Immunohistohemical expression of the chemokine fractalkine and its receptor in the human brain cortex after severe traumatic brain injury and brain hemorrhage.

AIM: Recent experimental studies have suggested that chemokines, a subclass of chemoattractant cytokines which play an important role in regulating leukocyte migration and intercellular communication, participate in brain responses of traumatic injury. Fractalkine (CX3CL1) is a peculiar chemokine, the only one with a CX3C motif, existing both as a soluble and a membrane-anchored molecule. In the brain, Fractalkine has been suggested to have a role in neuroprotection under experimental conditions of brain injury. METHODS: Eighteen human brain samples were obtained during surgery of decompressive craniotomy for severe traumatic brain injury (TBI) or after spontaneous intracranial haemorrhage (ICH). Five normal brain samples were obtained during surgery for unruptured intracranial aneurysms (standard gyrectomy). Immunohistochemistry of formalin fixed and paraffin embedded tissues was performed in order to verify the expression of fractalkine and its receptor (CX3CR1). The values of chemokine and receptor expression were correlated with the clinical parameters of the patients. RESULTS: The chemokine fractalkine was significantly upregulated in the neural compartment after brain injury, compared to normal brain samples. Intensity scores were significantly higher when the interval between injury and surgery was >5 h, (P=0.015). In the glial compartment, Fractalkine expression was significantly associated with less severe clinical conditions and lower intracranial pressure at surgery (P=0.014). Expression of the receptor CX3CR1 was detected, at low intensity, on both glial and neurons. Higher expression in neurons was associated with better clinical conditions (Glasgow score) of patients at admission (P=0.037). CONCLUSION: The results of this study highlights for the first time that fractalkine and its receptor CX3CR1 are expressed in the human brain after TBI and ICH and may be involved in the limitation of tissue damage.

[Cell therapy for stroke: from myth to reality]. / La thérapie cellulaire de l'accident vasculaire cérébral ischémique : du mythe à la réalité

INTRODUCTION: Stroke is one of the leading causes of death and disability worldwide. Intravenous recombinant tissue plasminogen activator is the only available therapy for acute ischemic stroke, but its use is limited by a narrow therapeutic window and cannot stimulate endogenous repair and regeneration of damaged brain tissue. Stem cell-based approaches hold much promise as potential novel treatments to restore neurological function after stroke. STATE OF THE ART: In this review, we summarize data from preclinical and clinical studies to investigate the potential application of stem cell therapies for treatment of stroke. Stem cells have been proposed as a potential source of new cells to replace those lost due to central nervous system injury, as well as a source of trophic molecules to minimize damage and promote recovery. Various stem cells from multiple sources can generate neural cells that survive and form synaptic connections after transplantation in the stroke-injured brain. Stem cells also exhibit neurorevitalizing properties that may ameliorate neurological deficits through stimulation of neurogenesis, angiogenesis and inhibition of inflammation. PERSPECTIVES/CONCLUSION: Performed in stroke, cell therapy would decrease brain damage and reduce functional deficits. After the damage has been done, it would still improve neurological functions by activating endogenous repair. Nevertheless, many questions raised by experimental studies particularly related to long-term safety and technical details of cell preparation and administration must be resolved before wider clinical use.

Priming of late endothelial progenitor cells with erythropoietin before transplantation requires the CD131 receptor subunit and enhances their angiogenic potential.

