovenRESUMEN
BACKGROUND: Non-ketotic hyperglycinemia (NKH) is a rare, devastating autosomal recessive disorder of glycine metabolism with a very poor prognosis. Currently, few studies have reported genetic profiling of Chinese NKH patients. This study aimed to identify the genetic mutations in a Chinese family with NKH. METHODS: A Chinese family of Han ethnicity, with three siblings with NKH was studied. Sanger sequencing and multiplex ligation-dependent probe amplification combined with SYBR green real-time quantitative PCR was used to identify potential mutations in the GLDC, AMT and GCSH genes. The potential pathogenicity of the identified missense mutation was analyzed using SIFT, PolyPhen-2, PROVEAN and MutationTaster software. RESULTS: All patients exhibited severe and progressive clinical symptoms, including lethargy, hypotonia and seizures, and had greatly elevated glycine levels in their plasma and CSF. Molecular genetic analysis identified compound heterozygous variants in the GLDC gene in these three siblings, including a novel missense variant c.2680A > G (p.Thr894Ala) in exon 23 and a heterozygous deletion of exon 3, which were inherited respectively from their parents. In silico analysis, using several different types of bioinformatic software, predicted that the novel variant c.2680A > G in the GLDC gene was pathogenic. Moreover, the deletion of exon 3 was identified for the first time in a Chinese population. CONCLUSIONS: A novel missense variant and a previously reported deletion in GLDC gene were identified. The two variants of GLDC gene identified probably underlie the pathogenesis of non-ketotic hyperglycinemia in this family, and also enrich the mutational spectrum of GLDC gene.
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Pueblo Asiatico/genética , Glicina-Deshidrogenasa (Descarboxilante)/genética , Heterocigoto , Hiperglicinemia no Cetósica/genética , Secuencia de Aminoácidos , China , Exones , Femenino , Variación Genética , Genotipo , Glicina/sangre , Glicina/líquido cefalorraquídeo , Humanos , Hiperglicinemia no Cetósica/enzimología , Recién Nacido , Masculino , Mutación Missense , Linaje , Eliminación de Secuencia , HermanosRESUMEN
BACKGROUND AND AIMS: The inhibitory pathways that play a role in spinal modulation include local interneurons and descending control. Clinical data regarding the role of these pathways in acute pain is lacking. Accordingly, the aim of this study was to evaluate cerebrospinal fluid (CSF) levels of noradrenaline, serotonin, gamma-aminobutyric acid (GABA), and glycine in parturients with labor pain compared to those without labor pain. METHODS: One hundred term uncomplicated pregnant women receiving spinal anesthesia for cesarean section were enrolled in this prospective cross-sectional study. CSF noradrenaline, serotonin, GABA, and glycine levels were analyzed by enzyme-linked immunosorbent assay. Labor pain score was assessed by numerical rating scale. RESULTS: Median CSF serotonin concentration in parturients with labor pain was significantly lower than in those without pain (p < 0.001). Median CSF glycine level in the labor pain group was significantly higher than in the control group (p < 0.001). There were no significant differences in median CSF level of noradrenaline or GABA between parturients with and without labor pain. Subsequent analysis showed labor pain scores to be negatively correlated with CSF serotonin (r = -0.217, p = 0.04) but positively correlated with CSF glycine (r = 0.415, p < 0.001). CONCLUSION: CSF serotonin and glycine were significantly correlated with labor pain scores. These findings suggest that the serotonergic and glycinergic systems may play a role in spinal modulation of visceral pain.
