Clinical improvement of the aggressive neurobehavioral phenotype in a patient with a deletion of PITX3 and the absence of L-DOPA in the cerebrospinal fluid.

The development of midbrain dopamine (DA) neurons is regulated by several transcription factors, including Nurr1, Wnt1, Lmx1a/1b, En1, En2, Foxa1, Foxa2, and Pitx3. PITX3 is an upstream co-activator of the TH (tyrosine hydroxylase) promoter. Pitx3(-/-) mice have a selective loss of dopaminergic neurons in the substantia nigra and ventral tegmental area, leading to the significantly reduced DA levels in the nigrostriatal pathway and in the dorsal striatum and manifest anomalous striatum-dependent cognitive impairment and neurobehavioral activity. Treatment with L-DOPA, dopamine, or dopamine receptor agonists in these mice reversed several of their sensorimotor impairments. Heterozygous missense mutations in PITX3 have been reported in patients with autosomal dominant congenital cataract and anterior segment (ocular) mesenchymal dysgenesis (ASMD) whereas homozygous missense mutations have been found in patients with microphthalmia and neurological impairment. Using a clinical oligonucleotide array comparative genomic hybridization (aCGH), we have identified an ∼317 kb hemizygous deletion in 10q24.32, involving PITX3 in a 17-year-old male with a Smith-Magenis syndrome-like phenotype, including mild intellectual impairment, sleep disturbance, hyperactivity, and aggressive and self-destructive behavior. Interestingly, no eye anomalies were found in our patient. Analysis of neurotransmitters in his cerebrospinal fluid revealed an absence of L-DOPA and significantly decreased levels of catecholamine metabolites. Importantly, L-DOPA treatment of our patient has led to mild mitigation of his aggressive behavior and mild improvement of his attention span, extended time periods of concentration, and better sleep.

Pyridoxal phosphate-responsive seizures in a patient with cerebral folate deficiency (CFD) and congenital deafness with labyrinthine aplasia, microtia and microdontia (LAMM).

We present an 8-year-old boy with folate receptor alpha (FRα) defect and congenital deafness with labyrinthine aplasia, microtia and microdontia (LAMM syndrome). Both conditions are exceptionally rare autosomal recessive inherited diseases mapped to 11q13. Our patient was found to have novel homozygous nonsense mutations in the FOLR1 gene (p.R204X), and FGF3 gene (p.C50X). While the FRα defect is a disorder of brain-specific folate transport accompanied with cerebral folate deficiency (CFD) causing progressive neurological symptoms, LAMM syndrome is a solely malformative condition, with normal physical growth and cognitive development. Our patient presented with congenital deafness, hypotonia, dysphygia and ataxia in early childhood. At the age of 6 years he developed intractable epilepsy, and deteriorated clinically with respiratory arrest and severe hypercapnea at the age of 8 years. In contrast to the previously published patients with a FOLR1 gene defect, our patient presented with an abnormal l-dopa metabolism in CSF and high 3-O-methyl-dopa. Upon oral treatment with folinic acid the boy regained consciousness while the epilepsy could be successfully managed only with additional pyridoxal 5'-phosphate (PLP). This report pinpoints the importance of CSF folate investigations in children with unexplained progressive neurological presentations, even if a malformative syndrome is obviously present, and suggests a trial with PLP in folinic acid-unresponsive seizures.

The effect of peripheral enzyme inhibitors on levodopa concentrations in blood and CSF.

Levodopa combined with a dopa-decarboxylase inhibitor, such as carbidopa, shifts the metabolism to the COMT pathway. Adding the peripheral acting COMT inhibitor entacapone provides improvement for patients with PD suffering from motor fluctuations. We studied the effects of the enzyme inhibitors entacapone and carbidopa on the levodopa concentrations in CSF and in blood. Five PD patients with wearing-off underwent lumbar drainage and intravenous microdialysis. Samples were taken 12 h daily for 3 days. Day 1; intravenous levodopa was given, day 2; additional oral entacapone 200 mg tid, day 3; additional oral entacapone 200 mg tid and carbidopa 25 mg bid. Levodopa in CSF and in dialysates was analysed. The AUC for levodopa increased both in blood and CSF when additional entacapone was given alone and in combination with carbidopa. The C(max) of levodopa in both CSF and blood increased significantly. Additional entacapone to levodopa therapy gives an increase of C(max) in CSF and in blood. The increase is more evident when entacapone is combined with carbidopa.

