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Biochemical and Pharmacological Studies with Asparaginase in Man

Takao Ohnuma, James F. Holland, Arnold Freeman, et al. Cancer Res 1970;30:2297-2305. Published online September 1, 1970.

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aacrjournals. and 1 each infusion in three patients for acute brain dysfunction with with CML in the blastic phase. It was difficult to correlate response in vivo prior chemotherapy at the time of the asparaginase trail. EC 3. and had converted from préexistentLSA. Asparagine was given as a "rescue" malignant melanoma. Portions of this work have been reported in a preliminary form elsewhere (32). injec tion. Even 0. 2297-2305. Ohio. and Pharmacological Arnold Freeman. Fairlawn. pyrexia (30). anhydrous. Cleveland. The antiResearch Laboratories. and carcinoma of the colon. New York State Department of Health. Buffalo. phenol loose ferent origins had different rates of clearance from plasma and crystals.1. Nevertheless.u.. choriocarcinoma. coli enzyme. Ml. M2. 30). general toxicity of the host was not regarded as a critical problem (14. 37. In a critically ill child with ALL. Five and 1 of 12 patients with acute myelocytic leukemia were patients with AML had received no prior treatment. or weekly. 1970. bone marrow rating 1. All and a requirement of the leukemic cells for asparagine in vitro. acute myelocytic leukemia. New York 14203 SUMMARY clinical trials in ALL2 in children with the E. Inc. Asparagine reappeared in plasma 23 to 33 days after single injections.v. 2 after the remaining leukemic patients were in frank relapse from single injections. Patients with leukemia were evaluated by the rating criteria of Acute Leukemia Group B (15). 2011 Copyright © 1970 American Association for Cancer Research . Sinks Departments of Medicine A /T.. Regression of transplanted mouse and rat lymphomas by Asparaginase was obtained from Merck. 35) provided a sodium nitroprusside. CML. Fisher Scientific Co. 12. Louis. 9. kidney. 40).31). Tris and tion of asparaginase from E. 5). The present paper reports our observations on bio chemical and pharmacological studies with E. 24. Asparaginase produced multiple manifestations of toxicity involving brain. the peak activity obtained was dose related. whereas induced to bone marrow rating 1 marrow remission. In the initial clinical trials with E. Response in solid tumors was defined INTRODUCTION by at least 25% decrease in the product of 2 diameters as a reflection of tumor size. Three of 10 patients with acute lymphocytic leukemia had marked aber ration in their amino acid levels. lymphosarcoma. Holland. The drug was given in three different schedules: as a single dose.1) (4. Ten of 24 patients with acute lymphocytic leukemia blastoma. and L-aspartic acid. and intestinal hemorrhage were observed after guinea pig serum enzyme administration (14). Roswell Park Memorial Institute. coli enzyme were encouraging (24. Will Scientific. and a half-life was about 14 to 22 on August 8.. West Point. asparaginase Cancer Institute. O. Enzyme activity was detectable 13 to 22 days after single injections. CA-5834 and CA-2599 from the National Cancer Institute.. sodium dissimilar biological activity (6.[CANCER RESEARCH 30. LSA.. through the National tumor factor was later shown to be an enzyme.2 i. Pa. 2 with hypersensitivity reactions. Sharp and Dohme guinea pig serum was first described in 1953 (25). When more enzyme became available. Received January 6. hemolysis. the initial clearance of the enzyme from plasma followed first-order kinetics. LDH. H./kg MATERIALS AND METHODS as a skin test dose produced a substantial fall of plasma Forty-five patients were studied: 24 with ALL of whom 4 asparagine. 12 with AML. Search for follows: L-asparagine.. uridine-5-3H. L. SEPTEMBER 1970 2297 Downloaded from cancerres. and Lucius F. hypotension. After enzyme administration. Mallinckrodt Chemical Works. multiple myeloma. St. Initial 'The abbreviations used are: ALL. After i. glutamic-oxaloacetic transa minase. Plasma asparagjne and aspartic acid levels in leukemia were compared with levels in 20 normal individuals. acute lymphocytic leukemia. 30). September 1970] Biochemical in Man1 Takao Ohnuma. bone marrow rating 2.5. N. GOT. and fatty liver (24) were reported. N. further studies were conducted and a wide spectrum of toxicity was recognized (2. S.scherichia E coli asparagjnase were studied in 45 patients with leukemia and solid tumors.1970. Patients with acute myelocytic leukemia as a group had lowered levels of asparagine. emesis. each had palpable or radiological evidence of tumor. pancreas. coli (11. plasma asparagine fell precipi tously to nearly unmeasureable levels. J. chills. 33. patients with solid tumors had previously been treated. Daily adminis tration of the enzyme caused cumulation in serum. perhaps in part because of the initial assumption that the drug acted on a specific metabolic defect in sensitive tumor cells. neuro benefit. 26. daily. Ur-3H. 20-23. Buffalo. Mo. practical source of the enzyme for clinical study.] and Pediatrics ¡A. 2 with osteogenic sarcoma... F. coli asparaginase in patients with leukemia and solid tumors. liver. 18. Nutri other sources of the enzyme revealed that enzymes of dif tional Biochemical Corp. accepted May 8. fingernail. The biochemical and pharmacological effects oÃ. chronic 'This investigation was supported by USPHS Research Grants myelocytic leukemia. Y. Isolation of an active frac hypochlorite. 28. 30). fever. lactic dehydrogenase. F. The sources of chemicals used were as (L-asparagine amidohydrolase. hypersensitivity reactions (24. AML.F. 17. J.. Studies with Asparaginase James F./.