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Glicina/líquido cefalorraquídeo , Dolor de Parto/diagnóstico , Norepinefrina/líquido cefalorraquídeo , Serotonina/líquido cefalorraquídeo , Ácido gamma-Aminobutírico/líquido cefalorraquídeo , Adulto , Estudios Transversales , Femenino , Humanos , Dolor de Parto/líquido cefalorraquídeo , Masculino , Embarazo , Estudios Prospectivos , Médula Espinal/metabolismoRESUMEN
BACKGROUND: Autosomal recessive or X-linked inborn errors of intracellular cobalamin metabolism can lead to methylmalonic aciduria and homocystinuria. In neonates, both increased cerebrospinal fluid glycine and cerebrospinal fluid/plasma glycine ratio are biochemical features of nonketotic hyperglycinemia. METHODS: We describe a boy presenting in the neonatal period with hypotonia, tonic, clonic, and later myoclonic seizures, subsequently evolving into refractory epilepsy and severe neurocognitive impairment. RESULTS: Increased cerebrospinal fluid glycine and cerebrospinal fluid to plasma glycine ratio were indicative of nonketotic hyperglycinemia. Early magnetic resonance imaging showed restricted diffusion and decreased apparent diffusion coefficient values in posterior limb of internal capsules and later in entire internal capsules and posterior white matter. Sequencing did not show a mutation in AMT, GLDC, or GCSH. Biochemical analysis identified persistently increased cerebrospinal fluid levels of glycine and methylmalonic acid and increased urinary methylmalonic acid and plasma homocysteine levels, which improved on higher parenteral hydroxocobalamin dose. Exome sequencing identified a known pathogenic sequence variant in X-linked cobalamin (HCFC1), c.344C>T, p. Ala115Val. In addition, a hemizygous mutation was found in the ATRX (c. 2728A>G, p. Lys910Glu). Retrospective review of two other patients with X-linked cobalamin deficiency also identified increased cerebrospinal fluid glycine levels. CONCLUSIONS: This boy had X-linked cobalamin deficiency (HCFC1) with increased cerebrospinal fluid glycine and methylmalonic acid and increased cerebrospinal fluid to plasma glycine ratio suggesting a brain hyperglycinemia. Putative binding sites for HCFC1 and its binding partner THAP11 were identified near genes of the glycine cleavage enzyme, providing a potential mechanistic link between HCFC1 mutations and increased glycine.
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Enfermedades Genéticas Ligadas al Cromosoma X/líquido cefalorraquídeo , Glicina/líquido cefalorraquídeo , Hiperglicinemia no Cetósica/diagnóstico , Ácido Metilmalónico/líquido cefalorraquídeo , Deficiencia de Vitamina B 12/líquido cefalorraquídeo , Deficiencia de Vitamina B 12/diagnóstico , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/orina , Encéfalo/diagnóstico por imagen , Diagnóstico Diferencial , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Glicina/sangre , Humanos , Recién Nacido , Masculino , Deficiencia de Vitamina B 12/tratamiento farmacológico , Deficiencia de Vitamina B 12/genéticaRESUMEN
Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia. Here, we report four individuals from two families who presented at birth with facial dysmorphism, encephalopathy, arthrogryposis, hypotonia progressing to hypertonicity with startle-like clonus, and respiratory failure. Only one individual survived the respiratory failure and was weaned off ventilation but has significant global developmental delay. Mildly elevated cerebrospinal fluid (CSF) glycine and normal serum glycine were observed in two individuals. In both families, we identified truncating mutations in SLC6A9, encoding GLYT1. We demonstrate that pharmacologic or genetic abolishment of GlyT1 activity in mice leads to mildly elevated glycine in the CSF but not in blood. Additionally, previously reported slc6a9-null mice and zebrafish mutants also display phenotypes consistent with the affected individuals we examined. Our data suggest that truncating SLC6A9 mutations lead to a distinct human neurological syndrome hallmarked by mildly elevated CSF glycine and normal serum glycine.
