Pharmacokinetics of CuGTSM, a Novel Drug Candidate, in a Mouse Model of Menkes Disease.

PURPOSE: Menkes disease is a rare hereditary disease in which systemic deficiency of copper due to mutation of the ATP7A gene causes severe neurodegenerative disorders. The present parenteral drugs have limited efficacy, so there is a need for an efficacious drug that can be administered orally. This study focused on glyoxal-bis (N(4)-methylthiosemicarbazonato)-copper(II (CuGTSM), which has shown efficacy in macular mice, a murine model of Menkes disease, and examined its pharmacokinetics. In addition, nanosized CuGTSM (nCuGTSM) was prepared, and the effects of nanosizing on CuGTSM pharmacokinetics were investigated. METHODS: CuGTSM or nCuGTSM (10 mg/kg) was administered orally to male macular mice or C3H/HeNCrl mice (control), and plasma was obtained by serial blood sampling. Plasma concentrations of CuGTSM and GTSM were measured by LC-MS/MS and pharmacokinetic parameters were calculated. RESULTS: When CuGTSM was administered orally, CuGTSM and GTSM were both detected in the plasma of both mouse strains. When nCuGTSM was administered, the Cmax was markedly higher, and the mean residence time was longer than when CuGTSM was administered for both CuGTSM and GTSM in both mouse strains. With macular mice, the AUC ratio (GTSM/CuGTSM) was markedly higher and the plasma CuGTSM concentration was lower than with C3H/HeNCrl mice when either CuGTSM or nCuGTSM was administered. CONCLUSION: Absorption of orally administered CuGTSM was confirmed in macular mice, and the nano-formulation improved the absorption and retention of CuGTSM in the body. However, the plasma concentration of CuGTSM was lower in macular mice than in control mice, suggesting easier dissociation of CuGTSM.

Menkes disease: Oral administration of glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) rescues the macular mouse.

BACKGROUND: Menkes disease is a copper metabolism disorder caused by mutations in ATP7A, a copper-transporting P-type ATPase. In this study, oral copper supplementation via glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) (CuGTSM), a lipophilic copper complex, was investigated in male hemizygous macular (MoMl/y) mice, a mouse model of Menkes disease. METHODS: CuGTSM was administered by oral gavage on postnatal days 5, 8, 11, 17, 23, and 32. The copper levels in the organs and serum, copper-dependent enzyme activities in the brain, and ceruloplasmin (Cp) activity in the serum were measured at 15 days and 3 and 8 months of age. Histological analysis of the intestines and the rotarod test were also performed. RESULTS: CuGTSM treatment extended the lifespan of MoMl/y mice and partly restored the copper concentrations and cytochrome oxidase and DBH activities in the brain; however, the rotarod test showed impaired motor performance. The treatment also increased copper concentrations and Cp activity in the serum. In suckling MoMl/y mice, CuGTSM treatment transiently induced diarrhea accompanied by copper accumulation and altered villus morphology in the ileum. CONCLUSION: Oral administration of CuGTSM extended the lifespan of MoMl/y mice. Oral administration is attractive, but pharmaceutical studies are needed to reduce the adverse enteral effects.

Reduction in glutamine/glutamate levels in the cerebral cortex after adrenocorticotropic hormone therapy in patients with west syndrome.

West syndrome (WS), an intractable epileptic encephalopathy of infancy, is refractory to many antiepileptic drugs; however, adrenocorticotropic hormone (ACTH) is an effective treatment for WS. The mechanism behind the efficacy of ACTH is mediated by biochemical processes that remain unknown. We examined the effects of ACTH therapy with tetracosactide (TCS), a synthetic ACTH analogue, on brain metabolism in patients with WS, using (1)H magnetic resonance spectroscopy (¹H-MRS). In six patients with cryptogenic WS, we performed single-voxel ¹H-MRS at the occipital lobe cortex. Measurements were taken prior to TCS treatment, a few days after therapy, and several months after therapy. Data were also compared with subjects having only mild psychomotor delays. The metabolites measured were glutamine plus glutamate (Glx), N-acetylaspartate (NAA), choline (Cho), and myoinositol (mI); each was expressed as a ratio with creatine plus phosphocreatine (total creatine: tCr). The Glx/tCr ratio was significantly reduced after the TCS treatment. The NAA/tCr ratio was also significantly reduced after the treatment compared with the control group, although the change in NAA signal was heterogeneous among patients, correlating with respective outcomes. The Cho/tCr and mI/tCr ratios were not affected by TCS treatment. The reduction in Glx suggests a decrease in the glutamate-glutamine cycle, which plays a pivotal role in synthesizing neurotransmitters such as glutamate and GABA. TCS-induced Glx reduction may induce changes in synaptic signal transduction, thereby accounting for the effect of TCS on WS. The change in NAA indicates altered neuronal activity, which may be correlated with outcome in WS patients.

