af6-(a2-isopentenyl)adenosine, a natural component of...

[CANCER RESEARCH 30, 1429 1436, May 1970]

Toxicological and Antiproliferative Effects ofAf6-(A2-Isopentenyl)adenosine, a Natural Component ofMammalian Transfer RNA1

D. Suk, C. L. Simpson, and E. Mihich

Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York Stale Department of Health, Buffalo, New York 14203

SUMMARY

A^-CA^-IsopentenylJadenosine inhibited the growth of

Ehrlich carcinoma ascites and leukemia L1210 but notthat of Sarcoma 180, adenocarcinoma 755, and Walker256 carcinosarcoma. Pancreatic atrophy, lymphoid depression, superficial gastric ulcers, and reduction of mitoses in intestinal crypts were seen in rats treated withthe compound. In rats and dogs, the drug caused hepa-totoxicity and granulocytosis. In rats, the granulocytosiswas related to a release of mature granulocytes from thebone marrow without alteration of differentiation andproliferation rates of hematopoietic cells. Regenerationof liver after partial hepatectomy was also inhibited bythe drug in rats. Hypotension was measured in dogs after single i.v. doses of the drug. Several of these effectswere comparable to those caused by adenosine or someof its analogs.

INTRODUCTION

An isoprene-containing nucleoside isolated from thetRNA of immature peas, spinach leaves, yeast, chick embryo, and calf and human liver (12, 24) was identifiedchemically as /V-6-(3-methylbut-2-enylamino)-a,/3-D-ribofuranosylpurine (10, 11). This compound is also calledIPA.2

In plant in vitro systems, both cell division and differentiation were stimulated by IPA, suggesting that thecompound exerts cytokinin-like activity (19, 28, 33). Incontrast, in mammalian tissue culture systems IPA wasfound to inhibit the multiplication of cells derived fromhuman myelogenous leukemia and that of S-180 cells,but not that of cells derived from human lymphocytic leukemia (9). Because of its effects in these systems, IPA wasstudied in vivo in order to evaluate its antitumor andpharmacological properties prior to its clinical trial.

In animals, in addition to hepatic damage and pancreatic atrophy, IPA caused antiproliferative effects in gas-

' This investigation was supported in part by USPHS Grants CA-04130 and CA-11047.

"The abbreviations used are: IPA, A^-(^"-isopentenyl)adenosine;

SGOT, serum glutamic oxaloacetic transaminase.Received August 15, 1969; accepted January 16, 1970.

trointestinal mucosa and lymphoid tissues and pronouncedgranulocytosis. In a child with acute promyelocytic leukemia, the drug induced repeatedly complete, but short-lasting, bone marrow remission (15). Because of the transient nature of the antileukemic effects seen in man, theantiproliferative actions of IPA were studied in rats in detail, particularly with a view to evaluate their duration.Moreover, the possibility was tested that the drug stimulates differentiation and proliferation of bone marrowcells.

Some of these data have been reported previously inpreliminary communications (30, 31).

MATERIALS AND METHODS

The structure of IPA is shown in Chart 1. The compound was synthesized by Hall et al. (12) and Robins etal. (24). The drug was kept in a freezer at â€”70Â°.Solutions of IPA were prepared shortly before use by dissolving the compound in 70 to 90% ethyl alcohol and thendiluting with 0.9% NaCl solution.

In the chemotherapy tests, the drug was given i.p. ata concentration of 10 mg/ml 0.9% NaCl solution, containing 10% by volume of 95% ethanol (10% alcohol-0.9%NaCl solution). Control mice received the highest amountof solvent given to any drug-treated animals. The experimental tumors used were Sarcoma 180 and Ehrlich carcinoma ascites in Swiss HalCR mice, adenocarcinoma755 in C57BL/6Ja mice, leukemia L1210 in DBA/2 Ha-DD mice, and spontaneous mammary tumors of DBA/2Ha-DD mice. Transplantable tumors in solid form wereimplanted subcutaneously following standard trocar procedures. Ascites tumors were inoculated i.p. (I X IO6

cells/mouse). Tumor diameters were measured once aweek for 6 weeks by a standard Vernier's caliper tech

nique. The total number of Ehrlich carcinoma cells present in the intraperitoneal cavity was counted 1 day afterthe end of a 7-day treatment with IPA. Average survivaltime was evaluated in mice bearing L1210.

