Carmen Belmonte

Polymorphisms associated with etanercept response in moderate-to-severe plaque psoriasis

AIM Few studies have evaluated the influence of pharmacogenetics in psoriatic patients treated with etanercept. MATERIALS & METHODS We evaluated the association between 124 polymorphisms with the response to etanercept in patients with moderate-to-severe plaque psoriasis at 3 months (n = 78) and 6 months of treatment (n = 68). RESULTS The results of the multivariate analysis showed an association between polymorphisms rs13437088 (HLA-B/MICA), rs96844 (MAP3K1), rs2431697 (PTTG1), rs9304742 (ZNF816A) and the response to etanercept at 3 months. Besides polymorphisms rs928655 (GBP6) and rs2546890 (IL12B) were associated to response at 6 months. CONCLUSIONS Nevertheless, these biomarkers should be validated in large-scale studies before its implementation in clinical practice.

Effective phospholipids removing microelution-solid phase extraction LC-MS/MS method for simultaneous plasma quantification of aripiprazole and dehydro-aripiprazole: Application to human pharmacokinetic studies

Graphical abstract Figure. No caption available. HighlightsMass spectrometry to monitor aripiprazole and its metabolite plasma levels.Fast microelution solid phase extraction as sample preparation method.Effective plasma phospholipids removal.Method validated according to the recommendations of regulatory agencies.Application to the pharmacokinetic study of healthy volunteers. ABSTRACT A simple liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro‐aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole‐D8, has been used as the internal standard (IS) for both analytes. Only 200 &mgr;l of human plasma was needed for analyte extraction, using effective phospholipids‐eliminating three‐step microelution‐solid‐phase extraction (SPE, Oasis PRiME HLB 96‐well &mgr;Elution Plate). An ACE C18‐PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2‐&mgr;m on‐line filter. A combination of ammonium formate (5 mM)‐acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re‐equilibration time of 3 min, to give a total run time of 8 min. Five &mgr;l of the sample was injected into the chromatographic system. Aripiprazole, dehydro‐aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18–110 ng/ml and 0.35–100 ng/ml for aripiprazole and dehydro‐aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry‐over. Our microelution‐SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies.

Evaluation of the relationship between pharmacokinetics and the safety of aripiprazole and its cardiovascular effects in healthy volunteers

Aims The aim of this study was the evaluation of the possible relationship between pharmacokinetics and the safety of aripiprazole as well as its influence on blood pressure (BP), heart rate (HR), and corrected QT (QTc) interval. Methods The study population comprised 157 healthy volunteers from 6 bioequivalence clinical trials. Subjects were administered a single 10-mg oral dose of each formulation separated by a 28-day washout period. Plasma concentrations were measured using high-performance liquid chromatography coupled to mass spectrometry. Blood pressure was measured at the following times: predose and 0.5, 2, 4, 6, and 8 hours postdose. An electrocardiogram was recorded at predose, 4, and 8 hours postdose. Results Area under the curve (AUC), maximum plasma concentration, half-life, and distribution volume corrected for weight were higher in women. Aripiprazole treatment produced a decrease of BP (9.3 mm Hg on systolic and 6.2 mm Hg on diastolic pressure) and an increase in HR (12.1 beats per minute) and QTc interval (9.1 milliseconds). There were sex differences in BP, HR, and QTc interval. Women and subjects with higher AUC and maximum plasma concentration values were more prone to experience adverse drug reactions and gastrointestinal adverse reactions. The AUC was related with systolic BP and diastolic BP decrease and HR increase but there was no relationship between aripiprazole concentrations and QTc increase. Conclusions Aripiprazole decreases BP and increases HR and QTc interval. Pharmacokinetics, pharmacodynamics, and safety of aripiprazole are affected by sex. There is a directly proportional relationship between pharmacokinetic parameters and adverse drug reactions and effect on BP and HR.

