cancer res-1970-ohnuma-2297-305

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1970;30:2297-2305. Published online September 1, 1970.Cancer Res

Takao Ohnuma, James F. Holland, Arnold Freeman, et al.

ManBiochemical and Pharmacological Studies with Asparaginase in

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[C AN CE R R ES EA RC H 30, 2297-2305, S eptem ber 1970]

Biochem ical and Pharm acological Studies with Asparaginasein M an1

Takao Ohnum a, Jam es F. Holland, Arnold Freem an, and Lucius F. Sinks

D epartm ents of M edicine A /T. O ., J. F. H .] and P ediatrics A .F., L. F. S./, Rosw ell Park M em orial Institute, N ew YH ealth, B uffalo, N ew York 14203

SUMMARY

T he b io ch em ical a nd p harm aco lo gical effec ts oEscherichiacoli asparagjnase w ere studied in 45 patients w ith leukem iaand solid tumors. The drug was given in three differentschedules: as a single dose, daily, or w eekly. A fter i.v. injection, the peak activity obtained was dose related; the initialclearance of the enzyme from plasma followed first-orderkinetics, and a half-life was about 14 to 22 hr. Daily administration of the enzyme caused cumulation in serum. Enzymeactivity w as detectable 13 to 22 days after single injections.Plasm a asparagjne and aspartic acid levels in leukem ia w erecompared with levels in 20 normal individuals. Three of 10patients with acute lymphocytic leukemia had marked aberration in their amino acid levels. Patients with acute myelo-cytic leukemia as a group had low ered levels of asparagine.A fter enzym e adm inistration, plasm a asparagine fell precipitou sly to n early un measu rea ble lev els. A sparag in e reap pea redin plasm a 23 to 33 days after single injections. E ven 0.2 i.u./kgas a skin test dose produced a substantial fall of plasmaasp ara gine. A sparag in ase p rod uced m ultiple m an ifestation s oftoxic ity invo lv ing b ra in , liver, pancreas , k idne y, finge rnail, andhy persen sitiv ity reac tio ns. A sp ara gine w as g iv en as a "rescu e"infusion in three patients for acute brain dysfunction withbenefit. Ten of 24 patients w ith acute lym phocytic leukem iaand 1 of 12 patients with acute myelocytic leukemia wereinduced to bone marrow rating 1 marrow remission, 2 aftersingle injections. It w as difficult to correlate response in vivoand a requirem ent of the leukem ic cells for asparagine in vitro.

INTRODUCTION

Regression of transplanted mouse and rat lymphomas byguinea pig serum was first described in 1953 (25). The anti-tumor factor w as later shown to be an enzyme, asparaginase(L-asparagine am idohydrolase, EC 3.5.1.1) (4, 5). Search forother sources of the enzyme revealed that enzymes of different origins had different rates of clearance from plasm a andd issim ilar biolog ical activ ity (6,3 1). Iso latio n o f an active fraction of asparaginase from E. coli (11, 28, 35) provided apractical source of the enzyme for clinical study. Initial

clinical trials in A LL 2 in children w ith the E . coli enzyencou ra gin g (24, 3 0).

In the initial clinical trials w ith E . coli enzym e, chillem esis, pyrexia (30), hypersensitivity reactions (24,fatty liver (24) were reported. In a critically ill chA LL , h yp oten sion , h em olysis, and inte stinal he morrhobserved after guinea pig serum enzym e adm inistratioN evertheless, general toxicity of the host w as not regacritical problem (14, 24, 30), perhaps in part becausinitial assum ption that the drug acted on a specific m

defect in sensitive tumor cells. W hen more enzymeavailable, further studies w ere conducted and a w ideof toxicity was recognized (2, 9, 12, 17, 18, 20-23,37, 40). The present paper reports our observationsch em ical an d ph arm aco lo gical stu dies w ith E . co li aspin patients w ith leukemia and solid tumors. Portionsw ork have been reported in a prelim inary form elsew h

