Therapeutic drug monitoring of rivastigmine and donepezil under consideration of CYP2D6 genotype-dependent metabolism of donepezil

Background: The efficacy of acetylcholinesterase inhibitors (AChE-I) might depend on blood concentration. While rivastigmine metabolism is independent of the cytochrome P450 system, its isoenzymes, especially CYP2D6, metabolize donepezil. CYP2D6 polymorphisms can cause altered enzyme activity resulting in lower or higher than expected drug concentrations of donepezil. Objective: We investigated correlations between clinical efficacy and serum concentrations of rivastigmine and donepezil under special consideration of CYP2D6 genotype or gene dose-dependent metabolism of donepezil. Methods: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR) and allele-specific PCR were performed to assess CYP2D6 genotype and gene dose. Results: Patients treated with rivastigmine (n=28) or donepezil (n=48) were included in the study. Both gene dose and metabolism type significantly predicted the level of donepezil serum concentration (p=0.019 and p=0.013, respectively). In the rivastigmine group, changes of the word list delayed recall subtest before treatment and under stable medication were significantly associated with rivastigmine serum levels (beta=0.465;p=0.018). Drug serum concentrations were outside the recommended range in a substantial percentage of participants, which might have contributed to poor correlations between changes in cognitive measures and drug concentrations. Donepezil serum concentrations significantly depended on CYP2D6 gene dose. Conclusion: Testing AChE-I serum concentration should be considered in patients without clinical response to treatment or those with severe side effects. Patients with donepezil drug levels outside the recommended range might additionally profit from CYP2D6 genotyping or treatment with an AChE-I independent of CYP metabolism

Serum Concentrations of Cholinesterase Inhibitors in Patients With Alzheimer's Dementia Are Frequently Below the Recommended Levels

Background: Acetylcholinesterase inhibitors (AChE-I) are recommended for the treatment of cognitive symptoms but also of behavioral and psychological symptoms in dementia. They are widely used not only in Alzheimer's disease, but also in other forms of dementia. Efficacy of treatment might depend on serum concentration of the respective AChE-I. Objective: In patients with mild to moderate Alzheimer's dementia, we measured serum concentrations of hepatically metabolized donepezil and renally excreted rivastigmine and investigated possible modifiers. Additionally, we looked at correlations between serum concentrations and efficacy for both drugs. Methods: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR). Allele specific PCR were performed to determine CYP2D6 genotype and gene dose. Clinical efficacy was assessed by changes of the subtest wordlist delayed recall of the Consortium to Establish a Registry for Alzheimer's Disease-Neuropsychological Assessment Battery (CERAD-NAB). Results: Sixty-seven patients treated with a stable dosage of donepezil 10 mg (n=41) or rivastigmine 9.5 mg (n=26) were included. Mean serum concentration of donepezil and rivastigmine were 41.2 and 6.5 ng/ml, respectively. Serum concentrations were below the recommended range in 73% of the subjects in the donepezil group and in 65% of the participants in the rivastigmine group. When applying a dose-related reference, ranges 63% of patients in the donepezil group and 32% in the rivastigmine group had concentrations below the expected range. Gene dose, sex, and duration of treatment significantly predicted donepezil serum concentration (p=0.046, p=0.001, p=0.030 respectively). Only for rivastigmine did the serum concentration significantly contribute to the regression model predicting changes on the subtest word list delayed recall (β=0.472; p=0.019). Conclusions: Serum concentrations of about two thirds of the patients were below the recommended range. When not looking at absolute values but at the dose-related reference ranges, these numbers improved but still 32%, respectively 63% of patients had low serum concentrations. High serum concentrations of rivastigmine predicted clinical response to cognition. Therapeutic drug monitoring might help to identify the cause of poor clinical response to cognition and behavioral and psychological symptoms in patients with AChE-I treatment

gDNA extraction yield and methylation status of blood samples are affected by long-term storage conditions.

