This study suggests that ABCB1 is involved in a previously unexplored blood-tissue barrier in the cardiac endothelium where it limits drug exposure in the heart. Interestingly, the observed associations with cardiac repolarization were found to be independent of an influence of ABCB1 on romidepsin plasma pharmacokinetic parameters in both mice and humans. Mice lacking the ABCB1-type P-glycoprotein had a greater intracardiac exposure to romidepsin. Although there was no difference in the QTcmax
, this difference in exposure may have altered cardiac repolarization since the time to maximum QTc was different between wild-type and knockout (31 vs. 16 hours). These data suggest that ABCB1 is a component of a blood-tissue barrier within the cardiac endothelium. Consistent with an impact of ABCB1 on cardiac exposure to romidepsin, we also show differences in the QT interval associated with common inherited variants in the ABCB1
gene. Unexpectedly, the variant alleles understood to be associated with reduced function of the ABCB1 transporter (12
) were in this study associated with a reduced impact of romidepsin on the QT interval. Taken together, these results are consistent with a hypothesis that patients carrying variant ABCB1
alleles have increased cardiac ABCB1
gene expression (11
), which in turn limits the exposure of cardiac tissue to romidepsin.
The human ether-a-go-go related gene (hERG
) encodes the a-subunit of the rapid delayed rectifier current IKr
in the heart, which contributes prominently to terminal repolarization in human ventricular myocytes. Romidepsin has been shown to alter terminal cardiac repolarization by inhibiting the hERG protein, potentially by acetylation (34
). Maximal QTc prolongation was delayed following romidepsin administration in mice despite the rapid distribution and clearance of the drug demonstrating that romidepsin has a delayed, rather than acute, effect on ΔQTc in mice, similar to the effect observed in humans (13
). This is inconsistent with direct hERG/IKr
); rather it is likely that the delay in QTc prolongation is related to an intracellular mechanism whereby romidepsin modulates certain pathways related to the hERG protein, such as by blocking hERG shuttling to the membrane by hsp90 as has been observed in several other drug treatments (i.e. fluoxitine, pentamidine, probucil, geldenamycin, radicicol, celastrol; reviewed in (37
)). Nonetheless, removal of Abcb1a/1b from the genome seems to be responsible for decreasing the time taken to ΔQTcmax
, most likely by increasing the overall exposure of the heart to romidepsin in mice. It is expected that this occurs because Abcb1a/1b is important for effluxing romidepsin from the heart, back into the systemic circulation thereby protecting the heart from QTc changes. In our hands, using whole heart homogenate, the exposure increase between wild-type and Abcb1a/1b
−/− mice was relatively modest (~1.35-fold increase in knockouts); however, it is unclear to what degree cardiac exposure to romidepsin must increase in order to see an effect on QTc. Moreover, romidepsin exposure could have been even higher in cardiac myocytes of knockout mice than was apparent from whole heart homogenate as Abcb1 is expressed in cardiac endothelial cells and is thus expected to greatly limit drug penetration into highly vascularized cardiac muscle tissue.
Meissner et al.
demonstrated that human individuals carrying the variant ABCB1
2677TT genotype have increased ABCB1
mRNA levels in cardiac endothelial cells. Although the results of Meissner et al.
contrast with the current understanding of ABCB1
polymorphic variation (i.e. wild-type alleles are most often related to higher expression in other tissues (38
)), the current data obtained with romidepsin are consistent with Meissner et al.