M ATE RIA LS A ND M E TH OD S

Forty-five patients were studied: 24 with ALL ofhad converted from pr existent LSA , 12 w ith AM Lmalignant melanoma, 2 with osteogenic sarcoma, an

with CML in the blastic phase, choriocarcinoma,blastom a, m ultiple m yelom a, and carcinom a of the copatients w ith A ML had received no prior treatment,the remaining leukemic patients w ere in frank relapprior chemotherapy at the time of the asparaginasepatients w ith solid tum ors had previously been treathad palpable or radiological evidence of tum or. Patieleukemia were evaluated by the rating criteria oL eukem ia G roup B (15). R esponse in solid tum ors w aby at least 25% decrease in the product of 2 diametre flectio n of tum or size.

A sparaginase w as obtained from M erck, Sharp andR esearch L aboratories, West Point, Pa., through theCancer Institute. The sources of chemicals usedfollow s: L -asparagine, anhydrous, and L -aspartic acitional Biochemical Corp., Cleveland, Ohio; phencrystals, M allin ck ro dt C hem ical Work s, S t. L ou is, M ohypochlorite, W ill Scientific, Inc., Buffalo, N . Y.;sodium nitroprusside, Fisher Scientific C o., Fairlaw

'This investigation was supported by USPH S Research GrantsCA-5834 and CA-2599 f rom the National Cance r Ins ti tu te .

Received Janua ry 6 , 1970; accep ted May 8 ,1970.

'The abbreviations used are: A LL , acute lym phocyticLSA, lymphosa rcoma; AML, acu te mye locy ti c l eukemia; CMmyelocyt ic leukemia; GOT, glutamic-oxaloacet ic t ransa minuridine-5-3H , M l, bone m arrow rating 1; M 2, bone m arrowLDH, lactic dehydrogenase.

SEPTEM BER 1970 2297

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T. Ohnuma , J . F . H olla nd, A. F reema n, a nd L. F . Sinks

a-ke toglutaric ac id, S igma Chemical Co ., S t. Louis , Mo.; GOT,malic dehydrogenase , and NADH, Boehringer Mannhe im Co.,New York, N. Y.; Ur-3H (specific activity 20.0 Ci/mmole),D L-lysine-4,5-3H (specific activity, 45.0 Ci/mmole), and L-valine-5-3H (specific activity 0.6 Ci/mmole), Schw arz Bio-research, Inc., Orangeburg, N. Y.; 6C3HED lymphoma wasobtained from Dr. T. Hauschka, Roswell Park MemorialInstitute , and was maintained in C3H mice in asc ite s fo rm.

A sparaginase A ctivities in Plasma. B lood plasma w as obtained e ithe r by finger prick into heparinized capillary tube orby venipuncture after i.v. adminis tration of asparaginase. Fromthe capillary tube, 5 to 10 pi of plasma were used directly orafter appro priate dilutio n fo r the assay (3 1 ). The ac tiv ity w asexpressed in i.u. The sensitivity of the method was such as todetect activity of 1 X IO"3 i.u./ml. Protein was measured by

the method of Low ry et al. (27).Assay of Plasma L-Asparag ine and L-Aspartic Acid. Assays

were carried out according to the method described byCooney et al. (13). S amples of plasma w ere placed in a boilingwater bath for 10 min to inactivate intrinsic enzymes. Thecongealed plasma was cooled and centrifuged at 105,000 X gat 4 for 1 hr. Clear supernatant, 0.9 ml, and 0.1 ml ofreaction mixture consisting of 10 mM a-ketoglutaric acid, 2mM NADH, 0.3 i.u. of GOT, and 500 mM Tris buffer, pH 8.0,w ere plac ed in a silica cuv et. M alic dehy dro genase, 0.72 i.u. in0.1 ml of 100 mM Tris buffer, pH 8.0, was added, and thedecrease in absorbance at 340 m/i was recorded until thereaction reached a plateau (40 to 50 min). This change inabsorbance represente d the plasma L-aspartic acid. Then 0 .8i.u. of asparaginase (E. coli) in 0.01 ml of 0.9%N aCl solutionw as added, and the further decrease in absorbance at 340 mp.was similarly recorded, which represented the amount ofL-asparag ine . The values of L-asparag ine and L-aspartic ac idwere calculated from concurrent standards made from knownconcentrations of the amino ac ids .

