Clinical Investigations of 6-Azathymine

In the search for agents which might prove useful in the chemotherapy of neoplastic disease, attention has been focused on the processes by whichcomponents of nucleic acids are synthesized. Attempts have been made both to inhibit the de novobiosyntheses of ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) and to interfere withthe utilization for nucleic acid synthesis of preformed purine precursors. It is hoped that analogsof naturally occurring purines, pyrimidines, orprecursors of either will preferentially inhibit theanabolic processes in the neoplastic cells.

Among the compounds studied in humans areseveral types of folic acid antagonists; i.e., the 4-amino-substituted derivatives of folic acid (aminopterin and Methotrexate) (17, 32), diaminodichiorophenyl pyrimidines (30), and dihydrotriazines (28). These substances are thought to interfere with de novo synthesis of the purine bases ofDNA and RNA and of the thymidine portion ofDNA. The purine analogs tested clinically include2,6-diaminopurine (6), 8-azaguanine (2, 9, 10, 20),

* These studies were supported by research grants C-1889,

CY-2329, and CY-8215 from the National Cancer Institute ofthe National Institutes of Health, Public Health Service; theAmerican Cancer Society, the Damon Runyon Memorial Fundfor Cancer Research, the Lasker Foundation, the GrantFoundation, and the Black-Stevenson Fund.

Received for publication September 25, 1959.

6-mercaptopurine (7), thioguanine (31), 6-chloropurine (15), and thioguanosine (25). These apparently inhibit incorporation into nucleic acid ofpreformed purine bases. Among the pyrimidineanalogs studied in man are 5-bromouracil,1 azauracil (19), a group of fluorinated pyrimidines including 5-fluorouracil (12, 13, 40) and 5-fluorodeoxyuridine (1, 29), and azathymine (16). Of thisgroup, azathymine is the only one that has notproduced any antitumor or antileukemic effects inanimals2'@ has not affected the development ofthe chick embryo.4

6-Azathymine (6-methyl-aryin-triazine-3,5-(2,4)-dione) (Chart 1) was shown by Prusoff,Holmes, and Welch (35) to inhibit competitivelythe growth of S. faecalis and L. ca@ei when theseorganisms are grown in media supplemented bythymine or thymidine. The bacteriologic findingssuggested a functional relationship of azathymineand the 4-amino analogs of folic acid as well as aneffect of azathymine on pyrimidine metabolism.Therefore, a study of the effects of azathymine inneoplastic disease in man was undertaken (16).

1 J@ H. Burchenal, M. L. Murphy, and C. T. Tan, unpub

lished observations.

2 J H. Burchenal, unpublished observations.

‘D.A. Clarke, unpublished observations.4D. A. Karnofsky, unpublished observations.

435

A Thymine Analog*

ROSE Rum ELLISON, CHARLOTrE T C. TAN, M. LOIS

MURPHY, AND IRWIN H. KRAKOFF

(Di.ision of Clinical Chemotherapy, Sloan-Kettering Institute for Cancer Research; Departments of Pe4iatrics and Medicine,Memorial and James Ewing Hospitals; and Cornell University Medical College, New York, N.Y.)

SUMMARY

Twenty-four patients received azathymine. All fourteen children and two of theadults had acute leukemia; the other adults had a variety of solid tumors. All of the children had previously responded to Methotrexate and had then become resistant to it.Azathymine did not produce any improvement in any patient.

The hepatotoxic effect of azathymine was striking in the adults and occurred in theabsence of objective regression of the neoplastic disease. Liver damage also occurred inone child.

Azathymine exerts a specific renal effect, causing hyperuricemia associated with a decrease in the excretion of uric acid. This occurs early in the course of therapy and withdoses that do not cause liver function changes.

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CaIu)annADULTSTreated

withasathyinine

aloneTreated

withasathymineand other

anti

metaboliteaTreated

withasathymine

aloneTreated

withazathyniineand other

anti

metabolitesTotal

no. patients:

No. courses of therapy:

Duration of therapy:Range—daysNo. treated

<7days7—l3days2wks.ormore

Total dose:Range—gm/kgNo. received:

>1gm/kg0.5—1gm/kg0.1—0.5gm/kg

<0.1gm/kg

Average daily doseRange mg/kg7

7

1—27

322

.06—3.3

1231

35—12%7

9

3—45

133

.07—6.7

3211

23—1813

4

8-22

..

