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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[CANCER RESEARCH 30, 2297-2305,

September 1970]

Biochemical in Man1
Takao Ohnuma,

and

Pharmacological
Arnold Freeman,

Studies

with

Asparaginase

James F. Holland,

and Lucius F. Sinks

Departments of Medicine A /T. O., J. F. H.] and Pediatrics A.F., L. F. S./, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203

SUMMARY

clinical trials in ALL2 in children with the E. coli enzyme were encouraging (24, 30). In the initial clinical trials with E. coli enzyme, chills, fever, emesis, pyrexia (30), hypersensitivity reactions (24, 30), and fatty liver (24) were reported. In a critically ill child with ALL, hypotension, hemolysis, and intestinal hemorrhage were observed after guinea pig serum enzyme administration (14). Nevertheless, general toxicity of the host was not regarded as a critical problem (14, 24, 30), perhaps in part because of the initial assumption that the drug acted on a specific metabolic defect in sensitive tumor cells. When more enzyme became available, further studies were conducted and a wide spectrum of toxicity was recognized (2, 9, 12, 17, 18, 20-23, 26, 33, 37, 40). The present paper reports our observations on bio chemical and pharmacological studies with E. coli asparaginase in patients with leukemia and solid tumors. Portions of this work have been reported in a preliminary form elsewhere (32).

The biochemical and pharmacological effects o- scherichia E coli asparagjnase were studied in 45 patients with leukemia and solid tumors. The drug was given in three different schedules: as a single dose, daily, or weekly. After i.v. injec tion, the peak activity obtained was dose related; the initial clearance of the enzyme from plasma followed first-order kinetics, and a half-life was about 14 to 22 hr. Daily adminis tration of the enzyme caused cumulation in serum. Enzyme activity was detectable 13 to 22 days after single injections. Plasma asparagjne and aspartic acid levels in leukemia were compared with levels in 20 normal individuals. Three of 10 patients with acute lymphocytic leukemia had marked aber ration in their amino acid levels. Patients with acute myelocytic leukemia as a group had lowered levels of asparagine. After enzyme administration, plasma asparagine fell precipi tously to nearly unmeasureable levels. Asparagine reappeared in plasma 23 to 33 days after single injections. Even 0.2 i.u./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. Asparaginase produced multiple manifestations of toxicity involving brain, liver, pancreas, kidney, fingernail, and had converted from prexistentLSA, 12 with AML, 2 with hypersensitivity reactions. Asparagine was given as a "rescue" malignant melanoma, 2 with osteogenic sarcoma, and 1 each infusion in three patients for acute brain dysfunction with with CML in the blastic phase, choriocarcinoma, neuro benefit. Ten of 24 patients with acute lymphocytic leukemia blastoma, multiple myeloma, and carcinoma of the colon. Five and 1 of 12 patients with acute myelocytic leukemia were patients with AML had received no prior treatment, whereas induced to bone marrow rating 1 marrow remission, 2 after the remaining leukemic patients were in frank relapse from single injections. It was difficult to correlate response in vivo prior chemotherapy at the time of the asparaginase trail. All and a requirement of the leukemic cells for asparagine in vitro. patients with solid tumors had previously been treated; each had palpable or radiological evidence of tumor. Patients with leukemia were evaluated by the rating criteria of Acute Leukemia Group B (15). Response in solid tumors was defined INTRODUCTION by at least 25% decrease in the product of 2 diameters as a reflection of tumor size. Regression of transplanted mouse and rat lymphomas by Asparaginase was obtained from Merck, Sharp and Dohme guinea pig serum was first described in 1953 (25). The antiResearch Laboratories, West Point, Pa., through the National tumor factor was later shown to be an enzyme, asparaginase Cancer Institute. The sources of chemicals used were as (L-asparagine amidohydrolase, EC 3.5.1.1) (4, 5). Search for follows: L-asparagine, anhydrous, and L-aspartic acid, Nutri other sources of the enzyme revealed that enzymes of dif tional Biochemical Corp., Cleveland, Ohio; phenol loose ferent origins had different rates of clearance from plasma and crystals, Mallinckrodt Chemical Works, St. Louis, Mo.; sodium dissimilar biological activity (6,31). Isolation of an active frac hypochlorite, Will Scientific, Inc., Buffalo, N. Y.; Tris and tion of asparaginase from E. coli (11, 28, 35) provided a sodium nitroprusside, Fisher Scientific Co., Fairlawn, N. J.; practical source of the enzyme for clinical study. Initial
'The abbreviations used are: ALL, acute lymphocytic leukemia; LSA, lymphosarcoma; AML, acute myelocytic leukemia; CML, chronic 'This investigation was supported by USPHS Research Grants myelocytic leukemia; GOT, glutamic-oxaloacetic transa minase; Ur-3H, uridine-5-3H, Ml, bone marrow rating 1; M2, bone marrow rating 2; CA-5834 and CA-2599 from the National Cancer Institute. Received January 6, 1970; accepted May 8,1970. LDH, lactic dehydrogenase.

