Spontaneous overexpression of the long form of the Bcl-X protein in a highly resistant P388 leukaemia.

A novel resistant variant of murine P388 leukaemia, P388/SPR, was identified by de novo resistance to doxorubicin (DOX) in vivo. This mutant displayed a similar level of cross-resistance to etoposide (VP-16) and other topoisomerase II (topo II) inhibitors. Further analysis of the phenotype revealed a broad cross-resistance to vinca alkaloids, alkylating agents, antimetabolites, aphidicolin and UV light. Low-level expression of mdr1 and P-glycoprotein (P-gp), as well as a modest impairment of cellular drug accumulation and partial reversion of resistance to DOX and VP-16 by cyclosporine, confirmed a moderate role of P-gp in conferring drug resistance in P388/SPR cells. Consistent changes in neither topo II expression or activity nor glutathione metabolism could be detected. Induction of apoptosis was significantly reduced in P388/SPR cells, as indicated by minimal DNA fragmentation. Analysis of oncogenes regulating apoptotic cell death revealed a marked decrease of bcl-2 in combination with a moderate reduction of bax protein, but a striking overexpression of the long form of the bcl-X protein. Transfection of human bcl-X-L into P388 cells conferred drug resistance similar to that of P388/SPR cells. The data suggest that overexpression of bcl-X-L results in an unusual phenotype with broad cross-resistance to non-MDR-related cytotoxins in vitro, and provide an interesting example of spontaneous overexpression of another member of the bcl-2 gene family in cancer.     

Combined treatment of Bcl-2 antisense oligodeoxynucleotides (G3139), p-glycoprotein inhibitor (PSC833), and sterically stabilized liposomal doxorubicin suppresses growth of drug-resistant growth of drug-resistant breast cancer in severely combined immunodeficient mice

We studied the possibility of increasing sensitization of drug-resistant MDA435/LCC6 multidrug-resistant (MDR) human breast cancer cells to doxorubicin (DOX) by increasing cellular drug retention with P-glycoprotein (P-gp) inhibitor PSC833 in combination with induction of cell death through down-regulation of Bcl-2 protein using Bcl-2 antisense (G3139). In in vitro cytotoxicity assays, the combination of G3139 with DOX exhibited 40% increased cytotoxicity in both wild-type (WT) and MDR cells. PSC833 increased the cytotoxicity of DOX and Taxol with complete and partial reversal of the resistance of MDR cells to DOX and Taxol, respectively. The presence of G3139 did not increase the cytotoxicity of PSC833 combined with DOX or Taxol in both cell lines. In vivo studies with WT and MDR cell lines transplanted into severely combined immunodeficient mice demonstrated that G3139 (5 mg/kg) was able to suppress the growth of both WT and MDR tumors to an equivalent extent. PSC833 (100 mg/kg) partially restored the sensitivity of resistant tumors to DOX, and the combination of G3139 and PSC833 with liposomal DOX showed maximum growth suppression of MDR tumors compared with individual treatments. The improved efficacy of this treatment was attributed to Bcl-2 antisense-induced apoptosis, combined with cellular retention of DOX in tumor cells via P-gp blockade.     

In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833.

The new nonimmunosuppressive cyclosporin analogue, SDZ PSC 833, is a very potent multidrug-resistance modifier. In vitro, it was shown to be at least 10-fold more active than cyclosporin A (Sandimmune), itself more active than verapamil, on most P-glycoprotein-expressing multidrug-resistant (MDR) tumor cell lines. In vivo, SDZ PSC 833 was tested in a few protocols of combined therapy with either Vinca alkaloids or doxorubicin as anticancer drugs, using the homologous tumor-host system (P388 cells of DBA/2 origin grafted into DBA/2 or B6D2F1 mice). Although these MDR-P388 tumor cells belong to a highly resistant variant that in vitro required about 150-fold more anticancer drug for 50% cell growth inhibition than the parental P388 cells, significant prolongation of survival times of the MDR-P388 tumor-bearing mice was obtained when treated with a combination of SDZ PSC 833 p.o. were otherwise ineffective doses of anticancer drugs given i.p. This chemosensitizing effect of SDZ PSC 833 was dose-dependent and was most effective in a protocol combining administration of SDZ PSC 833 p.o. 4 h before a doxorubicin i.p. injection: in comparison with the survival of MDR-P388 tumor-bearing mice treated with the anticancer drug alone, the pretreatment with SDZ PSC 833 at 25 and 50 mg/kg gave 2- to 3-fold increases of survival times. Since the MDR-P388 tumor cells used in our studies belong to a highly resistant variant, with a much higher degree of drug resistance than the one known to occur in cancer patients, SDZ PSC 833 appears to be a very promising chemosensitizer.     

