Lilianna Bartnicka 1, Mateusz Kurzawski 1, Agnieszka Drozdzik 2, Edyta Plonska-Gosciniak 3, Wanda Gornik 1, Marek Drozdzik 1.

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1 Pharmacological Reports 2007, 59, ISSN Copyright 2007 by Institute of Pharmacology Polish Academy of Sciences Effect of ABCB1 (MDR1) 3435C >T and 2677G >A,T polymorphisms and P-glycoprotein inhibitors on salivary digoxin secretion in congestive heart failure patients Lilianna Bartnicka 1, Mateusz Kurzawski 1, Agnieszka Drozdzik 2, Edyta Plonska-Gosciniak 3, Wanda Gornik 1, Marek Drozdzik 1! Department of Pharmacology, Department of Periodontology, Department of Cardiology, Pomeranian Medical University, Powstañców Wlkp. 72, PL Szczecin, Poland Correspondence: Marek DroŸdzik, Abstract: The aim of the present study was to evaluate the effects of ABCB1 (MDR1) gene polymorphism on P-glycoprotein model substrate, i.e. digoxin, salivary secretion. The study was carried out in 77 patients diagnosed with congestive heart failure administered digoxin, who were subdivided into two groups: 1) co-administered P-glycoprotein inhibitors and 2) without any known P-glycoprotein inhibitors. The ABCB1 2677G >A,T and 3435C >T polymorphisms were evaluated using PCR-RFLP methods. Steady-state digoxin concentrations were measured in blood serum as well as in unstimulated and stimulated saliva using FPIA method. It was found that values of Pearson s coefficient were significantly higher in patients co-administered P-glycoprotein inhibitors in comparison with subjects who were not administered any inhibitor both for stimulated (Pearson s coefficient r = 0.832, p < 0.01) and unstimulated saliva (r = 0.812, p < 0.01). Evaluation of the impact of ABCB1 2677G >A,T and 3435C >T polymorphism on salivary digoxin secretion revealed significant differences in digoxin stimulated ratio between patients stratified by 2677G >A,T genotype (TT, TA> GT, GA> GG, p < 0.01). The results from the present study suggest that administration of P-glycoprotein inhibitors as well as ABCB1 gene polymorphism may affect salivary digoxin secretion. Key words: ABCB1 polymorphism, P-glycoprotein, digoxin, salivary secretion Abbreviations: ABCB1 ATP-binding cassette, sub-family B, member 1, MDR1 multidrug resistance-1, PCR polymerase chain reaction, P-gp P-glycoprotein, SNP single nucleotide polymorphism Introduction Salivary drug secretion may have some potential clinical consequences. Rate of drug secretion into saliva may contribute to drug side effects seen in the oral cavity, e.g. gingival overgrowth after administration of cyclosporine A, phenytoin or amlodipine [15]. However, gingival overgrowth is not observed in all medicated patients. Another potential clinical consequence of salivary drug secretion is therapeutic drug monitoring guided by drug concentrations in saliva. Phenytoin, theophylline have been most often studied and most authors agree that the correlation between salivary and plasma concentrations is sufficient to be used for therapeutic monitoring in patients treated with those drugs [5]. Pharmacological Reports, 2007, 59,

2 For other drugs studied, e.g. carbamazepine, phenobarbital, gentamycin or digoxin, wide interindividual differences in blood/saliva drug concentration ratio were revealed, which limit clinical application of salivary drug measurements for therapeutic drug monitoring [5, 12]. Among many potential factors which might contribute to interindividual differences in salivary drug secretion is active drug transport. Some drug transporters were identified in salivary glands. As reported by Uematsu et al. [17] and our previous study [2], expression of P-glycoprotein (product of ABCB1, formerly MDR1 gene), multidrug resistanceassociated protein (MRP1), MRP2/canalicular multispecific organic anion transporter (cmoat) and lung-resistance-related protein (LRP) were detected in salivary glands. However, there is no available direct data on active drug transport in salivary glands. Digoxin, which is secreted into saliva can be used as a model drug to study P-glycoprotein (encoded by ABCB1 gene)-dependent transport [13]. Single