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T. Ohnuma, J. F. Holland, A. Freeman, and L. F. Sinks a-ketoglutaric acid, Sigma Chemical Co., St. Louis, Mo.; GOT, malic dehydrogenase, and NADH, Boehringer Mannheim Co., New York, N. Y.; Ur-3H (specific activity 20.0 Ci/mmole), DL-lysine-4,5-3H (specific activity, 45.0 Ci/mmole), and Lvaline-5-3H (specific activity 0.6 Ci/mmole), Schwarz Bioresearch, Inc., Orangeburg, N. Y.; 6C3HED lymphoma was obtained from Dr. T. Hauschka, Roswell Park Memorial Institute, and was maintained in C3H mice in ascites form. Asparaginase Activities in Plasma. Blood plasma was ob tained either by finger prick into heparinized capillary tube or by venipuncture after i.v. administration of asparaginase. From the capillary tube, 5 to 10 pi of plasma were used directly or after appropriate dilution for the assay (31 ). The activity was expressed in i.u. The sensitivity of the method was such as to detect activity of 1 X IO"3 i.u./ml. Protein was measured by days prior to 5 daily injections of the drug. One patient received 5 additional doses every other day after he reached Ml marrow, 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. The total dose range varied from 200 to 40,000 i.u./kg. The highest total dose given was 2,200,000 i.u. Immunological Study. The first 34 patients received 10 or 20 i.u. of asparaginase intradermally as a skin test prior to the commencement of treatment. This was later abandoned because initial skin tests were never positive and anaphylactoid reactions occurred in patients despite negative skin test. In some patients, presence of antibody against asparaginase in the serum was determined by the method described by Campbell et al. (IO).

the method of Lowry et al. (27). Assay of Plasma L-Asparagine and L-Aspartic Acid. Assays RESULTS were carried out according to the method described by Cooney et al. (13). Samples of plasma were placed in a boiling Asparaginase Activity in Plasma. Asparaginase vials of dif water bath for 10 min to inactivate intrinsic enzymes. The congealed plasma was cooled and centrifuged at 105,000 X g ferent lots were randomly selected, and the activity was at 4 for 1 hr. Clear supernatant, 0.9 ml, and 0.1 ml of measured. These enzymes were dissolved as long as 10 months reaction mixture consisting of 10 mM a-ketoglutaric acid, 2 100-1 mM NADH, 0.3 i.u. of GOT, and 500 mM Tris buffer, pH 8.0, were placed in a silica cuvet. Malic dehydrogenase, 0.72 i.u. in 0.1 ml of 100 mM Tris buffer, pH 8.0, was added, and the 50decrease in absorbance at 340 m/i was recorded until the reaction reached a plateau (40 to 50 min). This change in absorbance represented the plasma L-aspartic acid. Then 0.8 i.u. of asparaginase (E. coli) in 0.01 ml of 0.9%NaCl solution was added, and the further decrease in absorbance at 340 mp. was similarly recorded, which represented the amount of L-asparagine. The values of L-asparagine and L-aspartic acid were calculated from concurrent standards made from known concentrations of the amino acids. Test of Leukemic Cells for L-Asparagine Requirement in K;/ro.The white cells were separated from the peripheral blood or bone marrow aspirate by a method developed in this labora tory (16). 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, 1 /nCi/ml; final concen tration, 0.05 jLtM),L-valine-5-3H (final activity, 4 juCi/ml) or DL-lysine-4,5-3H (final activity, 1 iCi/ml)in the presence of L-asparagine (final concentration, 0.38 mM) or asparaginase (final activity, 0.083 i.u./ml) were carried out by the method described by Oettgen et al. (30). 6C3HED lymphoma cells in the same concentration were incubated concomitantly to ensure that the system was optimal. 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. Administration, Dosage, and Schedule. The lyophilized enzyme was dissolved with 0.9% NaCl solution (5 ml/vial) and 0.1 injected i.v. directly or through the side arm of a running infusion within 0.5 hr after being dissolved. The drug was given as single, daily, or weekly doses. Most patients on the Chart 1. 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.

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Clinical Studies prior to the testing date and stored at -20". Frozen storage of

with Asparaginase

this duration was found not to cause any loss of activity. Eleven vials from 5 different lots labeled as containing 10,000 i.u. were found to have activity ranging from 6,100 to 7,900 i.u./vial and specific activity of 116 to 182 i.u/mg of protein. The doses in this paper are expressed in terms of label potency to afford comparison with the data of others. The activity of asparaginase found in plasma, however, is reported as such. The clearance of plasma asparaginase is shown in Chart 1. It may be seen that the peak activity lasted for about 3 hr after the injection, it was dose related, and the clearance of the activity from plasma followed first-order kinetics with plasma half-times of 14 to 22 hr. When blood samples were obtained by finger prick, the activity in the 2-hr samples was con sistently higher than the preceding samples, but this finding was not confirmed in the samples obtained by venipuncture. In 2 patients, the plasma enzyme activity was measured serially for a longer period. The activity was detectable 13 and 22 days after single injection of 1000 and 5000 i.u. /kg, respectively, but not after 18 and 25 days, respectively. Daily administration of 400 or 1000 i.u./kg of the enzyme had a cumulative effect (Chart 2). Development of immunological reactions were associated with rapid clearance of plasma enzyme. Thus, in one patient who developed arthralgia, erythematous skin eruption, and positive intradermal test to 10 i.u. of the enzyme of Day 15, 5 days after the end of a 5-day course of 1000 i.u./kg/day, the plasma asparaginase level fell rapidly and became undetectable
SO-i

