Inhibition of cytarabine-induced MDR1 (P-glycoprotein) gene activation in human tumor cells by fatty acid-polyethylene glycol-fatty acid diesters, novel inhibitors of P-glycoprotein function.

Fatty acid ester surfactants Cremophor EL and Solutol HS 15 were described earlier as modulators of multidrug resistance mediated by MDR1 P-glycoprotein (Pgp). We have shown that the most active components of these polydisperse surfactants are fatty acid-polyethylene glycol-fatty acid diesters (FA-PEG-FA). A new generation of Pgp-surfactant inhibitors of defined structure was therefore synthesized. In the present study we show that these compounds are also able to inhibit up-regulation of MDR1 gene expression caused by cytarabine (ARA-C) and doxorubicin in human tumor cell lines H9 and KB 3-1, which express minimal levels of MDR1 mRNA. The surfactant inhibitors, however, had no effect on the induction of MDR1 gene expression by protein kinase C agonists. Using a set of FA-PEG-FA diesters with various fatty acids and different lengths of the PEG domain, we demonstrated that the activity of diester preparations as inhibitors of drug-induced MDR1 activation was in proportion to their activity as inhibitors of Pgp function. Oleic and stearic acid diesters with PEG 900 (20 ethylene oxide units) were the most potent. The poloxamer analogs of these diesters demonstrated similar effects. In contrast, the well-known, structurally unrelated inhibitors of Pgp activity, verapamil, cyclosporin A and PSC 833, had no inhibitory effect on drug-induced MDR1 activation. The ability of FA-PEG-FA diesters to inhibit both Pgp function and drug-induced MDR1 activation suggests that these chemomodulators may be uniquely useful for the prophylaxis of Pgp-mediated multidrug resistance in drug-treated tumors.

Reversal of multi-drug resistance in vitro by fatty acid-PEG-fatty acid diesters.

Fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, that modulate multi-drug resistance (MDR) have been described; however, the drug potential of these preparations is unclear because of the molecular heterogeneity of these and other commercial surfactants. In previous experiments, an active but still polydisperse preparation, designated CRL 1337, was synthesized by reacting purified oleic acid with a 20-fold molar excess of ethylene oxide. We have subjected this preparation to chromatographic separation, and infrared analysis of the active fractions revealed a significant component of diester structures (fatty acid-PEG-fatty acid). A new generation of diester compounds has now been synthesized. Preparations comprised of 99% diesters were significantly more potent than monoester preparations for MDR modification activity in vitro. As previously determined for ethylene oxide-derived preparations similar to CRL 1337, the nature of the fatty acid domains proved to be important for activity, as was the relative length of the polyethylene glycol domain (which determines the hydrophile-lipophile balance). The ester linkage appeared unimportant since homologous diethers and diamides had activity similar to that of diesters. Stearic acid diester was 1.5- to 7-fold more potent than CRL 1337 when tested in cell proliferation inhibition assays. In light of these structural restrictions on drug potentiation, and since these surfactants are active at relatively low concentrations (below 1 microgram/ml), investigations of their mechanism of action were initiated by exploring specific interactions with P-glycoprotein. Both active and inactive diesters inhibited azidopine labeling of P-glycoprotein, suggesting that fatty acid-PEG diesters can interfere with P-glycoprotein substrate binding. Other attributes of these preparations must contribute to their ability to reverse MDR.

Comparison of solutol HS 15, Cremophor EL and novel ethoxylated fatty acid surfactants as multidrug resistance modification agents.

Some well-known fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, are modulators of multidrug resistance in vitro and in vivo. Because they are polydisperse, and their active component(s) have not been identified, the therapeutic potential of such surfactants is unclear. To better define the active components of Solutol HS 15 and to make more potent surfactant multidrug resistance modulators, highly purified C-18 fatty acids were esterified with ethylene oxide at 5-200 molar ratios. Unexpectedly, ethylene oxide esters of pure 12-hydroxy stearic acid, the major components of Solutol HS 15, displayed negligible resistance modification activity compared with Solutol HS 15 itself or to stearic and oleic acid esters synthesized under identical conditions. Since oleic acid esters appeared to have good activity, a series of these compounds was prepared to determine the optimal ethylene oxide/fatty acid ratio. The optimal ratio was found to be 20 mole ethylene oxide: I mole fatty acid, with a steep decline in activity for products made with ratios above and below the optimum. The most active oleic acid ester, designated CRL 1337, was 8.4-fold as potent as Solutol HS 15 and over 19-fold as potent as Cremophor EL in promoting rhodamine 123 accumulation in multidrug-resistant KB 8-5-11 cells in vitro. Our results show that the structure of the hydrophobic domain (fatty acid) of surfactants as well as its hydrophile-lipophile balance are critical in determining the potency of surfactants as reversing agents.

Nucleotide sequence and promoter analysis of SPO13, a meiosis-specific gene of Saccharomyces cerevisiae.

The SPO13 gene, required for meiosis I segregation in Saccharomyces cerevisiae, produces two developmentally regulated transcripts (1.0 and 1.4 kilobases) that differ in length at their 5' ends. The shorter transcript is sufficient to complement the spo13-1 mutation and contains a major open reading frame encoding a highly basic protein of 33.4 kilodaltons. A fragment upstream (-170 to -8) of the open reading frame confers meiosis-specific transcription on a spo13-HIS3 fusion. Deletions at the 5' end of spo13-lacZ fusions define a region between -140 and -80 that is essential for meiosis-specific expression. This region acts in an orientation-independent manner and is responsive to the MAT-RME regulatory cascade. It contains a 10-base-pair sequence, TAGCCGCCGA, found in a number of meiosis-specific genes, that appears to be required for SPO13 expression. This sequence is identical to URS1, a ubiquitous mitotic repressor element.