In the toxicological studies, rodents and dogs weregiven IPA parenterally at concentrations of 10 mg/ml10% alcohol-0.9% NaCl solution and of 5 mg/ml 5% alcohol-0.9% NaCl solution, respectively. The drug wasgiven by the i.v. and p.o. routes in mongrel dogs, and bythe i.p., S.C., and i.v. routes in rats and mice. In dogs,

MAY 1970 1429

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NH-CH2 -CH3

CH3

OH OH

Chart 1. Chemical structure of IPA

treatment was given i.v. by infusion over a period of 1 to 2hr. Mice, weighing 16 to 22 g, were obtained from thebreeding colony of this Institute. Sprague-Dawley rats,(Charles River Breeding Laboratories, Cambridge,Mass.) weighing 100 to 250 g were used. The design ofthe toxicity experiments, as well as the procedures followed in the hematological and blood biochemical studies, were reported previously (17, 18). Histopathologicalstudies were carried out on rats and dogs treated withsingle and repeated doses of IPA in the LD5o range bystandard procedures except when otherwise stated (17,18).

In the study of the antiproliferative effects of IPA inintact rats, the drug was given i.p. at the single doses of175 mg/kg. Control animals were given injections of equi-molar doses of adenosine (219 mg/kg) or amounts of 10%ethyl alcohol-0.9% NaCl solution similar to those givento the test rats. Three to 9 rats/group were sacrificed atvarious intervals for the comparative study of the parameters evaluated.

In the hematological studies, standard cell counts wereperformed on blood taken from the tail vein. The bonemarrow was obtained from one femur for the total cellcounts and from the other femur for the differentialcounts, according to methods reported previously byothers (20, 22). In each bone marrow 300 to 500 nucleated cells were counted and divided into the following 4groups: (a) myelocytes and their precursors, (b) matureseries of granulocytes, (c) lymphocytes, and (d) nucleatedred blood cells. For measurement of the mitotic index,700 to 1000 cells were counted.

One- to 1.5-cm-long duodenal segments were taken 0.5cm from the pyloric ring. These were cut longitudinallyand fixed flat in 10% buffered neutral formalin. Thesespecimens were cut at 3 levels and embedded in paraffin.The paraffin blocks were serially cut at about 3 p, andthe average number of mitotic cells per crypt was determined after 10 to 30 crypts had been examined in eachrat (20, 22). Initially, adult to crypt cell ratios, number ofdegenerated cells, and height of villi were also measured(6); since the changes caused by IPA in these 3 parameters were very slight, these measurements were discontinued.

Partial hepatectomy was performed according to themethod of Higgins and Anderson (14). It was confirmedthat the first massive mitotic activity of regenerating

hepatic cells occurs about 31 hr after surgery (1, 5, 13).Groups of 3 to 23 rats were given injections of 53 or 105mg/kg IPA, of 132 mg/kg adenosine or of 10% ethylalcohol-0.9% NaCl solution, at 8, 16, 20, 24, 28, and 30hr posthepatectomy. Each rat also received 8 mg/kg col-chicine at 25 hr, and was sacrificed 31 hr after hepatectomy. Dry liver weights were obtained in samples kept at55Â°for 5 days. In 14 control operations, it was found that67.5% of the liver was being removed when evaluated ona dry weight basis. Therefore, the degree of regeneration of the liver, based upon dry weights, was estimatedaccording to the following formula (7):

% regeneration = [(weight of liver at autopsy â€”calculated weight of remnant)/(calculated weight of remnant)

X 100

The mitotic index in regenerating liver was determinedby counting 3,000 to 10,000 cells.

RESULTS

Antitumor Effects

After treatment with IPA, the growth of Ehrlich carcinoma ascites was significantly inhibited, as indicated bythe reduction of the total number of cells seen (Table 1).The survival time of DBA/2 Ha-DD mice bearing L1210was slightly prolonged at certain doses. At the higherdoses used, survival time was shortened, possibly as a result of toxicity (Table 1). At maximum tolerated doses,the drug had only minor inhibitory effects against spontaneous mammary tumors of the DBA/2 Ha-DD mouseand had no activity against Sarcoma 180, adenocarcinoma755, and Walker 256 carcinosarcoma (31).

