Mansour Tobaiqy
email: m.tobaiqy@abdn.ac.uk

Derek Stewart

Peter J Helms

Christine Bond

Amanda Jane Lee

Nick Bateman

Dorothy Mccaig

James Mclay
email: j.mclay@abdn.ac.uk

A pilot study

Keywords: Adverse drug reactions, Pharmacovigilance, Children, Questionnaire

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Electronic supplementary material

The online version of this article (doi:10.1007/s00228-010-0790-9) contains supplementary material, which is available to authorized users.

What was already known Current pharmacovigilance systems are limited by spontaneous reporting of adverse drug reactions, lack of a denominator, and lower than expected reporting rates. The majority of Yellow Card reports are submitted by healthcare professionals and little is known on the utility of parental, compared to professional, reporting on behalf of children.

What this study adds Community pharmacy-based, prospective adverse drug reaction monitoring scheme identifies the expected range of adverse drug reactions to the target drugs. Almost a quarter of parents approached responded appropriately to the ADR questionnaire. However the application of this approach to ADR monitoring is likely to be limited by low community pharmacist uptake.

Introduction

In the UK, the routine system of pharmacovigilance is the Yellow Card Scheme (YCS) operated by the Medicines Healthcare Products Regulatory Agency (MRHA). The YCS has acknowledged limitations including reliance on spontaneous reporting, lack of denominators, and lower than expected reporting rates [START_REF] Choonara | Direct reporting of suspected adverse drug reactions by patients[END_REF][START_REF] Rawlins | Pharmacovigilance: paradise lost, regained or postponed?[END_REF][START_REF] Belton | Attitudinal survey of ADR reporting by medical practitioners in the United Kingdom[END_REF]. This is particularly true for adverse drug reactions (ADRs) affecting children, who are frequently prescribed off label medications, known to be associated with an increased incidence of ADRs [START_REF] Carleton | Paediatric adverse drug reaction reporting: understanding and future directions[END_REF][START_REF] Mclay | A prospective questionnaire assessment of attitudes and experiences of off label prescribing amongst hospital based paediatricians[END_REF]. Data suggest that ADRs account for 1.5-2.1% of paediatric hospital admissions [START_REF] Impicciatore | Incidence of adverse drug reactions in paediatric in/out patients: a systematic review and meta-analysis of prospective studies[END_REF][START_REF] Turner | Adverse drug reactions to unlicensed and off-label drugs on paediatric wards[END_REF], affect 2.6-9.3% of paediatric inpatients, and 1.5-11.1% of paediatric outpatients [START_REF] Jonville-Bera | Frequency of adverse drug reactions in children: a prospective study[END_REF][START_REF] Weiss | Survey of adverse drug reactions on a paediatric ward: a strategy for early and detailed detection[END_REF][START_REF] Kramer | Adverse drug reactions in general paediatric outpatients[END_REF]. Routine prescribing data obtained from General Practice Prescribing databases suggest that in the UK between 120,000 and 880,000 children experience an ADR annually. However current reporting rates for children suggest that only 0.2-1.6% of paediatric ADRs appear to be reported in the UK via the YCS, with, on average, only 2,000 such ADRs reported annually.

Despite the introduction of patient reporting, the majority of Yellow Card reports are submitted by healthcare professionals and little is known on the utility of parental, compared to professional, reporting on behalf of children.

The aim of our study was to pilot and evaluate a community pharmacy-based monitoring system to identify ADRs associated with the use of three groups of high-risk medicines: selective serotonin reuptake inhibitors (SSRIs) [START_REF] International | Pilot schemes for patient reporting of suspected ADRs have been launched in the UK[END_REF][START_REF]Selective serotonin reuptake inhibitors: overview of regulatory status and CSM advice relating to major depressive disorder (MDD) in children and adolescents including a summary of available safety and efficacy data[END_REF][START_REF] Bennet | Overprescribing of antidepressants to children: pharmacoepidemiological study in primary care[END_REF], anticonvulsants [START_REF] Clarkson | Surveillance for fatal suspected adverse drug reactions in the UK[END_REF], and treatments for ADHD including melatonin [START_REF]Atomoxetine[END_REF].

Methods

Approval for the study was obtained from the Fife, Forth Valley and Tayside Research Ethics Service and by the NHS Research and Development Committees for all participating Health Board areas. We invited all (n=827) registered community pharmacies within the Grampian (n=127), Lothian (n=180), Tayside and Angus (n=93), Lanarkshire (n=116), and Greater Glasgow Clyde (n=311) Health Board areas to take part. Community pharmacists were asked to prospectively identify all children for whom a prescription for one or more of the target drugs was presented and issue the parent/guardian/child collecting the prescription with a study information leaflet and an ADR questionnaire. An anonymised dispensing label was attached to the ADR questionnaire to accurately identify the drug, strength, and dose.

The ADR questionnaire was based on one used previously [START_REF] Stewart | Monitoring adverse drug reactions in children using community pharmacies: a pilot study[END_REF] and revised for consistency with the newly introduced patient reporting forms for the YCS. The questionnaire consisted of 23 structured and open questions to collect information on child demography (age, sex, and postcode), duration of medicine use, medical indication, perceived side effects/ADRs (open text), and description and severity of any ADR. Details of any actions taken or outcomes, including discontinuation of medicine, involvement of any healthcare professional, or submission of a Yellow Card were requested. To ensure compatibility with the YCS, the Yellow Card descriptors were used throughout. A tick list of symptoms covering all the major body systems was also included. The questionnaire was reviewed for face and content validity by a panel including clinical pharmacologists, toxicologists, paediatricians, hospital and community pharmacists, and lay people. Parents were asked to return the completed questionnaire whether the child had had an ADR or not.

At the time of this study, routine primary care data indicated that more than 13,000 children were prescribed one of the drugs of interest (3,300 anticonvulsants, 3,100 SSRI, 6,900 treatments for ADHD) annually in Scotland, equating to an average of 11 candidate children per pharmacy (based on 1,150 community pharmacies in Scotland and a typical 4-to 8-week prescription cycle).

Data from the ADR questionnaire were entered into an SPSS (Statistical Package for Social Sciences, version 16.0) database. Descriptive statistics were used to summarise recruitment rate (by Health Board area and by pharmacy), questionnaire response rates, and frequencies of the questionnaire variables.

Results

Response rates

Eight hundred and twenty-seven community pharmacies were approached to take part in this study, and 72 pharmacies were recruited giving a participation rate of 8.7% (72/827). Participating pharmacies distributed 229 ADR questionnaires; 57 completed questionnaires were returned, 2 of which were non-evaluable, giving a response rate of 24%. Forty-two (76.3%) ADR questionnaires were for males, and the median age was 13 [interquartile range (IQR) 9-14] years.

ADHD medication

Twenty-seven (49%) of the questionnaires returned were for children prescribed methylphenidate, of which 19 (70.4%) reported 26 possible ADRs. In order of frequency the ADRs reported were reduced appetite and loss of appetite (n = 6, 23%), psychiatric disorders (5, 20%), stomach upset (3, 12%), sleep disturbance (3, 12%), headache (3, 12%). Seven returned ADR questionnaires were for melatonin, only one of which reported a possible ADR, "raised temperature at night". Three of the returned ADR questionnaires were for atomoxetine, one of which reported an ADR, "stomach problems".

Anticonvulsants

Thirteen returned ADR questionnaires were for the anticonvulsants sodium valproate (n=5), carbamazepine (n=3), topiramate (n=2), lamotrigine (n=2), and levetiracetam (n=1). Six (46.1%) of these questionnaires reported suspected ADRs, including drowsiness, dizziness, and weight gain.

SSRI

One ADR questionnaire for an SSRI was returned and this reported a suspected ADR of "nausea".

Severity of reported ADRs

Twenty-seven respondents gave 38 responses to the question "How bad would you say this side effect was?" using descriptors from the Yellow Card. The majority of reported ADRs (39.5%, 15/38) were described as mild-irritating but permitting normal daily function. However 36.8% of ADRs (n=14) were reported as affecting the child's everyday activities and 2.6% (n=1) as requiring hospitalisation.

Action taken and outcome following onset of ADR Twenty-three respondents gave 38 responses to the question "What action was taken following the onset of the suspected ADR?". For reported ADRs, 65.7% (25) were discussed with their doctor, 7.8% [START_REF] Belton | Attitudinal survey of ADR reporting by medical practitioners in the United Kingdom[END_REF] discussed with the community pharmacist, and 5.2% [START_REF] Rawlins | Pharmacovigilance: paradise lost, regained or postponed?[END_REF] did nothing; 21% (n=8) reported a variety of other actions.

Free text reports of ADRs, duration, and outcome Thirty of 55 (54.5%) questionnaires reported a total of 44 ADRs perceived to be due to the medication under study (Table 1). When asked the question "Has the side effect stopped?", 28 respondents gave a total of 40 responses to this question. At the time of reporting, 26 ADRs were ongoing, and 14 ADRs were reported as having resolved. The duration of the reported ADRs ranged from 1 week to 6 years.

