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Purpose: A lipid-based prodrug of mitomycin C [MMC; 2,3-(distearoyloxy)propane-1-dithio-4'-benzyloxycarbonyl-MMC] was designed for liposome formulation. The purpose of this study was to examine the in vitro cytotoxicity, pharmacokinetics, in vivo toxicity, and in vivo antitumor activity of this new lipid-based prodrug formulated in polyethylene glycol-coated (pegylated) liposomes., Experimental Design: MMC was released from the MMC lipid-based prodrug (MLP) by thiolytic-induced cleavage with a variety of thiol-containing reducing agents. MLP was incorporated with nearly 100% efficiency in cholesterol-free pegylated liposomes with hydrogenated phosphatidylcholine as the main component and a mean vesicle size of approximately 90 nm. This formulation was used for in vitro and in vivo tests in rodents., Results: In vitro, the cytotoxic activity of pegylated liposomal MLP (PL-MLP) was drastically reduced compared with free MMC. However, in the presence of reducing agents, such as cysteine or N-acetyl-cysteine, its activity increased to nearly comparable levels to those of free MMC. Intravenous administration of PL-MLP in rats resulted in a slow clearance indicating stable prodrug retention in liposomes and long circulation time kinetics, with a pharmacokinetic profile substantially different from that of free MMC. In vivo, PL-MLP was approximately 3-fold less toxic than free MMC. The therapeutic index and absolute antitumor efficacy of PL-MLP were superior to that of free MMC in the three tumor models tested. In addition, PL-MLP was significantly more active than a formulation of doxorubicin in pegylated liposomes (DOXIL) in the M109R tumor model, a mouse tumor cell line with a multidrug-resistant phenotype., Conclusions: Delivery of MLP in pegylated liposomes is a potential approach for effective treatment of multidrug-resistant tumors while significantly buffering the toxicity of MMC.

The Kyowa Hakko Kogyo and Bristol-Myers Squibb companies reported that select mitomycin C(7) aminoethylene disulfides displayed improved pharmacological profiles compared with mitomycin C (1). Mechanisms have been advanced for these mitomycins that differ from 1. Central to many of these hypotheses is the intermediate generation of 7-N-(2-mercaptoethyl)mitomycin C (5). Thiol 5 has been neither isolated nor characterized. Two efficient methods were developed for mitomycin (porfiromycin) C(7)-substituted thiols. In the first method, the thiol was produced by a thiol-mediated disulfide exchange process using an activated mixed mitomycin disulfide. In the second route, the thiol was generated by base-mediated cleavage of a porfiromycin C(7)-substituted thiol ester. We selected four thiols, 7-N-(2-mercaptoethyl)mitomycin C (5), 7-N-(2-mercaptoethyl)porfiromycin (12), 7-N-(2-mercapto-2-methylpropyl)mitomycin C (13), and 7-N-(3-mercaptopropyl)porfiromycin (14), for study. Thiols 5 and 12-14 differed in the composition of the alkyl linker that bridged the thiol with the mitomycin (porfiromycin) C(7) amino substituent. Thiol generation was documented by HPLC and spectroscopic studies and by thiol-trapping experiments. The linker affected the structure of the thiol species and the stability of the thiol. We observed that thiols 5 and 12 existed largely as their cyclic isomers. Evidence is presented that cyclization predominantly occurred at the mitomycin C(7) position. Correspondingly, alkyl linker substitution (13) or extension of the linker to three carbons (14) led to enhanced thiol stability and the predominant formation of the free thiol species. The dominant reaction of thiols 5 and 12-14 or their isomers was dimerization, and we found no evidence that thiol formation led to mitosene production and aziridine ring-opening. These findings indicated that thiol generation was not sufficient for mitomycin ring activation. The potential pharmacological advantages of mitomycin C(7) aminoethylene disulfides compared with 1 is discussed in light of the observed thiol cyclization pathway.

A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C18 columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-microm particle size, 150x6 mm I.D.), using a methanol-water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C18 extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients.

BMS-181174, a new mitomycin C (MMC) analog, showed more activity than the parent compound in tumor xenografts. In a phase I study with a 5-30 min slow bolus administration, hematologic and vascular toxicity were observed as major side effects. A prolonged infusion was suggested to circumvent the vascular toxicity. In this phase I study BMS-181174 was administered as a 6 h infusion every 4 weeks; the doses studied were 3.2, 6.4, 11.5, 19.0, 32.0, 50.0, 75.0 and 100 mg/m2. Twenty-eight patients were enrolled in the study, the majority with colorectal cancer. Hematologic side effects consisted of thrombocytopenia, and mild leuko- and granulocytopenia. The most distressing non-hematologic side effect was vascular toxicity consisting of phlebosclerosis and phlebitis, becoming dose limiting at 100 mg/m2. One patient developed a hemolytic uremic syndrome at a cumulative dose of 350 mg/m2. Pharmacokinetic data are available for 24 patients. The AUC ranged from 3.35 to 41.49 (microg x h/ml) and was highly correlated with the dose administered (r = 0.83). The kinetics appeared to be linear. One patient with metastatic colon cancer had a partial response of liver metastases. BMS-181174 is a MMC analog with a toxicity profile comparable to that of the parent compound. As doses above 50 mg/m2 are complicated by vascular toxicity precluding multiple administrations, further exploration of BMS-181174 will not be performed.

KW-2149 is a new, semisynthetic, C-7-N-Substituted, mitomycin C analog showing antitumor activity both in vitro and in vivo, equal or superior to mitomycin C. In a phase I study, KW-2149 was administered as an i.v. bolus injection every 21 days and at a dose of 100 mg/m2 pulmonary toxicity was dose limiting. Animal studies have indicated since that KW-2149-induced pulmonary toxicity can be prevented by pretreatment with corticosteroids. This paper presents the results of a further phase I study of KW-2149 with corticosteroid pretreatment. Patients were treated with oral dexamethasone 8 mg every 12 h, starting 24 h before KW-2149 administration, for 5 days. KW-2149 was given as an i.v. bolus injection every 21 days. Seventeen patients were treated with a total of 48 courses. Six patients received 60 mg/m2 and 11 patients 75 mg/m2. Two courses were not evaluable for toxicity. Significant lung toxicity was observed in at least three patients treated with a dose of 75 mg/m2 KW-2149 and pulmonary toxicity was therefore considered the dose-limiting toxicity at 75 mg/m2. No other important side effects were noted. One partial response was observed in a patient with colorectal cancer. Pretreatment with dexamethasone failed to suppress KW-2149-induced lung toxicity.