nucleotide polymorphisms (SNPs) of ABCB1 gene have been identified including one which was localized in the position 3435C >T of exon 26 [7]. This SNP is supposed to be related to altered expression of ABCB1 gene and P-gp activity. In homozygous TT-allele subjects the P-gp expression in digestive system is lower in comparison with CT heterozygous and homozygous non-mutated CC individuals. Homozygous wild- -type allele carriers have been reported to have higher plasma concentrations of digoxin, which was caused by the lack of active P-gp form in digestive tract [7]. The 3435C >T SNP is a silent mutation that does not cause amino acid substitution and is suggested to be linked, in a majority of subjects, with the mutation in exon 21, position 2677 (2677G >T,A), producing Ala893Thr and Ala893Ser, respectively [16]. Individuals who were homozygous for 2677A,T had significantly decreased intestinal P-gp expression and increased digoxin serum levels after oral administration. Another explanation of the functional role of 3435C > T polymorphism was provided by Wang et al., who demonstrated the effect of the polymorphism on mrna stability. The authors revealed that the T allele was associated with lower mrna levels [18]. Therefore, polymorphism in ABCB1 gene encoding P-glycoprotein may influence salivary concentrations of its substrate drugs, and thus, may affect drugrelated oral side effects or feasibility of therapeutic drug monitoring (using drug concentrations in saliva). The aim of the present study was to evaluate the effects of ABCB1 gene polymorphism on a model substrate, i.e. digoxin, salivary secretion. Materials and Methods Patients Seventy seven unrelated Polish subjects of Caucasian origin diagnosed with congestive heart failure, 40 males and 37 females, aged from 30 to 85 years were included in this study after giving informed consent. The patients were medicated with digoxin mg daily (Digoksyna, Polfa; Bemecor, Polfa) for at least 7 days to reach steady state. The subjects were subdivided into two groups: group 1) 46 patients (22 males, 24 females), aged years (mean 68.7 ± 11.3 years) co-administered P-glycoprotein inhibitors for at least 7 days (spironolactone, carvedilol, verapamil, famotidine, amiodarone, propafenone, prednisone, atorvastatin, simvastatin) and group 2) 31 subjects (15 males, 16 females), aged years (mean 69.5 ± 12.5 years) without any known P-glycoprotein inhibitors. The Ethics Committee of the Pomeranian Medical University in Szczecin, Poland approved protocol of the study. Digoxin measurement In all study subjects, digoxin concentrations were measured in blood serum as well as in stimulated and unstimulated (resting) saliva. Blood and saliva were sampled simultaneously in steady state (at least 7 days from the onset of digoxin medication), before morning drug administration. Blood was drawn from a peripheral vein. In preparation for saliva sampling, the mouth was rinsed with ml of tap water. Unstimulated saliva was collected on a cotton swab (Salivette, Sarstedt, Germany) placed sublingually (for s), which was then centrifuged. Stimulated saliva was collected by the use of swabs containing citric acid (Salivette, Sarstedt, Germany). Digoxin concentrations in serum and saliva were measured by the fluorescence polarization immunoassay (FPIA) method using TDx apparatus (Abbott, USA). 324 Pharmacological Reports, 2007, 59,

3 Salivary digoxin secretion Lilianna Bartnicka et al. Genotyping Genomic DNA was extracted from 450 l of whole blood samples using a non-organic and non-enzymatic extraction method [4]. Genotyping for the presence of 3435C >T and 2677G >A,T SNPs was performed using previously described PCR-RFLP methods applied by our laboratory [11]. in Table 1. It is seen that the ratio of the drug concentration approximates 1.0 in the case of stimulated saliva different from unstimulated sa- Statistical analysis Genotype frequencies were calculated by direct counting and then dividing by the number of subjects or the number of chromosomes to produce genotype and allele frequencies, respectively. The data were tested for their fit to