Symptom list

Eighty-four reports of symptoms possibly associated with medication were recorded by the respondents in 33 of the returned ADR questionnaires. A description of the symptoms is reported in Table 1. Forty-six symptoms were reported for methylphenidate of which the most frequent were mood problems (34.8%, 16), feeding problems (26%, 12), schooling problems (9.3%, 4), and headache (9.3%, 4). Twelve symptoms were reported for dexamfetamine, nine for sodium valproate, five for lamotrogine, four for levetiracetam, three for carbamazepine, three for melatonin, three for atomoxetine, and one for fluoxetine.

Discussion

The study demonstrated that a community pharmacybased, prospective adverse drug reaction monitoring scheme identified the expected range of adverse drug reactions to the target drugs, demonstrating its validity. To our knowledge this is the first pharmacovigilance study to target community-based paediatric populations prescribed specific medicines of interest via community pharmacies. Our study has several limitations including low pharmacy and pharmacist participation rates, a situation which was exacerbated by a lower than expected questionnaire distribution rate, factors which limit the generalisability of our findings.

The design of the current study did not permit the reasons for pharmacy non-participation to be determined, however a similar approach using financial incentivisation has been demonstrated to have a positive effect in other pharmacy-based research studies [START_REF] Armour | Pharmacists' views on involvement in pharmacy practice research: strategies for facilitating participation[END_REF][START_REF] Faber | Recruitment of parents and physicians of stimulant-using children via community pharmacies is successful[END_REF]. Although UK community pharmacists are encouraged to contribute to the YCS, it is not explicitly included in their NHS contractual framework. However the framework does include as a core component a public health service which comprises poster campaigns, smoking cessation advice and treatment, provision of emergency hormonal contraception and chlamydia detection and treatment. Consideration should possibly be given to including ADR monitoring and reporting in this framework with associated remuneration. However further research into those factors influencing pharmacy involvement in pharmacovigilance and pharmacovigilance related research is required.

Twenty-four percent of parents/guardians returned the ADR questionnaire, which is less than the predicted rate of 40% suggested in our previous study [START_REF] Stewart | Monitoring adverse drug reactions in children using community pharmacies: a pilot study[END_REF]. The medicines involved in the current study were prescribed for psychiatric, neurological, and behavioural conditions, and the nature of these conditions may have reduced parental response rates. Nonetheless, the return rate of 24% would, if scaled up to the whole population, produce significantly greater Our approach generated a number of responses, mostly for methylphenidate. Importantly, not all parents/guardians had been informed of these potential ADRs by their health professionals and, in some, the ADRs were still an issue of concern long after the start of therapy and the initial occurrence of the ADR. The ADR monitoring system we propose has potential for targeted monitoring and reporting of ADRs if parent/guardian recruitment and response can be enhanced.

Conclusions

The monitoring system utilised identified expected ADRs for the study medicines and produced frequencies which were similar to those reported in the literature, thus demonstrating face and content validity. Although the process was limited by the low community pharmacist and parent participation rate, such an approach could enhance and complement current ADR monitoring/reporting systems and generate new data likely to have a positive impact on paediatric patient safety.

Table 1

 1 Free text ADR reports, medicine-related symptoms, prescribed medicines, and child demographics In Scotland, paediatric Yellow Card ADR reports currently number approximately 200 per year, while in the UK a total of 906 ADR and 13 fatal ADR reports were received by the MHRA for methylphenidate between October 1964 and October 2009. Despite the low community pharmacy uptake in this study we generated 72 suspected ADRs or medicine-related symptoms for methylphenidate during a 3-month data collection period.

	Medicine(s)	Prescribed	Age	Sex Free text	Duration of	Severity ADR	Recovery of	Reported tick
	prescribed	daily dose	(years)		reported	ADRs		now	child from	list symptoms
					ADRs			resolved	ADR
	Methylphenidate 10 mg	5	M Sleeping	-	2	Yes	Recovered	-
					problems				completely
	Methylphenidate 54 mg	12	M Loss of	6 years	3	No	Recovered but	Constipation, headache,
					appetite				some lasting	very slow weight gain,
									effects	mood change, struggled
					Affect growth 6 years	3	No	Recovered but	at school
									some lasting
									effects
					Depression	On/off	-	Yes	-
	Methylphenidate 18 mg	12	M Weight gain	Always	2	No	No	-
									improvement
					Poor sleep	Always	3	No	No
									improvement
	Methylphenidate 36 mg	13	M Stomach	Everyday	1	No	Other	Mild cramping, difficulty
					cramp	(all)				getting to sleep
					Loss of		1	N o
					appetite
					Difficulty		1	N o
					getting to
					sleep
	Methylphenidate	10 mg	6	M Hallucination	2-3 months 2	Yes	Getting better	Irritation, bad mood,
	and sodium	300 mg			(hearing and					hearing voices and
	valproate				seeing things					noises, scared of
					that were not					classroom (seeing
					there)					monsters)
	Methylphenidate 18 mg	16	M Headache	1 week	2	Yes	Other	Nausea, no appetite
	Methylphenidate 20 mg	13	M Reduced	2 years	1	Yes	Recovered	Slight weight loss
					appetite				completely
	Methylphenidate 54 mg	13	M -	-	-	-	-	Weight loss, no appetite,
										can't set to sleep
	Methylphenidate 20 mg	12	M Stomach upset Day-day	2	No	Other	Sore head, weepy, doesn't
					Violent out	Frequently		No	Other	feel hungry, mood
					bursts					change
	Methylphenidate 30 mg	9	M Loss of	Most days	2	No	Other	Headache first week only,
					appetite					mild weight loss,
					Rebound	Most	2	-	Other	sleeping and schooling
					hyperactivity	evenings				problems, dryness of
										mouth
	Methylphenidate 5 mg	12	M Alopecia	-	-	-	Other	Headache
	Methylphenidate 18 mg	12	M Trouble	-	2	Yes	Recovered	-
					sleeping				completely
	Methylphenidate 36 mg	13	M Headache	6 months	3	Yes	Recovered	Bit tearful at times, lack of
					Stomach ache 6 months	3	Yes	completely	appetite
	Methylphenidate	10 mg	7	M Decreased	Continually 1	No	No
	and melatonin				appetite				improvement
	Methylphenidate 5 mg	9	F	Headache	1 week	1	Yes	Getting better	Mood problems
	Methylphenidate 20 mg	15	F	-	-	-	-	-	Low confidence, sleeping
										problems
	Methylphenidate 15 mg	7	F	Mood swings From	1	N o	-	Weight loss, schooling
						evening				problem
	Methylphenidate	70 mg	14	M Reduced	Years	-	No	-	Regular headache, gets
	and melatonin	18 mg			appetite					down, slow weight gain,
										sleeping problems, poor
										behaviour at school,
										mood change

Eur J Clin Pharmacol (2010) 66:627-632


Jan Gerard Maring

Floris M Wachters

Monique Slijfer

J Marina Maurer

H Marike Boezen

Donald R A Uges

Elisabeth G E Vries

Harry J M Groen

Jan Gerard

Marike Boezen

R A Donald

Elisabeth G E Uges

Harry J M De Vries

Groen

H M Boezen

E G E De Vries

Pharmacokinetics of gemcitabine in non-small-cell lung cancer patients: impact of the 79A>C cytidine deaminase polymorphism

Keywords: Gemcitabine, Cytidine deaminase, Pharmacogenetics, Pharmacokinetics, Genetic polymorphism

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abbreviations

Introduction

During the last decade, several new chemotherapeutic agents have been evaluated for their beneficial effect in patients with non-small-cell lung cancer (NSCLC). So far, gemcitabine plus cisplatin has emerged as one of the standard regimens for the treatment of advanced NSCLC [START_REF] Schiller | Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer[END_REF]. Nowadays, this regimen is mainly used in patients with squamous cell histology [START_REF] Scagliotti | Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer[END_REF]. Gemcitabine (2′,2′-difluorodeoxycytidine; dFdC) is a fluoropyrimidine antimetabolite that is transported into cells by equilibrative nucleoside transporters [START_REF] Mackey | Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines[END_REF]. Within the cell, gemcitabine is phosphorylated into gemcitabine monophosphate (dFdCMP) by deoxycytidine kinase (dCK) and thereafter into di-and triphosphates [START_REF] Heinemann | Comparison of the cellular pharmacokinetics and toxicity of 2′, 2′-difluorodeoxycytidine and 1-beta-D-arabinofuranosylcytosine[END_REF]. Gemcitabine triphosphate (dFdCTP) is incorporated in DNA, and subsequently only one deoxynucleotide molecule more can be inserted, which leads to the halt of DNA synthesis [START_REF] Huang | Action of 2′, 2′-difluorodeoxycytidine on DNA synthesis[END_REF]. This process is amplified through inhibition of the enzyme ribonucleotide reductase (RNR) by gemcitabine diphosphate (dFdCDP). Inhibition of RNR leads to intracellular depletion of dCDP and dCTP, which favours the incorporation of dFdCTP into DNA [START_REF] Heinemann | Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2′, 2′-difluorodeoxycytidine[END_REF][START_REF] Plunkett | Gemcitabine: metabolism, mechanisms of action, and self-potentiation[END_REF].

