Kinase and deaminase activity in a variety of subcutaneous mouse tumors.

Extracts of solid mouse tumors were examined for deoxycytidine kinase and deaminase activities. 1beta-D-Arabinofuranosylcytosine nucleotide was formed at a rate of 45 nmoles/hr by Glioma 26/57 and only 14 nmoles/hr by Ridgway osteogenic sarcoma. Deaminase activity was highest in Lewis lung (114 nmoles of 1-Beta-D-arabinofurano-syluridine formed per hr) and in CaD2 (104 nmoles of u-beta-D-arabinofuranosyluridine formed per hr). Deaminase activity in tumor extracts is sensitive to freezing, while deaminase activity in monkey serum is not. It was observed that kinase activity varies by as much as 50% in different cell lines of the same tumor. In the presence of tetrahydrouridine, kinase activity was significantly increased in most of the tumors studied.

Phase I trial of misonidazole (NSC#261037) plus cyclophosphamide in solid tumors.

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.

Kinetics of intravenous melphalan.

We have studied the disposition and elimination of melphalan after intravenous administration in 9 patients with cancer. High-pressure liquid chromatography and 14C-melphalan were used to assay drug concentration in plasma and urine. Composite plasma t1/2alpha was 7.7 +/- 3.3 and t1/2beta was 108 +/- 20.8 min for 8 of the patients. The mean 24-hr urinary excretion of melphalan was 13.0 +/- 5.4% of the administered dose. In 2 patients, 80% to 100% of the measured 14C counts in plasma and urine samples at each study interval, up to 24 hr after drug administration, could be accounted for by the sum of parent compound, monohydroxy and dihydroxy products, and methanol nonextractable radioactivity (i.e., protein-bound activity). These data and evidence of rapid disappearance from plasma at 37 degrees in vitro suggest that spontaneous degradation, and not enzymatic metabolism, is the major determinant of the t1/2 of melphalan in vivo.

Quadrupole-based mass spectrometric evaluation of isotope ratios of carbon dioxide in expired air from mice and men following the administration of (13)C-methyl methionine.

A method is described for the measurement of the isotopic ratio of (13)CO2/(12)CO2 in expired air from individual mice and from humans by means of a quadrupole-based mass spectrometer system. Following the administration of (13)C-methyl methionine or another appropriately labeled substrate, the (13)C portion of the molecule is converted to (13)CO2. The (13)CO2 enters the carbonate pool(s) and is ultimately eliminated in the expired air where it is available for analysis. The expired air is transported by a small pump from the subject to a digital valve which provides for the alternate influx of expired air and standard into the mass spectrometer for 30 or 60 seconds each, respectively. The inlet consists of a control valve connected to a microbore stainless steel tube, and can be adjusted manually to achieve a source pressure of 4 X 10(-5) torr. The correction factors for drift in sensitivity and in the mass axis are generated by repeated, automatic analysis of the running standard and relating those measurements to values generated for the standard during the first minutes of the experiment. Each measurement of an isotopic ratio in expired air is corrected by an amount determined by the standard immediately preceding it. Precision for the measurements of both sample and standard ratios is ±0.2%. The technique should prove useful in assessing the metabolism, of substrates that are converted to CO2 and may find utility as a diagnostic tool for certain diseases and metabolic disorders.

Immunochemical properties of junctional and extrajunctional acetylcholine receptor.

Immunological assays were performed to compare two distinct forms of the nicotinic acetylcholine receptor (AChR): junctional (JR) and extrajunctional receptor (EJR). Antibodies from myasthenia gravis patients' sera inhibited the binding of [125I]alpha-bungarotoxin (BGT), to EJR more effectively than binding to JR. Immunological differences between JR and EJR were confirmed by other assay methods. In all cases, EJR appeared to have antigenic determinants not found on JR. It was established that enzymatic removal of carbohydrates from EJR caused it to more closely resemble JR. Thus differences between JR and EJR may be due, in part, to carbohydrate residues found on EJR that are absent on JR. The extent of antibody binding to EJR was examined by gel filtration methods. Immunochemical studies of bands from SDS gels showed that antibodies are present in myasthenic serum which react with the 3 subunits (42, 53, 64 kdaltons) of AChR to varying degrees.

Structure of the group-specific polysaccharide of group E Streptococcus.

The structure of the group-specific polysaccharide of group E Streptococcus was determined by methylation, periodate oxidation, and partial methanolysis, and the configuration of the anomeric linkages by 1H- and 13C-n.m.r. spectroscopy. The trisaccharide repeating unit----2)-alpha-L-Rhap-(1----3)-[beta-D-Glcp-(1----2)]-alpha-L -Rhap-(1----was determined.

Structure of the serotype f polysaccharide antigen of Streptococcus mutans.

The structure of the serotype f polysaccharide antigen of Streptococcus mutans was determined by methylation analysis, periodate oxidation, and partial methanolysis, and the configuration of the anomeric linkages by 13C-n.m.r. spectroscopy, indicating the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Glcp-(1----3)]-alpha-L-+ ++Rhap- (1----. The structure of the backbone of the polysaccharide was confirmed by demonstrating immunological identity between the product of Smith degradation of the S. mutans serotype f antigen and the group A-variant streptococcal polysaccharide.