Hardy-Weinberg equilibrium by calculating expected frequencies of genotypes and comparing them to the observed values using a 2 test (Statistica 6.0, Statsoft). The 2 test (with Yate s correction when applicable) was used to compare the observed allele and genotype frequencies to the published data for general Polish population. Salivary and serum digoxin concentration ratios were compared using the Kruskal-Wallis and Mann-Whitney U tests (non-normal distribution). The data were also analyzed with Pearson s correlation coefficient. Results Mean values of digoxin serum/saliva concentration ratio and values of Pearson s coefficient are presented Fig. 1. Additive effect of )*+* 2677G >T,A variant alleles on digoxin stimulated and unstimulated ratios in congestive heart failure patients (vertical bars 95% CI) Tab. 1. Mean (± SD) values of digoxin serum/saliva concentration ratio and values of Pearson s coefficient All cases Group 1 (with P-gp inhibitors) Group 2 (without P-gp inhibitors) Statistical significance (p) Digoxin serum/saliva concentration ratio Stimulated ± ± ± Group 1/Group 2 p=0.461 Unstimulated ± ± ± Group 1/Group 2 p=0.090 Pearson s coefficient Stimulated Group 1/Group 2 p = Unstimulated Group 1/Group 2 p = Pharmacological Reports, 2007, 59,

4 Tab. 2. Mean (± SD) values of digoxin serum/saliva concentration ratio and values of Pearson s coefficient for specific )*+* genotypes All cases Group 1 (with P-gp inhibitors) Group 2 (without P-gp inhibitors) Genotype GG n=31 GT, GA n=29 TT, TA n=17 CC n=15 CT n=43 TT n=19 GG n=11 GT, GA n=10 TT, TA n=10 CC n=7 CT n=15 TT n=9 GG n=20 GT, GA n=19 TT, TA n=7 CC n=8 CT n=28 TT n=10 Digoxin serum/saliva concentration ratio Stimulated Unstimulated 0.85 ± 0.37* 1.50 ± 0.72** 1.07 ± 0.54* 1.8 ± 0.75** 1.28 ± 0.79* 2.10 ± 0.18** 0.87 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.46 Pearson s coefficient Stimulated Unstimulated * Statistically significant differences between different 2677G > A,T genotypes as evaluated by Kruskal-Wallis test (p < 0.01), between TT, TA LI. GT, GA (p < 0.01, Mann-Whitney test) and between 2677GT, GA LI. GG (p < 0.05, Mann-Whitney test); ** a trend towards higher values in TT, TA > GT, GA > GG (p < 0.1 Kruskall-Wallis test), GT, GA > GG and TT, TA > GT, GA (p < 0.1, Mann-Whitney test) Tab. 3. Mean (± SD) values of digoxin serum/saliva concentration ratio and values of Pearson s coefficient for specific MDR1 haplotypes All cases Group 1 (with P-gp inhibitors) Group 2 (without P-gp inhibitors) 2677 GT, GA 3435 CC n= GG 3435 CT n= TT, TA 3435 TT n= GT, GA 3435 CC n= GG 3435 CT n= TT, TA 3435 TT n= GT, GA 3435 CC n= GG 3435 CT n= TT, TA 3435 TT n=4 Digoxin serum/saliva concentration ratio Stimulated 0.91 ± ± ± ± ± ± ± ± ± 0.46 Unstimulated 1.77 ± ± ± ± ± ± ± ± ± 0.51 Pearson s coefficient Stimulated Unstimulated No statistically significant differences between relevant genotypes as evaluated by Kruskal-Wallis test 326 Pharmacological Reports, 2007, 59,

5 Salivary digoxin secretion Lilianna Bartnicka et al. liva, both when P-glycoprotein inhibitors were coadministered or not. However, the observed differences did not reach statistical significance due to high interindividual variability. The values of Pearson s coefficient were significantly higher in patients comedicated with P-glycoprotein inhibitors in comparison with subjects who were not administered any inhibitor, both for stimulated (0.832, p < 0.01) and unstimulated saliva (0.812, p < 0.01). The following ABCB1 allele frequencies have been found in the study subjects: 2677G 0.591, 2677T 0.370, 2677A 0.039, 3435C 0.474, 3435T 0.526, and these values did not differ significantly from frequencies in general population [11]. The genotype frequency distribution did not show a significant deviation from Hardy-Weinberg equilibrium for both studied SNPs. Evaluation of the impact of ABCB1 2677G>A,T and 3435C >T genotypes on salivary digoxin secretion revealed significant differences in digoxin stimulated ratio among patients stratified by 2677G >A,T genotype (TT, TA > GT, GA > GG, p < 0.01). Similar trend in digoxin unstimulated ratio (p < 