4-mer hyaluronan oligosaccharides stimulate inflammation response in synovial fibroblasts in part via TAK-1 and in part via p38-MAPK.

4-mer hyaluronan (HA) oligosaccharides stimulate pro-inflammatory effects in different cell types by interacting with both the toll-like receptor-4 (TLR-4) and -2 (TLR-2). This interaction induces the activation of the transforming growth factor activated kinase-1 (TAK-1) that activates the nuclear factor kappaB (NF-kB) either directly and/or through the activation of p38-mitogen-activated protein kinase (p38-MAPK). This in turn induces the transcription of proinflammatory mediators that prime inflammation. Our aim was to investigate the involvement of TAK-1 and p38-MAPK in 4-mer HA oligosaccharide-induced inflammatory response in mouse synovial fibroblasts obtained from normal DBA/J1 mice (NSF) and from mice subjected to collagen-induced arthritis (CIA). Treatment of NSF and rheumatoid arthritis synovial fibroblasts (RASF) with 4-mer HA showed a marked up-regulation of TLR-4, TLR-2, TAK-1 and p38-MAPK mRNA expression and of the related proteins, as well as NF-kB activation. High levels were also detected of TNF-α, IL- 1ß, MMP-13 and iNOS. Treatment of NSF and RASF, previously stimulated with 4-mer HA oligosaccharides, with TAK- 1 and/or p38-MAPK specific inhibitors significantly reduced all the parameters, although the inhibitory effect of p38- MAPK was less effective than that of TAK-1. The addition of CD44 antibody to both NSF and RASF showed that CD44 was not involved in 4-mer HA-induced inflammation.

Effect of cytokines on hyaluronan synthase activity and response to oxidative stress by fibroblasts.

Cytokines such as tumour necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma), and transforming growth factor-beta (TGF1beta) modulate hyaluronan synthase (HAS) gene expression and protein activity. The aim of this research is to evaluate the response of HAS gene expression and the related protein synthesis in fibroblasts after treatment with TNFalpha, IFNgamma and TGF1beta and to assess the potential protective effect of increased hyaluronan (HA) synthesis during oxidative stress. In this study, gene expression, protein synthesis, hyaluronan content, cell death, lactate dehydrogenase (LDH) activity, membrane lipid peroxidation and endogenous antioxidant depletion are determined for HAS1, HAS2 and HAS3. Messenger RNA (mRNA) expression and protein formation of the three HAS genes is modulated using different cytokines and various doses and correlated with increased HA synthesis. Protection of fibroblasts from injury induced by exposure to reactive oxygen species was significantly increased by TGF1beta and was associated with increased gene expression and protein formation of HAS1 and HAS2 enzymes synthesising high-molecular-weight HA. It is proposed that specific HAS enzyme activity and HA molecular weight specificity is involved in the protective mechanism.

The antioxidant activity of chondroitin-4-sulphate, in carbon tetrachloride-induced acute hepatitis in mice, involves NF-kappaB and caspase activation.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROC) are the main causes of carbon tetrachloride (CCl4)-induced acute liver injury. Chondroitin-4-sulphate (C4S) is known to inhibit lipid peroxidation through antioxidant mechanisms. Activation of nuclear factor (NF)-kappaB and caspases may strongly intensify inflammation and cell damage, in addition to that directly exerted by ROS. We investigated whether treatment with C4S, besides exerting antioxidant activity, was able to modulate NF-kappaB and apoptosis activation in CCl4-induced liver injury in mice. EXPERIMENTAL APPROACH: Acute hepatitis was induced in mice by an i.p. injection of CCl(4). Varying doses of C4S were administered i.p. 1 h before, 6 and 12 h after CCl4 injection. 24 h after CCl4 injection, the mice were killed for biochemical and histological analysis. KEY RESULTS: CCl4 injection produced: marked elevation of alanine aminotransferase and aspartate aminotransferase; hepatic membrane lipid peroxidation, assayed by 8-isoprostane levels; and depletion of reduced glutathione and superoxide dismutase. CCl4 also decreased NF-kappaB translocation and IkBalpha, and increased gene expression of mRNA and protein of metalloproteases (MMP)-2 and -9, and of pro- and cleaved forms of caspases-3 and -7. There was also increased liver polymorphonuclear infiltration, evaluated by elastase assay, and hepatic cell disruption.C4S treatment inhibited lipid peroxidation; blocked NF-kappaB activation and IkBalpha protein loss; decreased mRNA and proteins for MMPs and caspases; restored endogenous antioxidants; limited hepatic polymorphonuclear accumulation and tissue damage. CONCLUSIONS AND IMPLICATIONS: As antioxidants may inhibit NF-kappaB and caspase activation, we hypothesize that treatment with C4S was able to inhibit NF-kappaB and apoptosis activation in hepatic injury.

