Asparagine Depletion Research Articles

Changes in coagulation and fibrinolysis in childhood ALL: a two‐step dose reduction of one E. coli asparaginase preparation

The influence of two different E. coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood ALL was recently reported, demonstrating a clearly significant association between ASP activity and haemostatic changes. Since the Bayer preparation is no longer available for treatment of large series of patients with ALL, the present study was designed to prospectively evaluate coagulation and fibrinolytic changes in leukaemic children receiving different doses of Medac ASP, which is now available for treatment of childhood ALL. Leukaemic children in whom ASP Medac was administered at 3 d intervals in a two-step dose reduction (5000 IU/m2, n = 10; 2500 IU/m2, n = 15) were compared with children who had received Bayer ASP 10,000 IU/m2 in the same time schedule in a former randomized trial; at the same venipuncture, blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring. Compared with Bayer ASP 10,000 IU/m2, patients receiving Medac ASP 5000 IU/m2 showed significantly decreased values of fibrinogen, plasminogen, and alpha 2-antiplasmin, along with significantly enhanced thrombin generation. Improvement occurred in children treated with 2500 IU/m2 Medac ASP; alpha 2-antiplasmin and D-dimer no longer differed from the Bayer group. Since both patient groups showed complete asparagine depletion during the course of ASP administration, the lower dosage of 2500 IU/m2 administered at 3 d intervals should guarantee the specific metabolic therapy for ALL, leading to depletion of the circulating pool of asparagine.

Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations.

The antileukaemic enzyme L-asparaginase is used to achieve the greatest possible reduction in blood levels of the amino acid asparagine, an essential factor for the growth of leukaemic blasts. There are two main sources of the enzyme, E. coli and Erwinia. Faced with increasing reports of treatment complications, we established a programme to monitor enzyme activity and asparagine levels in serum, in children receiving treatment for acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL). Trough asparagine and asparaginase levels were measured in 49 children on induction treatment with different E. coli preparations (Asparaginase medac, Crasnitin) and in 52 children on re-induction (Asparaginase medac, Crasnitin, and, in the event of allergic reactions, Erwinase) just prior to each sequential application of 10000 U/m2 of asparaginase. Measurements were made by an enzyme assay and an HPLC method. During induction, both Escherichia coli preparations induced the desired reduction in asparagine, but the asparaginase activity with Asparaginase medac was significantly higher than with Crasnitin (median of trough levels 475 versus 74 U/l). Under re-induction treatment (median, Asparaginase medac 528 U/l, Crasnitin 49 U/l, and Erwinase < 20 U/l) complete asparagine depletion was recorded on day 3 in more than 90% of Asparaginase medac samples, more than 60% of Crasnitin samples and in 26% of Erwinase samples. The latter two groups included some children with unchanged asparagine levels and no measurable enzyme activity. Different asparaginase preparations are not readily interchangeable. When Asparaginase medac is used instead of Crasnitin, and identical dose will be associated with significantly higher enzyme activity, well above the level required for complete asparagine depletion. Clinical studies will need to specify both the preparation and the dose to be used. When substitution of an alternative drug is mandatory owing to allergic reactions, monitoring is advisable.

Asparaginase-induced derangements of glutamine metabolism: the pathogenetic basis for some drug-related side-effects.

Several side-effects of asparaginase therapy have been said to be a consequence of the glutaminase activity of Escherichia coli asparaginase, especially the deleterious influence on the liver function. We report here the drug-induced impairments of asparagine and glutamine metabolism in correlation to concentrations changes of plasma proteins, synthesized in the liver, in patients with acute lymphatic leukaemia. One hour after asparaginase application, plasma glutamine decreased to 5% (0-39%: median, range) of the initial values, with a subsequent rise to concentrations slightly lower than those prior to therapy. During the 14 days of drug application the fasting plasma concentrations of glutamine fell to a median of 63% of the pre-therapeutic levels, indicating a depletion of the glutamine pools. Two days after the end of asparaginase application, in one patient the glutamine concentrations increased to the pre-therapeutic range. Plasma concentrations of fibrinogen and antithrombin III decreased to 46% and 56%, respectively, of the initial values, with a slight increase 2 days after the end of therapy. The changes of plasma protein concentrations followed the course of plasma glutamine and asparagine. From that we deduce that the hepatic synthesis of the plasma proteins might be influenced by asparagine and glutamine depletion as a consequence of the therapy with E. coli asparaginase.

