SDZ PSC 833, a non-immunosuppressive cyclosporine: its potency in overcoming P-glycoprotein-mediated multidrug resistance of murine leukemia.

Cyclosporin A (CsA, Sandimmune) is known to reverse P-glycoprotein (P-gp170)-mediated multidrug resistance as efficiently as other prototype compounds of resistance modifiers. The immunosuppressive activity and nephrotoxicity of CsA, however, may limit its clinical use. PSC-833, a new cyclosporine, exerts a similar resistance-modifying activity but lacks toxicity or immunosuppressive activity. We have tested its potency in vitro and in vivo on the L1210 leukemia cell line transfected with a full-length cDNA copy of the human mdr I gene, which showed a stable 30-fold resistance towards adriamycin as compared to the parental cell line. In vitro growth of the transfected cell was unchanged. In vivo growth was less aggressive; the survival time of inoculated mice was prolonged. In vitro, PSC-833 was at least as potent as CsA or verapamil in reversing multidrug resistance. In vivo, the drug-resistant L1210 leukemia was completely unresponsive to i.v. monotherapy with adriamycin at its maximum tolerated dose (MTD). PSC-833 enhanced the activity and toxicity of adriamycin. The MTD of adriamycin was about 3 times lower than when given alone. On this basis, the MTD of i.v. adriamycin in combination with oral PSC-833 successfully overcame refractoriness to treatment. Survival times of the mice were considerably prolonged and even some cures of leukemic mice occurred.     

Bromocriptine reverses P-glycoprotein-mediated multidrug resistance in tumor cells.

One of the most important causes of anticancer treatment failure is the development of multidrug resistance (MDR). The main characteristics of tumor cells displaying the MDR phenomena are cross-resistance to structurally unrelated cytotoxic drugs having different mechanisms of action and the overexpression of the MDR1 gene, which encodes a transmembrane glycoprotein named P-glycoprotein (P-gp). This study evaluated whether bromocriptine, a D2 dopaminergic receptor agonist, influenced anticancer drug cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. The K(i) values for P-gp of cyclosporine and verapamil were 1.09 and 540 microM, respectively, and that of bromocriptine was 6.52 microM in a calcein-AM efflux assay using porcine kidney epithelial LLC-PK1 and L-MDR1 cells, overexpressing human P-gp. Bromocriptine at 10 microM reduced the IC50 of doxorubicin (DXR) in K562-DXR from 9000 to 270 ng/ml and that of vincristine (VCR) in K562-VCR from 700 to 0.30 ng/ml, whereas the IC50 values of DXR and VCR in the K562 subline were only marginally affected by these drugs. Bromocriptine restored the anticancer effect of DXR, VCR, vinblastine, vinorelbine and etoposide on MDR-tumor cells overexpressing P-gp. These observations suggest that bromocriptine has the potential to reverse tumor MDR involving the efflux protein P-gp in the clinical situation.     

SDZ 280-446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the P-glycoprotein-mediated tumour cell multidrug resistance.

SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species (mouse, human, Chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, ovary fibroblastoid carcinoma), and using four different drug classes (colchicine, vincristine, daunomycin/doxorubicin and etoposide). By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers (including verapamil, quinidine and amiodarone which have already entered clinical trials in MDR reversal). Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules.     

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.     

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     

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.     

Enhanced oral bioavailability of paclitaxel in mice treated with the P-glycoprotein blocker SDZ PSC 833.

Inhibition of intestinal P-glycoprotein might enhance the absorption of orally administered P-glycoprotein substrate drugs. We show here a 10-fold increased oral bioavailability of paclitaxel in mice treated with the P-glycoprotein blocker SDZ PSC 833. These results encourage further research on the development of a clinically useful oral formulation of paclitaxel.     

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.     

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.     

In vivo circumvention of human colon carcinoma resistance to bleomycin.