Chondroitin-4-sulphate inhibits NF-kB translocation and caspase activation in collagen-induced arthritis in mice.

OBJECTIVE: Free radical damage, inflammation, and apoptosis play a critical role in the onset and progression of cartilage erosion in arthritis. Many studies have demonstrated that glycosaminoglycans (GAGs), and chondroitin-4-sulphate (C4S) in particular, possess antioxidant activity that is able to inhibit lipid peroxidation which is the main mechanism of free radical-mediated biological injury. In addition to the effect directly exerted by reactive oxygen species (ROS), the activation of nuclear factor kB (NF-kB) and caspases may contribute substantially to increase inflammation and cell damage. We studied whether the antioxidant action of chronic C4S treatment to reduce ROS injury involves NF-kB and caspases modulation using an experimental model of collagen-induced arthritis in mice. METHODS: Arthritis was induced in mice via an intradermal injection at the base of the tail of 100 microl of emulsion containing bovine type II collagen in complete Freund's adjuvant. RESULTS: Arthritis provoked the following: severe oedema and inflammation in the hind paws; lipid peroxidation in the joints [measured by 8-isoprostane (8-IPE) levels]; reduction of the endogenous antioxidants catalase (CAT) activity and reduced glutathione (GSH) levels; induction of NF-kB translocation; a loss of cytoplasmic NF-kB inhibitor alpha (IkBalpha); an increase in metalloproteinase-13 (MMP-13), caspase-3 and caspase-7 gene expression and their related protein; the induction of cartilage polymorphonuclear (PMN) activation and infiltration [evaluated by elastase (ELA) assay] and cartilage alterations evaluated by histological analysis. Intraperitoneal administration of different doses of C4S (for 25 days), ameliorated all the symptoms of inflammation in the articular knee and paw joints, limited lipid peroxidation, inhibited NF-kB activation and IkBalpha protein loss, decreased mRNA MMP-13 and caspases expression and their related protein, restored endogenous antioxidants, and reduced PMN accumulation in the damaged cartilage. CONCLUSION: The evidence that C4S was able to inhibit NF-kB and apoptosis activation supports the hypothesis that the C4S effect depends on reduction of ROS production, although other direct effects cannot be excluded.

Differential effect of growth factors on hyaluronan synthase gene expression in fibroblasts exposed to oxidative stress.

The aim of this study was to evaluate how growth factors (PDGF-BB, EGF, and TGF-1beta) modulate hyaluronan synthase (HAS) activities in normal or stressed cultured human skin fibroblasts. The effects of concomitant treatment with cytokines and FeSO4 plus ascorbate on HAS mRNA expression, protein synthesis, and hyaluronic acid (HA) concentrations were also studied. Treatment of fibroblasts with growth factors up-regulated HAS gene expression and increased HAS enzymes and HA production. PDGF-BB induced HAS mRNA expression, protein synthesis, and HA production more efficiently than EGF and TGF-1beta. EGF was less effective than TGF-1beta. In addition, TGF-1beta reduced the expression and synthesis of HAS3, while PDGF-BB and EGF had the opposite effect. Concomitant treatment with growth factors and the oxidant was able to further increase HAS mRNA expression, once again with the exception of HAS3 with TGF-1beta. HAS protein synthesis was reduced, while HA levels were unaffected in comparison to those obtained from exposure to FeSO4 plus ascorbate alone. In conclusion, although growth factors plus the oxidant synergistically induced HAS mRNA expression in part, enzyme production was not correlated with this increase. Moreover, the increase in HAS mRNA levels was not translated into a consequent rise in HA concentration.