Alterations in the tyrosine and phenylalanine pathways revealed by biochemical profiling in cerebrospinal fluid of Huntington's disease subjects.

Huntington's disease (HD) is a severe neurological disease leading to psychiatric symptoms, motor impairment and cognitive decline. The disease is caused by a CAG expansion in the huntingtin (HTT) gene, but how this translates into the clinical phenotype of HD remains elusive. Using liquid chromatography mass spectrometry, we analyzed the metabolome of cerebrospinal fluid (CSF) from premanifest and manifest HD subjects as well as control subjects. Inter-group differences revealed that the tyrosine metabolism, including tyrosine, thyroxine, L-DOPA and dopamine, was significantly altered in manifest compared with premanifest HD. These metabolites demonstrated moderate to strong associations to measures of disease severity and symptoms. Thyroxine and dopamine also correlated with the five year risk of onset in premanifest HD subjects. The phenylalanine and the purine metabolisms were also significantly altered, but associated less to disease severity. Decreased levels of lumichrome were commonly found in mutated HTT carriers and the levels correlated with the five year risk of disease onset in premanifest carriers. These biochemical findings demonstrates that the CSF metabolome can be used to characterize molecular pathogenesis occurring in HD, which may be essential for future development of novel HD therapies.

The effect of L-dopa infusions with and without phenylalanine challenges in parkinsonian patients: plasma and ventricular CSF L-dopa levels and clinical responses.

We monitored the motor response and plasma and ventricular CSF (CSFv) concentrations of L-dopa during IV infusions of L-dopa in two patients with advanced Parkinson's disease. Concentrations of L-dopa in CSFv mirrored, but lagged behind, those in plasma. In the fasting state, the duration, but not the magnitude, of the motor response was greater with increasing plasma and CSFv levels of L-dopa. During IV infusions of L-dopa following oral administration of phenylalanine, a large neutral amino acid that shares a transport system into the brain with L-dopa, the duration of the motor response was markedly attenuated despite undiminished CSFv levels of L-dopa. These observations suggest that either L-dopa entry into CSFv and the brain are differentially affected by phenylalanine or that phenylalanine affects other steps in the motor response. These observations demonstrate that, except in the fasting state, L-dopa in CSFv is not a reliable predictor of motor response.

Dopamine sulfate in ventricular cerebrospinal fluid and motor function in Parkinson's disease.

We measured sequential plasma and CSF levodopa and CSF dopamine sulfate levels following a single dose of levodopa/carbidopa in two patients with advanced Parkinson's disease (PD). We obtained CSF from an Ommaya reservoir implanted in the lateral ventricle several months earlier at the time of transplantation of adrenal medulla to caudate nucleus and could detect dopamine only in its sulfconjugated form. Peak CSF levodopa and dopamine sulfate levels occurred 1 to 1.5 hours after peak plasma concentration of levodopa. The time course of clinical improvement and worsening correlated precisely with the appearance and disappearance of both levodopa and dopamine sulfate in the CSF. The precise correlation between CSF dopamine sulfate and levodopa indicates that in patients with advanced PD the brain retains some capacity to convert levodopa to dopamine. The transient nature of the correlation between motor fluctuations, CSF levodopa, and CSF dopamine sulfate is consistent with suggestions that in patients with advanced PD there is a diminished capacity to store dopamine synthesized from exogenous levodopa. Dopamine sulfate appears to be a useful index of the availability of dopamine in the parkinsonian brain.

Dopamine/L-dopa ratio in cerebrospinal fluid of parkinsonian patients treated with L-dopa + benserazide.

