Impact of Polymorphism of CYP2D6 on Equilibrium Concentration of Duloxetine in Patients Suffering from Major Depressive Disorder

Abstract

Introduction

Duloxetine is commonly prescribed to patients with recurrent depressive disorder. Some part of patients in this group do not respond adequately to treatment regimen containing duloxetine, while many of them experience dose-dependent adverse drug reactions. Previous research investigated that CYP2D6 is involved in the biotransformation of duloxetine, the activity of which is highly dependent on the polymorphism of the gene encoding it.

Objective

The objective of this study was to evaluate the influence of 1846G > A polymorphism of the CYP2D6 gene on the concentration/dose indicator of duloxetine, using findings on enzymatic activity of CYP2D6 (as evaluated by the 6M-THBC/pinoline ratio measurement) and on CYP2D6 expression level obtained by measuring the hsa-miR-370-3p plasma concentration levels in patients suffering from recurrent depressive disorder.

Material and Methods

This study enrolled 118 patients with recurrent depressive disorder (average age – 40.6±17.1 years). Therapy included duloxetine in an average daily dose of 103.7±37.1 mg per day. Treatment efficacy was assessed using the international psychometric scales. Therapy safety was assessed using the UKU Side-Effect Rating Scale. For genotyping we performed the real-time polymerase chain reaction (PCR Real-time). Therapeutic drug monitoring has been performed using HPLC-MS/MS.

Results

Our findings revealed the statistically significant results in terms of the treatment efficacy evaluation (HAMD scores at the end of the treatment course): (GG) 9.0 [7.0; 10.0] and (GA) 11.0 [8.5; 14.0], p < 0.001; at the same time, the statistical significance in the safety profile was obtained (the UKU scores): (GG) 3.0 [3.0; 4.0] and (GA) 4.0 [3.0; 4.0], p = 0.007. We revealed a statistical significance for concentration/dose indicator of duloxetine in patients with different genotypes: (GG) 0.776 [0.529; 1.067] and (GA) 1.388 [0.942; 1.732], p < 0.001.

Conclusion

Thus, the effect of genetic polymorphism of the CYP2D6 gene on the efficacy and safety profiles of duloxetine was demonstrated in a group of 118 patients with recurrent depressive disorder.

Introduction

Depressive disorders are among the most prevalent comorbid disorders in patients suffering from alcoholism.² Antidepressants are used to treat patients with depressive disorders,¹¹ and duloxetine is a representative of this drug group.¹¹ Despite the fact that depressive are included in guidelines for the treatment patients with patients suffering from depressive disorders, the amount of resistant patients and those with dose-dependent adverse drug reactions remains high.¹³

CYP2D6 participates in the pharmacokinetics of numerous medicationsused for therapy of patients suffering from major depressive disorder.⁴ Gene encoding CYP2D6, is highly polymorphic, which can affect the activity of CYP2D6.¹⁰ Changes in activity may affect the rate of metabolism of xenobiotics, including substrate drugs, which, in turn, may have an impact on their efficacy and safety profiles.¹⁷ There are four groups of metabolizers distinguished by their metabolic rates: extensive metabolizers with normal metabolic rate; poor metabolizers having mutations in the CYP2D6, gene, which may cause a decrease in the activity of the encoded protein, as well as a slowdown in the metabolism of substrate drugs, which may probably result in an increased risk of adverse drug reactions; intermediate ones having a mutation in only one of the homologous chromosomes, which reduces the activity of CYP2D6, however, to a lesser degree than in poor metabolizers; ultra-rapid metabolizers having the congenital increased activity of CYP2D6, which ,may lead to the accelerated biotransformation of substrate drugs along with the reduced treatment efficacy.^1,7,16 Consequently, changes in the profiles of the efficacy and safety of duloxetine may depend on the changes in genes, encoded pharmacodynamics and pharmacokinetics elements and, inter alia, CYP2D6.

The objective of this study was to investigate the effect of 1846G>A polymorphism of the CYP2D6 gene on the concentration/dose indicator of duloxetinepatients suffering from recurrent depressive disorder, using findings on activity of CYP2D6 (as evaluated by the 6M-THBC/pinoline ratio measurement) and on CYP2D6 expression level obtained by measuring the hsa-miR-370-3p plasma levelspatients diagnosed with recurrent depressive disorder.

