Uptake and retention of morpholinyl anthracyclines by adriamycin-sensitive and-resistant P388 cells

Summary Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracycines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes.

Section: Discussionsupporting

confidence: 92%

Human hepatoma cells rich in P-glycoprotein are sensitive to aclarubicin and resistant to three other anthracyclines

Lehne

Angelis²,

Clausen³

et al. 1996

“…Extraction with 10 M urea was performed by solubilising the cell pellet in 0.2 ml of the reagent and incubating for 1 h at 37°C. After the incubation period, the urea solution was extracted twice with 0.5 ml of chloroform, the phases were separated by centrifugation at 12,000g for 1 min and the chloroform extracts were combined and evaporated with nitrogen (Streeter et al, 1986). Northern blotting Northern blotting analysis was performed as described by Kingston (1988) and Selden (1988).…”

Section: Methodsmentioning

confidence: 99%

Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance

Sakata

Kwok²,

Murphy³

et al. 1991

Summary In this report, we investigate several examples of hypoxia-induced drug resistance and compare them with P-glycoprotein associated multidrug resistance (MDR). EMT6/Ro cells exposed to drugs in air immediately after hypoxic treatment developed resistance to adriamycin, 5-fluorouracil, and actinomycin D. However, these cells did not develop resistance to colchicine, vincristine or cisplatin. When the cells were returned to a normal oxygen environment, they lost resistance. There was no correlation between the content of adriamycin and the development of adriamycin resistance induced by hypoxia. There was no difference between the efflux of adriamycin from aerobic cells and that from hypoxia-treated cells. The mRNA for P-glycoprotein was not detected in the hypoxia-treated cells. These results suggest that hypoxia-induced drug resistance is different from P-glycoprotein associated multidrug resistance.As a tumour grows, heterogeneities of cellular microenvironments occur, such as the development of oxygen gradients in the tumour as a consequence of deficient vascularisation, and cause hypoxic cells that may be resistant to radiotherapy (Sutherland, 1988). Several studies using monolayer cultures (Smith et al., 1980;Teicher et al., 1981Teicher et al., , 1985 and the multicell spheroid system (Sutherland et al., 1979) Conditions of hypoxic exposure Before being gassed with N2, the cells were supplied with 5 ml of fresh complete medium and allowed to equilibrate in a humidified 37°C incubator. Cells undergoing hypoxic stress were isolated in specially designed hypoxic chambers at room temperature (Sutherland et al., 1982). The chambers were repeatedly evacuated and filled every 15 min for 2.25 h with the appropriate gas mixtures certified to contain less than 10 ppm 02. The sealed chambers were then removed to a warm room (37'C) at a time point, t, referred to hereafter as '0' hours of hypoxia.Preparation of drugs and conditions of drug exposure All drugs used were obtained from Sigma Chemical Company. Stock solutions of adriamycin (ADR), vincristine, and actinomycin D (ACTD) were prepared with phosphatebuffered saline (PBS). Solutions of 5-fluorouracil (5-FU), colchicine, and cisplatin were made before each experiment. The solvents used were PBS for ADR, ACTD and cisplatin and distilled water for 5-FU. Absolute ethanol was used as a solvent for colchicine in order to obtain a sufficiently high concentration for the experiments. The final concentration of alcohol in the medium was 1%. As a control for the alcohol solvent, we established that 1% of absolute ethanol in cultures for 2 h had no effect on plating efficiency. Drug treatment was started under aerobic conditions at 37'C in the incubator after culture dishes were removed from the chambers at the zero time. Exposure times for ADR, ACTD and cisplatin were 1 h. A 2 h exposure time was used for 5-FU, colchicine and vincristine to cause significant cell killing. Exposure times were limited to 1-2 h to avoid the effect of release from a ORPs-induced state,...

“…Unlike other anthracyclines, the morpholinyl derivates have been found to cause DNA damage not through stabilization of topoisomerase II-induced double-strand breaks but through topoisomerase I single-strand breaks (Wasserman et al, 1990). In vivo the morpholinyl anthracyclines are activated to highly potent metabolite(s) by cytochrome P450 (Streeter et al, 1986;Lau et al, 1989; Lewis et al, 1992;Ripamonti et al, 1992). Morpholinyl anthracyclines show no cross-resistance in doxorubicin-resistant Cycles 2-6 3.0 (0.1-9.8) 1.7 (< 0.1-6.8) 117 (9-372) 103 (69-153) n=82 n=81 n=80 n=82 P-glycoprotein-positive and multidrug resistance-associated protein positive cell lines (Streeter et al, 1986;Coley et al, 1989;Coley et al, 1991;Danesi et al, 1993), cell lines with an altered topoisomerase II and cell lines resistant to cisplatin and melphalan (Grandi et al, 1990;Ripamonti et al, 1992;Van der Graaf et al, 1995).…”

mentioning

confidence: 99%

Broad phase II and pharmacokinetic study of methoxy-morpholino doxorubicin (FCE 23762-MMRDX) in non-small-cell lung cancer, renal cancer and other solid tumour patients

Bakker

Droz²,

Hanauske³

et al. 1998

Summary The aim was to perform a broad phase 11 and pharmacokinetic study of methoxymorpholino-doxorubicin (MMRDX), a drug active against multidrug-resistant tumour cells in vitro when given by i.v. bolus at 1.5 mg m-2 every 4 weeks, in metastatic or unresectable solid tumour patients with known intrinsic drug resistance. Patients received a maximum of six cycles. Plasma, urine and leucocyte MMRDX and its 1 3-dihydro metabolite pharmacokinetic analysis was performed in patients without liver metastases. Patients (n = 48, 21 NSCLC, 19 renal cell, three head and neck tumour, three cervical cancer and two adenocarcinoma of unknown primary) received 132 cycles of MMRDX. Common toxicity criteria (CTC) grade III/IV thrombocytopenia (12% of cycles) and neutropenia (27% of cycles) occurred with median nadir on day 22. Transient transaminases elevation 2 grade III/IV was observed in 7% of cycles, late and prolonged nausea 2 grade 11 in 34% and vomiting . grade 11 in 39%. In two patients, the left ventricular ejection fraction was reduced 2 15%. Of 37 evaluable patients, one out of 17 NSCLC had a partial response. Mean (± s.d.) MMRDX AUCo, calculated up to 24 h after dosing was 20.4 ± 6.2 ,ug h 1-' (n = 11) and tf/2,3 was 44.2 h. Mean plasma clearance (± s.d.) was 37.2 ± 7.3 h-' m-2 and volume of distribution 1982 ± 64 m-2. MMRDX leucocyte levels 2 and 24 h after infusion were 450 to 600-fold higher than corresponding MMRDX plasma levels. In urine, 2% of the MMRDX dose was excreted unchanged, and 2% as metabolite. The main side-effects of 1.5 mg m-2 every 4 weeks of MMRDX are delayed nausea and vomiting and haematological toxicity. MMRDX is characterized by extensive clearance and rapid and extensive distribution into tissues. A low response rate was observed in patients with tumours with intrinsic chemotherapy resistance.