The CSF dopamine/L-dopa ratio was examined in 14 parkinsonian and six control patients. In parkinsonian patients the dopamine/L-dopa ratio showed significant correlation with the severity of the disease, as characterized by Yahr's scale. These data suggest some correlation between the dopamine synthesizing capacity and the degree of disability.

Effects of the catechol-O-methyltransferase inhibitor tolcapone in Parkinson's disease: correlations between concentrations of dopaminergic substances in the plasma and cerebrospinal fluid and clinical improvement.

We compared the concentrations of dopaminergic substances in the plasma and cerebrospinal fluid (CSF) with clinical severity in patients with Parkinson's disease (PD) under L-dopa/carbidopa treatment and under L-dopa/carbidopa+tolcapone treatment. Compared with treatment with L-dopa/carbidopa alone, the co-administration of tolcapone produced a significant decrease in clinical severity; a remarkable reduction in the 3-O-methyldopa (3-OMD) concentration and significant increase in the L-dopa concentration both in the plasma and CSF; and a significant increase in the dopamine concentration in the CSF. The clinical effects of tolcapone were closely correlated with the reduction in the 3-OMD concentration, but not with the increase in the dopamine and L-dopa concentrations in the CSF.

Development of poly(butylene succinate) microspheres for delivery of levodopa in the treatment of Parkinson's disease.

Parkinson's is a major neurodegenerative disorder that occurs due to loss of dopaminergic neurons in basal ganglia. Conventional therapy includes surgery that involves lot of risk and administration of levodopa which is accompanied by poor bioavailability, short half-life, and side effects. In the present study, poly(butylene succinate) (PBSu) microspheres-based drug delivery system to improve the bioavailability of the drug levodopa was evaluated for the first time. Biodegradable porous and smooth PBSu microspheres were prepared by double emulsion solvent evaporation technique (W/O/W) and the effect of solvent and surfactant was studied. The maximum encapsulation efficiency achieved was 53.93% and 62.28% for porous and smooth microspheres, respectively. In vitro drug release was studied in phosphate buffered saline and simulated CSF buffer of pH 7.4. Initially a burst effect followed by sustained release of drug was obtained for about 32 h and 159 h for porous and smooth microspheres, respectively. The release rate was higher in simulated CSF when compared with PBS, due to higher concentration of sodium ions and cations in simulated CSF.

L-DOPA pharmacokinetics in the MPTP-lesioned macaque model of Parkinson's disease.

OBJECTIVE: The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned non-human primate is widely used to model Parkinson's disease (PD) and to evaluate the efficacy of new therapies. However, some doubts have been raised about the translatability of findings in the MPTP-lesioned monkey, because the doses of L-3,4-dihydroxyphenylalanine (L-DOPA) required to alleviate parkinsonism and elicit dyskinesia are high, on a mg/kg basis, when compared to clinical practice. Thus, in the MPTP-lesioned macaque, doses ranging from 20 to 40 mg/kg might be used, while in the clinic single L-DOPA administrations ranging from 100 to 200 mg are more typical. However, bioavailability of drugs varies between species and it is unknown how plasma L-DOPA levels providing therapeutic benefit in the non-human primate compare to those having similar actions in PD patients. METHODS: We administered acute challenges of L-DOPA 30 mg/kg orally to MPTP-lesioned macaques with established dyskinesia, and determined plasma, brain and cerebrospinal fluid (CSF) levels of L-DOPA using high-performance liquid chromatography-mass spectrometry/mass spectrometry. RESULTS: The maximal plasma concentration of L-DOPA (C(max)) was 18.2 ± 3.8 nmol/ml and was achieved 1.6 ± 0.3 h after administration (t(max)). Half-life was 58.8 ± 22.7 min. L-DOPA levels in the caudate nucleus at peak behavioural effect were 3.3 ± 0.7 µg/g tissue protein while they were 1.5 ± 0.1 nmol/ml in the CSF. CONCLUSIONS: Although therapeutically-active doses of L-DOPA administered to the MPTP-lesioned macaque are higher on a mg/kg basis than those administered in clinical settings, they lead to L-DOPA C(max) similar to those achieved with 200 mg L-DOPA in clinic. L-DOPA t(max) and half-life are also similar to those reported in human.