Material and Methods

Clinical Characteristics of the Study Subjects

The study involved 118 male patients (average age — 40.63 ± 17.06 years). The inclusion criteria were the existence of two diagnoses: “Depressive episode (F32.x, according to ICD-10)”, and “Mental and behavioral disorders due to use of alcohol. Dependence syndrome. Currently abstinent but in a protected environment (F.10.212)”; signed informed consent, and 8-weeks duloxetine monotherapy. Exclusion criteria were presence of any other mental disorders; presence of severe somatic disorders (except alcoholic hepatitis and toxic encephalopathy); presence of any other psychotropic medications in treatment regimen; creatinine clearance values <50 mL/min, creatinine concentration in plasma >1.5 mg/dL (133 mmol/L), bodyweight less than 60 kg or greater than 100 kg, age of 75 years or more and presence of any contraindications for duloxetine use.

Therapy Efficacy and Safety Evaluation

In order to assess duloxetine efficacy, several international psychometric scales were used: Hospital Anxiety and Depression Scale (HADS),¹⁸ The assessment of depression states by rating (HAMD), The clinical global impressions scale,³ Psychometric properties of the Penn Alcohol Craving Scale.^5,6 The safety profile was evaluated using the UKU Side-Effect Rating Scale (UKU).⁹ Patients were examined on weeks 1, 4 and 8 of duloxetine therapy.

Genotyping

For genotyping, venous blood samples were collected into VACUETTE® (Greiner Bio-One, Austria) vacuum tubes on the 8 week of duloxetine therapy. The DNA amplifiers “Dtlite” by DNA Technology (Moscow, Russia), CFX96 Touch Real-Time System with CFX Manager software by Bio-Rad Laboratories Inc. (Hercules, CA, USA) and the “SNP-screen” sets by “Syntol” (Moscow, Russia) were used to perform the real-time polymerase chain reaction in order to determine the single-nucleotide polymorphisms (SNPs) 1846G>A of the CYP2D6 (rs3892097) gene. We used two allele-specific hybridizations in every “SNP-screen” set, which have enabled us to determine separately two alleles of the studied SNP on two fluorescence channels.

Therapeutic Drug Monitoring

For TDM, venous blood samples were collected on the week 8th of duloxetine therapy. The plasma calibration standards (St) and quality control samples (QC) were made from a stock solution prepared by consistent dissolving of substantial amounts in methanol with subsequent dilution to the relevant concentrations. Calibration curve was created using 5, 10, 20, 50, 100, 200, 500, 1000, 2000 ng/mL calibration standards along with 5 ng/mL (LLOQ), 15 ng/mL (Low QC), 1000 ng/mL (Medium QC), and 1500 ng/mL (High QC) quality control samples (QC). Diazepam (250 ng/mL in acetonitrile) was used as the internal standard.

Sample Preparation

Samples were prepared using a protein precipitation method. A 1.5 mL tube was filled with 200 mcL of analyzed plasma sample and 600 mcL of acetonitrile containing the internal standard. The mixture was shaken on Vortex for 10 minutes, and then samples were centrifuged at 14,500 g for 10 minutes at 4°C. Then the supernatant was transferred to an autosampler vial. Samples were analyzed using the HPLC system Agilent 1260 (Agilent Technologies, California, USA) and tandem mass selective detector Agilent 6460 (Agilent Technologies, California, USA) with Jet Stream Electrospray Ionization Source.

Conditions of Chromatographic Analysis

Stationary phase: column Agilent Poroshell 120 EC-C18 (2.7 μm, 3.0 mm × 50 mm) with the precolumn InfinityLab Poroshell 120 EC-C18 (2.7 μm, 3.0 mm × 5.0 mm) (Agilent Technologies, California, USA). The column temperature was 50°C. The mobile phase consisted of the A eluent (10 mM ammonium formate in 0.1% formic acid) and B eluent (methanol in 0.1% formic acid). The flow rate was 0.4 mL/min. Gradient elution process was performed; the gradient of the mobile phase is presented in Table 1. The analysis time was 9.0 minutes for every sample. The volume of the inserted sample was 2 mcL. Retention time under the given conditions was 4.75 min for duloxetine and 4.84 min for diazepam.

Table 1. Gradient of the Mobile Phase.

TIME OF ANALYSIS, MIN		ELUENT A VOLUME RATIO, %		ELUENT B VOLUME RATIO, %
0.00		95		5
0.50		95		5
1.00		50		50
1.50		5		95
3.00		5		95
3.01		95		5
5.00		95		5

Conditions of Mass-spectrometry Determination

We used positive mode electrospray ionization for mass-selective detection. Detector registered following MRM-transitions: from 349.0 m/z [M+H+] to 206.1 m/z (collision cell energy 40 V) and from 349.0 m/z [M+H+] to 184.0 m/z (collision cell energy 32 V) for duloxetine; from 285.1 m/z [M+H+] to 193.1 m/z (collision cell energy 32 V) and from 285.1 m/z [M+H+] to 154.1 m/z (collision cell energy 24 V) for diazepam (the internal standard). The voltage on phragmentor for duloxetine and diazepam was 156 V and 166 V, respectively. The voltage on capillary was 3.5 kV, the temperature of desiccant gas was 350°C, nitrogen flow was 6 L/min. Nebulizers pressure was 45 psi, sheath gas temperature was 375°C, sheath gas flow was 11 L/min.