was added.5-3H (final activity. Y. and 500 mM Tris buffer. and the 50decrease in absorbance at 340 m/i was recorded until the reaction reached a plateau (40 to 50 min). The congealed plasma was cooled and centrifuged at 105. 2298 CANCER RESEARCH VOL.0 Ci/mmole).u. Blood plasma was ob tained either by finger prick into heparinized capillary tube or by venipuncture after i.0./ml) were carried out by the method described by Oettgen et al. 0. Sigma Chemical Co. and L. St. In some patients. Asparaginase Activities in Plasma.u. pH 8. Freeman. The highest total dose given was 2. This change in absorbance represented the plasma L-aspartic acid. Mo. and the further decrease in absorbance at 340 mp. This was later abandoned because initial skin tests were never positive and anaphylactoid reactions occurred in patients despite negative skin test. 2011 Copyright © 1970 American Association for Cancer Research . Most patients on the Chart 1. New York. (13).. Orangeburg. Ur-3H (specific activity 20. Samples of plasma were placed in a boiling Asparaginase Activity in Plasma. 6C3HED lymphoma cells in the same concentration were incubated concomitantly to ensure that the system was optimal.u./ro.9%NaCl solution was added. 0.0.. The values of L-asparagine and L-aspartic acid were calculated from concurrent standards made from known concentrations of the amino acids. and Lvaline-5-3H (specific activity 0. and Schedule.1 ml of measured. Ohnuma./ml.L-valine-5-3H (final activity.v. Clear supernatant. (IO). 0. Sinks a-ketoglutaric acid. A. Roswell Park Memorial Institute. 1 /nCi/ml..9% NaCl solution (5 ml/vial) and 0. (27). DL-lysine-4. which represented the amount of L-asparagine. and the activity was at 4° for 1 hr. 45. 6C3HED lymphoma was obtained from Dr. the method of Lowry et al.u. Boehringer Mannheim Co. The first 34 patients received 10 or 20 i.01 ml of 0.3 i. in 0. 30 Downloaded from cancerres. 0. The concentration was adjusted to 6 X 10s leukemic cells/ml in Eagle's medium with Hanks' balanced salt solution containing 15% dialyzed fetal calf serum and the incorporation of Ur-3H (final activity. Percentage of incorpora tion was calculated by the area under the curve for uptake at several time points with asparaginase divided by that area under the curve for uptake when the cells were incubated with L-asparagine.083 i. The lyophilized enzyme was dissolved with 0. N. of asparaginase intradermally as a skin test prior to the commencement of treatment. Immunological Study.u. Assay of Plasma L-Asparagine and L-Aspartic Acid.T.5 hr after being dissolved.38 mM) or asparaginase (final activity. F. (30). The total dose range varied from 200 to 40. Louis. Test of Leukemic Cells for L-Asparagine Requirement in K..5-3H (specific activity. administration of asparaginase. pH 8.u. From the capillary tube. Malic dehydrogenase. were placed in a silica cuvet.05 jLtM). daily.72 i. final concen tration.200.000 on August 8. Then 0.9 ml.. 5 to 10 pi of plasma were used directly or after appropriate dilution for the assay (31 ). malic dehydrogenase. Y. Protein was measured by days prior to 5 daily injections of the drug.aacrjournals. 0. Administration. was similarly recorded. J.The white cells were separated from the peripheral blood or bone marrow aspirate by a method developed in this labora tory (16). coli) in 0. The drug was given as single.8 i. N. GOT. F./kg. 2 100-1 mM NADH. and 0. or weekly doses. Clearance of plasma asparaginase activity after single injec daily schedule received a single injection as a test dose 4 to 14 tions in 4 patients. of GOT.u. and NADH. Inc. These enzymes were dissolved as long as 10 months reaction mixture consisting of 10 mM a-ketoglutaric acid. and was maintained in C3H mice in ascites form. One patient received 5 additional doses every other day after he reached Ml marrow. Assays RESULTS were carried out according to the method described by Cooney et al. 1 ¿iCi/ml)in the presence of L-asparagine (final concentration. Dosage. The sensitivity of the method was such as to detect activity of 1 X IO"3 i. The activity was expressed in i. 0.u.1 injected i.000 i. 4 juCi/ml) or DL-lysine-4. T.v. another received the enzyme daily for 27 doses and a 3rd patient with ALL received a 5-day course after weekly treatment had been shown to be ineffective. presence of antibody against asparaginase in the serum was determined by the method described by Campbell et al.u. Holland.000 X g ferent lots were randomly selected.1 ml of 100 mM Tris buffer. Schwarz Bioresearch. Hauschka. directly or through the side arm of a running infusion within 0.0 Ci/mmole). of asparaginase (E.6 Ci/mmole). Asparaginase vials of dif water bath for 10 min to inactivate intrinsic enzymes..