Test of Leukemic Cells for L-A sparagine Requirement inK;/ro .The white cells were separated from the peripheral bloodor bone marro w aspirate by a metho d dev elo pe d in this labo ratory (16). The concentration was adjusted to 6 X 10sleukemic cells /ml in Eag le 's medium with Hanks ' balanced saltsolution containing 15% dialyzed fetal calf serum and theincorporation of Ur-3H (final ac tiv ity, 1 /nCi/ml; final concentration, 0 .05 jLtM),L-valine -5 -3H (final ac tiv ity, 4 juCi/ml) o rDL-lysine -4 ,5 -3H (final ac tiv ity, 1 iCi/ml)in the presence ofL-asparag ine (final concentration, 0 .38 mM) or asparag inase(final activity, 0.083 i.u./ml) w ere carried out by the methoddescribed by Oettgen et al. (30). 6C3HED lymphoma cells inthe same concentration were incubated concomitantly toe nsure that the sy stem w as o ptimal. Percentag e o f incorpo ra

tion was calculated by the area under the curve for uptake atseveral time points with asparaginase divided by that areaunder the c urv e fo r uptake w hen the cells w ere incubate d w ithL-asparagine.

A dministration, D osage, and S chedule. The lyophilizedenzy me w as dissolve d w ith 0 .9 % N aCl solutio n (5 ml/vial) andinjected i.v. directly or through the side arm of a runninginfusion within 0.5 hr after being dissolved. The drug wasgiven as single, daily, or weekly doses. Most patients on thedaily schedule received a single injection as a test dose 4 to 14

days prior to 5 daily injections of the drug. Onereceived 5 additional doses every other day after heMl marrow, another received the enzyme daily forand a 3rd patient with ALL received a 5-day courw eekly treatment had been show n to be ineffective.do se range v aried fro m 20 0 to 4 0,0 00 i.u./kg . The higdo se g iv en w as 2,20 0,0 00 i.u.

Immunological S tudy. The first 34 patients receiv2 0 i.u. o f asparag inase intradermally as a skin test pricommencement of treatment. This was later ababecause initial skin tests were never positive and anaphreactions occurred in patients despi te negative skin tes

In some patients, presence of antibody agains t aspain the serum was determined by the method descrCampbell e t al. ( IO).

R E S U LT S

Asparag inase Activ ity in Plasma. Asparag inase v iaferent lots were randomly selected, and the activmeasured. These enzy mes w ere dissolv ed as long as

100-1

5 0-

0 .1

Chart 1 . Clearance of plasma asparaginase activi ty af ter stions in 4 patients.

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prior to the testing date and stored at -20". Frozen storage ofthis duration was found not to cause any loss of activity.Elev en v ials from 5 differe nt lo ts labeled as co ntaining 1 0,0 00i.u. were found to have activity ranging from 6,100 to 7,900i.u./vial and specific activity of 116 to 182 i.u/mg of protein.The do se s in this paper are ex pressed in terms o f label po tencyto afford comparison with the data of others. The activity ofasparaginase found in plasma, however, is reported as such.

The clearance o f plasma asparag inase is sho wn in Chart 1 . Itmay be seen that the peak activity lasted for about 3 hr afterthe injection, it was dose related, and the clearance of theac tiv ity from plasma fo llowed firs t-o rde r kine tic s with plasmahalf-times of 14 to 22 hr. W hen blood samples w ere obtainedby finger prick, the activity in the 2-hr samples was consistently higher than the prece ding samples, but this findingw as no t co nfirmed in the sample s o btained by v enipuncture .In 2 patients, the plasma enzyme activity was measuredserially fo r a long er perio d. The activ ity w as detectable 1 3 and22 days after single injection of 1000 and 5000 i.u. /kg,re spectiv ely, but not after 1 8 and 2 5 day s, respectiv ely. D ailyadministration of 400 or 1000 i.u./kg of the enzyme had acumulative effect (Chart 2).