0.1), irrespectively of P-glycoprotein inhibitor co-administration, was also noted (Fig. 1, Tab. 2). Analysis of 3435C > T genotypes, as well as functional ABCB1 gene haplotypes, grouped for low and high activity genotypes, did not demonstrate any significant effects of the haplotypes on the drug secretion into saliva (Tab. 2, 3). Discussion Salivary drug secretion may affect oral pathology due to local drug-related side effects and measurement of salivary drug concentration can be applied for therapeutic drug monitoring. That is why it is important to define mechanisms implicated in salivary drug secretion. Among well-defined factors influencing drug concentration in saliva are: molecular mass, lipid solubility, degree of ionization, salivary ph and flow rate, protein binding, drug co-administration and mechanism of drug transport [5]. The latter factors may contribute to limitations of therapeutic monitoring using salivary drug concentration by producing wide interindividual differences in drug blood/saliva concentration ratio. Several active drug transporters have been identified in salivary glands [2, 17]. The best described is P-glycoprotein encoded by ABCB1 gene. It has been revealed that P-gp activity is genetically determined, with SNPs at positions 2677G > A,T and 3435C > T being the best established [7, 16]. Thus, the aim of the present study was to establish the effects of ABCB1 gene polymorphism on salivary digoxin secretion, which is a model substrate to study P-gp activity [7, 10]. Digoxin is a non-ionized, neutral and relatively lipophilic substance. Therefore, it should easily diffuse from the blood compartment into saliva, independently of variations in the ph-gradient. It was also postulated by Jusko et al. [9] as well as by Haeckel and Muhlenfeld [6] that digoxin concentration in stimulated saliva reflected the drug concentration in serum, whereas digoxin concentration in unstimulated saliva correlated with intracellular drug concentration. So, the latter concentration may parallel the pharmacologically relevant fraction. In the first step of the study, the effects of P-glycoprotein inhibitors on salivary digoxin secretion were evaluated. It was revealed that the values of Pearson s coefficient were significantly higher in patients coadministered P-gp inhibitors in comparison with subjects who were not administered any inhibitor, both for stimulated (0.832, p < 0.01) and unstimulated saliva (0.812, p < 0.01). These observations did suggest that administration of P-gp inhibitors might abolish genetically determined differences in P-gp transport by more prominent effects in the most active 2677GG and 3435CC P-gp forms. The same mechanisms of P-gp inhibition by clarithromycin was postulated by Kurata et al. [10]. Moreover, a wide interindividual variability of digoxin concentration ratio, irrespectively of the mode of saliva collection (stimulated vs. unstimulated) did suggest a role of genetic factors. Therefore, in a second step of the study, implication of ABCB1 gene polymorphism, encoding P-glycoprotein, in the regulation of salivary drug concentrations was analyzed. In the present study, two most important functional genotypes, i.e. 2677G > A,T and 3435C > T were analyzed [7, 16]. Evaluation of the impact of ABCB1 2677G > A,T and 3435C > T genotypes on salivary digoxin secretion revealed significant differences in digoxin stimulated ratio among patients stratified by 2677G > A,T genotype (TT, TA > GT, GA > GG, p < 0.01) with no significant effects of 3435C > T polymorphism. The above observations may suggest that there is an im- Pharmacological Reports, 2007, 59,

