Angela Avenoso

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

Abstract 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.

Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats

To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-induced arthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 μl of emulsion containing bovine type II collagen in complete Freunds adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting; high tumour necrosis factor-α (TNF-α) plasma levels; and synovial neutrophil accumulation. Treatment with HYA and C4S, starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase, decreased plasma TNF-α levels, and limited synovial neutrophil infiltration. These data confirm that erosive destruction of the joint cartilage in CIA is due at least in part to free radicals released by activated neutrophils and produced by other biochemical pathways. The beneficial effects obtained with the treatment suggest that HYA and C4S could be considered natural endogenous macromolecules to limit erosive damage in CIA or as a useful tool with which to study the involvement of free radicals in rheumatoid arthritis.

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

Abstract 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 (rs=0.371, P<0.02), but not for norclozapine (rs=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.

Effect of fluvoxamine on the pharmacokinetics of imipramine and desipramine in healthy subjects.

The effect of the selective serotonin reuptake inhibitor fluvoxamine (100 mg/day for 10 consecutive days) on the kinetics of a single oral dose of imipramine (50 mg) and desipramine (100 mg) was investigated in 12 healthy subjects. Compared with a control session, treatment with fluvoxamine caused a significant prolongation of imipramine half-life (from 22.8 ± 6.4 to 40.5 ± 5.0 h, means ± SD, p < 0.01) and a marked decrease in imipramine apparent oral clearance (from 1.02 ± 0.19 to 0.28 ± 0.06 L/h/kg, p < 0.0001). No significant changes in desipramine kinetics were observed during fluvoxamine treatment. These findings indicate that, at the dosage tested, fluvoxamine markedly inhibits the demethylation of imipramine without affecting significantly the CYP2D6-mediated hydroxylation of desipramine.

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.

Inhibition of risperidone metabolism by fluoxetine in patients with schizophrenia: A clinically relevant pharmacokinetic drug interaction

The effect of fluoxetine on the steady-state plasma concentrations of risperidone and its active metabolite 9-hydroxyrisperidone (9-OH-risperidone) was evaluated in 10 patients with schizophrenia or schizoaffective disorder. Patients stabilized on risperidone (4–6 mg/day) received additional fluoxetine (20 mg/day) to treat concomitant depression. One patient dropped out after 1 week due to the occurrence of akathisia associated with markedly increased plasma risperidone concentrations. In the other subjects, mean plasma concentrations of risperidone increased during fluoxetine administration from 12 ± 9 ng/mL at baseline to 56 ± 31 at week 4 (p < 0.001), while the levels of 9-OH-risperidone were not significantly affected. After 4 weeks of combined treatment, the levels of the active moiety (sum of the concentrations of risperidone and 9-OH-risperidone) increased by 75% (range, 9–204%, p < 0.01) compared with baseline. The mean plasma risperidone/9-OH-risperidone ratio also increased significantly. During the second week of adjunctive therapy, two patients developed Parkinsonian symptoms, which were controlled with anticholinergic medication. These findings indicate that fluoxetine, a potent inhibitor of the cytochrome P450 enzyme CYP2D6 and a less potent inhibitor of CYP3A4, reduces the clearance of risperidone by inhibiting its 9-hydroxylation or alternative metabolic pathways. This interaction may lead to toxic plasma risperidone concentrations. In addition to careful clinical observation, monitoring plasma risperidone levels may be of value in patients given adjunctive therapy with fluoxetine.

Relationship between plasma desipramine levels, CYP2D6 phenotype and clinical response to desipramine : a prospective study

ObjectiveThe clinical relevance of the CYP2D6 oxidation polymorphism in the treatment of depression with desipramine (DMI) was studied prospectively in depressed outpatients.MethodsAfter CYP2D6 phenotype determination with dextromethorphan, 31 patients were treated with oral DMI at a dosage of 100 mg per day for 3 weeks. At the end of the 3rd week of treatment, severity of depressive symptoms was assessed by the Hamilton Depression Rating Scale and steady-state plasma concentrations of DMI and its metabolite 2-hydroxydesipramine (2-OH-DMI) were measured by high-performance liquid chromatography (HPLC).ResultsPlasma DMI levels were significantly correlated with dextromethorphan metabolic ratio. The two patients with the poor metabolizer phenotype showed the highest plasma concentrations of DMI and complained of severe adverse effects, requiring dosage reduction. No significant correlation was found between plasma levels of either DMI or DMI plus 2-OH-DMI and antidepressant effect.ConclusionThese findings indicate that the dextromethorphan metabolic ratio has a great impact on steady-state plasma levels of DMI in depressed patients and may identify subjects at risk for severe concentration-dependent adverse effects. On the other hand, this index of CYP2D6 activity does not seem to predict the degree of clinical amelioration.

Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) -mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-alpha and IL-1beta levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation.

Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes

Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the cluster determinant 44 (CD44) receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-kappaB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1beta). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes. Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-kappaB translocation and increased the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta. The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected. The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression.

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.