..

3*

1.0-1.6

3..

..

..

65—1252

2

8—14

..

11

0.7—0.8

..

2....50—100

436 Cancer Research Vol. 20, May 1960

MATERIALS AND METHODSIn the initial trials, azathymine was adminis

tered to five adults and fourteen children. All ofthe children and two of the adults had acute leukemia resistant to other forms of therapy, includingthe folic acid antagonists. The other three adultshad carcinoma of the ovary, carcinoma of thebreast and rectum, and melanoma, all with metastases. The drug was given orally, and all toleratedit well initially. The three non-leukemic adults andseven of the children received the azathyininealone. The rest received it in combination withother antimetabolites such as 6-mercaptopurine,Methotrexate, Daraprim, and azaserine.

The amounts of azathymine received by thevarious groups of patients and the duration of theperiods of therapy are shown in Table 1. The daily

THYMINE

OH

N@ C—CH3

dose ranged from 2.5 to 5.0 gm. in the adults and0.5 to 10.0 gm. in the children. The largest dailydose of 10 gm. was given to a child weighing 45 kg.He received a total of 3.3 gm/kg over a 27-dayperiod. Another child had 6.7 gm/kg in 87 dayswhile also receiving Methotrexate. All but one ofthe adults had 5 gm. daily.

Urine uric acid was measured by a modificationof the method of Archibald (18) employing theenzyme, uricase. This method has been describedin detail eLsewhere (24). Serum uric acid was measured by the same method, except that only occasional specimens were subjected to incubationwith uricase. Urine total nitrogen was measuredby acid digestion and direct Nesslerization (23).Urine urea nitrogen was measured by incubationwith urease and direct Nesslerization.

RESULTS

No clinical or hematologic improvement wasnoted in any of the patients treated.

TOXICITY

Symptomatic.—Subjectively, no ill effects occurred in any of the children, whereas two of theadults developed anorexia and nausea while receiving 5 gm. of azathymine daily. These symp

TABLE 1

6-AZATHYMINE

OH

PjI#cH@HO_C.@N@.N

CHART 1.—Structural formula of thymine and the analog6-azathymine, 6-methyl-asym-triazine-S,5-(2,4)-dione.

TUE DOSE OF AZATI{YMINEAND THE DURATIONOF TREATMENT

* One patient received two courses of azathymine, lasting 8 and 9 days. in a 37-day

period.

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EU4Is0N ei al.—Clinical Investigations of 6-Azathymine 437

toms slightly preceded laboratory evidence of liverdamage. A third adult, a 58-year-old woman withpapillary and solid carcinoma of the ovary withabdominal metastases, became stuporous on twooccasions after receiving each time a total of 40gm. of azathymine, 1.0 gm/kg in 8 days. Therewas no change in the status of her primary diseaseor in hepatic function studies on these occasions.The mental depression cleared within 2—3daysafter the drug was stopped.

Hepatic.—Clinical as well as laboratory evidence of disturbed liver function occurred duringtreatment in five patients, four of the five adultsbut only one of the fourteen children. The fifthadult was the one noted above to have becomestuporous, necessitating interruption of therapytwice. Only five of the eight children who received

J.L 46 YRS.@ CA BREAST,RECTUM

BODANSKY

URIC ACID

MG./100 CC.

48 HR.

225fl CHOLESTEROL0ESTERSI FREEM@I@UU@ @U@uuUNITS@ THYMOLTURBIDITY

MGL/100CC.@

trol, 38.6 seconds), a normal accelerator globulin,and a proconvertin level 35 per cent of normal.The “true―prothrombin concentration was 20 percent of normal. The recalcification time was normal.

