Peter J. Wedlund

The cytochrome P450 2D6 (CYP2D6) enzyme polymorphism: Screening costs and influence on clinical outcomes in psychiatry

This study examined factors that affect cost, reliability, and the value of determining the cytochrome P450 2D6 (CYP2D6) polymorphism in clinical practice.

The CYP2C19 Enzyme Polymorphism

The genetic test is gradually replacing probe drugs as the primary tool for screening populations for the CYP2C19 polymorphism. A full appreciation for the clinical and toxicological relevance of this genetic variation is presently limited. Further research is needed in several areas. The development and use of 3-D models of the CYP2C19 enzyme to automate and increase the rate at which CYP2C19 substrates are identified could reap great benefits. Meanwhile, clinical research should begin to determine whether the CYP2C19 polymorphism affects therapeutic outcomes and toxicity of drugs in actual patient settings. Combining research efforts in molecular modeling, genetic testing, clinical and epidemiological research will be required if better appreciation of this genetic variation and its importance in the population at large is to emerge.

Dextromethorphan: Enhancing its systemic availability by way of low‐dose quinidine‐mediated inhibition of cytochrome P4502D6

There has been a substantial amount of interest in the anticonvulsant and neuroprotective actions of dextromethorphan. Its therapeutic efficacy, however, is limited by its extensive first‐pass elimination by way of the cytochrome P4502D6 enzyme in humans. The purpose of this research was to determine whether quinidine (a selective inhibitor of cytochrome P4502D6) could improve dextromethorphan systemic delivery in patients with amyotrophic lateral sclerosis (a neurodegenerative disease). In the absence of quinidine, 60 mg dextromethorphan every 12 hours resulted in plasma concentrations of only 12 ± 13 ng/ml (range, <5 to 40 ng/ml; n = 7). The same dose of dextromethorphan in the presence of 75 mg quinidine every 12 hours resulted in dextromethorphan plasma concentrations of 241 ± 94 ng/ml (range, 157 to 402 ng/ml; n = 5). The achievement of higher dextromethorphan plasma concentrations was also associated with an increased occurrence of adverse effects in some patients. Based on the brain/plasma ratio for dextromethorphan in rats, it is estimated that brain dextromethorphan concentrations of 1.0 to 10 µg/gm may be attainable in humans by inhibition of cytochrome P4502D6 activity with quinidine.

CYP2D6 genetic variation in healthy adults and psychiatric African-American subjects: implications for clinical practice and genetic testing.

Limited information is available on the frequency of the many CYP2D6 alleles found in African-Americans. DNA was isolated and genetic testing was performed on samples from 222 African-Americans, healthy controls (n=131), and psychiatric patients (n=91). Each DNA was tested for CYP2D6 alleles *2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *31, *35, *36, *37, *40, *41 and *43 and 8 multiple copy alleles (*1xn, *2xn, *4xn, *41xn, *2Lxn, *17xn, *35xn and *10xn) using the AmpliChip CYP450 prototype microarray assay, along with allele-specific-PCR and PCR restriction fragment length polymorphism methods. No significant difference was noted between controls and psychiatric patients in any CYP2D6 allele frequencies. Three subjects were genotyped as poor metabolizers (1.4%; 0.0–2.9%, 95% confidence intervals (CI)), and 10 were classified as ultrarapid metabolizers (4.5%; 1.8–7.2%, 95% CI). A new CYP2D6 allele (*58) and two new duplicated CYP2D6 alleles (*17xn and *2Lxn) not previously reported were also identified. The frequency of the CYP2D6 overexpression in African-Americans may represent a greater therapeutic challenge than its deficiency based on these results. The most common alleles found in African-Americans including CYP2D6*1, *17 and *41 need to be investigated more closely for race-specific allelic variations and the mechanism responsible for differences in allele function more closely examined. The diversity of CYP2D6 alleles suggests that nucleotide arrays or similar methods are needed to efficiently test for the most prominent/relevant CYP2D6 alleles in humans.