4 days after the last injection. In another patient whose plasma enzyme activity became undetectable 8 days after a 2nd dose of 5000 i.u./kg of enzyme, the plasma was found to have a positive precipitation reaction to asparaginase. Plasma L-Asparagine and L-Aspartic Acid. Plasma L-asparagine and L-aspartic acid values in 10 healthy men, 10 healthy women, 10 patients from 7 to 44 years with ALL, and 8 patients from 12 to 72 years with AML were plotted in Chart 3. In healthy adult controls, L-asparagjne values were 60 13 (mean 1 S.D.) and 54 nmoles/ml in men and in women, 16 respectively. L-Aspartic acid concentrations were 11 and 4 10 nmoles/ml in men and women, respectively. 4 Among 10 patients with ALL, there appear to be 2 groups: 1 group with L-asparagjne concentration within or near the normal range and with normal L-aspartic acid, the 2nd group with low L-asparagjne and reciprocally elevated L-aspartic acid. This subgroup of 3 patients was unique in that 2 patients died within 48 hr of injection. The 3rd patient was induced into complete remission. Patients with AML had lower levels of L-asparagjne with normal L-aspartic acid concentration. After enzyme administration, plasma L-asparagjne fell pre cipitously, with a reciprocal rise in L-aspartic acid. Even 0.2 i.u./kg of the enzyme, which is in fact the skin test done, caused substantial fall of plasma L-asparagine levels by the following day. In 6 measurements, L-asparagine fell from 31 7 (mean S.E.) to 4 and L-aspartic acid rose from 26 6 1, to 43 nmoles/ml. After a single injection of 1000 i.u./kg 3 i.v. in 1 patient, 16 measurements in 32 days revealed that

0.5-

0.1

1 72 HOURS

n
120

1 144

i
192

24

48

we

Chart 2. Plasma asparaginase activity during and after 5 daily injections of asparaginase in 3 patients, o o, enzyme level in a patient who, after a single injection of 400 i.u. 5 days previously, received 400 Lu. daily for 5 doses; a a, enzyme level in 2 patients who, after a single injection of 1000 i.u. 6 days previously, received 1000 i.u. daily for 5 doses. See text for discussion of the rapid fall in patient plotted as A.

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T. Ohnuma, J. F. Holland, A. Freeman, and L. F. Sinks been completed on 16 patients who died after asparaginase treatment. With the exception of 2 patients, 1 who died on the day of asparaginase administration, and 1 who died 10 days after receiving 200 i.u./kg, livers of all patients showed severe fatty metamorphosis. Albumin decreased an average of 1.8 g/100 ml during the first 2 weeks and then reached a plateau. Seven of 8 valuablepatients showed hypocholesterolemia with average decrease of 62 mg/100 ml. Eight of 10 valuable patients had a fall of fibrinogen level averaging 214 mg/100 ml. In 3 of those 10 patients, there was a concomitant defi ciency in Factor V, Factor VII, and Factor X. Bleeding, observed frequently in patients with leukemia, could not usually be ascribed only to fibinogenopenia or other coagula tion protein deficiencies, since thrombocytopenia often existed also. In contrast to the decrease in his serum albumin, one patient with IgA multiple myeloma showed no changes in immunoglobulin levels with serial determinations after 5000 i.u./kg of the enzyme was administered. Mild azotemia was commonly noted at about Day 7 of the study. We did not establish whether this was renal or prerenal in origin. The most striking side effect of treatment, when it oc curred, was cerebral dysfunction. Mild depression and drowsi ness were found in about 25% of the patients. Severe brain dysfunction, manifested as disorientation and confusion, was seen in 7 patients. In 2, it was noted within 24 hr; in the other 5, it did not appear until after 1 week. In 2 of the delayed dysfunction patients, disorientation and confusion were

80

60

I,
ao-

-8-

Sb

NORMAL MALE

NORMAL FEMALE

ALL

AML

Chart 3. Plasma L-asparagine (ASN) and L-aspartic acid (ASP) values in normal healthy men and women, and in patients with ALL and AML. u, *, v, T, ,and o, 3 patients with reverse correlation of the amino acids.