Influence of CYP2D6,CYP3A4,CYP3A5 and ABCB1 Polymorphisms on Pharmacokinetics and Safety of Aripiprazole in Healthy Volunteers

The aim of this study was to investigate the effect of polymorphisms in cytochrome P450 (CYP) 2D6, CYP3A4 and CYP3A5 enzymes and in P‐glycoprotein (P‐gp) on the pharmacokinetics and safety of aripiprazole and, its active metabolite, dehydro‐aripiprazole, in 148 healthy volunteers from six bioequivalence trials receiving a single oral dose of aripiprazole. The plasma concentrations of both analytes were measured by LC‐MS/MS. CYP2D6 (*3,*4,*5,*6,*7,*9 and copy number variations), CYP3A4 (*20 and *22), CYP3A5*3 and C3435T, C1236T and G2677T/A in ABCB1 gene were determined. As the number of active CYP2D6 alleles decreased, AUC0−t, Cmax and t1/2 of aripiprazole were higher and clearance of aripiprazole, AUC0−t of dehydro‐aripiprazole and ratio dehydro‐aripiprazole/aripiprazole were lower. AUC0−t of aripiprazole of poor metabolizer (PM) subjects was increased by 50% compared to extensive metabolizers (EM), and AUC0−t of dehydro‐aripiprazole was decreased by 33%. ABCB1 1236TT subjects had a lower clearance of aripiprazole (p = 0.023) and AUC0−t (p = 0.039) and Cmax of dehydro‐aripiprazole (p = 0.036) compared to C/C. CYP3A5*3/*3 subjects had a 10% lower ratio dehydro‐aripiprazole/aripiprazole than *1/*3 (p = 0.019). Adverse drug reactions (ADRs) had a directly proportional relationship with AUC0−t of aripiprazole (p = 0.001), especially nausea/vomiting, which were more common in women (p = 0.005). Women and CYP3A5*1/*1 subjects showed more often dizziness (p = 0.034; p = 0.009). Pharmacokinetics of aripiprazole is affected by CYP2D6 phenotype but also by sex and C1236T (ABCB1 gene), while dehydro‐aripiprazole pharmacokinetics is affected by CYP2D6 and C1236T. The ratio dehydro‐aripiprazole/aripiprazole was influenced by CYP2D6 phenotype and CYP3A5*3. Concentrations of aripiprazole, sex, CYP3A5*3 and CYP2D6 were involved in the development of ADRs.

Effect of Polymorphisms on the Pharmacokinetics, Pharmacodynamics and Safety of Sertraline in Healthy Volunteers

Sertraline is a selective serotonin reuptake inhibitor widely metabolized in the liver by cytochrome P450 (CYP) enzymes. Besides, it is a P‐glycoprotein substrate. Moreover, serotonin transporters and serotonin receptors are involved in its efficacy and safety. The aim of this study was to evaluate the role of polymorphisms of metabolizing enzymes, transporters and receptors on the pharmacokinetics, pharmacodynamics and tolerability of sertraline in healthy volunteers. Forty‐six healthy volunteers (24 men and 22 women) receiving a 100‐mg single oral dose of sertraline were genotyped for 17 genetic variants of CYP enzymes (CYP2B6, CYP2C9, CYP2C19, CYP2D6), ATP‐binding cassette subfamily B member 1 (ABCB1), solute carrier family 6 member 4 (SLC6A4), 5‐hydroxytryptamine receptor 2A (HTR2A) and 5‐hydroxytryptamine receptor 2C (HTR2C) genes. Pharmacokinetic and pharmacodynamic parameters were similar in men and women. Polymorphisms in CYP2C19 and CYP2B6 genes influenced sertraline pharmacokinetics, with a greater effect of CYP2C19. Individuals carrying defective alleles for CYP2C19 and CYP2B6 showed higher area under the curve (AUC) and half‐life (T1/2). Moreover, CYP2C19*17 was related to a decreased AUC and T1/2. No significant effect was found for polymorphisms in CYP2C9, CYP2D6 and ABCB1 on sertraline pharmacokinetics. Sertraline had a small heart rate‐lowering effect, directly related to maximum concentration (Cmax) and the presence of ABCB1 minor alleles. Sertraline had no significant effect on blood pressure and QTc. There was a tendency to present more adverse drug reactions in women and individuals with higher AUC of sertraline, such as CYP2C19 intermediate metabolizers and CYP2B6 G516T T/T individuals.

