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)

Re-use of mitomycin C in the treatment of superficial tumours of the urinary bladder: an evaluation in view of its chemical stability.

The possibility of re-using mitomycin C (MMC) containing solutions after intravesical instillation in the urinary bladder is evaluated in view of the chemical stability of the drug. Generalized positive conclusions about the re-use of these solutions are unjustified due to the possible variation in the urinary pH of individual patients. Only in cases where the urinary pH during treatment is greater than 6 may re-use be considered, provided that bacterial contamination can be avoided. In cases where the pH is too low (i.e. less than 6) a considerable degradation occurs during instillation (about 34% at pH 5) and storage during 1 week at 4 degrees C (about 25% at pH 5). On the other hand, considering the acid activation pathway, induction of a low urinary pH during treatment might enhance the efficiency, although re-use in this case is impossible.

The mutagenicity of urine fractions from patients administered antineoplastic therapy.

A concern among hospital personnel is their exposure to mutagenic drugs and in the incidental exposures that could occur in caring for the patients. In a recent published study the mutagenicity of urine from patients administered antineoplastic drugs was determined and techniques were developed to chemically inactivate the mutagenicity. A question still remained as to what components of the excreted urine were mutagenic. Urine samples from patients receiving mutagenic drugs were fractionated by high pressure liquid chromatography (HPLC) to then assay by the Ames test the collected and concentrated fractions to determine what were the mutagenic compounds in the urine. Urine samples from patients on single agent cancer treatment with cisplatin, cyclophosphamide, doxorubicin and mitomycin C were assayed. In general, all urine samples containing the cytotoxic agents studied were mutagenic because of the presence of the parent compound, except cyclophosphamide which requires activation and therefore an active metabolite was the major mutagenic constituent in the urine sample. This data indicates that the mutagenicity of urine from patients receiving these antineoplastic agents is the result of the parent compound or a single major metabolite.

Pharmacokinetics of mitomycin C in dogs: application of a high-performance liquid chromatographic assay.

A normal-phase high-performance liquid chromatographic (HPLC) assay was developed for the determination of mitomycin C in plasma and urine. The method involves extraction of mitomycin C from plasma or urine into ethyl acetate-2-propanol-chloroform (70:15:15) with UV detection at 365 nm. Quantitation was performed with an internal standard (porfiromycin) by the peak height ratio method. Excellent correlation was obtained between the HPLC assay and the established microbiological cup-plate bioassay. The pharmacokinetics of mitomycin C were investigated in beagle dogs following a 1-mg/kg iv (22-mg/m2) bolus dose. The plasma mitomycin C concentration versus time data were analyzed by using an open three-compartment model. The average volume of distribution was 1.90 L or 17% of body weight for the central compartment and 7.7 L or 68% of body weight for the terminal elimination phase. The volumes of distribution at steady state, calculated by model-dependent and -independent methods, compared very well with each other and were 6.5 L or 58% of body weight. Total body clearance averaged 112 mL/min, and the mean terminal plasma half-life was 53 min. The 0-24-h urinary excretion of intact mitomycin C accounted for 19% of the dose. The terminal half-life and percent urinary recovery of mitomycin C in dogs is similar to that in humans. Based on these observations, the dog appears to be a good model for studying the disposition of mitomycin C.

Detecting mitomycin C in human plasma during intravesical therapy.

Mitomycin C (MMC) concentrations in plasma and urine were measured in five patients with intact bladder during intravesical instillation therapy. No MMC was detected in plasma by a selective high performance liquid chromatographic (HPLC) method. The detection limit of the method is 1 ng/ml. Our results are in accordance with clinical experiences of the lack of systemic toxicity during MMC instillation therapy. There was a remarkable loss of MMC in the voided urine. The probable explanation could be that a considerable amount of MMC is absorbed into the bladder wall.

[Studies on the physiological disposition and pharmacokinetics of 7-N-(p-hydroxyphenyl)-mitomycin C].

A new mitomycin derivative, 7-N-(p-hydroxyphenyl)-mitomycin C (KW 2083), was labeled with 14C at 3 and 5 position of p-hydroxyphenyl moiety. Its radioactive purity and specific radioactivity were 97.6% and 3.42 mu Ci/mg, respectively. The labeled drug was administered intravenously to male rats or pregnant rats. Radioactivity was rapidly cleared from the plasma and transferred to the peripheral tissues. However low and sustained level of radioactivity was maintained in the plasma, even 48 to 72 hours after administration. About 29% of the radioactivity was recovered from the urine and 58% from the feces by 72 hours after administration. In the bile, 45% of the dose was excreted until 48 hours after administration, and the enterohepatic circulation was observed. The tissue levels of 14C-KW 2083 given to male rats were highest in the kidney, liver and intestinal contents. In the brain, eyes and spinal cord the radioactivity was very low. Autoradiography with 14C-KW 2083 in male or pregnant rats demonstrated that radioactivity distributed in kidney, liver and intestinal contents. The transfer of radioactivity was very low in the fetuses of rats. Radioactivity was observed to some extent in the fetal membrane. Plasma concentrations of KW 2083 or mitomycin C in dogs were determined by means of bioactivity and could be best described by a two-compartment open model. The total clearance of KW 2083 was higher than mitomycin C and hence the biological half life of KW 2083 was shorter. The result may suggest less toxicity of KW 2083 to the host cells.