Enzyme-induced asparagine and glutamine depletion and immune system function

Depletion of nonessential amino acids and its effect on the immune system can be studied by the administration of bacterial enzymes. Escherichia coli asparaginase hydrolyzes both asparagine and glutamine: administration of this enzyme to mice is rapidly immunosuppressive. Vibrio succinogenes asparaginase hydrolyzes only asparagine and has no apparent effect on immune system function. When the enzymes are rendered nonantigenic and nonimmunogenic by covalent attachment of polyethylene glycol, the effects on immune system function remain the same as described above with the native (nonmodified) enzymes. We believe the data reviewed justify the conclusion that glutamine deficiency is specifically immunosuppressive whereas asparagine deficiency is not. We further believe that enzymatic depletion of nonessential amino acids can be a useful tool for nutritional investigations.

Glutaminase-free asparaginase from vibrio succinogenes: an antilymphoma enzyme lacking hepatotoxicity.

AbstractSuccessful treatment of neoplastic disease has been impeded by the lack of therapeutic agents which specifically destroy tumor cells. Enzymes which selectively deplete substrates required by tumor cells, but not by normal tissue, could improve therapeutic indices dramatically. Presently, microbial asparaginases are used clinically for treating acute lymphocytic leukemia. While these enzymes should destroy neoplastic cells and spare normal tissues, their use is accompanied by many toxic effects and immunosuppression. The administration of Escherichia coli or Erwinia carotovora asparaginase depletes circulating glutamine as well as asparagine. It has been suggested that this glutaminase activity may be responsible for the observed toxicities. We have isolated a glutaminase‐free asparaginase from Vibrio succinogenes with potent antilymphoma activity. Previously, we demonstrated that administration of Vibrio asparaginase does not cause the immunosuppression of humoral or cell‐mediated responses associated with treatment by other microbial enzymes. In the present communication we have evaluated the hepatotoxic effects of different asparaginases since liver damage is the major toxicity associated with treatment by these microbial enzymes. BALB/c mice treated with 50 IU of E. coli asparaginase daily for 4 days exhibited diffuse microfatty infiltration within hepatocytes throughout the liver. Cross‐sections of liver from V. succinogenes asparaginase‐treated mice appeared normal as compared to specimens from control animals. Quantitation of the total amount of extractable lipid from the livers of E. coli asparaginase‐treated animals indicated a 45% and 127% increase in lipid concentration as compared to controls after 4 and 5 days of treatment, respectively. The Vibrio enzyme did not cause a change in extractable lipid concentration as compared to control animals. Plasma antithrombin III activity and albumin, triglyceride and cholesterol concentrations all decreased in E. coli asparaginase‐treated mice, confirming hepatotoxicity. No modifications in plasma proteins were observed in mice treated with asparaginase from V. succinogenes. The plasma lipids did decrease minimally but only the levels of cholesterol were shown to be statistically significant from those of controls. The data strongly support the concept that specific asparagine depletion is not significantly hepatotoxic. More importantly, the asparaginase from V. succinogenes may serve as a potent antileukemic agent without causing damage to normal tissues.

Characterization of the effects of asparaginase from escherichia coli and a glutaminase‐free asparaginase from vibrio succinogenes on specific cell‐mediated cytotoxicity

AbstractAsparaginases isolated from Escherichia coli and Erwinia carotovora are effective in the treatment of acute lymphoblastic leukemia in man. During treatment with either of these enzymes, patients frequently experience pronounced toxicity, including liver and pancreatic dysfunction and immunosuppression. The capabilities of these enzymes to hydrolyze L‐glutamine, an important amino acid in mammalian intermediary metabolism, have led investigators to suggest that the glutaminase activity may be responsible for the observed toxicities. Unlike other asparaginases, an enzyme isolated in our laboratory from Vibrio succinogenes, has been shown to be a potent antilymphoma agent and to be highly specific for L‐asparagine. Our previous work has established that the glutaminase‐free asparaginase from V. succinogenes does not suppress the in vivo humoral or cell‐mediated immune responses of mice to sheep red blood cells, whereas the E. coli enzyme abolishes these responses. In this study, we describe the mechanisms by which E. coli asparaginase suppresses specific antibody‐dependent cell‐mediated cytotoxicity. An analysis of the kinetics of appearance of the specific cell‐mediated cytotoxic response of spleen cells after immunization with sheep red blood cells revealed that it paralleled the IgG plaque‐forming cell response. In addition, 18‐h culture supernatants from immune spleen cells conferred upon non‐immune cells the ability to specifically lyse sheep red blood cells. Pretreatment of immune supernatants with protein A‐Sepharose 4B absorbed out the soluble arming factor. An analysis of E. coli asparaginase‐induced suppression of specific antibody‐dependent cell‐mediated cytotoxicity revealed that a decreased synthesis of specific IgG was responsible for the observed reduction in cytotoxic reactivity. When culture supernatants from spleen cells of immunized animals not treated with asparaginase were added to spleen cells from E. coli asparaginase‐treated mice, however, there was an increased cytotoxic response. This suggested that there was an increase in the number or ratio of effector cells for the specific antibody‐dependent cell‐mediated cytotoxic response in spleens of enzyme‐treated animals. Through the characterization of the immunosuppressive effects of two asparaginases—one having the catalytic capability to hydrolyze L‐glutamine and the other lacking this catalytic activity—we now have evidence that the immunosuppressive effects of E. coli asparaginase on specific antibody‐dependent cell‐mediated cytotoxicity cannot be the result of asparagine depletion alone. Our data demonstrate that the glutaminase‐free asparaginase from V. succinogenes is not immunosuppressive and suggest that an asparaginase with high substrate specificity for L‐asparagine may be a safer treatment for leukemia patients.

