Elton R. Homan

Studies on Poly I:C toxicity in experimental animals☆☆☆

Abstract Polyinosinic:polycytidylic acid copolymer (Poly I:C), a synthetic double stranded RNA, has been proposed as a candidate for clinical introduction as an antitumor agent. In preclinical toxicologic evaluation, acute lethality studies were undertaken in 3 mouse strains, anaphylaxis studies in guinea pigs, and acute and subchronic toxicities were evaluated in rhesus monkeys and beagle dogs following daily parenteral dosing for up to 28 days. Poly I:C is more lethal to mice following iv administration than ip, and there appear to be differences in mouse strain susceptibility. No evidence of anaphylaxis was seen in guinea pigs. Signs of toxicity in dogs and monkeys were dose related, were qualitatively similar in the 2 species, and included hypoactivity, anorexia, emesis, diarrhea, ataxia and weight loss. Clinical changes included anemia, elevations of transaminases, lactic dehydrogenase, and alkaline phosphatase, decreased clotting rate and increased prothrombin time. Pathologic changes consisted of focal hemorrhages, infarction or congestion of a variety of organs, vasculitis and decreased erythroid hematopoiesis. The effects were more severe in dogs than in monkeys for equivalent mg/kg doses. In the monkey iv administration produced greater toxicity than im or ip administration. Signs of toxicity and pathologic changes appeared to be reversible in both dogs and monkeys after cessation of dosing.

Don, conv and donv--I. Inhibition of L-asparagine synthetase in vitro.

Abstract The inhibition in vitro of l -asparagine synthetase ( l -glutamine hydrolyzing, EC 6.3.5.4) from leukemia 5178Y rendered resistant to l -asparaginase (L5178Y/AR), and from mouse pancreas by the ketoamino acids DON ( l -DON; 6-diazo-5-oxo- l -norleucine), CONV ( l -CONV; 2-amino-5-chlorolevulinic acid; 5-chloro-4-oxo- l -norvaline) and DONV ( l -DONV; 5-diazo-4-oxo- l -norvaline) was investigated using both l -glutamine and ammonium chloride as substrates. At a concentration of 1 mM, DON and CONV almost completely inhibited the utilization in vitro of l -glutamine by l -asparagine synthetase of L5178Y/AR and of mouse pancreas, whereas DONV inhibited both enzymes only by 50 per cent. DON, CONV and DONV did not affect the utilization in vitro of ammonium chloride by l -asparagine synthetase of L5178Y/AR, while DON and CONV modestly inhibited the utilization of ammonium chloride by the pancreatic enzyme. The inhibition produce by DONV was fully and rapidly reversed by dialysis, whereas that produced by DON and CONV was essentially irreversible. The utilization in vitro of l -glutamine by eight fetal rat liver amidotransferases was strongly inhibited by DON and CONV, while DONV exerted modest inhibition on only two of these enzymes. In a survey of other enzymes which use l -asparagine and l -glutamine as substrates, DONV was shown to be the best inhibitor of l -asparagine-utilizing enzymes, DON was the best inhibitor of enzymes utilizing l -glutamine, and CONV affected both groups of enzymes to a variable degree. DON irreversibly inhibited l -asparaginase from Erwinia carotovora (EC 3.5.1.1). This enzyme also was found to catalyze the decomposition of DON. From these findings and also from kinetic studies, it is clear that all three ketoamino acids are capable of behaving as antagonists of l -glutamine in vitro . DON is the most universally active antagonist of this amino acid, followed by CONV, and last by DONV. Similarly, all three agents can function as l -asparagine antagonists under appropriate conditions.