BACKGROUND: Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases. Erythropoietin (EPO) is a cytokine that promotes angiogenesis after ischemic injury. EPO receptors (EPORs) classically include two EPOR subunits, but may also associate with the ß-common chain (CD131) in a newly identified receptor involved in EPO cytoprotective effects. OBJECTIVE: The aim was to take advantage of the proangiogenic properties of EPO to enhance ECFC graft efficiency. We postulated that priming ECFCs by adding epoietin α in culture medium prior to experiments might increase their angiogenic properties. We also explored the role of the CD131 subunit in EPO priming of ECFCs. METHODS AND RESULTS: By western blotting on cord blood ECFC lysates, we showed that EPOR and CD131 expression increased significantly after EPO priming. These proteins coimmunoprecipitated and colocalized, suggesting that they are covalently bound in ECFCs. EPO at 5 IU mL(-1) significantly stimulated proliferation, wound healing, migration and tube formation of ECFCs. EPO priming also increased ECFC resistance to H2 O2-induced apoptosis and survival in vivo. Similarly, in vivo studies showed that, as compared with non-primed ECFC injection, 5 IU mL(-1) EPO-primed ECFCs, injected intravenously 24 h after hindlimb ischemia in athymic nude mice, increased the ischemic/non-ischemic ratios of hindlimb blood flow and capillary density. These effects were all prevented by CD131 small interfering RNA transfection, and involved the phosphoinositide 3-kinase-Akt pathway. CONCLUSION: These results highlight the potential role of EPO-primed ECFCs for cell-based therapy in hindlimb ischemia, and underline the critical role of CD131 as an EPO coreceptor.

Erythropoietin 2nd cerebral protection after acute injuries: a double-edged sword?

Over the past 15 years, a large body of evidence has revealed that the cytokine erythropoietin exhibits non-erythropoietic functions, especially tissue-protective effects. The discovery of EPO and its receptors in the central nervous system and the evidence that EPO is made locally in response to injury as a protective factor in the brain have raised the possibility that recombinant human EPO (rhEPO) could be administered as a cytoprotective agent after acute brain injuries. This review highlights the potential applications of rhEPO as a neuroprotectant in experimental and clinical settings such as ischemia, traumatic brain injury, and subarachnoid and intracerebral hemorrhage. In preclinical studies, EPO prevented apoptosis, inflammation, and oxidative stress induced by injury and exhibited strong neuroprotective and neurorestorative properties. EPO stimulates vascular repair by facilitating endothelial progenitor cell migration into the brain and neovascularisation, and it promotes neurogenesis. In humans, small clinical trials have shown promising results but large prospective randomized studies failed to demonstrate a benefit of EPO for brain protection and showed unwanted side effects, especially thrombotic complications. Recently, regions have been identified within the EPO molecule that mediate tissue protection, allowing the development of non-erythropoietic EPO variants for neuroprotection conceptually devoid of side effects. The efficacy and the safety profile of these new compounds are still to be demonstrated to obtain, in patients, the benefits observed in experimental studies.

The microvascular network of the pituitary gland: a model for the application of fractal geometry to the analysis of angioarchitecture and angiogenesis of brain tumors.

In geometrical terms, tumor vascularity is an exemplary anatomical system that irregularly fills a three-dimensional Euclidean space. This physical characteristic, together with the highly variable vessel shapes and surfaces, leads to considerable spatial and temporal heterogeneity in the delivery of oxygen, nutrients and drugs, and the removal of metabolites. Although these biological features have now been well established, quantitative analyses of neovascularity in two-dimensional histological sections still fail to view tumor architecture in non-Euclidean terms, and this leads to errors in visually interpreting the same tumor, and discordant results from different laboratories. A review of the literature concerning the application of microvessel density (MVD) estimates, an Euclidean-based approach used to quantify vascularity in normal and neoplastic pituitary tissues, revealed some disagreements in the results and led us to discuss the limitations of the Euclidean quantification of vascularity. Consequently, we introduced fractal geometry as a better means of quantifying the microvasculature of normal pituitary glands and pituitary adenomas, and found that the use of the surface fractal dimension is more appropriate than MVD for analysing the vascular network of both. We propose extending the application of this model to the analysis of the angiogenesis and angioarchitecture of brain tumors.

Focal extra-axial hemorrahagic mass with subdural hemorrhage secondare to extramedullary hematopoiesis in idiopathic myelodysplastic sindrome.

Idiopathic myelodysplastic syndrome is a disease characterized by a clonal stem cell disorder in which megacaryocitic and granulocytic lineages are mainly involved; extramedullary myeloid metaplasia is due to abnormal location of myeloid tissue in other organs than bone marrow. Rarely the central nervous system is involved. When it happens, it is typical to find masses around the brain and pachymeningeal thickening, but it is very rare to find it associated with subdural haemorrhage, as in the case we describe in the present article. Considering our case and the literature we can suggest that radiological images associated with the clinical history of the patient suggestive for extramedullary hematopoiesis can be sufficient for a correct diagnosis and for a radiotherapy treatment, demanding surgery in the case of diagnostic doubts, massive hemorrahages or neurological decifits caused by the focal lesions.