L-asparagine levels remained immeasurable until Day 21 but returned by Day 23. In another patient, 14 measurements in 33 days after a single injection of 5000 i.u./kg showed that L-asparagine was unmeasurable until Day 29 but returned by Table 1 Day 33. In both cases, 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. These results indicate the prolonged activity patients with adequate observations to determine toxicity in question. of exogenous enzyme in man. No. of No. of Toxicity. Toxicity observed after asparaginase administra valuable affected affected tion is summarized in Table 1. Chart 4 shows the relation between dose range and composite mean score for toxicity 1. Immediate ratings of 0 to 4+, implying none, mild, moderate, severe, and Nausea, vomiting, and chills 27 38 71 life threatening. Thirteen of 15 patients who received a single 2. Delayed 1. Hepatic 32 33 97 dose were scheduled to receive additional drug in weekly Increase in bilirubin, GOT, courses, but because of death from advanced disease in 8, and LDH, alkaline phosphatase, because of toxicity in 5, no further drug was given. It is thus decrease in albumin, not unanticipated that the mean toxicity ratings for the group cholesterol, fibrinogen, and other hepatic coagulation that received only single doses would be higher than for the factorsfatty metamorphosis. other regimens. In the other dosage regimens, mean toxicity 2. 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. Azotemia 25 38 68 L-asparagine starvation. 4. Neurological: 13 39 33 Headache, drowsiness, Immediate reactions usually occurred 0.5 to 1 hr after injec depression, disorientation, tion and lasted approximately another hr. These reactions confusion included nausea and vomiting, fever, and chills. In some 5. Pancreatic: 6 39 15 patients, nausea or emesis lasted a few days. In patients who Abdominal pains, increase in amylase or lipase, hyperglycemia, received weekly injections, premedication with antihistaminics hypoinsulinemia, malabsorption and hydrocortisone often modified or eliminated these im syndrome mediate reactions. 6. Immunological: Hepatic toxicity was observed more frequently than any Positive skin test, anaphylactoid 5 39 13 other. Increase in bilirubin, GOT, LDH, and alkaline phosreaction 7. Miscellaneous: phatase and, decrease in albumin, cholesterol, fibrinogen, and Transverse banding of the other coagulation factors occurred. At death, severe fatty fingernails, hypotension metamorphosis was seen (Fig. 1). Microscopic observation has

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Clinical Studies
lu gW 15yZ 2 On

with Asparaginase

05,i0

u8
Ul2

5,000-"

u u u u

;1POPODOI]DDDGDoUUU JUn1^TiDULiuiD
DDDDDAILY u
HttM.I

10,000tiu/kgS]j[11erenD|uuSINGLEi]Jrm!ay

DDDODUUDODDDDDDDDD

Chart 4. Composite mean toxicity score versus dose and schedule of asparaginase. The first 7 patients receiving weekly treatment were given 1000 i.u./kg/week, for example, and the next 3 patients received 4000 i.u./kg. Under daily treatment, gaps in dose schedule schema represent observation intervals of 4 to 14 days. Three patients from the single injection group and 1 patient from the daily injection group were omitted because of inability to evaluate toxicity scores. Toxicity score: 0, none; 1, mild; 2, moderate; 3, severe; 4, life-threatening toxicity.

accompanied by loss of recent memory, confabulation, and had been injected. Three other patients developed anaphy easy suggestibility which resembled Korsakow's psychosis. The lactoid reactions. The reaction includes shaking chills, hypo syndrome lasted nearly 1 month in each. In one case, even a tension, stridor, numbness, cyanosis and, in one patient, coma, single dose of 1000 i.u./kg of asparaginase produced late- beginning within 2 min. All 3 patients had had negative skin tests before their repeat weekly injections: the 2nd dose of appearing dysfunction (33). Pancreatic toxicity was manifest in varying degree in 4 5000 i.u./kg, and the 3rd and 4th, respectively, of 1000 patients as abdominal pain, upper abdominal tenderness, diar i.u./kg. These patients were treated with epinephrine, antirhea, increase in serum and urinary amylase, hyperglycemia, histaminics, and steroid and recovered without complication. hypoinsulinemia and hypocalcemia. Development of a malabOne patient with AML, who after 1,000 i.u./kg of the sorption syndrome was suggested in 2 of these patients by a enzyme developed acute brain dysfunction, pancreatitis, positive Schilling test done with and without intrinsic factor. malabsorption, and muscle wasting, was later found to have Nonketotic hyperglycemia occurred in 3 adults and 2 transverse banding of the nails (33). Another patient with children, of whom 2 showed no clinical evidence of pan AML developed hypotension of 70 mm Hg systolic and 45 mm creatitis. Blood glucose rose as high as 955 mg/100 ml in 1 Hg diastolic occurring 3 days after 10,000 i.u./kg of the patient. In 2 patients, the hyperglycemia was accompanied by enzyme. He received metaraminol and steroid for 3 days and polyuria of 11,000 and 8,000 ml, respectively, with 4+ urine recovered. sugar tests. Polyuria responded to insulin but not to antiBone marrow aplasia was not observed in nonleukemic diuretic hormone. Blood sugar levels fell back to normal range patients. Although we have seen a variety of unusual toxic effects, in approximately 2 weeks. The response of hyperglycemia to insulin was appropriate, showing neither unusual sensitivity the drug was fairly well tolerated by most of the patients and nor resistance. Hyperglycemia occurred in one man only after the toxicities were essentially reversible. Two patients died on the 1st dose and was not seen after the 2nd, 3rd, or 4th Day 1 who had far-advanced neoplasms with overwhelming weekly dose of asparaginase, possibly because of depletion of infections, and causal relation to asparaginase was not found at hepatic glycogen, as suggested by negative periodic acid-Schiff autopsy. Another patient with ALL who received 1000 i.u./kg stain of the liver at autopsy. died on Day 2 with disorientation, confusion, fever, and Positive skin test or anaphylactoid reaction were not ob increase of blood urea nitrogen from 4 to 40 mg/100 ml. At served at the first exposure to the enzyme. Intradermal injec autopsy, bronchopneumonia was seen. L-Asparagine "Rescue" Infusion. L-Asparagine rescue in tion of 10 i.u. evoked a positive response 18 hr later in a 44-year-old man who received a single injection of 1000 i.u./kg fusion was given to 3 patients for the acute brain dysfunc 1 week previously. He had no evidence of clinical allergy but tional syndrome in hopes of reversing the abnormalities. The was not retreated. 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.u./kg/day was followed by periment (38). Pertinent data are summerized in Table 2. No an illness resembling serum sickness with generalized arthralgia adverse reaction was observed from the amino acid infusion. In and paravenous erythema near vein sites where asparaginase 2 patients, definite improvement ensued, and in 1, probable

