The clinical pharmacology of intermediate-dose oral methotrexate (MTX) was studied in nine patients receiving 18 courses of treatment. Serum and urine MTX concentrations were measured by means of a competitive protein binding assay after oral aqueous solution (3 courses), 50-mg tablets (13 courses) or IV drug (2 courses) had been administered in four doses of 100 mg/m2 at 6-h intervals or in four doses of 200 mg/m2 at 6-h intervals and followed by citrovorum factor rescue. Levels above 150 ng/ml (3.3 x 10(-7) M) were maintained throughout all treatment cycles, with rapid disappearance of drug after the last dose. A 100% increase in administered dose resulted in only a 42% increase in the concentration-time level. Methotrexate was absorbed well from both aqueous solutions and 50-mg tablets, but serum levels after 50-mg tablets were only 20% of those achieved after IV administration. We conclude that significant serum MTX concentrations can be achieved for prolonged periods of time after oral administration of intermediate doses, but that the proportion of drug absorbed is much less than is seen with lower doses.
To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 1251 These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.
Misonidazole, a hypoxic cell sensitizer, enhances the antitumor effects of cyclophosphamide in preclinical studies. Several studies also showed increased cytotoxicity for normal tissues. We undertook a phase I study of this combination. The regimen consisted of oral administration of misonidazole at one of two dose levels, 1 g/m2 and 2 g/m2, followed by an intravenous (IV) injection of cyclophosphamide four hours later. The cycle was repeated every twenty-one days. The dose of misonidazole remained constant for each regimen, but the dose of cyclophosphamide ranged from 0.4 g/m2 to 1.3 g/m2. Thirty-eight trials in 35 patients with advanced solid tumors were considered evaluable. Dose-limiting toxicity was granulocytopenia at 1 g/m2 of cyclophosphamide without significant thrombocytopenia or anemia. Peripheral neuropathy was negligible. Two patients received cumulative doses of 8 and 16 g/m2 of misonidazole without neurotoxicity. One patient developed hemorrhagic cystitis. Nausea and vomiting was mild to moderate. Possible evidence of tumor stabilization was seen in three patients, and one patient had a mixed response. The mean serum half-life for misonidazole was 11.3 hours (range, 8.4 to 20.0) and for cyclophosphamide 8.3 hours (range, 3.2 to 15.5), both within the previously reported ranges. In conclusion, it appears that this combination is well tolerated and that misonidazole does not significantly potentiate myelotoxicity caused by cyclophosphamide or alter its pharmacokinetics. The recommended starting doses for misonidazole and cyclophosphamide in phase II trials using this schedule of administration should be 2 g/m2 and 1 g/m2, respectively, with escalation for cyclophosphamide to individual tolerance.
Ametantrone acetate is an intensely blue anthracenedione undergoing clinical trials in man. In this Phase I study, 20 patients received 39 courses of drug as a single IV dose given daily for five days and repeated every three weeks (21 days). Dosage escalations proceeded from 15 mg/m2 to 35 mg/m2. Predictable and reversible leukopenia was the dose limiting toxicity. One previously untreated patient with renal cell carcinoma metastatic to the lungs and right arm experienced a partial response of 51 days duration. Nine patients had pharmacokinetic studies performed during the study. Ametantrone was extensively distributed (apparent volume of distribution, 26.3 l/m2) and demonstrated a short half-life (harmonic mean half-life, 0.38 hour). The maximum tolerated dose in this study was 35 mg/m2. Recommended doses for Phase II trials are 30 mg/m2 in good risk patients and 25 mg/m2 in poor risk patients. Because of the partial response seen in one patient with renal cell carcinoma, Phase II trials should include this tumor category in order to better define the activity of ametantrone in this disease. In addition, since the total amount of drug that could be given to patients receiving the five day schedule (125-150 mg/m2) was approximately the same amount that could be administered as a single dose (140 mg/m2), it would appear that the only advantage of the daily times five day dosage schedule would be in the lower incidence of bluish skin discoloration.
Despite the fact that autoantibodies react with body constituents of the animals that produced them, they are best induced by immunization with cross-reacting antigen from a foreign (xenogenic) species (1, 2). We have come across a situation in which a pancreas-specific autoantibody is regularly induced in rabbits by immunization with antigenic material prepared from dog pancreatic zymogen membranes.Materials and methods. Pancreases were obtained from dogs which had been fasted for 24 hr. These animals were anesthetized with pentobarbital; the pancreas was removed and immediately placed in ice cold buffer containing lOmM tris-maleate, 0.25 M sucrose and 5 X M EGTA, pH 5.5 (TMSE buffer). The tissue was then washed and homogenized in 200 ml of cold TMSE buffer with a Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenate was filtered through a 1 10-mesh cheesecloth and fractionated by differential centrifugation into nuclear, zymogen granule (ZG), mitochondrial, and microsomal subfractions. ZG membranes were prepared from ZG's by lysis in three volumes of 0.2 M sodium borate in 0.9% NaC1, pH 8.0 followed by centrifugation to remove soluble components, and flotation of the membranes on a discontinuous sucrose density gradient as described by Meldolesi et al. ( 3 ) .The various fractions were assayed for succinate dehydrogenase (SDH), trypsin, and amylase activity, and, as judged by these determinations, the membrane subfraction contained only trace amounts of nonmembrane contaminants. The ZG fraction, however, was usually contaminated by mitochondia to the extent of 10%.Proteins of the different subfractions were compared by electrophoresis on 10% polyacrylamide gels containing 0.1 % sodium dodecyl sulfate (SDS) according to the method of Lamli (4), except that the samples were treated with 1% SDS at room temperature approximately 10 min before application to the gels. Where applicable 3 % P-mercaptoethanol was also added. Coomassie Brilliant Blue R was used for visualization of proteins, while the periodic acid Schiff stain (PAS) (5) was used for sugar residues.Antibody was prepared by combining the purified ZG membrane preparation (100 pg/ml) with an equal volume of complete Freunds' adjuvant (Difco Laboratories, Detroit, Michigan) ; the resulting emulsion was injected subcutaneously into the nape of the neck and dorsal thoracic skin of three albino rabbits. The antigen with the addition of incomplete Freunds' adjuvant was injected at 2-week intervals for two more doses. After a rest of 3 weeks, a second series of five inoculations was applied. Final bleedings were performed 7-10 days after the last immunization and collected antiserum was stored at -20°C until use.The antiserum was evaluated by Ouchterlony double-diffusion in 1 % agarose gel. Protein content of the various antigens was routinely determined by the method of Lowry (6), using bovine serum albumin as a standard. Protein concentrations of the pancreatic subfractions were 23.5, 10.5, and 1 mg/ml, for the crude pancreatic homogenate, th...
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