D ev elo pment o f immuno lo gical reactions w ere asso ciatedw ith rapid clearance of plasma enzyme. Thus, in one patientw ho developed arthralgia, erythematous skin eruption, andpositive intradermal test to 10 i.u. of the enzyme of Day 15, 5days after the end of a 5-day course of 1000 i.u./kg/day, theplasma asparaginase level fell rapidly and became undetectable

4 day s after the last injectio n. In another patient w ho seenzyme activity became undetectable 8 days after a 2nof 5000 i.u./kg of enzyme, the plasma was found topositive precipitation reaction to asparaginase.

Plasma L-Asparagine and L-Aspartic Acid. Plasma L-gine and L-aspartic acid values in 10 healthy men, 10women, 10 patients from 7 to 44 years with ALL,patients from 12 to 72 years with A ML w ere plotted in3. In healthy adult contro ls, L-asparag jne v alues w ere 6(mean 1 S .D .) and 54 16 nmoles/ml in men and inrespectively. L-Aspartic ac id concentrations were 11 10 4 nmoles/ml in men and women, respectively.

Among 10 patients w ith ALL, there appear to be 2 g1 group w ith L-asparagjne concentration w ithin or neno rmal rang e and w ith no rmal L-aspartic acid, the 2 ndw ith lo w L-asparag jne and re cipro cally elev ated L-aacid. This subg ro up o f 3 patients w as unique in that 2 pdied w ithin 48 hr of injection. The 3rd patient w as ininto complete remission. Patients w ith A ML had low eof L-asparagjne with normal L-aspartic acid concentration

Afte r enzyme administration, plasma L-asparag jne fecipito usly, w ith a recipro cal rise in L-aspartic acid. Evi.u./kg of the enzyme, which is in fact the skin testcaused substantial fall of plasma L-asparagine levelsfo llo wing day. In 6 measurements, L-asparag ine fell fro7 (mean S .E.) to 4 1, and L-aspartic acid rose fromto 43 3 nmoles/ml. After a single injection of 1000i.v. in 1 patient, 16 measurements in 32 days reveale

SO-i

0 . 5 -

0.1

24

1

48

1

72

H O U R S

n120

1

14 4

1we

i192

Chart 2 . Plasma asparag inase ac tiv ity during and af te r 5 dai ly injections of asparag inase in 3 pati ents , o o , enzyme leve l ina sing le injec tio n o f 40 0 i.u. 5 day s pre vio usly, re ce iv ed 4 00 Lu. daily fo r 5 do ses ; a a, e nzy me le vel in 2 patie nts w ho , afte1000 i .u. 6 days previous ly, rece ived 1000 i.u. dai ly for 5 doses. See text for discussion of the rapid fal l in pati ent plotted as A

S EPTEM BER 1 970 2299



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C linica l Stu dies with Asp ar a gina se

lu 2 Ong W15yZ

05,i0

-Ul2

5,000-"

u u u u8

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

;1POPODOI]DDDJUn1^TiDULiuiDDDODUUDODD

DDDDDAILY HttM.I

Chart 4. Composite mean toxicity score versus dose and schedule of asparaginase. The first 7 patients receiving weekly treatme1000 i.u./kg/week, for example, and the next 3 patients received 4000 i.u./kg. Under daily treatment, gaps in dose schedule s

observation intervals of 4 to 14 days. Three patients from the single injection group and 1 patient from the dai ly injection groupbecause 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, andeasy suggestibility which resembled Korsakow's psychosis. Thesyndrome lasted nearly 1 month in each. I n one case, even asingle dose of 1000 i.u./kg of asparaginase produced late-appearing dysfunction (33).

