Lactobacillus acidophilus versus placebo in the symptomatic treatment of irritable bowel syndrome: the LAPIBSS randomized trial.

Irritable bowel syndrome is a chronic functional gastrointestinal disorder characterized by abdominal pain/discomfort and altered bowel habits. The use of Lactobacilli as probiotics during irritable bowel syndrome is based on their interesting mechanisms of action and their excellent safety profile but little is known about their clinical efficacy due to the lack of adequately designed clinical trials. The current clinical trial protocol aims to determine the effects of a mixture of Lactobacillus acidophilus NCFM and LAFTI L10 as probiotics to improve irritable bowel syndrome symptoms (LAPIBSS). Eighty patients with a positive diagnosis of irritable bowel syndrome according to Rome III criteria were recruited to a multicentre, double-blinded, in parallel groups, placebo-controlled randomized trial. Patients were provided with a daily dose of two capsules with two strains of Lactobacilli (5x109cfu/capsule) or placebo for 8 weeks on a 1:1 ratio. The primary outcome is to obtain scores of abdominal pain/discomfort assessed with a 100-mm visual analogue scale. The secondary outcome is to obtain scores of bloating, flatus and rumbling tested with a 100-mm visual analogue scale, composite score, stool frequency and stool consistency/appearance assessed with the Bristol Stool Form scale. According to the hypothesis that abdominal pain is mainly the result of a visceral hypersensitivity, the current study protocol aims to provide high quality proof of concept data to elucidate the efficacy of a consumption of a mixture of Lactobacillus acidophilus probiotic strains after 8 weeks, for decreasing abdominal pain. Ethical approval was given by ethics committee French Consultative Committee for the Protection of Individuals in Biomedical Research of the South West (Number CPP08-014a) and ANSM (French National Agency for Medicines and Health Products Safety - Number B80623-40). The findings from LAPBISS will be disseminated through peer-reviewed publications and at scientific conferences. TRIAL REGISTRATION: EudraCT N°2008-A00844-51.

Sodium calcium exchanger operates in the reverse mode in metastatic human melanoma cells.

Cytosolic Ca2+ ([Ca2+]cyt) is important in the regulation of several cellular functions involved in metastasis. We hypothesize that distinct [Ca2+]cyt regulation explains the acquisition of a more metastatic phenotype. To test this hypothesis, we used highly and lowly metastatic human melanoma cells and [Ca2+]cyt was monitored using Fura—2AM and fluorescence spectroscopy. Stimulation with ATP elicited a sustained increase in [Ca2+]cyt in highly metastatic cells, but a transient increase in lowly metastatic cells. Na+ substitution revealed Na+/Ca2+ exchanger (NCX) activity in reverse mode in highly, but not in lowly metastatic cells. In highly metastatic cells, addition of Na+ in the plateau phase of [Ca2+]cyt increase elicited with ATP, in the absence of Na+, resulted in a rapid return to basal, indicating that NCX can operate in both reverse and forward modes. Inhibition and knockdown of NCX, using KB—R7943 and siRNA NCX—1 respectively, supported the significance of NCX in [Ca2+]cyt regulation in highly metastatic cells. Stimulation with UTP triggered a rapid increase in highly metastatic cells [Ca2+]cyt, but not in lowly metastatic cells suggesting that highly and lowly metastatic cells exhibit distinct purinergic receptors. These data indicate that following agonist—stimulation, NCX operates preferentially in the reverse mode to enable a sustained [Ca2+]cyt increase in highly metastatic cells. The forward mode of NCX operation to extrude Ca2+ is preferred in lowly metastatic cells. The acquisition of a more metastatic phenotype involves a switch in NCX activity from forward to reverse mode that is favorable to maintain elevated [Ca2+]cyt in response to agonist stimulation.

Vacuolar H+-ATPase is down-regulated by the angiogenesis-inhibitory pigment epithelium-derived factor in metastatic prostate cancer cells.

