Search

In vitro experiments showed that 6-diazo-5-oxo-L-norleucylglycine, a dipeptide analog of L-glutaminylglycine, inactivates gamma-glutamyl transferase bound to renal brush border membrane vesicles but does not inactivate the purified transferase. The rate of inactivation of the membrane-bound enzyme decreased markedly in the presence of dipeptides, such as L-leucylglycine and L-alanylglycine, or in the presence of o-phenanthroline, an inhibitor of renal peptidases. The presence of L-cysteinylglycine S-acetyldextran polymer (Mr 500,000), which does not permeate membranes, protected the membrane-bound transferase from inactivation by 6-diazo-5-oxo-L-norleucyglycine. This and other findings suggest that the norleucylglycine derivative was hydrolyzed by peptidase(s) bound to the outer surface of the brush border membranes and that the 6-diazo-5-oxo-L-norleucine thus released acts as an affinity-labeling reagent for the membrane-bound transferase. Similar effects were observed in vivo. Intravenous administration of 6-diazo-5-oxo-L-norleucylglycine to mice resulted in a marked decrease in renal transferase activity. Mice thus pretreated with 6-diazo-5-oxo-L-norleucylglycine, but not an untreated group, excreted significant amounts of S-carbamido[14C]methylglutathione in their urine within 30 min of intravenous administration of this compound. This finding suggests that the renal transferase was involved in the hydrolysis of the glutathione S-conjugate in the glomerular filtrate in vivo and that the administered 6-diazo-5-oxo-L-norleucylglycine underwent hydrolysis peptidase(s)-catalyzed to liberate 6-diazo-5-oxo-L-norleucine that reacted with the membrane-bound gamma-glutamyl transferase.

DON, a glutamine antagonist, was administered iv to 26 patients with advanced cancer either once every 3 wks or daily for 3 days every 3 wks to determine toxicity and to look for evidence of therapeutic effect. Total doses ranged from 150 to 600 mg/m2. The single-day schedule produced intolerable nausea and vomiting and no evidence of cytotoxicity at 450-550 mg/m2 given over 10 mins or over 4 hrs. On the 3-day schedule, patients had tolerable gastrointestinal toxic effects at total doses up to 480 mg/m2 given in three equally divided doses by 10-min infusion. This dose also produced cytotoxic activity manifested as transient mild leukopenia and, rarely, thrombocytopenia. No objective responses were seen. Analysis of the plasma elimination of DON demonstrated dose-dependent pharmacokinetic behavior. The parent compound was not detectable in the urine of any patient, indicating extensive metabolism of the drug.

The diazo antibiotic 6 diazo-5-oxo-L-norleucine is transported into Escherichia coli using the general aromatic amino acid transport system and a specific phenylalanine transport system. Resistance to this antibiotic is the result of a decreased rate of transport of the antibiotic and also the result of a more rapid rate of efflux of the antibiotic from the cell.

The uptake system for 6-diazo-5-oxo-L-norleucine (DON) was studied in mouse P388 leukemia cells. The DON transport system was found to resemble that of another glutamine antimetabolite, Acivicin, in its strong temperature dependence, utilization of the "L" transport system, inhibition by glutamine but not by glutamate, potent inhibition by p-chloromercuribenzene sulfonate, Na+, and only minimal inhibition by various energy poisons. A Km of approximately 70 microM and a Vmax of 3.4 nmoles/10(6) cells/min was calculated for this cell line. The accumulated DON was not metabolized by P388 cells and moderate efflux occurred at 37 degrees C. The DON transport characteristics of a DON-resistant P388 cell line (100 times ID50 of parent line) were similar to those of the DON-sensitive parent line, indicating that altered drug transport may not be involved in development of resistance to this antimetabolite. The finding that an Acivicin-resistant subline of P388 cells which exhibited good transport of DON showed negligible transport of Acivicin suggests different modes of resistance towards the two glutamine antimetabolites.