The prothrombin time reverted to normal within several days in all but one adult with acuteleukemia who had a continued rise, to almost 2minutes, despite the administration of vitamin Kand blood. In two of the four cases the return of

ES. 4 YRS. d ACUTELEUKEMIA8@

MG./1OOCC@j URIC ACID

@EPHALINFLOCcULATION@@@,

TP@YMOLTURBIDITY

32@2T-@

SECONDS 22-I@17.1

VITAMINK1O@ i2@f @..PL,L@TS 50 MG.

GMS.

x )Q3

1954 OCt ‘ NOV ‘ DEC.nCORTISONE AZATHYMINE @IDARAPRIMflHYDROCORTISONEU15@JS/DAY 1.5 GM./DAY @25 MG/DAY @a100 MG/DAY

CHAWr 3.—Appearance of jaundice and hypoprothrombinemia in a child with acute leukemia treated with azathymineand Daraprim.

the prothrombin time to normal followed the administration of vitamin K. In four cases the prothrombin time elevation was followed by the development of jaundice, and other liver functiontests became abnormal in three of the adults.

Charts 2, 3, and 4 show the course of three patients who developed impairment of liver function.The patient depicted in Chart 2 received a total of60 gm., 1.4 gm/kg, of azathymine during a 3-weekperiod. At the end of this time the prothrombintime, cephalin flocculation, thymol turbidity, andbromsulfalein retention became elevated, andthere was a fall in the total cholesterol level witha marked rise in the percentage of free cholesterol.

Chart 3 shows the findings in a 4-year-old boywith acute leukemia who was treated with azathymine and Daraprim. This combination was

SECONDS

A ATHYMINE-5GM/DAY Th'IM@7,. rn• @5GM./DAY1954 MAY JUNE JULY@

CaABT 2.—Appearanceofliverfunctiontestabnormalitiesand hyperuricemiaafterthe administrationof 60 gm. ofazathymine.

total doses of at least 0.5 gm/kg had adequatechecks of liver functions throughout the period oftreatment. A fifth child had normal liver functiontests during the first course of therapy (6.7 gm/kg)but was not checked during the second course(1.4 gm/kg).

The earliest chemical change noted was elevation of the prothrombin time with progressiveelevation continuing for as long as 5 days after thelast dose of azathymine. Studies performed on onepatient at a time when the prothrombin time ofhis undiluted plasma was 47.5 seconds (control,15.2 seconds) showed a prothrombin time of 146.6seconds with 12.5 per cent diluted plasma (con

3+48 HR.

NEG.

UNITS

121

MG./100CC. BILIRUBIN

oJ

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PATIENTCoaTSoL

PERIOD*TR@4TMENT.PERIODSerum

uric acid,mg. %(mean)Urine

uric acid,mg/day(mean)Blood

ureanitrogen.

mg. %(mean)Done

(gm/day)Daysof

therapySerumuric acid,

mg. %(max.)Urine

uric acid,mg/day(mean)Blood

ureanitrogen,

mg. %(max.)1

2

3

4

5

6

7

85.1

3.83.7

3.74.03.9

4.56.4

2.5

6.0

4.8

5.7418

37%

371600

1006

640

394

39612.0

7.97.6

12.37.58.7

6.55.2

8.7

15.6

5.2

11.15.0

6.05.0

5.0SOt

5.02.5

2.5

2.5

5.0

6.05

88

201213

55

5

4

4

69.2

10.415.2

10.912.04.3

10.39.4

5.4

9.7

11.4

16.4189

368

226288

580

337

208

22315.6

14.614.5

14.511.714.3

6.66.0

8.2

19.0

5.2

438 Cancer Research Vol. 20, May 1960

used because of the synergistic action noted byElion and Hitchings (14) for these two drugs inL. casei. He had a fall in the leukocyte level from7,000 to 700/cu mm after 3 weeks of this treat

ment. At the end of 6 weeks, after receiving 40gm., 2.3 gm/kg, of azathymine, there was elevation of the prothrombin time to 26 seconds associated with icterus and cephalin flocculation abnormality. The prothrombin time returned to normalafter he was given vitamin K intravenously for 2days. The icterus cleared in several days.

A third patient (Chart 4), a 53-year-old manwith melanoma who received a total dose of 90gm., 1.6 gm/kg, of azathymine in a 19-day period,developed marked changes in all the liver functiontests studied. These reverted to pretreatment 1evels within a month after the last dose of the drug.

