Phase I randomized clinical trial of N-acetylcysteine in combination with an adjuvant probenecid for treatment of severe traumatic brain injury in children.

BACKGROUND: There are no therapies shown to improve outcome after severe traumatic brain injury (TBI) in humans, a leading cause of morbidity and mortality. We sought to verify brain exposure of the systemically administered antioxidant N-acetylcysteine (NAC) and the synergistic adjuvant probenecid, and identify adverse effects of this drug combination after severe TBI in children. METHODS: IRB-approved, randomized, double-blind, placebo controlled Phase I study in children 2 to 18 years-of-age admitted to a Pediatric Intensive Care Unit after severe TBI (Glasgow Coma Scale [GCS] score ≤8) requiring an externalized ventricular drain for measurement of intracranial pressure (ICP). Patients were recruited from November 2011-August 2013. Fourteen patients (n = 7/group) were randomly assigned after obtaining informed consent to receive probenecid (25 mg/kg load, then 10 mg/kg/dose q6h×11 doses) and NAC (140 mg/kg load, then 70 mg/kg/dose q4h×17 doses), or placebos via naso/orogastric tube. Serum and CSF samples were drawn pre-bolus and 1-96 h after randomization and drug concentrations were measured via UPLC-MS/MS. Glasgow Outcome Scale (GOS) score was assessed at 3 months. RESULTS: There were no adverse events attributable to drug treatment. One patient in the placebo group was withdrawn due to adverse effects. In the treatment group, NAC concentrations ranged from 16,977.3±2,212.3 to 16,786.1±3,285.3 in serum and from 269.3±113.0 to 467.9±262.7 ng/mL in CSF, at 24 to 72 h post-bolus, respectively; and probenecid concentrations ranged from 75.4.3±10.0 to 52.9±25.8 in serum and 5.4±1.0 to 4.6±2.1 µg/mL in CSF, at 24 to 72 h post-bolus, respectively (mean±SEM). Temperature, mean arterial pressure, ICP, use of ICP-directed therapies, surveillance serum brain injury biomarkers, and GOS at 3 months were not different between groups. CONCLUSIONS: Treatment resulted in detectable concentrations of NAC and probenecid in CSF and was not associated with undesirable effects after TBI in children. TRIAL REGISTRATION: ClinicalTrials.gov NCT01322009.

CSF monoamine metabolites in child and adult psychiatric patients. A developmental perspective.

Concentrations of acid metabolites of dopamine and serotonin were measured in lumbar CSF of a diagnostically heterogeneous group of 154 psychiatric patients following oral probenecid loading. The patients ranged in age from 2 to 67 years old. No patients had received psychoactive medications for at least two weeks prior to the lumbar puncture. Children had higher mean CSF homovenillic acid (HVA) levels, higher mean CSF HVA-probenecid ratios, higher CSF HVA-log probenecid ratios, and lower mean CSF probenecid levels than adults. Age was negatively correlated with CSF HVA level and with CSF HVA-probenecid ratio. These correlations were more pronounced in male patients than in female patients.

Probenecid-induced norepinephrine elevations in plasma and CSF.

Probenecid administered in divided oral doses totaling 100 mg/kg increased levels of norepinephrine (NE) in plasma and cerebrospinal fluid (CSF). This technique is commonly used to measure the rate of accumulation of acidic metabolites of certain brain neurotransmitter biogenic amines in CSF after blockade of their transport into blood. Since levels of 3-methoxy-4-hydroxy-phenylethyleneglycol, a neutral metabolite of NE, are also elevated after high oral doses of probenecid, the increases of CSF and plasma NE levels may be directly related to probenecid-induced release of this amine from noradrenergic neurons. In patients who experienced nausea or vomiting there were lower levels of probenecid in CSF, probably secondary to diminished absorption of the medication. These patients also had lower levels of NE in plasma than did patients who remained asymptomatic.

Dopamine and serotonin metabolism in neuropsychiatrically disturbed children. CSF homovanillic acid and 5-hydroxyindoleacetic acid.

Lumbar cerebrospinal fluid homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and probenecid were measured in four subgroups of neuropsychiatrically disturbed children and a contrast group of pediatric patients. With the exception of a serotonin metabolite difference between autistic and nonautistic psychotic children, there were no significant differences in metabolite concentrations among autistic, nonautistic psychotic, aphasic, and cognitively and attentionally impaired groups, or between the developmentally disabled and contrast groups of children. Younger children had higher concentrations of HVA than older children. Girls had significantly lower HVA/probenecid ratios than boys, which did not appear to be related to underlying neuropsychiatric disorder. Significant probenecid-metabolite correlations indicate the importance of measuring probenecid in the cerebrospinal fluid in clinical studies.