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T. Ohnuma, J. F. Holland, A. Freeman, and L. F. 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, sterilized by Nalgene Filter Unit (Nalgene Labware Division, Rochester, N. Y.), and infused in 24 hr.

L-asparagineduringinfusion(nmoles/ml)160Serumalbuminaft ofL-asparagine(mmoles/kg/24 ofaspar mentalstatusDefiniteProb aginase(i.u./kg infusion(nmoles/ml)0043Serumalbuminbeforeinfusion2.6 hr)121Daysgiven12-179-1438-44Maximum PatientD.W.J.C.D.D.DiagnosisAMLCML(blastic)AMLDose 1)1,0005,00010,000PlasmaL-asparaginebefore X 7)2.6(Day 10)1.7 (Day 9)1.3 (Day 10)0.9 (Day 27)1.5 (Day (Day 38)Dose Table 3 Response in leukemia LSA-*ALL indicates ALL converted from prexistent SA. L 18)2.0 (Day 21)2.1 (Day 15)1.9 (Day 17)2.0 (Day 48)2.2 (Day (Day 56)Improvementin

Ur-3H and L-valine-5-3H (or DL-lysine-4,5-3H) by leukocytes

in 20 patients as an attempt at predictive behavior. 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. In the presence of Adult Child Adult Child Adult Child asparaginase, incorporation of uridine and lysine was always less than 30% of controls in the mouse lymphoma. The inhibi MlM2Hematologicaleffect tions in human leukemic cells were never this pronounced. Furthermore, incorporation of precursors by control human culture was always less than the mouse lymphoma cells ranging onlyNo response0013100060100000311530002 from 0.45 to 20.8%, with an average of 6.6%. Biochemical prediction of therapeutic outcome was not reproducibly suc Total 16 10 cessful, since indistinguishable biochemical results were found in ALL and AML, and within diseases no good correlation improvement was noted. The relation to infusion was such with hematological response occurred. that clinical observers considered the phenomena to be caus ally related (33). Therapeutic Effect. The response to asparaginase in patients DISCUSSION with leukemia is summarized in Table 3. 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. These patients enzyme. Systematic increase in enzyme levels for the first 2 hr were classified as "hematological effect only." Decrease in in finger prick blood, as contrasted to blood taken by veniperipheral leukocytes alone was not included. The enzyme is puncture, 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. This implies an AML of adults as well. Although the numbers are small, there early and appreciable extravascular space of distribution. appear to be no differences in the response rate in children Initial peak activities of 5, 12, and 60 i.u./ml obtained after with ALL between those who received daily injections injections of 400, 1000, and 5000 i.u./kg, respectively, are (Ml/total, 5/8) and those with weekly treatment (Ml/total, reasonably close to estimated values of 5.6,14, and 70 i.u./ml 4/8). 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.u./kg (33). Another labeled dose, and distribution in the plasma volume estimated woman with AML whose marrow was induced to M2 died as 5% of body weight. After asparaginase administration, sus from massive pulmonary embolism. All the 10 ALL patients tained depression of L-asparagine concentration was observed. 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- E. coli enzyme is cleared from plasma within 24 hr and where cristine and/or Daunorubicin with prednisone. Among these depressed L-asparagine returned to normal in 48 hr (7, 8). We 10 ALL patients who were induced into Ml, 3 relapsed on have previously shown, by using avian and guinea pig enzymes, Days 14, 15, and 21; 1 died from empyema despite the Ml an inverse correlation between the rate of clearance of exog marrow; and 6 went into a Phase II maintenance program on enous enzyme in the plasma and the therapeutic effects in other drugs. No patient with solid tumor responded to asparag mouse tumors (31). From this point of view, the prolonged clearance of E. coli enzyme in man may be regarded as a inase. L-Asparagine Requirement by Leukemic Cells In Vitro. favorable factor in its effectiveness. Prolonged half-life of E. Chart 5 shows the 36 studies of in vitro incorporation of coli enzyme in man [8 to 30 hr (E. Frei, III, personal com-

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Clinical Studies

with Asparaginase

L^IsJJjxAA - SH O
lOO-80-.60-40"2O-0-Ur.