Chondroitin sulphate: antioxidant properties and beneficial effects.

Most biological molecules exhibit more than one function. In particular, many molecules have the ability to directly/indirectly scavenge free radicals and thus act in living organisms as antioxidant. During oxidative stress, the increase of these molecules levels seems to be a biological response that in synergism with the other antioxidant defence systems may protect cells from oxidation. Among these structures, chondroitin sulphate is a biomolecule which has increasingly focused the interest of many research groups due to its antioxidant activity. This review briefly summarises the action of chondroitin sulphate in reducing molecular damage caused by free radicals and associated oxygen reactants.

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 +/- 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 +/- 13.23 U/L), and restored the hepatic concentrations of VE (9.52 +/- 3.21 nmol/g tissue), inhibited OH* production (2,3-DHBA= 3.54 +/- 1.31 microM; 2,5-DHBA= 7.37 +/- 2.46 microM), restored the endogenous antioxidant GSH (12.77 +/- 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-alpha concentrations (31.47 +/- 18.25 IU/ml) and hepatic TNF-alpha mRNA levels (11.65 +/- 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4 -induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Plasma concentrations of risperidone and 9-hydroxyrisperidone during combined treatment with paroxetine.

SUMMARY: The effects of paroxetine on steady-state plasma concentrations of risperidone and its active metabolite 9-hydroxyrisperidone (9-OH-risperidone) were studied in 10 patients with schizophrenia or schizoaffective disorder. Patients stabilized using risperidone therapy (4-8 mg/d) also received paroxetine (20 mg/d) for 4 weeks. During paroxetine administration, mean plasma concentrations of risperidone increased significantly (P < 0.01), whereas levels of 9-OH-risperidone decreased slightly but not significantly. After 4 weeks of paroxetine treatment, the sum of the concentrations of risperidone and 9-OH-risperidone (active moiety) increased significantly by 45% (P < 0.05) over baseline. The mean plasma risperidone/9-OH-risperidone ratio was also significantly modified (P < 0.001) during paroxetine treatment. The drug combination was generally well tolerated with the exception of one patient who developed Parkinsonian symptoms in the second week of adjunctive therapy. In this patient total plasma levels of risperidone and its active metabolite increased by 62% during paroxetine co-administration. The authors' findings indicate that paroxetine, a potent inhibitor of CYP2D6, may impair the elimination of risperidone, primarily by inhibiting CYP2D6-mediated 9-hydroxylation and to a lesser extent by simultaneously affecting the further metabolism of 9-OH-risperidone or other pathways of risperidone biotransformation. Careful clinical observation and possibly monitoring of plasma risperidone levels may be useful whenever paroxetine is co-administered with risperidone.

Relationship between plasma risperidone and 9-hydroxyrisperidone concentrations and clinical response in patients with schizophrenia.

RATIONALE: Evaluation of relationships between serum antipsychotic drug concentrations and clinical response may provide valuable information for rational dosage adjustments. For risperidone, this relationship has been little investigated to date. OBJECTIVE: To assess the relationship between plasma concentrations of risperidone and its active 9-hydroxy-metabolite (9-OH-risperidone) and clinical response in schizophrenic patients who experienced an acute exacerbation of the disorder. METHODS: Forty-two patients (30 males, 12 females, age 24-60 years) were given risperidone at dosages ranging from 4 to 9 mg/day for 6 weeks. The design of the study was open and risperidone dosage could be adjusted individually according to clinical response. Steady-state plasma concentrations of risperidone and its 9-hydroxymetabolite were measured after 4 and 6 weeks using a specific HPLC assay. Psychopathological state was assessed at baseline and at weeks 2, 4, and 6 by means of the positive and negative syndrome scale (PANSS), and patients were considered responders if they showed a greater than 20% reduction in total PANSS score at final evaluation compared with baseline. RESULTS: Mean plasma concentrations of risperidone, 9-OH-risperidone, and active moiety (sum of risperidone and 9-OH-risperidone concentrations) did not differ between responders (n = 28) and non-responders (n = 14). No correlation between plasma levels and percent decrease in total PANSS score was found for risperidone (rs = -0.187, NS), 9-OH-risperidone (rs = 0.246, NS), and active moiety (rs = 0.249, NS). Active moiety concentrations in plasma were higher (P < 0.001) in patients developing clinically significant parkinsonian symptoms (n = 7) than in those with minimal (n = 7) or no drug-induced parkinsonism (n = 28). CONCLUSIONS: In chronic schizophrenic patients experiencing an acute exacerbation of the disorder, plasma levels of risperidone and its active metabolite correlate with the occurrence of parkinsonian side effects, whereas no significant correlation appears to exist with the degree of clinical improvement.