Protein synthesis in stimulated lymphocytes: II. Amino acid requirements

The amino acids required for protein synthesis by phytohemagglutinin (PHA) and antigen stimulated lymphocytes have been determined by measuring the kinetics of protein synthesis in amino acid deficient media. Stimulated lymphocytes have a partial requirement for serine in addition to the 13 amino acids which Eagle (1) found to be essential for mammalian cell cultures. Asparagine is not required for the lymphocyte response. This observation supports the suggestion that l-asparaginase inhibits lymphocyte blastogenesis by its action on the membrane rather than by depletion of exogenous asparagine. With the exception of serine, tryptophan, and isoleucine, the omission of an essential amino acid results in complete inhibition of PHA induced protein synthesis. In the absence of either tryptophan or isoleucine, the rate of protein synthesis is unaffected during the first 30 hr of incubation but subsequently declines rapidly to that of unstimulated cultures, suggesting a possible role for tryptophan and isoleucine in the regulation of the lymphocyte cell cycle. The inhibition of protein synthesis by amino acid deficiencies is reversible upon replacement of the missing amino acid.

Nutritional effects on precursor uptake and compartmentalization of intracellular pools in relation to RNA synthesis

The effects of nutritional variables on the processing of exogenous precursors into RNA was examined. General nutritional deprivation, or asparagine depletion, led to significant changes in the absolute pool sizes, especially of ATP, UTP and CTP. Fluctuations were found depending on the elapsed time after the nutritional perturbations occurred, and the cell density of the cultures. Depletion of the medium by 28 h of growth, or 1 h of guinea pig asparaginase action, led to considerable inhibition of the conversion of exogenous uridine to CTP by the cells. A series of experiments indicated that in 6C3HED lymphoma cells the uridine nucleotide pool which provides the immediate precursors to RNA (denoted UTP N A) behaves as a small compartment in rapid equilibrium with exogenously supplied nucleosides. The resemblance to the compartmentation model described by Plagemann (Plagemann, P.G.W. (1972) J. Cell Biol. 52, 131–146 and (1971) J. Cell. Physiol. 77, 241–258) for rat hepatoma cells was close. The UTP N A pool of the 6C3HED cells constitutes no more than 5% of the cellular UTP pool and is relatively slow in equilibrating with the general cell pool. Correction of the rates of incorporation of isotope into RNA by using some function of the whole cell UTP specific activity to normalize the pool effects, was shown to be invalid.

Concentrations of asparagine in tissues of prepubertal rats after enzymic or dietary depletion of asparagine.

Growth of weanling rats was significantly depressed after 8 days of asparagine depletion produced by dietary means or by asparaginase treatment. Moreover, the concentration of free asparagine was significantly lowered in forebrain, skeletal muscle, liver, kidney, spleen and small intestines 3 h after an asparaginase injection, but remained lowered only in forebrain and skeletal muscle after 8 days of enzymic or dietary depletion of asparagine.

Some clinical observations on the treatment with L-asparaginase of the acute leukemias.

The history of this new type of enzymatic depletion of the amino acid asparagine, which has been shown to be indispensable for the growth most particularly of the early cellular stages of the lymphatic system and, to a lesser degree, of some other tissues, has passed through several distinct investigational phases. The first stage was, characteristically, purely biological, starting with Clementi’s [17] preliminary report in 1922 of the richness in asparaginase of guinea pig serum, comprising Kidd’s [30] pioneering observations in 1953 on the suppression of certain transplanted lymphomas of mice and rats by guinea pig serum and finally including Broome’s [13] experimental demonstration that the enzyme L-asparaginase (L-ase) was the growth-inhibitory fraction of guinea pig serum, a finding later confirmed by the demonstration by Mashburn and Wriston [34] that L-ase derived from Escherichia coli had the same effect as that from guinea pig serum. This important discovery, together with the additional demonstration that L-ase derived from E. coli exists in two forms, known as EC-1 and EC-2 [16], of which only the second was active against 6C3HED lymphoma in mice [39, 40], opened up the possibility of large-scale production for clinical trials.