Only a small part of the gemcitabine dose is responsible for the cytotoxic effects, since more than 90% of the dose is inactivated by the enzyme cytidine deaminase (CDA) into 2′,2′-difluorodeoxyuridine (dFdU). CDA is ubiquitous in the human body, catalyzing the hydrolytic deamination of (deoxy-) cytidine to (deoxy-)uridine. The total enzyme capacity of CDA in all organs and tissues determines the biotransformation rate of gemcitabine into dFdU and thereby indirectly the duration of exposure to gemcitabine. Interestingly, cloning of human CDA has revealed a 79A>C nonsynonymous coding single nucleotide polymorphism (cSNP) in exon 1 of the CDA gene corresponding with two protein variants with more than twofold difference in vitro deamination rates [START_REF] Kirch | Recombinant gene products of two natural variants of the human cytidine deaminase gene confer different deamination rates of cytarabine in vitro[END_REF][START_REF] Gilbert | Gemcitabine pharmacogenomics: cytidine deaminase and deoxycytidylate deaminase gene resequencing and functional genomics[END_REF]. The A genotype corresponds to the wild type Lys-carrying enzyme and the C genotype to a Gln-carrying variant (Gln27 CDA). The wild type CDA enzyme has been reported to exert a 1.3to 3.3-fold higher deamination rate of cytarabine than Gln27 CDA [START_REF] Kirch | Recombinant gene products of two natural variants of the human cytidine deaminase gene confer different deamination rates of cytarabine in vitro[END_REF]. Thus, the pharmacokinetics of gemcitabine may also be affected by this genetic polymorphism.

The aim of the current study was to evaluate the impact of the common 79A>C cSNP in the CDA gene on the pharmacokinetics of gemcitabine and its metabolite dFdU in gemcitabine-treated NSCLC patients.

Patients and methods

Patient selection

This study was performed as a site study of a phase III trial of gemcitabine plus epirubicin versus gemcitabine plus cisplatin in advanced NSCLC patients [START_REF] Wachters | First-line gemcitabine with cisplatin or epirubicin in advanced non-small-cell lung cancer: a phase III trial[END_REF]. Patients had to meet the inclusion and exclusion criteria of the main study. In short, patients were included if they had stage III/IV NSCLC. No prior chemotherapy was allowed. Radiotherapy should have been completed at least 4 weeks before inclusion, and patients should have recovered from any toxic side effect. All patients had to have a performance status≤2 according to the Eastern Cooperative Oncology Group (ECOG) scale and a life expectancy of at least 12 weeks. An adequate bone marrow reserve and normal renal (creatinine clearance≥ 60 ml/min) and liver function were required. The glomerular filtration rate (GFR) was calculated according to the formula of Cockcroft and Gault [START_REF] Cockcroft | Prediction of creatinine clearance from serum creatinine[END_REF]. Patients were excluded if they had active infections, second primary malignancies, uncorrected hypercalcemia, or an LVEF≤45% measured by multiple gated acquisition (MUGA) scan. A detailed description of the inclusion and exclusion criteria is published elsewhere [START_REF] Wachters | First-line gemcitabine with cisplatin or epirubicin in advanced non-small-cell lung cancer: a phase III trial[END_REF]. The local medical ethics committee of the hospital approved the protocol. All patients gave written informed consent before study entry including genotyping procedures.

Treatment and sample collection

Gemcitabine (Gemzar®, Lilly, Nieuwegein, the Netherlands) in a dose of 1,125 mg/m 2 in 250 mL 0.9% NaCl solution was administered intravenously as a 30 min infusion. Patients cotreated with epirubicin received the epirubicin dose after completion of the gemcitabine infusion. The cisplatin dose was administered the day after the gemcitabine infusion.

Blood sampling Blood sampling was carried out on the first day of the first chemotherapy cycle. For pharmacokinetic sampling, a cannula was placed intravenously in the arm of the patient contralateral to the side of drug administration. Blood samples of 9 mL were collected in heparinized tubes containing 0.25 mg tetrahydrouridine (THU) in 50 μL water, just before chemotherapy and at t=25, 40, 50, 60, 75, 90, 105, 120, 150, 180, 270, and 360 min after the start of the gemcitabine infusion. Blood samples were immediately placed on ice and centrifuged at 2,500g for 10 min within 1 h after collection. Plasma samples were stored at -80°C until further analysis.

Genotyping material Harvesting of oral mucosa cells was performed before chemotherapy by thorough mouth rinsing for 30 s with 5 mL 0.9 % NaCl solution. The cell suspension was collected in plastic cups and subsequently placed on ice. Within 1 h after collection, the cell suspension was centrifuged for 10 min at 190g at 4°C. The supernatant was discarded and cells were washed in 10 mL ice cold phosphate buffered saline, pH=7.40 (PBS) and centrifuged for 10 min at 190g at 4°C. The pellet was resuspended in 1 mL ice cold PBS. The remaining suspension was transferred to a microcentrifuge cup and centrifuged briefly (15 s) at 10,000g. The supernatant was discarded, and the pellet was kept frozen at -80°C until further analysis.

Pharmacokinetic analysis

Gemcitabine hydrochloride and dFdU were obtained from Eli Lilly (Indianapolis, IN). Tetrahydrouridine was purchased from Calbiochem (La Jolla, CA). All other chemicals were of standard analytical grade. The analysis of gemcitabine and dFdU in plasma was carried out by high performance liquid chromatography as described by Freeman et al. [START_REF] Freeman | Validated assays for the determination of gemcitabine in human plasma and urine using high-performance liquid chromatography with ultraviolet detection[END_REF].

Cytidine deaminase genotyping

The CDA exon 1 polymorphism (C/A) at codon position 27 was genotyped by direct sequencing, in both directions, of PCR-amplified genomic DNA.

First, the CDA region flanking the polymorphic site was amplified using PCR with forward primer 5′-AGTAG CTTCCCCTTCCAGTAGC and reverse primer 5′-CCTC TTCCTGTACATCTTCCTCTG. The 25 μL reactions contained 2.5 units Taq polymerase (Amersham Biosciences, Uppsala, Sweden); 0.5 mM dNTP mix (Roche Diagnostics, Mannheim, Germany); 1x PCR buffer (Roche Diagnostics), 0.05 mM MgCl 2 ; 0.2 μM of each primer; and approximately 50 ng genomic DNA. The amplification was performed on a PTC-225 thermal cycler (MJ Research, Waltham, MA), using a stepdown protocol. The first five cycles were carried out at 94°C, 65°C, and 72°C, each for 30 s. The next five cyles were carried out at 94°C, 63°C, and 72°C, each for 30 s. The last 25 cyles were at 94°C, 60°C, and 72°C, each for 30 s. Following cycling the PCR products were purified with the Qiagen Qiaquick PCR purification kit (Westburg, Leusden, the Netherlands). Subsequently, 100 ng of the purified PCR product was cycle sequenced with a Dyeterminator kit (US81090, Amersham Biosciences, Roosendaal, the Netherlands) in a thermal cycler (MJ Research), using 0.05 mM sequencing primer. For the reverse reaction, the same primer was used as in the PCR, but for the forward reaction an internal primer was used (5′-GGTACCAA CATGGCCCAGAAG). After the cycle reaction the sequencing products were cleaned on a Sephadex plate (Amersham Biosciences) by centrifugation for 5 min at 910g. The eluted sample was vacuum dried for 45 min at 65°C. Finally, 20 μL of loading solution (Amersham Biosciences) was added to dissolve the sequencing products. The samples were analyzed on a MegaBACE 1000 capillary sequencer (Amersham Biosciences) by injecting the samples for 45 s at 3 kV and running them for 5 h at 4 kV. The data were processed with Sequence Analyzer 3.0 (Amersham Biosciences) and Seqman II (DNASTAR, Madison, WI).

Pharmacokinetic modelling

Pharmacokinetic data were analyzed with the Mw\Pharm software package (version 3.5; MediWare, Groningen, the Netherlands) using the KinFit module. For gemcitabine and its metabolite dFdU, the AUC (using the trapezoid rule), clearance, distribution volume, and elimination half-lives were calculated by non-compartmental analysis.

Statistical analysis

Patient data were grouped according to genotype. Differences in laboratory and demographic data between groups were tested using the Mann-Whitney test.

CDA genotype-related differences in pharmacokinetic data were investigated by analyzing the wild type AA genotype patient group against the combined heterozygote AC and homozygote CC genotype patient group using the Mann-Whitney test. We assumed an effect of 30% of the alternative genotype on the gemcitabine AUC as clinically relevant. From the literature, the allele frequency of the wild type genotype was estimated at 0.7, and therefore an almost equal distribution of patients in AA wild type (49%) and combined homozygote CC plus heterozygote AC (51%) patient groups was expected. We calculated that group sample sizes of 10 and 10 were needed to achieve 92% power to detect a difference of 30% between the null hypothesis that both group means are equal and the alternative hypothesis that the mean AUC of the mutant group is 30% larger, with group standard deviations of 20% and with a significance level of 0.05 using a two-side Mann-Whitney test assuming that the actual distribution is logistic.

