Potential usefulness of quinine to circumvent the anthracycline resistance in clinical practice.

Quinine, the widely used antimalaria agent, was found to increase the cytotoxicity of epideoxorubicin (epiDXR) in resistant DHD/K12 rat colon cancer cells in vitro. Quinine appeared as slightly less effective than quinidine or verapamil for anthracycline potentiation but its weaker cardiotoxicity could counterbalance this disadvantage in vivo. Serum from six patients treated by conventional doses of quinine (25-30 mg kg-1 day-1) was demonstrated to enhance the accumulation of epiDXR in DHD/K12 cells as judged by fluorescence microscopy and HPLC assay (1.6 to 6-fold compared with control serum). In this patients quinine concentrations in serum ranged from 4.4 to 10.1 micrograms ml-1. Our results suggest that quinine could be safely used as anthracycline resistance modifier in clinical practice.

Primary or acquired resistance to anthracyclines of human cancers is partly associated with the overexpression of a membrane glycoprotein (P 170) that effluxes drugs out of cancer cells (Goldstein et al., 1989;Dalton et al., 1989). Anthracycline resistance may be altered in vitro by a variety of agents such as verapamil (Tsuruo et al., 1982), quinidine (Tsuruo et al., 1984), amiodarone (Chauffert et al., 1986) or cyclosporine (Slater et al., 1986). However, use of resistance modifiers in clinical practice is still a problem due to the toxicity of these agents that precludes the achievement of effective concentrations in patient serum (Gottesman & Pastan, 1989;Genne et al., 1990).
In this paper we report that quinine, the widely used antimalaria drug, enhanced in vitro the cytotoxicity of epidoxorubicin in resistant colon cancer cells. Moreover, serum of quinine treated patients was demonstrated to increase the cellular accumulation of the anthracycline in resistant cells.

Patients
The first patient (no. 1) was treated for a chloroquineresistant malaria; the other patients (nos2-6) were treated after informed consent with a combination of quinine and doxorubicin for anthracycline resistant tumours. Quinine was given either per os or intravenously at a daily dose ordinarily used for malaria treatment (24-30mgkg-' day-'). When given per os, the daily dose of quinine was supplied in three regular intakes. Intravenous treatment was given as continuous infusion. Patient serum was collected at steady state 48 h at least after starting quinine administration. Peak and trough plasma concentrations of quinine were determined in two patients 2 and 8 h respectively after an oral intake. Serum of one of the authors was used as control. After blood collection, serum was centrifugated and stored at -80C until assay.

Cancer cells
The DHD/K12 cancer cell line was established in our laboratory from a chemically induced colon cancer in syngeneic BDIX rats (Martin et al., 1975). Inherent resistance of DHD/ K12 cells to anthracyclines is partly related to a drug efflux mechanism (Chauffert et al., 1984) which is efficiently inhi-bited by amiodarone or verapamil (Chauffert et al., 1986).
Cells were grown as a monolayer adherent to the surface of culture flasks. Culture medium was a mixture of Ham's FIO medium and fetal bovine serum (10:1; V/V). For experiments, cells were detached from the culture flasks by a 10 min treatment with EDTA (0.2 mg ml-') and tryspin (2.5mg ml-') in Hank's medium without Ca2+ or Mg2+. Drugs Epidoxorubicin (epiDXR) was obtained from Farmitalia Carlo Erba laboratories (Milan, Italy). EpiDXR was preferred to doxorubicin because of its greater penetration and cytotoxicity in vitro in DHD/K12 cells. Daunorubicin used as internal standard for HPLC assay was purchased by Roger Bellon laboratories (Neuilly, France). Quinine sulphate was used for oral treatment and was obtained from Cooperation Pharmacologique Franqaise (Melun, France). Quinine formiate was used for intravenous treatment and was obtained from Vaillant-Defresne laboratories (Quinoforme, Paris, France). Quinine hydrochloride, quinidine hydrochloride and hydroquinidine hydrochloride used for in vitro experiments were obtained from Sigma (La Verpilliere, France). Verapamil hydrochloride was purchased from Biosedra laboratories (Malakoff, France).

Cytofluorescence study
The intracellular accumulation of epiDXR in DHD/K12 cells was studied by UV illumination which induced a yelloworange fluorescence at the intracellular sites of anthracycline localisation. We previously reported that low accumulation of anthracyclines in nucleus of primary resistant DHD/K12 cells was related to a drug efflux mechanism (Chauffert et al., 1984). In the presence of sufficient concentration of a drug efflux inhibitor in incubation medium, anthracycline accumulation increased in cancer cells and then a bright fluorescence was observed in nuclei. For microscopic examination, DHD/ K12 cells were cultivated for 24 h on glass coverslips then exposed for I h to epiDXR (5 jg ml-') diluted in patient or control serum. After rinsing with cold phosphate buffered saline (PBS), cells were examined under an UV fluorescence microscope (Leitz, Weitzlar, FR Germany).
EpiDXR uptake in cancer cells DHD/K12 cells in suspension were incubated for 1 h at 37°C with epiDXR (5 or g ml-') diluted in patient or control serum. After rinsing twice with cold PBS and centrifugation, cells pellets were mixed with daunorubicin diluted in borate buffer, pH!9.4. Anthracyclines were extracted by a chloro-form-methanol mixture (4:1, V/V). The organic phase was evaporated under a nitrogen stream; the dry residue was diluted in mobile phase and injected into an HPLC apparatus; the mobile phase was a mixture of acetonitrile and formiate buffer, pH 4 (1:2, V/V); the stationary phase was a microbondapak C18 column (Waters Associates, Millford, USA). Drugs were detected by fluorimetry at excitation and emission wavelengths of 480 and 560 nm respectively.
In vitro drug-sensitivity test Enhancement of epiDXR cytotoxicity induced by verapamil, quinidine or quinine was compared by an in vitro test using a long exposure to anthracycline. DHD/K12 cells (I x 104 cells in 200 jlI culture medium) were seeded in the wells of a microculture plate (9 x 12 wells) in presence of epiDXR (0.25 jg ml-') combined with various concentrations of the resistance modifier agents. After 72 h, cell survival was determined by a methylene blue colorimetric assay soon described elsewhere (Martin et al., 1982;Oliver et al., 1989). Surviving cells remained adherent to the well bottom whereas dead cells were detached in culture medium. After rinsing wells with PBS, adherent cells were fixed for 15 min by pure ethanol then stained by methylene blue (1% in PBS); dye in excess was flushed away with abundant tap water. Dye fixed to cell proteins was eluted by a mixture of HCI 0.1 N and pure ethanol (1: 1. V/V). Optical density (OD) was measured in each well at 630 nm by an automatic spectrophotometer; it has been previously demonstrated that OD is proportional to the number of living cells remaining attached on the bottom of each well at the end of experiment.