Pancreatic toxicity was manifest in varying degree in 4patients as abdominal pain, upper abdominal tenderness, diarrhea, increase in serum and urinary amylase, hyperglycemia,hypoinsulinemia and hypocalcemia. Development of a malab-sorption syndrome was suggested in 2 of these patients by a

positive Schill ing test done with and without intrinsic factor.N onketotic hyperglycemia occurred in 3 adults and 2children, of whom 2 showed no clinical evidence of pancreatitis. Blood glucose rose as high as 955 mg/100 ml in 1patient. In 2 patients, the hyperglycemia was accompanied bypolyuria of 11,000 and 8,000 ml, respectively, with 4+ urinesugar tests. Polyuria responded to insulin but not to anti-diuretic hormone. Blood sugar levels fell back to normal rangein approximately 2 weeks. The response of hyperglycemia toinsulin was appropriate, showing neither unusual sensitivitynor resistance. Hyperglycemia occurred in one man only afterthe 1st dose and was not seen after the 2nd, 3rd, or 4thweekly dose of asparaginase, possibly because of depletion ofhepatic glycogen, as suggested by negative periodic acid-Schiff

stain of the liver at autopsy.Positive skin test or anaphylactoid reaction were not observed at the fi rst exposure to the enzyme. Intradermal injection of 10 i.u. evoked a positive response 18 hr later in a44-year-old man who received a single injection of 1000 i.u./kg1 week previously. He had no evidence of clinical allergy butwas not retreated. A similar reaction in a 16-year-old girl 4days af ter a 5-day course of 1000 i.u./kg/day was followed byan illness resembling serum sickness with generalized arthralgiaand paravenous erythema near vein sites where asparaginase

had been injected. Three other patients developed anaphlactoid reactions. The reaction includes shaking chills, hytension, stridor, numbness, cyanosis and, in one patient, combeginning within 2 min. All 3 patients had had negativetests before their repeat weekly injections: the 2nd dose5000 i.u./kg, and the 3rd and 4th, respectively, of 1i.u./kg. These patients were treated with epinephrine, ahistaminics, and steroid and recovered without complicatio

One patient with AM L, who after 1,000 i.u./kg ofenzyme developed acute brain dysfunction, pancreatit

malabsorption, and muscle wasting, was later found to htransverse banding of the nails (33). A nother patient wAML developed hypotension of 70 mm Hg systolic and 45H g diastolic occurring 3 days after 10,000 i.u./kg ofenzyme. He received metaraminol and steroid for 3 daysrecovered.

Bone marrow aplasia was not observed in nonleukempatients.

Although we have seen a variety of unusual toxic effethe drug was fairly well tolerated by most of the patientsthe toxicities were essentially reversible. Two patients diedDay 1 who had far-advanced neoplasms with overwhelminfections, and causal relation to asparaginase was not foundautopsy. Another patient with ALL who received 1000 i

died on D ay 2 with disorientation, confusion, fever,increase of blood urea nitrogen from 4 to 40 mg/100 mlautopsy, bronchopneumonia was seen.L-Asparagine "Rescue" Infusion. L-Asparagine rescuefusion was given to 3 patients for the acute brain dysfutional syndrome in hopes of reversing the abnormalities.dose of L-asparagine was chosen from a previous humanperiment (38). Pertinent data are summerized in Table 2adverse reaction was observed from the amino acid infusion2 patients, def inite improvement ensued, and in 1, proba

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Table 2L -aspa rag ine r e scue infu sion fo r b r a in dys func ti ona l syndrome

One day 's dose of the amino acid was dissolved in 2 l iters of 5%glucose, s teri lized by Nalgene Fil ter Uni t (Nalgene LabN . Y.), and infused in 24 hr.

PatientD.W.J.C.D.D.DiagnosisAMLCML(blastic)AMLDose

ofasparaginase(i.u./kg

X)1,0005,00010 ,000PlasmaL-asparaginebefore

infusion(nmoles/ml)0043Serumalbuminbeforeinfusion2.6

(Day)2.6(Day0)1.7(Day)1.3(Day0)0.9(Day7)1.5(Day 38)Dose

ofL-asparagine(mmoles/kg/24

hr)121Daysgiven12-179-1438-44Maximum

L-asparagineduringinfusion(nmoles/ml)

(Day8)2.0(Day1)2.1(Day5)1.9(Day7)2.0(Day8)2.2(Day 56)Improvemen

mentalstat

Table 3Response in leukemia

LSA-*ALL indicates ALL converted from prexistentLSA.

