Purvalanol A, olomoucine II and roscovitine inhibit ABCB1 transporter and synergistically potentiate cytotoxic effects of daunorubicin in vitro.

Cyclin-dependent kinase inhibitors (CDKi) have high potential applicability in anticancer therapy, but various aspects of their pharmacokinetics, especially their interactions with drug efflux transporters, have not yet been evaluated in detail. Thus, we investigated interactions of five CDKi (purvalanol A, olomoucine II, roscovitine, flavopiridol and SNS-032) with the ABCB1 transporter. Four of the compounds inhibited efflux of two ABCB1 substrates, Hoechst 33342 and daunorubicin, in MDCKII-ABCB1 cells: Olomoucine II most strongly, followed by roscovitine, purvalanol A, and flavopiridol. SNS-032 inhibited ABCB1-mediated efflux of Hoechst 33342 but not daunorubicin. In addition, purvalanol A, SNS-032 and flavopiridol lowered the stimulated ATPase activity in ABCB1 membrane preparations, while olomoucine II and roscovitine not only inhibited the stimulated ATPase but also significantly activated the basal ABCB1 ATPase, suggesting that these two CDKi are ABCB1 substrates. We further revealed that the strongest ABCB1 inhibitors (purvalanol A, olomoucine II and roscovitine) synergistically potentiate the antiproliferative effect of daunorubicin, a commonly used anticancer drug and ABCB1 substrate, in MDCKII-ABCB1 cells as well as in human carcinoma HCT-8 and HepG2 cells. We suggest that this pronounced synergism is at least partly caused by (i) CDKi-mediated inhibition of ABCB1 transporter leading to increased intracellular retention of daunorubicin and (ii) native cytotoxic activity of the CDKi. Our results indicate that co-administration of the tested CDKi with anticancer drugs that are ABCB1 substrates may allow significant dose reduction in the treatment of ABCB1-expressing tumors

Efavirenz reduces renal excretion of lamivudine in rats by inhibiting organic cation transporters (OCT, Oct) and multidrug and toxin extrusion proteins (MATE, Mate).

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor used in first-line combination antiretroviral therapy (cART). It is usually administered with nucleoside reverse transcriptase inhibitors (NRTI), many of which are substrates of OCT uptake solute carriers (SLC22A) and MATE (SLC47A), P-gp (MDR1, ABCB1), BCRP (ABCG2), or MRP2 (ABCC2) efflux transporters. The aim of this study was to evaluate the inhibitory potential of efavirenz towards these transporters and investigate its effects on the pharmacokinetics and tissue distribution of a known Oct/Mate substrate, lamivudine, in rats. Accumulation and transport assays showed that efavirenz inhibits the uptake of metformin by OCT1-, OCT2- and MATE1-expressing MDCK cells and reduces transcellular transport of lamivudine across OCT1/OCT2- and MATE1-expressing MDCK monolayers. Only negligible inhibition of MATE2-K was observed in HEK-MATE2-K cells. Efavirenz also reduced the efflux of calcein from MDCK-MRP2 cells, but had a rather weak inhibitory effect on Hoechst 33342 accumulation in MDCK-MDR1 and MDCK-BCRP cells. An in vivo pharmacokinetic interaction study in male Wistar rats revealed that intravenous injection of efavirenz or the control Oct/Mate inhibitor cimetidine significantly reduced the recovery of lamivudine in urine and greatly increased lamivudine retention in the renal tissue. Co-administration with efavirenz or cimetidine also increased the AUC0-∞ value and reduced total body clearance of lamivudine. These data suggest that efavirenz is a potent inhibitor of OCT/Oct and MATE/Mate transporters. Consequently, it can engage in drug-drug interactions that reduce renal excretion of co-administered substrates and enhance their retention in the kidneys, potentially compromising therapeutic safety

Effect of the tested CDKi on intracellular accumulation of DNR in MDCKII-ABCB1 cells.