Additionally, differences between uncombined CDA genotype groups regarding gemcitabine AUC, clearance, and half-life were also analyzed using the Kruskall-Wallis test. Statistical analyses were performed using the SPSS 14.0 statistical package (SPSS 2005, Chicago, IL). Power calculations were performed using NCSS 2004 Statistical & Power Analysis Software (NCSS, Kaysville, UT).

Results

Patients

A total number of 20 patients were included. Patients were grouped according to CDA genotype. Patient characteristics are presented in Table 1.

The groups were comparable with respect to demographic parameters, renal function, and liver enzymes. All patients, except one, had AST levels below the upper limit of normal. Two patients had a slightly reduced creatinine clearance (CrCL), between 50 and 60 ml/min. Coadministration of epirubicin and cisplatin was equally distributed within and between groups.

Pharmacokinetics

Mean pharmacokinetic curves of gemcitabine and dFdU in plasma are presented in Fig. 1 according to the different genotypes. Gemcitabine was rapidly cleared from plasma and undetectable after 3 h in all patients. The pharmacokinetic data are presented in Table 2.

The influence of the presence of epirubine on gemcitabine pharmacokinetics was checked by grouping the patients according to treatment schedule (gemcitabine plus cisplatin or gemcitabine plus epirubicine). No difference in gemcitabine pharmacokinetics was measured between the two groups using the Mann-Whitney U-test.

Pharmacogenetic analysis

The gemcitabine pharmacokinetic data of all 20 patients were analyzed with respect to the impact of the 79A>C polymorphism in the CDA gene. The frequencies of the A/A, A/C, and C/C genotypes were 0.40, 0.40, and 0.20 respectively. The allele frequency of the 79A>C polymorphism was 0.40 in our study. Detailed data are presented in Table 2. The mean pharmacokinetic curves of gemcitabine and dFdU according to CDA genotype are presented in Fig. 1. No differences in pharmacokinetic parameters were observed between the wild type AA genotype group and the combined heterozygote AC and homozygote CC genotype group, using the Mann-Whitney test. Additionally, in the Kruskal-Wallis analysis, we found no significant differences in gemcitabine AUC, clearance, or serum halflife among the different CDA genotypes (see Table 2).

Time (h)

Fig. 1 Pharmacokinetics of gemcitabine and its metabolite dFdU after a 30 min infusion of 1,125 mg/m 2 gemcitabine grouped according to genotype of the cytidine deaminase 79A>C polymorphism

Discussion

In this study, we investigated the impact of the common 79A>C nonsynonymous single nucleotide polymorphism in the cytidine deaminase gene on gemcitabine and dFdU pharmacokinetics in NSCLC patients. In all patients gemcitabine was rapidly cleared from plasma, as was expected from previous reports [START_REF] Abbruzzese | A phase I clinical, plasma, and cellular pharmacology study of gemcitabine[END_REF].

The most important factor that determines the clearance rate of gemcitabine is the activity of CDA. This enzyme rapidly catabolizes gemcitabine. The total enzyme capacity of CDA in all organs and tissues determines the biotransformation rate of gemcitabine into dFdU and thus the duration of exposure to gemcitabine. Despite increasing interest in DNA polymorphisms related to gemcitabine metabolism, so far only a few reports have been published on the relation between CDA polymorphisms and gemcitabine pharmacokinetics.

The 79A>C cSNP is the most common CDA polymorphism with previously reported allele frequencies of 0.20 in Japanese, 0.11 in African Americans, 0.04 in Africans, 0.30 in Caucasian Americans, and 0.36 in European Caucasians [START_REF] Kirch | Recombinant gene products of two natural variants of the human cytidine deaminase gene confer different deamination rates of cytarabine in vitro[END_REF][START_REF] Sugiyama | Pharmacokinetics of gemcitabine in Japanese cancer patients: the impact of a cytidine deaminase polymorphism[END_REF][START_REF] Yue | A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity[END_REF][START_REF] Fukunaga | Identification and analysis of single-nucleotide polymorphisms in the gemcitabine pharmacologic pathway[END_REF]. The polymorphism at nucleotide position 79 corresponds to codon27, which is located within an Nterminal core domain and might be involved in binding pocket loops that cooperate with zinc ligands [START_REF] Yue | A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity[END_REF][START_REF] Betts | Cytidine deaminase. The 2.3A crystal structure of an enzyme: transition-state analog complex[END_REF]. The A genotype corresponds to the wild type Lys-carrying enzyme and the C genotype to a Gln-carrying variant (Gln27 CDA). Kirch et al. found that the deamination rate of cytarabine by bacterial wild type CDA enzyme was a 1.3-3.3 fold higher than by Gln27 CDA [START_REF] Plunkett | Gemcitabine: metabolism, mechanisms of action, and self-potentiation[END_REF]. Gilbert et al. recently reported in a functional genomics study in mammalian cells that the activity of human recombinant Gln27 CDA was about 66% of the wild type CDA activity for gemcitabine [START_REF] Kirch | Recombinant gene products of two natural variants of the human cytidine deaminase gene confer different deamination rates of cytarabine in vitro[END_REF]. Thus, assuming highest CDA activity in A/A, intermediate activity in A/C, and lowest in C/C diplotypes, we studied the consequences of this polymorphism on gemcitabine AUC, clearance, and serum half-life.

All patients in our study were Caucasian Europeans, and in this group we found an allele frequency of 0.40, which is in line with the allele frequencies of 0.30 and 0.36 earlier reported for Caucasians [START_REF] Kirch | Recombinant gene products of two natural variants of the human cytidine deaminase gene confer different deamination rates of cytarabine in vitro[END_REF][START_REF] Fukunaga | Identification and analysis of single-nucleotide polymorphisms in the gemcitabine pharmacologic pathway[END_REF]. We did, however, not observe an impact of the CDA 79A>C genotype on gemcitabine pharmacokinetics. Obviously, our study was powered to detect differences of at least 30% in population means and smaller differences in pharmacokinetic parameters may have gone unnoticed. In the post-hoc statistical analysis, we found a much larger variation in gemcitabine AUC values, ranging from 30-50%, than the initially expected 20%. As a result we achieved only 25% power to detect a difference. We reasoned however that, due to the fact that the data in both groups largely overlap, it seems very unlikely that dose adjustment according to CDA genotype will become clinically feasible, if a difference might exist. Interestingly, this matter has recently been elucidated, as, in parallel with our study, Sugiyama et al. investigated the 79A>C polymorphism in a population of 251 Japanese patients. In line with our data, they could not detect any effect of the 79A>C polymorphism on the gemcitabine pharmacokinetics after a 30 min 800-1,000 mg/m 2 infusion [START_REF] Sugiyama | Pharmacokinetics of gemcitabine in Japanese cancer patients: the impact of a cytidine deaminase polymorphism[END_REF]. It is important to realize that the two studies differ with respect to administered gemcitabine dose and patient ethnicity. The mean dose in our study was 38% higher as a result of both a higher standard dose (1,125 mg/m 2 ) and generally higher body surface area (BSA) values in Caucasians. Obviously, these factors do not change or modulate the observed lack of impact on pharmacokinetic outcomes of the 79A>C polymorphism.

One explanation for our findings may be a far more complex regulation of CDA gene transcription, leading to substantial interindividual differences in quantities of CDA enzyme, unrelated to the 79A>C polymorphism. Although Kirch et al. [8] and Gilbert et al. [START_REF] Gilbert | Gemcitabine pharmacogenomics: cytidine deaminase and deoxycytidylate deaminase gene resequencing and functional genomics[END_REF] demonstrated reduced enzyme activity with the 79A>C polymorphism using in vitro deamination of ara-C and dFdC, respectively, it is possible that additional polymorphisms within CDA also influence enzyme activity or that variants in other enzymes in gemcitabine metabolism play a greater than expected role. Other studies have shown that the CDA '5 flanking region contains numerous potential transcription factor binding sites that can cause variations in transcription [START_REF] Demontis | Isolation and characterization of the gene coding for human cytidine deaminase[END_REF]. Unfortunately, we did not perform direct measurement of CDA activity in our patients and thus could not check the genotype-phenotype relation at the enzyme level.

Moreover, we hypothesized that possibly the total capacity of CDA in the body may far exceed the amount needed to cope with plasma levels resulting from 800-1,125 mg/m 2 gemcitabine doses. In that case, differences in pharmacokinetics may only become manifest at much higher plasma concentrations when Michaelis-Menten enzyme saturation is becoming relevant.

Finally, the pharmacokinetic results might have been dispersed as a result of co-administration of other chemotherapeutic agents. Indeed, all patients in our study received combined chemotherapy, but cisplatin was administered on day 2 and therefore did not interact with our results. We analyzed that co-administration of epirubicin did not influence the pharmacokinetics of gemcitabine, which is in line with previously reported findings regarding the pharmacokinetics of both drugs when combined in treatment schedules [START_REF] Perez-Manga | Gemcitabine in combination with doxorubicin in advanced breast cancer: final results of a phase II pharmacokinetic trial[END_REF][START_REF] Fogli | Gemcitabine, epirubicin and paclitaxel: pharmacokinetic and pharmacodynamic interactions in advanced breast cancer[END_REF][START_REF] Conte | Gemcitabine plus epirubicin plus taxol (GET) in advanced breast cancer: a phase II study[END_REF].