The Vacuolar H+-ATPases (V-ATPases), a multi-subunits nanomotor present in all eukaryotic cells resides in the endomembranes of exocytotic and endocytotic pathways. Plasmalemmal V-ATPases have been shown to be involved in tumor cell metastasis. Pigment epithelium-derived factor (PEDF), a potent endogenous inhibitor of angiogenesis, is down-regulated in prostate cancer cells. We hypothesized that the transduction of PEDF in prostate cancer cells will down-regulate V-ATPase function; that in turn will decrease the expression of the V-ATPase accessory protein ATP6ap2 and a-subunit isoforms that target V-ATPase to the cell surface. To test these hypotheses, we used the human androgen-sensitive prostate cancer cells LNCaP, and its castration-refractory-derivative CL1 that were engineered to stably co-express the DsRed Express Fluorescent Protein with or without PEDF. To determine if PEDF down-regulates the function of V-ATPase, we measured the rate of proton fluxes (JH+) of the cytosolic and endosome/lysosome compartments. The mRNA levels for subunit-a isoforms and the ATP6ap2 were measured using quantitative reverse transcription-PCR. The results showed that PEDF expression decreased the rate of JH+ in metastatic CL1 cells without affecting JH+ in non-metastatic LNCaP cells, when studying pH(cyt). Interestingly, PEDF did not affect JH+ in endosomes/lysosomes either in metastatic cells or in non-metastatic cells. We also showed that PEDF significantly decreases the levels of a4 isoform and ATP6ap2 in metastatic CL1 cells, without affecting the levels of a4 isoform in the non-metastatic LNCaP cells. These data identify PEDF as a novel regulator of V-ATPase suggesting a new way by which PEDF may inhibit prostate tumor growth.

V-ATPase regulates communication between microvascular endothelial cells and metastatic cells.

To metastasize distant organs, tumor cells and endothelial cells lining the blood vessels must crosstalk. The nature of this communication that allows metastatic cells to intravasate and travel through the circulation and to extravasate to colonize different organs is poorly understood. In this study, we evaluated one of the first steps in this process­the proximity and physical interaction of endothelial and metastatic cells. To do this, we developed a cell separator chamber that allows endothelial and metastatic cells to grow side by side. We have shown in our previous studies that V-ATPases at the cell surface (pmV-ATPase) are involved in angiogenesis and metastasis. Therefore, we hypothesized that the physical proximity/interaction between endothelial and metastatic cells expressing pmV-ATPase will increase its activity in both cell types, and such activity in turn will increase pmV-ATPase expression on the membranes of both cell types. To determine pmV-ATPase activity we measured the proton fluxes (JH+) across the cell membrane. Our data indicated that interaction between endothelial and metastatic cells elicited a significant increase of JH+ via pmV-ATPase in both cell types. Bafilomycin, a V-ATPase inhibitor, significantly decrease JH+. In contrast, JH+ of the non-metastatic cells were not affected by the endothelial cells and vice-versa. Altogether, our data reveal that one of the early consequences of endothelial and metastatic cell interaction is an increase in pmV-ATPase that helps to acidify the extracellular medium and favors protease activity. These data emphasize the significance of the acidic tumor microenvironment enhancing a metastatic and invasive phenotype.

The standardized Ginkgo biloba extract Egb-761 protects vascular endothelium exposed to oxidized low density lipoproteins.

Dietary antioxidants are frequently proposed as protective agents for the vascular endothelium during the onset of atherosclerosis. This protection may occur at two distinct levels. First, they prevent oxidative modification of atherogenic lipoproteins (LDL). Second, they can provide a cellular protection against oxidized LDL-mediated endothelium dysfunction, although this mechanism remains poorly considered in many instances. To gain insight into the mechanism underlying such cellular protection against oxidized LDL, we examined the impact of a popular traditional medicine, an extract from Ginkgo biloba with well-known antioxidant properties, on two endothelial cells properties: cell adhesion and ionic homeostasis. Cellular lipoperoxides levels were also measured as a marker of cellular oxidative stress. Human umbilical-vein endothelial cells were exposed to native (nat-) or oxidized (ox-) LDL, the latter prepared to be compatible with clinically observed levels of oxidation. Although nat-LDL had little effect, ox-LDL increased endothelial adhesive properties (35%, p<0.01) and lipoperoxidation (45%, p<0.01). Na,K-ATPase activity, a key regulator of ionic homeostasis, was significantly decreased after exposure to nat-LDL (30%, p<0.01) and dramatically depressed after exposure to ox-LDL (65%, p<0.001). The standardized preparation of Ginkgo biloba EGb-761 totally protected adhesive properties and endothelial lipoperoxide levels. Moreover, it limited the decrease in Na,K-ATPase activity induced by ox-LDL to levels similar to nat-LDL. This suggests that EGb-761 protects endothelial adhesive properties and helps prevent the disruption of ionic homeostasis. The EGb-761-mediated inhibition of ox-LDL-induced lipoperoxide levels in endothelial cells appears to be an important mechanism by which Ginkgo biloba extract protects endothelial properties.