Hematopoietic.—There was little hematologiceffect in the leukemic patients studied. Two of theadults, however, showed marked decrease in hemoglobin with slight thrombocytopenia, 1—2weeksafter the last dose of azathymine. The platelet decrease was very brief in duration, but the hemoglobin required several months to return to normal.

In one of these two adults the marrow showedchanges in the erythroid elements to a picture re

TABLE 2

E.R.53 YRS.d'MELANOMAWEIGHTLBS. 129 123 121 123 122 118 129126122 116

@@1OTALPROTEIN •BALBUMIN

GMJIOOCC.

MG.I100CC. 11]@

BODANSKY _____ _________________

48 HR.@CHOLESTEROL0 ESTERS •FREE

MG.I@10OCC. 14@] gggg@ggg@g gggggg@g

UNITS 3....@TFI@v'M0L.@TURBIDITY

MG./100CC. _______ _________

S

SECONDS I1LJc@BI,ZME

THYMINEAZATHYMINE- I 5GMJDAY

5GM/DAY I @flD

1954 OCT. NOV DEC. 1955 JAN. FEa

CHAnr 4.—Appearance of liver function test abnormalitiesand hyperuricemiaafterthe administrationof 90 gm. ofazathymine and recurrence of these abnormalities after thesimultaneous administration of 70 gm. of azathymine andthymine.

EFFECT OF AZATHYMINE ON URIC ACID EXCRETION

* These were periods identical in duration with and, in most cases, immediately preceding the

treatment periods.t Plusthymine,5.0gm/day.

@ Thymine alone, 5.0 gm/day.

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Eu.IsoN ci al.—Clinical Investigations of 6-Azatliymine 439

sembling megaloblasts. In the other, during treatment with azathymine, there was a marked decrease in the percentage of nucleated red bloodcells in the marrow. After treatment there was atemporary increase of these cells above normallevels with no morphologic change.

Renal.—A11 of the five adults treated initiallywith azathymine were found to have elevatedserum uric acid levels during the course of treatment. The data are difficult to evaluate in two ofthese patients (both ofwhom had acute leukemia),since one had no pretreatment determination ofuric acid and the other had a rise in the blood ureanitrogen later to 68 mg. per cent. The serum uricacid was not determined regularly in most of thechildren. In one, there was a transient rise in theuric acid level from 1.4 to 7.1 mg. per cent (without blood urea nitrogen elevation) at the time ofhepatotoxicity. Four others showed elevations ofuric acid levels to 6.5—11.6mg. per cent. The uricacid level reverted to normal in all patients withina week after the last dose of azathymine. Uric acidexcretion was studied in five additional patients(Table 2) to determine the mechanism of the hyperuricemia. Urine uric acid was found to fallmarkedly in each instance while the serum uricacid level was rising. The change in urate excretionwas noted as early as 24 hours after the first doseof azathymine. Chart 5 illustrates an example ofthe influence of azathymine on uric acid excretion.The inhibition of urate excretion occurred withoutsignificant change in blood urea nitrogen. Urineurea nitrogen and urine total nitrogen were measured in two patients, and no changes were observed. In one patient simultaneous determinations of urea and urate excretion and phenolsulfonephthalein excretion were made during a control period and repeated during chronic azathymine administration. Of these parameters, urateexcretion was the only one affected. The simultaneous administration of equal doses of azathymine and thymine to one patient resulted in inhibition of uric acid excretion similar to that whichoccurred when azathymine was given alone.(Hepatotoxicity was not noted in this group ofpatients, but all were treated for periods of lessthan a week and received less than 0.5 gm. ofazathymine/kg.)

REVERSAL Ex1B.mII@s

In view of the competitive antagonism of thymine and azathymine in some bacteria, azathymine administration was repeated cautiously inone patient (Chart 4) with the simultaneous administration of an equal amount of thymine orally(5 gm. daily). The drugs were both discontinued

as soon as there were signs of liver damage (when atotal dose of 70 gm. of each had been given), sothat the marked hepatotoxicity which was notedfollowing 90 gm. of azathymine alone was not repeated. There was, however, a definite rise in theprothrombin time to 21 seconds, a decrease in theplasma albumin, a decrease in the total cholesterol, a rise in the cephalin flocculation, and a slightrise in bromsulfalein retention. The hemoglobinlevel, which had begun to rise, again fell, as did thepercentage of nucleated red cells in the marrow.

