Steady state plasma anastrozole and anastrozole metabolite trough concentrations were determined in 196 patients while chronically on a one mg/day dosage. Five patients were excluded from the analyses because one had no detectable plasma anastrozole or anastrozole metabolite (despite reporting that she was taking the drug), three patients had the second blood draw obtained less than four weeks after the initiation of therapy, and one patient was excluded because of technical problems with the comparison hormone assays. Thus, 191 patients were evaluable in these analyses and their characteristics are listed in .
Patient Characteristics (n=191)
Plasma anastrozole and anastrozole metabolite concentrations
The three major metabolites detected in plasma were anastrozole conjugates, hydroxy-anastrozole and hydroxy-anastrozole conjugates (). The median plasma concentration of free anastrozole was 32.2 ng/ml, with a range from 0.0 to 98.8 ng/ml. Two patients without detectable anastrozole were included in the analysis because they had measurable hydroxy-anastrozole and hydroxy-anastrozole-conjugates. These patients will be discussed in more detail subsequently. The frequency distribution for anastrozole concentrations shown in demonstrates the wide variation among patients.
Figure 1 Steady state plasma anastrozole and anastrazole conjugate concentrations in breast cancer patients treated with one mg/day oral dose of anastrozole. Observed anastrozole metabolism (A); frequency distribution histograms for levels of anastrozole (B) and (more ...)
The median plasma concentration of anastrozole conjugates was 4.2 ng/ml (range: 0.0-54.4 ng/ml) and the frequency distribution shown in demonstrates wide variation in their plasma concentrations. Anastrozole and anastrozole conjugate concentrations were not statistically correlated (Spearman correlation = 0.10, p-value = 0.18).
The majority (over 80%) of the hydroxy-anastrozole was recovered as conjugates. As noted previously, a lack of internal standards prevented absolute quantitation of these two metabolites, but there was a 29-fold range in hydroxy-anastrozole conjugate concentrations. Hydroxy-anastrozole and hydroxy-anastrozole conjugates were positively correlated (Spearman correlation =0.63, p-value < 0.001), implying that subjects with higher hydroxylated metabolites formed more conjugates. However, two outliers had high hydroxy-anastrozole but low hydroxy-anastrozole-conjugates, indicating a possible deficiency in their ability to catalyze the conjugation reaction for the hydroxylated metabolite. Conversely – and more important clinically – two different patients had undetectable anastrozole concentrations, very high anastrozole conjugate concentrations and very little drug response in terms of change in their estrogen hormone levels. These observations raised the possibility that these latter two patients might represent “ultrarapid” conjugators of the drug, and, as a result, might fail to have the desired therapeutic response. These latter two patients will be discussed in greater detail subsequently.
There was no statistical association between time to second blood draw and anastrozole concentration (Spearman correlation = -0.004, p-value = 0.95), or for anastrazole conjugate concentration (Spearman correlation = 0.07, p-value = 0.36). However, there were significant correlations between time to second blood draw and hydroxy-anastrozole (Spearman correlation = 0.18, p-value = 0.008) and hydroxyanastrozole-conjugate (Spearman correlation = -.20, p-value = 0.005) concentrations.
Estrone, estradiol and estrone-conjugate concentrations pre-treatment and after anastrozole therapy
Pre- and post-treatment plasma levels of E1, E2, E1-conjugates, androstenedione and testosterone are listed in . Pre-treatment levels for all of these hormones demonstrated substantial variability. Among patients considered to be clinically postmenopausal by their oncologist, 28 (15%) had E2 levels greater than10 pg/ml, the conventional concentration separating premenopausal from postmenopausal women, with a range of 10.2-40.3 pg/ml (median: 13.55 pg/ml). Sixteen of these patients had been entered from Mayo and 12 from MD Anderson. The median age of these 28 patients was 58.5 years, with a range from 47 to 80 years. Only one patient in this group had received prior tamoxifen, nine (32%) had received prior chemotherapy, and 15 (54%) were active smokers. The median BMI for these 28 patients was 36.3 (range: 19.9-45.0), and nine (32%) had a BMI greater than 40.0. All but one of these patients had a decrease in their E2 levels after anastrozole therapy, with 18 (64%) dropping to undetectable levels.
Pre-treatment hormone levels (pg/ml) and during anastrozole therapy
Relationship of anastrozole concentrations to estrone, estradiol and estrone-conjugate concentrations
(panels A-C) displays changes in E1, E2, and E1-conjugate concentrations, color-coded for quartile of anastrozole level. demonstrates that only a small proportion of the patients (17%) had E1 concentrations above the LLQ while on anastrozole, with a median of 2.87 pg/ml but a very wide range (1.58-45.2 pg/ml). Likewise for E2, shows that only a small proportion of patients (17%) had concentrations above the LLQ on anastrozole, with a median of 1.26 pg/ml but once again with a very wide range (0.65-97.0 pg/ml). The findings with respect to E1-conjugates were quite different, with the vast majority of patients (93%) having levels above the LLQ with a median of 12.95 pg/ml but an exceedingly wide range (3.50-2990 pg/ml).
