The aim of our study was to investigate the role of uptake transporters and their genetic variations in pharmacokinetics and pharmacodynamics of raloxifene in postmenopausal osteoporosis.
Our in vitro experiments with OATP1B1 and OATP1B3 transfected CHO cell lines showed that M2 and M3 interact with both, OATP1B1 and OATP1B3 transporters, raloxifene interacts only with OATP1B1 and M1 does not interact with any of the studied transporters. This does not diminish the physiological importance of the tested transporters, because as we have demonstrated, M1 in vivo concentrations are much lower compared to the concentrations of M2 and M3. A direct transport of raloxifene species into the transfected cell lines could not be measured, because the radiolabelled raloxifene species were not available. In addition, affinities or IC 50s could not be determined due to the very small amount of available raloxifene metabolites.
On the basis of this in vitro data we decided to study the effect of SLCO1B1 and SLCO1B3 genetic variants on the pharmacokinetics and pharmacodynamics of raloxifene in a group of Caucasian postmenopausal osteoporotic women. Therefore, genotyping of three polymorphisms, SLCO1B1 c.388A > G, SLCO1B1 c.521T > C and SLCO1B3 int7C > G which were previously shown to alter transporter activity of some drugs [20–25] was performed. The SLCO1B1 c.388A > G polymorphism is responsible for the amino acid substitution (p.N130D) in extracellular loop 2 of the OATP1B1 protein and was shown to affect substrate specificity suggesting that this loop is involved in substrate recognition [43, 44]. This polymorphism could be responsible for unchanged, increased or reduced transport activities of OATP1B1 [23, 45–47]. On the other hand, it was found that SLCO1B1 c.521T > C (p.V174A) polymorphism decreases the membrane expression of OATP1B1 protein and consequently lowers its transport activity . Because of the high allelic frequencies in Caucasian population, 41% for c.388A > G and 18% for c.521T > C , and influence on transport activities, the c.388A > G and c.521T > C polymorphisms could contribute to the high inter-individual variability in pharmacokinetics and pharmacodynamics of raloxifene. Additionally, a third polymorphism located in intron 7 of SLCO1B3 gene was studied because of its strong association with unconjugated bilirubin , whose levels were found to correlate with those of raloxifene.
Serum concentrations of raloxifene, M1, M2 and M3 were determined using a validated method. To our knowledge, the developed LC/MS/MS method is the first method for detection of M1, M2, M3 and raloxifene. The novelty of this contribution is the observation that the main in vivo metabolite of raloxifene is M3. Our in vivo study showed that the average raloxifene species serum levels are in ratio of 1 : 19.5 : 87.7 : 141.7, for RAL, M1, M2 and M3, respectively.
Serum levels of raloxifene, M3 and TR increase with the number of SLCO1B1 c.388A > G mutated alleles. Levels of M1 and M2 were visibly elevated as well, but due to the small sample size and the pronounced data variability, a statistical confirmation of this observation could not be made. Analyses of possible combinations of both SLCO1B1 gene variants (haplotype) were consistent with the results of individual testing of SLCO1B1 polymorphisms and showed that haplotype *1b caused higher concentrations of raloxifene, M3 and TR. These results could indicate that the uptake of raloxifene in hepatocytes is lower and consequently, the serum concentrations are higher. Moreover, concentrations of metabolites also increase with the number of mutated alleles.
It is well known that only unconjugated raloxifene can bind with high affinity to estrogen receptors to evoke a pharmacodynamic response , although it represents <1% of total serum raloxifene . This small fraction of unconjugated raloxifene found in systemic circulation originates partly directly from the parent compound absorbed from the gut and partly from the systemic cleavage of the circulating glucuronides into raloxifene . Hence, the raloxifene metabolites actually represent transport forms and a depot of active raloxifene. Given the extremely low and highly variable raloxifene concentrations, the serum levels of raloxifene metabolites can be used as a measure of raloxifene exposure.
Our correlation analysis between serum levels of raloxifene species with clinical parameters showed higher levels of M3 and TR in patients with a higher decrease in serum CTX, one of the most reliable bone resorption markers , thus implicating a better antiresorptive effect of raloxifene in those patients. In addition, higher M2 concentrations were found to correlate with a greater increase in BMD at lumbar spine (BMD-LS) but not with BMD values at other sites of skeleton. This is in concordance with the already known “site specific” bone effects during raloxifene treatment, which is therefore predominantly used for the prevention of vertebral osteoporotic fractures and not for hip or wrist or any other common osteoporotic fractures. Based on this correlation, the influence of SLCO1B1 and SLCO1B3 genetic variants on clinical outcomes was tested: a lowering of bone turnover markers, an increase of bone quality (QUI measurements) and an increase in bone mineral density. The CTX was shown to be influenced by a number of *1b haplotype copies, which was shown also to increase the raloxifene species plasma concentration, that most probably caused a higher decrease in CTX during treatment. A statistical significance was observed also in change of BMD-FN regarding SLCO1B1 c.388A > G polymorphism and *1a haplotype but due to the small sample size, only modest changes in BMD-FN in one year and the inherent measurement error by DXA  the direction could not be determined. Additionally, no influence of SLCO1B1 and SLCO1B3 genetic variants on HOL, HDL, LDL and TG was observed.
Beside the influence of SLCO1B1 and SLCO1B3 genetic variants on raloxifene pharmacokinetics and pharmacodynamics the influence of studied polymorphisms on the probability of raloxifene adverse effects was also evaluated. The statistical analysis did not establish any correlation of studied polymorphisms with the probability of raloxifene adverse effects because the study was not designed for this purpose and number of patients was too low. Further studies with higher number of patients are needed to discover the possible associations of genetic polymorphisms with raloxifene adverse effects.
Our results indicate that a significant part (up to 20%) of the observed high inter-individual variability in pharmacokinetics and pharmacodynamics can be explained by a genetic influence of the SLCO1B1 c.388A > G polymorphism. Therefore, pharmacogenetics of SLCO1B1 could be taken into account to individualize raloxifene therapy.
The main limitation of the current in vivo study is a rather small number of the enrolled participants. Additionally, the duration of our study should be prolonged due to very minimal changes in BMD observed in one year. Other pharmacodynamic parameters and concentrations of raloxifene species should also be monitored in such prolonged study. Moreover, multiple comparisons may be the cause for the reported positive associations. As such, our results are exploratory in nature and should be interpreted as hypothesis generating and confirmed by the accomplishment of a lager study. However, it is our opinion that the present in vivo study in connection with the in vitro data allows to make stated interpretations.