EurJ Cancer, Vat. 29A, No. 4, pp. 559-561, 1993. 0964-1947/9356.00 + 0.00 Pnnted m Great Britain ~) 1993 Pergamon Press Ltd Effect ofD,L-Verapamil, Verapamil Enantiomers and Verapamil Metabolites on the Binding of Vincristine to or1-Acid Glycoprotein Barry G. Woodcock, Mahran S. Abdel-Rahman, Frank Wosch and Sebastian Harder Vincristine binding to solutions of or 1-acid glycoprotein (AGP, 2 mg/ml) and the effect of D,L-verapamil, verapamil enantiomers and the verapamil metabolites norverapamil and D617 were investigated in v/tro using equilibrium dialysis and 3H-labelied vincristine. Vincristine binding to AGP (52.3 _+ 3.6%) was concentration independent over the range 0.002-2.0 p.g/ml. The displacement of vincdstine from AGP varied between 25.1 and 81.3% with D,L-verapamil and verapamil enantiomers added at concentrations in the range 5-50 I, tg/mi. In contrast, the displacement by D617 (5-100 v.g/ml) was weaker and varied between 0 and 47%. The displacement at 20 vtg/ml produced by D,L-verapamil, R-verapamil, S-verapamil and norverapamil was 53.1%, 56.8%, 58.9% and 53.9%, respectively, was more than double that for D617 (25%; P = 0.002). It is concluded that vincristine, O,L-verapamil and verapamil isomers and metabolites interact at binding sites on AGP. These interactions may be clinically important in multidrug resistance, for example in cancer patients with elevated levels of AGP undergoing treatment with verapamii and vinca alkaloids. EurJ Cancer, Vol. 29A, No. 4, pp. 559-561, 1993. INTRODUCTION MULTIDRUGRESISTANCE (MDR) is the commonly used term for crossover-resistance of turnout cells to structurally unrelated cytostatic drugs including vinca alkaloids. One of the mechan- isms causing this incurred resistance is the enhanced expression of transmembraneous P-glycoprotein (P-gp) in the tumour cell. P-gp is known to act as an outward flow pump (multidrug- carrier) lowering the intracellular concentration of the cytotoxic drugs [1-3]. Several investigators have reported that verapamil is able to reverse MDR in animals and humans [4-11]. The precise mode of action is not known but it is possible that verapamil binds to P-gp, thereby competitively inhibiting efflux of the cytostatic drug and raising its intracellular concentration. This property is not related to the ability of verapamil to inhibit the transmem- braneous flux of z~ Ca since reversal of MDR can be effected using both R- and S-enantiomers of verapamil and the demethyl- ated verapamil metabolite norverapamil. The dealkylated metabolite of verapamil D617 is inactive in MDR [11]. Chatterjee et al. [9, 12] have reported that the acute phase protein st-acid glycoprotein (AGP) interferes with verapamil in experimental MDR. At concentrations similar to those in cancer patients, where the concentrations of AGP in plasma is 2- to 3- fold higher than normal [13, 14], AGP completely abolishes the ability of verapamil and toremifene to reverse MDR in Chinese hamster ovary cell lines resistant to doxorubicin. Since AGP strongly binds vinblastine and verapamil [ 15-17], the possibility Correspondence to B.G. Woodcock. B.G. Woodcock, F. Wosch and S. Harder are at the Department of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor- Stern-Kai 7, D 6000 Frankfurt/Main 70, F.R.G.; and M.S. Abdel- Rahman is at the Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt. Revised 24 Aug. 1992; accepted 21 Oct. 1992. exists that binding interactions on AGP modify the pharmaco- logical activity of these agents and that these interactions have clinical relevance in cancer chemotherapy. The purpose of this study was therefore to investigate the effect of racemic verapamil (D,L-verapamil), verapamil metab- olites and verapamil enantiomers on the binding of vincristine to AGP in vitro and to assess the clinical importance of inter- actions with this glycoprotein in patients undergoing chemo- therapy. MATERIALS AND METHODS AGP was obtained from Fluka GmbH, Neu-Ulm or the Sigma Chemical Co., and vincristine sulphate from Eli Lilly. [3H]Vincristine sulphate, with specific activity 296 MBq/mg, was supplied by Amersham (U.K.). n,L-Verapamil-HCl, R- verapamil-HCl and the verapamil metabolites norverapamil- HCI and D617-HCI [2-methyl-3-cyano-3-(Y ,4'-dime- thoxyphenyl)-6-methylamino-hexane HCI] were supplied by Knoll AG (Ludwigshafen, F.R.G.). S-Verapamil-HC1 was obtained from Research Biochemicals Incorporated (U.S.A.). Stock solutions of labelled vincristine in phosphate buffer were stored at -20°C. Unlabelled vincristine sulphate and solutions of other reagents were prepared on the day of the experiment and used immediately. Binding studies were carried out at 37°C in phosphate buffer 0.16 mol/l, pH 7.4 using equilibrium dialysis (Dianorm system). The 1-ml chambers were separated by a cellulose semipermeable membrane (Diachema, Munich, F.R.G.) having a cut-off at 5000 Da. The membrane was soaked in 15% ethanol for 10 min, in distilled water overnight and washed with phosphate buffer before use. The dialysis time required to establish equilibrium was 4 h at a rotation speed of 4 rpm [ 17, 18]. Individual binding experiments were carried out in duplicate. The concentration of AGP was 2 mg/ml. Each experiment contained between 500 and 1000 Bq [3H]vincristine. In the first set of experiments with 559