Pharmacological and Pathological Effects

Toxicity in Rodents. The LD5o values of IPA by thei.p. route in Swiss HalCR mice and Sprague-Dawley rats

Table IEffects of Â¡PAagainst Ehrlich ascites carcinoma and leukemia LI2IO

Treatment"(mg/kg/day)None5102040100200Ehrlichascitescarcinoma*

(no.ofcellsXIO6)6161452226611591Leukemia

L12IOCaverage

survival(days)7.98.39.58.27.97.63.7

"Treatment given i.p. for 7 (Ehrlich) or 6 (L1210) consecutive daysstarting the day after i.p. tumor inoculation (1 X IO6cells/mouse).

' Total cell counts performed 1 day after the end of treatment (5 to

10 mice/group).' Average survival counted from the day of tumor inoculation which

was Day 0 (15 to 30 mice/group).

1430 CANCER RESEARCH VOL. 30



Toxicological and Antiproliferative Effects of Â¡PA

are shown in Table 2. The compound had no appreciablecumulative toxicity. In mice, the LDso values by the i.v.route were between 275 and 400 mg/kg X 1 and between225 and 250 mg/kg X 5. By the s.c. route, these doseswere between 375 and 400 mg/kg X 1 and between 250and 275 mg/kg X 5. In rats the LD50 values by the i.v.route were 200 to 214 mg/kg X 1 and 150 to 175 mg/kg X5; s.c. these doses were 225 to 250 mg/kg X 1 and 150 to160 mg/kg X 5. The drug p.o. was not lethal up to doses of800 mg/kg. Thus, both in mice and in rats IPA is wellabsorbed after i.p. or s.c. administration. This contentionis further supported by the observation that acute symptoms of intoxication occurred in both species a few minutes after i.p. injection.

In mice, after i.p. injections of IPA doses in the LD5orange, shock developed within 5 min and lasted up to 2hr. This acute effect was not seen in mice given corresponding amounts of alcohol-NaCl solution mixtures. Recovery from this effect was complete and in no case wereincreased reactions seen upon repeated daily doses. Lossof body weight was pronounced, but recovery occurredupon discontinuation of treatment. Death of lethally intoxicated animals usually occurred shortly after the endof treatment and no later than Day 15. No sign of localtoxicity was observed after s.c. injection. No diarrheawas noted. The slight hair changes seen were consistentwith malnutrition in lethally intoxicated mice.

In rats, the course of intoxication was similar to thatobserved in mice, except that only sedation occurredshortly after i.p. doses, without deep shock, and thatfrank alopecia occurred in about one-half of the animalstreated with doses in the LDso range. Alopecia developedshortly after the end of 5-day treatments.

In routine tests, it was found that, in rats given IPAi.p., a transient but pronounced increase in peripheralneutrophils occurred within 2 days after single doses.This increase was seen in 18 of 21 rats. A decrease of lymphocytes was noticed in 12 of 14 rats treated with dosesin the LDso range. No significant changes in hematocrit,hemoglobin, or erythrocytes were found in the peripheralblood. In view of these findings, a detailed hematologicalstudy was carried out.

Table 2Lethal effects of IPA in Swiss HalCR mice

and Sprague-Dawley ratsFemale animals were used.

No. of successive dailyi.p.dosesTo

mice15To

rats15LD5â€žÂ°(mg/kg/day)37524917212719/20

confidencelimits"(mg/kg/day)352-392238-261162-182120-134Slope"1.191.131.221.14

Hematological Effects in Rats. Following a single IPAinjection, the number of granulocytes increased in peripheral blood and decreased in the bone marrow (Chart2). Most of the cells involved in these changes were neutrophils.

In the peripheral blood, granulocytosis occurred 4 to24 hr after drug injection, and it was most marked at 8 to16 hr. Normal numbers of granulocytes were found in ratssacrificed 2 days after IPA administration. The numberof lymphocytes increased only slightly within 1 to 2 hrafter drug injection, and decreased slightly during the following 2 to 4 days (data not shown).