A high calorie diet induces type 2 diabetes in the desert sand rat (Psammomys obesus).

Diabetes mellitus type 2 is a major factor for cardiovascular diseases. The toxic effects of chronic hyperglycemia involve many alterations in the vascular tissue, including atherosclerosis. The pathogenesis of atherosclerosis in the diabetic syndrome (DS-II) has not been fully elucidated. A better understanding of the progress of DS-II at the level of the aorta could help us to identify inhibitors of atherosclerosis. Studies have shown that obesity and high calorie diet (HCD) are associated with the development of DS-II, however the lack of naturally occurring experimental models of DS-II have impaired to directly address these issues. We hypothesize that a HCD induces DS-II. This study (15 months duration) is designed to determine if HCD induces DS-II in the desert sand rat (Psammomys obesus; P. obesus). We also evaluated the histopathology of the aorta in animals fed with a laboratory chow pellets ad libitum (hypercaloric) and in control (animals fed with the naturally occurring hypocaloric diet; halophile plants). The weight and blood chemistry (glucose, lipids, and insulin levels) were evaluated periodically (once per week), and the histology of the aortas of these animals were assessed every 3 months for up to 12 months, during the development of DS-II. This study demonstrates that 40% of the animals in HCD develop DS-II at 3 months. Histological characterization demonstrates the typical alterations observed in atherosclerosis, i.e., alteration of the elastic fibers of the media layer and enrichment in collagen and glucosaminoglycans. This study demonstrates that P. obesus is an excellent animal model to study the progression of DS-II and the development of atherosclerosis.

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling.

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells.

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3--based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.

Protective effect of cicletanine on renal function in diabetic spontaneously hypertensive rats.

Hypertension and diabetes are commonly associated and strongly predispose to renal injuries. In general, antihypertensive therapies protect from these damages, but the effect of cicletanine, a new type of antihypertensive drug, is unknown. This study examines the effects of cicletanine on renal failure in spontaneously hypertensive rats with diabetes (SHRD). Diabetes mellitus was induced with streptozotocin in uninephrectomized SHR. Rats received the vehicle, 10 mg or 50 mg/kg per day of cicletanine for 6 weeks. Age-matched untreated Wistar-Kyoto rats were used as controls. Systolic blood pressure (SBP), microalbuminuria and proteinuria were assessed throughout the treatment. At the end of the study, creatinine clearance measurements and histological analysis of kidneys were performed. Cicletanine did not affect SBP but decreased the elevated albuminuria of diabetic SHR in a dose-dependent manner. Similar results were obtained for proteinuria. Treatment with the high dose of cicletanine also normalized the altered creatinine clearance of diabetic SHR. These results indicate that cicletanine has a renal-protective action, probably blood pressure-independent, in a model combining hypertension and diabetes. The mechanism of renal-protection of cicletanine is not clearly established but may be due to the stimulation of arterial prostacyclin synthesis and/or to the reduction of intraglomerular capillary pressure.

RT-PCR detection of the Na,K-ATPase beta3-isoform in human heart.

The Na,K-ATPase is a heterodimer composed of an alpha-catalytic and a beta-glycoprotein subunit. At present, three different alpha-polypeptides (alpha1, alpha2, alpha3) and two distinct beta-isoforms (beta1 and beta2) have been detected in human heart. The aim of the present study was to determine whether or not the beta3-isoform of the Na,K-ATPase can be detected in human heart. Using the highly sensitive method of RT-PCR, we here show that human heart expresses the beta3-isoform of the Na,K-ATPase. Given the differences in pharmacological properties of the nine different Na,K-ATPase isoenzymes (containing all combinations of the subunit isoforms), the study of beta3-isoform regulation in human heart may be of interest in understanding the altered response of human myocardium to digitalis therapy during heart failure.

Altered erythrocyte n-3 fatty acids in Mediterranean patients with coronary artery disease.

