COMMENTARY

Virtual Journal in Pharmacogenetics-Pharmacogenomics

The MDR1 C3435T polymorphism: Effects on P-glycoprotein expression/function and clinical significance

Corresponding author:
Mark J. Dresser, Ph.D.
Clinical Pharmacology
ALZA Corporation
1950 Charleston Road
Mountain View, CA 94043
Telephone: 650-564-2716
Facsimile: 650-564-2996
E-mail: mark.dresser@alza.com

The MDR1 gene product P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. P-gp utilizes the energy derived from ATP hydrolysis to pump a wide range of compounds, including numerous clinically used drugs, out of cells; this activity has important pharmacokinetic and pharmacodynamic consequences. For example, P-gp is expressed within the apical membranes of intestinal, renal, and hepatic epithelial cells, where it affects the absorption and elimination of its substrates. P-gp is also located within the apical membranes of capillary endothelial cells of the brain, where it can limit the penetration of drugs to the CNS. In addition to the roles of P-gp in absorption, distribution, and elimination, the overexpression of P-gp is implicated in the development of the multi-drug resistance (MDR) phenotype of some tumor cells. Consequently, P-gp inhibitors are now being developed as MDR reversal agents.

Interestingly, a number of P-gp substrates, including digoxin and cyclosporin A, exhibit substantial interindividual variability in their pharmacokinetics. Some of this variability could be attributed to environmental factors, but it is also reasonable to predict that some of this variability arises due to genetic factors, including mutations in genes involved in drug metabolism and transport, such as MDR1. Understanding the functional and clinical consequences of MDR1 variants is important—if this variability could be assigned to a mutation in the MDR1 gene, patients could be screened and appropriate dose adjustments could be made on the basis of their MDR1 genotype. Furthermore, MDR1 variants could have important pharmacodynamic consequences: patients carrying null MDR1 alleles, if such alleles exist, might not respond to P-gp inhibitors used as MDR reversal agents in cancer treatment.

Recently, a number of papers have reported the discovery and initial characterization of MDR1 variants; to date, more than 20 mutations in the MDR1 gene have been identified (1-5). Until now, however, the functional and clinical consequences of only one common MDR1 variant, C3435T, have been investigated (1-3). Originally identified in a German Caucasian population by Hoffmeyer et al., C3435T was found to correlate with P-gp expression in the duodenum as determined by Western blots and quantitative immunohistology (P=0.056) (1). Individuals with the CC genotype (n=6) had higher levels of P-gp expression, approximately 2-fold, compared with individuals with the TT genotype (n=5); heterozygotes had intermediate expression levels (n=10). The mechanism by which the T allele results in lower duodenal P-gp expression is unknown, because C3435T is a silent mutation and does not result in changes in the P-gp sequence. However, Hoffmeyer et al. hypothesize that C3435T may be linked to other variants in the MDR1 gene (1).

Hoffmeyer et al. only examined the effects of the MDR1 C3435T polymorphism on P-gp expression in the duodenum. However, because the MDR1 gene is expressed in many normal tissues and cell types, it is important to establish whether the mutation alters P-gp expression exclusively in the duodenum, thereby affecting only drug absorption, or whether expression is altered in other tissues as well, leading to changes in distribution, elimination, or both of these processes. Using a rhodamine efflux assay as a measure of P-gp activity, Hitzl et al. examined P-gp activity in CD56+ natural killer cells from healthy subjects with the different genotypes at the 3435 locus (2). Rhodamine is a P-gp substrate, thus CD56+ cells with higher P-gp activity would be predicted to have lower intracellular rhodamine fluorescence. Hitzl et al. found that CD56+ cells from individuals with the CC genotype (n=10) had lower rhodamine fluorescence (51.1 ± 11.4%) compared with CD56+ cells from individuals with the TT genotype (n=11) (67.5 ± 9.5%), indicating that cells from CC carriers have higher P-gp activity compared with cells isolated from TT carriers (2). Although this difference was statistically significant, the consequences of a functional difference of this magnitude are debatable. In addition to these functional studies, Hitzl et al. quantified MDR1 RNA transcript levels in leukocytes (2). They did not find a correlation between RNA levels and genotype at position 3435. Hitzl et al. hypothesize that the lack of a correlation was due to their use of leukocytes as the RNA source; leukocytes are a heterogeneous pool of cells that include CD56+ cells, but also other cell types. Although the results of the RNA expression experiments of Hitzl et al. do not necessarily invalidate the results of their functional studies, further experiments examining P-gp transcript levels, and ideally P-gp protein levels, in CD56+ cells are needed to resolve this issue.