Copper-trafficking efficacy of copper-pyruvaldehyde bis(N4- methylthiosemicarbazone) on the macular mouse, an animal model of Menkes disease.

BACKGROUND: Menkes disease (MD) is a disorder of copper transport caused by ATP7A mutations. Although parenteral copper supplements are partly effective in treating MD, the copper level in the brain remains insufficient, whereas copper accumulates in the kidney. We investigated the copper-trafficking efficacy of copper-pyruvaldehyde bis(N4-methylthiosemicarbazone) (Cu-PTSM), a lipophilic copper complex, in macular mice, an animal model of MD. METHODS: Macular mice were treated with cupric chloride (CuCl2) or Cu-PTSM on postnatal days 4, 10, and 17. At 4 wk of age, the copper levels in major organs and cytochrome oxidase (CO) activity in brain tissue were measured. Hematology, blood biochemistry, and urinary ß2-microglobulin (ß2-M) secretion were also assessed. RESULTS: The copper levels in the brains of the Cu-PTSM-treated group remained low, but CO activity in the cerebral and cerebellar cortices in the Cu-PTSM-treated group were higher than those in the CuCl2-treated group. There were no significant differences in hematological or biochemical findings or in urinary ß2-M secretion among the groups. CONCLUSION: Although the copper-trafficking efficacy of Cu-PTSM was limited, the improved CO activity in the brain suggests that Cu-PTSM delivered copper more effectively to neuronal CO than did CuCl2. Reduced renal copper accumulation may be beneficial in prolonged copper supplementation.

A patient with a 6q22.1 deletion and a phenotype of non-progressive early-onset generalized epilepsy with tremor.

We report a patient with a 6q22.1 deletion, who presented with a rare syndrome of generalized epilepsy, myoclonic tremor, and intellectual disability. There was no clinical progression after follow-up for more than 10 years. Our report presents the genetic basis for a phenotype involving a non-progressive generalized epilepsy with tremor. The efficacy of valproic acid for seizure control and the partial efficacy of deep brain stimulation with propranolol for myoclonic tremor is detailed.

The nutrient formula containing eicosapentaenoic acid and docosahexaenoic acid benefits the fatty acid status of patients receiving long-term enteral nutrition.

Currently, various formulas with different fatty acid compositions are used for enteral nutrition (EN). All formulas contain various concentrations of essential fatty acids: linoleic acid (LA) and alpha-linolenic acid (ALA); LA is biotransformed into arachidonic acid (AA) and ALA into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in vivo. Some formulas contain preformed EPA and DHA. However, the effects of the differences in the fatty acid composition on the fatty acid status of patients receiving long-term EN is not clear. We measured serum fatty acid concentrations in 50 patients with neurological diseases receiving long-term EN. The data were then compared retrospectively with reference to the fatty acid compositions of the formulas used. All of the patients received almost their entire nutritional intake via EN for at least 1 year. Blood samples were obtained just before injecting the EN solution. Among the formulas that did not include EPA or DHA, formulas with low ALA concentrations were associated with low serum EPA and DHA. Conversely, the ALA-enriched formulas with reduced LA concentrations significantly increased EPA and DHA levels, although the levels remained lower than the control values. With the formula containing EPA and DHA, the EPA and DHA levels reached control values. Therefore, the fatty acid composition of the EN formulas affected the fatty acid status of patients receiving long-term EN. Formulas containing preformed EPA and DHA with suitable amounts of essential fatty acids may benefit these patients.

Residual effect of a 7-amino metabolite of clonazepam on GABAA receptor function in the nucleus reticularis thalami of the rat.