Use of transduced adipose tissue stromal cells as biologic minipumps to deliver levodopa for the treatment of neuropathic pain: possibilities and limitations.

Subarachnoidal grafting of monoamine-producing cells has been used with success to treat chronic pain in animal models. In the search for a source of autologous transplantable cells, capable of delivering neuroactive substances to the cerebrospinal fluid (CSF) to treat pain, we have tested adipose tissue-derived stromal cells (ADSCs) transduced to produce levodopa. Intrathecally grafted ADSCs survive for long term adhered to spinal cord and nerve root meninges. Cultured ADSCs were retrovirally transduced with tyrosine hydroxylase (TH) and/or GTP cyclohydroxylase 1 (GCH1) genes and stably expressed them for at least 6 weeks in culture. Singly transduced cultures did not produce measurable levodopa but doubly transduced or a mixture of singly transduced ADSCs were able to efficiently synthesize and release levodopa. When 0.5-1 x 10(6) TH- and GCH1-expressing ADSCs were intrathecally grafted in rats, elevated levels of levodopa and dopamine metabolites were found in CSF at 3 days, although at lower concentrations than expected. Unexpectedly, no levodopa was measurable in CSF at 6 days. In a rat model of neuropathic pain, intrathecal grafting of doubly transduced cells did not produce antiallodynic effects at 2 or 6 days, even when histological analysis revealed the presence of weak TH-immunoreactive subarachnoidal cell clusters. These results suggested that doubly transduced cells could indeed function as biological minipumps to enhance the dopaminergic neurotransmission at the spinal cord level but transgenes were rapidly silenced after intrathecal grafting. Transgene silencing was mimicked in culture by serum deprivation for 3 days. Serum addition at this point recovered transgene expression in just 6 h, as did, to a smaller degree, dbcAMP or histone deacetylase inhibitors. Transgene expression silencing in serum deprivation conditions was prevented by 5'-terminal IRES sequences. The present study does not discard the use of transduced cells as a strategy to treat chronic pain but shows that controlling transgene silencing in implanted cells needs to be achieved first.

Development of electrochemical biosensor based on tyrosinase immobilized in composite biopolymeric film.

An electrochemical enzyme electrode for dopa and dopamine was developed via an easy and effective immobilization method. The enzyme tyrosinase was extracted from a plant source Amorphophallus companulatus and immobilized in a novel composite of two biopolymers: agarose and guar gum. This composite matrix-containing enzyme forms a self-adhering layer on the active surface of glassy carbon electrode, making it a selective and sensitive phenol sensor. Dopa and dopamine were determined by the direct reduction of biocatalytically liberated quinone species at -0.18V versus Ag/AgCl (3M KCl). The analytical characteristics of this sensor, including linear range, lower detection limit, pH, and storage stability, are described. It has reusability up to 15 cycles and a shelf life of more than 2 months.

L-dopa pharmacokinetics in plasma and cisternal and lumbar cerebrospinal fluid of monkeys.

The pharmacokinetics of levodopa (L-dopa) in plasma and in cisternal and lumbar cerebrospinal fluid (CSF) were studied in Rhesus monkeys that were given 2- to 3-hour intravenous infusions of L-dopa. Steady-state L-dopa concentrations in cisternal CSF correlated well with plasma levels, and yielded a CSF:plasma ratio of 0.17. The disappearance of L-dopa from plasma and cisternal CSF compartments fits an open, two-compartment pharmacokinetic model. Although slower, the distribution and elimination half-lives for L-dopa from cisternal CSF (8.9 and 49.2 minutes, respectively) were of a similar magnitude to those from plasma (4.9 and 33.2 minutes, respectively). If cisternal CSF reflects brain extracellular fluid, then plasma pharmacokinetics of L-dopa are a reasonable approximation of those in the brain. In contrast to cisternal CSF, the disappearance of L-dopa from lumbar CSF fits an open, one-compartment model with an elimination half-life of 100 minutes. This indicates that the lumbar CSF compartment is unsuitable for investigation of the pharmacokinetics of L-dopa in the brain.

