Safe and effective use of flavopiridol will require a thorough understanding of the factors influencing the onset of hyper-acute tumor lysis syndrome and inter-individual variability in response. We previously reported that at least some of the variability in outcomes may be explained by PK, yet significant portions of this variability remain unexplained. In this first evaluation of pharmacogenetics using the flavopiridol PK-derived schedule, we identified a novel flavopiridol-transporter interaction and confirmed its functional relevance in vitro. Furthermore, a subset of the polymorphisms evaluated in this study was significantly associated with outcomes. The striking activity of this dosing schedule in CLL and flavopiridol's emerging activity with combination chemotherapy in other diseases demand these pharmacogenetic and other factors be identified and further characterized.
To expand on our previous evaluation of flavopiridol using the PK-directed schedule, we sought to characterize the role of PGx in flavopiridol disposition. We combined focused and exploratory approaches, whereby PK data from a phase I dataset was evaluated against SNPs in candidate genes previously shown to metabolize or transport flavopiridol in in vitro
studies, as well as SNPs in genes not known to interact with flavopiridol. The candidate genes evaluated included ABCC2, ABCG2, UGT1A1 and UGT1A9. Indeed, at least one SNP in each of these genes was significantly associated in univariate analysis with flavopiridol and flavo-G PK. In multivariate analysis with the phase I dataset, a single SNP in ABCC2 (rs8187710) remained significant. This is a non-synonymous SNP resulting in a C1515Y alteration and is part of a haplotype with the rs17222723 SNP (also a non-synonymous SNP with a V1188E residue change). This haplotype has been reported to be associated with PBMC accumulation of lopinavir 
, susceptibility to nonalcoholic fatty liver disease 
, and doxorubicin-induced cardiotoxicity 
. This is a relatively infrequent SNP, and only 5 of 35 and 4 of 51 individuals were genotyped for the polymorphic A allele in the phase I and II datasets, respectively. Significant associations between this SNP and PK parameters were not observed in the phase II dataset.
Although UGT1A1 SNPs were not included in the final popPK model, both flavopiridol and flavo-G were correlated with the UGT1A1*28 promoter polymorphism, and fewer TA repeats (6 and 7) were associated with lower flavo-G concentrations and AUC in both the phase I and II datasets. One of the most broadly studied substrates of UGT1A1 and 1A9 is irinotecan/SN-38, which shares metabolic pathways and has a similar toxicity profile (diarrhea) with flavopiridol. Interestingly, the *3 polymorphism of 1A9 was previously shown to be associated with SN-38 but not flavopiridol disposition 
. Innocenti and colleagues measured ratios of flavo-G
flavopiridol and identified an indirect relationship between flavo-G levels and diarrhea 
. Zhai and colleagues reported PK and UGT1A1 polymorphism data in 49 patients with refractory neoplasms treated with 1-hour IV flavopiridol 
. However, their results indicated no correlation of the TATA box promoter UGT1A1*28 genotype with flavopiridol PK or diarrhea severity. While we did not identify correlations with diarrhea for flavopiridol and flavo-G PK 
, we observed one association with PGx and diarrhea.
