Decreased biotolerability for ivermectin and cyclosporin a in mice exposed to potent P-glycoprotein inhibitors

SDZ PSC 833 or SDZ 280-446 are strong blockers of the function of class 1 mdr gene-encoded P-glycoprotein molecules, which were developed for the reversal of multi-drug-resistance of tumor cells. When treated with such drugs, normal mice may display hypersensitivity to cyclosporin A and ivermectin. The recorded signs of acute toxicity are compatible with alterations of the murine central nervous system functions and with earlier data suggesting that P-glycoprotein expressed at the murine blood-brain barrier might be involved in the exclusion of cyclosporin A or ivermectin from brain tissue.     

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.     

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.     

Distribution and activity of doxorubicin combined with SDZ PSC 833 in mice with P388 and P388/DOX leukaemia.

SDZ PSC 833 (PSC 833) is a non-immunosuppressive analogue of cyclosporin A and is a potent modifier of P-glycoprotein (P-gp)-mediated multidrug resistance. The present study was undertaken to evaluate whether doxorubicin (DOX) pharmacokinetic and anti-tumour activity on P388- and P388/DOX-resistant leukaemia was modified by PSC 833 pretreatment. P388- or P388/DOX-bearing mice were given PSC 833 intraperitoneally 30 min before an intravenous injection of DOX. The levels of DOX were determined by a high-performance liquid chromatography method in leukaemic cells and in normal tissues (heart, lung, liver, small intestine, kidney and spleen). In all tissues, DOX concentrations were significantly increased in mice pretreated with PSC 833. The difference was greatest in P-gp-overexpressing P388/DOX cells, the DOX area under the curve being approximately seven times greater after PSC 833 and DOX than after DOX alone. In P388 cells the difference was approximately 2.5 times, as in the majority of normal tissues. As expected DOX levels in P388 cells were higher than in P388/DOX cells in mice treated with DOX alone, whereas after PSC 833 and DOX the levels of DOX were similar in the two leukaemic lines. In spite of this PSC 833 was unable to reverse the resistance to DOX of P388/DOX leukaemia in vivo, suggesting that mechanisms other than P-gp expression are responsible for resistance.     

Effect of the nonimmunosuppressive cyclosporin analog SDZ PSC-833 on colchicine and doxorubicin biliary secretion by the rat in vivo

Colchicine and doxorubicin are secreted into bile as a major pathway of their elimination. Colchicine and doxorubicin are also substrates for P-glycoprotein, and P-glycoprotein has been demonstrated to be present at the liver canalicular membrane. Cyclosporin (CsA) inhibits colchicine biliary secretion in vivo. In the present study, the effects of SDZ PSC-833, a nonimmunosuppressive cyclosporin D analog, on the biliary secretion of colchicine and doxorubicin were investigated. SDZ PSC-833 given at a bolus dose of 2 mg/kg promptly decreased colchicine biliary clearance from 9.05±0.2 to 2.41±0.43 ml min^–1 kg^–1 (P<0.001) and the colchicine bile/plasma ratio from 146±8 to 35±5 (P<0.001). SDZ PSC-833 also inhibited doxorubicin biliary clearance (basal: 10.5±3 vs post-SDZ PSC-833: 2.48±0.94 ml min^–1 kg^–1;P=0.06) and the doxorubicin bile/plasma ratio (basal: 228±64 vs post-SDZ PSC-833: 48±22;P<0.01). Colchicine renal secretion was completely inhibited by SDZ PSC-833. Thus, SDZ PSC-833 inhibits the constitutive transport of the multidrug-resistance substrates colchicine and doxorubicin and is more potent than cyclosporin in this regard. The possibility of increased toxicity to normal tissues because of impaired elimination of cytotoxic agents will need to be considered if SDZ PSC-833 is used to chemosensitize cancer cells.This work was supported in part by the Research Service, Department of Veterans Affairs     

Dose-dependent brain penetration of SDZ PSC 833, a novel multidrug resistance-reversing cyclosporin,in rats