Racial differences in parathyroid hormone levels in patients with secondary hyperparathyroidism.

AIM African-Americans (AA) with normal renal function have higher parathyroid hormone (PTH) levels than Caucasians (C). This difference was also noted in cross-sectional studies of patients on dialysis. In this study, we evaluated patients with end-stage renal disease who have just began dialysis and who were not receiving any vitamin D therapy. METHODS A total of 363 patients were recruited (C: 260; AA: 103). All patients had serum calcium, phosphorus, alkaline phosphatase and intact PTH (iPTH) levels measured within 3 months of initiating dialysis. RESULTS Serum PTH levels were significantly higher in AA vs. C (383 +/- 33 vs. 246 +/- 19, p < 0.001). This difference was present despite similar calcium, phosphorus and alkaline phosphatase levels between the 2 groups and regardless of gender or diabetes status. However, PTH levels in patients younger than 47 years of age were similar in both groups. CONCLUSION PTH levels in ESRD patients over 47 years of age are higher in AA compared to C. The difference is, in part, due to an age-dependent reduction in PTH seen only in C. Further studies are needed to understand the mechanisms of these racial differences and to verify whether they mirror similar alterations at the level of the end-organ tissue.

Simplified phenotyping with dextromethorphan by thin-layer chromatography: application to clinical laboratory screening for deficiencies in oxidative drug metabolism

Identifying individuals with a deficient capacity for oxidative drug metabolism is of increasing clinical importance. Dextromethorphan (DM) is gaining wide acceptance as a probe drug to characterize individual expression of a specific cytochrome P-450 isozyme. The thin-layer chromatography (TLC) technique described in the present study is a rapid and inexpensive alternative to the methods currently available for assessing the urinary metabolic profile of DM. Sixty-five healthy volunteers participated in the study by ingesting 213 mmol DM and collecting all urine for the ensuing 8 h. Urine samples were analyzed by TLC and high-performance liquid chromatography (HPLC) after treatment with β-glucuronidase. Based on the relative color intensities of DM and its O-demethylated metabolite, dextrorphan, the TLC analysis provided an accurate phenotype assessment. A greater intensity of the parent drug relative to the metabolite indicates a poor metabolizer phenotype whereas a reversed relative intensity indicates the extensive metabolizer phenotype. The phenotype assignments made by TLC were verified by comparison with the quantitative results (based on metabolic ratios) obtained from HPLC analysis. Complete agreement was found between the two methods. The routine implementation of phenotype determination into clinical protocols can be realized with this facile TLC technique.

Age and propranolol stereoselective disposition in humans

The apparent oral clearance of S(−)‐ and R(+)‐propranolol as a function of age was evaluated in 53 healthy male volunteers (age range, 21 to 84 years) after a single 40 mg oral dose of the racemic mixture. No significant age‐associated change in the total (bound plus unbound) and unbound S(−) and R(+) apparent oral clearance was observed (p > 0.05). Stereoselectivity in apparent oral clearance (both total and unbound) remained unaffected by advancing age (p > 0.05). The relationship between age and propranolol enantiomer plasma protein binding was also evaluated in 70 subjects, 53 of whom were from this study (age range, 21 to 89 years). Plasma free fractions for S(−)‐ and R(+)‐propranolol were unchanged with increasing age (p > 0.05), even though the binding was stereoselective (plasma free fractions for R(+) > plasma free fractions for S(−); p < 0.05). The findings from this relatively large and extensive study indicate that age does not influence the stereoselective disposition of propranolol.

Genetic aspects of drug disposition and therapeutics.