As noted in Table 2, elevation of the serum uricacid level again occurred. During the period of

JK @.WYRSP/880$.4RCOMA

AZAThYMINE5 GM/DAY

+@10 BI@OOD UREA NITROGEN

MG/IOOCC

0 @, 4I2@ SERUM URICACID

MG@OOCC

DAYSO 4 8 12 6 20

CHART 5.—Inhibition of urinary urate excretion and development of hyperuricemia following administration of azathymine.

biochemical abnormalities, the patient experiencedanorexia and nausea. When both compounds werediscontinued, the patient was seen to improve subjectively first, and, following this, all chemicalvalues reverted to pretreatment levels. During theperiod that the serum albumin was low the patientsuddenly gained 17 pounds in weight and developed ankle edema and ascites. There was a rapiddiuresis as soon as the plasma albumin rose.

No clinical or biochemical change followed subsequent administration of 90 gm. of thymine aloneorally to this same patient over a 21-day period.

DISCUSSIONNo improvement was noted in any patient

treated with azathymine. The leukemic patientswere all known to be resistant to Methotrexate,and the three patients with solid tumors would nothave been expected to respond to folic acid antagonists. In order to check further on the activityof azathymine, it should be tested in patients expected to be sensitive to Methotrexate. However,

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440 Cancer Research Vol. 20, May 1960

the hepatotoxicity observed in the adults as well asthe rapid development of hyperuricemia precludedfurther clinical trials of azathymine. The simultaneous administration of thymine did not preventthese toxic effects of azathymine. The mechanismof the development of liver damage is not known.The liver function test abnormalities suggest hepatocellular damage rather than intrahepatic obstruction by bile stasis. The rapid clearing of theabnormalities when drug administration wasstopped indicates that this was not activation of alatent virus. There is no histologic material presently available to clarify this point.

The finding of hyperuricemia is due to a specific renal effect of azathymine on the excretion ofuric acid. This was shown by the concomitant decrease in the total urinary excretion of uric acid inthe absence of any change in the urinary volume orthe blood urea nitrogen levels. Several compoundshave been found to influence the urinary excretionof uric acid. Among these are probenecid (39),phenylbutazone (43), and large doses of salicylates(4), whichaugmenturate excretion,and pyrazineamide (11), chlorothiazide (26), and smalldoses of salicylates (42), which inhibit excretion.Azathymine appears to belong to the latter groupof compounds which selectively interfere with theexcretion of uric acid.

Acute and chronic toxicity Studies in mice andrats (27)@indicated the lethal action of the drug tobe associated entirely with the acute effects, i.e.,production of paralysis at very high doses (3200mg/kg). Cats receiving 500 mg/kg developed anascending paralysis. Similar effects did not occurin two dogs who received 20 mg/kg/day for a totalof ten doses. The dogs receiving this amount, however, became anoretic and very weak and developed an elevated hematocrit and increase in prothrombin time and bromsulfalein retention as wellas hind-quarter weakness. These abnormalities disappeared when the drug was stopped. There wereno hematopoietic effects in the dogs other thansome reticulocytopenia. The only neurological abnormality noted in the patients was stupor, whichdeveloped twice in one woman.