 This study quantitatively assessed the brain penetration of a potent P-glycoprotein inhibitor, SDZ PSC 833, and its effect on the blood-brain barrier (BBB) permeability (PS) of an anticancer agent, vincristine. At lower doses of SDZ PSC 833 the brain penetration, defined as the brain-to-blood partition coefficient (Kp), was very low in spite of the high lipophilicity of this compound. At higher doses, however, the brain penetration of SDZ PSC 833 was markedly increased. Since the blood pharmacokinetics of SDZ PSC 833 proved to be linear in the dose range studied, these results demonstrated a dose-dependent brain passage of SDZ PSC 833. The brain passage of cyclosporin A was also found to be dose-dependent. However, the potency of SDZ PSC 833 in inhibiting the efflux mechanism at the BBB was higher than that of cyclosporin A since 10 times higher doses of cyclosporin A were required to obtain the same Kp values recorded for SDZ PSC 833. Moreover, the coadministration of SDZ PSC 833 increased the brain penetration of cyclosporin A, whereas the latter did not modify that of SDZ PSC 833. The increase in SDZ PSC 833 and vincristine PS values observed at high blood levels of SDZ PSC 833 are consistent with the hypothesis of a saturation of the P-glycoprotein pump present at the BBB. The involvement of P-glycoprotein in the brain passage of SDZ PSC 833 could be of great significance for clinical application of the drug in the treatment of brain cancers when it is given in combination with anticancer agents. Received: 8 October 1995/Accepted: 25 January 1996     

Inhibitory Effects of a Cyclosporin Derivative, SDZ PSC 833, on Transport of Doxorubicin and Vinblastine via Human P-Glycoprotein

The inhibitory effects of SDZ PSC 833 (PSC833), a non-immunosuppressive cyclosporin derivative, on the P-glycoprotein (P-gp)-mediated transport of doxorubicin and vinblastine were compared with those of cyclosporin A (Cs-A). The transcellular transport of the anticancer drugs and PSC833 across a monolayer of LLC-GA5-COL150 cells, which overexpress human P-gp, was measured. Both PSC833 and Cs-A inhibited P-gp-mediated transport of doxorubicin and vinblastine in a concentration-dependent manner and increased the intracellular accumulation of doxorubicin and vinblastine in LLC-GA5-COL150 cells. The values of the 50%-inhibitory concentration (IC₅₀) of PSC833 and Cs-A for doxorubicin transport were 0.29 and 3.66 μ M , respectively, and those for vinblastine transport were 1.06 and 5.10 μ M , respectively. The IC₅₀ of PSC833 for doxorubicin transport was about 4-fold less than that for vinblastine transport, suggesting that the combination of PSC833 and doxorubicin might be effective. PSC833 itself was not transported by P-gp and had higher lipophilicity than Cs-A. These results indicated that the inhibitory effect of PSC833 on P-gp-mediated transport was 5- to 10-fold more potent than that of Cs-A, and this higher inhibitory effect of PSC833 may be related to the absence of PSC833 transport by P-gp and to the higher lipophilicity of PSC833.     

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.     

Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents.

The multidrug transporter P-glycoprotein (Pgp), which is frequently overexpressed in multidrug resistant leukemia, has many proposed physiological functions including involvement in transmembraneous transport of certain growth-regulating cytokines. Therefore, we studied cell growth of three pairs of drug resistant and sensitive leukemia cell lines (KG1a, K562 and HL60) exposed to three different inhibitors of Pgp. The resistant KG1a and K562 sublines, which expressed high levels of Pgp, responded to low doses of the cyclosporin SDZ PSC 833, the cyclopeptolide SDZ 280-446, and the cyclopropyldibenzosuberane LY335979 with a dose-dependent growth inhibition. In the resistant variants of KG1a and K562 cells the mean half-maximal growth inhibitory doses (GI50) of SDZ PSC 833 were 312 (SE 41) and 414 (SE 50) nM, those of SDZ 280-446 were 685 (SE 51) and 578 (SE 54) nM, and those of LY335979 were 66 (SE 1) and 48 (SE 8) nM, respectively. Exposure to 1 microM SDZ PSC 833 resulted in tetraploidization, cytokinesis failure and apoptosis of the KG1a and K562 MDR variants. Conversely, parental cells with no or low levels of Pgp and the non-Pgp resistant variant of HL60 cells were not receptive to these cytotoxic effects. We conclude that inhibition of Pgp may exercise selective cytotoxicity in Pgp-rich leukemia cells indicating a possible therapeutic target in multiresistant leukemia.     