as individuals with higher copy numbers of variant ABCB1
2677T alleles exhibited reduced lengthening of the QT-interval following romidepsin, compared to patients carrying increasing numbers of ABCB1
wild-type alleles. The association between ΔQTc is strongest when all three of the studied alleles are considered together in a haplotype, consistent with functional studies of ABCB1
allelic variation (12
). Meissner et al
. did not evaluate haplotypes in their study of mRNA expression. Since we determined that ABCB1
variant alleles confer a phenotype with lowered transport efficiency, the data suggest that gene expression in the cardiac endothelium may be a more important factor than altered protein folding and function induced by the non-synonymous 2677G>T/A (A893S/T) SNP, or the synonymous 1236C>T and 3435C>T transitions (12
The increase in time taken to achieve ΔQTcmax
in mice suggests that the human data may result from ABCB1
expression status modulating an early or later ΔQTcmax
and not necessarily a greater magnitude of ΔQTc induced by romidepsin. This study found that ABCB1
alleles have an effect after 4 hours such that individuals carrying wild-type alleles, where ABCB1 expression is presumably low in the cardiac endothelium (11
), also have a larger ΔQTc at that time point. We did not find a relationship between ABCB1
SNPs and ΔQTc at 24 hours or during subsequent administrations of the drug. However, since QTc was not monitored continuously in clinical trials, we were unable to assess ΔQTcmax
in patients, and future studies in humans must evaluate QTc prolongation with more resolution in order to determine whether or not ABCB1 expression is responsible for a greater magnitude, or a delayed ΔQTcmax
induced by romidepsin in humans. Moreover, romidepsin may induce ABCB1 in humans (23
) thereby facilitating its own elimination from the heart; this is possibly the reason that the difference in ΔQTc based on genotype is only apparent following the first administration, but not subsequent administrations of the drug. It also remains unclear whether other ABCB1 substrate drugs that prolong the QT interval (risperidone, clozapine, and potentially tamoxifen) are also limited from penetrating the heart tissue in a similar fashion, and whether genetic variation influences their intracardiac concentration. A single, small study in patients treated with doxorubicin appears to indicate that cardiomyopathy is more prevalent in patients with low ABCB1 mRNA and protein expression in cardiac endothelium (8
). Thus, further research is required to ascertain the clinical importance of ABCB1 expression on substrate drugs that mediate QT-prolongation and cardiotoxicity.
This study contains several limitations and inconsistencies with the literature. First, the functional impact of allelic variation in ABCB1
gene expression presented herein add to a body of literature that has been controversial. Many investigations have demonstrated that in liver, intestine, and blood cells, the ABCB1
2677G>T/A and 3435C>T wild-type alleles actually predict higher ABCB1 expression relative to variant alleles (39
). However, functional studies have not consistently confirmed an impact of this higher gene expression on systemic drug exposure, with investigators reporting both increased and decreased drug clearance associated with these alleles. Nonetheless, our results are consistent with the observations set forth by Meissner et al
. in human cardiac endothelial cells; cardiac expression of ABCB1 may be regulated in a tissue-dependent fashion (11
). Second, due to data limitations, we were not able to ascertain QTcF in most patients, and QTcF appears to be a better measure of romidepsin-induced QTc prolongation than QTcB in some patients (13
). Third, this study didn't evaluate the pharmacokinetics of reduced romidepsin, the active metabolite, and we are not able to conclude whether or not exposure to the active metabolite is greater. Thus our results should be considered exploratory and in need of further validation.
In conclusion, this study provides evidence that QTc interval changes following treatment with the ABCB1 model substrate romidepsin might be linked to the expression of the ABCB1 drug transporter in the cardiac endothelium. We believe that this is the first demonstration that ABCB1 expression alters QT-prolongation both in preclinical and clinical settings. This study is also the first to suggest that ABCB1 allelic variation may serve as a marker for prolonged QT-interval mediated by ABCB1 substrate drugs. These results warrant further evaluation for both romidepsin, and for other ABCB1 substrates that have the potential to cause cardiac side effects.
STATEMENT OF TRANSLATIONAL RELEVANCE
Using romidepsin as an ABCB1 model substrate, this study shows that ABCB1 participates in a blood-tissue barrier in the cardiac endothelium. This blood-heart barrier limits the intracardiac concentration of romidepsin thereby reducing QT-prolongation induced by romidepsin. Analysis of clinical samples revealed that genetic variants in ABCB1 that were previously linked to increased expression of ABCB1 in the blood-cardiac barrier may be responsible for a less severe QT-prolongation phenotype in individuals receiving romidepsin. Therefore, a putative blood-heart barrier should be further explored with specific focus on ABCB1-substrate drugs that prolong the QT-interval, and ABCB1 alleles may serve as markers for QT prolongation induced by romidepsin.