The role of the MDR1 gene in the development of multidrug resistance in human hepatoblastoma: clinical course and in vivo model

BACKGROUND: The P-glyprotein (P-gp), which is a membrane channel encoded by the MDR1 gene, represents a possible explanation for multidrug resistance in human hepatoblastoma (HB). P-gp shows up-regulation in tumor cells after chemotherapy; however, to date, its exact role in HB has not been described. The authors investigated the role of the MDR1 gene in the clinical course of patients with HB and in an in vivo model of HB. They also studied the effects of the MDR1 antagonizer PSC 833 on chemotherapy in mice xenotransplanted with HB. METHODS: Resected tumor specimens, including both primary tumors and recurrent tumors, from a child suffering from HB were investigated histologically. Cell suspensions from the originally removed tumor were incorporated subcutaneously into nude mice. Animals were treated with cisplatin (CDDP) plus PSC 833. MDR1 gene expression levels in the different resected tumors from the patient and in the xenotransplants after treatment were determined with polymerase chain reaction analysis. RESULTS: MDR1 gene expression was increased in the patient's tumors after every course of chemotherapy from 30% to > 190%. In the xenotransplants, MDR1 gene expression was enhanced significantly after chemotherapy (P(CDDP) = 0.008; P(CDDP+PSC) = 0.002). Tumor volumes (P < 0.001) and serum alpha-fetoprotein levels (P = 0.0002) were significantly lower in the animals that were treated with CDDP + PSC compared with the animals that were treated with CDDP alone. CONCLUSIONS: The current results suggest that MDR1 gene expression and P-gp are a potential mechanism of drug resistance in HB. The chemosensitizer PSC 833 significantly improved the effects of chemotherapy in animals xenotransplanted with HB. These data encourage further studies concerning the role of chemosensitizers in overcoming multidrug resistance in patients with HB.     

In vivo response of mitoxantrone and doxorubicin with dipyrone in parental and doxorubicin-resistant P388 leukemia

The efficacy of dipyrone to modulate antitumor activity of mitoxantrone (MTN) and doxorubicin (DOX) was studied in vivo in mice bearing P388 murine lymphocytic leukemia sensitive (P388/S) and resistant P388/DOX) to DOX. P388/DOX-bearing mice demonstrated marginally higher sensitivity to dipyrone at 200 mg/kg when compared to P388/S-bearing mice. However, dipyrone could significantly enhance the antitumor activity of MTN and DOX in both P388/S and P388/DOX-tumor-bearing mice. MTN was cross-resistant to P388/DOX.     

SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein

SDZ PSC 833 (PSC 833) is a new multidrug resistance modulator. Recent studies have shown that the principal mechanism of action of PSC 833 is to bind P-glycoprotein (P-gp) and prevent cellular efflux of chemotherapeutic drugs. We previously reported that PSC 833 increases cellular ceramide levels. The present study was conducted to determine whether the impact of PSC 833 on ceramide generation is dependent on P-gp. Work was carried out using the drug-sensitive P-gp-deficient human breast adenocarcinoma cell line, MCF-7, and drug resistant MCF-7/MDR1 clone 10.3 cells (MCF-7/MDR1), which show a stable MDR1 P-gp phenotype. Overexpression of P-gp in MCF-7/MDR1 cells did not increase the levels of glucosylceramide, a characteristic which has been associated with multidrug resistant cells. Treatment of MCF-7 and MCF-7/MDR1 cells with PSC 833 caused similar ceramide elevation, in a dose-responsive manner. At 5.0 microM, PSC 833 increased ceramide levels 4- to 5-fold. The increase in ceramide levels correlated with a decrease in survival in both cell lines. The EC50 (concentration of drug that kills 50% of cells) for PSC 833 in MCF-7 and MCF-7/MDR1 cells was 7.2 +/- 0.6 and 11.0 +/- 1.0 microM, respectively. C6-Ceramide exposure diminished survival of MCF-7 cells; whereas, MCF-7/MDR1 cells were resistant to this short chain ceramide analog. Preincubation of cells with cyclosporine A, which has high affinity for P-gp, did not diminish the levels of ceramide generated upon exposure to PSC 833. These results demonstrate that PSC 833-induced cellular ceramide formation occurs independently of P-gp. As such, these data indicate that reversal of drug resistance by classical P-gp blockers may be modulated by factors unrelated to drug efflux parameters.     