Liquid chromatographic determination of mitomycin C in human plasma and urine.

A method is given for the determination of the antineoplastic drug mitomycin C in plasma and urine samples. Mitomycin is isolated from the biological matrix with the aid of a Sep-Pak C18 extraction column and eluted with methanol. The methanol is evaporated and the residue is redissolved in the chromatographic mobile phase (methanolic phosphate buffer). Mitomycin C is separated from coextracted compounds by reversed-phase liquid chromatography on a LiChrosorb RP-8 column. A high detection sensitivity and selectivity was obtained by photometric measurements at 365 nm. The precision of the determinations was better than 6% relative standard deviation for plasma samples within the range 2-1000 ng/ml, and for urine samples within the range 0.5-4.4 micrograms/ml. The pH-dependent stability of mitomycin in buffer solutions has been studied.

Determination of mitomycin C in plasma, serum and urine by high-performance liquid chromatography with ultra-violet and electrochemical detection.

The performance of a number of normal phase and reversed-phase systems, with ultraviolet detection at 360 nm, has been investigated with respect to their applicability to pharmacokinetic studies of mitomycin C (MMC). The reversed-phase system developed was also combined with a polarographic detector in order to compare the sensitivity and selectivity of ultraviolet and electrochemical detection. A simple isolation procedure, based on the adsorption of MMC on a non-ionogenic resin, has been developed. The developed assay is applied to a pharmacokinetic study from which some examples are given.

Urinary excretion characteristics of a polymeric prodrug of mitomycin C, mitomycin C-dextran conjugate.

Urinary excretion characteristics of a polymeric prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), following intravenous administration was studied in rats. Three types of MMC-D, conjugates with dextrans of molecular weights of 10000, 70000 and 500000, were tested and urine concentration of MMC, dextran and spacer were determined by three analytical methods, i.e., bioassay, anthrone method and radioactivity counting. MMC was assayed separately as a free form and conjugated form based on antimicrobiological activity. MMC administered as a free form was excreted rapidly into urine but only a small amount of MMC was excreted following the administration of MMC-D. The excreted amount of MMC in a conjugated form varied with the size of carrier dextran while similar sustained excretion was observed regardless of the carrier size. The excretion of carrier dextran determined by anthrone method was confined as the molecular weight was increased. The effect of molecular weight was also observed in the case of spacer-introduced dextran (dextran-C6 spacer) and original dextran. Compared with neutral dextran, cationic MMC-D and anionic dextran-C6 spacer exhibited diminished excretion, indicating the effect of charge on urinary excretion. The urinary recovery of radioactivity was almost in accordance with that of carrier dextran. However, the urinary recovery of MMC based on biological activity was considerably lower than that of carrier dextran. It was suggested that MMC-D underwent inactivation to a great extent before releasing active MMC in the body. The effect of physicochemical properties such as molecular weight and electric charge on the urinary excretion of the polymeric prodrug was thus elucidated.

The stability and antitumor activity of recycled (intravesical) mitomycin C.

Although intravesical mitomycin C (MMC) is effective in the treatment of superficial bladder cancer, its expense is a major factor limiting its use. These authors have analyzed the antitumor activity and stability of MMC following 2-hour intravesical instillation in consideration of recycling the drug or using a smaller dose over a longer retention time. The first voided urine samples from 11 patients who received 40 mg MMC intravesically were measured for MMC content by high performance liquid chromatography (HPLC). An average of 50% of the parent drug was recovered. MMC from the urine samples inhibited the growth of a transplantable murine transitional cell carcinoma as effectively as stock drug. Moreover, MMC is relatively stable in human urine at body temperature. These findings suggest that recovery and reuse of the intravesically administered drug is possible and if sterility and appropriate concentrations can be established for the initial and subsequent doses, the drug may be able to be recycled.

Bladder cancer induced by noncarcinogenic substances.

Chemical carcinogenesis is currently regarded as a complicated series of events requiring initiation and promotion in specific sequences. Carcinogens are thought to be chemicals which can induce both initiation and promotion so that few if any additional host or environmental factors are required in the production of neoplasms. Most experimental studies to date have investigated bladder cancer using these highly active agents and the role of other substances in urothelial neoplasia has not been emphasized. We have examined the role of a variety of solutions, including water and saline, in pilot studies of urothelial carcinogenesis using the ALZA mini-pump for continuous infusion. Bladder tumors indistinguishable morphologically from papillary transitional cell carcinomas were induced. Although experimental induction of bladder cancer with powerful carcinogens may completely overwhelm host defenses and result in more and higher grade neoplasms, similar tumors may occur after exposure to substances not generally considered to be carcinogenic. This process, which probably requires cofactors and host-chemical interaction, may be more representative of environmental carcinogenesis than systems using powerful carcinogens and should be further investigated.