In the bone marrow, a marked decrease of the numberof mature granulocytes was noted in animals sacrificedfrom 1 to 72 hr after IPA injection. Normal values werefound on Days 5 to 7. The number of myelocytes and theirprecursors changed in parallel to that of mature granulocytes, but the extent of the variation was slight. The maximum decrease in the number of nucleated red cells wasfound on Day 2, and consisted of a drop from average control values of 24 X IO6cells/femur to that of 14 X IO6cells.

The number of lymphocytes decreased 2 to 4 days afterdrug injection from an average value of 19 X IO6cells/femoral marrow to that of 7 X IO6cells. Return to control

values occurred by Day 4 for the nucleated red cells, and

30

?0 20

Z_1CDfei"Ã¯o3OU.

0

O2I20161284

OM''(23.9):<,4l

1 i 1 . ,hhi:

01 2 4 8 12 16HOURS

234567

DAYS

(16).

' Calculated according to the method of Litchfield and Wilcoxon

Chart 2. Number of mature granulocytes in the peripheral blood(lower) and bone marrow (upper) of rats at various times following asingle, i.p. injection of 175 mg/kg IPA. Each group consisted of 3 to 9rats. Each point represents average cell count values: vertical bars,S.D. from these values: numbers in parentheses, upper values of suchlimits at 8 and 16 hr. In each rat, cell counts in peripheral blood werealso obtained prior to IPA injection, and fell within the range of control values indicated on the chart.

MAY 1970 1431




by Day 7 for the lymphocytes. After injection of adeno-sine or of control volumes of 10% alcohol-0.9% NaCl solution, a 2-fold increase in granulocytes was the only changeobserved and it occurred 8 hr after injection.

The number of cells in mitosis and the mitotic index inthe bone marrow were also measured in order to assesspossible stimulatory effects of IPA. No significant increase in these parameters was noted 8 to 16 hr and 4 daysafter drug injection. Thus the data suggested that, underthe experimental conditions used, IPA does not stimulatecellular proliferation in rat bone marrow.

Pathological Effects in Rats. The pathological effectswere studied in 44 rats given a single i.p. injection of 170to 200 mg/kg and in 17 rats given 5 daily i.p. injectionsof 130 mg/kg/day. Four rats were sacrificed 1, 2, 4, 8,and 16 hr and 1, 2, 4, 8, and 17 days after single doses,and 3 to 4 rats on Days 5, 8, 11, 14, and 28 after the beginning of 5-day treatments.

No significant gross lesions were observed followingsingle i.p. injections except for multiple minute hemor-rhagic spots in the stomach mucosa seen in animals sacrificed 24 hr after the injection. Changes in the liver wereslight and had disappeared by Day 4. Pyknosis in lym-phoid cells was seen in all lymphatic organs starting at4 hr after treatment; recovery was evident within 1 or 2days.

Pyknosis was evident in chief cells at the base of gastric glands at 4 hr, and by 8 hr was present in chief cellsthroughout the mucosa. By 24 hr multiple superficial gastric ulcers were present in most rats, but at 48 hr nonewere seen. Very few pyknotic cells were seen in intestinalmucosa at 4 and 8 hr. In 4 rats treated with 200 mg/kgIPA, the number of pyknotic nuclei seen at 8 hr wasclearly greater than that observed after 170 mg/kg. Inview of the transient toxic effects seen in the gastrointestinal mucosa, the antiproliferative effects in intestinalmucosa were studied in detail. The number of mitoticcells per crypt was significantly reduced, relative to control levels, 12 to 16 hr after the injection of 175 mg/kgIPA (Chart 3). A return to normal values was apparentat 24 hr. The number of mitotic cells appeared to beslightly increased in rats sacrificed on Days 4 and 6. Adecrease in the number of cells in mitosis comparable tothat caused by IPA was also seen in rats treated withadenosine, but not in those given control amounts of 10%alcohol-0.9% NaCl solution.