AOOO

AA.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. Bacterial endotoxin is known to cause chills, fever, vomiting, hypotension, fibrinogenopenia, hepatic injuries (39), depletion of liver glycogen, renal failure, coagulopathies, and delayed hypersensitivity reaction (41). These effects resemble many of the toxicities observed after asparaginase. Probably the most intriguing finding to explain much of the toxicity is the deficiency in protein biosynthesis produced by L-asparagine depletion. Thus, after asparaginase, incorporation of L-methionine-35S
*AlA

XPX6C3HEDLYMPHOMAOfPAA0AAOA0

Mz IMP ALL

NR

Ml

M2 IMP AML

NR

Chart 5. L-Asparagine requirement by leukemic cells m vitro Ml and M2 indicate eventual marrow status achieved after treatment. IMP, improvement; NR, no response, DL-Lysine-4,5-3H was used in the mouse experiments, and valine-5-3H was used in all human studies.

munication); 7 to 19.5 hr (21)] compared to that in mice [2.5 hr (3)] has also been observed by others. This provided basis for the weekly administration of the enzyme rather than daily, with indistinguishable therapeutic effects. The enzymatic assay of L-asparagine and L-aspartic acid developed by Cooney et al. (13) was found to be reliable and convenient compared to older methods, and reproducible with as little as 5 nmoles/ml. No other method of protein precipi tation was studied to ascertain whether an artifact of transient heat activation of enzyme occurred. Further refinement may, however, be necessary to prevent in vitro amidohydrolysis which may take place while the sample is being processed. Standard L-asparagine solution was found to be hydrolyzed slowly during storage at 0-4". Over a period of 3 months, there is a loss of about 1%/10 days. The basis for aberration of L-asparagine and L-aspartic acid values in leukemic patients is not clear. As a group, patients with AML had low L-asparagine levels and only 1 complete remission occurred. Most patients with ALL had normal L-asparagine and L-aspartic acid levels, and the complete remission rate was high. Three patients with ALL had an inversion of normal L-asparagine and L-aspartic acid ratios, reminiscent of effects of asparaginase per se. This may be due to overwhelming infection or functional abnormalities in the dying patient. Many factors have been considered as possible contributors to the wide spectrum of toxicity, such as contamination by glutaminase activity (29), or the effects of liberation of ammonia from L-asparagine, by the depression of L-asparagine, by the administration of an exogenous protein or large molecule per se, but particularly by the possible endotoxin contaminant. Since the amount of asparaginase given to man is 1,000 to 10,000 times as much as was tested for pyrogenicity in the rabbit, it is possible that a proportionate

into albumin was markedly inhibited (J. F. Holland, L. Walter, and T. Ohnuma, unpublished data). Clinical findings suggest that synthesis of albumin, insulin, fibrinogen, and fingernail protein were defective, whereas that of globulin was apparently unaffected. The failure to find hypoglobulinemia suggests some systematic differential requirement for L-aspara gine between liver cell and plasma cell. Since immunoglobulin contains the same general amounts of L-asparagine as most other proteins, the plasma cell presumptively has a welldeveloped system to supply its own L-asparagine. Together with the evidence of patients who developed immunological reactions, these findings suggest that asparaginase treatment is not immunosuppressive, a situation dissimilar to data from the mouse (36). Since skin testing did not indicate the patients who sustained anaphylactic response, we have abandoned it. We are aware of 1 fatal instance of anaphylaxis encountered by a colleague, despite immediate and vigorous therapeutic measures. L-Asparagine has been shown to protect against ethionine- or carbon tetrachloride-induced fatty liver, which presumably resulted from defective removal of fat because of inhibition of protein biosynthesis (1). 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. We have observed 2 forms of diffuse brain dysfunctional syndrome: one occurred within 24 hr and then cleared rapidly; the other occurred insidiously about 1 week later and lasted approximately 1 month. In all instances, central nervous sys tem leukemia, massive central nervous system bleeding, meningitis, and electrolyte imbalance and other iatrogenic causes were excluded. We have not measured ammonia on each occasion, 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. 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). 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. Clinical observation of improvement in mental status after L-asparagine rescue infusion in all 3 patients studied is of interest. Mental abnormalities did not apparently increase after interruption of L-asparagine infusion in 2 patients. In the 3rd

