CYP 2 D 6 basic genotyping as a potential tool to improve the antiemetic efficacy of ondansetron in prophylaxis of postoperative nausea and vomiting

Background. Postoperative nausea and vomiting (PONV) is a common complication after anesthesia and surgery. Ondansetron is one of the most widely used drugs in the prophylaxis of PONV and is extensively metabolized in humans. In vitro metabolism studies have shown that ondansetron is a substrate for human hepatic cytochrome P450 enzymes. The cytochrome P450 (human hepatic cytochrome [CYP]) 2D6 inhibitor quinidine reduced in vitro hydroxylation of ondansetron, which indicates the important role of CYP2D6 in ondansetron metabolism. Genotyping these alleles allows the prediction of the extensive metabolizer (EM) and poor metabolizer (PM) phenotypes with approx. 90–96% accuracy.


Introduction
Postoperative nausea and vomiting (PONV) is a common complication after anesthesia and surgery.Pain, nausea and vomiting are frequently listed by patients as their most pressing perioperative concerns.With the change in emphasis from inpatient hospitals to outpatient hospitals and office-based medical/surgical settings, there has been an increased interest in the problem of PONV.The etiology and consequences of PONV are complex and multifactorial, with patient-, medicine-, and surgeryrelated factors; the important predictors are female gender, a history of PONV and a history of motion sickness.The vomiting center can be triggered by the activation of dopamine, serotonin (type 3), histamine (type 1), and muscarinic cholinergic receptors in the chemoreceptor trigger zone and the nucleus tractus solitarius, as well as acetylcholine receptors in the vestibular apparatus, vagal afferents from the periphery and the endocrine environment.][3] Ondansetron is one of the most widely used drugs in the prophylaxis of PONV. 4 While its mechanism of action has not been fully characterized, ondansetron is not a dopamine-receptor antagonist.Serotonin receptors of 5-HT3 type are present both peripherally on vagal nerve terminals and centrally in the chemoreceptor trigger zone of the area postrema.It is not certain whether ondansetron's antiemetic action is mediated centrally, peripherally or in both sites.Approximately 95% of ondansetron is extensively metabolized in humans: only 5% of a radiolabeled dose is recovered from the urine in the form of the parent compound.The primary metabolic pathway is hydroxylation on the indole ring, followed by subsequent glucuronide or sulfate conjugation.Although some non-conjugated metabolites have pharmacological activity, these are not found in plasma at concentrations likely to significantly contribute to the biological activity of ondansetron. 5,6n vitro metabolism studies have shown that ondansetron is a substrate for human hepatic cytochrome (CYP) P450 enzymes, including CYP1A2, CYP2D6, CYP3A4, and CYP2E1.According to Fisher et al. as well as Janicki and Sugino, the CYP2D6 inhibitor quinidine reduced the in vitro hydroxylation of ondansetron, which indicates the important role of CYP2D6 in ondansetron metabolism. 5,7,9he in vitro study by Sanwald et al. confirmed that ondansetron is both CYP2D6-and CYP2E1-dependent. 8he enzymes belonging to the cytochrome P450 family, especially CYP2D6, have an important role in the metabolism of over 25% of drugs. 9,102][13][14] The polymorphism of CYP2D6 is relatively well-known and more than 20 mutations of the CYP2D6 gene have been described.CYP2D6*3 (formerly A-mutation), CYP2D6*4 (formerly B-mutation) and CYP2D6*5 (gene deletion, formerly D-mutation) are the most common non-functional CYP2D6 alleles. 15Therefore, the genotyping of these alleles allows the prediction of the extensive metabolizer (EM) and poor metabolizer (PM) phenotypes with about 90-96% accuracy. 16,17

Objectives
The aim of our study was to evaluate whether the efficacy of antiemetic treatment with ondansetron is influenced by mutations of the most common CYP2D6 genotype, hypothesizing that extensive metabolizers of this drug are at risk of being undertreated.This study sought to investigate whether the efficacy of antiemetic treatment with ondansetron could be improved by adjusting for the most common CYP2D6 genotype, using basic, widely available and inexpensive (less than $100) polymerase chain reaction (PCR), and polymerase chain reaction amplification and restriction fragment length polymorphism (PCR-RFLP) tests.

Subjects
The study was performed on a group of 93 women with thyroid diseases (neutral struma nodosa and struma nodosa toxica), hospitalized for thyroid gland surgery (strumectomy).They were patients of the 1 st Department of Surgery, Wroclaw Medical University, Poland.Their ages ranged from 18 to 84 years, with a mean of 49.85 years (standard deviation [SD]: 14.41).None had significant renal, hepatic or cardiovascular disease, insulin-dependent diabetes, chronic obstructive pulmonary disease, or cerebral vascular accident.Patients were excluded if any potential CY-P2D6 inhibitor, such as other agents metabolized by isoenzyme CYP2D6, i.e., beta-adrenergic blocking agents, tricyclic antidepressants, antiarrhythmic Ic class drugs, debrisoquine, codeine, or dextromethorphan, had been administered 14 days prior to surgery.
Informed consent was obtained in every case.The protocol for the study was approved by the Bioethics Committee of the Wroclaw Medical University, Poland.

Study design
Ondansetron was administered to patients orally in doses of 8 mg before surgery, for the prevention of PONV.