Expression of P-glycoprotein on normal lymphocytes: enhancement of the doxorubicin-sensitivity of concanavalin A-responding mouse spleen cells by P-glycoprotein blockers.

The in vitro proliferative response of mouse spleen cells (SC) to the T-cell mitogen, concanavalin A (ConA), displays a doxorubicin (DOX)-resistant component. This T-cell proliferative response displays a much higher DOX sensitivity in the presence of novel potent inhibitors of P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), the cyclosporin (Cs) derivative, SDZ PSC 833, and the semi-synthetic cyclopeptolide, SDZ 280-446. Another resistance modulator, verapamil, might share this property, but its detection was impaired by the intrinsic toxicity of this calcium channel blocker for T-cell proliferation. A CD8+ cell-depleted SC suspension displayed a higher sensitivity to DOX alone, as well as a different sensitivity profile to SDZ 280-446. The CD8+ cells that are sensitized to DOX by the resistance modulating agents (RMA) might correspond to a formerly described T-cell subpopulation with the MDR phenotype, which seems to be essentially constituted of CD8+ (cytotoxic) T cells. Our results may open the way to a novel form of immunomodulation combining classical antineoplastic agents with Pgp-blocking Cs analogs (even non-immunosuppressive ones), which may be particularly useful when treating acute graft rejection.     

Pharmacologic interactions between the resistance-modifying cyclosporine SDZ PSC 833 and etoposide (VP 16-213) enhance in vivo cytostatic activity and toxicity.

Cyclosporin A reverses multidrug resistance (MDR) and increases the in vivo cytostatic activity and toxicity of the anticancer agent etoposide (VP 16-213). SDZ PSC 833 (PSC 833), a non-immunosuppressive, non-toxic cyclosporin and very active modifier of P-gp 170-mediated MDR, elicits similar effects when administered with adriamycin. The underlying mechanisms, however, are not yet understood. The present pharmacological interaction study with PSC 833 and VP 16-213 was carried out to reveal the nature of this enhancement of cytostatic activity and toxicity. Rats pre-treated with either PSC-833 or solvent received a single dose of VP 16-213. Plasma levels of VP 16-213 were measured by high-performance liquid chromatography (HPLC). The resulting increase in cytostatic activity and toxicity of VP 16-213 mediated by PSC 833 was paralleled by marked changes in the pharmacokinetic parameters of VP 16-213 in vivo. Bioavailability and blood levels of VP 16-213 were significantly increased 30 min after administration if PSC 833 had been given before. The disappearance rate of VP 16-213 from the intravascular compartment was considerably slowed down by PSC 833. In drug-sensitive xenografts of human colon carcinoma, the PSC-833-induced pharmacologic changes in vivo could be counteracted by dose reduction of VP 16-213 while a full therapeutic potential was maintained. Doses of VP 16-213, 1.5 to 2 times smaller, combined with PSC 833, were as effective in terms of tumor-growth inhibition as the maximum tolerated dose of VP 16-213 alone. Thus, pharmacologic interactions between PSC 833 or other resistance modifiers and VP 16-213 and other cytostatic agents require careful attention if they are to be used in humans to overcome MDR.     