No pathological changes were found in the gastrointestinal mucosa after a 5-day course of treatment. Necroticlesions were noted in the liver. These necrotic foci wereirregular and randomly located and consisted of infiltrating lymphocytes and large mononuclear cells with occasional necrotic hepatic cells. Kupffer cells were swollenand contained phagocytized debris in some areas. Complete recovery had occurred in all rats by the 28th day.Lymphoid depletion was seen at the end of treatment andwas reversed in about 3 days. Marked atrophy of the exocrine pancreas was seen at the end of treatment. Although recovery occurred in many acini, extensive fattyreplacement was frequently present at Day 28.

a 2fe

0124 8 12

HOURS

3 4

DAYS

Chart 3. Changes in number of mitotic cells in duodenal epitheliumof rats at various times after a single i.p. injection of 175 mg/kg IPA.Each point represents the average number of mitotic cells per crypt for1 rat. This number was determined by counting 10 to 30 crypts in eachanimal. Complicating effects of possible diurnal variations were excludedby scheduling the sacrifice of rats at various times of the day.

Effects on Regenerating Rat Liver. After partial hep-atectomy, the weight of regenerating liver of control ratsincreased rapidly, and at 56 hr was 170% of the calculatedremnant. A large number of mitoses appeared suddenly31 to 32 hr after hepatectomy, whereas a second but muchsmaller wave of mitoses was observed at 48 hr. The patterns of regeneration seen were consistent with those observed by others (14).

As is shown in Chart 4, a marked reduction of bothweight of regenerating liver and the percentage of mitosiswas observed in rats treated with 105 mg/kg IPA between8 and 24 hr after partial hepatectomy and sacrificed at31 hr. This dose of IPA was the approximate LDso in hep-atectomized rats. In animals treated with 50% of this dosebetween 8 and 24 hr after surgery, the average percentageof mitoses was reduced to 4.7 to 5.0, whereas the weightof regenerating liver was not affected significantly. In animals treated with adenosine or with control volumes of10% alcohol-0.9% NaCl solution, both average weight ofregenerating liver and the percentage of mitotic cells weresimilar to those found in untreated rats.

Toxicity in Dogs. The lethal effects of IPA in dogs aresummarized in Table 3. After treatment by the p.o. route,1 dog died the day after the administration of 75 mg/kg,whereas 2 dogs survived after receiving 170 mg/kg X 1or 90 mg/kg X 10. By the i.v. route of administration, thedrug was lethal at doses of 62.5 mg/kg X 1or higher. However, 3 dogs survived 16 to 25 days after the beginning ofa 10-day treatment with 60 to 75 mg/kg/day; the reasonfor this is not evident. Thus lethality of IPA was somewhat erratic in dogs, perhaps depending upon unrecognized variations in the rate of infusion.

In 6 of the 7 lethally intoxicated dogs death occurredwithin 2 days of treatment. These deaths might have beenrelated to the hypotension caused by IPA (Table 4). Itshould be noted that 1 animal died in severe hypotension




Toxicological and A ntiproliferative Effects of IPA

100905

86S

70I

60|

50Â§

401

5 30otuÅ“20a*IOOâ€¢

""â€”It REGENERATION OF LIVER

TfcMITOTIC CELLS

Table 4Hypolensive effects of Â¡PAin mongrel dogs

16 20-24 28-30

HOURS AFTER HEPATECTOMY

AT WHICH IPA WAS INJECTED

Chart 4. Effects of IPA on liver regeneration after partial hepatec-tomy. All the animals received 8 mg/kg colchicine 25 hr after surgeryand were sacrificed 6 hr later (31 hr after surgery). Rats were given asingle i.p. injection of 105 mg/kg IPA at the various times after hepa-tectomy indicated on the abscissa. The average percentage regenerationwas based upon dry weights and was calculated according to a standardformula (7). The average mitotic index was determined after counting3,000 to 10,000 hepatic cells/rat. Three to 23 rats were in each group.

Table 3Lethal effects of IPA in mongrel dogs

Treatment"(mg/kg/day

Xno.170759050502001007562.55075605050X

1X1X10X9X30XXXXXX

10X10X10X

24Route

ofadministration*p.o.p.o.p.o.p.o.p.o.i.v.i.V.Â¡.V.i.v.i.v.i.v.i.v.i.v.i.v.No.

dead/no. treated0/1I/I0/10/11/32/2I/I1/31/10/20/20/10/30/1Dayofdeath'(S.