the activity in the 2-hr samples was con sistently higher than the preceding samples.100 to 7. o o.u. 5 days after the end of a 5-day course of 1000 i. 10 healthy women. Patients with AML had lower levels of L-asparagjne with normal L-aspartic acid concentration./kg of the enzyme. enzyme level in a patient who. there appear to be 2 groups: 1 group with L-asparagjne concentration within or near the normal range and with normal L-aspartic acid./kg/day.u. In 2 patients. 16 measurements in 32 days revealed that 0./vial and specific activity of 116 to 182 i.900 i. respectively. is reported as such.u. of the enzyme of Day 15. after a single injection of 1000 i. the plasma enzyme activity was measured serially for a longer period./kg 3 i. 5 days previously. Daily administration of 400 or 1000 i. however.2 i. and the clearance of the activity from plasma followed first-order kinetics with plasma half-times of 14 to 22 hr. This subgroup of 3 patients was unique in that 2 patients died within 48 hr of injection. In healthy adult controls. Even 0. Eleven vials from 5 different lots labeled as containing 10.5- 0.u. and positive intradermal test to 10 i.u. SEPTEMBER 1970 2299 Downloaded from cancerres. the 2nd group with low L-asparagjne and reciprocally elevated L-aspartic on August 8./kg of enzyme. 16 respectively. Plasma L-Asparagine and L-Aspartic Acid. the plasma was found to have a positive precipitation reaction to asparaginase.E.000 i. Thus. it was dose related. After a single injection of 1000 i. in 1 patient. It may be seen that the peak activity lasted for about 3 hr after the injection. Plasma L-asparagine and L-aspartic acid values in 10 healthy men. /kg. In another patient whose plasma enzyme activity became undetectable 8 days after a 2nd dose of 5000 i. Plasma asparaginase activity during and after 5 daily injections of asparaginase in 3 patients. L-asparagine fell from 31 ± 7 (mean ± S. which is in fact the skin test done. 4 Among 10 patients with ALL.u. The clearance of plasma asparaginase is shown in Chart 1.1 —1— —1— 72 HOURS n— 120 —1 144 —1— —i— 192 24 48 we Chart 2. the plasma asparaginase level fell rapidly and became undetectable SO-i 4 days after the last injection. but this finding was not confirmed in the samples obtained by venipuncture. after a single injection of 400 i. The 3rd patient was induced into complete remission.aacrjournals. enzyme level in 2 patients who. to 43 ± nmoles/ml.u/mg of protein.D. plasma L-asparagjne fell pre cipitously. respectively. daily for 5 doses. 2011 Copyright © 1970 American Association for Cancer Research . received 400 Lu. The doses in this paper are expressed in terms of label potency to afford comparison with the data of others. respectively. in one patient who developed arthralgia.u.) to 4 ± and L-aspartic acid rose from 26 ±6 1. When blood samples were obtained by finger prick. received 1000 i. L-asparagjne values were 60 ± 13 (mean ±1 S.Clinical Studies prior to the testing date and stored at -20". 10 patients from 7 to 44 years with ALL. 6 days previously. L-Aspartic acid concentrations were 11 ± and 4 10 ± nmoles/ml in men and women. The activity was detectable 13 and 22 days after single injection of 1000 and 5000 i. The activity of asparaginase found in plasma. caused substantial fall of plasma L-asparagine levels by the following day.u.u.u. but not after 18 and 25 days./kg of the enzyme had a cumulative effect (Chart 2). daily for 5 doses. In 6 measurements. See text for discussion of the rapid fall in patient plotted as A—¿.v. and 8 patients from 12 to 72 years with AML were plotted in Chart 3. Frozen storage of with Asparaginase this duration was found not to cause any loss of activity. a a. After enzyme administration. with a reciprocal rise in L-aspartic acid. erythematous skin eruption.u. Development of immunological reactions were associated with rapid clearance of plasma enzyme. were found to have activity ranging from 6.u.) and 54 ± nmoles/ml in men and in women.

GOT./kg of the enzyme was administered. Immunological: Hepatic toxicity was observed more frequently than any Positive skin test. 4. drowsiness. observed frequently in patients with leukemia. and L. *. Mild azotemia was commonly noted at about Day 7 of the study. but because of death from advanced disease in 8. ao- O -8- Sb NORMAL MALE NORMAL FEMALE ALL AML Chart 3. Seven of 8 évaluablepatients showed hypocholesterolemia with average decrease of 62 mg/100 ml.T. Bleeding. was cerebral dysfunction. vomiting. and other hepatic coagulation that received only single doses would be higher than for the factors—fatty metamorphosis.5 to 1 hr after injec depression. nausea or emesis lasted a few days. hypotension metamorphosis was seen (Fig. Hepatic 32 33 97 dose were scheduled to receive additional drug in weekly Increase in bilirubin. J. disorientation. 2011 Copyright © 1970 American Association for Cancer Research . Immediate ratings of 0 to 4+. In 3 of those 10 patients. Loss of weight (more than 24 30 80 score seems not to be dose dependent. This suggests difference 5% of pretreatment level) in the susceptibility of host cells of different individuals to 3. fever. it took about 10 days after the enzyme Asparaginase toxicity was no longer detectable by the techniques used for L-aspara The percentage in each category was calculated on the basis of gine levels to rise. and chills. tion and lasted approximately another hr. We did not establish whether this was renal or prerenal in origin. In 2 of the delayed dysfunction patients. received weekly injections. was seen in 7 patients. u. T. At death. 3 patients with reverse correlation of the amino acids. Toxicity observed after asparaginase administra évaluable affected affected tion is summarized in Table 1. could not usually be ascribed only to fibinogenopenia or other coagula tion protein deficiencies. In 2. Holland. Albumin decreased an average of 1. implying none. moderate. Severe brain dysfunction. Increase in bilirubin. In the other dosage regimens. GOT.8 g/100 ml during the first 2 weeks and then reached a plateau. decrease in albumin. Neurological: 13 39 33 Headache. other regimens. livers of all patients showed severe fatty metamorphosis. F. courses. because of toxicity in 5. severe fatty fingernails. Delayed 1. v./kg showed that L-asparagine was unmeasurable until Day 29 but returned by Table 1 Day 33. malabsorption and hydrocortisone often modified or eliminated these im syndrome mediate reactions. L-asparagine levels remained immeasurable until Day 21 but returned by Day 23. Sinks been completed on 16 patients who died after asparaginase treatment. A. Eight of 10 évaluable patients had a fall of fibrinogen level averaging 214 mg/100 ml. mean toxicity 2. manifested as disorientation and confusion. and 1 who died 10 days after