Method Validation

The methodology used in the study met FDA Guidance for Industry: Bioanalytical method validation. Calibration dependence was linear for diapason at 0.5–200 ng/mL. Correlation coefficients were normal (at least 0.99). We evaluated the intra- and inter-cycles precision and accuracy rates. Precision and accuracy rates were normal (no more than 20% at LLOQ, no more than 15% for other points). The matrix effect had no influence.

Local Ethical Committee

The local ethical committee of the Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation approved the research (The protocol No. 6 from 5/16/2017).

Statistical Analysis

Statistical analysis of the results was performed with non-parametric methods using the “Statsoft Statistica v. 10.0” (Dell Statistica, Tulsa, OK, USA). The normality of sample distribution was evaluated using the W-Shapiro-Wilk test and taken into account when choosing a method. The differences were considered statistically significant at p < 0.05 (power above 80%). Two samples of continuous independent data were compared using the Mann-Whitney U-test, which takes into account the abnormal nature of data distribution, with further correction of the obtained p-value using the Benjamin-Hochberg test, due to the multiple comparison procedure. Several samples of continuous data were analyzed using the Kruskal-Wallis H-test. Correlation analysis was performed using the Spearman nonparametric test, taking into account the abnormal nature of sample distribution. Pearson’s Chi2 test for evaluation of the sampling distribution of the alleles (Hardy Weinberg equilibrium) has been used. Research data are presented in the form of the median and interquartile range (Me [Q1; Q3]), or in case of their normal distribution, as the arithmetic mean and standard deviation (Mean ± SD).

Study Results

The CYP2D6 genotyping by polymorphic marker 1846G>A (rs3892097) performed in 118 subjects have revealed the following:

• The amount of patients with the GG genotype was 95 (80.5%);

• The number of patients with the GA genotype was 23 (19.5%);

• There were no subjects with the AA genotype.

Genotype distributions followed a Hardy–Weinberg equilibrium (Chi2 = 1.38, p-value = 0.24).

The results of data analysis performed for psychometric assessments (HADS, HAMD) and side-effect rating scale (UKU) on weeks 1, 4 and 8 in patients who were administered duloxetine can be found in Table 2.

Table 2. Data from the Psychometric Assessments and Side-Effect Rating Scale in Patients who Received Duloxetine, on Weeks 1, 4 and 8 of the Study.

SCALE		GG		GA		P-VALUE
Week 1
HADS		37.0 [36.0; 39.0]		36.0 [35.0; 38.0]		0.098
HAMD		22.0 [22.0; 23.0]		22.0 [21.0; 22.0]		0.384
UKU		0.0 [0.0; 0.0]		0.0 [0.0; 0.0]		0.281
Week 4
HADS		22.0 [19.0; 24.0]		25.0 [22.5; 27.0]		<0.001
HAMD		14.0 [12.0; 15.0]		14.0 [13.0; 15.0]		0.211
UKU		3.0 [2.0; 3.0]		3.0 [1.0; 5.0]		0.351
Week 8
HADS		16.0 [11.0; 21.5]		19.0 [15.5; 23.0]		0.123
HAMD		9.0 [7.0; 10.0]		11.0 [8.5; 14.0]		<0.001
UKU		3.0 [3.0; 4.0]		4.0 [3.0; 4.0]		0.007

p – p-value obtained in Benjamini-Hochberg multiple testing correction (based on the results of Mann-Whitney U test), Data are presented as Me and IQR.

Dynamics of changes in HAMD scale scores among the patients with different genotypes are demonstrated in Figure 1 the compared groups hadn’t statistically significant differences: (GG) 22.0 [22.0; 23.0], (GA) 22.0 [21.0; 22.0], p = 0.384. By week 4 statistically significant differences were not observed, as well as like on the 1st visit in patients with different genotypes: (GG) 14.0 [12.0; 15.0], (GA) 14.0 [13.0; 15.0], p = 0.211. On the last 8th week of the study, statistically significant differences remained: (GG) 9.0 [7.0; 10.0], (GA) 11.0 [8.5; 14.0], p < 0.001. For other psychometric scales, the same dynamics of changes in scores as in the HAMD scale was obtained.