25")1(S.

3611)(S.IS')36"

(S. 71."1\d)0,

111

(S.8,43'')2(S.

39,44")(S.16,'25")(S.

18')(S.15, 32,"57")(S.39")

" Unless otherwise specified, treatment was given once daily, the

daily dose being infused i.v. over a 1- to 2-hr period. The drug was notadministered during weekends.

The drug was given p.o. in gelatin capsules or in the form of 250-mg pills, and it was administered i.v. in a 5% alcohol solution.

'Counted from the 1st day of treatment which was considered Day

0; S, sacrificed.dDogs in which hematological and blood biochemical studies were

performed.

5.5 hr after the beginning of the infusion. After single injection, however, the drug was less potent than adenosinein causing a transient hypotensive response.

The course of intoxication in the dogs which survivedthe acute lethal effects of IPA included: (a) sedation,which lasted for several hr after each infusion, (b) vomiting, which occurred during the i.v. infusion or as late as

DogAcBCInjectionIPAAdenosine

(mg/kg)(mg/kg)0.5120.512482It)20250*Minimum

di-astolic blood

pressure"

(mmHg)110504035757560403011060252510050Period

oftimetorecovery*

(min)0.511.50.511.32.04.3349.3330"

Â°Femoral artery blood pressure was measured by a standard can-

nulation procedure in dogs anesthetized with Nembutal. Grass Model5D polygraph recorder was used.

*Period of time from the injection to the return of blood pressure to

preinjection levels.'This dog was treated with adenosine and IPA at 1-week intervals.d Infused for 150 min: died in hypotension.

24 hr after administration p.o., and (c) marked anorexiaand about 20% transient body weight losses.

The hematological and blood biochemical changescaused by IPA are shown in the 3 typical examples presented in Table 5. Anemia was seen in 7 of 8 dogs treatedi.v. and in 3 of 6 dogs treated p.o. Reticulocytopenia occurred to a variable extent in 12 dogs. Reticulocytosis developed during recovery from anemia in 6 dogs. A 2- to3-fold increase in neutrophils was seen in 12 dogs. This increase seemed to occur rapidly. The increase of nonseg-mented neutrophils was relatively greater than that ofthe segmented ones in each case. In 1 dog, almost a 10-fold increase of nonsegmented neutrophils occurred within24 hr after the 1st dose of IPA. In 2 dogs moderate lym-phopenia was noted. Thrombocytopenia occurred in 9 ofthe 14 dogs studied and it was evident 3 to 6 days afterthe beginning of treatment. Thrombocytosis was notedin 6 dogs following thrombocytopenia.

The blood biochemical changes caused by IPA consisted of alterations in parameters of hepatic function,and were seen in each of the dogs. Increase of serum alkaline phosphatase activity seemed to be the first sign tooccur and was the most marked one. Increases of SCOTactivity were consistently seen in each of the dogs studied,but they were relatively moderate. Borderline prolongation of prothrombin time, slight elevation of bilirubin-emia, and a slight decrease of cholesterolemia occurredin 5 animals. The drug caused no significant alteration ofblood urea nitrogen levels and glycemia.

Pathological Effects in Dogs. The pathological effectsof IPA were assessed in 17 dogs, in 14 of which hema-

MAY 1970 1433




Table 5Blood changes caused by IPA in mongrel dogs

Treatment"(mg/kg/day

DayDogX no.)no.'1

50 X 10 i.v. 0112932

S2

90 X 10 p.o. -33710152136

S3

50 X 27 p.o. 029446271

SBodyweight(kg)8.98.29.111.510.911.210.410.911.211.39.38.79.28.4Hemoglobin(g/100ml)17.913.713.521.023.721.020.618.716.417.921.716.914.317.4Reticulo-cytes(%)0.800.50.10001.70.60.60.31.2Neutro-phils(X