De novo Formation of the Ventriculus Terminalis? A Brief Report of a Special Case.

The ventriculus terminalis is an ependymal cystic cavity in the conus medullaris, sometimes found in children. Persistence of the ventriculus terminalis in adults can cause lower back pain or neurological disturbances. However, there are no literature reports of de novo formation of a ventriculus terminalis in the conus medulallaris, as in the case we illustrate here.

Lack of toxicity of hydroethanolic extract from Mitragyna inermis (Willd.) O. Kuntze by gavage in the rat.

In traditional medicine in Mali, extracts derived from Mitragyna inermis (Willd.) O. Kuntze (Family: Rubiaceae) are commonly used to treat malaria. The antimalarial activity and the lack of genotoxicity in vitro and in vivo have been demonstrated in previous studies. Acute and chronic evaluation of the toxicity of the hydroethanolic extract of Mitragyna inermis leaves was performed in this study, according to the recommendations (cahier de l'Agence no. 3) of the French Drug Office. Two dosages (300 mg/kg and 3 g/kg) were given in one single administration by gavage to male and female rats. No animal died and no behavioral signs of acute toxicity were observed. Chronic toxicity studies over 28 days showed no changes in body weight and no macroscopic abnormality in the 14 organs examined after the animals were sacrificed. With the 3 g/kg/d drug dosage (100-fold higher than those proposed in man), only slight histological abnormalities were observed. Statistically significant differences, compared to control animals, in the weight of some organs and the values of some haematological or biochemical parameters were observed. However, these values always remained in the range given by the breeder for naive animals of the same strain. These investigations thus seemed to indicate the safety of repeated oral administration (up to 3 g/kg/d) of the hydroethanolic extract of Mitragyna inermis leaves, which can therefore be continuously used with safety by the African population in traditional treatment of malaria.

Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures.

This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol.

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.

Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures.

The expression and activity of glutamate transporters (EAAC1, GLAST and GLT1) were examined during the development of cortical neuron-enriched cultures. Protein content and mitochondrial respiration both increased during the first 7 days, later stabilized and decreased from DIV14. Glutamate transport and extracellular concentration were relatively constant from DIV3 to 18. The kinetic parameters of glutamate transport were at DIV7: K(m)=19+/-3 microM and V(max)=1068+/-83 pmol/mg protein/min and at DIV14: K(m)=40.8+/-9.3 microM and V(max)=1060+/-235 pmol/mg protein/min. The shift in K(m) towards higher values suggest a more important participation of GLAST after DIV14. At DIV7 and 14, glutamate transport was poorly sensitive to dihydrokaïnate (DHK) suggesting a weak participation of GLT1 in glutamate transport. Western blot experiments and immunocytochemistry showed that EAAC1 was expressed by neurons whatever the stage of the culture. GLAST was found in astrocytes as soon as DIV3 and labeling increased during the development of the culture. There was little neuronal GLT1 immunoreactivity at DIV7, only detected by immunocytochemistry. From DIV10 to 18, an increasing astrocytic expression of GLT1 was observed, also detected by Western blotting. These results show that: (1) glutamate uptake remains stable all along the development of the cultures although the pattern of expression of the different transporters is changing, suggesting that glutamate transport is highly regulated; (2) neuronal EAAC1 may play a critical role during the early stages of the culture when it is expressed alone; and (3) the developmental expression pattern of glutamate transporters in cortical neuron-enriched cultures is quite similar to that observed in vivo during early postnatal development.

Pharmacokinetic and pharmacodynamic analysis of sedative and amnesic effects of lorazepam in healthy volunteers.