receiving 200 i. it was noted within 24 hr. and alkaline phosreaction 7. of exogenous enzyme in man.and o. In some 5./kg. Ohnuma.u. and Factor X. In contrast to the decrease in his serum albumin. and in patients with ALL and AML. Miscellaneous: phatase and. It is thus decrease in albumin. Plasma L-asparagine (ASN) and L-aspartic acid (ASP) values in normal healthy men and women. one patient with IgA multiple myeloma showed no changes in immunoglobulin levels with serial determinations after 5000 i. In patients who Abdominal pains. No. 14 measurements in 33 days after a single injection of 5000 i. increase in amylase or lipase.u. Immediate reactions usually occurred 0. and Transverse banding of the other coagulation factors occurred. and LDH. mild. of Toxicity. and Nausea. alkaline phosphatase. Freeman.aacrjournals. Thirteen of 15 patients who received a single 2. fibrinogen. severe. Factor VII. In both cases. hyperglycemia. The most striking side effect of treatment. when it oc curred. LDH. in the other 5. no further drug was given. and chills 27 38 71 life threatening. In another patient. These reactions confusion included nausea and vomiting. there was a concomitant defi ciency in Factor V. fibrinogen. Pancreatic: 6 39 15 patients. 1 who died on the day of asparaginase administration. With the exception of 2 patients. Chart 4 shows the relation between dose range and composite mean score for toxicity 1. it did not appear until after 1 week. since thrombocytopenia often existed also. Mild depression and drowsi ness were found in about 25% of the patients. not unanticipated that the mean toxicity ratings for the group cholesterol. cholesterol. 30 Downloaded from cancerres.u. F. Azotemia 25 38 68 L-asparagine starvation. premedication with antihistaminics hypoinsulinemia. ». disorientation and confusion were 80 • • O 60 I. 1). anaphylactoid 5 39 13 other. on August 8. of No. These results indicate the prolonged activity patients with adequate observations to determine toxicity in question. Microscopic observation has 2300 CANCER RESEARCH VOL.

hyperglycemia. Three patients from the single injection group and 1 patient from the daily injection group were omitted because of inability to evaluate toxicity scores. of whom 2 showed no clinical evidence of pan AML developed hypotension of 70 mm Hg systolic and 45 mm creatitis. moderate.u. was later found to have Nonketotic hyperglycemia occurred in 3 adults and 2 transverse banding of the nails (33). Intradermal injec autopsy. and had been injected. diar i. evoked a positive response 18 hr later in a 44-year-old man who received a single injection of 1000 i. The response of hyperglycemia to insulin was appropriate. No an illness resembling serum sickness with generalized arthralgia adverse reaction was observed from the amino acid infusion.000-•"• u u u u .aacrjournals. The first 7 patients receiving weekly treatment were given 1000 i. Toxicity score: 0.u. and the next 3 patients received 4000 i./kg/day was followed by periment (38). Another patient with children. life-threatening toxicity.000 and 8. cyanosis and. malabsorption. Development of a malabOne patient with AML. gaps in dose schedule schema represent observation intervals of 4 to 14 days. severe.u.beginning within 2 min. in one patient. confusion. who after 1./kg. L-Asparagine "Rescue" Infusion. 2011 Copyright © 1970 American Association for Cancer Research . mild. and steroid and recovered without complication.u.i0 u8 Ul2 5. In 2 patients. positive Schilling test done with and without intrinsic factor. stridor. in approximately 2 weeks. Under daily treatment. 3rd. Blood sugar levels fell back to normal range patients.u. In one case./kg stain of the liver at autopsy. sugar tests. and Positive skin test or anaphylactoid reaction were not ob increase of blood urea nitrogen from 4 to 40 mg/100 ml. 4. died on Day 2 with disorientation. numbness.000 i. the hyperglycemia was accompanied by enzyme. histaminics. 1.u. showing neither unusual sensitivity the drug was fairly well tolerated by most of the patients and nor resistance. single dose of 1000 i. 3. Polyuria responded to insulin but not to antiBone marrow aplasia was not observed in nonleukemic diuretic hormone. increase in serum and urinary amylase. Pertinent data are summerized in Table 2.u. even a tension.I 10. hypoinsulinemia and hypocalcemia. These patients were treated with epinephrine. accompanied by loss of recent memory.000 ml.u./kg/week. none. He received metaraminol and steroid for 3 days and polyuria of 11. respectively. with 4+ urine recovered. The lactoid reactions. as suggested by negative periodic acid-Schiff autopsy. bronchopneumonia was seen. and muscle wasting. of 1000 patients as abdominal pain. In and paravenous erythema near vein sites where asparaginase 2 patients. L-Asparagine rescue in tion of 10 i. Another patient with ALL who received 1000 i. antirhea. probable SEPTEMBER 1970 2301 Downloaded from cancerres. Two patients died on the 1st dose and was not seen after the 2nd. A similar reaction in a 16-year-old girl 4 dose of L-asparagine was chosen from a previous human ex days after a 5-day course of 1000 i. 2.u. possibly because of depletion of infections. Hyperglycemia occurred in one man only after the toxicities were essentially reversible./kg. The was not retreated. for example. and the 3rd and 4th. coma./kg of asparaginase produced late. Although we have seen a variety of unusual toxic effects.u. Pancreatic toxicity was manifest in varying degree in 4 5000 i. All 3 patients had had negative skin tests before their repeat weekly injections: the 2nd dose of appearing dysfunction (33). Blood glucose rose as high as 955 mg/100 ml in 1 Hg diastolic occurring 3 days after 10.000 i. and in 1. The reaction includes shaking chills.Clinical Studies lu gW 15yZ 2 On with Asparaginase 05.u./kg fusion was given to 3 patients for the acute brain dysfunc 1 week previously. At served at the first exposure to the on August 8. respectively. definite improvement ensued. or 4th Day 1 who had far-advanced neoplasms with overwhelming weekly dose of asparaginase. upper abdominal tenderness. pancreatitis. He had no evidence of clinical allergy but tional syndrome in hopes of reversing the abnormalities./kg.1POPODOI]DDDGDoUUU JUn1^TiDULiuiD DDDDDAILY u HttM. hypo syndrome lasted nearly 1 month in each.000tiu/kgS]j[11erenD|uuSINGLEi¡¡]Jrm!ay DDDODUUD§ODDDDDDDDD Chart 4./kg of the sorption syndrome was suggested in 2 of these patients by a enzyme developed acute brain dysfunction. Three other patients developed anaphy easy suggestibility which resembled Korsakow's psychosis. Composite mean toxicity score versus dose and schedule of asparaginase. fever./kg of the patient. confabulation. and causal relation to asparaginase was not found at hepatic glycogen.