Myeloid and lymphoid cell alterations in normal mice exposed to chemotherapy with doxorubicin and/or the multidrug-resistance reversing agent SDZ PSC 833

The cyclosporin SDZ PSC 833 (PSC) is a potent in vivo chemosensitizer for tumor cells with P-glycoprotein(Pgp)-dependent multidrug resistance (MDR). However, Pgp expression also occurs in CD8⁺ T cells, NK cells, macrophages and stem cells. In order to find whether PSC might display specific myelotoxicity or potentiate the toxicity of anti-cancer drugs, healthy mice were exposed to single doxorubicin (DOX) and combined (DOX + PSC) chemotherapy protocols known to be near or above the borderline of toxicity for tumor-bearing mice. Mice treated with DOX alone or with (DOX + PSC) showed transient spleen hypoplasia, with a general decrease of all leucocyte lineages and a persistent fall in the numbers of B cells in the bone marrow. In (DOX + PSC)-treated mice, PSC only potentiated the DOX effects without inducing specific depletions of the Pgp-expressing leukocytes (CD8⁺ and Mac-l⁺ cells). Hematopoietic cell grafts from normal mice to (DOX ± PSC)-treated mice did not correct their B-cell lineage deficiency. When lethally irradiated mice were rehabilitated with hematopoietic cells from (DOX ± PSC)-treated mice (including those with very reduced survival), all chimeras survived for at least 8 months after the cell graft, at which time their leucocyte population profiles were similar to those of control chimeras.     

Modulation of Multidrug Resistance by SDZ PSC 833 in Leukemic and Solid-tumor-bearing Mouse Models

P-Glycoprotein inhibitors, including the nonimmunosuppressive cyclosporin D analog SDZ PSC 833 (PSC 833), have been developed to circumvent multidrug resistance. In the present study, the potential of PSC 833 in reversing multidrug resistance was evaluated in various systemic treatment models with leukemic and solid-tumor-bearing mice. Having a relatively wide therapeutic window of daily p.o. doses from 12.5 to 75 mg/kg, PSC 833 significantly improved the antileukemic activity of the anticancer drugs adriamycin (ADM), vincristine (VCR) and etoposide (VP-16) given i.p. or i.v. against i.p.-inoculated vincristine-resistant P388 tumor (P388/VCR). PSC 833 in combination with i.p.-injected anticancer drugs in optimal schedule and dosage induced apparent cures in some leukemic mice, whereas no cures were obtained with the cyclosporin A/anticancer drug combinations. PSC 833 combined with i.v.-injected anticancer drugs was highly active, but not curative, against P388/VCR and parental P388 tumors (maximum T/C>175%). PSC 833 in combination with intravenous treatment with ADM showed prominent anti-solid-tumor activity against s.c.-inoculated colon adenocarcinoma 26 and human colorectal adenocarcinoma HCT-15. Against colon adenocarcinoma 26, the PSC 833/ADM combinations induced cure in two or three of six mice. PSC 833/ADM combinations significantly inhibited the growth of the tumor with maximum percent inhibitions of 83 and 73% in the early and advanced stages of the HCT-15 tumor models, respectively. The present study demonstrated that PSC 833 is highly active in potentiating the antitumor activity of systemically administered ADM, VCR and VP-16 against four murine and human tumors with a relatively wide therapeutic window of daily p.o. dose range of 12.5–100 mg/kg.     