Clinical application of the probenecid test for measurement of monoamine turnover in the CNS.

The probenecid-induced accumulation of the acidic metabolites of dopamine and serotonin is widely used to estimate the CNS turnover of these amines in human subjects. The theoretical basis of the probenecid test is discussed, including the assumptions on which the test is based, and the limitations of the procedure. Suggestions are offered on appropriate methods for analyzing test data. Available evidence suggests that maximal inhibition of the efflux of the acidic metabolites of dopamine and serotonin from the CSF is not achieved with probenecid doses up to 100 mg/kg. Therefore, before a comparison can be made between the accumulation of CSF metabolites from different groups of subjects, a correction for CSF probenecid concentrations is necessary. Moreover, in addition to the measurement of dopamine and serotonin turnover, the probenecid test can be extended to include comparisons of CNS turnover of other monoamines including octopamine, p-tyramine, and tryptamine.

Cerebrospinal fluid probenecid studies: a reinterpretation.

Probenecid is used to block the transport of acid monoamine metabolites from cerebrospinal fluid (CSF), on the assumption that the resultant rise in CSF concentrations of the metabolites will reflect presynaptic "turnover" of the parent monoamine. However, CSF levels of probenecid correlate with CSF levels of the metabolite, suggesting that the blockade is incomplete at the probenecid levels obtained in human studies. This article reviews the literature on CSF probenecid-metabolite correlations and presents data demonstrating variations in the correlations across diagnostic groups. These cross-diagnostic variations may be due to group differences in membrane transport characteristics and and confound attempts to "correct for" CSF probenecid concentrations in studies of monoamine turnover.

Thioridazine: central dopamine turnover and clinical effects of antipsychotic drugs.

Thioridazine was administered to 14 patients diagnosed as within the "schizophrenic spectrum" with the result that substantial improvement in psychotic symptoms was achieved while significant extrapyramidal side effects occurred in only 1 patients. Under these conditions lumbar cerebrospinal fluid homovanillic acid following probenecid was significantly elevated (48%). Cerebrospinal fluid probenecid and 5-hydroxyindoleacetic acid declined significantly during treatment with thioridazine. Taken in conjunction with the results of other studies, it appears that cerebrospinal fluid homovanillic acid can be moderately elevated in man during successful treatment with antipsychotic drugs when extrapyramidal side effects are virtually absent. Possible interpretations of these results are discussed with regard to the clinical effects of antipsychotic drugs as they relate to central dopaminergic activity.

CSF monoamine metabolites in depression and schizophrenia.

The authors report cerebrospinal fluid (CSF) concentrations of five monoamine metabolites before and after probenecid administration in normal subjects and patients with depression and schizophrenia. No differences were found in baseline metabolite concentrations among the three groups. CSF metabolite and CSF probenecid concentrations were significantly correlated in depressed patients for all metabolites, and there was decreased postprobenecid accumulation of homovanillic acid in the CSF of depressed patients compared with schizophrenic patients and with normal subjects. The authors propose a method for correcting for probenecid concentrations. Data from normal subjects should be of value for other investigators using the probenecid technique.

Relationship between estimated premorbid adjustment and CSF homovanillic acid and 5-hydroxyindoleacetic acid levels.

The authors completed ratings of premorbid sexual and social adjustment and assays of the CSF homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and probenecid concentrations in 108 psychiatric patients. Among the 30 patients diagnosed as schizophrenic, poor premorbid sexual adjustment was associated with higher accumulations of HVA in the CSF. No relationship between the estimated premorbid social adjustment and CSF HVA levels was observed. These findings suggest that 1) sexual and social premorbid adjustment are partially independent variables, and 2) one of the biological correlates of sexual adjustment in schizophrenic patients may be the functional maturity of central dopamine systems.

Neuroendocrine and neurochemical measurements in depression.

The authors performed dexamethasone suppression tests (DST), TRH infusions, 72-hour urine collections, and lumbar punctures on a group of male depressed patients. Approximately 60% of the patients were DST positive and 33% had a blunted TSH response. Two biologic variables, the 8 a.m. postdexamethasone cortisol and the postprobenecid CSF 5-hydroxyindoleacetic acid (5-HIAA), accounted for over half of the variance in the behavioral measure, the Hamilton score. Plasma cortisol elevation was associated with high 3-methoxy-4-hydroxyphenyl glycol (MHPG) excretion; TSH blunting was associated with low urinary MHPG excretion. Comprehensive biologic measures showed certain significant interrelationships and correlations with the severity of depression.