that clinical observers considered the phenomena to be caus ally related (33).6(Day 10)1.u. appear to be no differences in the response rate in children Initial peak activities of 5. and 5000 i.1 (Day 15)1.u. there early and appreciable extravascular space of distribution. Systematic increase in enzyme levels for the first 2 hr were classified as "hematological effect only. These values are arranged on the abscissa according to order of decreasing antileukemic effect and were compared with the LSA-+ALL ALL AML values of a sensitive mouse lymphoma. and L. suggests that tissue fluids were squeezed from the most effective against ALL in children but has some value in finger pad in obtaining the capillary samples./ml 4/8).aacrjournals. Prolonged half-life of E. reasonably close to estimated values of 5. sus from massive pulmonary embolism. These patients enzyme./kg infusion(nmoles/ml)0043Serumalbuminbeforeinfusion2./kg. 5/8) and those with weekly treatment (Ml/total.8%. and within diseases no good correlation improvement was noted. who were induced into Ml with asparaginase had had exten These findings are in contrast to the results in mice where the sive prior chemotherapy and had failed to remit again on vin. Although the numbers are small. favorable factor in its effectiveness. and distribution in the plasma volume estimated woman with AML whose marrow was induced to M2 died as 5% of body weight. 1000./kg (33). coli enzyme is cleared from plasma within 24 hr and where cristine and/or Daunorubicin with prednisone. 3 relapsed on have previously shown. One patient with AML was induced into complete which are calculated from the actual dose given being 70% of remission after a single dose of 1000 i. This implies an AML of adults as well. with an average of 6./ml obtained after with ALL between those who received daily injections injections of 400. N.5 (Day (Day 38)Dose Table 3 Response in leukemia LSA-*ALL indicates ALL converted from préexistent SA. 15." Decrease in in finger prick blood. A. 30 Downloaded from cancerres.). J.00010. 8). L-asparagineduringinfusion(nmoles/ml)160Serumalbuminaft ofL-asparagine(mmoles/kg/24 ofaspar mentalstatusDefiniteProb aginase(i. Holland. We have frequently seen definite decrease or disappearance of leukemic cells from Serial measurements of asparaginase after a single injection periphery and/or rise in platelets after asparaginase with bone of the enzyme has shown prolonged clearance rates of the marrow changes within the same classification.3 (Day 10)0.DiagnosisAMLCML(blastic)AMLDose 1)1. and 21.000PlasmaL-asparaginebefore X 7)2. sterilized by Nalgene Filter Unit (Nalgene Labware Division. In the presence of Adult Child Adult Child Adult Child asparaginase. All the 10 ALL patients tained depression of L-asparagine concentration was observed. personal com- 2302 CANCER RESEARCH VOL.6 hr)121Daysgiven12-179-1438-44Maximum PatientD. No patient with solid tumor responded to asparag mouse tumors (31). Ohnuma.9 (Day 27)1. incorporation of precursors by control human culture was always less than the mouse lymphoma cells ranging onlyNo response0013100060100000311530002 from 0. Frei.9 (Day 17)2. 1 died from empyema despite the Ml an inverse correlation between the rate of clearance of exog marrow. Therapeutic Effect.7 (Day 9)1. 12. Sinks Table 2 L-asparagine rescue infusion for brain dysfunctional syndrome One day's dose of the amino acid was dissolved in 2 liters of 5% glucose. The enzyme is puncture. respectively.0 (Day 21)2. Y. The inhibi MlM2Hematologicaleffect tions in human leukemic cells were never this pronounced. since indistinguishable biochemical results were found in ALL and AML. 2011 Copyright © 1970 American Association for Cancer Research . Another labeled dose.u. and 70 i. and 60 i.J. Chart 5 shows the 36 studies of in vitro incorporation of coli enzyme in man [8 to 30 hr (E.W. incorporation of uridine and lysine was always less than 30% of controls in the mouse lymphoma. F. Among these depressed L-asparagine returned to normal in 48 hr (7.5-3H) by leukocytes in 20 patients as an attempt at predictive behavior. Days 14.D. Furthermore.2 (Day (Day 56)Improvementin Ur-3H and L-valine-5-3H (or DL-lysine-4. are (Ml/total.45 to 20. The response to asparaginase in patients DISCUSSION with leukemia is summarized in Table 3.14. the prolonged clearance of E. The relation to infusion was such with hematological response occurred.0 (Day 48)2. L-Asparagine Requirement by Leukemic Cells In Vitro.0005.u. After asparaginase administration. and 6 went into a Phase II maintenance program on enous enzyme in the plasma and the therapeutic effects in other drugs.D.u. Rochester.6%.E. III. by using avian and guinea pig enzymes. as contrasted to blood taken by veniperipheral leukocytes alone was not included. From this point of view. L 18)2. F. Biochemical prediction of therapeutic outcome was not reproducibly suc Total 16 10 on August 8. coli enzyme in man may be regarded as a inase.C.T. and infused in 24 hr. Freeman.6. We 10 ALL patients who were induced into Ml.