ALL LSA-+ALL AML

Adult Child Adult Child Adult Child

MlM2Hematologicaleffect

onlyNoresponse0013100060100000311530002

Total 16 10

improvement was noted. The relation to infusion was suchthat clinical observers considered the phenomena to be causally related (33).

Therapeutic Effect. The response to asparaginase in patientsw ith leukemia is summarized in Table 3. We have frequentlyseen definite decrease or disappearance of leukemic cells fromperiphe ry and/o r rise in plate le ts afte r asparag inase with bonemarrow changes within the same c lassification. These patientsw ere classified as "hematological effect only." D ecrease inperipheral leukocytes alone w as not included. The enzyme ismost effective against A LL in children but has some value inA ML o f adults as w ell. A ltho ug h the numbers are small, thereappear to be no differences in the response rate in childrenwith ALL between those who received daily injections(Ml/total, 5/8) and those w ith w eekly treatment (Ml/total,4/8). One patient with AML was induced into completeremission after a single dose of 1000 i.u./kg (33). Anotherwoman with AML whose marrow was induced to M2 diedfrom massive pulmonary embolism. A ll the 10 A LL patientswho w ere induced into Ml with asparaginase had had extensive prior chemotherapy and had failed to remit again on vin-cristine and/or D auno rubicin w ith predniso ne. A mo ng these10 ALL patients who were induced into Ml, 3 relapsed onDays 14, 15, and 21; 1 died from empyema despite the Mlmarrow; and 6 went into a Phase II maintenance program onothe r drugs. No patient with so lid tumor responded to asparaginase.

L-Asparagine Requirement by Leukemic Cells In Vitro.Chart 5 shows the 36 studies of in vitro incorporation of

Ur-3H and L-valine-5-3H (or DL-lys ine-4 ,5-3H) byin 20 patients as an attempt at predictive behaviovalues are arranged on the abscissa according todecreasing antileukemic effect and w ere comparedvalues of a sensitive mouse lymphoma. In the prasparaginase, inco rpo ratio n o f uridine and ly sine wless than 30% of controls in the mouse lymphoma.tions in human leukemic cells w ere never this proFurthermo re, inco rpo ration o f precursors by co ntroculture was always less than the mouse lymphoma cfrom 0.45 to 20.8%, with an average of 6.6%. Bpredictio n o f the rapeutic o utco me w as no t reproducessful, s ince indis tinguishable biochemical resultsin ALL and AML, and within diseases no good cwith hematological response occurred.

DISCUSSION

Serial measurements of asparaginase af ter a s ingleof the enzyme has shown prolonged clearance raenzy me. S ystematic increase in e nzy me leve ls fo r thin finger prick blood, as contrasted to blood takenpuncture, suggests that tissue fluids w ere squeezedfing er pad in o btaining the capillary samples. Thisearly and appreciable extravascular space of disInitial peak activities of 5, 12, and 60 i.u./ml obtainjections of 400, 1000, and 5000 i.u./kg, respecreasonably close to estimated values of 5.6,14, andw hich are calculated from the actual dose given belabe led dose , and distribution in the plasma volumeas 5 % o f bo dy w eig ht. A fter asparag inase administratained depress ion of L-asparagine concentration wa

These findings are in contrast to the results in miceE. coli enzyme is clea red from plasma within 24 hr a nd wdepressed L-asparagine returned to normal in 48 hrhave previous ly shown, by using av ian and guinea pan inverse correlation betw een the rate of clearanceenous enzyme in the plasma and the therapeuticmouse tumors (31). From this point of view, theclearance of E. coli enzyme in man may be regfavo rable facto r in its effectiv eness. Pro lo nged halcoli enzyme in ma n [8 to 30 hr (E. F rei, III, persona l c

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lOO-80-.60-40"2O-0-Ur.