Homovanillic acid and 5-hydroxyindoleacetic acid cerebrospinal fluid levels. A study with and without probenecid administration of their relationship to the state of consciousness after head injury.

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were measured in the lumbar cerebrospinal fluid of 98 patients after closed head injury. The HVA levels decreased in patients, whether or not they were given the drug probenecid, which inhibits the active transport of these acids from the brain. The decline of HVA was more notable in patients with the longest duration of unconsciousness. The HVA levels showed no correlation with the state of consciousness at the moment of the lumbar puncture. The 5-HIAA levels were below normal in the conscious patients, but paradoxically, at about normal levels in unconscious patients. The overall results suggest a decreased cerebral dopamine and serotonin metabolism after head injury.

Neurochemical analysis of cerebrospinal fluid.

The use of cerebrospinal fluid (CSF) analysis for studying in vivo alterations in central neuronal activity requires a relatively sophisticated knowledge of CSF physiology and pathology. Ventriculospinal concentration gradients, circadian rhythms, physical activity, stress, medications, precursor intake, concomitant illness, obstructed CSF circulation, age, and sex alter the baseline neurochemical composition of CSF. Differential probenecid blockade of the egress of acidic monoamine metabolites and cyclic nucleotides from the CSF may complicate interpretations of their accumulations. Degradation of CSF constituents during collection, storage, and analysis may introduce errors in quantification. These sources of CSF variability can be minimized with proper methodolology.

Intravenous probenecid loading. Effects on plasma and cerebrospinal fluid probenecid levels and on monoamine metabolites in cerebrospinal fluid.

Probenecid blocks the active transport from cerebrospinal fluid to blood of homovanillic acid and 5-hydroxyindoleacetic acid, thus increasing cerebrospinal fluid levels of these products of central monoamine metabolism. The half-life in plasma of probenecid given as a single intravenous infusion (40 mg per kilogram of body weight) to patients with either Huntington's chorea or Parkinson's disease averaged about 6.6 hours. In cerebrospinal fluid, peak values for homovanillic acid and 5-hydroxyindoleactic acid occurred in samples collected 8 hours after the 1-hour probenecid infusion was started. Even after 4 hours, however, levels of both monoamine metabolites were significantly increased. There was a positive correlation between cerebrospinal fluid levels of probenecid and the increase in 5-hydroxyindoleacetic acid but not homovanillic acid. Compared with the oral administration of probenecid, the intravenous infusion technique produced more consistent elevations in plasma and cerebrospinal fluid probenecid levels, greater increases in cerebrospinal fluid homovanillic acid values, and fewer gastrointestinal side effects.

Therapy of intention myoclonus with L-5-hydroxytryptophan and a peripheral decarboxylase inhibitor, MK 486.

Three patients with postanoxic intention myoclonus, two patients with intention tremor, and one patient with cerebral palsy were administered L-5-hydroxytryptophan (L-5HTP), the precursor of serotonin, in combination with MK 486, a peripheral amino acid decarboxylase inhibitor. L-5HTP combined with MK 486 were potent long-term therapeutic agents for postanoxic intention myoclonus, but had no effect on intention tremor or cerebral palsy. These drugs were well-tolerated by the patients, and more effective than any other known therapy for intention myoclonus. Cerebrospinal fluid concentration of 5-hydroxyindoleacetic acid, the main catabolite of serotonin, appeared low in two patients with intention myoclonus and increased markedly during drug therapy. Postanoxic intention myoclonus may be causally related to a deficiency of brain serotonin.

Reduced cerebrospinal fluid 5-hydroxyindoleacetic acid and homovanillic acid in children with epilepsy.

Cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and probenecid were examined in 14 children with epilepsy (ages 6 months to 17 years) and 17 controls (ages 14 months to 16 years). The concentrations of amine metabolites were significantly correlated with probenecid concentrations in both groups of children. Cerebrospinal fluid concentrations of 5-HIAA were 63.6 ng per milliliter plus or minus 8.23 S.E.M. and 117 ng per milliliter plus or minus 11.6 S.E.M. for the epilepsy and control groups respectively. HVA averaged 89.1 ng per milliliter plus or minus 15.2 S.E.M in epilepsy and 172 ng per milliliter plus or minus 19.2 S.E.M. in the control group. These findings indicate a significant difference between epilepsy and control groups. Probenecid concentrations were similar in each group. The reduced cerebrospinal fluid amine metabolite concentrations in children with epilepsy were not related to age, anticonvulsant medication, cerebrospinal fluid folate or protein concentration, or cerebrospinal fluid cell count. Our findings suggest a relationship between brain amines and epilepsy.