coagulopathies. (13) was found to be reliable and convenient compared to older methods. It may be speculated that acute brain disease is related to ammonia or L-aspartic acid intoxication while the delayed brain syndrome is associated with defective protein synthesis in the brain produced fundamentally by L-asparagine defici ency. fever. whereas that of globulin was apparently unaffected. Thus. such as contamination by glutaminase activity (29). Most patients with ALL had normal L-asparagine and L-aspartic acid levels. The failure to find hypoglobulinemia suggests some systematic differential requirement for L-aspara gine between liver cell and plasma cell. Many factors have been considered as possible contributors to the wide spectrum of toxicity. but particularly by the possible endotoxin contaminant.5 hr (21)] compared to that in mice [2. hepatic injuries (39). Walter. AOOO AA. and T. however. after asparaginase. In all instances. Three patients with ALL had an inversion of normal L-asparagine and L-aspartic acid ratios. and electrolyte imbalance and other iatrogenic causes were excluded. depletion of liver glycogen. As a group. hypotension. L-Asparagine requirement by leukemic cells m vitro Ml and M2 indicate eventual marrow status achieved after treatment. Over a period of 3 months. The similarity of patho logical changes in the liver after these chemical toxins and after asparaginase suggests that the hepatic injury is indeed due to inhibition of protein synthesis because of L-asparagine deficiency.aacrjournals. improvement. the plasma cell presumptively has a welldeveloped system to supply its own L-asparagine. patients with AML had low L-asparagine levels and only 1 complete remission occurred. be necessary to prevent in vitro amidohydrolysis which may take place while the sample is being processed. Methionine sulfoximine-induced seizures in cats are related to inhibition of L-glutamine synthesis and adminis tration of L-glutamine and L-asparagine to epileptic patients resulted in improvement in electroencephalographic records (38). but liberation of large amounts of ammonia by enzyme were estimated from the increases in L-aspartic acid values or "blank" ammonia values in asparaginase determina tion. We have not measured ammonia on each occasion. a situation dissimilar to data from the mouse (36). and delayed hypersensitivity reaction (41). fibrinogen. the other occurred insidiously about 1 week later and lasted approximately 1 month. Ohnuma.AAMl increase in endotoxin dose exceeded the pyrogenic threshold in man but might have been tested as a negative in rabbits based on dose concentrations alone. IMP. and reproducible with as little as 5 nmoles/ml. We are aware of 1 fatal instance of anaphylaxis encountered by a colleague. This provided basis for the weekly administration of the enzyme rather than daily. 7 to 19. DL-Lysine-4. massive central nervous system bleeding. and fingernail protein were defective.000 times as much as was tested for pyrogenicity in the rabbit. Bacterial endotoxin is known to cause chills. vomiting. no response. and valine-5-3H was used in all human studies. NR. despite immediate and vigorous therapeutic measures. Since the amount of asparaginase given to man is 1. there is a loss of about 1%/10 days. renal on August 8. This may be due to overwhelming infection or functional abnormalities in the dying patient. unpublished data). by the administration of an exogenous protein or large molecule per se.5-3H was used in the mouse experiments. central nervous sys tem leukemia. meningitis. by the depression of L-asparagine. it is possible that a proportionate into albumin was markedly inhibited (J. or the effects of liberation of ammonia from L-asparagine. Probably the most intriguing finding to explain much of the toxicity is the deficiency in protein biosynthesis produced by L-asparagine depletion. Holland. which presumably resulted from defective removal of fat because of inhibition of protein biosynthesis (1).or carbon tetrachloride-induced fatty liver. These effects resemble many of the toxicities observed after asparaginase. The basis for aberration of L-asparagine and L-aspartic acid values in leukemic patients is not clear. No other method of protein precipi tation was studied to ascertain whether an artifact of transient heat activation of enzyme occurred. We have observed 2 forms of diffuse brain dysfunctional syndrome: one occurred within 24 hr and then cleared rapidly. fibrinogenopenia.000 to 10. In the 3rd SEPTEMBER 1970 2303 Downloaded from cancerres.60-40"2O-0-Ur. The enzymatic assay of L-asparagine and L-aspartic acid developed by Cooney et al. Mental abnormalities did not apparently increase after interruption of L-asparagine infusion in 2 patients. insulin. munication). reminiscent of effects of asparaginase per se. these findings suggest that asparaginase treatment is not immunosuppressive. Further refinement may.5 hr (3)] has also been observed by others. we have abandoned it. and the complete remission rate was high. F.SH • O lOO-80-.Clinical Studies with Asparaginase •L^IsJJjxAA• . Clinical findings suggest that synthesis of albumin. with indistinguishable therapeutic effects. L-Asparagine has been shown to protect against ethionine. Since skin testing did not indicate the patients who sustained anaphylactic response. Standard L-asparagine solution was found to be hydrolyzed slowly during storage at 0-4". incorporation of L-methionine-35S *AlA• XPX6C3HEDLYMPHOMAOfPAA0AAOA0 Mz IMP ALL NR Ml M2 IMP AML NR Chart 5. Together with the evidence of patients who developed immunological reactions. Since immunoglobulin contains the same general amounts of L-asparagine as most other proteins. 2011 Copyright © 1970 American Association for Cancer Research . L. Clinical observation of improvement in mental status after L-asparagine rescue infusion in all 3 patients studied is of interest.