Structure of the group G streptococcal polysaccharide.

The structure of the group-specific polysaccharide of group G Streptococcus was determined by means of methylation analysis and selective chemical degradations. The anomeric configurations and conformations of the sugar residues were studied by 1H- and 13C-n.m.r. spectroscopy. The tetrasaccharide repeating unit, ----3)-alpha-D-Galp-(1----2)-[alpha-L-Rhap-(1----3)-beta-D-GalpNAc - (1----4)]-alpha-L-Rhap-(1----, was determined.

A method for differentiation and analysis of quinine and quinidine by gas chromatography/mass spectrometry.

A method is presented for the positive identification of quinine and quinidine. The method requires extraction of the compounds from an alkaline solution into an organic solvent and concentration of the sample by evaporation of the solvent. The sample is chromatographed without derivatization and may be detected by the flame ionization detector (FID) or mass spectrometry (MS). Limits of sensitivity are less than 5 ng per assay for the underivatized compounds. Analysis of urine extracts is discussed.

Inhibition of deamination of 14C-cytosine arabinoside (NSC-63878): a useful biologic assay for tetrahydrouridine (NSC-112907).

Inhibition of the deamination of 14C-cytosine arabinoside by two lots of tetrahydrouridine was studied in monkey serum. The average inhibition of deaminase activity was 78% for tetrahydrouridine lot AJ39 (1.0 muM) when the concentration of cytosine arabinoside ranged from 44.2 to 170.7 muM; under the same conditions tetrahydrouridine lot AJ22 inhibited deamination by an average of 68%. Apparent Ki values were 0.26 muM for AJ39 and 0.43 muM for AJ22. The assay may be used to check the relative biologic activity of various lots of tetrahydrouridine.

Pharmokinetics of 2'-deoxycoformycin in normal and L1210 leukemic mice.

Following intravenous administration, 2'-deoxycoformycin (0.25 mg/kg) was rapidly distributed to tissues of both normal mice and mice bearing L1210 leukemia cells and readily eliminated, primarily by urinary excretion. Elimination of 2'-deoxycoformycin from plasma was biphasic, and half-lives for the alpha- and beta-phases of 10 and 33 min for normal mice and 7 and 40 min for L1210-bearing animals. The volume of distribution at steady state was approximately 20 ml, suggesting that the drug was distributed in the total body water for both groups of mice. The kidney, liver, small intestine, spleen, thymus, and L1210 tumor had tissue/plasma ratios greater than or equal to 1 at 15 min after dosing. In both groups, greater than 90% of the dose of 2'-deoxycoformycin was recovered in the urine within 3 hr. As determined by bioautography of urine samples, no detectable metabolism occurred. The presence of the L1210 tumor caused changes in the tissue distribution of 2'-deoxycoformycin. At later time periods, tissues from tumor-bearing mice contained significantly higher levels of this drug when compared to normal mice. However, the tumor was without significant effect on blood levels or urinary excretion of 2'-deoxycoformycin.

Influence of tetrahydrouridine on the phosphorylation of 1-beta-D-arabinofuranosyl-cytosine (ara-C) by enzymes from solid tumors in vitro.

The phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in cell-free extracts from a variety of solid mouse tumors, L1210 ascites and in normal liver and spleen. Two apparent Michaelis constants were observed for kinase activity in Lewis lung (Km1, 4.15 muM; Km2 58.1 muM), sarcoma 180 (Km1 6.66 muM; Km2 56.18 muM), adenocarcinoma 755 (Km1 4.34 muM; Km2 50.0 muM) and l1210 (Km1 29.41 muM; Km2 41.67 muM). The Km1 values generally ranged from 5 to 20 muM 3H-ara-C while the Km2 values ranged from 20 to 60 muM 3H-ara-C. Normal spleen (Km 47.6 muM), normal liver (Km 10.0 muM) and Ridgway osteogenic sarcoma (Km 31.2 muM) had single Km values. In the presence of tetrahydrouridine (H4U), the in vitro phosphorylation of ara-C was increased as much as 91% in cell-free extracts from adenocarcinoma 755; lesser increases were observed in other tumor extracts. At low substrate concentrations, the apparent Km decreased or did not change in the presence of H4U, while at higher substrate concentrations the apparent Km was increased or did not change in the presence of H4U. In the presence of H4U, Vmax for kinase activity increased most in those tumors possessing deaminase activity.

Pharmacokinetics of the absorption, distribution, and elimination of melphalan in the dog.

[14C]melphalan ([14C]L-PAM) was rapidly absorbed from the gut of dogs after oral dosing and reached a maximum concentration in the serum by 30 minutes. The disappearance of L-PAM (intact drug) from the serum was biphasic after iv administration, with half-lives of 14 and 66 minutes for the alpha and beta phases, respectively. The urinary excretion accounted for 44% of the total radioactivity and 25% appeared in the feces. Approximately 8% of the dose was excreted unchanged in the urine. Biliary excretion was rapid, with 11% of the dose being accounted for in the bile after 30 minutes; approximately 80% of these drug equivalents (materials containing radioactivity) was parent compound. Since large amounts of L-PAM appeared in the bile, the agent may prove to be active against cancers of the gall bladder, bile ducts, and duodenum.