No effect of reboxetine on plasma concentrations of clozapine, risperidone, and their active metabolites.

The effect of reboxetine on steady-state plasma concentrations of the atypical antipsychotics clozapine and risperidone was studied in 14 patients with schizophrenia or schizoaffective disorder with associated depressive symptoms. Seven patients stabilized on clozapine therapy (250-500 mg/day) and seven receiving risperidone (4-6 mg/day) were given additional reboxetine (8 mg/day). After 4 weeks of reboxetine therapy, mean plasma concentrations of clozapine, norclozapine, and risperidone active moiety (sum of concentrations of risperidone and 9-hydroxyrisperidone) increased slightly but not significantly by 5%, 2%, and 10%, respectively. The mean plasma clozapine/norclozapine and risperidone/9-hydroxyrisperidone ratios were not modified during reboxetine treatment. Reboxetine coadministration with either clozapine or risperidone was well tolerated. These findings indicate that reboxetine has minimal effects on the metabolism of clozapine and risperidone and may be added safely to patients receiving maintenance treatment with these two antipsychotics.

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection.

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether-isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 microl potassium phosphate (0.1 M, pH 2.2) and 60 microl of the acid solution was injected into a C18 BDS Hypersil analytical column (3 microm, 100x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H3PO4)-acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5-100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.

Plasma concentrations of risperidone and 9-hydroxyrisperidone: effect of comedication with carbamazepine or valproate.

To evaluate the pharmacokinetic interaction between risperidone and the mood-stabilizing agents carbamazepine and valproic acid, steady state plasma concentrations of risperidone and 9-hydroxyrisperidone (9-OH-risperidone) were compared in patients treated with risperidone alone (controls, n = 23) and in patients comedicated with carbamazepine (n = 11) or sodium valproate (n = 10). The three groups were matched for sex, age, body weight, and antipsychotic dosage. Plasma concentrations of risperidone and 9-OH-risperidone did not differ between valproate-comedicated patients and controls. By contrast, the concentrations of both compounds were lower in patients taking carbamazepine, although the difference reached statistical significance only for the metabolite (p < 0.001). The sum of the concentrations of risperidone and 9-OH-risperidone in patients receiving carbamazepine (median 44 nmol/L) was also significantly lower than in patients receiving valproate (168 nmol/L) and in controls (150 nmol/L). In five patients assessed with and without carbamazepine comedication, dose-normalized plasma risperidone and 9-OH-risperidone concentrations were significantly lower when the patients received combination therapy than when they received risperidone alone. In three patients assessed with and without valproate, no major changes in the levels of risperidone and its metabolite were observed. These findings demonstrate that carbamazepine markedly decreases the plasma concentrations of risperidone and its active 9-OH-metabolite, probably by inducing CYP3A4-mediated metabolism. This interaction is likely to be clinically significant. Conversely, valproic acid does not cause any major change in plasma antipsychotic levels.

Relationship between plasma concentrations of clozapine and norclozapine and therapeutic response in patients with schizophrenia resistant to conventional neuroleptics.