Epigenetics is believed to provide great chances for a better understanding of the development and treatment of many diseases where the analysis of genomic DNA has so far failed to provide conclusive answers. Methylcytosine is a frequently used quantitative marker of epigenetic studies. Since immediate analysis of sampled material is in most cases not possible, storage time and conditions are critical aspects regarding the quality of genomic DNA and reliability of analysis. Blood is frequently used for such analyses. We, therefore, collected blood samples of ten volunteers and stored them under various conditions for ten months: -70°C, -20°C, 2-8°C and room temperature. An additional aliquot was frozen at -70°C and thawed once a week at room temperature. We then compared the DNA extraction yields and methylation status in relation to storage time and conditions. We found significantly lower DNA extraction yields (up to -97.45%; p ≤ 0.001) as well as significantly higher methylation levels after ten months of storage (up to +42.0%; p ≤ 0.001). These results suggest that storage time has an important influence on DNA analyses of blood samples for all storage conditions. This might be due to differences in stability of methylated and non-methylated DNA. Our study indicates that storage conditions and time may be a critical factor for epigenetic methylation studies and require rigorous validation. For reliable analyses we, therefore, recommend to perform epigenetic analysis directly after sample collection

Bland-Altman plots of the measured methylation levels according to the different storage conditions.

<p>The differences between the measured values at the time T₁ (green), T₃ (yellow) and T₁₀ (red) and the start time T₀ and the corresponding mean are plotted. Further the mean differences (M; solid lines) and the 95% confidence intervals (M ± 2SD, doted lines) are shown. For all storage conditions there is a shift toward higher methylation levels after prolonged storage time.</p

Mean relative gDNA extraction yields and standard deviations (SD) according to different storage conditions and storage times.

<p>The relative values were calculated with regard to the gDNA extraction yield at T₀ directly after sample collection: The gDNA yields for all storage conditions were decreasing. After ten month of storage the yields of gDNA for all storage conditions were significantly less compared to time T₀ (p ≤ 0.001).</p

Overview of the mean methylation levels and absolute changes (dif abs.) in methylation compared to T₀ for the sum of all CpG sites after storage for one, three and ten months as well as p-values.

<p>Methylation levels for all storage conditions are significantly increasing with storage time. Only blood samples stored at -70°C for one month show in mean lower methylation levels compared to T₀, however, this correlation was not significant.</p

Mean methylation levels ± SD for the sum of all CpG sites at the start time T₀ and after storage for one, three and ten months.

<p>Methylation status for all storage conditions are significantly increasing with storage time. Only blood samples stored at -70°C for one month show in mean lower methylation levels compared to T₀, however, this correlation was not significant.</p

Therapeutic Drug Monitoring of Rivastigmine and Donepezil Under Consideration of CYP2D6 Genotype-Dependent Metabolism of Donepezil

Background: The efficacy of acetylcholinesterase inhibitors (AChE-I) might depend on blood concentration. While rivastigmine metabolism is independent of the cytochrome P450 system, its isoenzymes, especially CYP2D6, metabolize donepezil. CYP2D6 polymorphisms can cause altered enzyme activity resulting in lower or higher than expected drug concentrations of donepezil. Objective: We investigated correlations between clinical efficacy and serum concentrations of rivastigmine and donepezil under special consideration of CYP2D6 genotype or gene dose-dependent metabolism of donepezil. Methods: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR) and allele-specific PCR were performed to assess CYP2D6 genotype and gene dose. Results: Patients treated with rivastigmine (n=28) or donepezil (n=48) were included in the study. Both gene dose and metabolism type significantly predicted the level of donepezil serum concentration (p=0.019 and p=0.013, respectively). In the rivastigmine group, changes of the word list delayed recall subtest before treatment and under stable medication were significantly associated with rivastigmine serum levels (beta=0.465;p=0.018). Drug serum concentrations were outside the recommended range in a substantial percentage of participants, which might have contributed to poor correlations between changes in cognitive measures and drug concentrations. Donepezil serum concentrations significantly depended on CYP2D6 gene dose. Conclusion: Testing AChE-I serum concentration should be considered in patients without clinical response to treatment or those with severe side effects. Patients with donepezil drug levels outside the recommended range might additionally profit from CYP2D6 genotyping or treatment with an AChE-I independent of CYP metabolism