Measurement of quinine concentration in patient serum
Patient serum was mixed with borate buffer, 0.5 M, pH 9.8, and hydroquinidine used as internal standard. Extraction was performed by a mixture of dichloromethane and isoamylic alcohol (98:2, V/V). The organic phase was evaporated under a nitrogen stream. The dry residue was dissolved in the mobile phase and injected into an HPLC apparatus. The mobile phase was a mixture of acetonitrile and potassium phosphate buffer, pH 3.8 (1:4, V/V). The stationary phase was a Novapak C1 8 5 j column. Drugs were detected by fluorimetry at excitation and emission wavelengths of 350 and 440 nm respectively.
Only a weak and inhomogenous fluorescence was seen in cell nuclei after a 1 h incubation of DHD/K12 cells in control serum supplemented with epiDXR (5 jig ml-'). In contrast, we observed an intense and homogenous nuclear fluorescence when cells were treated with epiDXR 5 j.g ml-' diluted in all the sera of quinine treated patients (Figure 2).
Fluorescence microscopy allowed the demonstration of the rapid reversibility of the inhibition of anthracycline efflux by quinine. When DHD/K12 cells were incubated for 1 h in Ham's FIO medium supplemented with quinine 5jigml-l and epiDXR 5fjgml-', cell nuclei were brightly fluorescent; however, nuclear fluorescence disappeared almost completely in less than I h when cells were incubated again in Ham's FIO medium supplemented with epiDXR 5 fig ml-' but without quinine.
EpiDXR content in DHD/K12 cells was from 1.6 to 5.4fold greater after a 1 h incubation in serum of quinine treated patients comparatively to control serum (Table I). Quinine concentrations in patient serum ranged from 4.4 to 10.1 jig ml-' (Table II) Resistance modifier concentration (,g ml 1) Figure 1 Cytotoxicity of epiDXR (0.25fjgmIm 1 for 72h) on DHD/K12 cells in presence of quinine (m), quinidine (-) or verapamil (*). No cytotoxicity was registered at considered concentrations for verapamil, quinidine or quinine used alone without epiDXR. Each point is the mean of three determinations (maximal standard deviation = 3%). a b Figure 2 Fluorescence microscopic study of DHD/K12 cells after 1 h incubation with 5 jug ml ' epiDXR in presence of control serum (a) or serum from a quinine treated patient (b) ( x 520).
serum was demonstrated by the weak difference between peak and trough concentrations in two patients treated with oral quinine.

Discussion
Although quinine appeared to be slightly less effective than quinidine or verapamil for the circumvention in vitro of the anthracycline resistance of rat colon cancer cells, its lower cardiotoxicity could be a considerable advantage for its use as multidrug resistance modifier in clinical oncology. Serum  Control  170  210  1  430  870  2  280  390  3  580  1060  4  930  1250  5  800  1320  6 260 480 aMean of three determinations (maximal standard deviation = 9%). of quinine treated patients was demonstrated to increase the epiDXR accumulation in DHD/K12 anthracycline resistant cells; such an enhancing effect was obtained for quinine concentrations ranging from 4.4 to 1O. I g mlh' in patient serum. In this work, the daily dose of quinine was 25 or 30 mg kg-' day-' as usually recommended for the treatment of malaria (Hall, 1976). Higher quinine concentrations (10-15 fig ml-') could be reached with a daily dose of 35 mg kg-' as for patients with cerebral malaria (White et al., 1982). Risk of severe poisoning, mainly transient or permanent visual deficit, occurs only when serum levels exceeded 15 tg ml-' (Boland, 1985). Comparatively serum concentrations of quinidine that are recommended for treatment of cardiac dysrytmias ranged from 3 to 5 tLg ml-' with risk of severe blockade of auriculoventricular conduction above 8 tLg ml-' (Holford et al., 1981). In the study of Benson et al. (1985), which evaluated the tolerance of verapamil given by continuous infusion (0.12 tg kg-' h-') in association with vinblastine, the maximal tolerated concentration was 0.29 jig ml-' in serum. Binding of resistance modifiers to serum proteins must also be considered before extrapolating the results of in vitro studies to clinical practice. However, no dramatic difference is registered in the binding of the present resistance modifiers to serum proteins: 90% for verapamil (Schomerus et al., 1976), 75-95% for quinidine (Ochs et al., 1980) and quinine (Silamut et al., 1985). Stability of quinine concentration in patient serum related to its long half-life (10 h) appears also as a propitious property for its use as a circumventing agent of the anthracycline resistance in future clinical studies.