Microbiologic studies with azathymine (35) indicated that this compound competitively antagonized the utilization of exogenous thymine orthymidine, thus inhibiting the growth of 8. faecaii..,, L. casci, and L. t.eichmanii. The drug interrupted the utilization of the endogenously formedmetabolites as well, as demonstrated by the inhibition of the growth of these organisms by azathymine when they are grown in media containingfolic acid or vitamin B12, but no thymine or thy

‘P.S.Philips, unpublished observations.

midine. The inhibitory effect of azathymine onthese organisms seems to depend on the enzymaticformation first of azathymidine (33, 34, 37, 41).Azathymidine is a much more potent inhibitor ofthe growth of these microorganisms and, unlikeazathymine, produces the same degree of inhibition with either thymine or thymidine present.The azathymidine is inhibitory not only when it ispresent in the medium initially but also when it isadded during the rapid log phase of growth of theorganism (37, 41). This inhibition can be prevented by simultaneous addition of excess thymidine. If the thymidine is added after the rapidlyreproducing cells are exposed to azathymidine for1—2hours, however, the inhibition cannot be reversed unless the inhibited cells are removed fromthe azathymidine medium.

Although in S. faee@aKs azathymine-C'4 is incorporated into DNA to the extent of 12—18per centof the cell complement of thymine (34), the inhibition is not considered to be due to such an irreversible reaction. It is thought that the inhibitory effect may be exerted on a reversible system;i.e., inhibition of formation of a precursor of DNAor of a hypothetical coenzyme which containsthymidine or which requires thymidine for its production.

Studies in two mammalian in vitro systems, rabbit bone marrow and mouse Ehrlich ascites tumor(2.1, 36), agreed with the microbiological findingsin that azathymidine but not azathymine markedly depressed the uptake of C'4 formate during invitro incubation. (Under the conditions used, theuptake of formate-C'4 is primarily into the methylgroup of thymine, relatively little isotope appearing in the purines.)

Some of the findings with azathymine are analogous to those observed with other pyrimidine analogs. Evidence suggests that the active form ofazathymine is the deoxyribotide, and similar evidence exists about the need for enzymatic transdeoxyribosidation of 5-bromouracil (3) and the5-fluorinated pyrimidines (5). In addition, the incorporation of these various pyrimidine analogsinto the cell nucleic acid in vivo does not seem to bethe only mechanism by which some of these antimetabolites affect growth. Thus, azauridine orazauridylic acid acts by inhibiting the enzymeorotidylic decarboxylase (22), thus inhibiting formation of uridine-5'-phosphate, and 5-fluoro-2'-deoxyuridine (after phosphorylation) prevents themethylation of deoxyuridine-5'-phosphate (5).

Although the active form of azathymine appearsto be the deoxyribotide, the clinical trials wereperformed with azathymine. It is possible but doesnot seem likely that the renal and hepatotoxicity

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EUAISON ci al.—Clinical Investigations of 6-Azathymine 441

observed were related to the presence of the freebase and would not occur with the deoxyriboside.These abnormalities were limiting factors in theadministration of the drug and occurred withoutany evidence of therapeutic effect, suggesting that

no further benefit would be obtained by the use ofazathymidine alone. (It would also prove difficultto produce enough of this deoxyriboside to beclinically useful.) There is evidence, however, ofsynergism with other antimetabolites in microbiologic systems (14) and of potentiation of theeffects of fluorinated pyrimidines in mouse leukemia (8), suggesting that such combination therapyis the next step to be tried clinically.

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2. ARMI8TEAD, G. C., Ja. ; BURCHENAL, J. H. ; KARNOFSKY,D. A.; and Soum@t@, C. M. Preliminary Studies on theClinical Toxicity of 5-Amino-7-hydroxy-1-v-triazolo(d)pyrimidine (Guanazolo), Cancer, 2: 1087—88,1949.

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6. BUIRCHENAL,J. H. ; KARNOFSKY,D. A. ; KINGSLEY-PILLEES, E. M.; SOtYTHAM, C. M.; MYmts, W. P. L.; ESCEER.G. C.; Ca@vurt,L. F.; DARGEON,H. W.; and RHOADS,C. P. The Effects of the Folic Acid Antagonists and 2,6-Diaminopurine on Neoplastic Disease. Cancer, 4:549—69,1951.

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Occurrence of Hyperuricemia during Pyrazinamide-isoniazidTherapy.Am. Rev. Tub.,74:289—92,1956.

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Vol. 20, May 1960

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1960;20:435-442. Cancer Res   Rose Ruth Ellison, Charlotte T. C. Tan, M. Lois Murphy, et al.   Clinical Investigations of 6-Azathymine A Thymine Analog

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