This study describes for the first time the pharmacokinetic and pharmacodynamic modeling of the psychomotor and amnesic effects of a single 2-mg oral dose of lorazepam in healthy volunteers. Twelve healthy volunteers were included in this randomized, double-blinded, placebo-controlled two-way crossover study. The effect of lorazepam was examined for a battery of tests that explored mood, vigilance, psychomotor performance, and memory. The pharmacokinetic and pharmacodynamic modeling of these tests was performed using the indirect response model. Vigilance and psychomotor performance were significantly impaired. Short-term memory was not affected, but a paradoxical tendency to improvement of the score was observed 0.4 hours after drug intake. Significant impairment was observed for immediate and delayed cued verbal recall, for immediate and delayed free recall, and for picture recognition as well as for visual-verbal recall, but not for cued visual-spatial recall or priming. Globally, the different effects were greatest between 0.4 to 3 hours after dosing. However, the time course profile of the recovery period suggests a possible dissociation between the kinetics of the effects of lorazepam on vigilance, psychomotor performance, and visual episodic memory, on the one hand, and on verbal episodic memory, on the other. The pharmacokinetic and pharmacodynamic model used two compartments with first-order absorption to describe the lorazepam concentrations and an indirect response model with inhibition or stimulation of Kin to describe the effects. The mean values for calculated median effective concentration (EC50) derived from the pharmacokinetic and pharmacodynamic modeling of the different tests ranged from 11.3 to 39.8 ng/mL. According to these EC50 values, lorazepam seemed to be more potent on the delayed-recall trials than on the immediate-recall trials; similar observations were made concerning the free-recall versus cued-recall trials. The previously stated results suggest that the tests performed in this study represent sensitive measurements of the effects of lorazepam on the central nervous system. Moreover, the parameter values derived from pharmacokinetic and pharmacodynamic modeling, especially, the EC50 values, may provide sensitive indices that can be used to compare the central nervous system effects of benzodiazepines.

Pronounced effect of caprylocaproyl macrogolglycerides on nasal absorption of IS-159, a peptide serotonin 1B/1D-receptor agonist.

OBJECTIVES: This double-blind, randomized, two-way crossover study in 12 healthy male subjects investigated the influence of caprylocaproyl macrogolglycerides on the pharmacokinetics of IS-159 (serotonin-carboxylmethyleneoxy-L-tyrosylglycinamide), a peptide serotonin 1B/1D-receptor agonist, after intranasal administration. METHODS: A dose of 4 mg IS-159 was administered in a volume of 200 microL, once in the presence and once in the absence of 2% caprylocaproyl macrogolglycerides. Plasma concentrations of IS-159 were measured over a period of 12 hours for determination of pharmacokinetic parameters. Systemic and local tolerability were assessed at regular time points, the latter by rhinoscopy and visual analog scales. RESULTS: Caprylocaproyl macrogolglycerides significantly increased the maximum plasma concentration (from 4.7 +/- 1.7 to 48 +/- 17 ng/mL) and the area under the plasma concentration-time curve (from 12 +/- 4.7 to 56 +/- 22 ng x h/mL) of IS-159. The time to maximum concentration (15 to 20 minutes) and the elimination half-life (2.0 to 2.3 hours) were not different between the two treatments. Rhinoscopic examination revealed no differences between treatments, but in the presence of caprylocaproyl macrogolglycerides subjects reported more local and systemic adverse events and on the visual analog scales greater nasal obstruction and rhinorrhea. CONCLUSION: 2% caprylocaproyl macrogolglyceride markedly increased the absorption of IS-159 through the nasal mucosa and elicited only mild irritant effects.

Pharmacokinetic-pharmacodynamic analysis of mnesic effects of lorazepam in healthy volunteers.

AIMS: To describe the pharmacokinetic-pharmacodynamic modelling of the psychomotor and mnesic effects of a single 2 mg oral dose of lorazepam in healthy volunteers. METHODS: This was a randomized double-blind, placebo-controlled two-way cross-over study. The effect of lorazepam was examined with the following tasks: choice reaction time, immediate and delayed cued recall of paired words and immediate and delayed free recall and recognition of pictures. RESULTS: The mean calculated EC50 values derived from the PK/PD modelling of the different tests ranged from 12.2 to 15.3 ng ml-1. On the basis of the statistical comparison of the EC50 values, the delayed recall trials seemed to be more impaired than the immediate recall trials; similar observations were made concerning the recognition vs recall tasks. CONCLUSIONS: The parameter values derived from PK/PD modelling, and especially the EC50 values, may provide sensitive indices that can be used, rather than the raw data derived from pharmacodynamic measurements, to compare CNS effects of benzodiazepines.