In addition to the in vitro studies described above that examined P-gp expression and the functional consequences of the C3435T polymorphism, three clinical studies have been conducted to investigate the clinical relevance of this mutation in the MDR1 gene. Hoffmeyer et al. reported an association between the genotype at the 3435 locus and in vivo P-gp activity as measured by digoxin plasma concentration and pharmacokinetic parameters from two clinical studies in healthy volunteers (1). In the first study, Hoffmeyer et al. genotyped volunteers who had participated in a previous clinical trial, in which eight healthy male volunteers received a single 1 mg oral dose of digoxin or an intravenous infusion of 1 mg of digoxin with and without rifampin (a potent P-gp and CYP3A4 inducer) pretreatment in a crossover fashion (1, 6). Plasma and urine digoxin concentrations were determined and pharmacokinetic parameters, including AUC, bioavailability, Tmax, Cmax, renal CL, and half-life were calculated or derived (6). Hoffmeyer et al. found that individuals with the CC genotype (n=3) had higher duodenal P-gp expression and lower digoxin AUC values after rifampin pretreatment compared with the one subject who was homozygous for the TT genotype (1). Heterozygotes had intermediate duodenal P-gp expression levels and AUC values. While these data indicate that CC carriers had a lower exposure to digoxin compared with CT carriers or the single TT carrier, it is not possible to attribute the observed differences in exposure levels to differences in digoxin intestinal absorption, because AUC is dependent on both absorption (bioavailability) and clearance. To assign a cause to the differences in AUC values would require bioavailability and clearance values. And before one can establish a relationship between duodenal P-gp expression levels and absorption, a significant correlation between bioavailability and P-gp expression is required. Furthermore, it is unclear why the authors chose to discuss only the data from rifampin-induced treatments. Were the differences in pharmacokinetic parameters among genotype groups similar with and without pretreatment with rifampin? For in this case, rifampin pretreatment only complicates the analysis, since one cannot rule out the possibility that the TT homozygote carries a mutant protein involved in the rifampin-dependent induction pathway.

In the second clinical study, Hoffmeyer et al. determined the MDR1 genotype at the 3545 locus in 14 healthy volunteers who had participated in a clinical study in which they had received 0.25 mg digoxin daily until steady-state digoxin plasma concentrations were reached (1). A 38% difference was found in the mean steady-state digoxin Cmax values between individuals carrying the CC genotype (n=7) and the TT genotype (n=7) (1). But again, in the absence of additional information, it is not possible to assign a cause for the observed differences in Cmax values between the CC and TT genotypes, because Cmax is dependent on both absorption and elimination. Hence, the only definitive conclusion we can draw from the data is that a small, albeit significant, difference exists in the steady-state Cmax values between CC and TT carriers.

In a third clinical investigation, von Ahsen et al. determined the allele frequency for the C3435T allele in 124 stable Caucasian renal transplant patients receiving cyclosporin A (CsA) therapy (3). In contrast to the findings of Hoffmeyer et al. for digoxin, von Ahsen et al. did not find significant differences among individuals carrying the CC (n=31), CT (n=54), or TT (n=44) genotypes for the three parameters they measured: the CsA dose required to maintain therapeutic concentrations, dose-adjusted CsA trough concentrations, and incidence of acute rejection. Hence, the C3435T polymorphism does not appear to be clinically relevant for CsA therapy. However, the reason von Ahsen et al. did not observe significant differences between genotypes could have been that the true effect was caused by a particular haplotype, not the C-to-T change at position 3435.

While the C3435T polymorphism clearly appears to be correlated with P-gp expression in duodenal tissue and functional activity in CD56+ cells, the clinical relevance of this particular mutation in the MDR1 gene appears to be drug-dependent. Hoffmeyer et al. found genotype-associated differences in digoxin AUC values after rifampin pretreatment in a small group of subjects and a small genotype-associated difference in digoxin's steady-state Cmax (1). In contrast, von Ahsen et al. found no differences in either CsA plasma concentrations or outcome in stable renal transplant patients with different MDR1 genotypes at the 3435 locus, indicating that this particular MDR1 variant does not appear to be relevant for CsA therapy (3). While the results of these studies suggest that the C3435T mutation in the MDR1 gene does not have substantial clinical consequences, at least for digoxin and CsA, the clinical importance of the other mutations and haplotypes in the MDR1 gene has yet to be determined.

References

1. Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, Johne A, Cascorbi I, Gerloff T, Roots I, Eichelbaum M, Brinkmann U. 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 U S A 2000 Mar 28;97(7):3473-8.

2. Hitzl M, Drescher S, van der Kuip H, Schaffeler E, Fischer J, Schwab M, Eichelbaum M, Fromm MF. The C3435T mutation in the human MDR1 gene is associated with altered efflux of the P-glycoprotein substrate rhodamine 123 from CD56+ natural killer cells. Pharmacogenetics 2001 Jun;11(4):293-8.

3. von Ahsen N, Richter M, Grupp C, Ringe B, Oellerich M, Armstrong VW. No influence of the MDR-1 C3435T polymorphism or a CYP3A4 promoter polymorphism (CYP3A4-V allele) on dose-adjusted cyclosporin A trough concentrations or rejection incidence in stable renal transplant recipients. Clin Chem 2001 Jun;47(6):1048-52.

4. Cascorbi I, Gerloff T, Johne A, Meisel C, Hoffmeyer S, Schwab M, Schaeffeler E, Eichelbaum M, Brinkmann U, Roots I. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther 2001 Mar;69(3):169-74.

5. Tanabe M, Ieiri I, Nagata N, Inoue K, Ito S, Kanamori Y, Takahashi M, Kurata Y, Kigawa J, Higuchi S, Terakawa N, Otsubo K. Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene. J Pharmacol Exp Ther 2001 Jun;297(3):1137-43.

6. Greiner B, Eichelbaum M, Fritz P, Kreichgauer HP, von Richter O, Zundler J, Kroemer HK. The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest 1999 Jul;104(2):147-53.