Objective: Assess the efficacy of intratumoral chemotherapy in a colonic tumor model implanted subcutaneously in the BD IX rat., Method: In order to determine their antitumoral effect, 5 anticancer drugs were administered via intravenous and direct intratumoral routes 2 or 10 days after subcutaneous inoculation of tumoral cells. Intratumoral diffusion was evaluated using Patent blue injected directly into the tumoral tissue. Cisplatinum was administered via intratumoral, intravenous and intra-arterial routes to determine the intratumoral and intrarenal concentrations achieved with each of these administration routes. Cisplatinum was also administered via intravenous and intratumoral infusion for 30 minutes to determine the antitumoral effect of each of these routes., Results: Mitomycin and cisplatinum inhibited growth of tumors which had not yet become established and caused advanced stage tumors to regress. For early stage tumors, the intratumoral route was always more effective than the intravenous route. Patent blue diffusion demonstrated a nonhomogeneous intratumoral distribution. Compared with intravenous or intra-arterial infusion, intratumoral infusion gave much higher concentrations of cisplatinum within the tumors and reduced systemic diffusion. At 7 weeks, the antitumoral effect was equivalent for the 2 administration routes while at 13 weeks, the intratumoral treatment was more effective than the intravenous treatment., Conclusion: These findings in an experimental animal model demonstrate that intratumoral chemotherapy is more effective than intravenous chemotherapy. It is however still impossible to consistently cure tumors induced in animals.

The progress of mitomycin C (MMC) bioreduction was studied in vivo in the rat Sp 107 mammary carcinoma after intra-tumoural injection of either 100 micrograms or 1 mg. 2,7-Diaminomitosene (2,7-DM) was utilised as a primary bioreductive metabolite and 10-decarbamoyl 2,7-diaminomitosene (DC 2,7-DM) served as a secondary bioreductive metabolite, both of which were measured by high-performance liquid chromatography. 2,7-DM and DC 2,7-DM were produced rapidly, achieving close to their maximal concentrations at the earliest time point studied [5 min]. 2,7-DM was cleared rapidly from the tumour with apparent half-lives of 5 and 35 min after the low and high drug doses, respectively. DC 2,7-DM had a longer apparent half-life of 130 min at the higher dose but, as compared with 2,7-DM, was only a minor metabolite [the area under the curve (AUC) of 2,7-DM was 5.6-fold that of DC 2,7-DM]. At the lower drug dose, DC 2,7-DM was not detectable. Rapid formation and disappearance of bioreductive metabolites of MMC may account for the failure of previous studies to detect these products in vivo.

Hepatic extraction rates (ER) of anticancer drugs during hepatic arterial infusion were investigated with the aid of direct hemoperfusion (DHP) under hepatic venous isolation (HVI). Using mongrel dogs (n = 6), adriamycin (ADR), mitomycin C (MMC) and cisplatin (CDDP) were simultaneously administered to the hepatic artery at each dosage of 1 mg/kg in 10 minutes under HVI.DHP. Hepatic venous flow and plasma concentrations of each drug at the carotid artery, the inlet and outlet sides of DHP were periodically determined during HVI.DHP. Based on these data, drug adsorption and removal rates were estimated. In addition, hepatic tissue uptakes of each drug were calculated from the amounts of drug administered and leaked in the hepatic effluent. Subsequently, the percentage of tissue uptake of each drug to the amount of drug administered was determined as ER of each drug. Drug adsorption rates during the first 10 minutes after infusion showed no significant difference among three drugs. Drug removal rate of CDDP tended to be higher than those of other two drugs. ER of CDDP (54.8 +/- 18.3%) were significantly lower (p < 0.01) as compared to ADR (84.4 +/- 16.2%) and MMC (83.1 +/- 15.7%). These results indicate that ER of each drug should be taken into consideration to determine appropriate drug for hepatic arterial chemotherapy.

The mitomycin antibiotics, because of their preferential toxicities for hypoxic cells, have significant potential as adjuncts to ionizing radiation in the treatment of solid tumors. To gain information on the mechanism by which these agents exert their cytotoxicities to hypoxic and aerobic cells, the effects of MC, POR and several of their analogs were studied in EMT6 mammary carcinoma cells. The rate of uptake of POR by these cells was directly correlated with the cytotoxicity produced by this agent under both hypoxia and aeration. At equivalent concentrations, uptake of POR into hypoxic cells was more rapid than into aerobic cells. Hypoxic cells also accumulated the antibiotic in concentrations well in excess of that present in the extracellular medium, presumably as a result of reductive activation and covalent binding of POR to cellular structures. Such activation and binding occur to a much lesser degree in aerated cells, resulting in the rapid efflux of POR from these cells when the antibiotic is removed from the extracellular environment. To gain information on the reaction of POR with DNA, mono- and bis-adducts formed in EMT6 cells exposed to this agent were measured. Three major adducts were formed. Two were mono-adducts consisting of deoxyguanosine linked at its N2-position to the C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct in which POR was cross-linked to two deoxyguanosines at their N2-positions. More adducts were formed in hypoxia than in air, and more bis-adducts were present in hypoxic cells. Simultaneous exposure of cells to both POR and DIC reduced the total adduct level and a new unknown adduct was formed, primarily under hypoxia. Several mitomycins were evaluated for their capacity to kill EMT6 cells and to produce DNA cross-links in both hypoxia and aeration. The number of cross-links required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation. The findings support the concept that DNA is a critical target in the action of the mitomycins and that cross-linking of the DNA creates an important lesion for cytodestruction.