Although we could not demonstrate an effect of the CDA 79A>C polymorphism on gemcitabine pharmacokinetics, the polymorphism still may be relevant at the cellular level. Bathla et al. recently demonstrated that the post-induction treatment-related mortality in children treated for acute myeloid leukemia with high dose cytarabine was significantly higher in children bearing the CC compared to the AC or AA genotype [START_REF] Bathla | Cytidine deaminase genotype and toxicity of cytosine arabinoside therapy in children with acue myeloid leukemia[END_REF]. Cytarabine and gemcitabine share a common metabolic pathway. Furthermore, Tibaldi et al. showed that the wild type CDA Lys27Lys (AA) genotype was associated with better clinical outcome, longer time to progression, and overall survival [START_REF] Tibaldi | Correlation of CDA, ERCC1, and XPD polymorphisms with response and survival in gemcitabine/cisplatin-treated advanced nonsmall cell lung cancer patients[END_REF]. Interestingly, they also found an increased CDA enzymatic activity in red blood cells associated with the CDA Gln/Gln (CC) genotype. Although these findings contradict previous results from in-vitro functional genomics experiments by Kirch et al. and Gilbert et al., this clinical study illustrates that the association between genotype and treatment efficacy may be quite complex.

Recently two new polymorphisms in the CDA gene were reported. These concern the 208G>A and 435T>C polymorphism with allele frequencies of 0.04 and 0.30 in Japanese patients, respectively [START_REF] Yue | A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity[END_REF]. The 208G>A polymorphism produces an alanine-to-threonine substitution (Ala70Thr) within the conserved catalytic domain. Introduction of this gene in yeast null mutants resulted in a 20% reduction of the 50% inhibitory concentration value for cytarabine [START_REF] Yue | A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity[END_REF]. Sugiyama et al. also studied both variants and found a decreased gemcitabine clearance in patients with the 208G>A polymorphism [START_REF] Sugiyama | Pharmacokinetics of gemcitabine in Japanese cancer patients: the impact of a cytidine deaminase polymorphism[END_REF]. However, the 208G>A polymorphism has not been detected so far in Caucasians and was therefore not included in our study. The 435T>C polymorphism (Thr145Thr) does not encode for an amino acid change. Since an impact of this silent 425T>C polymorphism on CDA enzyme activity was expected to be less probable, we did not include this polymorphism in our investigations.

Conclusions

We conclude that the common 79A>C CDA polymorphism does not have a major consistent, significant effect on gemcitabine plasma clearance. The full pharmacogenetic picture of CDA in relation to gemcitabine clearance and gemcitabine pharmacodynamics may nevertheless be quite complex and can only be elucidated in large clinical trials.

Table 1

 1 Patient characteristics

		All patients (n=20)	Cytidine deaminase genotype subgroups
			A/A (n=8)	A/C (n=8)		C/C (n=4)
	Gender (M/F)	17/3	8/0		6/2		3/1
	Age (years)	65 (44-73)	67 (55-73)	65 (57-73)		59 (44-73)
	Weight (kg)	77 (58-97)	81(60-97)	75 (58-87)		85 (63-88)
	Length (cm)	175 (161-192)	177 (168-193)	173 (161-185)	182 (169-192)
	BSA (m 2 )	1.93 (1.61-2.27)	2.00 (1.69-2.27)	1.90 (1.61-2.03)	2.09 (1.72-2.13)
	Creatinine (μmol/l)	78 (49-119)	80 (60-119)	75 (49-99)		81 (66-92)
	CrCl (ml/min) (normal: >60)	97 (51-134)	97 (51-130)	88 (64-106)	104 (59-134)
	AST (U/l) (normal: <45)	22 (15-35)	22 (12-35)	22 (15-28)		21 (17-27)
	ALT (U/l) (normal: <45)	27 (7-47)	23 (7-32)	29 (15-47)		26 (12-36)
	Bilirubin (μmol/l) (normal: <20)	10 (6-17)	10 (6-16)	10 (6-17)		10 (9-11)
	Co-treatment
	Epirubicin	10	5		3			2
	Cisplatin	10	3		5			2
	Values are medians with the ranges in parentheses
				100
			Concentration (mg/L)	10 1				gem A/A gem A/C gem C/C
								dFdU A/A
								dFdU A/C
								dFdU C/C
				0 0
				0	1	2	3	4	5	6

Table 2

 2 Gemcitabine pharmacokinetics

	Cytidine deaminase genotype subgroups
	All patients (n=20)

Conflict of interest An unrestricted research grant from Eli Lilly, Nieuwegein, the Netherlands, was received for this study.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


Áine Merwick

Niamh Hannon

Peter J Kelly

O ' Killian

Rourke

Killian O'rourke

Warfarin-flucloxacillin interaction presenting as cardioembolic ischemic stroke

Keywords: Warfarin, Flucloxacillin, Antibiotic, Interaction, Sub-therapeutic anticoagulation, Stroke

come

Warfarin-flucloxacillin interaction presenting as cardioembolic ischemic stroke

Both warfarin and flucloxacillin are frequently prescribed medications which have the potential to have life threatening interactions. We describe a case of sub-therapeutic anticoagulation following a course of oral flucloxacillin, resulting in an ischemic stroke. A 68-year-old female on long term anti-coagulation for atrial fibrillation presented with an acute left hemiparesis. International normalised ratio (INR) was 1.4 in the emergency department. She had commenced a 14-day course of oral flucloxacillin 500 mg qid and oral phenoxymethylpenicillin 999 mg qid for a soft tissue infection of her foot 17 days prior to admission. The most recent INR taken 19 days prior to admission was 3.5.

The patient's anti-coagulation regimen had been managed by a hospital based clinic. A target INR of 2-3 had been maintained with doses of 4 and 3 mg on alternate days for a period of over 6 months with monthly INR checks. Past medical history included diabetes mellitus type 2, tissue mitral valve replacement, myocardial infarction and hypercholesterolaemia. Her concomitant medications were quinapril, atorvastatin, metoprolol, lercardipine, spironolactone, bumetanide, digoxin, metformin, gliclazide and biphasic isophane insulin. The patient did not report any other recent change in medication, any change in diet or alcohol intake and no change in warfarin brand. She had not experienced any gastrointestinal illness and reported good compliance with her medication regimen.

No alternative stroke mechanism was identified following extensive investigation including carotid duplex, magnetic resonance brain imaging (MRI), magnetic resonance angiography (MRA), and trans-thoracic echocardiography.

Using the Drug Interaction Probability Scale, a rating of 7 in this case indicates a probable causative interaction [START_REF] Horn | Proposal for a new tool to evaluate drug interaction cases[END_REF]. On review of the patient's pharmacy, medical and anticoagulation clinic records, it was noted that oral flucloxacillin and phenoxymethylpenicillin had previously been prescribed for the patient 6 weeks prior to admission. On that occasion an INR of 1.8 was recorded 5 days after completing the course of antibiotics.

A case of suspected interaction between oral flucloxacillin and warfarin has been recently reported [START_REF] Garg | Decreased INR response secondary to warfarin-flucloxacillin interaction[END_REF]. Review of the literature reveals one further description of subtherapeutic anti-coagulation when flucloxacillin and a coumarin were prescribed concurrently. The authors in that case attributed the fall in INR to concomitant rifampicin, however the role of flucloxacillin may have been underestimated [START_REF] Harmsze | Prolonged diminished effect of coumarin derivatives after use of rifampicin[END_REF]. Flucloxacillin has the potential to induce expression of cytochrome P450 3A4 which is the enzyme which catalyzes the 10-hydroxylation of R-warfarin [START_REF] Huwyler | Induction of cytochrome P450 3A4 and P-glycoprotein by the isoxazolylpenicillin antibiotic flucloxacillin[END_REF][START_REF] Tang | Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions[END_REF].

The inflammatory response to infection and individual genetic variation may influence warfarin metabolism by pharmacokinetic or pharmacodynamic mechanisms [START_REF] Horn | Proposal for a new tool to evaluate drug interaction cases[END_REF][START_REF] Kamali | Genetic influences on the response to warfarin[END_REF]. In this case the infection treated was a localised skin infection with no evidence of a systemic inflammatory response or underlying osteomyelitis. Sub-therapeutic anticoagulation has been reported previously with warfarin and nafcillin and dicloxacillin, other narrow spectrum betalactam antibiotics [START_REF] Kim | Interaction between warfarin and nafcillin: case report and review of the literature[END_REF][START_REF] Lacey | Interaction of dicloxacillin with warfarin (letter)[END_REF].

This case further demonstrates the potential for interaction between warfarin and flucloxacillin resulting in subtherapeutic anti-coagulation. Amoxicillin has been reported to enhance the effects of warfarin rather than to diminish its effects, and the role of phenoxymethylpenicillin in the case is not clear [START_REF] Davydov | Warfarin and amoxicillin/clavulanate drug interaction[END_REF][START_REF] Holbrook | Systematic overview of warfarin and its drug and food interactions[END_REF]. We suggest vigilance when any medication is begun or discontinued in a patient on warfarin therapy by health care professionals involved in prescribing and monitoring of anti-coagulation.

