Flavopiridol pharmacogenetics: clinical and functional evidence for the role of SLCO1B1/OATP1B1 in flavopiridol disposition

Abstract

Background: Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.

Methodology/principal findings: Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.

Conclusions/significance: Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.

Trial registration: ClinicalTrials.gov NCT00058240.

Conflict of interest statement

Competing Interests: A patent has been filed on this method of administering flavopiridol. Inventors on this patent include J.C.B., M.R.G., T.S.L., and J.T.D. It has no financial value now. The remaining authors declare no competing financial interests. The patent does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Individual estimate of Q vs. baseline total bilirubin (with full phase I dataset).

Regression analysis; Q = 11.21 * Bilirubin level+0.7422 (P

Figure 2. Relationships between the most significant genetic covariates and estimates for each of the 4 PK parameters from univariate analysis in the phase I dataset.

Box plots represent the scatter of individual V1 estimates vs. SLCO1B1 rs11045819 (Panel A), CL estimates vs. SLCO1B1 rs4149056 (Panel B), and V2 estimates vs. ABCC2 rs8187710 (Panel C). For rs11045819, data is displayed with V1 estimates using BSV on V1.

Figure 3. Uptake of flavopiridol, flavo-G, SN-38 and lenalidomide in OATP1B1 transfected cells.

The bar graph indicates means + SD for triplicate determinations of 10 µM flavopiridol, flavo-G, SN-38 and lenalidomide uptake rates in cell lines (HEK-293 or MDCK-II) transfected with empty control vector or vector cloned with the OATP1B1 gene (SCLO1B1). Incubations with flavo-G were for 30 min., and all other drugs were for 10 min. Transport rates are expressed as percentages normalized to empty vector control. * p<.05 p t-test.>

Figure 4. Uptake of flavopiridol in nonsynonymous polymorphic variants of OATP1B1.

The bar graph indicates means + SD for triplicate determinations of 10 µM flavopiridol uptake rates in MDCK-II cells transfected with empty control vector or vector cloned with the reference or polymorphic SLCO1B1 genes. All incubations were for 10 min. Variants are indicated with respect to their amino acid change; T155P = rs11045819, D130N = rs2306283, V174A = rs4149056. Transport rates were normalized to empty vector control. Differences in uptake rates were compared to the reference OATP1B1 transporter. * p<.05 p student t-test.>

Figure 5. Association of PGx, PK and bilirubin from the phase I dataset.

Flavo-G PK parameter estimates from 27 patients were evaluated for relationships with PGx. Fewer TA repeats in the UGT1A1 promoter were weakly associated with lower flavo-G Cmax (Panel A) and AUC (Panel B). For SLCO1B1, the rs2306283 SNP correlated with flavo-G plasma concentrations (the total time flavo-G plasma concentrations were below 1.5 µM, p = .019; Panel C, one outlier removed). For ABCG2, the rs1564481 SNP was associated with flavo-G AUC (p = .050; Panel D) and the rs2231142 SNP was associated with flavopiridol AUC (p = 0.08, Panel E). The SLCO1B1 rs3829310 minor C allele was associated with higher baseline total bilirubin (p = .011; Panel F). For plots A and B, the x-axis indicates the number of promoter TA repeats for both gene copies (6.7 = 6 and 7 TA repeats; 7 = 7 TA repeats for each gene copy; 7.8 = 7 and 8 TA repeats; 8 = 8 TA repeats for each gene copy).

Figure 6. Association of PGx and PK from the phase II dataset.

Flavopiridol and flavo-G PK parameter estimates from the phase II study was evaluated for relationships with PGx. Presented are flavopiridol CL associations with ABCG2 rs2622624 (p = 0.008, Panel A) and rs3114018 (p = 0.004, Panel B); UGT1A1*28 (promoter) polymorphisms associated with flavo-G Cmax (p = 0.02, Panel C) and AUC (p = 0.04, Panel D); the ABCG2 rs2231142 SNP was weakly associated with flavopiridol AUC (Panel E, p = .08); For plots C and D, the x-axis indicates the number of promoter TA repeats for both gene copies (6.7 = 6 and 7 TA repeats; 7 = 7 TA repeats for each gene copy; 7.8 = 7 and 8 TA repeats; 8 = 8 TA repeats for each gene copy).