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T. Ohnuma, J. F. Holland, A. Freeman, and L. F. Sinks patient (J. C.), recrudescence of brain dysfunction followed after discontinuation of L-asparagine infusion. Serum albumin levels did not correlate well with abnormal mentality, sug gesting differences in chemical abnormalities of liver and ner vous system. The close correlation of L-asparagine infusion with benefit suggests that the relation is causal and not coin cidental to spontaneous recovery. It should be added, how ever, 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. Even 12 days after aspara ginase administration in Patient D. W., abnormally high L-aspartic acid levels resulted from L-asparagine infusion (33). Because L-asparagine given early may thus give rise to toxic levels of ammonia, its use is contraindicated when asparaginase levels are high. A nontoxic inhibitor of asparaginase would be of value to allow restitution of L-asparagine levels. The in vitro estimation of RNA and protein biosynthesis and its inhibition by L-asparagine deficiency evolved by asparaginase did not provide reliable prognostication. Possible factors which might influence clinical results are the develop ment of an antibody that rapidly clears the enzyme from plasma. We observed this on one occasion. When patients developed anaphylactoid responses or positive skin reaction to the drug, we terminated treatment. The derepression or induction of an L-asparagjne-synthesizing system in leukemic cells after asparaginase administration has been reported (19). The rapid selection of a variant animal tumor cell population which does not require L-asparagine can occur/ vitro even in the presence of L-asparagjne (34). All these factors may con tribute to the discrepancy of in vitro estimation and thera peutic effect. One of the most difficult clinical problems is that the toxicity is apparently not dose related and some manifesta tions such as anaphylaxis, pancreatitis, brain dysfunctional syndrome, and coagulopathies can be lethal. Proper and close clinical observation of patients receiving the enzyme is there fore essential.
Guinea Pig Serum Is Responsible for Its Antilymphoma Effects. Nature,/p;.-1114-1115,1961. Broome, J. D. Evidence That the L-Asparaginase of Guinea Pig Serum Is Responsible for Its Antilymphoma Effects. I and II. J. Exptl. Med., 118: 99-120, 121-148, 1963. Broome, J. D. Antilymphoma Activity of L-Asparaginase in Vivo. Clearance Rates of Enzyme Preparations from Guinea Pig Serum and Yeast in Relation to Their Effect on Tumor Growth. J. Nati. Cancer Inst., 35: 967-974, 1964. Broome, J. D. Studies on the Mechanism of Tumor Inhibition by L-Asparaginase. J. Exptl. Med., 727: 1055-1072, 1968. Broome, J. D. Factors Which May Influence the Effectiveness of L-Asparaginase as Tumor Inhibitors. Brit. J. Cancer, 22: 595-602, 1968. Burchenal, I. H., Karnofsky, D. A., Murphy, M. L., and Oettgen, H. F. Effects of L-Asparaginase on Leukemias and Other Neoplasms (Abstr.). Program of the XII Congress of the International Society of Hematology, New York, p. 7, 1968. Campbell, D. H., Garvey, J. S., Cremer, N. E., and Sussdorf, D. H. Antigen-Antibody Reactions. Ring or Interfacial Test. In: D. H. Campbell, J. S. Garvey, N. E. Cremer, and D. H. Sussdorf (eds.), Methods in Immunology, pp. 131-135. New York: W. A. Ben jamin, 1964. Campbell, H. A., Mashburn, L. T., Boyse, E. <\., and Old. L. J. Two L-Asparagjnases from Escherichia coli B. Their Separation, Purifica tion and Anti-Tumor Activity. Biochemistry, 6: 721-730, 1967. Canellos, G. P., Haskell, C. M., Arseneau, J., and Carbone, P. P. Hypoalbuminemic and Hypocholesterolemic Effect of L-Asparag inase (NSL-109229) Treatment in Man-Preliminary Report. Cancer Chemotherapy Rept.,55: 67-69,1969. Cooney, D. A., Capizzi, R. A., and Hand Schumacher, R. E. Evalua tion of L-Asparagine Metabolism in Animals and Human Subjects. Cancer Res., 30: 929-935, 1970. Dolowy, W. C., Henson, D., Cornet, J., and Seilin, H. Toxic and Anti-Neoplastic Effects of L-Asparaginase. Study of Mice with Lymphoma and Normal Monkeys and Report on a Child with Leukemia. Cancer, 19: 1813-1819, 1966. Ellison, R. R.. Holland, J. F., Weil, M., Jacquillat, C., Borion, M., Bernard, J., Sawitsky, A., Rosner, F., Gussoff, B., Silver, R. T., Karanas, A., Cuttner, J., Spurr, C. L., Hayes, D. M., Blom, J., Leone, L. A., Haurani, F., Kyle, R., Hutchison, J. L., Forcier, J., and Moon, J. H. Arabinosyl Cytosine: A Useful Agent in the Treat ment of Acute Leukemia in Adults. Blood, 32: 507-523, 1968. Gailani, S. Pyridoxal Phosphate Determination in Isolated Leuko cytes and Tissue by E. coli Apotryptophanase. Anal. Biochem., 13: 19-27, 1965. Gralnick, H. R., and Henry, P. H. L-Asparaginase Induced Coagulopathy (Abstr.). Proc. Am. Assoc. Cancer Res., 10: 32, 1969. Harris, J. E., Whitecar, J., Bodey, G. P.. and Freireich, E. I. L-Asparaginase in the Treatment of Adult Acute Leukemia (Abstr.). Proc. Am. Assoc. Cancer Res., 10: 35, 1969. Haskell, C. M., and Canellos, G. P. Asparagine Synthetase in Human Leukemia. A Mechanism of Resistance to L-Asparaginase (Abstr.). Clin Res., 17: 402, 1969. Haskell, C. M., Canellos, G. P., Leventhal, B. G., Carbone, P. P., Block, J. B., Serpick, A. A., and Selawry, O. S. L-Asparaginase: Effects in Patients with Neoplastic Disease. New Engl. J. Med., 281: 1028-1034, 1969. Haskell, C. M., Canellos, G. P., Leventhal, B. G., Carbone, P. P., Serpick, A. A., and Hansen, H. H. L-Asparaginase Toxicity. Cancer Res.,2P.' 974-975, 1969.