Chemocoagulation therapy was evaluated in an experimental model of metastasis of murine lymph nodes following injection of a suspension of mitomycin C--containing activated carbon particles in 80% ethanol (MMC-CH-ET) into the popliteal lymph node. Lymph node metastasis was induced in the left popliteal and the lumbar lymph nodes 8 days after injection of mouse leukemia P388 cells into the footpad of the left hindleg of BDF1 mice. When MMC-CH-ET was injected into the left popliteal lymph node, it immediately left this site and entered the lumbar lymph node via lymphatic vessels. When compared with tissue concentrations of mitomycin C following injection of an aqueous solution of this drug, the mitomycin C concentration of MMC-CH-ET was maintained at significantly higher levels for 2 hr following injection both at the site of injection and at secondary lymph nodes. Furthermore, coagulative necrosis was identified histologically throughout the injected lymph node and the secondary lymph node, including the metastatic site. The mortality of mice treated with MMC-CH-ET injection was significantly reduced and lymph node metastasis was controlled with MMC-CH-ET when compared with the results for mice treated with an aqueous solution of mitomycin C or treated by surgical lymph node dissection. In this report, we suggest that the use of MMC-CH-ET as a therapeutic agent may be useful in targeting lymph node metastasis.

Purpose: Intrapleural cisplatin-based chemotherapy has been used in the treatment of patients with malignant pleural mesothelioma and malignant pleural effusions, but the pharmacokinetics of this form of chemotherapy have not been previously evaluated. We performed pharmacokinetic studies on 12 patients who received both intrapleural cisplatin and mitomycin immediately following pleurectomy/decortication for malignant pleural mesothelioma., Patients and Methods: Simultaneous pleural fluid and plasma samples were collected at 15 and 30 minutes, and at 1, 2, 3, 4, and 24 hours after administration of the intrapleural chemotherapy (cisplatin 100 mg/m2 and mitomycin 8 mg/m2), and after cisplatin (total and free) and mitomycin levels were measured. The mean peak levels, the areas under the concentration-time curve (AUC) and the drug half-lives (t1/2s) in plasma and pleural fluid were compared using the paired t test. Differences were considered significant if P less than or equal to .05., Results: Systemic absorption was rapid, with peak plasma levels being reached within 1 hour of administration of the intrapleural chemotherapy. Peak plasma levels measured after intrapleural chemotherapy approximated those reportedly attained during systemic administration of these drugs at similar doses. However, the mean peak cisplatin and mitomycin levels, and their mean AUCs, were significantly higher in the pleural fluid than in the plasma. There was a three- to fivefold advantage (on a logarithmic scale) for pleural to plasma AUCs for both cisplatin and mitomycin. The mean t1/2s for cisplatin and mitomycin were significantly longer in the plasma than in the pleural fluid., Conclusions: The pharmacokinetics of intrapleural cisplatin-based chemotherapy are analogous to those of intraperitoneal chemotherapy. Our findings show that intrapleural cisplatin-based chemotherapy has a distinct local pharmacologic advantage, but also produces significant and sustained drug plasma levels.

We examined the kinetics of penetration of mitomycin C (MMC) in the dog bladder wall after intravesical instillation of 20 mg/40 ml, a dose used in patients. Bladder tissues were harvested and concentration-depth profiles were established by analysis of thin tissue slices cut parallel to the urothelial surface of the bladder. Tissue concentrations after a dwell time of 5-7 min were similar to those after 30-120 min. In tissues harvested 60 and 75 min after removal of the dose, MMC was not detected in 5 of 6 samples and was less than 1 micrograms/g at the mucosa in the remaining sample, suggesting a rapid "washout" of the drug. The rapid equilibrium between the drug in urine and bladder tissue indicates that the duration of exposure of the bladder wall tissue was approximately equal to the dwell time of intravesical therapy. Tissue concentrations declined log-linearly with respect to the depth of penetration. The concentration immediately underneath the urothelium (C0) showed considerable intra- and interanimal variability. Bladder distention appeared to increase C0 by several fold. C0 ranged from 2 to 275 micrograms/g wet tissue weight, with a median value of 24 micrograms/g, or 11 micrograms/g when two animals with distended bladders were excluded. MMC concentrations in 3 different sites of the same bladder varied up to 5-fold. Within the capillary-perfused mucosa and muscularis (between 50 and 2000 microns from the urothelial surface), concentrations decreased by 50% for each 500-microns distance. The median concentration at 2000 microns was 1 microgram/g (n = 24). At 2000-3000 microns, tissue concentrations in most (18 of 24) specimens either declined to an asymptotic value or were lower than the detection limit of 0.1 microgram/g. Concentrations in the bladder contents were 200-500 micrograms/ml, the average tissue concentration from 50 to 3000 microns was 10 micrograms/g, and plasma concentrations were less than 0.1 microgram/ml. This supports the therapeutic advantage of intravesical therapy of high local drug concentrations while minimizing systemic exposure. A comparison of the urine concentration and C0 indicated a 30-fold decline in concentration across the urothelium. This suggests the importance of the urothelium as a barrier to MMC absorption. A separate study in our laboratories showed that 16 micrograms/ml of MMC was needed to produce a 90% inhibition of the labeling index of explants of human bladder cancers located in the urothelium (Ta tumor, TNM classification), 25 micrograms/ml in the lamina propria (T1 tumors), and 43 micrograms/ml in the muscle layer (T2 tumors).(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical predictability of preclinical test for antitumor agents has not been significantly improved even after the use of a human tumor/nude mouse model. Such different antitumor activities between preclinical and clinical tests probably due to the fact that therapeutic used in both tests usually each maximum tolerated dose (MTD), are pharmacokinetically not equivalent. Therefore, we introduced a new concept of "clinically equivalent dose (CED)", which can reproduce in the nude mouse the blood level of a given drug observed with human patients received its therapeutic dose. Treatment of human tumors implanted in the nude mice with CEDs of several drugs exhibited much better correlation with their clinical efficacies than those with MTDs. The feasibility of use of CED predicted by animal scale-up procedure as a therapeutic dose in the preclinical test was discussed.

Desialylated fetuin (asialofetuin) was adopted as a carrier for introducing drugs in parenchymal liver cells. Mitomycin C, as a model guest-compound, was covalently attached to asialofetuin through a spacer of the succinyl group. The asialofetuin-mitomycin C conjugate contained 4.4 w/w% of mitomycin C and liberated it gradually at physiological conditions (t1/2 = 37 h). The survival time of the conjugate in rat blood circulation was significantly smaller than that of the non-desialylated fetuin conjugate; the elimination half-life was 7.3 min after intravenous injection. At 30 min after injection of the conjugate in rats, 40% of the dose was present in the liver. Parenchymal cells in the liver selectively took up the conjugate, which was highly distributed to the lysosomal fraction in the cell. The greater uptake of the conjugate by hepatocytes reflected the increased excretion in the bile; totally 10.4% of the dose was recovered.