Temporal relationships between plasma and cerebrospinal fluid pharmacokinetics of levodopa and clinical effect in Parkinson's disease.

We studied the pharmacokinetics of levodopa and its metabolites in plasma and ventricular cerebrospinal fluid in 4 parkinsonian patients with indwelling Ommaya reservoirs placed at the time of previous adrenal-medullary to caudate nucleus transplantation. Cerebrospinal fluid levodopa levels were 11.9% of those in plasma. Motor performance and dyskinesia correlated more closely with the time course of the appearance of levodopa in the ventricular cerebrospinal fluid than with the plasma levodopa concentration and did not correlate with plasma 3-O-methyldopa or cerebrospinal fluid 3-O-methyldopa or homovanillic acid. Our data confirm that the wearing off of the levodopa effect in patients with advanced Parkinson's disease is a function of drug concentration in the central nervous system.

Levodopa and 3-O-methyldopa in cerebrospinal fluid after levodopa-carbidopa association.

Since motor fluctuations in Parkinsonian patients might be, at least in part, explained by an antagonism between levodopa (LD) and its metabolite 3-O-methyldopa (3-OMD) at blood-brain-barrier (BBB), we decided to study LD and 3-OMD plasma and cerebrospinal fluid (CSF) levels in subjects undergoing lumbar puncture for diagnostic purposes. After informed consent, 70 subjects took a tablet of carbidopa-levodopa association (Sinemet or Sinemet-CR) 0.5, 1, 2, 4, 8, 12 h before blood and lumbar cerebrospinal fluid collection. LD and 3-OMD were determined by an HPLC-electrochemical method. The subjects treated with Sinemet-CR had lower LD cerebrospinal fluid concentrations along with lower LD and higher 3-OMD plasma concentrations. This pattern of LD cerebrospinal fluid concentrations may be explained by means of a transport competition between LD and 3-OMD at blood brain barrier level.

Assessment of pointer dog behavior. Drug effects and neurochemical correlates.

Studies of behavioral traits of the "Arkansas Line of nervous pointer dogs" are well documented (Murphree et al. 1969). Demonstrations of drug effects on these traits, as measured on the standardized behavior test and expressed as "Morbidity Score," have been disappointing, despite other evidence of effectiveness of drug treatments such as modification of operant performance (Murphree et al. 1974c). A simple reproducible test with high interrater reliability is presented here. Subjects were nine nervous dogs that received placebo, pimozide, and chlordiazepoxide in a double-blind crossover design. Both drugs significantly attenuated "human avoidance," a nervous trait. Placebo had no effect. The chlordiazepoxide effect was maximal on the first day of administration, returning to baseline three to four days after the last dose. The pimozide maximal effect was on day 4, with return to baseline six to nine days after the last dose. In addition to the aberrant behavior of the nervous pointer, hyperresponsiveness to dopaminergic stimulation and increased levels of L-DOPA in the cisternal spinal fluid were observed.

Aromatic L-amino acid decarboxylase deficiency: diagnostic methodology.

Aromatic L-amino acid decarboxylase (EC. 4.1.1.28) deficiency is a newly described inborn error of metabolism that affects serotonin and dopamine biosynthesis. The major biochemical markers for this disease are increases of L-dopa, 3-methoxytyrosine, and 5-hydroxytryptophan in urine, plasma, and cerebrospinal fluid together with decreased cerebrospinal fluid concentrations of homovanillic acid and 5-hydroxyindoleacetic acid. In addition, concentrations of vanillactic acid are increased in the urine. Specific HPLC and gas chromatography-mass spectrometry methods are described that permit the identification and measurement of these metabolites in the above body fluids. Simplified assays for human plasma L-dopa decarboxylase and liver L-dopa and 5-hydroxytryptophan decarboxylase, used to demonstrate the enzyme deficiency, are also reported.