Potentiation of the reversal activity of SDZ PSC833 on multi-drug resistance by an anti-p-glycoprotein monoclonal antibody MRK-16

SDZ PSC833 (PSC833), an analogue of cyclosporines, is one of the most potent modulators of multi-drug resistance (MDR). We previously reported that MRK-16, an anti-P-glycoprotein MAb, enhanced MDR reversal activity of cyclosporin A (CsA) through inhibition of P-glycoprotein-mediated CsA transport. We have examined here whether MRK-16 can enhance MDR reversal activity of PSC833. We found that MRK-16 potentiated the MDR reversal activity of PSC833, and of CsA, in MDR sublines of human myelocytic leukemia K562 and human ovarian cancer A2780 cells. Like MRK-16 combined with CsA, MRK-16 enhanced the effect of a sub-optimum dose of PSC833 on vincristine accumulation in MDR cells. However, MRK-16 could not increase cellular accumulation of PSC833 in MDR tumor cells, yet it could increase cellular accumulation of CsA. P-glycoprotein could not transport PSC833 but could transport CsA. Our results indicate that MRK-16 potentiates the MDR reversal activity of both PSC833 and CsA, yet also suggest that the molecular mechanism of the potentiation differs between the two substances. © 1996 Wiley-Liss, Inc.     

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.     

Comparison of cyclosporin A and SDZ PSC833 as multidrug-resistance modulators in a daunorubicin-resistant Ehrlich ascites tumor

Summary Recent studies by Boesch et al. have demonstrated that a nonimmunosuppressive cyclosporin analog, SDZ PSC 833 (an analog of cyclosporin D), is an active multidrug-resistance modifier that is at least 10 times more potent than cyclosporin A. In vitro accumulation and cytotoxicity experiments using daunorubicin (DNR) and vincristine (VCR) under the influence of SDZ PSC 833 and cyclosporin A were performed in wild-type (EHR2) and the corresponding highly DNR-resistant (about 80-fold) Ehrlich ascites tumor cells (EHR2/DNR+). In accumulation experiments, both SDZ PSC 833 and cyclosporin A were found to reverse the multidrug-resistant (MDR) phenotype, but to the same degree at equimolar concentrations. Thus, in EHR2/DNR+ cells, both cyclosporins at 5 g/ml enhanced DNR and VCR accumulation to sensitive levels, but only a negligible effect on DNR accumulation in the drug-sensitive cells was seen. In the clonogenic assay, the cytotoxicity of the two modulators was equal. The lethal dose for 50% of the cell population (LD₅₀) was approx. 7 g/ml for both compounds, and no toxicity was observed at concentrations below 2 g/ml. At nontoxic doses, both cyclosporins effectively increased the cytotoxicity of DNR and VCR in a concentration-dependent manner. The dose-response curves were nearly identical and did not demonstrate differences in modulator potency. These data permit the conclusion that cyclosporin A and SDZ PSC 833 do raise the intracellular accumulation of DNR and VCR to the same levels and that SDZPSC 833 does not potentiate cytotoxicity better than cyclosporin A in EHR2/DNR+ cells. However, since the new compound is nonimmunosuppressive and causes less organ toxicity, clinical studies of its MDR modulating effect seem highly relevant.     

Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil,cyclosporine A and SDZ PSC 833 in dependence on extracellular pH

The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transepithelial flux of tritiated vinblastine was investigated using tight and highly resistant (R>1,400 cm²) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts. Apical addition of these chemosensitisers inhibited drug flux (137 pmol h^–1 cm^–2; range, 133–142 pmol h^–1 cm^–2) in the basal to apical secretory direction at clinically relevant concentrations, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM). Acidification of the modulator-containing apical compartment to an extracellular pH (pHo) of 6.8 had no influence on MDR reversal by CsA at 1 g/ml (0.9 M; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 M; flux inhibition, 60%), in contrast to R,S- and R-VPL, which showed decreased inhibition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5). P-gp-mediated rhdamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison with pHo 7.5 in standard medium, but at low pHo the inhibitory activity of r-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished significantly, again without a reduction in the effect of PSC 833 (rhodamine 123 flux inhibition, 75%). In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epithelia and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for single-cell suspensions, resulting in increased (2.2- to 3.7-fold) intracellular drug accumulation. Functional apical P-gp expression, the absence of paracellular leakage and modulator-sensitive rhodamine 123 efflux in single HCT-8 cells indicate a P-gp-mediated transcellular efflux in HCT-8 monolayers. In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracellular conditions, which reduce the VPL-induced drug retention significantly. As far as MDR contributes to the overall cellular drug resistance of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.Abbreviations CsA cyclosporine A - HEPES [N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid)] - MDR multidrug resistance; PBS, phosphate-buffered saline - P-gp P-glycoprotein - pHo extracellular pH - PSC 833 SDZ PSC 833 - R resistance - TEM transmission electron microscopy - VPL verapamil     