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C-dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4-3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (Ess) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The Ess for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.

A prospective, randomized, double-masked, and placebo-controlled study was performed to evaluate the effects of a localized and sustained release of mitomycin on the success of glaucoma filtration surgery in rabbits. A bioerodible polymer was used as the drug carrier. Full-thickness filtration surgeries were performed and data from 22 rabbits were collected. One eye received a polymer impregnated with mitomycin (0.02 mg or 0.06 mg), while the fellow eye received a drug-free polymer. Intraocular pressure, bleb survival, and postoperative complications were investigated. Intraocular pressures remained lower for a longer period of time (P less than 0.004) and filtration blebs lasted longer (P less than 0.05) in experimental eyes than in control eyes. Conjunctivitis and sectorial corneal haze occurred more frequently in eyes treated with the higher dosage mitomycin compared with control eyes. The use of mitomycin-C in a polymer delivery system appeared to promote the success of glaucoma filtration surgery in rabbits. With the lower dosage of mitomycin, clinically significant ocular toxicity was not noted.

The influence of the vasopressin-pro-drug glycylpressine (GP) and of a chemoembolisation with Spherex starch-particles (SP) on the availability of mitomycin (CAS 50-07-7, mitomycin C, MMC) was investigated in 30 patients with liver metastases, MMC administration was performed after blocking of the common hepatic artery by different concentrations of SP and after different time-intervals of GP. The comparison of plasma-levels after bolus injection without GP and after administration of MMC after 2, 5 and 15 min of vasoconstriction and after chemoembolisation with 450 mg or 900 mg SP, respectively, showed a remarkable reduction in the systemic circulation of MMC in the blood vessel system at about 40% (GP) and 45% (SP). A statistically significant influence on the pharmacokinetics of MMC with regard to CO, Vd, T 1/2zp, AUC and Cl(tot) was found, but not in t1/2el, t1/2biol and Vl. Both methods cause a distinctly accelerated diffusion of MMC into the tissue of the tumor region by change of the hemodynamics, leading to lowered side-effects. Thus the clinical picture was improved by MMC.

Microencapsulated mitomycin C has been used for the treatment of intrahepatic tumors in Japan, but there have been no reports of its use in western countries. In this study, the pharmacokinetic profile of intrahepatic arterial mitomycin C microcapsules is reported. Regional mitomycin C was administered to six patients, both in the microencapsulated form and in solution. The clearance (140 +/- 31 1/hour, mean +/- SD) and half-life of drug in plasma (0.39 +/- 0.03 hours), and volume distribution (246 +/- 23 1) were significantly higher, and peak drug concentrations (80 +/- 75 ng/ml) significantly lower with the microencapsulated preparation than with the free drug (clearance, 46 +/- 8 1/hour; half-life, 0.11 +/- 0.02 hours; volume distribution, 33 +/- 4; peak drug concentration, 812 +/- 423 ng/ml), on Student's t testing (P less than 0.05). The results show that very little systemic exposure is associated with the microencapsulated form of mitomycin C. Dose escalation should be feasible without increasing systemic toxicity.

Determination of the depth of penetration of locally applied drug therapy and evaluation of possible mechanisms of drug transport require knowledge of drug concentration-versus-tissues depth profiles. A method to determine the drug concentration-depth profile is needed. We have devised such a method and used it to determine the penetration of mitomycin C (MMC) in the dog bladder wall after intravesical drug instillation. This method is based on sectioning of frozen tissue into 40-microns segments, followed by drug extraction and high-pressure liquid chromatography analysis. Tissue concentrations could be detected with a sensitivity of 1 ng/sample, or 20 ng/g for tissue samples of approximately 2 x 2 cm. This sensitivity was sufficient to describe the penetration of MMC in the bladder wall of dogs, using an identical instillation technique, dwell time, and MMC concentration as in human patients. Tissue concentrations were expressed relative to tissue weight or tissue protein contents. For MMC, standardization to tissue weight yielded a better mathematical fit of the concentration-versus-depth profiles than standardization to protein content. The time interval between tissue harvesting and freezing was critical. The MMC concentration at the urothelial side of dog bladders was 2- to 10-fold higher in samples processed immediately after harvesting, compared to samples processed after 1 hr or longer. This significant decrease was not due to drug metabolism in situ. In separate in vitro experiments, we found that the degradation of MMC in 8% tissue homogenate was relatively slow, with only a 30% decline in concentration over 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

During an intra-arterial chemotherapy of patients with liver metastases the plasma concentration-time curves of Mitomycin C (1) after occlusion of the common hepatic artery by the vasoconstricting agent Glycylpressin (2) were investigated. Vasoconstriction by 2 leads to significantly (p less than 0.01) lower blood levels of 1 in contrast to bolus injection of 1 without 2 under identical dosage regimens. This reduction of systemic availability of about 50% leads to a corresponding higher availability for the tumor region.

To enable the treatment of hepatic metastasis with higher, theoretically more effective, doses of systemically toxic anticancer drugs, an isolated liver perfusion (ILP) technique was developed in WAG/Ola rats. First, in a toxicity study the maximally tolerated dose (MTD) of mitomycin C (MMC) was determined for a 25-min ILP and for hepatic artery infusion (HAI) after the administration of a bolus dose. The MTD in the ILP setting (4.8 mg/kg) was 4 times that using HAI (1.2 mg/kg). Subsequently, in a rat colorectal hepatic-metastasis model, concentrations of MMC in tumour, liver, plasma and perfusate were measured during a 25-min ILP to investigate the expected pharmacokinetic advantage of ILP. The mean plasma level determined after ILP (1.2 as well as 4.8 mg/kg MMC) was significantly lower (P less than 0.001) than that obtained following HAI. This may explain both the absence of severe systemic toxicity and the higher MTD in ILP-treated groups. No significant difference in mean tumour and liver tissue concentrations of MMC were found when the groups treated with 1.2 mg/kg drug via HAI vs ILP were compared. The mean MMC concentration in tumour tissue was significantly higher (almost 5 times; P less than 0.05) in rats treated by ILP with the MTD (4.8 mg/kg) than in those treated via HAI with the MTD (1.2 mg/kg). ILP of MMC can be safely performed using a dose 4 times higher than the MTD in the HAI setting, leading to an almost 5-fold concentration of MMC in hepatic metastasis. ILP of MMC may therefore represent a promising therapy for metastasis confined to the liver.