Pharmacogenetics of trazodone in healthy volunteers: association with pharmacokinetics, pharmacodynamics and safety

AIM The aim was to evaluate the effect of polymorphisms in metabolizing enzymes and transporters on the pharmacokinetics, pharmacodynamics and adverse effects of trazodone in healthy volunteers. MATERIALS & METHODS 36 healthy volunteers receiving a single 100-mg oral dose of trazodone were genotyped for 11 variants in CYP3A4, CYP3A5, CYP2D6 and ABCB1 by real-time PCR. Plasma concentrations were measured using liquid chromatography-tandem mass spectrometry method. RESULTS & CONCLUSION Sex affected the pharmacokinetics of trazodone with higher clearance in women. Polymorphisms in ABCB1, but not in CYP3A or CYP2D6, influenced trazodone pharmacokinetics. Trazodone decreased blood pressure and prolonged the corrected QT interval interval. CYP2D6 and ABCB1 polymorphisms were associated with the incidence of dizziness and prolonged corrected QT interval, respectively. Subjects with adverse drug reactions had lower concentrations of trazodone suggesting its metabolite (m-chlorophenylpiperazine) could be responsible for these effects.

Influence of CYP2C19 Phenotype on the Effect of Clopidogrel in Patients Undergoing a Percutaneous Neurointervention Procedure

This observational retrospective study assessed the antiplatelet response and clinical events after clopidogrel treatment in patients who underwent percutaneous neurointervention, related to CYP2C19 metabolizer status (normal (NM), intermediate/poor (IM‐PM), and ultrarapid (UM); inferred from *2, *3, and *17 allele determination). From 123 patients, IM‐PM had a higher aggregation value (201.1 vs. 137.6 NM, 149.4 UM, P < 0.05) and lower response rate (37.5% vs. 69.8% NM, 61.1% UM), along with higher treatment change rate (25% vs. 5.7% NM, 10.5% UM). The highest ischemic events incidence occurred in NM (11.3% vs. 6.3% IM, 10.5% UM) and hemorrhagic events in UM (13.2% vs. 0% IM and 3.8% NM). No differences were found regarding ischemic event onset time, while hemorrhagic event frequency in UM was higher with shorter onset time (P = 0.047). CYP2C19 no‐function and increased function alleles defined the clopidogrel response. UM patients had increased bleeding risk. Therapeutic recommendations should include dose reduction or treatment change in UM.

Effects of aripiprazole on pupillometric parameters related to pharmacokinetics and pharmacogenetics after single oral administration to healthy subjects

Background: Pupillometry is used for the detection of autonomic dysfunction related to numerous diseases and drug administration. Genetic variants in cytochrome P450 (CYP2D6, CYP3A4), dopamine receptor (DRD2, DRD3), serotonin receptor (HTR2A, HTR2C) and ATP-binding cassette subfamily B (ABCB1) genes were previously associated with aripiprazole response. Aims: Our aim was to evaluate if aripiprazole affects pupil contraction and its relationship with pharmacokinetics and pharmacogenetics. Methods: Thirty-two healthy volunteers receiving a 10 mg single oral dose of aripiprazole were genotyped for 15 polymorphisms in ABCB1, CYP2D6, DRD2, DRD3, HTR2A and HTR2C genes by reverse transcription polymerase chain reaction. Aripiprazole and dehydro-aripiprazole plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Pupil examination was performed by automated pupillometry. Results: Aripiprazole caused pupil constriction and reached the peak value at Cmax. HTR2A rs6313 T allele carriers and HTR2C rs3813929 C/T subjects showed higher maximum constriction velocity and maximum pupil diameter. Besides, Gly/Gly homozygotes for DRD3 rs6280 showed significantly lower maximum constriction velocity values. A/G heterozygotes for DRD2 rs6277 showed higher total time taken by the pupil to recover 75% of the initial resting size values. CYP2D6 intermediate metabolisers showed higher area under the curve, Cmax and T1/2 than extensive metabolisers. ABCB1 G2677T/A A/A homozygotes had greater T1/2 in comparison with C/C homozygotes. ABCB1 C3435T T allele carriers and C1236T C/T subjects showed greater area under the curve than C/C homozygotes. Conclusions: Aripiprazole affects pupil contraction, which could be a secondary effect through dopamine and serotonin receptors. Pupillometry could be a useful tool to assess autonomic nervous system activity during antipsychotic treatment.

