Tamoxifen is an anticancer prodrug effective against breast tumors for women that is activated through hepatic metabolism. Although this drug has been proven to be effective for reducing recurrences, inter-individual genetic variation has increased the number of adverse effect cases in patients taking this drug. In recent studies, tamoxifen has been found to be mainly metabolized by two enzymes: Cytochrome P450 2D6 (CYP2D6) and Sulphotransferase 1A1 (SULT1A1). CYP2D6 demethylates tamoxifen on the nitrogen region and hydrolyzes the benzene ring cis to the ethyl group, forming 4-hydroxytamoxifen (4OHTam) and N-demethyltamoxifen (NDTam), respectively. NDTam is further metabolized by CYP2D6 into 4-hydroxydemethyltamoxifen (4OHNDTam), the primary active metabolite. The two active metabolites, NDTam and 4OHNDTam, are then further metabolized, which decreases activity through various other Phase II metabolizers such as UDP-glucoronyltransferase (UGT) and, most importantly, SULT1A1.
It has been found that different subpopulation are characterized by certain genetic polymorphism, such as gene deletion and duplication, in CYP2D6 and SULT1A1, which give way to altered enzymatic changes. These different enzymatic activities can give different plasma level for different metabolites. In order to evaluate the relationship between these genotypes and plasma levels, this study intended to study how the CYP2D6 and SULT 1A1 genotype and SULT1A1 gene copy number influenced the pharmacokinetics of tamoxifen in breast cancer patients. Gjerde et al. hypothesized that although the genotype of both enzymes influenced tamoxifen pharmacokinetics, the gene copy number of SULT1A1 had no effect.Methods
The patient’s characteristics were Caucasian women with breast cancer whom were estrogen and/or progesterone receptor positive from either Haukland University Hospital, St. Olav University Hospital or Ferde Central Hospital. These patients were between the ages of 32 and 85 and took 20 mg of tamoxifen every day for 80 days.
To measure the concentration of tamoxifen and metabolites, the investigators were able to develop a specific methods used to measure their levels in the serum called high-pressure liquid chromatography-tandem mass spectrometry system.
Determining the genotype of both CYP2D6 and SULT1A1 was tested for poor (PM) and ultra-rapid (UM) metabolizer using long Polymerase Chain Reaction (PCR) and CYP2D6-specific primers. PM genotypes were denoted as *3, *4, *5, and *6 while UM genotypes were denoted as *2 X2. The products of the long PCR was used as a template in another technique called PCR-restriction length polymorphism assay (RFLP) which was used to determine the genotype for the poor metabolizers using gel electrophoresis. For the rest of the genotypes, genotype *5 and *6 were determined using a nuclease assay with Taqman probes and the UM genotypes were determined by long PCR.
Determining SULT1A1 genotypes was a more difficult process for the investigator. Their first set of primers gave the team mismatches and classification. Therefore, they had to find another set of primers and verified the result products using a PCR-based RFLP assay. Gene copy number was determined by comparing height and area ratios of the amplicons (PCR products) to a reference 205 bp amplicon.
Statistical analysis was conducted using logistic regression in relation to the age of the patients, which were then used to examine their altered enzymatic activity. Logarithmic conversion was used to normalize their distribution and tests statistics were determined using two-sided testing.
Figure 1a. provides a schematic to understand the metabolic pathway of tamoxifen and the enzymes involved. The enzymes in bold along the arrows of each pathway denote the primary enzyme involved in the process. Figure 2b. displays the relationship between the genotypes of both SULT1A1 and CYP2D6. Each ratio is in the format of Ln (metabolite/drug), which can be understood as the ratio of product and substrate. When this log ratio increases, the amount of metabolite formation increases as well. In this figure, not only are tamoxifen and the primary active metabolites compared, but the metabolites from other pathways are compared as well. What is seen in these graphs are proportional relationships between the metabolites and the different genotypes of the perspective enzymes.
In the graph depicting Ln (NDtam/tamoxifen) for CYP2D6 genotypes, an inverse relationship can be seen as the genotype’s enzymatic activity increases. Because the enzymatic activity of the enzyme is increases, CYP2D6 is metabolizing faster and therefore, there is less substrate for the pathway that demethylates tamoxifen into NDTam. According to the investigators, the three ratio, 4OHNDTam/Tamoxifen, 4OHNDTam/NDTam, and NDDTam/NDTam, displayed a gene-dose relationship where the increase in enzymatic activity accompanied an increase in metabolite formation. In terms of SULT1A1, an inverse relationship of the ratio of NDTam/tamoxifen was seen associated with an increase in enzymatic activity of SULT 1A1.
Table 1 displays the characteristics of the total study population separate by CYP2D6 genotypes and SULT1A1 genotypes and gene copy numbers. Gjerde et al. state that overall CYP2D6 distribution displayed an equilibrium that resembled Hardy-Weinberg principle, where the distribution can be described by the following equation: p
2. On the other hand, SULT 1A1 had no relationship with tamoxifen or any of its metabolites. However, when comparing ratios of NDTam to tamoxifen, enzymatic activity and serum levels displayed a proportional relationship, with an inverse relationship with NDDTam and NDTam.
In Table 2., median (and range) tamoxifen serum levels are compared with the different genotypes and gene number of the enzymes in question. For example, when measuring and analyzing NDTam concentration, the demethylated metabolite for tamoxifen mediated by CYP2D6, one can see a decrease in this metabolite as enzymatic activity increases. This occurs due to CYP2D6 working well enough to hydroxylate tamoxifen into 4OHTAm more than it will demethylate. Consequently, when comparing SULT1A1 genotype and gene copy number, there is no significant change in the serum level of each metabolite.
Determining the clinical relevance of these tamoxifen-metabolizing enzymes is important discussing a new step for tamoxifen and metabolite serum monitoring of patients with distinct polymorphism. Although Gjerde states that the NDTam pathway is the most predominant metabolite in terms of concentration, it seem more to the contrary that 4OHTam is the more predominant pathway according to their own figure. In Figure 1., the distinction of the bold enzymes as the primary enzymes in that pathway can be made since it makes sense for the rest of the schematic.
Their claim that serum concentrations of 4OHTam, 4OHDTam, and NDTam are affect by CYP2D6 genotypes seen legitimate, but there are some factors that are left vulnerable in their results. For instance, their sample sizes for these metabolite ratios were rather small. The range for sample sizes were very broad, ranging from 86 to 5. There should have been more recruitment of patients from each hospital or increase the number of hospitals to recruit patients from.
On the other hand, the researchers claim that the serum levels of tamoxifen and metabolites not being influenced by SULT 1A1 genotypes and gene copy number can be explained due to the other Phase II metabolizers such as glucoronidation. This makes sense because glucoronidation is not dependent on the genetic polymorphism of SULT1A1 and there will metabolize the same substrate throughout the experiment. In addition, Gjerde et al. propose that another reason for the scattered data, especially in Figure 1., would be the concomitant drug therapies that were taken adjuvant with tamoxifen. To conclude, their study supports the claim that tamoxifen administration should be monitored amongst breast cancer patients, especially those with distinct polymorphisms. Further studies should be conducted to evaluate the treatment outcome of different CYP2D6 and SULT1A1 with new tamoxifen drug monitoring proposed from this study.