- SH OL^IsJJjxAA

XPX6C3HEDLYMPHOMAOfPAA0AAOA0

AOOO

*AlA

AA.AAMl

Mz IMP NR Ml M2 IMP NRALL AML

Chart 5. L-Asparagine requirement by leukemic cellsm vitro Ml and

M2 indicate eventual marrow status achieved after treatment. IMP,improvement; NR, no response, DL-Lysine-4,5-3H was used in themouse experiments, and valine-5-3H was used in all human studies.

munication); 7 to 19.5 hr (21)] compared to that in mice [2.5hr (3)] has also been observed by others. This provided basisfor the weekly administration of the enzyme rather than daily,with indistinguishable therapeutic effects.

The enzymatic assay of L-asparagine and L-aspartic aciddeveloped by Cooney et al. (13) was found to be reliable andconvenient compared to older methods, and reproducible withas little as 5 nmoles/ml. No other method of protein precipitation was studied to ascertain whether an artifact of transientheat activation of enzyme occurred. Further refinement may,however, be necessary to prevent in vi tro amidohydrolysiswhich may take place while the sample is being processed.Standard L-asparagine solution was found to be hydrolyzedslowly during storage at 0-4". Over a period of 3 months,there isa loss of about 1%/10 days.

The basis for aberration of L-asparagine and L-aspartic acidvalues in leukemic patients is not clear. As a group, patientswith AML had low L-asparagine levels and only 1 completeremission occurred. M ost patients with A LL had normalL-asparagine and L-aspartic acid levels, and the completeremission rate was high. Three patients with A LL had aninversion of normal L-asparagine and L-aspartic acid ratios,reminiscent of effects of asparaginase per se. This may be dueto overwhelming infection or functional abnormalities in the

dying patient.M any factors have been considered as possible contributorsto the wide spectrum of toxicity, such as contamination byglutaminase activity (29), or the effects of liberation ofammonia from L-asparagine, by the depression of L-asparagine,by the administration of an exogenous protein or largemolecule per se, but particularly by the possible endotoxincontaminant. Since the amount of asparaginase given to manis 1,000 to 10,000 times as much as was tested for pyro-genicity in the rabbit, it is possible that a proportionate

increase in endotoxin dose exceeded the pyrogenic thresin man but might have been tested as a negative in rabbased on dose concentrations alone. Bacterial endotoxiknown to cause chills, fever, vomiting, hypotension, figenopenia, hepatic injuries (39), depletion of liver glyrenal failure, coagulopathies, and delayed hypersensitireaction (41) . These effects resemble many of the toxiobserved after asparaginase. Probably the most intrigfinding to explain much of the toxicity is the deficienprotein biosynthesis produced by L-asparagine depleThus, after asparaginase, incorporation of L-methionineinto albumin was markedly inhibited (J. F. HollanWalter, and T. Ohnuma, unpubl ished data) . Clinical f isuggest that synthesis of albumin, insul in, f ibrinogen,fingernail protein were defective, whereas that of globuliapparently unaffected. The failure to find hypoglobulinsuggests some systematic differential requirement for L-agine between liver cell and plasma cell. Since immunoglocontains the same general amounts of L-asparagine asother proteins, the plasma cell presumptively has adeveloped system to supply its own L-asparagine. Togwith the evidence of patients who developed immunoloreactions, these findings suggest that asparaginase treatmennot immunosuppressive, a situation dissimilar to data frommouse (36). Since skin testing did not indicate the patwho sustained anaphylactic response, we have abandonedWe are aware of 1 fatal instance of anaphylaxis encountby a colleague, despite immediate and vigorous therapemeasures. L-Asparagine has been shown to protect agethionine- or carbon tetrachloride-induced fatty liver,presumably resulted from defective removal of fat becauinhibition of protein biosynthesis (1). The similarity oflogical changes in the liver after these chemical toxinsafter asparaginase suggests that the hepatic injury is indeedto inhibition of protein synthesis because of L-asparadeficiency.