D. 32: 507-523. C.. 2304 CANCER RESEARCH VOL. J. P. S. Himelstein. 1963. Cancer Chemotherapy Rept. F. W. Guinea Pig Serum Is Responsible for Its Antilymphoma Effects. Scheig. and Selawry. and Hansen. abnormally high L-aspartic acid levels resulted from L-asparagine infusion (33). Cremer. 9.. Oettgen. L-Asparaginase Induced Coagulopathy (Abstr. 20. L. E.. 727: 1055-1072. 17. Carbone.. 14. Ohnuma. We observed this on one occasion. E. Hill.. Broome. New Engl. O. C. 20: 1574—1580. P.. C. Ring or Interfacial Test. Canellos.. F. and Henry. Two L-Asparagjnases from Escherichia coli B. Broome. 30 Downloaded from cancerres. J. Evidence That the L-Asparaginase Activity of 19. A. F. P. Weil. J. A Mechanism of Resistance to L-Asparaginase (Abstr. and D.. 11. recrudescence of brain dysfunction followed after discontinuation of L-asparagine infusion. O.). S. D. Evidence That the L-Asparaginase of Guinea Pig Serum Is Responsible for Its Antilymphoma Effects. Hypoalbuminemic and Hypocholesterolemic Effect of L-Asparag inase (NSL-109229) Treatment in Man-Preliminary Report. H. Murphy. 35: 967-974. D. G. P. Block. Leone.-1114-1115. Campbell. Karanas. 1969. coli Apotryptophanase. New York: W. Possible factors which might influence clinical results are the develop ment of an antibody that rapidly clears the enzyme from plasma.. 13: 19-27. When patients developed anaphylactoid responses or positive skin reaction to the drug. Their Separation.. M.. A. Exptl. A.. F. Cancer Res. Med. H. Holland... Nati. D. New York.1969. Clearance Rates of Enzyme Preparations from Guinea Pig Serum and Yeast in Relation to Their Effect on Tumor Growth.. Gralnick. Broome. 1969. E. Borion. Cancer Inst...55: 67-69. Cornet. Serum albumin levels did not correlate well with abnormal mentality. B. 7. 17: 402. 1969.. R. C. J. J.. Dolowy. 1968. Brit. R. Even 12 days after aspara ginase administration in Patient D. Canellos. 10: 32. its use is contraindicated when asparaginase levels are high. 1964. Assoc. J. and Klatskin. L-Asparaginase Toxicity.. 19: 1813-1819. C. 1965.. M. Broome. we terminated treatment.. Hypofibrinogenemia due to L-Asparaginase: Studies Using 13 ' I-Fibrinogen (Abstr. 6: 721-730. Assoc. M. A. 10. J. and Freireich. Clin Res. R. A.. Cancer Res. A. L. Proc. and Carbone. J.aacrjournals... L-Asparaginase: Effects in Patients with Neoplastic Disease. N. E. O. 17: 399. 16. Antigen-Antibody Reactions.. Response to Highly Purified L-Asparaginase during Therapy of Acute Leukemia. that rescue effect would not be expected in the days immediately after asparaginase administration because exog enous L-asparagine may be rapidly catalyzed by large amounts of the enzyme still circulating. Pyridoxal Phosphate Determination in Isolated Leuko cytes and Tissue by E.. Asparagine Synthetase in Human Leukemia. Ben jamin.. J. R. Arseneau..).. and Hand Schumacher.). H. E. Pharmacol.. One of the most difficult clinical problems is that the toxicity is apparently not dose related and some manifesta tions such as anaphylaxis. L. D. Cuttner... Antilymphoma Activity of L-Asparaginase in Vivo. Toxic and Anti-Neoplastic Effects of L-Asparaginase.1961. M.. Carbone. The derepression or induction of an L-asparagjne-synthesizing system in leukemic cells after asparaginase administration has been reported (19). Haskell. A. P. Haskell.. G.. p. H. 3. ACKNOWLEDGMENTS We are grateful to Mrs. sug gesting differences in chemical abnormalities of liver and ner vous system. A. J. J. G.. M. A. R. Sylvia Grew and Mr. John Gawoski for theii skillful technical assistance. Proper and close clinical observation of patients receiving the enzyme is there fore essential. P. 5. H. 10: 35. and Canellos. 1968. Canellos. Burchenal. Cancer.. N... D. Sussdorf (eds. Alexander. J. Because L-asparagine given early may thus give rise to toxic levels of ammonia. R. J. 21. 1969. J. Capizzi. Boyse.. N. 1969. Exptl. I. Mashburn. G.. S. Broome. J. H.2P. J. Med.. Serpick. Forcier.. Incidence of Sensitivity among Leukemias of Various Types: Comparative Inhibitory Activities of Guinea Pig Serum L-Asparaginase and Escherichia coli L-Asparaginase. Silver. F.. pp. A. 1967. Jacquillat. B. L... Sinks patient (J.... A. Campbell. Cancer. 