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ACKNOWLEDGMENTS
We are grateful to Mrs. Sylvia Grew and Mr. John Gawoski for theii skillful technical assistance.

16. 17. 18.

REFERENCES
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Clinical Studies
23. Hill, J. M., Loeb, E., Roberts, J., MacLellan, A., Khan, A., and Hill, N. O. L-Asparaginase Therapy of Leukemia (Abstr.). Program of the XII Congress of the International Society of Hematology, New York, p. 8,1968. 24. Hill, J. M., Roberts, J., Loeb, E., Han, A., MacLellan, A., and Hill, R. W. L-Asparaginase Therapy for Leukemia and Other Malignant Neoplasms. J. Am. Med. Assoc., 202: 882-888,1967. 25. Kidd, J. G. Regression of Transplanted Lymphomas Induced in Vivo by Means of Normal Guinea Pig Serum-I and II. J. Exptl. Med., 98: 565-582,583-606, 1953. 26. Lash, E., Schwartz, M. K., Tallal, L., and Oettgen, H. F. Serum Lipids in Patients Receiving L-Asparaginase (Abstr.). Program of the XII Congress of the International Society of Hematology, New York, p. 10,1968. 27. Lowry, O. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.,;PJ: 265-275, 1952. 28. Mashburn, L. T., and Winston, J. C., Jr. Tumor Inhibitory Effect of L-Asparaginase from Escherichia colt. Arch. Biochem., 105: 451-452, 1964. 29. Miller, H. K., Salser, J. S., and Balis, M. E. Amino Acid Levels following L-Asparagine Amidohydrolase (EC 3.5.1.1.) Therapy. Cancer Res., 29: 183-187,1969. 30. Oettgen, H. F., Old, L. J., Boyse, E. A. Campbell, H. A., Philips, F. S., Clarkson, B. D., Tallal, L., Leeper, R. D., Schwartz, M. K., and Kim, J. H. Inhibition of Leukemias in Man by L-Asparaginase. Cancer Res., 27: 2619-2631,1967. 31. Ohnuma, T., Bergel, F., and Bray, R. C. Enzymes in Cancer. Asparaginase from Chicken Liver. Biochem. J., 103: 238-245, 1967.

with Asparaginase

32. Ohnuma, T., Holland, J. F., Freeman, A., and Sinks, L. Studies of Asparaginase and Asparagine in Acute Leukemia (Abstr.). Proc. Am. Assoc. Cancer Res., 10: 66,1969. 33. Ohnuma, T., Holland, J. F.. St. Arneault, G., and Nagel, G. Effects of Asparaginase in Acute Myelocytic Leukemia. J. Am. Med. Assoc., 210: 1919-1921,1969. 34. Patterson, M. K., Jr., Maxwell, M. D., and Conway, E. Studies on the Asparagine Requirement of the Jensen Sarcoma and the Devia tion of Its Nutritional Variant. Cancer Res., 29: 296-300,1969. 35. Roberts, J., Prager, M. D., and Bachynsky, N. The Antitumor Activity of Escherichia coli L-Asparaginase. Cancer Res., 26: 2213-2217,1966. 36. Schwartz, R. S. Immunosuppresion by L-Asparaginase. Nature, 224: 275-276, 1969. 37. Tallal, L., Tan, C., Oettgen, H. F., McCarthy, M., Helson, L., and Murphy, L. L-Asparaginase in 111 children with Leukemias and Solid Tumors (Abstr.). Proc. Am. Assoc. Cancer Res., 10: 92, 1969. 38. Tower, D. B. Nature and Extent of the Biochemical Lesions in Human Epileptogenic Cerebral Cortex. Neurology, 5: 113-130, 1955. 39. Weil, M. H. The Animal Model and the Human Patient in Bacterial Shock. In M. Landy and W. Braun (eds.), Bacterial Endotoxins, pp. 187-210. New Brunswick, N. J.: The Institute of Microbiology, Rugers-The State University, 1964. 40. Whitecar, J. P., Jr., Harris, J. E., Bodey, G. P., and Freireich, E. J. Host Effects of L-Asparaginase Treatment (Abstr.). Clin. Res., 17: 408, 1969. 41. Zweifach, B. W., and Jenoff, A. Bacterial Endotoxemia. Ann. Rev. Med., 16: 201-220, 1965.

Fig. 1. Fatty metamorphosis of the liver. This severe fatty metamorphosis was seen in a patient whose liver function 5 days prior to death showed only slight abnormalities (bilirubin, 2.0 mg/100 ml; serum GOT, 32 units; LDH, 137 units; alkaline phosphatase, 4.6 Bessey-Lowry units; total protein, 8.0 g/100 ml). H & E.

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