IO3)7.410.113.04.911.53.77.57.114.68.07.18.616.45.8PlateletsX10:i23028378256182184202168716582272210292198Serumalkalinephospha-tase(mg/100

ml)5.385.612.94.214.514.128.138.019.59.13.360.055.313.9SCOT(units/ml)12.639.49.010.216.589.119.18.6111.140.923.3Bilirubintotal(mg/100ml)O.I0.20.250.150.800.400.400.250.25Negative0.050.10

' Treatment was given once daily, 5 days a week for the number of administrations indicated.bCounted from the day of the 1st administration, which is Day 0.

tological and blood biochemical changes had also beenstudied. The pathological changes were largely confinedto the liver and lymphoid tissue. Liver necrosis was moresevere and prolonged than in rats. Marked lymphoid depletion was seen only in dogs autopsied early after treatment. No lesions were seen in the stomach or pancreascomparable to those seen in rats. Active or healing hepatic necrosis was seen in all dogs which died or were sacrificed less than 30 days after treatment and in 7 of 9 dogskilled 35 days or more after administration of the drug.The lesions varied markedly in severity and were unevenly distributed, never involving the lobules uniformly.In general, the severity correlated well with the amountof drug given and the length of time which had elapsedsince treatment was completed. Nevertheless, there weresome striking exceptions in this regard. For instance, 1dog died within 24 hr after administration of a single doseof 75 mg/kg p.o. and had massive liver necrosis associatedwith widespread hemorrhages in other organs, whereasanother dog survived after 90 mg/kg X 10 p.o. and hadonly scattered collections of pigmented histiocytes in theliver when sacrificed on Day 36 (Dog 2, Table 5). The 4dogs that received 24 doses or more of 50 mg/kg all hadmultiple, irregular nodules up to 1.5 cm in diameter inthe liver when sacrificed 36 to 71 days from the start oftreatment. Microscopically, the nodules consisted of hy-perplastic parenchymal cells and were associated withincreased fibrous tissue and proliferating bile ducts. Theonly 2 dogs which had no evidence of liver damage weresacrificed 35 to 57 days after treatment with 50 mg/kg X10 i.v. or 75 mg/kg X 6 i.v.

DISCUSSION

The effects of IPA against tumors in rodents are relatively modest as indicated, for instance, by the fact thattoxic doses were required to reduce the growth of Ehrlichcarcinoma ascites in Swiss mice significantly and thatonly moderate prolongation of survival of DBA/2 micewith L1210 was caused by the drug at optimal doses (seeTable 1). In spite of its slight antitumor effects in mice,IPA was considered to be worth testing for possible activity in cancer patients because, in tissue culture systems,the compound was found to inhibit the growth of Sarcoma180 cells and of cells derived from human myelogenousleukemia but not that of cells derived from human lym-phoblastic leukemia (9). In initial clinical studies, IPA reportedly caused hematological and clinical remission on4 occasions in a child with acute promyelocytic leukemia(15), thus substantiating the validity of the decision totest the drug in man.

The toxicological effects of IPA consisted of hepato-toxicity, pancreatic atrophy, and antiproliferative effectsin the lymphoid tissue, the gastrointestinal mucosa, andthe regenerating liver. Moreover, the drug caused granu-locytosis in both rats and dogs. Hypotension was observedin dogs.

Signs of liver toxicity ranging from inflammatorychanges to focal or massive necrosis were seen in mostrats after 5 doses of IPA in the LDso range, and in 14 ofthe 17 dogs studied. In both species, evidence of hepaticnecrosis was found as late as 15 to 20 days after the end




Toxicological and Antipmliferative Effects of Â¡PA

of treatment. A direct correlation was not always apparent between the histopathological findings and the previously observed changes of blood biochemical parameters of hepatic function. For instance, of the 6 dogs whichhad shown marked elevation of both alkaline phospha-tase and SCOT activities, one had very slight liverchanges. Thus, in some cases, hepatotoxicity seemed tobe reversible. In contrast, 1 dog, which died 1 day aftera single p.o. administration of 75 mg/kg IPA, had evidence of acute hepatic necrosis and hemorrhages in manyorgans.

Atrophy of the acinar portion of the pancreas wasnoted only in rats. It is of interest that pancreatitis wasobserved in rats after treatment with 2-chloroadenosine(29).