Figure 2 Plasma concentrations of estrone (A), estradiol (B), and estrone conjugates (C) according to quartile of anastrozole concentration in breast cancer patients before and after treatment with one mg/day oral dose of anastrozole. Key for line color: black, (more ...)
As anticipated, the majority of patients experienced a drop in E1, E2 and E1-conjugate concentrations after anastrozole therapy, but unexpected rises were identified in three (2%) patients for E1, in five (3%) patients for E2, and in six (3%) patients for E1-conjugates (, panels A, B, C). Eight patients (4%) had a rise in at least one of the estrogenic compounds (E1, E2, E1-conjugates), and the ages of those patients were 47, 50, 52, 52, 57, 57, 58 and 66 years, indicating that the rises did not occur only in the younger postmenopausal women. All eight patients had FSH and LH levels in the postmenopausal range at the time of the rise in one of the estrogenic compounds. The median BMI in these eight patients was 25.2 with a range of 18.3 to 38.7. The BMIs for these eight patients was not significantly different from the other 183 patients in this study (Wilcoxon p=0.20). As can be seen in , the majority of patients with a rise in the level of at least one of these estrogenic compounds fell within the highest quartile for anastrozole concentrations.
The statistical association of clinical variables with pre-treatment concentrations of E1, E2 and E1-conjugate, as evaluated by step-wise regression, showed that BMI was positively correlated with all three hormone concentrations (in addition, smoking status was associated with E1, and stage and age were associated with E1-conjugate). Hence, it was critical to adjust for clinical variables when evaluating the association of anastrozole concentrations with changes in E1, E2 and E1-conjugate concentrations. In addition to clinical variables, pre-treatment hormone levels and anastozole concentration were evaluated in step-wise regression. This allowed us to evaluate the contribution of each variable, adjusted for the others, in case variables might be correlated (such as pre-treatment hormone level and BMI). In no instance was the level of anastrozole statistically associated with change in E1, E2 or E1-conjugates, although changes were often associated with BMI and pre-treatment hormone concentrations. However, whereas this was true of the entire group, there were two outliers in whom the lack of detectable anastrozole was associated with lack of change in E1-conjugates (see the subsequent section).
Similar to the regression analyses for change in hormone concentrations, we also evaluated actual post-treatment concentrations. In no instance was the concentration of anastrozole statistically associated with the final concentrations of E1, E2 or E1-conjugates.
Relationships among anastrozole, anastrozole conjugate and estrone conjugate concentrations
displays the relationship among anastrozole, anastrozole conjugate and E1-conjugate concentrations. Two patients (red open circles) had extremely low concentrations of anastrozole but very high levels of anastrozole conjugates. Those same two patients showed a relatively small change in plasma estrone conjugate levels after receiving anastrozole, i.e., a decrease by 58 pg/ml and 124 pg/ml, which are substantially less than the median decrease of 208 pg/ml. These observations raise the possibility that these two patients might represent “ultra-rapid” conjugators of anastrozole, accounting for the low parent drug concentrations and relatively small changes in hormone levels after drug. Neither of these patients was a current smoker and neither were being treated with drugs known to induce microsomal drug-metabolizing enzymes.
Figure 3 Correlation of anastrozole, anastrozole conjugates and changes in estrone conjugates (A) and of hydroxyl-anastrozole, hydroxyl-anastrozole-conjugates and changes in estrone conjugates (B) in breast cancer patients treated with one mg/day oral dose of (more ...)
displays the relationship among hydroxy-anastrozole, hydroxyanastrozole conjugate and E1-conjugate concentrations. Two different patients (red closed circles) displayed high levels of hydroxy-anastrozole, but low hydroxy-anastrozole conjugates, which raises the possibility of a relative decrease in the ability to conjugate the hydroxy metabolite. The E1-conjugates in these two patients decreased by 234 pg/ml and 214 pg/ml, which are near the median change of -208 pg/ml, as anticipated since they had measurable concentrations of the parent drug.
Androstenedione and testosterone concentrations pre-treatment and after anastrozole therapy
displays the relationship between pre- and post-treatment androstenedione concentrations. Substantial variability is evident in pre-treatment androstenedione concentrations but with no consistent change in androstenedione concentrations after treatment with anastrozole. In 187 patients, 55% showed a rise and 43% showed a drop in androstenedione after treatment with anastrozole
Concentrations of androstenedione (A) and testosterone (B) in breast cancer patients before and after treatment with one mg/day oral dose of anastrozole.
displays the relationship between pre- and post-treatment testosterone concentrations. Again, there was substantial variability in pre-treatment testosterone concentrations. In 189 patients, 61% showed a rise and 38% showed a drop in testosterone after treatment with anastrozole. Finally, there was not a consistent relationship between the changes in androstenedione and testosterone concentrations after anastrozole therapy (data not shown).