In this study we used two highly sensitive assays for measuring the serum levels of tamoxifen and its metabolites and estrogens [28, 29]. As hypothesized, we observed positive associations between the serum levels of estrogens (E1 and E2) and tamoxifen, as well as two of its' non-hydroxylated metabolites (NDDtam and tamNox). Interestingly, the concentrations of the more potent hydroxylated tamoxifen metabolites (4OHtam and 4OHNDtam) were not correlated to estrogen levels.
Metabolites of tamoxifen that are not hydroxylated may also contribute to the effects or adverse effects of tamoxifen. NDtam, NDDtam and tamNox have estrogen receptor affinity that is the same as for tamoxifen itself but which is 100-fold lower than 4OHtam and 4OHNDtam [2, 31, 32]. Notably, high levels of NDtam and NDDtam have been reported in patients experiencing tamoxifen-related side effects [33, 34]. In animals NDDtam appears to decrease the rate of tamoxifen metabolism  whereas tamNox may represent a pool for its' reconversion back to tamoxifen . The roles of NDtam, NDDtam, and tamNox in the clinical situation remain to be explored more in detail.
Dowsett et al suggests that tamoxifen effectively saturates ER during tamoxifen of postmenopausal women . This may suggest that estrogen levels during tamoxifen therapy may have limited importance. However, long term exposure to tamoxifen has been shown to induce a state of adaptive hypersensitivity in breast tumors to E2 . Thus, upon development of tamoxifen resistance, low levels of estrogen may stimulate tumor growth. Moreover, clinical studies have shown that the expression of 17β-hydroxysteroid dehydrogenase 1 (17HSD1), which converts E1 to the active estrogen E2, and 17HSD2, which converts E2 to E1, are predictive factors for treatment response to tamoxifen in both premenopausal and postmenopausal patients [37, 38]. These suggest that estrogen levels may be of importance during long term tamoxifen therapy.
Tamoxifen and estrogens are both partly metabolized by the enzymes CYP2C19, 2D6, 3A5, and SULT1A1 . Therefore, we screened for associations between these genotypes and estrogen serum levels and observed that the predicted CYP2C19 activity was related to the level of E1. This should be expected as only E1 and not E2 or E1S is converted by CYP2C19 (figure 1B). Patients carrying the CYP2C19*17 allele predicting high enzyme activity had lower serum levels of E1 compared with those carrying CYP2C19 defective alleles. This finding is in agreement with a recent report suggesting that an increased catabolism of estrogens by CYP2C19 may lead to decreased estrogen levels and therefore reduced breast cancer risk , and the observation that CYP2C19*17 identifies patients likely to benefit from tamoxifen treatment .
The 4OHtam to tamoxifen ratio was positively associated with increasing CYP2C19 predicted enzymatic activity, whereas no such correlation was observed for the ratio of 4OHNDtam to NDtam. This is consistent with previous reports showing that CYP2C19 hydroxylates tamoxifen, but not NDtam . Of note is the finding that the metabolic ratio NDtam/tamNox was highly significantly related to the predicted activity of CYP2C19. This is in line with the suggestion of Jordan et al that tamNox represents an intermediate metabolic step between tamoxifen and NDtam .
The predicted CYP2D6 and CYP3A5 activities did not influence estrogen levels, whereas SULT1A1 gene copy number was positively related to the levels of E2, E1, and E1S, but not to those of tamoxifen or its metabolites. A novel finding in the present study was the observation of lower serum levels of tamNox in patients homozygous for the low activity allele CYP3A5*3 compared to patients heterozygous for this allele. Although not significant there was also a trend for lower levels of 4OHtam, NDtam and NDDtam. This was expected since CYP3A5 hydroxylates and demethylates tamoxifen (figure 1A). Wegman et al observed, in contrast to Goetz et al, that patients homozygous for CYP3A5*3 have improved recurrence free-survival [18, 25]. The clinical importance of CYP3A5 in this matter remains unanswered.
The observed associations between the kinetics of tamoxifen and the levels of estrogens may be due to competition between tamoxifen or its metabolites and estrogens to be processed by identical enzymes such as CYP2C19. An alternative explanation is that tamoxifen and its metabolites inhibit enzymes that are involved in the degradation of estrogens. This is line with the finding of Meltzer et al who demonstrated that tamoxifen and NDtam may act both competitively and non-competitively on microsomal mixed function oxidases . Another explanation is that the associations may also be a consequence of the observed increase of serum levels of dehydroepiandrosterone (DHEA) during tamoxifen treatment therapy [43, 44]. DHEA can be converted to androstendione which is a precursor of estrogens. Both tamoxifen and estrogens are also excreted in bile as conjugates. After deconjugation by intestinal microflora they are partly reabsorbed resulting in pronounced enterohepatic cycling [45, 46]. Accordingly, the deconjugation capacity of the microflora in the intestines may be a common determinant for their re-absorption and bioavailability [6, 47].
We have observed estrogen agonistic effects of tamoxifen in the liver and in the pituitary gland of postmenopausal patients . The serum level of SHBG was more than doubled and the level of FSH was almost halved. To be certain that tamoxifen levels were in steady-state in the present study all patients included were treated with tamoxifen for at least 80 days. After this treatment period we believe that also estrogen levels were at steady state levels. Although tamoxifen exerts an estrogen agonistic effect on the pituitary of postmenopausal women, we do not know the separate effects of each of its metabolites on the pituitary. Therefore, it is interesting that of the five tamoxifen metabolites examined, only the serum concentrations of 4OHNDtam was positively associated with the serum levels of FSH in this situation of steady-state tamoxifen kinetics. This suggests that 4OHNDtam has an anti-estrogenic effect in the pituitary gland.
Hot flashes have been suggested as a predictor of tamoxifen efficacy . Of note, FSH serum concentrations correspond better with the frequency of hot flashes than the levels of E2 . Interestingly, some results from clinical studies suggest that patients carrying functional CYP2D6 alleles have a higher incidence of hot flashes, higher levels of 4OHNDtam and better outcome during tamoxifen treatment [25, 26]. Accordingly, relations between the frequency of hot flashes, serum concentrations of 4OHNDtam and outcome during tamoxifen therapy may exist.
Limitations of the present study are that the correlations observed are weak and we have not had admittance to patient journals and do not know of any additional drug use. However, we and others have observed that breast cancer patients often use several additional drugs that may interact with tamoxifen [49–52]. Accordingly, the weak correlations observed are not surprising as drugs, enzymes and other factors in addition to those analyzed in the present study may interact with the metabolism of tamoxifen as well as that of estrogens.