A considerable amount of 7-aminoclonazepam (ACZP), a major metabolite of clonazepam (CZP), is present in the brain during CZP treatment, yet the pharmacological properties of ACZP remain unknown. We investigated the effects of ACZP on the GABA(A) receptor-mediated currents (I(GABA)) in neurons from the nucleus reticularis thalami (NRT) of the rat, using a nystatin-perforated patch technique. Neurons in which CZP (10 nM) exerted prominent augmentation (>100% augmentation) of I(GABA), which comprised 32% of the neurons tested, were included for the analysis of ACZP. In these neurons, ACZP augmented I(GABA), which was blocked by 10 microM flumazenil, a benzodiazepine receptor (BZR) antagonist. The half-maximal effective concentration of ACZP was 124 nM, whereas that of CZP was 1.8 nM. The maximal enhancements induced by ACZP and CZP were 38% and 170%, respectively. In neurons from the ventrobasal complex of the thalamus, the effect of ACZP was negligible. Our results suggest that ACZP was a weak partial BZR agonist and that ACZP may competitively modify the effect of CZP, leading to clinical consequences for patients with high levels of ACZP.

Beta-phenylethylamine inhibits K+ currents in neocortical neurons of the rat: a possible mechanism of beta-phenylethylamine-induced seizures.

beta-Phenylethylamine (beta-PEA), an endogenous amine synthesized in the brain, serves as a neuromodulator and is involved in the pathophysiology of various neurological disorders such as depression, schizophrenia, and attention-deficit hyperactivity disorder. beta-PEA fully exerts the physiological effects within the nanomolar concentration range via the trace amine receptors, but beta-PEA also causes convulsions at much higher concentrations via an as yet unknown mechanism. To investigate the electrophysiological mechanism by which beta-PEA induces convulsions, we examined the effect of beta-PEA on ionic currents passing through the cell membrane of dissociated rat cerebral cortical neurons, using a patch-clamp technique. The external application of beta-PEA suppressed ionic currents which continuously flowed when the membrane potential was held at -25 mV. The suppression was in a concentration-dependent manner and a half-maximal effective concentration was 540 muM. These currents suppressed by beta-PEA consisted of two K(+) currents: a time- and voltage-dependent K(+) current (M-current) and a leakage K(+) current. The suppression of the M-current reduces the efficacy of the current in limiting excessive neuronal firing, and the suppression of the leakage K(+) current can cause membrane depolarization and thus promote neuronal excitation. Reducing both of these currents in concert may produce neuronal seizing activity, which could conceivably underlie the convulsions induced by high-dose beta-PEA.

Olfactory stimulation using black pepper oil facilitates oral feeding in pediatric patients receiving long-term enteral nutrition.

Patients with severe neurological disorders often require enteral nutrition (EN). Since long-term EN can cause multiple complications, reinstating the oral intake of food is beneficial. Olfactory stimulation using black pepper oil (BPO), a strong appetite stimulant, was reported to facilitate swallowing in older people. Therefore, the effects of olfactory stimulation with BPO were investigated in pediatric patients receiving long-term EN due to neurological disorders. The effects of scenting with BPO for 1 min immediately before every meal were evaluated in ten patients: 4 boys and 6 girls, aged 19-97 months (51 +/- 26 months). The neurological disorders included periventricular leukomalacia (3 patients), hypoxic ischemic encephalopathy (3), Costello syndrome (1), Russell-Silver syndrome (1), Miller-Dieker syndrome (1), and cerebral palsy of unknown etiology (1). In eight of these patients, BPO intervention was continued for 3 months. Five of these eight patients showed increases in the amount of oral intake with desirable effects including facilitated swallowing movement, although complete elimination of the need for EN was not achieved. In the other three patients, BPO intervention was not effective; severe cerebral tissue loss, profound malformation or intractable seizures seemed to reduce the efficacy of BPO. In two cases, BPO intervention was discontinued due to cough or because the odor of BPO was unbearable to the family. In conclusion, olfactory stimulation with BPO facilitated oral intake in a subset of patients on long-term EN. BPO stimulation may be useful for facilitating oral intake when used in combination with conventional methods.

Morphofunctional organization in three patients with unilateral polymicrogyria: combined use of diffusion tensor imaging and functional magnetic resonance imaging.

We examined the fiber organization of the brain in three patients with unilateral polymicrogyria (PMG) using diffusion tensor imaging (DTI) in combination with functional magnetic resonance imaging (fMRI). DTI revealed altered fiber tract architecture in patients with PMG. Long projection fibers, such as the corticospinal tract, were reduced the most, whereas long association fibers were less affected. The diminution of the fiber tracts was relevant to the loss of functionality of the PMG-affected cortex. Our preliminary study suggests that the combination of DTI and fMRI reinforces the clinical assessment of functionality in PMG.