RATIONALE: Monitoring plasma clozapine concentrations may play a useful role in the management of patients with schizophrenia, but information on the relationship between the plasma levels of the drug and response is still controversial. OBJECTIVE: The purpose of this study was to assess the relationship between plasma concentrations of clozapine and its weakly active metabolite norclozapine and clinical response in patients with schizophrenia resistant to conventional neuroleptics. METHODS: Forty-five patients, 35 males and ten females, aged 19-65 years, were given clozapine at a dosage up to 500 mg/day for 12 weeks. Steady-state plasma concentrations of clozapine and norclozapine were measured at week 12 by a specific HPLC assay. Psychopathological state was assessed at baseline and at week 12 by using the Brief Psychiatric Rating Scale, and patients were considered responders if they showed a greater than 20% reduction in total BPRS score compared with baseline and a final BPRS score of 35 or less. RESULTS: Mean plasma clozapine concentrations were higher in responders (n=18) than in non-responders (n=27) (472+/-220 versus 328+/-128 ng/ml, P<0.01), whereas plasma norclozapine levels did not differ between the two groups (201+/-104 versus 156+/-64 ng/ml, NS). A significant positive correlation between plasma levels and percent decrease in total BPRS score was found for clozapine (r(s)=0.371, P<0.02), but not for norclozapine (r(s)=0.162, NS). A cutoff value at a clozapine concentration of about 350 ng/ml differentiated responders from non-responders with a sensitivity of 72% and a specificity of 70%. At a cutoff of 400 ng/ml, sensitivity was 67% and specificity 78%. The incidence of side effects was twice as high at clozapine concentrations above 350 ng/ml compared with lower concentrations (38% versus 17%). CONCLUSIONS: These results suggest that plasma clozapine levels are correlated with clinical effects, although there is considerable variability in the response achieved at any given drug concentration. Because many patients respond well at plasma clozapine concentrations in a low range, aiming initially at plasma clozapine concentrations of 350 ng/ml or greater would require in some patients use of unrealistically high dosages and imply an excessive risk of side effects. Increasing dosage to achieve plasma levels above 350-400 ng/ml may be especially indicated in patients without side effects who failed to exhibit amelioration of psychopathology at standard dosages or at lower drug concentrations.

Plasma concentrations of clozapine and its major metabolites during combined treatment with paroxetine or sertraline.

The effect of paroxetine or sertraline on steady-state plasma concentrations of clozapine and its major metabolites was studied in 17 patients with schizophrenia or schizoaffective disorder stabilized on clozapine therapy (200-400 mg/day). In order to treat negative symptomatology or concomitant depression, 9 patients received additional paroxetine (20-40mg/day) and 8 patients sertraline (50-100 mg/day). After 3 weeks of paroxetine administration, mean plasma concentrations of clozapine and norclozapine increased significantly by 31% (p<0.01) and by 20% (p<0.05), respectively, while levels of clozapine N-oxide remained almost unchanged. The mean plasma norclozapine/clozapine and clozapine N-oxide/clozapine ratios were not modified during paroxetine treatment. No significant changes in plasma concentrations of clozapine and its major metabolites were observed after 3 weeks of combined therapy with sertraline. Clozapine coadministration with either paroxetine or sertraline was well tolerated. Our findings suggest that the metabolism of clozapine is not affected by sertraline treatment at typical therapeutic doses, while paroxetine, a potent inhibitor of CYP2D6, appears to inhibit the metabolism of clozapine, possibly by affecting pathways other than N-demethylation and N-oxidation. While sertraline may be added safely to patients on maintenance treatment with clozapine, careful clinical observation and monitoring of plasma clozapine levels may be useful whenever paroxetine is coadministered with clozapine.

No effect of the new antidepressant reboxetine on CYP2D6 activity in healthy volunteers.