Eur J Clin Pharmacol (2010) 66:643-644 DOI 10.1007/s00228-010-0802-9

Conflict of interest

No conflict of interest is reported.


Helene Puehringer
email: puehringer@viennalab.co.at

Ralph M Loreth

Gudrun Klose

Brigitte Schreyer

Walter Krugluger

Barbara Schneider

Christian Oberkanins

VKORC1 -1639G>A and CYP2C9*3 are the major genetic predictors of phenprocoumon dose requirement

Keywords: Coumarins, CYP2C9, Dose variability, Phenprocoumon, VKORC1 H, Puehringer

Introduction

The vitamin K antagonists (VKAs) warfarin, acenocoumarol and phenprocoumon are widely prescribed and highly effective oral anticoagulants for the long-term treatment and prophylaxis of a number of thromboembolic disorders. The management of anticoagulation with coumarins is complicated by their narrow therapeutic window, the high inter-individual dose variability and the increased risk of hemorrhagic side effects in patients requiring a low dose. To assure an efficient and safe therapy, patients require an individual dose adjustment and constant monitoring of the international normalised ratio (INR). Warfarin is the oral anticoagulant of choice in most countries, particularly in North America, UK and Scandinavia, while acenocoumarol and phenprocoumon are frequently prescribed in many continental European countries.

Since the discovery of the target enzyme VKORC1 [START_REF] Li | Identification of the gene for vitamin K epoxide reductase[END_REF][START_REF] Rost | Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2[END_REF], several rare amino acid substitutions leading to warfarin resistance have been described to date [START_REF] Rost | Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2[END_REF][START_REF] Harrington | Pharmacodynamic resistance to warfarin associated with a Val66Met substitution in vitamin K epoxide reductase complex subunit 1[END_REF][START_REF] Bodin | A vitamin K epoxide reductase complex subunit-1 (VKORC1) mutation in a patient with vitamin K antagonist resistance[END_REF][START_REF] Loebstein | A coding VKORC1 Asp36Tyr polymorphism predisposes to warfarin resistance[END_REF]. Furthermore, common single nucleotide polymorphisms (SNPs) in noncoding regions of the VKORC1 gene are known to be associated with altered drug sensitivity [START_REF] D'andrea | A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin[END_REF][START_REF] Rieder | Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Doses[END_REF][START_REF] Wadelius | Common VKORC1 and GGCX polymorphisms associated with warfarin dose[END_REF]. Among these, the tightly linked promoter variant -1639G>A and the 1173C>T substitution in intron 1 are the most useful tag-SNPs for the identification of the low-dose haplotype group A (variant) and the high-dose haplotype group B (wildtype) described by Rieder et al. [START_REF] Rieder | Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Doses[END_REF].

Whereas VKORC1 polymorphisms have a relatively similar pharmacodynamic effect on all VKAs, the contribution of cytochrome P450 2C9 (CYP2C9) to the clearance of each anticoagulant appears to be substantially different. CYP2C9 is mainly responsible for the metabolic clearance of the S-enantiomere, which shows the highest anticoagulation activity in the racemic mixture of coumarins. About 40% of ingested phenprocoumon are excreted unchanged, whereas warfarin and acenocoumarol are almost completely metabolised mainly by CYP2C9-driven hydroxylation. The biologically more active S-phenprocoumon is cleared to 65% only by CYP2C9 and to 35% by the less polymorphic CYP3A4 [START_REF] Ufer | Comparative pharmacokinetics of vitamin K antagonists. Warfarin, phenprocoumon and acenocoumarol[END_REF]. The defective CYP2C9*2 and *3 variants are causally linked to a reduced dose requirement for warfarin [START_REF] Margaglione | Genetic modulation of oral anticoagulation with warfarin[END_REF][START_REF] Higashi | Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy[END_REF][START_REF] Voora | Prospective dosing of warfarin based on cytochrome P-450 2C9 genotype[END_REF][START_REF] Hillman | A prospective, randomized pilot trial of model-based warfarin dose initiation using CYP2C9 genotype and clinical data[END_REF], and a similar effect of CYP2C9*3 on acenocoumarol has been repeatedly reported [START_REF] Hermida | Differential effects of 2C9*3 and 2C9*2 variants of cytochrome P-450 CYP2C9 on sensitivity to acenocoumarol[END_REF][START_REF] Tassies | Pharmacogenetics of acenocoumarol: cytochrome P450 CYP2C9 polymorphisms influence dose requirements and stability of anticoagulation[END_REF][START_REF] Bodin | Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genotypes as determinants of acenocoumarol sensitivity[END_REF]. The metabolism of phenprocoumon seems to be less influenced by CYP2C9 genotypes [START_REF] Kirchheiner | Effects of CYP2C9 polymorphisms on the pharmacokinetics of R-and Sphenprocoumon in healthy volunteers[END_REF][START_REF] Ufer | Genetic polymorphisms of cytochrome P450 2C9 causing reduced phenprocoumon (S)-7hydroxylation in vitro and in vivo[END_REF]. The combined effect of the VKORC1 and CYP2C9 alleles on dose variability, severe overanticoagulation and time to achieve stable anticoagulation has been extensively studied in patients treated with warfarin [START_REF] Carlquist | Genotypes of the cytochrome p450 isoform, CYP2C9, and the vitamin K epoxide reductase complex subunit 1 conjointly determine stable warfarin dose: a prospective study[END_REF][START_REF] Schwarz | Genomic determinants of response to warfarin during initial anticoagulation[END_REF][START_REF] Meckley | An analysis of the relative effects of VKORC1 and CYP2C9 variants on anticoagulation related outcomes in warfarintreated patients[END_REF][START_REF] Wadelius | The largest prospective warfarin-treated cohort supports genetic forecasting[END_REF] and to a lesser extent in acenocoumaroltreated patients [START_REF] Tassies | Pharmacogenetics of acenocoumarol: cytochrome P450 CYP2C9 polymorphisms influence dose requirements and stability of anticoagulation[END_REF][START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and acenocoumarol anticoagulation status: interaction between both genotypes affects overanticoagulation[END_REF][START_REF] Spreafico | Effects of CYP2C9 and VKORC1 on INR variations and dose requirements during initial phase of anticoagulation therapy[END_REF]. However, there is still only limited and somewhat controversial information available on the relative contributions of VKORC1 and CYP2C9 polymorphisms to the clinical outcome of phenprocoumontreated patients [START_REF] Schalekamp | Effects of cytochrome P450 2C9 polymorphisms on phenprocoumon anticoagulation status[END_REF][START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: Interaction between both genotypes affects dose requirement[END_REF][START_REF] Hummers-Pradier | Determination of bleeding risk using genetic markers in patients taking phenprocoumon[END_REF][START_REF] Visser | The risk of overanticoagulation in patients with cytochrome P450 CYP2C9*2 or CYP2C9*3 alleles on acenocoumarol or phenprocoumon[END_REF]. Moreover, in contrast to warfarin, for which several dosing algorithms have been developed in recent years [START_REF] Sconce | The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen[END_REF][START_REF] Zhu | Estimation of warfarin maintenance dose based on VKORC1 (-1639G>A) and CYP2C9 genotypes[END_REF][START_REF] Anderson | Randomized trial of genotyped-guided versus standard warfarin dosing in patients initiating oral anticoagulation[END_REF][START_REF] Gage | Use of pharmacogenetic and clinical factors to predict the therapeutic dose of warfarin[END_REF][START_REF]Estimation of the warfarin dose with clinical and pharmacogenetic data[END_REF], no pharmacogenetic-based predictive rules for determining the initial dose of phenprocoumon treatment are currently established.

Here, we report the results of our investigation on the extent to which genetic polymorphisms in the VKORC1 promoter (-1639G>A) and the 3′-untranslated region (3730G>A)-the latter defining a subtype of the high-dose haplotype B [START_REF] Rieder | Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Doses[END_REF][START_REF] Wadelius | Common VKORC1 and GGCX polymorphisms associated with warfarin dose[END_REF]-as well as CYP2C9*2 and *3 contribute to individual dose variability in phenprocoumon therapy.

Methods

Patients

For this retrospective study, we recruited 185 patients on long-term maintenance therapy with phenprocoumon (Marcumar) between April 2007 and December 2008. All outpatients ≥18 years with a stable anticoagulation status who were being monitored in clinics in Austria (Rudolfstiftung Hospital, Vienna; n=76) and Germany (Westpfalz-Klinikum, Kaiserslautern; n =109) were eligible for the study. No exclusion criteria were applied. At the time of admission most study participants were being co-treated with ß-blockers, angiotensin-converting enzyme inhibitors, digitalis glycosids, diuretics, simvastatin or antidiabetic drugs due to various age-related co-morbidities (mean age of patients69.6 years). Mean weekly phenprocoumon doses were calculated after stable anticoagulation was achieved, which was defined as INR measurements within the target INR of 2.5±0.5 at constant phenprocoumon dosing for at least 3 consecutive clinic visits. For the vast majority of patients, INR values were recorded every 3 weeks. Blood samples were taken during routine controls, and data on age, gender, weight and required phenprocoumon dose were collected for each patient. All subjects provided informed consent to participate in the study.