A preliminary analysis of trace elements in the scalp hair of patients with severe motor disabilities receiving enteral nutrition.

The concentrations of essential trace elements (copper, zinc, selenium, manganese, chromium, molybdenum, cobalt, and iodine) in the scalp hair of 21 patients with severe motor disabilities receiving enteral nutrition were measured using inductively coupled plasma-mass spectrometry. Preliminary results show that copper, selenium, and molybdenum concentrations in the patients' hair were significantly lower than those in an age-matched control group (p<0.01). This suggests that intake of these elements may be reduced in patients receiving restricted enteral nutrition, although the clinical significance of these results should be discussed.

Histamine H1 antagonists block M-currents in dissociated rat cortical neurons.

We investigated the effects of histamine H1 antagonists on acutely dissociated neurons from the rat cortex using the patch-clamp technique. First-generation antihistamines, such as pyrilamine, d-chlorpheniramine, diphenhydramine, and ketotifen, suppressed M-currents in a concentration-dependent manner with respective half-inhibition concentrations (C50) of 35.9, 48.5, 34.8, and 47.8 microM at a holding potential of -26.5 mV. Astemizole, a second-generation antihistamine, inhibited M-currents with a C50 of 18.1 microM, but cetirizine did not do so, up to a concentration of 300 microM. Neither ranitidine nor cimetidine, both H2 antagonists, suppressed M-currents. The C50 of pyrilamine significantly decreased with membrane hyperpolarization, suggesting that it acts directly on M channel pores. The inhibition of M channels may be involved in the neurotoxic effects of histamine H1 antagonist overdose.

The effects of copper-histidine therapy on brain metabolism in a patient with Menkes disease: a proton magnetic resonance spectroscopic study.

We report on metabolic changes in the brain of a boy with Menkes disease. He was treated with parenteral copper (Cu)-histidine supplementation, from 5 months of age, and assessed with proton magnetic resonance spectroscopy ((1)H-MRS). The single-voxel (1)H-MRS before treatment revealed an accumulation of lactate and a reduced N-acetyl aspartate (NAA)/total creatine (tCr) ratio with a z-score of -3.0. During treatment, the lactate signal faded away, whereas the NAA signal gradually increased to a z-score of -1.5 at 120 days of treatment. The choline/tCr ratio did not deviate much initially (z-score +0.5), but the ratio increased markedly during treatment (z-score +4.8). Consequently, the Cu-histidine therapy initiated after the critical period still improved the neuronal metabolism, suggesting that some Cu was delivered to neurons. Nevertheless, the brain atrophy, impaired myelination, and severe neurological symptoms were not ameliorated.

[Development of a therapeutic agent for Menkes disease: solubilization of a copper-disulfiram complex].

Menkes disease (MD) is a neurodegenerative disorder characterized by copper deficiency. It is caused by defective intestinal absorption of copper resulting from a deficiency of a copper-transporting ATPase, ATP7A. We investigated the effects of combination therapy with copper and disulfiram, a known lipophilic chelator. We synthesized a copper-disulfiram complex (Cu-DSF) and determined its crystal structure by X-ray crystallographic analysis. Unfortunately, Cu-DSF was not orally bioavailable due to its lipophilicity. We therefore planned to use cyclodextrin as a solubilizing agent to increase the water solubility of Cu-DSF. After comparisons of the effects of cyclodextrins (α, ß, γ), it was found that addition of ß-cyclodextrin (ß-CyD) increased the solubility of Cu-DSF. Moreover, the modified ß-CyD, hydroxypropyl-ß-cyclodextrin, was yet more effective as a solubilizing agent. For the development of a convenient method to determine the concentration of Cu-DSF included by ß-cyclodextrins, a standard curve based on UV-visible(VIS) absorption was derived.

Elevated serum levels of neutrophil elastase in patients with influenza virus-associated encephalopathy.