Effect of ABCB1 C3435T Polymorphism on Pharmacokinetics of Antipsychotics and Antidepressants

P‐glycoprotein, encoded by ABCB1, is an ATP‐dependent drug efflux pump which exports substances outside the cell. Some studies described connections between C3435T polymorphism T allele and lower P‐glycoprotein expression; therefore, homozygous T/T could show higher plasma levels. Our aim was to evaluate the effect of C3435T on pharmacokinetics of 4 antipsychotics (olanzapine, quetiapine, risperidone and aripiprazole) and 4 antidepressants (trazodone, sertraline, agomelatine and citalopram). The study included 473 healthy volunteers receiving a single oral dose of one of these drugs, genotyped by real‐time PCR. Multivariate analysis was performed to adjust the effect of sex and genotype of the main cytochrome P450 enzymes. C3435T polymorphism had an effect on olanzapine pharmacokinetics, as T/T individuals showed lower clearance and volume of distribution. T/T individuals showed lower T1/2 of 9‐OH‐risperidone, but this difference disappeared after multivariate correction. T/T homozygous individuals showed lower dehydro‐aripiprazole and trazodone area under the concentration‐time curve, along with lower half‐life and higher clearance of trazodone. C/T genotype was associated to higher citalopram maximum concentration. C3435T had no effect on quetiapine, sertraline or agomelatine pharmacokinetics. C3435T can affect the elimination of some drugs in different ways. Regarding risperidone, trazodone and dehydro‐aripiprazole, we observed enhanced elimination while it was reduced in olanzapine and citalopram. However, in quetiapine, aripiprazole, sertraline and agomelatine, no changes were detected. These results suggest that P‐glycoprotein polymorphisms could affect CNS drugs disposition, but the genetic factor that alters its activity is still unknown. This fact leads to consider the analysis of ABCB1 haplotypes instead of individual variants.

Influence of tpmt polymorphisms in mercaptopurine pharmacokinetics in healthy volunteers

Mercaptopurine is a drug commonly used in the treatment of different types of cancer, especially acute lymphoblastic leukaemia, and autoimmune diseases such as ulcerative colitis or Crohns disease and in patients receiving organ transplants. It is metabolized by three cytosolic enzymes. One of them, thiopurine S‐methyltransferase (TPMT), is responsible for catalysing the methylation reaction of mercaptopurine to 6‐methylmercaptopurine, thus inactivating the drug. Individuals with TPMT loss‐of‐function alleles (*2, *3A, *3B or *3C) can be extremely sensitive to the effect of mercaptopurine, since it can be accumulated, therefore producing haematological toxicity. The objective of this study was to evaluate the role of TPMT polymorphisms on the pharmacokinetics of mercaptopurine. For that purpose, we used collected pharmacokinetic data from 48 healthy volunteers (all males) who received a single oral dose of mercaptopurine 50 mg in two bioequivalence studies. The volunteers were subsequently genotyped for TPMT *2, *3A, *3B and *3C alleles by real‐time PCR. There were four carriers (8.3%) of TPMT*2 and TPMT*3A alleles. Mercaptopurine elimination was affected by TPMT loss‐of‐function polymorphisms, since heterozygous subjects show 18% higher half‐life compared to wild‐type individuals. This fact is consistent with the expected since the presence of loss‐of‐function alleles decreases TPMT enzymatic activity and, thus, affects mercaptopurine elimination. Moreover, mercaptopurine pharmacokinetic parameters were different among races, since Latins showed higher plasma concentrations and lower clearance compared to Caucasians. This fact might be due to a different distribution of polymorphisms in genes, other than TPMT, that also influence the pharmacokinetics of mercaptopurine.