The effect of the new antidepressant reboxetine on the activity of the cytochrome P450 (CYP) 2D6 isoenzyme was investigated in 10 healthy volunteers using dextromethorphan as a model CYP2D6 substrate. Each volunteer received a single 30 mg oral dose of dextromethorphan on three different occasions separated by an interval of at least 4 weeks: a) in a control session; b) after 1 week of treatment with reboxetine, 8 mg/day; and c) after 1 week of treatment with paroxetine (an inhibitor of CYP2D6 activity) 20 mg/day. Urine was collected over the next 8 hours for the determination of the dextromethorphan/dextrorphan metabolic ratio. All subjects were classified as extensive metabolizers (EM) with a dextromethorphan/dextrorphan ratio < 0.3. There were no notable changes in the urinary dextromethorphan/dextrorphan ratio in the reboxetine phase as compared to the control session. By contrast, there was a statistically significant increase in the metabolic ratio in the paroxetine phase (p < 0.001), with 4 subjects switching to poor metabolizer (PM) phenotype. These results suggest that reboxetine is unlikely to cause clinically significant interactions with substrates of CYP2D6.

Small effects of valproic acid on the plasma concentrations of clozapine and its major metabolites in patients with schizophrenic or affective disorders.

Two separate studies were carried out to assess the effect of valproic acid on the steady-state plasma concentrations of clozapine and its major metabolites norclozapine and clozapine N-oxide in psychotic patients. In the first study, concentrations of clozapine and metabolites were compared between patients treated with clozapine in combination with sodium valproate (n = 15) and control patients treated with clozapine alone (n = 22) and matched for sex, age, body weight, and antipsychotic dosage. Patients comedicated with valproate tended to have higher clozapine levels and lower norclozapine levels, but the differences did not reach statistical significance. In a subsequent study, plasma concentrations of clozapine and its metabolites were determined in 6 patients with schizophrenia stabilized on clozapine therapy (200-400 mg/d) before and after treatment with sodium valproate (900-1200 mg/d) for 4 weeks. Mean plasma concentrations of clozapine and its metabolites did not change significantly throughout the study, but there was a trend for clozapine levels to be higher and for norclozapine levels to be lower after valproate. Overall, these findings suggest that valproic acid may have an inhibiting effect on the CYP1A2- or CYP3A4-mediated conversion of clozapine to norclozapine. However, the interaction is unlikely to be clinically significant.

Cytochrome P450 2D6 genotype and steady state plasma levels of risperidone and 9-hydroxyrisperidone.

The role of the polymorphic cytochrome P450 2D6 (CYP2D6) in the metabolism of risperidone to its major active metabolite, 9-hydroxyrisperidone (9-OH-risperidone), has been documented after single oral doses of the drug. In this study, the influence of the CYP2D6 polymorphism on the steady-state plasma concentrations of risperidone and 9-OH-risperidone was investigated. Thirty-seven schizophrenic patients on monotherapy with risperidone, 4-8 mg/day, were genotyped by RFLP and PCR for the major functional variants of the CYP2D6 gene. Steady state plasma levels of risperidone and 9-OH-risperidone were analysed by HPLC. Based on the genotype analysis, three patients were classified as ultrarapid metabolizers (UM) with an extra functional CYP2D6 gene, 16 were homozygous extensive metabolizers (EM), 15 heterozygous EM and three poor metabolizers (PM). The median steady-state plasma concentration-to-dose (C/D) ratios of risperidone were 0.6, 1.1, 9.7 and 17.4 nmol/l per mg in UM, homozygous EM, heterozygous EM and PM, respectively, with statistically significant differences between PM and the other genotypes (P < 0.02). The C/D of 9-OH-risperidone also varied widely but was not related to the genotype. The risperidone/9-OH-risperidone ratio was strongly associated with the CYP2D6 genotype, with the highest ratios in PM (median 0.79). Heterozygous EM also had significantly higher ratios than homozygous EM (median value 0.23 versus 0.04; P < 0.01) or UM (median 0.03; P < 0.02). No significant differences were found in the C/D of the sum of the plasma concentrations of risperidone and 9-OH-risperidone between the genotype groups. In conclusion, the steady-state plasma concentrations of risperidone and the risperidone/9-OH-risperidone ratio are highly dependent on the CYP2D6 genotype. However, as risperidone and 9-OH-risperidone are considered to have similar pharmacological activity, the lack of relationship between the genotype and the sum of risperidone and 9-OH-risperidone indicates that the CYP2D6 polymorphism may be of limited importance for the clinical outcome of the treatment.