The decrease of the intracellular concentration of drug in resistant cells compared to sensitive cells is, in most cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein responsible for an active efflux of the drug. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, we have examined the cellular accumulation of 4'-O-tetrahydropyranyl-adriamycin, alone and in conjunction with various ionophores on the one hand and with cyclosporin A on the other hand. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. Using experimental conditions in which these ionophores were unable to modify either the intracellular pH, or the transmembrane potential, or to induce an intracellular ATP depletion, we have shown that mobile ionophores as well as cyclosporin inhibit the P-glycoprotein-mediated efflux of 4'-O-tetrahydropyranyl-adriamycin in K562 resistant cells, whereas gramicidin, a channel-forming ionophore, does not. The concentration that must be used to inhibit 50% of the efflux was 0.7 pM for valinomycin, 0.4 pM for nonactin, 0.2 pM for nigericin, 1.1 pM for monensin, 0.4 pM for lasalocid, 1.2 pM for calcimycin and 0.4 pM for cyclosporin. Due to the high toxicity of the ionophores, the observation that they increased 4'-O-tetrahydropyranyl-adriamycin accumulation in the multidrug-resistant cells is not correlated with an effect of these compounds on drug resistance. However, the correlation exists in the case of cyclosporin. From our data showing that lipophilic neutral complexes, formed between carboxylic ionophores and metal ions, as well as lipophilic cationic complexes, formed between neutral ionophores and metal ions, are both able to inhibit the P-glycoprotein-mediated efflux of anthracycline we can infer that the lipophilicity but not the cationic charge is an important physical property.One of the major limitations in the treatment of cancer is the development of resistance to chemotherapy in patients who initially responded. It has been estimated that among the one million deaths each year due to cancer in Europe, 90% of them were influenced by the problem of resistance to chemotherapeutic agents. This problem is further exacerbated by the fact that these tumors are often cross-resistant to other drugs which were not used during the treatment and which are structurally and functionally unrelated. This is termed multidrug resistance.Multidrug resistance is a well-characterized phenomenon (see recent review by Bradley et al., 1988;Gottesman and Pastan, 1993;Beck, 1987). However, the mechanisms of multidrug resistance are far from being elucidated and, in fact, modifications of several cellular functions have been observed. Thus, multidrug resistance is frequently associated with decreased drug accumulation resul...

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Hydroxyrubin (OH-Dox), a neutral doxorubicin derivative that is only slightly cross-resistant to doxorubicin (Dox), can be actively pumped out of resistant K562 cells by P-glycoprotein (P-gp). This efllux is saturable and can be inhibited by verapamil. The Michaelis constant is equal to 2 f 0.5 PM. However, the efficiency of P-gp in pumping out the drugs is 2.5 times less for OH-Dox than for Dox. This shows that in order to be pumped out by P-gp a molecule does not necessarily have to have a basic center. The mean influx coefficient for the drug is 5 times higher for OH-Dox than for Dox. In conclusion, the degree of resistance of analogs is related not only to their ability to be recognized and transported by P-gp but also, and probably essentially, to their kinetics of uptake. Both parameters have to be taken into account in the rational design of new compounds capable of overcoming multidrug resistance.Key words: Multidrug resistance; P-Glycoprotein; Doxorubicin; Michaelis constant rntroductionThe multidrug resistance (MDR) cell phenotype reflects the capacity of some cell strains to display cross-resistance to certain cytotoxic drugs (typically anthracyclines, vinca alkaloids, colchicine, etc.) [ 1,2]. It is now well established that the MDR phenotype is strongly correlated with the over-expression of P-gp, a 170-kDa membrane glycoprotein encoded by mdr class genes, that actively pumps drugs out of resistant cells. This effiux is energy dependent.It is now generally accepted that the uptake of drugs, such as anthracyclines, by cells occurs by passive diffusion of the neutral form of the drug [3-71. However, the mechanism according to which the drugs are removed from the cells is still unknown. It has been reported that (i) a wide variety of drugs recognized by P-gp are positively charged [8]; (ii) an ideal modulator of P-gp would have at least two planar aromatic rings, a tertiary nitrogen that would be charged at physiological pH, and be relatively lipophilic [9]; and (iii) P-gp specifically pumps out the protonated form of daunorubicin [lo].A better comprehension of the molecular requirements for drug-protein interactions is a necessary prerequisite to the rational design of new compounds capable of overcoming MDR. To get some insight into that problem, we have studied the uptake and the efflux in reistant cells of a neutral molecule, OH-Dox, a synthetic analog of Dox in which the amine group of the sugar portion has been replaced by a hydroxyl group (Scheme 1) [l l-l 31. At neutral pH, about 96% of the amine group of Dox is protonated and the molecule carries a net positive charge of + 1 [6]. Substitution of a hydroxyl group for the amine group results in the loss of the positive charge.We present here data that clearly demonstrate (i) the existence of P-gp-mediated efflux of the uncharged molecule (OH-Dox), and (ii) the fact that the kinetics of uptake must be taken into account when searching for new non-cross-resistant compounds. *Corresponding author. Fax: (33) (1) 48 38 77 77.Abbreviations; M...