Comparative Study on Reversal Efficacy of SDZ PSC 833, Cyclosporin a and Verapamil on Multidrug Resistance in vitro and in vivo

A non-immunosuppressive cyclosporin, SDZ PSC 833 (PSC833), shows a reversal effect on multidrug resistance (MDR) by functional modulation of MDR1 gene product, P-glycoprotein. The objective of the present study was to compare the reversal efficacy of three multidrug resistance modulators, PSC833, cyclosporin A (CsA) and verapamil (Vp). PSC833 has approximately 3-10-fold greater potency than CsA and Vp with respect to the restoring effect on reduced accumulation of doxorubicin (ADM) and vincristine (VCR) in ADM-resistant K562 myelogenous leukemia cells (K562/ADM) in vitro and also on the sensitivity of K562/ADM to ADM and VCR in in vitro growth inhibition. The in vivo efficacy of a combination of modifiers (PSC833 and CsA: 50 mg/kg, Vp 100 mg/kg administered p.o. 4 h before the administration of anticancer drugs) with anticancer drugs (ADM 2.5 mg/kg i.p., Q4D days 1, 5 and 9, VCR 0.05 mg/kg i.p., QD days 1-5) was tested in ADM-resistant P388-bearing mice. PSC833 significantly enhanced the increase in life span by more than 80%, whereas CsA and Vp enhanced by less than 50%. This reversal potency, which exceeded that of CsA and Vp, was confirmed by therapeutic experiments using colon adenocarcinoma 26-bearing mice. These results demonstrated that PSC833 has signficant potency to reverse MDR in vitro and in vivo, suggesting that PSC833 is a good candidate for reversing multidrug resistance in clinical situations.     

Restoration of doxorubicin responsiveness in doxorubicin-resistant P388 murine leukaemia cells by dipyrone

The effect of dipyrone along with doxorubicin and mitoxantrone was studied alone and in combination on the 3H-thymidine (3H-TdR) incorporation in P388 leukaemia sensitive (P388/S) and resistant (P388/ADR) to doxorubicin. Dipyrone 10(-4) M demonstrated minimal inhibitory effect on DNA biosynthesis in both the sensitive and resistant cells. Doxorubicin and mitoxantrone at equimolar concentrations, indicated time and dose-dependent inhibition in 3H-TdR incorporation in the sensitive cells. The inhibition was more at the higher drug concentrations at 4 h drug exposure. Mitoxantrone showed cross-resistance in P388/ADR compared to P388/S. Both the drugs along with 10(-4) M dipyrone in the incubating medium revealed synergistic inhibitory activity in P388/S and P388/ADR. Observations indicate circumvention of doxorubicin and mitoxantrone resistance in P388/ADR by dipyrone.     

PSC833逆转人骨肉瘤细胞株多药耐药性的研究

目的 探讨PSC 833对骨肉瘤耐药细胞株U2OS/DOX的逆转作用及其机制。方法 用MTT法进行PSC 833逆转MDR活性测定;用流式细胞仪技术，观察PSC833对细胞内Rh123积聚和外排的影响；用免疫荧光技术定量检测逆转剂对P-gp表达水平的影响。结果 PSC833能显著增加DOX对U2OS/DOX的细胞毒性作用，逆转效果明显强于VPL和 CSA，且存在剂量依赖关系,对敏感株U2OS无明显作用；PSC 833能使耐药株细胞内Rh123外排减少、积聚增加，而对P-gp的表达水平没有明显影响。结论 PSC833能增强DOX对U2OS/DOX的细胞毒性作用，逆转MDR效果明显优于VPL和CSA，其机理在于抑制耐药株细胞膜上P-gp的功能，而对P-gp的表达水平没有影响。     