The pharmacokinetics of bronchial artery infusion of 20 mg (11.4-14.0 mg/m2) mitomycin was studied in 14 patients with non-small cell lung cancer (NSCLC). The mean elimination half-life was 34.3 min (range 6-72), and the area under the plasma concentration-time curve (AUC) was 166 ng h/ml (39-312). The mean maximum plasma drug concentration (Cmax) was 178 ng/ml (12-540) and back-extrapolated plasma drug concentration was 308 ng/ml (17-1423). The mean volume of distribution in the one-compartment model was 0.183 l/kg (0.010-0.887) and the rate constant for unchanged drug appearing in the urine was 1.91/min (0.57-7.27). There was considerable variation among individuals with respect to the pharmacokinetics of mitomycin, and the mean Cmax and AUC were lower than those reported after intravenous administration.

Systemic toxicity is usually the dose-limiting factor in cancer chemotherapy. Regional chemotherapy is therefore an attractive strategy in the treatment of liver metastasis. Two ways of regional chemotherapy, hepatic artery infusion (HAI) and isolated liver perfusion (ILP), were compared investigating the difference in toxicity with tissue and biofluid concentrations of mitomycin C (MMC). In wistar derived WAG rats the maximally tolerated dose of mitomycin C via HAI was 1.2 mg kg-1. Body weight measurements after HAI with doses higher than 1.2 mg kg-1 suggest both an acute and delayed toxic effect of mitomycin C since the time weight curves were triphasic: a rapid weight loss, a steady state and a second fall in weight phase. These rats died due to systemic toxicity. ILP with 4.8 mg kg-1 was associated with no signs of systemic toxicity and only transient mild hepatotoxicity. ILP with 6.0 mg kg-1 was fatal mainly due to hepatic toxicity. The four times higher maximally tolerated dose in ILP resulted in a 4-5 times higher peak concentration of mitomycin C in liver tissue, while the plasma concentration remained significantly lower than in the HAI treated rats. In the tumour tissue a 500% higher concentration of mitomycin C was measured in the ILP with 4.8 mg kg-1 than in HAI with 1.2 mg kg-1 treated rats. We demonstrated that when mitomycin C was administered by ILP a 400% higher dose could be safely administered and resulted in a five times higher tumour tissue concentration. In view of the steep dose-response curve of this alkylating agent this opens new perspectives for the treatment of liver metastasis.

A new dosage form of mitomycin C (MMC-CH) comprising 0.75 mg/ml of activated carbon particles adsorbing mitomycin C at 124 micrograms/mg and 7 micrograms/ml of mitomycin C in a free state was injected intraperitoneally to male rats of Donryu strain transplanted intraperitoneally with 10(7) cells/rat of Yoshida sarcoma 4 days before injection. The rats were subjected to autopsy within 60 min after injection. MMC-CH adhered selectively to the tumor surface of Yoshida sarcoma growing intraperitoneally rather than to the surface of organs such as small intestines which the surface cancer did not affect. Within 120 min after injection, mitomycin C concentration in the tissue samples was bioassayed. Intraperitoneally injected MMC-CH distributed high levels of mitomycin C to the tumor rather than to the unaffected organ located intraperitoneally. Animals killed by cancer after the treatment of MMC-CH were autopsied. Histological effects of MMC-CH were studied microscopically. Mitomycin C adsorbed on activated carbon particles induced degenerative changes in the tissues of tumors to which the activated carbon particles had adhered.

Gastrointestinal malignancy may spread to peritoneal surfaces in the absence of lymphatic or hematogenous metastases. To treat peritoneal carcinomatosis, a uniformly lethal disease process, extensive cytoreductive surgery and i.p. chemotherapy were combined. Early postoperative i.p. chemotherapy was instilled in the first few days after the surgical procedure in an attempt to treat anatomic sites that would be sealed off by postoperative adhesions. Mitomycin C was given on the first postoperative day at two doses, 10 and 12 mg/m2. 5-Fluorouracil was given on postoperative days 2-5 at 15 and 20 mg/kg, respectively. Median area under the curve ratio i.p./i.v. was 117 for 5-fluorouracil and 21.6 for mitomycin C. Elevated intraportal levels of drug were observed for i.p. 5-fluorouracil but not for mitomycin C. The marked pharmacokinetic advantage of postoperative i.p. suggests that this treatment strategy should be considered in a clinical trial in patients at risk for progression of peritoneal carcinomatosis.

In order to prevent peritoneal metastasis after gastrectomy for gastric cancer, we conducted a study to find a clinical dose of MMC which would result in IC50, the drug concentration which inhibits the growth of colonies of cells by 50%. After closure of the abdomen, MMC dissolved in 1,000 ml of physiological saline was administered. In the 40 mg group, the mean intraperitoneal MMC concentration after 60 min was 8.2 micrograms/ml, which exceeded IC50 of 7.6 micrograms/ml. In the group, peak serum concentration of MMC was 0.29 micrograms/ml. Seventy to 80% of intraperitoneally administered MMC was transferred in blood. Serious adverse effects were not observed. One-year survival rate of absolute noncurative resection with peritoneal metastasis was 33% in the control group and 78% in the treated group. Two-year survival rate of curative resection with free cancer cells in the peritoneal cavity was 11% in the control group and 60% in the treated group.

Intraperitoneal hyperthermic perfusion (IPHP) was performed right after surgery for 31 advanced gastric cancer patients with serosal invasion and/or peritoneal dissemination. The survival rate in the IPHP group, was compared with that in the control group, 30 patients who underwent surgery alone within the same period of time. The 1-year survival rates for IPHP group and control group were 81.9% and 40.3%, respectively. The 2-year survival rates were 52.1% and 11.8%, and the 3-year survival rates were 26.1% and 0%, respectively. The survival rate for IPHP group was better than that for control group, with p = 1.46 x 10(-4). IPHP and control group with peritoneal dissemination included 21 and 9 patients respectively, and 50% survival rates were 18 months and 4.2 months, respectively. IPHP and control group with serosal invasion included 10 and 21 patients, respectively, and 8 out of 10 patients survived in the IPHP group, while 17 out of 21 patients died in the control group. These results suggest that IPHP is effective for gastric cancer patients with peritoneal dissemination and/or serosal invasion.