Broad variability in drug activity is frequently observed and often is associated with interindividual differences in genetic makeup and exposure to environmental factors. Although it is recognized that these factors are to some extent interdependent, the role of genetics as a determinant of drug response can sometimes be isolated. In particular, when an enzyme or protein is spawned by a single gene locus having two different alleles, this protein may either be expressed (e.g., a homozygous dominant or heterozygous trait) or be functionally absent (e.g., homozygous recessive) in different individuals. Clearly, if this protein/enzyme plays a critical role in determining the pharmacologic activity of a drug, two or more distinct groups of individuals (phenotypes) may be discerned on the basis of their response to the particular agent. The therapeutic and toxicologic implications of this phenomenon are potentially profound. The polymorphic expressions of several enzymes or proteins linked to drug response have been identified. In the following overview, representative examples of genetic polymorphisms in the expression of drug receptors and metabolic enzymes are presented, with particular emphasis on polymorphic biotransformations. Several reviews have been written on these topics and the interested reader is referred to them for further details.1-8

Nicotine increases dopamine clearance in medial prefrontal cortex in rats raised in an enriched environment

Environmental enrichment results in differential behavioral and neurochemical responsiveness to nicotine. The present study investigates dopamine clearance (CLDA) in striatum and medial prefrontal cortex (mPFC) using in vivo voltammetry in rats raised in enriched (EC) or impoverished conditions (IC) and administered nicotine (0.4 mg/kg) or saline. Baseline CLDA in striatum or mPFC was not different between EC and IC. Across repeated DA application, striatal CLDA increased in saline‐control EC and IC. CLDA increased in mPFC in saline‐control IC; CLDA did not change in saline‐control EC. Thus, enrichment differentially alters dynamic responses of the dopamine transporter (DAT) to repeated DA application in mPFC, but not in striatum. In EC, nicotine increased mPFC CLDA compared to saline‐control, but had no effect on CLDA in IC; nicotine had no effect in striatum in EC or IC. Compared to respective saline‐controls, nicotine increased dihydroxyphenylacetic acid content in striatum and mPFC in EC, but not in IC. Nicotine also had no effect on DA content in striatum or mPFC in EC or IC. Results indicate that enrichment eliminated the dynamic response of mPFC DAT to repeated DA application in saline‐control and augmented the nicotine‐induced increase in DAT function in mPFC, but not in striatum.

Pharmacogenomic testing: the cost factor

INTRODUCTION A number of reports in the literature have characterized the clinical relevance of genetic variations on therapeutic response and outcomes to drugs. These and other reports support the general belief that genetic testing could be used to improve patient therapy. The urgency of moving in this direction has been further underscored by reports that adverse drug reactions add billions of dollars per year to our health care bill, and that over 100 000 patients per year may die from taking prescription medications properly. This has convinced many researchers that genetic tests could improve how drugs are selected and prescribed to patients, and that such tests should become clinically available for this purpose within the next 10–20 years. Beyond the ethical and legal questions surrounding genetic testing, the clinical use of patient genomic information to improve therapeutic treatment often ignores the most fundamental question about this technology—is it cost effective? The clinical implementation of genomic testing hinges on answering a very simple question: ‘Under what condition(s)/situation(s) are the savings provided by a pharmacogenomic test greater than the cost of genetic testing?’ The answer to this question requires two pieces of information: (1) the cost of performing a genetic test; and (2) the savings that could be realized if genetic testing were employed in place of current clinical methods. When the savings realized are greater than the cost of performing the test, it makes economic sense to apply pharmacogenomic testing. If pharmacogenomic testing had been proven to save money by now, it would already be a routine clinical tool. It is the lack of adequate data to answer this simple question that has delayed the introduction of pharmacogenomic testing into therapeutic practice. There is a progression of steps that must be followed to both understand when and how to perform a cost analysis of pharmacogenomic testing. This starts with a description of where genetic testing has already been shown to be cost effective and why, and proceeds by defining the elements that are required before one can begin to apply similar efforts to a cost analysis of pharmacogenomic testing. There must also be an appreciation for the size of the savings that pharmacogenomic testing must generate in order to be a cost-effective tool. Equally important is the need for researchers to understand that such studies must be performed not in selected populations, but in the routine clinical environment. Finally, there are many different approaches to cost analysis, but one must distinguish between what is ideal and what is practical. It is hoped the following points and ideas will stimulate discussion in this area and help focus attention on what needs to be done to move pharmacogenomic testing from the bench to the bedside.