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex.

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Ketamine effects on bupivacaine local anaesthetic activity and pharmacokinetics of bupivacaine in mice.

This study was designed to document possible changes in bupivacaine (B) local anaesthetic activity and pharmacokinetics in mice after a ketamine (K) injection. In the experiments, bupivacaine (8.25 mg.kg(-1)), was injected into the popliteal space of the right posterior limb: the local anaesthetic activity was assessed according to a sciatic nerve blockade method with three different doses (2, 10 and 40 mg/kg) of ketamine and the kinetics were studied after a 10 mg/kg dose. When ketamine was associated, the local anesthetic activity of bupivacaine was significantly enhanced as well as its elimination half-life. Significantly lower levels of the main metabolite, PPX, were observed, when ketamine was associated, suggesting a metabolic inhibition phenomenon. The ketamine-induced increase in the total anaesthetic effect of bupivacaine may thus be explained by kinetic modifications i.e. a possible inhibiting effect of ketamine on the metabolism of bupivacaine.

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats.

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (10(-3) or 4 x 10(-3) M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K+ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine) (L-CCG) (10(-3) M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1-1000 microM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.

Activation of the adenylate cyclase-dependent protein kinase pathway increases high affinity glutamate uptake into rat striatal synaptosomes.

This study examined the effects of various agents known to alter protein phosphorylation through protein kinase A or C on high affinity glutamate uptake measured in vitro on rat striatal homogenates. Incubation of synaptosomes with the phosphatase inhibitor, okadaic acid, dramatically increased glutamate uptake indicating that underlying phosphorylation mechanisms may be involved in the regulation of this transport process. The protein kinase C activator, phorbol-12,13-dibutyrate, or inhibitor, staurosporine, did not significantly modify glutamate uptake. In contrast, forskolin, which activates adenylate cyclase, induced a dose-dependent increase in glutamate uptake. Saturation kinetic analysis indicated that forskolin increased the Vmax without modifying the Km of the transport process as compared to control values. The effect of forskolin was mimicked by dibutyryl adenosine monophosphate, an analog of cAMP which activates protein kinase A, and counteracted by high doses of N-[2-(methylamino) ethy1]-5-isoquinoline sulfonamide, a protein kinase A inhibitor. These results suggest that an adenylate cyclase-dependent protein kinase may be involved in the post-translational regulation of glutamate transporters.

Plasma concentrations and pharmacokinetics of idebenone and its metabolites following single and repeated doses in young patients with mitochondrial encephalomyopathy.

OBJECTIVE: The pharmacokinetics and tolerance of idebenone after single or repeated doses have been studied in young patients with mitochondrial encephalomyopathy. RESULTS: No significant adverse effects were noted. In 3 out of 7 patients idebenone induced overall stimulation and improvement in arousal. Plasma concentrations of idebenone and its main metabolites were determined and the pharmacokinetic parameters of idebenone after single and repeated doses were estimated. During the single dose study, the mean plasma concentrations of idebenone and its main metabolites and mean pharmacokinetic parameters were comparable to published results (Cmax = 452.2 ng.ml-1, tmax = 2.3 h, AUC = 26 micrograms. ml-1.h, t1/2 beta = 16.5 h). During the repeated doses study, no significant difference was found between mean residual plasma concentrations of idebenone on Day 2 (47 ng.ml-1) and Day 5 (70.6 ng.ml-1), and mean t1/2 beta of idebenone after the single and after repeated dose studies, i.e., there was no evidence of accumulation. Although idebenone did not appear to accumulate during this study, the coadministration of anticonvulsants, often prescribed during mitochondrial encephalomyopathy, can affect its pharmacokinetics.