We examined serum levels of various cytokines, chemokines, growth factors, and adhesion molecules in patients with uncomplicated influenza (n=20) and influenza virus-associated encephalopathy (IE) (n=18) to understand the underlying mechanism of IE. We found that IL-1ß, IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, G-CSF, GM-CSF, TNF-α, TIMP-1, MMP-9, sE-selectin, and neutrophil elastase were elevated significantly in sera from patients with uncomplicated influenza and those with IE, compared with normal controls (n=20). Of note, neutrophil elastase, sE-selectin, IL-8, and IL-13 were elevated significantly in IE as compared with uncomplicated influenza. In the present study, for the first time, we found that serum levels of neutrophil elastase were increased in patients with IE compared with uncomplicated influenza, which suggested that cerebral endothelial damage in the development of IE was mediated by neutrophil elastase. The present study implied that anti-elastase agents are possibly an effective therapeutic protocol for IE, but this needs further elucidation.

Acute dysautonomia: complete recovery after two courses of IVIg.

An 11-year-old boy presented with acute dysautonomia manifesting as severe orthostatic hypotension following fever. Serial orthostatic tests with measurement of the coefficient of variation in the R-R intervals showed improvement after one course and complete recovery after two courses of intravenous high-dose immunoglobulin therapy (IVIg). Repeated courses of IVIg should be considered to treat this disorder if spontaneous remission does not occur.

Two successful cases of bromide therapy for refractory symptomatic localization-related epilepsy.

Potassium bromide was tried for two children with daily convulsive focal motor seizures with unconsciousness and focal motor seizure status. The treatment resulted in complete cessation of the attacks. It has been reported that bromide is effective for generalized tonic-clonic seizures and not for complex partial seizures, such as convulsive focal motor seizures with unconsciousness. However, our experiences provide evidence that bromide is one of the useful therapeutic agents for intractable symptomatic localization-related epilepsy.

[Treatment with fluvoxamine against self-injury and aggressive behavior in autistic children].

Five autistic children underwent fluovoxamine administration. Their self-injury and aggressive behaviors did not respond psychotherapy and other medication with haloperidol, carbamazepine. The improvement of the behaviors was excellent in two patients, and partial in one patient. In a patient who received a combination of haloperidol and fluvoxamine, fluvoxamine treatment was discontinued because of severe drowsiness and could not continue. The other patients showed no obvious side effects. These results suggest that fluvoxamine treatment may be indicated for self-injury and aggressive behaviors in autistic children.

Increased Ki-67 immunoreactivity in the white matter in hemimegalencephaly.

Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged unilateral hemisphere with cortical malformation comprising abnormal hypertrophic cells. To address the proliferative status of HMG, Ki-67 immunoreactivity was investigated in HMG specimens obtained during epilepsy surgery. Nine HMG tissues were stained with a Ki-67 antibody and Ki-67 labeling index in the malformed cortex, and the underlying white matter was measured separately and compared with tissues from focal cortical dysplasias and normal brains from autopsy. In HMG tissues, Ki-67-positive cells were scattered in both the gray and white matter, with a significantly higher Ki-67 labeling index in the white matter compared with gray matter. No dysmorphic neuron or balloon cell was stained for Ki-67. As Ki-67 immunoreactivity overlapped with that of ionized calcium-binding adaptor protein-1, Ki-67-positive cells were identified as microglia. In HMG, microglia were activated and entered into a proliferative status with higher distribution in the white matter, implying an ongoing neuroinflammatory process involving the white matter.

The usefulness of subtraction ictal SPECT and ictal near-infrared spectroscopic topography in patients with West syndrome.

The recent findings on subtraction ictal SPECT and ictal near-infrared spectroscopic topography in patients with West syndrome were summarized and its availability for presurgical evaluation was discussed. The subtraction ictal SPECT study in patients with West syndrome demonstrated the cortical epileptic region and subcortical involvement, which may consist of epilepsy networks related to the spasms. Moreover, subtraction ictal SPECT may have predictive power for short-term seizure outcome. Patients with a symmetric hyperperfusion pattern are predicted to have a better seizure outcome, whereas patients with asymmetric hyperperfusion pattern may develop poor seizure control. Importantly, asymmetric MRI findings had no predictive power for seizure outcome. Multichannel near-infrared spectroscopic topography applied to the patients with West syndrome detected an increase in regional cerebral blood volume in multiple areas which were activated either simultaneously or sequentially during spasms. Topographic changes in cerebral blood volume were closely correlated with spasm phenotype, suggesting that the cortex is involved in the generation of spasms. In conclusion, subtraction ictal SPECT may be considered as a useful tool for presurgical evaluation of patients with West syndrome and investigation of the pathophysiology of spasms. The ictal near-infrared spectroscopic topography should be more investigated to see if this is useful tool for presurgical evaluation.