Metabolic inactivation of bleomycin (BLM) by cysteine proteinase-like enzymes is thought to be a major mechanism of BLM tumor resistance. We now report that the human colon carcinoma COLO-205 is highly resistant to BLM and that E-64, a cysteine proteinase inhibitor, sensitizes COLO-205 to BLM. Treatment of COLO-205-bearing nude mice with either E-64 (40 mg/kg) or BLM (10 mg/kg) alone did not inhibit COLO-205 growth. However, pretreatment with E-64 prior to BLM prevented these xenografts from growing. Analysis by high performance liquid chromatography of in vivo BLM metabolism following [3H]BLM A2 treatment of COLO-205-bearing nude mice showed a different metabolic profile among the various organs and the tumor. Whereas [3H]BLM A2 was the only major radioactive peak detected in sera and tumors, several metabolites, including deamido-BLM A2, were found in kidney, liver, and lung as early as 15 min. Pretreatment of mice with E-64 inhibited tumor, kidney, and lung BLM A2 metabolism. Furthermore, pretreatment with E-64 increased BLM A2 accumulation in tumors (6.1-fold), kidney (4.0-fold), lung (2.8-fold), liver (1.8-fold), and serum (1.7-fold). E-64 pretreatment did not enhance the major toxicity of BLM, pulmonary fibrosis, as determined by both lung hydroxyproline levels and histopathology. Thus, the cysteine proteinase inhibitor E-64 affects the metabolic fate and the levels of accumulation of BLM in vivo. These results demonstrate that resistance of human COLO-205 tumors to BLM can be circumvented by E-64 without enhancement of the major side effect of BLM, suggesting a possible clinical use of this combination therapy.     

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.     

Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids

Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas. We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm). Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells. The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr. Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil. Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants. Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS. Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA). Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89. Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity.     

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.     

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     

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     

Phase I study of paclitaxel in combination with a multidrug resistance modulator, PSC 833 (Valspodar), in refractory malignancies.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics of paclitaxel when given with PSC 833 (valspodar) to patients with refractory solid tumors. PATIENTS AND METHODS: Patients were initially treated with paclitaxel 175 mg/m(2) continuous intravenous infusion (CIVI) over 3 hours. Subsequently, 29 hours of treatment with CIVI PSC 833 was started 2 hours before paclitaxel treatment was initiated. In this combination, the starting dose of paclitaxel was 52.5 mg/m(2). Paclitaxel doses were escalated by 17.5 mg/m(2) increments for four subsequent cohorts. Each cohort consisted of three patients with the exception of the last cohort, which consisted of six patients. Data for the pharmacokinetics of paclitaxel with and without concurrent PSC 833 administration were obtained. RESULTS: All 18 patients completed at least one course of concurrent treatment (median, two courses; range, one to six) and were evaluable for toxicity. The MTD for paclitaxel with PSC 833 was 122.5 mg/m(2). Neutropenia was the DLT. All patients had PSC 833 blood concentrations greater than 1, 000 ng/mL before, during, and 24 hours after the paclitaxel infusion. PSC 833 produced small increases in the paclitaxel peak plasma concentrations and areas under the concentration-time curve. However, PSC 833 greatly prolonged the terminal phase of paclitaxel, resulting in plasma paclitaxel concentrations of more than 0.05 micromol/L for much longer than expected. As a result, myelosuppression was comparable to that produced by full-dose paclitaxel given without PSC 833. Of the 16 patients who were assessable for response, one patient experienced a partial response and an additional nine patients experienced disease stabilization after paclitaxel treatment alone. CONCLUSION: Treatment with paclitaxel 122.5 mg/m(2) as a 3-hour CIVI concurrent with a 29-hour CIVI of PSC 833 results in acceptable toxicity. The addition of PSC 833 alters the pharmacokinetics of paclitaxel, which explains the enhanced neutropenia experienced by patients treated with this drug combination.     

Modulator activity of PSC 833 and cyclosporin-A in vincristine and doxorubicin-selected multidrug resistant murine leukemic cells

Multidrug resistance (MDR) lines from a murine T-cell leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Daunorubicin (DNR) efflux was evidenced after 25 additional passages with constant 160 ng ml(-1) of either VCR or DOX, an effect that was inhibited by verapamil, cyclosporin-A (CsA) and PSC 833. The expression of Pgp was not evidenced in the resistant cell lines using anti-human Pgp antibodies. Cell proliferation assay showed that cell lines resistant to VCR (LBR-V160) or DOX (LBR-D160) required higher doses of either drug to produce GI50 compared with control cell line obtained after culture in the absence of VCR or DOX. When resistant cell lines were maintained during 60 days in the absence of either VCR or DOX, MDR phenotype reversal was obtained in LBR-D160 while LBR-V160 remained resistant to the drug, as shown by cell proliferation assays and by drug efflux pump functionality. When VCR or DOX were used together with either CsA or PSC 833, the latter was more effective to produce reversal of resistance than the former, whereas CsA presented greater cytotoxic effect than PSC 833 for sensitive and resistant cells. Cross-resistance was found between VCR, DOX and other antineoplasic agents on murine leukemic cell line. VCR was more effective to induce MDR since the resistant cell lines were more stable to the MDR phenotype.     

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576

The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.     

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.