<p>(A) Inhibition, expressed as the ratio between the median fluorescence with and without the indicated inhibitors during the efflux period, normalized to the effect in the parental cell line MDCKII (see <i>Materials and Methods</i> for details). LY (1 µM) was used as a model inhibitor. Presented data are means ± SD obtained from three independent experiments performed in duplicate. <i>P</i> values of differences between observed inhibition ratios and the null hypothetical value of 1 (no ABCB1 inhibition) were determined by unpaired two-tailed <i>t</i> tests: *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. (B) Representative histograms of data obtained in assays with each compound at 20 µM: control with no inhibitor (light grey), tested compound (black), 1 µM LY (dark grey).</p

Cytotoxicity and combination experiments in HCT-8 and HepG2 cell lines.

<p>Cytotoxic effect of (A, E) purvalanol A (▪), (B, F) olomoucine II (▾), (C, G) roscovitine (▴) or daunorubicin (•) and their combination (⧫) on HCT-8 cells or HepG2. For combinations, concentrations corresponding to particular points in the graph are sums of concentrations of the individual drugs administered in fixed concentration ratios (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083467#pone-0083467-t002" target="_blank">Table 2</a>), based on the ratio of their respective EC₅₀ values. Presented data are means ± SD obtained from at least three independent experiments performed in triplicate. (D, H) The cytotoxic effect (combination index, CI, plot) of CDKi and daunorubicin combinations on MDCKII-ABCB1 or MDCKII parent cells, obtained using CompuSyn software. Fractional effects (Fa) were calculated from the cell viability values of individual compounds; Fa  =  0 means no antiproliferative effect, Fa  =  1 means 100% antiproliferative effect. CI  =  0.9–1.1 indicates additive effect, CI <0.9 synergism and CI >1.1 antagonism.</p

Cytotoxicity and combination experiments in MDCKII-ABCB1 and parental cell lines.

<p>Cytotoxic effect of (A, E) purvalanol A (▪), (B, F) olomoucine II (▾), (C, G) roscovitine (▴) or daunorubicin (•) and their combination (⧫) on MDCKII-ABCB1 or MDCKII parent cells. For combinations, concentrations corresponding to particular points in the graph are sums of concentrations of the individual drugs administered in fixed concentration ratios (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083467#pone-0083467-t002" target="_blank">Table 2</a>), based on the ratio of their respective EC₅₀ values. Presented data are means ± SD obtained from at least three independent experiments performed in triplicate. (D, H) The cytotoxic effect (combination index, CI, plot) of CDKi and daunorubicin combinations on MDCKII-ABCB1 or MDCKII parent cells, obtained using CompuSyn software. Fractional effects (Fa) were calculated from the cell viability values of individual compounds; Fa  =  0 means no antiproliferative effect, Fa  =  1 means 100% antiproliferative effect. CI  =  0.9–1.1 indicates additive effect, CI <0.9 synergism and CI > 1.1 antagonism.</p

Effects of CDKi on the ATPase activity of ABCB1-Sf9 membrane preparations.

<p>Vanadate-sensitive ATPase activity in the presence of purvalanol A, olomoucine II, roscovitine, flavopiridol, or SNS-032 in activation (•) and inhibition (□) experiments. Presented data are means ± SD representative of at least two experiments performed in duplicate. The significance of differences linked to the absence and presence of CDKi in the basal activity of the transporter in activation assays (†<i>P</i><0.05; ††<i>P</i><0.01; †††<i>P</i><0.001) and the activity of the activated transporter in inhibition assays (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001) were determined using unpaired <i>t</i> tests.</p

Dose reduction index (DRI) values for drug combinations scheduled after 72 h of simultaneous treatment.

<p>ND, not determined.</p

Effects of CDKi and the model ABCB1 inhibitor LY on ABCB1-mediated efflux of HOE in MDCKII-ABCB1 cells.

<p>0% and 100% respectively indicate the fluorescence of unaffected control cells and the maximal fluorescence observed in assays with a particular CDKi. Presented data are means ± SD obtained from three independent experiments performed in triplicate.</p