6 pact of ABCB1 2677G > A,T polymorphism on digoxin transport in salivary glands. The results indicate that similarly to the intestine, salivary P-gp mediates its substrates excretion from the body [7]. It is evidenced by higher serum/saliva concentration ratio (both for stimulated and unstimulated saliva) and higher values of Pearson s coefficient in carriers of ABCB1 2677TT, TA genotype as compared to ABCB1 2677GG subjects. The results from the present study do also support Tanabe et al. [16] report, in which the authors postulated that ABCB1 2677G > A,T polymorphism is a better predictor of P-glycoprotein activity than 3435C > T as it involves amino acid substitution contrary to the latter. So, interindividual differences in salivary digoxin secretion may be, at least in part, associated with ABCB1 polymorphism and thus constitute a limitation of salivary digoxin analysis for therapeutic drug monitoring. However, negative findings on association between ABCB1 3435C > T polymorphism and P-glycorpotein-mediated digoxin transport are in keeping with observations of Gerloff et al. [3] and Becquemont et al. [1] who reported no effects of ABCB1 3435C > T polymorphism on digoxin kinetics. Some authors have postulated that analysis of ABCB1 gene haplotypes is superior to unphased SNP analysis to predict MDR1 phenotype, i.e. P-gp activity [8, 10]. However, in the present study, analysis of the functional ABCB1 gene haplotypes, grouped for low and high activity genotypes, did not demonstrate significant effects of the haplotypes on the drug secretion into saliva. So, the results from the present study and literature data are not always univocal. In the recent, very extensive review by Sakaeda [14], it was concluded that ABCB1 genotypes were generally little associated with the pharmacokinetics of drugs, since effects of intestinal P-glycoprotein on the intestinal absorption of substrates was minimal in case of commercially available oral drugs, including digoxin. So, further studies are needed to fully define the role of ABCB1 gene polymorphism in salivary drug secretion. Conclusions The results from the present study suggest that administration of P-glycoprotein inhibitors affects salivary digoxin secretion, and thus influences the drug concentration in saliva which limits its application for therapeutic drug monitoring. It can be further stated that ABCB1 polymorphism may affect salivary digoxin secretion, and thus contribute to interindividual variability of the drug concentration in saliva. Acknowledgments: The study was supported by grant 6P05E12029 from the State Committee for Scientific Research (Warszawa, Poland). Dr M. Kurzawski is supported by the Foundation for Polish Science (Warszawa, Poland). References: 1. Becquemont L, Verstuyft C, Kerb R, Brinkmann U, Lebot M, Jaillon P, Funck-Brentano C: Effect of grapefruit juice on digoxin pharmacokinetics in humans. Clin Pharmacol Ther, 2001, 70, Drozdzik M, Mysliwiec K, Lewinska-Cheustowska M, Banach J, Drozdzik A, Grabarek J: P-glycoprotein drug transporter MDR1 gene polymorphism in renal transplant patients with and without gingival overgrowth. J Clin Periodontol, 2004, 31, Gerloff T, Schaefer M, Johne A, Oselin K, Meisel C, Cascorbi I, Roots I: MDR1 genotypes do not discriminate between absorptive pharmacokinetic parameters of a single oral dose of 1 mg digoxin in healthy white volunteers. Br J Clin Pharmacol, 2002, 54, Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P: A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques, 1991, 11, Haeckel R, Hanecke P: Application of saliva for drug monitoring an in vitro model for transmembrane transport. Eur J Clin Chem Clin Biochem, 1996, 34, Haeckel R, Muhlenfeld HM: Reasons for intraindividual inconstancy of the digoxin saliva to serum concentration ratio. J Clin Chem Clin Biochem, 1989, 27, Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, Johne A, Cascorbi I et al.: Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA, 2000, 97, Johne A, Kopke K, Gerloff T, Mai I, Rietbrock S, Meisel C, Hoffmeyer S et al.: Modulation of steady-state kinetics of digoxin by haplotypes of the P-glycoprotein MDR1 gene. Clin Pharmacol Ther, 2002, 72, Jusko WJ, Gerbracht L, Golden LH, Koup JR: Digoxin concentrations in serum and saliva. Res Commun Chem Pathol Pharmacol, 1975, 17, Kurata Y, Ieiri I, Kimura M, Morita M, Irie S, Urae A, Ohdo S et al.: Role of human MDR1 gene polymorphism in bioavailability and interpretation of digoxin, a substrate of P-glycoprotein. Clin Pharmacol Ther, 2002, 72, Pharmacological Reports, 2007, 59,

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