Genotyping

Genomic DNA was extracted from peripheral blood leukocytes using the GEN X TRACT Blood DNA Extraction System (ViennaLab Diagnostics, Vienna, Austria). Genotyping for the VKORC1 -1639G>A and 3730G>A SNPs as well as for the CYP2C9 allelic variants *2 (430C>T) and *3 (1075A>C) was performed by PCR followed by reversehybridisation as described for teststrip assays [START_REF] Oberkanins | A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations[END_REF]. Briefly, DNA fragments spanning the four SNPs were amplified and biotinylated in a multiplex PCR using primers described elsewhere [START_REF] D'andrea | A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin[END_REF][START_REF] Hillman | A prospective, randomized pilot trial of model-based warfarin dose initiation using CYP2C9 genotype and clinical data[END_REF][START_REF] Montes | The c.-1639G>A polymorphism if the VKORC1 gene is a major determinant of the response to acenocoumarol in anticoagulated patients[END_REF]. Amplicons were hybridised for 30 min at 45±0.5°C to a membrane teststrip presenting parallel aligned allele-specific oligonucleotides for each of the two VKORC1 [-1639G>A: TTGGCC(G/A)GGTGCG; 3730G>A: ATACCC(G/A)CACATG] and CYP2C9 [*2: GAGGAC(C/T)GTGTTC; *3: AGATAC(A/C)TTGACC] polymorphisms. After a series of stringent washes, specifically bound PCR fragments were detected using a streptavidin-alkaline phosphatase conjugate and colour substrates (BCIP/NBT reaction). The entire hybridisation and detection procedure was carried out either manually or fully automated using a temperature-controlled teststrip processor (profiBlot II T 48; TECAN, Groedig, Austria). Reference DNA samples previously typed by direct DNA sequencing were available for all SNPs and were used for performance control of the assay.

Statistical analysis

For a description of age, weight and dose, we computed the mean, standard deviation (SD), median and minimum and maximum values. Pearson correlation coefficients were calculated to estimate the associations between age, weight and dose. The quality of the genotype data was assessed by testing for Hardy-Weinberg equilibrium. Genotypes were coded according to the number of variant alleles per patient (0, 1, 2). Associations between different genotypes or between genotypes and sex were analysed by contingency tables in combination with a χ 2 test. The dependency of age, weight and dose on different genotypes or sex was analysed using one-way analysis of variance (ANOVA) with Tukey's post hoc test, if appropriate. We used a stepwise linear regression model to estimate the impact of genotypes, age, weight and sex on the dose. Due to the skewed distribution, the square root-transformed dose values were used as the dependent variable in the model. For all tests a p value <0.05 was considered to be statistically significant. Because of the retrospective nature of the data, all statistical test results have to be interpreted with caution. SAS ver. 9.2 statistical software (SAS Institute, Cary, NC) was used for all statistical analyses.

Results

Population characteristics

The study population consisted of 185 European Caucasians with stable control of phenprocoumon therapy. Age, weight and sex distribution, indications for anticoagulation and VKORC1 and CYP2C9 genotype frequencies of the study participants are summarised in Table 1. Among these patients, the mean phenprocoumon dose required to maintain a therapeutic INR of 2-3 was 12.9 mg/week, but individual doses ranged from 1.5 to 34.5 mg/week, demonstrating a 23fold inter-patient variability. Testing for VKORC1 SNPs among the study participants revealed a frequency of 41.4% for the low-dose haplotype A-determining -1639A allele and 37.3% for the minor 3730A allele. The allele frequencies for CYP2C9*2 and *3 were 11.9 and 8.1%, respectively. The observed genotype frequencies for the VKORC1 and CYP2C9 SNPs showed no deviation from Hardy-Weinberg equilibrium (data not shown). Pairwise comparison of genotype frequencies revealed a significant linkage between the -1639G>A and 3730G>A loci in the VKORC1 gene (χ 2 test p<0.0001). All patients homozygous for -1639AA (n= 33) were found to be wild-type (GG) at the 3730 locus, whereas all homozygous 3730AA carriers (n=29) were wildtype (GG) at the -1639 locus. All other genotypes did not show any statistically significant distribution. Demographic factors, such as age, weight and gender, did not vary significantly among different VKORC1 and CYP2C9 genotypes except for VKORC1 3730AA, which was significantly over-represented in younger patients (p<0.02).

Impact of demographic variables and VKORC1 and CYP2C9 polymorphisms on phenprocoumon dose

Correlation analysis of the weekly phenprocoumon dose revealed a statistical significant negative correlation with age (r=-0.35) and a positive correlation with body weight (r=0. [START_REF] Wadelius | The largest prospective warfarin-treated cohort supports genetic forecasting[END_REF]. No significant differences were present between males and females.

The influence of genetic polymorphisms on the mean weekly dose needed to achieve stable anticoagulation was initially investigated by univariate analysis in patients not adjusted for age and weight (Fig. 1). Homozygous carriers of the VKORC1 -1639A allele required a significantly lower dose (8.8±5.3 mg) than heterozygous GA (12.5± 5.3 mg) or wild-type GG (15.5±6.8 mg) carriers (Fig. 1a). An inverse correlation was observed for the VKORC1 3730G>A polymorphism, with the average dose for homozygous AA carriers being higher (15.9±7.5 mg) than that for heterozygous GA (13.2±6.2 mg) or homozygous GG (11.5 ±5.5 mg) carriers (Fig. 1b). However, the difference was only significant between homozygous GG and AA genotypes and could be explained by (1) the high linkage of both VKORC1 loci and (2) the significantly higher proportion of AA genotypes in younger patients needing a higher dosage.

Irrespective of the VKORC1 genotype the presence of at least one CYP2C9*3 allele in patients substantially decreased the weekly administered mean phenprocoumon dose to 10.1 ± 4.8 mg compared to 13.9 ± 6.2 mg in CYP2C9*1*1 wild-types. In carriers with one CYP2C9*2 allele, the dose reduction, to 12.3±6.8 mg, was much less pronounced. Only one homozygous patient for each CYP2C9 variant and two compound heterozygous patients were found in our cohort (Fig. 1c; Table 2). However, none of the dose differences between CYP2C9 genotypes reached statistical significance due to the relatively small sample size.

The impact of the combined VKORC1 -1639G>A and CYP2C9 genotypes on the mean weekly phenprocoumon dose requirement is shown in Table 2. In all VKORC1 genotype groups, carriers of the CYP2C9*3 allele required a considerable lower dose than *2 carriers compared to carriers of the CYP2C9 wild-type. The dose-reducing effect conferred by the *3 variant was more evident in -1639GG patients (-4.8 mg) than in those with the GA (-3.2 mg) or AA (-1.8 mg) genotypes.

Multiple linear regression modelling

A multiple linear regression analysis was performed to test for individual contributions of genotypes and demographic factors to the variance in maintenance dosage. Variables found to be associated with phenprocoumon dose by univariate analysis were entered into the regression model. The stepwise analysis revealed a statistically significant contribution of VKORC1 -1639G>A, age and CYP2C9*3 to the variability of the square root-transformed phenprocoumon dose (Table 3). The model demonstrated that among all investigated parameters VKORC1 -1639G>A most strongly determined the weekly dose (r 2 =0.142), followed by patient age (r 2 =0.091) and CYP2C9*3 (r 2 = 0.047), jointly explaining 28% of the observed variability. CYP2C9*2 and weight turned out to have a marginal and insignificant influence on individual dose requirement, adding only 1.4 and 1.2%, respectively. VKORC1 3730G>A and gender were excluded, as they did not meet the significance criteria for entry into the model. Altogether, the full model comprising five covariates explained 31% of the variability observed in the square root-transformed phenprocoumon doses (r 2 =0.31; Table 3).

Discussion

Among our ethnically homogenous patient group we observed an up to 23-fold variation in dose requirement to achieve stable anticoagulation, which reflects the known high inter-individual variability. Our aim was to achieve an understanding of the basis for this large difference in sensitivity to phenprocoumon by evaluating the main genetic polymorphisms implicated in the drug's action and metabolism along with non-genetic parameters.

Our data corroborate the predominant role of VKORC1 haplotypes in the anticoagulation process with all classes of coumarin derivatives. As expected, the VKORC1 tag-SNP -1639G>A haplotype showed the greatest impact on phenprocoumon dose requirement and explained 14.2% of the dose response variability in our study. Two copies of the -1639A allele conferred the highest sensitivity to the drug, leading to a 43% lower dose requirement compared to two wild-type alleles. There is some evidence that this SNP is indeed the causal polymorphism for the haplotype A-induced sensitivity due to a reduced transcriptional activity of the promoter [START_REF] Yuan | A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity[END_REF], and, consequently, a lower mRNA transcript level [START_REF] Rieder | Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Doses[END_REF] that translates into decreased VKORC1 activity rendering the patient more susceptible to VKAs.