PSC833逆转人骨肉瘤细胞系多药耐药性的研究

目的 探讨PSC833对骨肉瘤耐药细胞系U2OS/DOX和SaOS/DOX的逆转作用及其机制。方法 用MTT法和细胞计数法测定PSC833逆转MDR的活性;用流式细胞仪观察PSC833对细胞内Rh123积聚和外排的影响;用免疫荧光技术定量检测逆转剂对P-gp表达水平的影响。结果 PSC833对DOX敏感系U2OS和SaOS无明显作用。PSC833能显著增加DOX对U2OS/DOX和SaOS/DOX的细胞毒性作用,逆转效果明显强于VPL和CSA,且存在剂量依赖关系。PSC833能减少耐药细胞系内Rh123的外排,增加Rh123的积聚,而对P-gp的表达水平没有明显影响。结论 PSC833能逆转U2OS/DOX和SaOS/DOX的MDR,其效果明显优于VPL和CSA,逆转机理在于抑制耐药系细胞膜上P-gp的功能,而对P-gp的表达水平没有影响。     

Identification of two distinct intracellular sites that contribute to the modulation of multidrug resistance in P388/ADR cells expressing P-glycoprotein

Although the ability of chemosensitizers to modulate P-glycoprotein (PGP)-based multidrug resistance (MDR) has been extensively studied, relatively little is known about the cellular pharmacology of the PGP inhibitors themselves in MDR cells. The studies described here have correlated the in vitro accumulation and retention properties of verapamil (VRP) in murine P388 (sensitive) and P388/ADR (MDR) cells with doxorubicin (DOX) uptake and cytotoxicity modulation characteristics in order to better understand VRP-tumor cell interactions that give rise to MDR modulation. VRP is rapidly taken up by DOX-sensitive and -resistant P388 cells where greater than 50% maximal VRP uptake occurs within 10 min of initial exposure at 37 degrees C. Whereas chemosensitization and DOX uptake in P388/ADR cells increase with increasing VRP concentration until a plateau is achieved at approximately 5 microM VRP, cellular modulator levels increase proportionally with increasing VPR concentrations beyond 20 microM. Subsequent to removal of noncell-associated modulator, VRP levels in both sensitive and resistant cells rapidly fall below 10% of those obtained at uptake equilibrium. However, a residual amount of VRP remains associated with the cells for extended time periods after the cells are washed. Pulse exposures of P388/ADR cells to high concentrations of VRP (50-100 microM) are capable of providing extended cell-associated VRP levels comparable to those obtained with continuous exposure at biologically active VRP concentrations (1-3 microM) and this leads to chemosensitization. These results are consistent with the existence of high- and low-affinity intracellular VRP pools in P388 MDR cells, both of which can contribute to the reversal of drug resistance. It is suggested that these properties should be taken into consideration during the design and evaluation of preclinical in vivo MDR models where pulsed exposure to high concentrations of resistance modulators often occurs. Special attention must be given to whether such high concentration pulses are desirable and/or achievable in relevant clinical settings.     

Biliary transport of irinotecan and metabolites in normal and P-glycoprotein-deficient mice

PURPOSE: The extensive and unpredictable biliary excretion of CPT-11 and its metabolites, SN-38 and SN-38 glucuronide (SN-38G) may contribute to the wide interpatient variability reported in the disposition and gastrointestinal toxicity of CPT-11. We studied the role of P-glycoprotein (P-gp) in in vivo biliary excretion of CPT-11, SN-38 and SN-38G in mice lacking mdr1-type P-gp [ mdr1a/1b(-/-)] in the presence of the multidrug resistance (MDR) reversal agent, PSC833. METHODS: Wild-type (Wt) and mdr1a/1b(-/-) mice ( n=3 or 4) were treated intragastrically with PSC833 (50 mg/kg) or vehicle 2 h prior to i.v. CPT-11 dosing (10 mg/kg), and bile samples were collected. RESULTS AND CONCLUSIONS: P-gp was found to play an important role in CPT-11 biliary excretion, as there was a significant (40%, P<0.05) decrease in its biliary recovery in 90 min in mdr1a/1b(-/-) mice (6.6+/-0.6% dose) compared with Wt mice (11+/-1.2%). This also implied a major role of other undetermined non-P-gp-mediated mechanism(s) for hepatic transport of CPT-11, which was inhibited by PSC833 (1.8+/-0.8% with PSC833, 6.6+/-0.6% without PSC833) in mdr1a/1b(-/-) mice. SN-38 and SN-38G biliary transport was unchanged in mice lacking P-gp after vehicle treatment, indicating a lack of P-gp mediation in their transport. PSC833 significantly reduced (56-89%) SN-38 and SN-38G biliary transport in Wt and mdr1a/1b(-/-) mice, suggesting that PSC833 may be a candidate to modulate biliary excretion of SN-38 with potential use in reducing CPT-11 toxicity.     