4. D. and Old. 1969. D. and Clifford. R. 281: 1028-1034. Biochem. Ellison. L. 18. Haskell. L. Roberts. J. F. M.. A.. Kyle. 1970. R. D. and L. J.. Bernard. A. 7. M... P. Clin. 22: 595-602. Factors Which May Influence the Effectiveness of L-Asparaginase as Tumor Inhibitors. Arabinosyl Cytosine: A Useful Agent in the Treat ment of Acute Leukemia in Adults. A. It should be added. 2. 30: 929-935. B.. 1968. J. J.. 6. C. Program of the XII Congress of the International Society of Hematology. E. 12. Boyse. <\. 1966. and Sussdorf. B.. Haskell. and Seilin. Sawitsky. 15. brain dysfunctional syndrome. 121-148. 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A. P. M. J. J. L. A. J. Nature and Extent of the Biochemical Lesions in Human Epileptogenic Cerebral Cortex. G. A.1968.. E.. Biochem. T. J. 31. M.. K. 98: 565-582.. Prager. E. R. K.5.: The Institute of Microbiology. Ann. 30. W. E. L.. SEPTEMBER 1970 2305 Downloaded from cancerres.0 mg/100 ml. D.583-606.. 5: 113-130. 10: 66. Farr. 1967. 2. M. Biol.. 187-210. H. Holland. F.. Tallal.. Chem. 26: 2213-2217. J.. Clin. Tumor Inhibitory Effect of L-Asparaginase from Escherichia colt.. 36. McCarthy. 2011 Copyright © 1970 American Association for Cancer Research . 1969. G... Loeb. Cancer Res.. with Asparaginase 32. Jr.1967. A. Bacterial Endotoxins. This severe fatty metamorphosis was seen in a patient whose liver function 5 days prior to death showed only slight abnormalities (bilirubin. 224: 275-276. J.Clinical Studies 23. Cancer Res. Host Effects of L-Asparaginase Treatment (Abstr.. Proc. 25. Cancer Res. Mashburn.. J. R..). C. Oettgen. 29: 296-300.. L. 210: 1919-1921.1969.). Proc. H. Protein Measurement with the Folin Phenol Reagent. 29: 183-187.).1966. D. on August 8. L. Bergel.. R.1967. Tallal. Res. and Bachynsky. Khan. N.. E. and Hill. Am..PJ: 265-275. Med. Harris. p.. 35.. Fatty metamorphosis of the liver. J. L-Asparaginase in 111 children with Leukemias and Solid Tumors (Abstr. and Murphy. Assoc. Arch.. Helson.. 40.. Med. O. Hill.. New York. A. Salser.. T. In M. Landy and W.) Therapy. MacLellan. Program of the XII Congress of the International Society of Hematology. S. 1953. R.. Neurology. N. and Bray. 27: 2619-2631.. J. Roberts.0 g/100 ml). H. LDH. E. Braun (eds. E. Program of the XII Congress of the International Society of Hematology. Rev. Miller. E. Ohnuma..).1968.. Exptl. F. total protein. 34. and Conway. Amino Acid Levels following L-Asparagine Amidohydrolase (EC 3. L-Asparaginase Therapy for Leukemia and Other Malignant Neoplasms.).. L.. 103: 238-245. J. Bacterial Endotoxemia. Effects of Asparaginase in Acute Myelocytic Leukemia.1. 1955.1969. G. J. J. Lash. Weil. 32 units. F. alkaline phosphatase. 28. Tower. Studies on the Asparagine Requirement of the Jensen Sarcoma and the Devia tion of Its Nutritional Variant.. J.. St. J. Am. J. B. A. J. C. Med. 38. 8. 24.. L. p. M. 137 units. Whitecar. Cancer Res.. Clarkson. Cancer Res. 105: 451-452. Lowry. D... Jr. 29. Han. Med.. Assoc. 27. S. Maxwell. A. Tan. Assoc. and Oettgen. 8. Patterson. M. Studies of Asparaginase and Asparagine in Acute Leukemia (Abstr. Schwartz. P.1969. L. Bodey. pp. Inhibition of Leukemias in Man by L-Asparaginase. 1969.aacrjournals. Hill. F.. J. 1969. N.6 Bessey-Lowry units.). A.. and Sinks. Am. Loeb. and Randall. H. H. and Balis. W. 4. The Animal Model and the Human Patient in Bacterial Shock. C. Arneault. L. Schwartz. B. 33. 1. T. M. 41. serum GOT. M. MacLellan. 202: 882-888. Enzymes in Cancer.1969.. J. and Freireich. F. L-Asparaginase Therapy of Leukemia (Abstr. J. R. H. - Fig.. T. Freeman. Tallal. N.. Rugers-The State University... Nature. Schwartz. H. Assoc.. D. Cancer Res. 10: 92. Philips. 10. Old... D. Boyse. J. and Jenoff.1. L. Oettgen.. Roberts. H & E. Ohnuma. Leeper.... H. E. 16: 201-220.. G. Serum Lipids in Patients Receiving L-Asparaginase (Abstr. Asparaginase from Chicken Liver. The Antitumor Activity of Escherichia coli L-Asparaginase.. Kidd. J. S. Am. Roberts. 37. Regression of Transplanted Lymphomas Induced in Vivo by Means of Normal Guinea Pig Serum-I and II.... 39. M. K. 1964.. and Winston.. New York. Ohnuma. Zweifach. K. and Hill. J.. Holland. O. 1964. F. and Nagel. 1965. B. and Kim. F. New Brunswick. 17: 408. Biochem. Immunosuppresion by L-Asparaginase. Jr.. Campbell. M. A. 26. J.. 1952. M..