A low frequency of ischaemic heart diseases in Eskimos has been related to polyunsaturated fatty acids. We therefore studied fatty acid patterns associated with coronary artery disease (CAD) for a possible relationship between fatty acid profile and CAD diagnosis in Mediterranean patients. The gas chromatography method was used to analyze the membranes of patients' erythrocytes. The patients without coronary stenosis were used as controls. Patients with CAD showed increased percentages of saturated fatty acids (35.8 vs. 34.2%, P<0.001) and monounsaturated fatty acids (14.6 vs. 13.6%, P<0.01), as well as reduced percentages of polyunsaturated fatty acids (38.5 vs. 41.3%, P<0.001). The decrease in polyunsaturated fatty acids percentages was due to the series of n-3 fatty acids (9.2 vs. 11.4%, P<0.001), mainly at the expense of docosahexaenoic acid [C22:6 (n-3)] (4.9+/-0.25% vs. 6.4+/-0.23%, P<0.001) and docosapentaenoic acid [C22:5 (n-3)] (3.0+/-0.19% vs. 3.9+/-0.12%, P<0.001). The study shows altered n-3 fatty acids in Mediterranean patients with CAD. Our data suggest that the percentage of docosahexaenoic and docosapentaenoic acids in erythrocytes could be used as indicators of an independent risk factor for coronary artery disease.

Specific up-regulation of mitochondrial F0F1-ATPase activity after short episodes of atrial fibrillation in sheep.

INTRODUCTION: Ventricular fibrillation induced by either digitalis intoxication or electrical stimulation is reported to alter myocardial energy by impairing the sarcolemmal Na,K-ATPase or the receptor for digitalis and the mitochondrial ATPase synthase or F0F1-ATPase. However, little is known about these membrane functions during atrial fibrillation (AF). METHODS AND RESULTS: We analyzed the effects of electrically induced AF on biochemical activities of atrial F0F1-ATPase and Na,K-ATPase in sheep. A group of six sheep was subjected to direct short electrical stimulation of the right atrium to induce AF. Sham-operated sheep served as a control group. Microsomal and mitochondrial membranes of atrial muscle were isolated and tested for enzymatic activity. All paced sheep developed multiple episodes of sustained AF, with a mean total duration of 110 minutes over a 2-hour period. Data showed that short-term pacing-induced AF significantly activated membrane F0F1-ATPase activity (P < 0.05) without changes in cytochrome-c oxidase activity, Na,K-ATPase activity, ouabain sensitivity, and alpha1-subunit expression. CONCLUSION: Specific activation of F0F1-ATPase activity is an early molecular consequence of sustained AF in sheep.

Inhibition of Na,K-ATPase by external electrical cardioversion in a sheep model of atrial fibrillation.

INTRODUCTION: Electrical external cardioversion commonly used to treat atrial fibrillation (AF) is associated with myocardial membrane damage and disturbances in ionic homeostasis (hemodynamically unstable). The present study was designed to investigate whether alterations in ionic homeostasis observed were due in part to changes in the myocardial activity of Na,K-ATPase. METHODS AND RESULTS: AF was induced by pacing in ten anesthetized sheep divided into two groups. Group I (n = 4) received a single external countershock of 360 J after three episodes of AF lasting 10 minutes. Group II (n = 6) served as controls. Activity, responsiveness to ouabain, and membrane expression of catalytic alpha and beta subunits of Na,K-ATPase in sarcolemmal myocardial membrane fractions were investigated. Membrane fluidity and fatty acid composition, and plasma levels of atrial natriuretic factor (ANF) also were measured. One shock after episodes of AF significantly decreased ventricular Na,K-ATPase activity up to 50% (P < 0.001) without modification of atrial activity at the membrane level. Sites with low affinity to ouabain showed a fivefold lower affinity for ouabain in the cardioversion group than in the control group (IC50 = 7.9 micromol/L vs 40 micromol/L ouabain, P < 0.05). Plasma levels of ANF were significantly increased in the cardioversion group compared with the control group. These changes were independent of membrane modulation in terms of expression of Na,K-ATPase, membrane fluidity, and fatty acid composition. CONCLUSION: This study suggests that left ventricular perturbation of ionic homeostasis subsequent to transthoracic cardioversion could result from inactivation of Na,K-ATPase activity.

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.

Localization of Na,K-ATPase alpha/beta isoforms in rat sciatic nerves: effect of diabetes and fish oil treatment.