P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.     

Differential effect of the calmodulin inhibitor trifluoperazine on cellular accumulation, retention, and cytotoxicity of anthracyclines in doxorubicin (adriamycin)-resistant P388 mouse leukemia cells

Calmodulin inhibitors enhance cytotoxic effects of doxorubicin (DOX) in DOX-resistant (P388/DOX) P388 mouse leukemia cells by increasing cellular accumulation and retention of drug. In P388/DOX cells treated for 3 hr, cytotoxic effects (based on colony formation in soft agar) of daunorubicin (DAU) in the presence of trifluoperazine (TFP) were DAU concentration-dependent and enhanced 2- to 100-fold. Additionally, in the presence of TFP, on a molar basis, equitoxic doses of DAU were 4-fold lower than DOX for P388/DOX cells. However, in P388/DOX cells treated for 3 hr with other anthracyclines, except for a slight enhancement in the cytotoxic effects of aclacinomycin A (ACM) with TFP, colony formation in soft agar of cells treated with N-trifluoroacetyladriamycin-14-valerate (AD32) and N-trifluoroacetyladriamycin were similar in the absence and presence of TFP. In DOX-sensitive (P388/S) P388 mouse leukemia cells treated for 3 hr, some enhancement in the cytotoxic effects due to TFP were observed with DAU and DOX but not with ACM, AD32, or N-trifluoroacetyladriamycin. Although accumulation of ACM and AD32 in P388/S and P388/DOX cells was similar and unaffected by TFP, the retention of ACM but not AD32 was enhanced 1.5-fold only in TFP-treated P388/DOX cells. In contrast, DAU accumulation in P388/S cells was 4-fold higher than in similarly treated P388/DOX cells, and the 2- and 4-fold increase due to TFP in the accumulation and retention, respectively, of DAU in P388/DOX cells was not observed in P388/S cells. Results from this study indicate that in P388/DOX cells, the calmodulin inhibitor TFP is more effective with DAU than DOX, significantly less effective with ACM, and ineffective with AD32 and N-trifluoroacetyladriamycin.     

Evaluation of quinidine effect on the antitumor activity of adriamycin and mitoxantrone in adriamycin-sensitive and -resistant P388 leukemia cells

Utilizing the P388 murine leukemia cells sensitive (P388/S) and resistant (P388/ADR) to Adriamycin (ADR), we evaluated the effect of quinidine, an anti-arrhythmic agent, on the cytotoxic activity of ADR and Mitoxantrone (MITO), both in vitro as well as in vivo. Quinidine enhanced the cytotoxicity of both ADR and MITO in P388/S and P388/ADR cells, as assessed by the decrease in color intensity of formazan crystal in the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. A dose dependent inhibition of 3H-thymidine and 3H-uridine incorporation was observed when the P388/S and P388/ADR cells were exposed to quinidine alone. A non-toxic concentration of quinidine (5 microM) enhanced the DNA biosynthesis inhibition induced by ADR (55 to 65%) and MITO (37 to 44%) in P388/ADR cells, indicating reversal of resistance, while in P388/S cells only a minimal increase in DNA biosynthesis inhibition was observed. The combination of quinidine at doses of 50 to 100 mg/kg significantly potentiated the antitumor activity of ADR and MITO in P388/ADR bearing mice, whereas the potentiation of ADR and MITO antitumor response was lower in P388/S bearing mice. Quinidine increased the cellular levels of ADR by 53 to 126% in P388/ADR cells in vitro, but failed to indicate such elevated levels of cellular ADR in P388/S cells. This enhanced intracellular accumulation of ADR in P388/ADR cells, explains the therapeutic efficacy of ADR and MITO in P388/ADR, both in vitro as well as in vivo. Results suggest the efficacy of quinidine to ameliorate the antitumor effects of ADR and MITO in drug resistant tumor cells.     