[Pharmacokinetics of mitomycin C in cancer patients during prolonged administration of the preparation]. / Farmakokinetika mitomitsina C u rakovykh bol'nykh pri prolongirovannom vvedenii preparata.

The mitomycin C levels in the blood of patients subjected to prolonged intravenous injection of the drug in 200-300 ml of isotonic sodium chloride solution for 15-50 minutes were determined in the microbiological test system consisting of E. coli and 1.5 per cent of agar in the meat-peptone broth with restricted contents of the nutrients. Such administration of the drug usually provided lower blood levels than intravenous injections of the drug in analogous doses. However, the drug renal excretion was also less intensive. It suggested that the drug administered for a prolonged period was more completely absorbed by the host tissues. This was confirmed by much lower blood levels of the drug, when the tumors were large, as compared to those in patients with insignificant residues of the tumor tissue after surgical resections. The curves of mitomycin C distribution in the blood indicate that the pharmacokinetics of the drug in patients with tumors is a multi-factorial function.

Pharmacokinetics of mitomycin C in patients receiving the drug alone or in combination.

We have characterized plasma pharmacokinetics in 30 patients receiving mitomycin C (mitomycin) in doses ranging from 6 to 20 mg/m2 by iv bolus injection, either alone or in combination with other chemotherapeutic drugs. Plasma elimination of the drug was described by a two-compartment model in all but three cases, giving mean values for alpha-half-life of 8.2 mins, beta-half-life of 51.8 mins, and total body clearance of 332.9 ml/min/m2. The mean urinary excretion was 8.1% of the total dose administered. We did not detect alterations in pharmacologic parameters related to liver dysfunction, dose of mitomycin, and concomitant administration of other cytotoxic drugs.

Detection of mutagenic activity in human urine using mutant strains of Salmonella typhimurium.

Histidine-requiring mutants of Salmonella typhimurium that can be reverted to prototrophy by a variety of mutagens were used mutagenic activity in the urine of patients receiving chemotherapeutic agents. Patients given cyclophosphamide and BCNU had detectable urinary mutagenic activity over a 24-hour period, with maximal levels occurring 12 to 21 hours after drug injection. Whereas native cyclophosphamide required the presence of a rat liver extract to be mutagenic in the test system, the cyclophosphamide metabolites in the urine were fully active in the absence of added liver extract. Mutagenic activity was detected in only the first voided urine specimen of patients receiving fluorouracil. Patients receiving Adriamycin, methotrexate, Mitomycin C, and low doses or oral melphalan did not have detectable mutagenic activity in their urines. One thousand and ten random urine speciments were screened for mutagenic activity. Only eight had greater than 26 revertant colonies per plate. Four of the eight had received metronidazole (Flagyl) for vaginitis while two others had received chemotherapeutic drugs. We were unable to detect increased mutagenic metabolites in the urine of 43 patients with known malignancies, using the standard assay conditions.

Mitomycin C reused: an in vitro cost-effectiveness study.

The use of mitomycin C, an effective agent in the intravesical treatment of superficial bladder cancer, is limited by its high cost. An in vitro study was done to determine whether mitomycin C could be recovered after intravesical administration and reused in the same patient. Cultured human transitional carcinoma cells from line 253-J were exposed to: medium alone; fresh mitomycin C (1 mg./ml.); mitomycin C stored for 1, 2 and 4 weeks; and mitomycin C recovered from patients after intravesical use, tested immediately and stored and tested after serial dilution after 1, 2, and 3 weeks. Results were assessed by cell counts 2 and 5 days after 2-hour exposure to the test solutions. With fresh mitomycin C and that stored up to 4 weeks, cell growth was less than 1 per cent of control (medium alone) at 2 and 5 days. Mitomycin C was also effective when recovered after intravesical use for up to 2 weeks, at which time cell growth was 9.7 and 3.1 per cent of control at 2 and 5 days, respectively. However, at 3 weeks, cell counts were 11.5 and 18.3 per cent of control at 2 and 5 days, suggesting that mitomycin C loses potency with dilution. We conclude that mitomycin C might be recovered, stored and reused for at least 2 weeks in individual patients, a practice that could result in a substantial reduction of treatment cost.

Urine recovery experiments with quercetin and other mutagens using the Ames test.

Recovery from urine of the mutagenic activity of 2-anthramine, cyclophosphamide, 7,12-dimethylbenz[a]anthracene, 6-chloro-9-((3-(2-chloroethylamino)-propyl)amino)-2-methoxyacridin e dihydrochloride (ICR-191), mitomycin-C, nitrofurantoin, and quercetin was studied with several of the Ames tester strains using acetone-extracted XAD-2 columns with yields ranging from 27% to 79%. Dose responses of the pure chemicals were also studied, and results showed TA 97 to be far more susceptible to quercetin mutagenesis than TA 1537. Reducing pour plate agar volume enhanced mutagenesis.