Genotyping for VKORC1 3730G>A, which defines a subtype of the high-dose haplotype B [START_REF] Rieder | Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Doses[END_REF][START_REF] Wadelius | Common VKORC1 and GGCX polymorphisms associated with warfarin dose[END_REF], had no additional predictive power for individual dose variability in our study. The effect of 3730G>A on phenprocoumon dose seen in the univariate analysis may be largely caused by its linkage to -1639G>A. According to Wadelius et al. [START_REF] Wadelius | Common VKORC1 and GGCX polymorphisms associated with warfarin dose[END_REF], the linkage disequilibrium for both SNPs is 0.39 (r 2 measurement). The fact that SNP 3730G>A failed the entry into our regression model underlines its negligible role in the phenprocoumon dose requirement. This is in contrast to findings of two other studies in which the 3730G>A SNP contributed 1.2 and 9% to the variability in warfarin dose, respectively [START_REF] Herman | The influence of sequence variations in factor VII, γglutamyl carboxylase and vitamin K epoxide reductase complex genes on warfarin dose requirement[END_REF][START_REF] Borgiani | Allelic variants in the CYP2C9 and VKORC1 loci and interindividual variability in the anticoagulant dose effect of warfarin in Italians[END_REF].

Higher age is an independent predictor of lower phenprocoumon needs. In our patient group, the factor age was the second-most important predictor and explained a greater percentage of the variability in dosage than the CYP2C9 genotype, which is in line with previous reports on coumarin anticoagulants [START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: Interaction between both genotypes affects dose requirement[END_REF][START_REF] Sconce | The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen[END_REF]. Drug metabolism decreases with age and is probably related to a reduced ability to metabolise coumarin derivatives. Furthermore, food intake and concomitantly the intake of vitamin K decreases in elderly patients, thereby increasing the individual's coumarin sensitivity.

Somewhat conflicting results have been reported in the past in terms of the impact of CYP2C9 genotypes on phenprocoumon dosing. While no differences were found in two studies [START_REF] Hummers-Pradier | Determination of bleeding risk using genetic markers in patients taking phenprocoumon[END_REF][START_REF] Visser | The risk of overanticoagulation in patients with cytochrome P450 CYP2C9*2 or CYP2C9*3 alleles on acenocoumarol or phenprocoumon[END_REF], Schalekamp et al. [START_REF] Schalekamp | Effects of cytochrome P450 2C9 polymorphisms on phenprocoumon anticoagulation status[END_REF] reported a significant dose difference between carriers of a CYP2C9*2 or *3 variant and CYP2C9*1*1 wild-type patients. In a follow-up study, this effect was shown to be modified by the VKORC1 C1173T genotype, being most pronounced in CC carriers [START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: Interaction between both genotypes affects dose requirement[END_REF]. In our cohort, a small but significant dosereducing effect by a single copy of the CYP2C9*3 allele was still apparent after patients were stratified for VKORC1 -1639G>A and age. For the CYP2C9*2 allele, the effect was much smaller and failed to reach statistical significance. The rare occurrence of homozygous *2 and *3 (n= 1) and compound heterozygous patients (n =2) in our cohort did not allow us to determine a gene-dose effect.

In the setting of our study, we were able to assign 31% of the observed inter-individual variability in phenprocoumon dose to specific factors, suggesting the presence of additional unidentified environmental and/or genetic factors that modify drug response. Several comprehensive screening studies, including genome-wide association analyses, have recently been undertaken in an attempt to identify as yet unknown genetic factors influencing warfarin response [START_REF] Wadelius | The largest prospective warfarin-treated cohort supports genetic forecasting[END_REF][START_REF] Cooper | A genomewide scan for common genetic variants with a large influence on warfarin maintenance dose[END_REF][START_REF] Takeuchi | A genomewide association study confirms VKORC1, CYP2C9, and CYP4F2 as principal Genetic Determinants of Warfarin Dose[END_REF]. However, insufficient evidence was obtained in all of these studies to support the inclusion of genetic variants other than VKORC1 haplotype A/B and CYP2C9*2 and *3 in algorithms for the prediction of coumarin maintenance dose. The only SNP found to be independently associated with warfarin dose was identified in the CYP4F2 gene, explaining only a further 1-2% of variability [START_REF] Takeuchi | A genomewide association study confirms VKORC1, CYP2C9, and CYP4F2 as principal Genetic Determinants of Warfarin Dose[END_REF][START_REF] Caldwell | CYP4F2 genetic variant alters required warfarin dose[END_REF]. The pharmacogenetic effect of the CYP4F2 V433M polymorphism was recently confirmed in acenocoumarol-treated patients [START_REF] Perez-Andreu | Pharmacogenetic relevance of CYP4F2 V433M polymorphism on acenocoumarol therapy[END_REF].

There are some limitations to this study that may partially account for the rather high percentage of unexplained variability. Owing to the retrospective study design, insufficient documentation of concomitant medication interfering with phenprocoumon efficacy was available. Furthermore, no data were recorded about destabilising comorbidities, hepatic or renal dysfunctions and smoking status. Therefore, all of these potentially confounding factors could not be considered in the prediction model.

In clinical practice, the initial dose finding process for phenprocoumon is still largely empiric, and the critical issue is how closely the induction dose matches the final maintenance dose. A clinical algorithm was recently published that includes weight, age, alcohol consumption and a surgery within 1 week as the main predictors for the loading dose of phenprocoumon. This model had an explanatory power of 33% [START_REF] Good | A clinical algorithm to predict the loading dose of phenprocoumon[END_REF]. Our data endorse a fundamental role of VKORC1 haplotypes and age and a minor role of CYP2C9 variants in the variability of dose requirement, which is in agreement with previous reports on phenprocoumon [START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: Interaction between both genotypes affects dose requirement[END_REF][START_REF] Werner | Pharmacogenetic characteristics of patients with complicated phenprocoumon dosing[END_REF] and other coumarins [START_REF] Rieder | Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Doses[END_REF][START_REF] Wadelius | Common VKORC1 and GGCX polymorphisms associated with warfarin dose[END_REF][START_REF] Tassies | Pharmacogenetics of acenocoumarol: cytochrome P450 CYP2C9 polymorphisms influence dose requirements and stability of anticoagulation[END_REF]. With respect to more severe clinical outcomes of VKAs, such as increased bleeding risk and retarded stabilisation, the roles may be switched to a stronger association with CYP2C9 polymorphisms [START_REF] Meckley | An analysis of the relative effects of VKORC1 and CYP2C9 variants on anticoagulation related outcomes in warfarintreated patients[END_REF][START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and acenocoumarol anticoagulation status: interaction between both genotypes affects overanticoagulation[END_REF][START_REF] Schalekamp | VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: Interaction between both genotypes affects dose requirement[END_REF]. In order to make the

Fig. 1

 1 Fig. 1 Box and whisker plots showing the distribution of weekly phenprocoumon doses based on different VKORC1 -1639G>A (a), VKORC1 3730G>A (b) and CYP2C9 (c) genotypes. Boxes Median and upper and lower quartiles, respectively. The maximum length of each whisker is 1.5× the interquartile distance. Points falling outside the plots show up as outliers. Asterisks above the median indicate statistically significant differences

Table 2

 2 Impact of the VKORC1 -1639G>A and CYP2C9 genotype combinations on mean phenprocoumon dose a Standard deviation is given in parenthesis

	VKORC1 -1639G>A	CYP2C9	Number of carriers	Mean phenprocoumon dose (mg/week) a
	GG	*1*1	41	16.5 (±7.4)
		*1*2	14	15.4 (±5.6)
		*1*3	10	11.7 (±5.3)
	GA	*1*1	58	13.3 (±4.6)
		*1*2	17	11.7 (±7.3)
		*1*3	9	10.1 (±3.0)
		*2*3	2	10.9 (±3.7)
		*3*3	1	6.8
	AA	*1*1	16	9.6 (±5.5)
		*1*2	9	8.7 (±5.1)
		*1*3	7	7.8 (±4.7)
		*2*2	1	5.3

Table 3

 3 Multiple linear regression model considering the square-root transformed weekly phenprocoumon dose as dependent variable a

	Variable	Parameter estimate Model r 2 p value
	Intercept	4.8230		<0.0001
	VKORC1 -1639G>A b -0.4184	0.1424	<0.0001
	Age (years)	-0.0187	0.2334	<0.0001
	CYP2C9*3 c	-0.5535	0.2806	0.0007
	CYP2C9*2 c	-0.2503	0.2944	0.0622
	Weight (kg)	0.0057	0.3068	0.0758

a √Dose=4.823-0.4148 (VKORC1 -1639G>A) -0.0187 (years) -0.5535 (CYP2C9*3) -0.2503 (CYP2C9*2) + 0.0057 (kg) b Value 0 for GG, 1 for GA, 2 for AA c Value 0, 1 or 2 for the number of CYP2C9*2 and *3 alleles √Dose square root-transformed dose

individual loading dose of phenprocoumon more predictable, genetic testing for both pharmacogenetic markers should be considered in algorithms developed in the future.