In vivo circumvention of vincristine resistance in mice with P388 leukemia using a novel compound, AHC-52

A novel compound partially analogous to nifedipine, AHC-52, was found to sensitize multidrug-resistant tumor cells. AHC-52 at 0.5 microgram/ml completely reversed the in vitro resistance to vincristine (VCR) in VCR-resistant P388 cells (P388/VCR). Of various regimens examined for the in vivo treatment of P388/VCR-bearing mice, the combination of 0.05 mg/kg of VCR with 100 mg/kg twice a day of AHC-52 daily demonstrated the best result with a 206% increase in life prolongation. This result was comparable with that observed in parental P388-bearing mice treated with the optimal dose of VCR alone, indicating almost complete circumvention of resistance by combination VCR-AHC-52 therapy. In addition, the combination of both agents exhibited therapeutic effects in the treatment of P388-bearing mice with some long term survivors. This result was presumably due to the elimination of heterogeneity of VCR sensitivity in this cell population. These results suggest that combination chemotherapy using a sensitizing agent such as AHC-52 will be effective in not only circumvention of multidrug resistance but also retardation of its occurrence.     

Modulation of resistance to idarubicin by the cyclosporin PSC 833 (valspodar) in multidrug-resistant cells

Idarubicin (IDA) is an anthracycline anticancer drug utilized in the treatment of acute leukemias. There are conflicting data published with regard to the cross-resistance of IDA in multidrug-resistant (MDR) cells expressing P-glycoprotein (P-gp). We evaluated the cytotoxicity and cellular accumulation of IDA in a panel of anthracycline-selected MDR cell lines. Leukemia K562/R7 cells and sarcoma MES-SA/Dx5 cells expressing high levels of the MDR1 (ABCB1) gene were resistant to IDA (42-fold and 150-fold, respectively). In both of these cell lines, resistance to IDA was equivalent to that for doxorubicin, the drug used to select for the MDR variants. The P-gp inhibitor PSC 833 (valspodar) at 2 microM completely restored sensitivity to IDA. IDA accumulation was decreased 12-fold in MES-SA/Dx5 cells vs parental cell line, and drug uptake was restored to control levels by PSC 833. Reduced intracellular IDA was correlated with P-gp content by flow cytometry. Experiments in NIH3T3 murine cells transfected with the human MDR1 gene substantiated the findings of cross-resistance to IDA and reversal of resistance by PSC 833. Our data indicate that IDA is a high-affinity substrate for P-gp.     

Changes in doxorubicin distribution and toxicity in mice pretreated with the cyclosporin analogue SDZ PSC 833

SDZ PSC 833 (PSC 833) is a cyclosporin A analogue that is under clinical investigation in combination with doxorubicin (Dx) or other anticancer agents as a type-1 multidrug resistance (MDR-1)-reversing agent. The present study was focused on the effects of PSC 833 on the distribution and toxicity of Dx in non-tumor-bearing CDF1 male mice. Mice were given PSC 833 i.p. at 30 min before i.v. Dx treatment. Dx levels were determined by a high-performance liquid chromatography (HPLC) assay at different times during a 72-h period following Dx treatment in the serum, heart, intestine, liver, kidney, and adrenals of mice. In all tissues, Dx area under the concentrationtime curve (AUC) values were much greater in mice receiving 10 mg/kg Dx in combination with 12.5 or 25 mg/kg PSC 833 than in mice receiving Dx alone. The highest increase in Dx concentrations was found in the intestine, liver, kidney, and adrenals. Lower, albeit significant, differences were found in the heart. PSC 833 did not appear to influence either urinary or fecal Dx elimination or Dx metabolism to a great extent. Doses of PSC 833 devoid of any toxicity potentiated the acute and delayed toxicity of Dx dramatically. The mechanism responsible for this enhanced toxicity has not yet been elucidated but is likely to be related to an increased tissue retention of Dx due to inhibition of the P-glycoprotein (Pgp) pump by PSC 833, as has recently been proposed for cyclosporin A.Abbreviations MDR Multidrug resistance - mdr-1 gene multidrug resistance-1 gene - Pgp P-glycoprotein - PSC 833 SDZ PSC 833 - Dx doxorubicin - HPLC high-performance liquid chromatography - AUC area under the concentration-time curve