The localization of the Na,K-ATPase isoenzymes in sciatic nerve remains controversial, as well as diabetes-induced changes in Na,K-ATPase isoforms. Some of these changes could be prevented by fish oil therapy. The aim of this study was to determine by confocal microscopy the distribution of Na,K-ATPase isoforms (alpha1, alpha2, alpha3, beta1, and beta2) in the sciatic nerve, the changes induced by diabetes, and the preventive effect of fish oil in diabetic neuropathy. This study was performed in three groups of rats. In the first two groups, diabetes was induced by streptozotocin and rats were supplemented daily with fish oil or olive oil at a dosage of 0.5 g/kg of body weight. The third one was a control group that was supplemented with olive oil. Five antibodies against specific epitopes of Na,K-ATPase isoenzymes were applied to stained dissociated nerve fibers with fluorescent secondary antibodies. The five isoenzymes were documented in nonspecific regions, Schwann cells (myelin), and the node of Ranvier. The localization of the alpha1, alpha2, and beta1 isoenzymes was not affected by diabetes. In contrast, diabetes induced a decrease of the alpha2 subunit (p < 0.05) and an up-regulation of the beta2 subunit (p < 0.05). These modifications were noted in both regions for alpha2 and were localized at the myelin domain only for the beta2. Fish oil supplementation prevented the diabetes-induced changes in the alpha2 subunit with an additional up-regulation. The beta2 subunit was not modified. A phenotypic change similar to nerve injury was induced by diabetes. Fish oil supplementation partially prevented some of these changes.

A quantitative immunocytochemical study of Na+,K+-ATPase in rat hepatocytes after STZ-induced diabetes and dietary fish oil supplementation.

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+,K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+,K+-ATPase catalytic alpha1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic alpha1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the alpha1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+,K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.

Steady state pharmacokinetics of carbamazepine-phenobarbital interaction in patients with epilepsy.

Two carbamazepine (CBZ) tablet formulations (conventional, CBZ-CO, or controlled release, CBZ-CR) are commonly prescribed in monotherapy or in comedication with phenobarbital (PB) in the treatment of epilepsies. This study compares the pharmacokinetics of CBZ-CO against CBZ-CR in patients with epilepsies chronically treated with CBZ in monotherapy or CBZ-PB in bitherapy, the effect of PB on CBZ-CO and CBZ-CR pharmacokinetic parameters, and the effect of the two formulations of CBZ on PB pharmacokinetic parameters. The absorption rate constant (Ka), apparent steady state volume of distribution (Vdss/F), and apparent total clearance (CL/F) were computed with the APIS software using blood level profiles from 34 patients divided into four groups: patients receiving either CBZ-CO or CBZ-CR in monotherapy, or CBZ-CO or CBZ-CR in comedication with PB. The results show that the lowest dispersion of pharmacokinetic parameters was in patients receiving CBZ-CR in monotherapy. The CBZ formulation alters CBZ Ka, (Vdss/F) and (CL/F) values. CBZ (CL/F) also depends on the treatment (presence or absence of comedication by PB). In patients receiving PB in comedication with CBZ, the formulation of CBZ has no effect on PB pharmacokinetic parameters. These changes may be clinically significant and should be taken into account.

Interactions between clobazam and standard antiepileptic drugs in patients with epilepsy.

We retrospectively collected plasma level assessments performed in 96 adult patients with epilepsy on stable monotherapy, including 9 patients on clobazam (CLB), 34 on carbamazepine (CBZ), 24 on phenobarbital (PB), 9 on phenytoin (PHT), and 20 on valproate (VPA); these results were compared to those obtained in 54 adult patients on stable bitherapy with the association of CLB with either CBZ (n = 17), PB (n = 17), PHT (n = 5), or VPA (n = 15). Our results show that CLB has no significant effect on the level to dose ratio (LDR) of CBZ, PB, PHT, or VPA. Conversely, CBZ, PB, and PHT significantly decrease the LDR of CLB. CBZ and PHT significantly increase the LDR of N-desmethylclobazam (NCLB), the major metabolite of CLB. A significant increase in the NCLB/CLB ratio was found in CBZ + CLB, PB + CLB, and PHT + CLB bitherapies. These findings are of clinical significance: clobazam is useful as adjunctive treatment in human epilepsy and is often chosen as the benzodiazepine adjunctive drug in chronic resistant epilepsy. Sedative side effects may occur, especially in patients treated by a CBZ + CLB or PHT + CLB bitherapy, and both CLB and NCLB plasma levels should be monitored in such patients.