DON, CONV and DONV—III. Pharmacologic and toxicologic studies

Abstract The pharmacologie, toxicologic and oncolytic properties of the ketoamino acids DON ( l -DON; 6-diazo-5-oxo- l -norleucine), CONV ( l -CONV; 5-chloro-4-oxo- l -norvaline; 2-amino-5-chloro-levulinic acid) and DONV ( l -DONV; 5-diazo-4-oxo- l -norvaline) were examined. DON was found to be the most active therapeutic agent of the three drugs against leukemia 1210 and also the most potent cytocidal agent against KB tumor cells in culture. The acute ld 50 values of the agents were dissimilar: CONV was the most toxic drug of the three after single intraperitoneal injections, and DONV the least toxic. Only DON showed evidence of prominent cumulative toxicity. In studies with isolated cells of leukemia 5178Y rendered resistant to l -asparaginase (L5178Y/AR), all three agents appeared to compete both with l -asparagine and with l -glutamine for transport into the cell. DONV competed most effectively with l -glutamine and CONV most effectively with l -asparagine. In mice, all three drugs were cleared from the plasma and excreted into the urine at a rapid rate. None was bound to the proteins of mouse plasma. After an intraperitoneal injection of 100 mg/kg, the concentration of DONV in the pancreas was approximately ten times that of CONV or DON; after comparable intravenous injections, only DONV could be identified in this tissue. Although the metabolism of all three ketoamino acids was found to be minor in degree, evidence is presented that they can be degraded in vitro by organ homogenates and also that purified enzymes can catalyze their transamination. In addition, DON was a good substrate for renal γ-glutamyl transferase (EC 2.3.2.2). In the case of DONV, some conversion to CO 2 by isolated tumor cells also was observed. From these and previous studies it is concluded that, of these analogs of l -glutamine and l -asparagine, DON is the most “ l -glutamine-like” agent of the three, DONV the most “ l -asparagine-like,” while CONV has important attributes of both amino acids.

A facile radiometric technique for measuring ATP

Abstract A facile radiometric technique for measuring ATP in samples of biologic origin is presented. The d -glucose-6-phosphate produced by the phosphorylation of excess tritiated d -glucose with crystalline hexokinase is stoichiometrically equivalent to the ATP present. Product is selectively precipitated with ethanolic barium acetate, washed with ethanol and the tritium label counted in a scintillation spectrometer. The technique is especially suitable for the measurement of ATP in large numbers of samples (50–100) and offers acceptable sensitivity down to 62 fmoles.

Hemostatic toxicity of poly I-C

Abstract Intravenous therapy in beagles with Poly I-C resulted in a dose-related toxicity. In animals receiving 3.0 mg/kg, the clinical and pathological evidence indicated that disseminated intravascular coagulation (DIC) led directly to their death. In animals receiving 0.3 mg/kg evidence for DIC was present both by clinical and pathological studies; however, the DIC seemed to be “compensated,” since animals survived the entire treatment schedule. Animals receiving 0.03 mg/kg displayed coagulation abnormalities only without postmortem evidence of thrombotic lesions.

Inhibition of L asparagine synthetase by mucochloric and mucobromic acids

Mucochloric and mucobromic acids are powerful inhibitors of tumoral and pancreatic L-asparagine synthetases. Two nitrogen donors, L-glutamine and ammonia, can be used by these enzymes; at a concentration of 1 mmol/l, mucochloric and mucobromic acids preferentially inhibit the utilization of ammonia as opposed to L-glutamine in vitro. Using the tumoral enzyme, kinetic analysis revealed that mucochloric acid produced inhibition which was apparently noncompetitive with ammonia but competitive with L-glutamine. In molar excess, L-glutamine and dithiothreitol effectively antagonized such inhibition; dialysis, however, failed to reverse established inhibition. These findings, suggest that the drugs operate by covalent attachment to crucial sulfhydryl functions on the enzyme.

Ethacrynic acid-an inhibitor of l-asparagine synthetase

Abstract Ethacrynic acid, a clinically useful diuretic, has been shown to inhibit l -asparagine synthetase from leukemia 5178Y resistant to l -asparaginase (L5178Y/AR) in vitro. This inhibition is thought to involve the formation of an adduct between ethacrynic acid and sulfhydryl functions on the enzyme; the adduct is not readily reversible even when another thiol, such as dithiothreitol, is used to displace the acid. A series of analogs of ethacrynic acid were examined as inhibitors of l -asparagine synthetase. Only one proved to be superior to the title compound; it was 2 2-(1-carboxymethoxy-4-chloro-2-naphthyl)3,6-diethyl-6-(1-carboxymethoxy-4-chloro-2-naphthoyl)-5,6-dihydro-[4H]-pyran (Compound D). There was no correlation between the diuretic or natriuretic potency of the series of compounds examined and their ability to inhibit l -asparagine synthetase activity. There was also no correlation between the ability of these agents to inhibit l -asparaginase from Dasyprocta agouti (another sulfhydryl enzyme) and l -asparagine synthetase from L5178Y/AR. In every case, ethacrynic acid and its analogs interrupted the utilization of ammonia by l -asparagine synthetase to a greater degree than the utilization of l -glutamine. In vivo, the inhibition by l -asparagine synthetase from L5178Y/AR by ethacrynic acid was feeble, while the analogous enzyme from pancreas was inhibited to a significant degree.