Figure 1.

Dynamics of Changes in the HAMD and UKU Scales Scores Across Patients with Different Genotypes by the Polymorphic Marker 1846G>A Gene CYP2D6 (rs3892097)

Data are presented as Me and IQR (colored lines connect the medians on different week of the study).

Dynamics of changes in UKU scores among the patients are presented at Figure 1. As can be seen, on week 1 of the research the compared groups had no statistically significant differences: (GG) 0.0 [0.0; 0.0], (GA) 0.0 [0.0; 0.0], p = 0.281. By week 4, a statistically significant differences were not observed, as well as like on the 1st visit: (GG) 3.0 [2.0; 3.0], (GA) 3.0 [1.0; 5.0], p = 0.211. A statistically significant difference remained on week 8 of therapy: (GG) 3.0 [3.0; 4.0], (GA) 4.0 [3.0; 4.0], p = 0.007.

Table 3 summarizes the data on concentration/dose ratio (C/D) values obtained for duloxetine through pharmacokinetic studies in terms of quantitative and nominal units. We revealed a statistical significance for C/D indicator in patients with different genotypes: (GG) 0.776 [0.529; 1.067], (GA) 1.388 [0.942; 1.732], p < 0.001 (Figure 2).

Table 3. 6M-THBC/Pinoline in Patients with Different Genotypes by the Polymorphic Marker 1846G>A Gene CYP2D6 (rs3892097).

PARAMETER		GG		GA		P-VALUE
THBC/Pinoline ratio (n.u)		7.55 [6.36; 10.03]		6.54 [5.12; 7.75]		0.009
Pinoline (pg/ml)		1427.60 [1069.88; 1788.85]		1547.34 [1259.55; 1747.91]		0.415
6M-THBC (pg/ml)		10580.34 [7091.31; 15765.41]		8832.02 [6813.43; 11897.04]		0.221

p – p-value obtained in Benjamini-Hochberg multiple testing correction (based on the results of Mann-Whitney U test), Data are presented as Me and IQR.

Figure 2.

Differences in CYP2D6 Enzymatic Activity Rates (as Evaluated by the 6M-THBC/Pinoline Ratio Measurement) Across Patients with Different Genotypes by the Polymorphic Marker 1846G>A of the CYP2D6 Gene (rs3892097)

Data are presented as Me and IQR.

Discussion

The findings of this study revealed a statistically significant difference between the values of duloxetine equilibrium concentration in patients with different genotypes of the CYP2D6 gene by polymorphic marker 1846G>A (rs3892097): patients carrying the A allele have a lower level of drug equilibrium concentration than those with the G allele (p < 0.001). This appears to be due to the reduced biotransformation and elimination rates of duloxetine in patients carrying the G allele, which in turn leads to a cumulation of the drug in plasma. It may lead to an increased risk of adverse drug reactions and pharmacoresistance, as we have concluded in our previous research.

Statistical analysis of the data on the clinical efficacy profile of duloxetine in patients with different genotypes by the polymorphic marker 1846G>A of the CYP2D6 gene (rs3892097) also revealed the statistically significant differences in the efficacy rates (p < 0.001). The analysis of duloxetine safety data also revealed the existence of a statistically significant difference (p = 0.007). The value of these parameters is statistically significantly higher for carriers of the minor allele than for carriers of the dominant allele. This may indicate that the carriage of this polymorphic marker may lead to an increased risk of the adverse drug reactions of duloxetine in comparison with carriers of nonmutant alleles, which is probably due to a reduction of the activity of CYP2D6 in these patients, the substrate of which is duloxetine, a decrease in the rate of its biotransformation and elimination, and accumulation in plasma level of the drug.

Thus, based on the results obtained that the genetic polymorphism 1846G>A affects the efficacy and safety rates of duloxetine therapy in patients with recurrent depressive disorder, it is possible to assume that before prescribing duloxetine to such patients it is necessary to take into account the results of genotyping by the loci of this gene. According to the results of our earlier studies, it was also shown that polymorphism of the CYP2D6 gene should be taken into account when administering duloxetine, as this may have an impact on the efficacy and safety profile of duloxetine in this category of patients.^8,12,14,15

Funding

This work was financially supported by the project 16-15-00227 entitled “Fundamental research and exploratory research in priority areas of research” of the Russian Science Foundation.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Conclusion

The effect of genetic polymorphism of the CYP2D6 gene on the efficacy and safety profiles of duloxetine was demonstrated in a group of 118 patients with recurrent depressive disorder.