The property of pluronic F127 (PLF) as a base for an anticancer agent, mitomycin C (MMC) was evaluated. A weighed amount of PLF and MMC were slowly added to cold water and mixed completely. The solution was warmed to 37 degrees C and MMC containing PLF gel was formed. The cumulative amount of MMC which was released from the gel during a 3-hour period was calculated. 5.5 percent of MMC was released from PLF-MMC gel which contained 20 or 25% PLF and 3.8% MMC from PLF-MMC gel containing 30% PLF. Intraperitoneal (i.p.) administration of large amount of PLF gel alone in rabbits or mouse revealed severe hepatorenal toxicity, and acute i.p. administered LD50 of PLF was supposed to be between 1.7 g and 5.0 g/kg body weight in mouse. Further evaluation of gradual releasing property of PLF is needed.

Targeting chemotherapy to the metastatic lesions of the lymph nodes after surgical removal of the main tumor, a drug delivery system using ultrafine particles of activated charcoal was developed and examined for its ability to adsorb mitomycin C (MMC) and to transport MMC to the lymph system. Satisfactory results were obtained. In animal experiments, serial sampling of thoracic duct lymph by cannulation and serial samplings of peripheral vein blood after injection of MMC-charcoal solution into the subserosal space of the intestinal wall were made to study the pharmacodynamics of the lymph-blood system through analysis of MMC regression curves. Clearance (K) and half-life (t1/2) are calculated from the formula; C(t) = C(t0) X e-kt, and compared with that of MMC-saline. Activated charcoal has a proven ability to transport MMC to the lymphatic system. With activated charcoal, MMC is retained for much longer in the lymph nodes than with saline. Congestion of the lymph system was produced by ligation of the thoracic duct in CH-40, with 25% prolongation of t1/2. In conclusion, these drug delivery systems are suitable for targeting chemotherapy at lymph node metastases, and it is proven that 1500 AA Mitsubishi ultrafine charcoal is a good carrier of drug and a slow drug-release material in the lymphatics. Minimal side effects are expected because of the constant low concentrations in the general circulation.

Biliary excretion characteristics of polymeric prodrugs of mitomycin C (MMC), mitomycin C-dextran conjugates with cationic or anionic charge (MMC-Dcat, MMC-Dan) and with different molecular weights of dextran (10,000, 70,000, and 500,000) were studied in rat. Following intravenous injection, bile was periodically collected and concentrations of free and dextran-conjugated MMC in it were determined by bioassay. MMC administered as a free form was excreted rapidly into bile and 1.8% of the dose was recovered within 2 h. A small amount of MMC was gradually excreted into bile after administration of all MMC-Ds and total recovery at 8 h was less than 1% of dose. In this case, a major part of excreted MMC was recovered as a conjugated form. MMC-Dcat gave a larger total excretion of MMC than MMC-Dan and excretion was also affected by the molecular weight of carrier dextran. The biliary recovery of MMC-Dcat labeled with 14C at a spacer moiety was significantly higher than that of conjugated MMC determined by bioassay suggesting release and/or inactivation of MMC in MMC-D during the circulation in the body. These results were compared with biliary excretion of model macromolecules with the same molecular weight but different electric charge in order to clarify the effect of electric charge on the biliary excretion of macromolecules. Cationic macromolecules exhibited higher biliary excretion in relation to greater hepatic uptake.

The current treatment of pancreatic cancer with resection and/or radiation is considered unsatisfactory because of a high incidence of failure and a moderate incidence of complications. A sizable number of these patients present with localized or regional disease. Regional high-dose chemotherapy, such as with isolated perfusion, may offer an alternative therapy with low treatment-related morbidity and mortality and better end results in this group of patients. In an effort to develop such a treatment modality, we evaluated the pharmacokinetics and toxicity of mitomycin C (MMC) during isolated perfusion of pancreas in a canine model. From this study, it appears that a dose of 0.25 mg MMC/kg body weight is most suitable for isolated perfusion of pancreas at 39 degrees C, maintaining flow rate and pressure within physiologic range. Isolated perfusion with a dose of 0.25 mg/kg body weight has very mild short- and long-term toxicities and markedly increases drug delivery to the pancreas, duodenum, and peripancreatic lymph nodes, making it the most suitable dose for possible clinical application.

The authors performed 68 liver embolizations in 51 patients. As selectively as possible, Lipiodol, to block the capillary bed, and a chemotherapeutic agent were injected into the liver tumors. A CT performed 24 hours after treatment showed the distribution of the contrast medium which is also an indicator of the distribution of the chemotherapeutic agents. It was found that the contrast medium had accumulated not only in the liver but also in the lungs. Here, four different degrees of accumulation were found, according to the amount of Lipiodol used. Embolization of the liver thus involves potential hazards for the lungs, such as microembolisms, pneumonia, and toxic effects of the chemotherapeutic agents.

Mouse monoclonal antibody A 7, which was raised against human colon cancer, was used for preparing the conjugates with neocarzinostatin, mitomycin C and adriamycin. The tissue distribution of these three conjugates were examined in athymic nude mice transplanted with human colon cancer. The distribution in tumor was different in these three conjugates. The route of administration of the conjugate affected its distribution in tumor tissues. In the case of tumor transplanted in the back of mice, intravenous administration seemed to superior to intraperitoneal one. In clinical trials of immunoconjugate composed of A 7 and polypeptide anticancer drug neocarzinostatin (A 7-NCS), human anti-mouse antibody (HAMA) was observed in most cases without serious immune response such as anaphylactic shock. Human antibody against neocarzinostatin could not be detected in any case receiving A 7-NCS.