We have observed 2 forms of diffuse brain dysfunctisyndrome: one occurred within 24 hr and then cleared rapthe other occurred insidiously about 1 week later and lapproximately 1 month. In all instances, central nervoutem leukemia, massive central nervous system bleedmeningitis, and electrolyte imbalance and other iatrogcauses were excluded. We have not measured ammonia onoccasion, but liberation of large amounts of ammoniaenzyme were estimated from the increases in L-asparticvalues or "blank" ammonia values in asparaginase determtion. Methionine sulfoximine-induced seizures in catrelated to inhibi tion of L-glutamine synthesis and admtration of L-glutamine and L-asparagine to epileptic pa

resulted in improvement in electroencephalographic re(38). It may be speculated that acute brain disease isrelateammonia or L-aspartic acid intoxication while the debrain syndrome is associated with defective protein syntin the brain produced fundamentally by L-asparagine dency.

Clinical observation of improvement in mental statusL-asparagine rescue infusion in all 3 patients studiedinterest. M ental abnormalities did not apparently increaseinterruption of L-asparagine infusion in 2 patients. In th

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patient (J. C.), recrudescence of brain dy sfunctio n fo llo wedaf ter discontinuation of L-asparagine infus ion. Serum albuminlevels did not correlate well with abnormal mentality, sugges ting diffe rences in chemical abnormalitie s o f liver and nervo us sy ste m. The clo se co rrelation o f L-asparag ine infusio nw ith benefit suggests that the relation is causal and not coincidental to spontaneous recovery. It should be added, however, that rescue effect would not be expected in the daysimmediate ly afte r asparag inase administration because exogenous L-asparag ine may be rapidly catalyzed by large amountsof the enzyme still circulating. Even 12 days after asparaginase administration in Patient D. W., abnormally highL-aspartic acid levels resulted from L-asparagine infus ion (33).Because L-asparagine given early may thus give rise to toxiclevels of ammonia, its use is contraindicated when asparaginaselev els are hig h. A no nto xic inhibito r of asparag inase w ould beof value to allow resti tution of L-asparagine levels .

The in vitro estimation of RN A and protein biosynthesisand its inhibition by L-asparagine deficiency evolved byasparaginase did not provide re liable prognostication. Possiblefac to rs which might influence c linical re sults are the deve lopment of an antibody that rapidly clears the enzyme fromplasma. We observed this on one occasion. When patientsdeve loped anaphy lac to id responses or positive skin reac tion tothe drug, we terminated treatment. The derepression orinduc tion of an L-asparag jne -synthesiz ing system in leukemiccells after asparaginase administration has been reported (19).The rapid selection of a v ariant animal tumor cell populationwhich does no t require L-asparag ine can occur/ v itro even inthe presence of L-asparagjne (34). A ll these factors may contribute to the discrepancy of in vitro estimation and therapeutic effect.

One of the most difficult clinical problems is that thetoxicity is apparently not dose related and some manifestations such as anaphy lax is , panc reatitis , brain dysfunc tionalsyndrome, and co ag ulopathie s can be lethal. Pro per and clo seclinical o bserv atio n o f patients receiv ing the enzy me is therefore essential.

A C K N O W L E D G M E N T S

We are grate ful to Mrs . Sylv ia Grew and Mr. John Gawoski for the iiskillful technical assistance.

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23. H ill, J. M ., L oeb, E ., R oberts, J., M acL ellan, A ., K han, A ., and H ill,N . O . L -A sparaginase T herapy of L eukem ia (A bstr.). P rogram ofthe X II C ong re ss o f th e In te rn atio nal S oc ie ty o f H em ato lo gy, N ewYork, p . 8 ,1968.

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F ig. 1. Fatty m etam orphosis of the liver. This severe fatty m etam orphosis w as seen in a patient w hose liver functionsh ow ed on ly slig ht ab no rm alities (b iliru bin , 2.0 m g/1 00 m l; seru m G OT, 3 2 u nits; L DH , 13 7 u nits; alk alin e p hos ph ata se ,total protein, 8.0 g/100 m l). H & E .

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