Determinations of 5-hydroxyindoleacetic acid and homovanillic acid in human CSF with monitoring of probenecid levels in CSF and plasma.

The accumulation of 5-HIAA and HVA in cerebrospinal fluid (CSF) was studied in eight healthy volunteers after oral administration of probenecid. Simulation indicated that a dose of 4.5 g probenecid should be used to achieve probenecid plasma concentrations between 200 and 400 micrograms/ml. Almost complete inhibition of the active transport of the acidic metabolites was assumed to be obtained at these concentrations. Probenecid 4.5 g was administered in two doses (2.5 g and 2 g), separated by 4 h. Plasma samples were drawn at varying intervals over a period of 46 h and lumbar puncture (LP) was performed at either 14 h or 20 h after the first administration of probenecid. The concentration of probenecid, 5-HIAA and HVA in CSF was estimated and the probenecid-induced accumulation of 5-HIAA and HVA was compared with their baseline values. There were no statistically significant differences (P greater than 0.05) in the accumulation of the monoamine metabolites between the two LP (14 h and 20 h), neither were there any differences in CSF concentrations of probenecid at the time of LP. There were only small differences in probenecid plasma concentrations, although statistically significant. Due to maximum blockade of the active transport system no correlation was observed between the CSF concentration of probenecid and the induced accumulation of 5-HIAA and HVA, respectively. The range of probenecid-induced accumulation for 5-HIAA and HVA in these volunteers was 156-429% and 183-600%, respectively. The suggested monitoring of probenecid plasma levels is proposed as a suitable model to investigate central neuronal activity of dopamine and serotonin in the central nervous system.

Alterations in cerebrospinal fluid dopamine metabolites following physostigmine infusion.

The effect of IV physostigmine administration on the cerebrospinal fluid (CSF) levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in normal subjects was determined. After an adjustment for differing CSF concentrations of probenecid, physostigmine was found to elevate CSF HVA and DOPAC concentrations. The authors discuss these changes in CSF HVA and DOPAC and their possible relationship to the ability of physostigmine to decrease the symptoms of some patients with tardive dyskinesia.

Probenecid in CSF and plasma of rabbits and dogs measured by radioimmunoassay.

Probenecid (P) in CSF of rabbits and dogs was investigated using a new radioimmunoassay technique. In rabbits, under steady-state conditions, CSF/free plasma concentration ratios of P were found to be similar to those reported in man. The ratios were concentration dependent and less than 0.5 suggesting an inhibition of transport of the drug in CNS. Under nonsteady-state conditions, no clear evidence of a transport system was found in dogs.

Cerebrospinal fluid acetylcholinesterase in neuropsychiatric disorders.

Acetylcholinesterase (AChE) was measured in the cerebrospinal fluid (CSF) of patients with a diagnosis of Huntington's disease, depression, schizophrenia, or mania and also in the CSF of normal subjects. No significant differences in CSF AChE were found between any diagnostic group and normal subjects. Furthermore, the administration of choline chloride, physostigmine, or probenecid did not significantly alter CSF AChE. No diurnal variation in CSF AChE activity was apparent. These findings, combined with the unclear relationship of brain AChE to CSF AChE, suggest that this measurement does not reflect the relative cholinergic underactivity presumed to exist in some neuropsychiatric conditions.

The effect of L-tryptophan administration on the concentration of probenecid in plasma and cerebrospinal fluid in patients.

The administration of large doses of probenecid has been used to study the central nervous system metabolism of catecholamines and indoleamines in patients with affective disease. It has been reported that alterations of the binding of L-tryptophan to plasma albumin binding sites occur during probenecid administration. The present study sought to determine if the administration of large doses of L-tryptophan affected probenecid concentrations in cerebrospinal fluid and/or plasma. The data indicate that during L-tryptophan treatment, plasma probenecid concentrations are reduced but that no significant alterations in cerebrospinal fluid probenecid concentrations occur. This would suggest that the kinetics of the probenecid blockade of transport of acidic biogenic amine metabolites out of cerebrospinal fluid are not altered by L-tryptophan loading.