Disposition characteristics of various anticancer drugs in a tissue-isolated tumor preparation were studied in Walker 256 carcinosarcoma-bearing rats using an in situ single-pass vascular perfusion technique. Three anticancer drugs, 5-fluorouracil, mitomycin C, and Adriamycin, and two lipophilic prodrugs of mitomycin C were tested in the tumor preparation perfused with Tyrode's solution containing 4.7% bovine serum albumin. After bolus arterial injection of test drugs, their outflow concentration-time curves were analyzed based on statistical moment theory. In each tumor preparation, the injection of drug was paired with that of vascular reference substance, Evans' blue-labeled bovine serum albumin, and disposition parameters of the drug were corrected with those of vascular reference substance. From the mean transit time values of vascular reference substance, the average vascular volume of the tumor preparation was calculated to be 0.063 ml/g, which decreased with tumor growth. All drugs showed significant extraction by the tumor tissue, depending on their physicochemical properties. Distribution volumes of tested drugs were from 1.53 to 3.33 times larger than the vascular volume. Calculated intrinsic clearance values for the protein-unbound fractions increased as the lipophilicity of the drug increased. The potential increase in tumor uptake was observed in lipophilic prodrugs of mitomycin C. The present experimental system is thus suggested to be useful for analyzing drug disposition in tumor tissue.

The cross-linking reaction of N-methylmitomycin A to 175-bp calf thymus nucleosomes and to reconstituted core particles having known DNA sequences was examined using gel-electrophoresis techniques. The nucleosome structure, as the CAP-DNA complex, leads to a decreased covalent binding in comparison to protein-free DNA, suggesting, in addition to sequence specificity, precise geometrical requirements for bifunctional covalent addition of the drug to the nucleic acid.

An emulsion method was developed for the incorporation of water-soluble mitomycin C into polylactic acid biodegradable microcapsules. With an average particle size of about 95 microns, microcapsules with a desired loading of from 3.65 to 13.80 per cent were prepared. These microcapsules, which contained both crystalline and finely dispersed drug particles, showed a dose-dependent drug release pattern with microcapsules of higher drug loading having a faster release rate than those of lower drug loading. Effective sterilization of the microcapsules for parenteral use was achieved by 60Co gamma-ray irradiation, which did not affect the microcapsule structure, release rate or drug stability. Mitomycin C showed dose-dependent antiproliferative activity against the growth of the K562 human erythroleukaemia cells. The microencapsulated dosage form of mitomycin C was found to enhance the drug's activity through sustained drug release. In experiments where drug concentrations in the cell medium were reduced according to the drug's biological half-life, the microcapsule systems showed a distinct advantage over the non-capsulated dose for the kinetic inhibition of K562 cell growth.

The effect of a new dosage form for anti-cancer agents was studied on tissue distribution and compared with the aqueous solution form. The new dosage form, developed in order to distribute a greater amount of the agent to regional lymph nodes, comprises polyvinylpyrrolidone and small activated carbon particles adsorbing pepleomycin (PEP-CH) or mitomycin C (MMC-CH) in saline. Pepleomycin at 500 micrograms/kg or mitomycin C at 250 micrograms/kg were injected into the gastric wall of dogs in the new dosage form or the aqueous solution form. The activity level of the agents in tissues was bioassayed within 24 h after injection by the thin agar plate method. Statistically, the new dosage form maintained an activity level in lymph nodes that was significantly higher than the aqueous solution form (ratio of PEP-CH to solution form, 6-32 times in 24 h; MMC-CH, 100-1000 times in 6 h; p less than 0.05-0.01), and a concentration in blood that was significantly lower (PEP-CH, about 1/2 times in 2 h; MMC-CH, 1/10-1/5 times in 1 h; p less than 0.05).

The uptake of tumor-inhibitory antibiotics by human K562 erythroleukemia cells was examined in the presence of leukoregulin to determine if the lymphokine's ability to increase the plasma membrane permeability of tumor cells facilitates concurrent entry of pharmacologically active molecules. K562 cells were exposed to 0.25-2.0 micrograms of doxorubicin/mL for up to 60 minutes at 37 degrees C. Commencing within 15 minutes, leukoregulin increased the entry of doxorubicin approximately twofold and the uptake of mitomycin, mithramycin, and propidium iodide twofold to tenfold. This finding indicates the potential biotherapeutic value of leukoregulin in promoting the selective entry of pharmacologically active molecules into leukoregulin-sensitive target cells.

The effects of degradable starch microspheres (DSM) on mitomycin C pharmacokinetics and bone marrow toxicity were studied in a phase II multicenter study. Sixty-three patients with non-resectable primary or secondary liver cancer were randomized to receive either i.a. mitomycin C 15 mg/m2 first, followed 5 weeks later by mitomycin C 15 mg/m2 plus DSM 360 mg administered into the hepatic artery (group I) or the same treatments in the opposite sequence (group II). In 36 out of 47 patients who received at least 2 treatments, peripheral venous blood samples were analyzed for mitomycin C pharmacokinetics on a minimum of 2 paired courses. In all patients, the area under the concentration time curve (AUC) was significantly lower when the drug was co-administrated with DSM, but the terminal half-life (t1/2) of mitomycin C was unchanged. In group I the addition of DSM resulted in a significantly lowered AUC, but not in group II. The discrepancy between the 2 groups is probably due to differences in DSM-induced intra-hepatic shunting. The addition of DSM resulted in significantly higher platelet nadir values, but unchanged white blood cell count nadir value. In conclusion, DSM reduce the systemic exposure of mitomycin C and seem to lessen the haematologic toxicity judged from a less pronounced decrease in platelets.

A fully automated liquid chromatographic system for the bioanalysis of mitomycin C has been described. The isolation of the analyte from the biological matrix (plasma, ascites and urine) is performed using a continuous-flow system equipped with a dialysis membrane in order to remove proteins. The samples are concentrated on a reversed-phase pre-column and subsequently introduced on to a reversed-phase analytical column by applying column-switching techniques. The drug is detected by absorbance measurements at 360 nm. Using the described system up to 100 samples a day can be analysed with determination limits of the order of 1 ng/ml, with a linear dynamic range of at least three decades for plasma and urine samples. The procedure was applied to pharmacokinetic studies of ovarian cancer patients treated intraperitoneally with mitomycin C.