Effect of PSC 833 on the cytotoxicity and pharmacodynamics of mitoxantrone in multidrug-resistant K562 cells

We examined the effect of PSC 833, a nonimmunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of an anthraquinone antileukemia drug mitoxantrone (MIT). This was done in P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1-9 cells and compared with the effect of cyclosporin A (CsA). We also compared MIT with the effect of PSC 833 on the cytotoxicity of daunorubicin (DNR) and doxorubicin (DOX). While PSC 833 and CsA had no effect on the cytotoxicity, accumulation and retention of MIT in the parent K562 cells, PSC 833 and CsA restored accumulation and retention of MIT in K562/D1-9 cells dose-dependently. Consequently, there was increased sensitivity of K562/D1-9 cells to MIT. The reversing activity of PSC 833 on the cytotoxicity of MIT was stronger than that of CsA, and was almost the same as the reversing activity of PSC 833 on the cytotoxicity of DNR and DOX. The resistance index of MIT decreased from 43.9-fold to 2.8-fold by 0.4 microM PSC 833, which is a clinically achievable plasma concentration. These results suggest that the combination of PSC 833 with MIT could be a promising treatment in reversing PGP-mediated MDR in leukemia patients.     

SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and [3H]palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis. In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, [3H]ceramide was >3-fold that of control cells. A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells. Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response. Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis. The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells. The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblastine. Whereas each agent at a concentration of 1.0 microM reduced cell survival by approximately 20%, when PSC 833 and vinblastine were coadministered, cell viability fell to zero. In parallel experiments measuring ceramide metabolism, it was shown that the PSC 833/vinblastine combination synergistically increased cellular ceramide levels. Vinblastine toxicity, also intensified by PSC 833 in wild-type KB-3-1 cells, was as well accompanied by enhanced ceramide formation. These data demonstrate that PSC 833 has mechanisms of action in addition to P-glycoprotein chemotherapy efflux pumping.     

Reversal of typical multidrug resistance by cyclosporin and its non-immunosuppressive analogue SDZ PSC 833 in Chinese hamster ovary cells expressing themdr1 phenotype

Summary The new non-immunosuppressive cyclosporin derivative SDZ PSC 833 (PSC) is a potent agent used to overcome typical multidrug resistance (MDR) associated with overexpression of themdr1 gene encoding for a P-170 glycoprotein. In the present study, the efficacy of PSC as compared with cyclosporin was determined in Chinese hamster ovary cell lines exhibiting different levels of resistance to colchicine (0, 0.1, 0.2 and 10 g/ml, respectively). Low concentrations of PSC (8.2nm) increased the cytotoxicity of colchicine in cell lines expressing low levels of drug resistance. The concentration resulting in 50% cell survival (LC₅₀ value) found for colchicine alone or in combination with PSC in the CHO-A3 cell line that was resistant to 100 ng colchicine/ml decreased from >500 to 200 ng/ml at 8.2nm PSC and to <100 ng/ml at 82 and 820nm PSC. In the CHO-A3 cell line that was resistant to 200 ng colchicine/ml, the LC₅₀ values decreased from >500 ng/ml for colchicine alone to 500 ng/ml for colchicine used in combination with 8.2nm PSC and to <100 ng/ml for colchicine combined with 82 or 820nm PSC. At a concentration of 82nm PSC, the maximal effect in MDR reversal was observed in the cell lines exhibiting moderate resistance. In the highly resistant cell line, PSC (820nm) also reversed colchicine resistance. In drug-accumulation experiments, we obtained a 4-fold increase in intracellular doxorubicin accumulation using 820nm PSC. A comparison of PSC with cyclosporin revealed that a cyclosporin concentration 20-fold that of PSC was required to obtain the same sensitising effect. On the basis of these data, it may be concluded that PSC is a most promising chemosensitiser.     

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.     

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

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 anthracyclines 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.