Genotyping methods
DNA was arranged from leukocytes extracted from peripheral blood.Genotyping for the defective CYP2D6*3, CYP2D6*4 and CYP2D6*5 alleles (a single base-pair deletion in exon 5 for CYP2D6*3, G1934 to A point mutation for CYP2D6*4, and a gene deletion for CYP2D6*5) was performed by PCR-RFLP method, based on the method described by Smith et al. 18 For the CYP2D6*3 mutation, PCR was performed using the primers E -GATGAGCT-GCTAACTGAGCCC and F -CCGAGAGCATACTC-GGGAC.For the CYP2D6*4 mutation, PCR was performed using the primers C -GCCTTCGCCAACCACTCCG and D -AAATCCTGCTCTTCCGAGGC.
The PCR amplification product (after verification by electrophoresis in 2% agarose gel) was identified by the RFLP method.The restriction enzymes BstNl (for CY-P2D6*4 mutation) and HpaII (for CYP2D6*3 mutation) were used.The separation of restriction products was performed by electrophoresis in 3% agarose gel with the addition of ethidium bromide.The results were observed under UV light and recorded in a graphic form.
A lack of CYP2D6 amplification, suggesting CYP2D6 gene deletion, was confirmed by an external laboratory (Research Institute of Medicinal Plants, Poznań, Poland).
The number of nausea and vomiting episodes was counted, and PONV intensity was evaluated with the Postoperative Nausea and Emetic Scale (PNES) (Table 1) during the first 5 h after surgery.
The PNES results were calculated immediately after surgery (0 h), during the 1 st hour (1 h) and during the next 4 h (5 h).

Statistical analysis
The statistical analysis of the results was performed using the χ 2 test, with or without Yates's correction, and the Fisher's exact test.For the statistical analysis of the antiemetic efficacy of ondansetron among different CYP2D6 genotype groups, the relative risk (odds ratio [OR]) was calculated.The pharmacoeconomic value of genotyping was determined by calculating the Number Needed to Diagnose (NND): the number of people who should be screened in order to prevent 1 adverse end point.
Out of all 93 patients, 23 (24.7%) experienced nausea and vomiting.Sixty subjects genetically defined as EMs and UMs of CYP2D6 had statistically significant higher frequency of nausea and vomiting (Yates corrected χ 2 = 5.48, p < 0.02, Fisher's exact test p < 0.011) than IMs and PMs.As many as 20 of the 60 EMs and UMs (33.3%) experienced PONV, while only 3 of the 29 IMs (10.3%) and none of the 4 PMs (0%) reported this condition (Fig. 1).
Moreover, PONV intensity (measured by the PNES) was higher among EMs and UMs (mean PNES value = 3.97) than among IMs and PMs (mean PNES value = 0.19).Detailed information regarding the frequency and intensity of PONV immediately after surgery (0 h), during the 1 st hour (1 h) and during the next 4 h (5 h) is shown in Table 2.
The NND -the number of people who should be screened in order to prevent 1 episode of PONV -was 3.35 (CI = 2.35-20.85),which suggests a relatively high pharmacoeconomic value for CYP2D6 genotyping.

Discussion
The evaluation of the metabolic polymorphism of CY-P2D6 and the frequency of PONV among surgical patients treated with ondansetron has rarely been reported until now.The antiemetic efficacy of ondansetron has only been studied during cancer chemotherapy.Our results are parallel to the results of Kaiser et al., who analyzed the effects of antiemetic treatment with ondansetron and tropisetron during cancer chemotherapy and revealed that genetically defined UMs of CYP2D6 had a higher frequency of vomiting within the first 4 h (p < 0.001) and within the period of 5-24 h (p < 0.03) after treatment than all the other patients; the tendency for nausea was similar. 21The results of both studies confirm conclusions of Fisher et al. and Dixon et al. from in vitro studies, endorsing the key role of CYP2D6 in the metabolism of ondansetron, despite the doubts of Ashforth et al. 5,22,23 The latter questioned the importance of CYP2D6 phenotype in the in vivo metabolism of ondansetron by showing that there was no significant difference in area under curve (AUC), maximal concentration (C max ), clearance (CL), or half-life time (t 1/2 ) between the 6 PMs and the 6 EMs. 23he involvement of the CYP2D6 system in the sensitivity to PONV was further confirmed by Wesmiller et al. and Dong et al. [24][25][26] Patients who were classified as PMs had fewer PONV episodes and higher pain scores.These findings suggest variability in CYP2D6's impact on susceptibility to PONV. 27anicki and Sugino suggested that genotyping for CYP2D6 UM polymorphisms (gene multiplications) for PONV or other medications "currently may be prohibitively costly and inconvenient" because "only a small proportion of Caucasians carry the CYP2D6 allele duplication and hence are at risk for PONV treatment failure". 28,29he frequency of the UM genotype varies by population, ranging from 1% to 8% among Caucasians. 14,26Basic, inexpensive genotyping for CYP2D6*NxN, CYP2D6*3, CYP2D6*4, and CYP2D6*5, definitely the most common mutations among Caucasians, could be useful for dividing patients into 2 subpopulations.The combined group of EMs and the rare UMs (66% of the Caucasian population) requires higher doses of ondansetron.The combined group of IMs and PMs (34%) could be treated with standard doses.The NND is low (3.35), which suggests a relatively high (in comparison with other tests) pharmacoeconomic value for basic CYP2D6 analysis. 30We suggest that this approach is quite affordable and could be clinically useful.
Our results, and those of other studies, strongly suggest that antiemetic treatment with ondansetron could be improved by adjusting the drug dosage according to the patient's CYP2D6 genotype, by using a basic, inexpensive and widely available PCR-RFLP test.
Genetically defined CYP2D6 EMs and UMs present a statistically significant higher risk of PONV than IMs and PMs.
Postoperative nausea and vomiting treatment with ondansetron could be improved by adjusting the drug dosage based on CYP2D6 genotyping, using simple, inexpensive and widely available PCR and PCR-RFLP tests.

Table 2 .
Postoperative nausea and vomiting (PONV) frequency and intensity (measured by the Postoperative Nausea and Emetic Scale [PNES]) in patients during the 1 st 5 h after surgical treatment