Mitomycin C-Dextran conjugates (MMC-D) were intravenously (iv) injected in mice bearing subcutaneous sarcoma 180. The tissue distribution was determined for three 14-C-labeled anionic conjugates (MMC-Dan) with molecular weights of 10, 70, and 500 kd and one cationic 70-kd 14C-conjugate (MMC-Dcat). The anionic conjugates were slowly cleared from the plasma, and their elimination rate decreased with increasing molecular weight. Radioactivity accumulated in liver, spleen, lymph nodes, and tumor but not in heart, lung, intestines, kidney, or muscle after iv injection of all types of 14C-MMC-Dan. In contrast, the cationic conjugate was rapidly cleared from the plasma and accumulated mostly in the liver and spleen, while tumor levels remained low. The antitumor effect of the 70-kd MMC-Dan, which afforded the highest tumor concentration, was superior to that of free MMC. Therefore, anionic mitomycin C-dextran conjugates with a high molecular weight may be useful for tumor targeting in cancer chemotherapy.

Although several pharmacological data of intravesical mitomycin C (MMC) are available now, data about resorption following subsequent intravesical instillations with different instillation times are lacking. We have analyzed MMC concentrations in blood plasma and urine following eight subsequent instillations, with 0.5- and 1.0-h instillation times. The relationships between urinary pH, urinary MMC concentrations, and MMC blood plasma concentrations were determined, as well as the stability of MMC in urine at pH 5-8 at 37 degrees C. An average of 40.3 and 46.4% of the total parent drug was recovered for the 0.5- and 1.0-h instillations, respectively. Blood plasma concentrations of MMC could be measured in nearly all patients and were independent of instillation times, urine concentration, or urinary pH. Resorption of MMC and recovery was stable during eight subsequent instillations. It was demonstrated that MMC can be degradated in urine at physiological conditions (pH less than 6; 37 degrees C). However, neither an influence of prolongation of the instillation time on MMC recovery from urine, nor a significant correlation between urinary pH and urinary MMC concentrations could be demonstrated. Since MMC can be degradated at pH less than 6 within 0.5 h, buffering of instillation fluids containing MMC is recommended. Reuse of instilled MMC, therefore, cannot be advised.

Mitomycin C and its methylated analogue porfiromycin (Por) have significant potential as adjuncts to regimens presently used for treating solid tumors because of their preferential toxicity to cells existing in an hypoxic environment. An understanding of the factors producing the differential activity of these drugs under aerobic and hypoxic conditions would facilitate the development of new agents of this class. Previous studies have focused on the enzymes that reductively activate the mitomycins and on the interaction of these drugs with DNA; none of these studies has fully explained the differences in cytotoxicity observed under hypoxic and aerobic conditions. The present investigation demonstrates that the rate of Por uptake is directly correlated with cytotoxicity under both aerobic and hypoxic conditions. Uptake of Por into hypoxic cells is more rapid than into aerobic cells at equal drug concentrations. Hypoxic cells also accumulate drug in concentrations well in excess of those in the extracellular medium; this is apparently a reflection of drug sequestration in these cells. This sequestration of Por, which affects the rate and extent of uptake in hypoxic cells, does not take place in aerobic cells. The failure of aerobic cells to sequester drug is evidenced by the very rapid efflux of Por from these cells upon removal of extracellular Por and by the fact that aerobic cells attain a state of equilibrium between the intracellular and extracellular drug concentrations. The findings demonstrate that differences in the uptake and retention of Por are associated with the preferential toxicity of Por to hypoxic cells.

Twenty mg of mitomycin C was administered to patients with colo-rectal cancer through various routes of administration. The values of pharmacokinetic parameters were as follows Intra-arterial administration (3 patients with rectal cancer): T1/2 alpha 8.4 min, T1/2 beta 63.8 min, maximum concentration (5 min after ia injection) 0.83 microgram/ml, AUC0-120 25.0 micrograms/ml/min. Intra-portal administration (7 patients with colon cancer): T1/2 alpha 6.1 min, T1/2 beta 61.0 min, maximum concentration (5 min after iv.) 0.75 microgram/ml, AUC0-120 25.3 micrograms/ml/min. Intra-peritoneal administration (14 patients with colon cancer): maximum concentration (20-30 min after i.p.) 0.23 microgram/ml, AUC0-120 21.1 micrograms/ml/min. Intra-pelvic administration (7 patients with rectal cancer): maximum concentration (20-30 min after i.p.) 0.06 micrograms/ml, AUC0-120 7.3 micrograms/ml/min. The reduction curve of intra-arterial administration closely resembles to that of intra-portal administration. Maximum blood level and AUC of intracavitary administration were relatively low compared with those of administration into blood vessels. Bone marrow suppression was seen in patients given mitomycin C into blood vessels and wound healing disturbance such as anastomotic leakage were noted in patients by intra-cavitary administration.

A mitomycin C-resistant (MMCR) strain of L1210 mouse leukemia was developed by continuous drug exposure in vitro. MMC concentrations were increased in a stepwise fashion beginning at 0.033 microM and ending at 0.34 microM. This produced a 10-fold resistant cell line over the parental line. Resistance simultaneously developed to anthracene and anthracycline DNA intercalators, to vinca alkaloids and epipodophyllotoxins but not to cisplatin, bleomycin, fluorouracil or ionizing X-rays. MMC resistance was reversed using the membrane-active agent verapamil. The level of non-protein sulfhydryls was increased 2-fold in the MMCR cells. Intracellular uptake of unchanged MMC was reduced by 40% in the MMCR cells. Cytogenetic analyses demonstrated no recognizable clonal chromosomal alterations unique to the resistant subline and no evidence of double minutes or homogeneously staining regions in the DNA. Gel renaturation analysis failed to document the presence of an amplified DNA domain. Southern blotting of parental and MMCR DNA using a cDNA probe (CHP1) for the P-glycoprotein gene also failed to demonstrate amplification or rearrangement of P-glycoprotein-related homologous sequences. However, an Mr 180,000 glycoprotein was detected in the plasma membranes from MMCR cells. This protein also specifically reacted with a monoclonal antibody (C219) to the P-glycoprotein of Ling and co-workers [Kartner et al., Nature, Lond. 316, 820 (1985)]. These results suggest a pleiotropic drug resistance pattern in the MMCR cells, associated with membrane glycoprotein alterations, enhanced non-protein sulfhydryl levels, and reduced MMC accumulation. This is a novel observation for a resistant cell line selected with an alkylating agent.