The 35-patient dataset evaluated in this study was small with regard to exploratory PGx, and therefore an additional dataset from 51 patients was used to evaluate the validity of the findings. While the associations identified in each dataset were not identical, similar trends were observed between PK parameters and SNPs in the candidate genes. Furthermore, the clinical associations we observed with SLCO1B1 were functionally validated in vitro
. SLCO1B1 is important for the disposition of statin drugs 
and various anti-cancer agents including irinotecan 
. It was recently highlighted by the International Transporter Consortium as one of the seven most relevant transporters for drug development due to its broad substrate specificity, potential for drug-drug interactions, and clinically relevant polymorphisms 
. Importantly, functional transport data indicated this gene may play a role in hepatic uptake of both flavopiridol and flavo-G. All five of the SLCO1B1 SNPs evaluated in the phase I dataset were significant in univariate analysis, and two of these (rs11045819 on V1 and rs4149056 on CL) were the strongest associations observed of any covariates evaluated for flavopiridol PK in multivariate analysis. These are non-synonymous SNPs affecting the amino acid sequence in OATP1B1. Previous studies indicated these variants were associated with reduced uptake activity of the OATP1B1 substrates estrone-3-sulfate, estradiol-17β-d-glucuronide, atorvastatin, cerivastatin, pravastatin, the SN-38 metabolite of irinotecan and rifampicin in 
. These reports are consistent with our in vitro
data which indicated these SNPs significantly reduced flavopiridol transport, while the other nonsynonymous SNP evaluated (rs2306283) had no measureable effect. Interestingly, this was the only SLCO1B1 SNP significantly associated with flavo-G PK. A recently reported study in MDCK-II cells with this SNP indicated increased transport of bromosulfophthalein and decreased transport of cholyltaurine 
. Collectively, the data may suggest that the altered protein sequence affects OATP1B1 transport of flavopiridol and flavo-G differently. Further in vitro
evaluations of these and other SNPs will be required to characterize their functional impact with respect to flavopiridol and flavo-G disposition. Additionally, the data presented suggests flavopridol may be a weak substrate of SLCO1B1 in comparison with SN-38 under the evaluated experimental conditions. However, characterization of the kinetics of flavopiridol and flavo-G transport via SLCO1B1 will be necessary for understanding the full impact this gene has on overall flavopiridol disposition.
The observed associations between PGx and outcomes in this study are encouraging as potential indicators for patient response. While no SNP met the significance criteria for association with TLS, the polymorphism most closely associated with this toxicity was SLCO1B1 rs4149056 (ANOVA p
.056). The strength of the associations between PK, outcomes and multiple SNPs in SLCO1B1 and the confirmation of functional OATP1B1 transport of both flavopiridol and flavo-G support the potential for this gene to be clinically relevant in patients receiving flavopiridol treatment. Overall, analysis of the phase II dataset revealed significant associations with the candidate genes that were supportive of the findings from the phase I dataset. In particular, trends in flavopiridol AUC with respect to the ABCG2 rs2231142 SNP and flavo-G Cmax and AUC with respect to the UGT1A1*28 polymorphism were strikingly similar between the two datasets (see and ). Again, further validation of these associations will be essential in larger datasets.
This work modifies and develops further our previously reported population model for the PK-directed schedule of flavopiridol. We demonstrate the most significant covariates in the phase I dataset to be polymorphisms in two transporter genes, SCLO1B1 and ABCC2, and the phase II validation set revealed significant associations with polymorphisms in ABCG2. While larger data sets are necessary to fully characterize the clinical impact of polymorphisms in SLCO1B1 and other genes on flavopiridol PK, our findings are supportive of a clinically significant role for PGx in flavopiridol disposition. The composite data to date suggests inter-individual variability in outcomes from flavopiridol therapy will be due to a combination of pharmacokinetics, pharmacogenetics and potentially other factors related to tumor cell sensitivity to flavopiridol's cytotoxic effects. Inter-individual variability is of particular concern for chemotherapeutic drugs with relatively narrow therapeutic windows. The developing model for flavopiridol PK may ultimately help to explain this variability by incorporating pharmacogenetic and other significant factors. With sufficient data and validation, this model may ultimately serve as a tool for predicting PK and associated outcomes in individuals prior to therapy. If sufficiently robust, such a tool would be clinically useful in identifying individuals likely to respond and/or experience severe TLS or other toxicities upon receiving flavopiridol. As a phase II registration study of flavopiridol in relapsed CLL nears completion and additional combination studies in hematologic and solid tumor diseases begin accruing patients, further exploration and characterization of the factors influencing inter-individual variability in outcomes from therapy will be necessary to insure the broad and safe clinical use of this drug.