Reduced toxicity and superior therapeutic activity of a mitomycin C lipid-based prodrug incorporated in pegylated liposomes.

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.

7-N-(mercaptoalkyl)mitomycins: implications of cyclization for drug function.

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.

Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography.

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.

Phase I and pharmacologic study of BMS-181174 given as a 6 h infusion every 4 weeks to patients with advanced solid tumors.

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-induced pulmonary toxicity is not prevented by corticosteroids: a phase I and pharmacokinetic study.

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.

[Intratumoral chemotherapy in an experimental animal model: another therapeutic possibility in cancerology]. / La chimiothérapie intratumorale dans un modèle expérimental animal: une autre possibilité thérapeutique en cancérologie.

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.

Conversion of mitomycin C to 2,7-diaminomitosene and 10-decarbamoyl 2,7-diaminomitosene in tumour tissue in vivo.

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.

[Comparison of hepatic tissue extraction rates of cytotoxic anticancer drugs during hepatic arterial chemotherapy--evaluation using direct hemoperfusion under hepatic venous isolation].

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.

Studies on the mechanism of the cytotoxic action of the mitomycin antibiotics in hypoxic and oxygenated EMT6 cells.

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 of metastatic lymph nodes using a suspension of mitomycin C-adsorbing activated carbon particles in 80% ethanol.

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.

Intrapleural cisplatin and mitomycin for malignant mesothelioma following pleurectomy: pharmacokinetic studies.

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.

Bladder wall penetration of intravesical mitomycin C in dogs.

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)

[Pharmacokinetic approach to the improvement of clinical predictability in the preclinical test for antitumor agents].

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.

Targeting of mitomycin C to the liver by the use of asialofetuin as a carrier.

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.

Hepatic disposition characteristics of electrically charged macromolecules in rat in vivo and in the perfused liver.

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.

Use of bioerodible polymers impregnated with mitomycin in glaucoma filtration surgery in rabbits.

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.

[Pharmacokinetics and local availability of mitomycin. The influence of vasoconstriction and chemoembolization]. / Pharmakokinetik und lokale Verfügbarkeit von Mitomycin. Einfluss von Vasokonstriktion und Chemoembolisation.

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.

A method to study drug concentration-depth profiles in tissues: mitomycin C in dog bladder wall.

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)