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Artrogriposis/genética , Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Glicina/líquido cefalorraquídeo , Hiperglicinemia no Cetósica/genética , Animales , Artrogriposis/diagnóstico , Preescolar , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Glicina/sangre , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Humanos , Hiperglicinemia no Cetósica/diagnóstico , Lactante , Recién Nacido , Masculino , Ratones , Ratones Noqueados , LinajeRESUMEN
We previously disclosed the discovery of rationally designed N-((1-(4-(propylsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1 (GlyT-1), represented by analogues 10 and 11. We describe herein further structure-activity relationship exploration of this series via an optimization strategy that primarily focused on the sulfonamide and benzamide appendages of the scaffold. These efforts led to the identification of advanced leads possessing a desirable balance of excellent in vitro GlyT-1 potency and selectivity, favorable ADME and in vitro pharmacological profiles, and suitable pharmacokinetic and safety characteristics. Representative analogue (+)-67 exhibited robust in vivo activity in the cerebral spinal fluid glycine biomarker model in both rodents and nonhuman primates. Furthermore, rodent microdialysis experiments also demonstrated that oral administration of (+)-67 significantly elevated extracellular glycine levels within the medial prefrontal cortex (mPFC).
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Benzamidas/química , Benzamidas/farmacología , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Animales , Benzamidas/síntesis química , Benzamidas/farmacocinética , Glicina/líquido cefalorraquídeo , Glicina/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Macaca fascicularis , Masculino , Metilación , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
A LC-MS/MS method has been developed and validated for the determination of glycine in human cerebrospinal fluid (CSF). The validated method used artificial cerebrospinal fluid as a surrogate matrix for calibration standards. The calibration curve range for the assay was 100-10,000ng/mL and (13)C2, (15)N-glycine was used as an internal standard (IS). Pre-validation experiments were performed to demonstrate parallelism with surrogate matrix and standard addition methods. The mean endogenous glycine concentration in a pooled human CSF determined on three days by using artificial CSF as a surrogate matrix and the method of standard addition was found to be 748±30.6 and 768±18.1ng/mL, respectively. A percentage difference of -2.6% indicated that artificial CSF could be used as a surrogate calibration matrix for the determination of glycine in human CSF. Quality control (QC) samples, except the lower limit of quantitation (LLOQ) QC and low QC samples, were prepared by spiking glycine into aliquots of pooled human CSF sample. The low QC sample was prepared from a separate pooled human CSF sample containing low endogenous glycine concentrations, while the LLOQ QC sample was prepared in artificial CSF. Standard addition was used extensively to evaluate matrix effects during validation. The validated method was used to determine the endogenous glycine concentrations in human CSF samples. Incurred sample reanalysis demonstrated reproducibility of the method.
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Líquido Cefalorraquídeo/química , Glicina/líquido cefalorraquídeo , Glicina/química , Calibración , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos , Humanos , Control de Calidad , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
RATIONALE: Hypofunction of NMDA receptors has been implicated in neuropsychiatric disorders including schizophrenia. NMDA receptor neurotransmission can be enhanced through inhibition of glycine reuptake by the glycine transporter type 1 (GlyT1). OBJECTIVES: The primary objective of these studies was to explore the relationship between plasma exposure and glycine cerebrospinal fluid (CSF) concentrations following administration of bitopertin and RG7118 in healthy volunteers. METHODS: The bitopertin study comprised four dose levels (3, 10, 30 and 60 mg) administered once daily for 10 days. In the RG7118 study, placebo, 15 or 30 mg RG7118 was administered once daily for 28 days. CSF samples were taken on day -2 and day 10, and day -1 and day 26 for bitopertin and RG7118, respectively. RESULTS: Twenty-two and 24 subjects participated in the bitopertin and RG7118 study, respectively. In the bitopertin study, CSF glycine concentrations showed a dose-dependent increase from baseline to day 10. The geometric mean ratios (coefficient of variation) of AUC0-12 h on day 10 over baseline were 1.3 (17 %), 1.3 (49 %), 1.7 (18 %) and 2.3 (14 %) after 3, 10, 30 and 60 mg, respectively. In the RG7118 study, the geometric mean ratio of glycine concentration (CV) on day 26 at 6 h post-dose over time-matched baseline was approx. 1.9 (24 and 15 %) for 15 and 30 mg. CONCLUSIONS: The mechanism of action of bitopertin and RG7118, i.e. inhibition of glycine reuptake in the brain, was confirmed. The maximal increase observed in healthy volunteers was similar to the one observed in animals showing the good translatability of this biomarker.