The antiproliferative effects of IPA were transient, asindicated by the rapid recovery from the toxic effects seenin both lymphoid tissue and gastrointestinal mucosa. Gastric lesions, possibly similar in their etiology to the gastricerosions caused by IPA, were also seen after treatmentwith adenosine or 2-chloroadenosine (29). The possibilityshould be considered that the action of IPA is somewhatsimilar to that of adenosine. In fact, the mitotic activityin the crypts of the duodenal mucosa was inhibited byboth IPA and adenosine. Also, consistent with this possibility is the observation that hypotension, comparable tothat caused by adenosine, was induced by IPA in dogs.

Transient granulocytosis was observed after treatmentwith the drug in rats and dogs. In rats, after IPA treatment, there was a rapid and marked peripheral granulocytosis with a simultaneous decrease in the number ofmature granulocytes in the femoral bone marrow. Thenumber of granulocytes returned to normal at 2 days inthe blood and at 5 to 7 days in the marrow. These data,together with the fact that no significant increase of mitotic activity was seen in the marrow, suggest that IPAacts by facilitating the release of granulocytes from thebone marrow. Other treatments were reported to causegranulocytosis, such as those with bacterial toxins (3, 13,25, 32) or X-irradiation (8, 23, 27). In particular, it is ofinterest that purine derivatives with weak antitumor activity, such as 6-methylpurine, also caused granulocytosisin rats (21). In contrast, other compounds of this typewhich had marked antitumor and antiproliferative effectscaused granulocytopenia by suppressing hematopoieticactivity (21). Low doses of X-irradiation may cause granulocytosis followed by granulocytopenia, whereas higherdoses induce granulocytopenia only (8, 23). Anemia,thrombocytopenia, and reticulocytopenia were also seenafter repeated administration of toxic doses of IPA.Therefore, the fact that single doses of IPA cause granulocytosis and only transient depression of hematopoiesissuggests that the action of the drug is weak and/or shortlasting. In this respect, the activity of IPA resemblesthat of other purine derivatives with weak antitumoreffects or that of low doses of X-irradiation.

The regeneration of the liver after partial hepatectomywas markedly inhibited by IPA. Similarly to the antipro

liferative effects in intestinal mucosa, about 6 hr werealso required for the effect to become evident in the liver.In fact, the drug did not inhibit mitoses if given 3 hr priorto the 31-hr mitotic wave. It should be noted that adenosine had no effect in this system when it was given atdoses which affected the gastrointestinal mucosa. Several antiproliferative treatments have been shown to affect rat liver regeneration. For instance, L-asparaginasecaused 100% suppression of the mitoses seen 31.5 hr after surgery (2). Hydroxyurea (26) caused about 90% inhibition of the incorporation of thymidine-2-14C and phosphate-1"? into the DNA of regenerating rat liver 24 hr

after partial hepatectomy.As the results of this study indicate, IPA has antipro

liferative effects in normal and tumor tissues in vivo, consistent with the antiproliferative action found in vitro byothers (9). In no case was evidence obtained suggestingthat the drug stimulates cell differentiation or proliferation. The hematological and antiproliferative effects ofIPA in rats are similar to those of adenosine and/or ofcertain adenosine analogs. In this respect, it is of interest that adenine increased the effects of IPA againstEhrlich ascites carcinoma (4). With the possible exceptionof the effects seen in regenerating liver, the action of IPAwas weak or transient. Transient antiproliferative effectsmay occur also in man upon treatment with IPA, as suggested by the transient remissions seen repeatedly in achild with acute promyelocytic leukemia (15). Furtherclinical trials are required before the potential value ofIPA as an antileukemic agent in man can be adequatelyassessed.

ACKNOWLEDGMENTS

We are indebted to Dr. A. Mittelman and Dr. R. Jones for the IPAused in this study. The excellent assistance of Mrs. L. Loth and Mr. L.Krajewski is gratefully acknowledged.

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1970;30:1429-1436. Cancer Res D. Suk, C. L. Simpson and E. Mihich Transfer RNA-Isopentenyl)adenosine, a Natural Component of Mammalian

2∆-(6NToxicological and Antiproliferative Effects of

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