Studies with the Murine Leukemogenic Rauscher Virus II

Studies with the Murine Leukemogenic Rauscher Virus II. Chemotherapy of Virus-induced Lymphoid Leukemia

MICHAEL A. CHIRIGOS, FRANK J. RAIJSCHER, ISMAIL A. KAMEL,* GEORGE R. FANNING, AND ABRAHAM GOLDIN

(Biochemical Pharmacology, Laboratory of Viral Oncology, and Pathologic Anatomy Branch, National Cancer Inatitute, Bethesda, Maryland)

SUMMARY Eight drugs were tested against the Rauscher virus-induced leukemia. In virus inoculated mice a limited course of drug therapy was initiated after the mice had be come leukemic. Netropsin, Melphalan, 6-mercaptopurine, and 5-fluorouracil produced a twofold increase in the survival time of leukemic animals. Cytoxan and Vincristine were moderately effective; p-anisaldehyde thiosemicarbazone and methylglyoxalbis guanylliydrazone were only minimally effective. Inhibition of splenomegaly was ob served in animals subjected to effective therapy.

In a previous report assay systems were de In view of the importance of host-tumor-virus scribed for the chemotherapeutic testing of drugs interrelationships in chemotherapy, it was con in mice given inoculations of Rauscher virus (1). sidered desirable to extend the study of the Rau The parameters of drug effectiveness employed scher virus-induced disease with the use of a variety were survival time of the animals, extent of spleno of potentially effective chemotherapeutic agents. megaly, and transmissibility of the Rauscher dis This report presents the results of an investigation ease. In this study the drugs investigated were of Melphalan, Cytoxan, Vincristine, 5-fluorouracil, methotrexate (MTX), 6-mercaptopurine (6-MP), 6-mercaptopurine, and methylglyoxalbisguanylhy triethylenemelamine (TEM), and 2-chloro-4', drazone tested in a population of mice with Ran 4â€•-di-2-imidazolin-2-yl-terephthalathllde (NSC scher virus-inducedleukemia.Two antiviral agents, 60,889). 6-MI5, TEM, and NSC-60,889 were effec Netropsin and p-anisaldehyde thiosemicarbazone, tive in increasing survival time when a limited were also tested in this system. Netropsin protected course of treatment was initiated at S or 17 days mice infected with vaccinia virus (9), and thiosem following virus inoculation. icarbazones have been reported to be effective MTX was ineffective when treatment was initi against virus disease (5, 12). ated 3 days following virus inoculation but did increase the number of long-term survivors when MATERIALS AND METHODS treatment was initiated at the later time. Patholo Preparation ofatandard virus.â€”The preparation gy studies had indicated that at 3 days following of standard pools of virus has been described pre virus inoculation no leukemia was apparent. How viously (3, 8). ever, definite evidence of leukemia occurred by the Virus inocu!um.â€”Preparation of the virus in 17th day following virus inoculation. It was con oculum was carried out as follows: sidered, in the latter instance, that increases in A frozen aliquot of the virus concentrate was survival time produced by drug therapy could diluted with sterile 0.9 per cent saline to a reflect inhibition of established leukemic disease (0.05 gui equivalent of spleen/mi) concentration. as well as any inhibition of the etiological agent, Two-tenths ml. of diluted virus was inoculated in the virus. traperitoneally into 7- to 8-week-old BALB/c'

S Present address, Department of Pathology, Georgetown male or female mice weighing between 28 and University Medical School, Washington, D.C. 25gm.

ReceivedforpublicationMay 15,1963. 1 Obtained from NIH breeding colonies. 1046

RandomiztUion of test animok.â€”In all experi In the second experiment control virus-inocu ments mice were randomized shortly after inocu lated mice were sacrificed 88 days after virus in lation and were then distributed into appropriate oculation. Histological examination of the spleen groups. revealed massive areas of hemorrhage and fibrosis. Drugs and treatment.â€”Netropsin, Melphalan, Numerous foci of lymphoid leukemic cells fre Cytoxan (cyclophosphamide), Vincristine, and 5- quently interspersed with immature erythrocytes fluorouracil (5-FU) were diluted in 0.9 per cent were prevalent. An examination of tail blood saline. 6-Mercaptopurmne (6-MP) was dissolved smears showed the presence of an appreciable in dilute alkali ; p-anisaldehyde thiosemicarbazone number of lymphoblasts, bands, myelocytes, and (NSC-712) and methylglyoxalbisguanylhydrazone metamyelocytes. diacetate trihydrate (NSC-30,689)2 were sus Chemotherapy of virus-inoculated mice.â€”The re pended in 0.5 per cent methyl cellulose. The drugs suits of treatment on the survival time of BALB/c were administered subcutaneously in the axillary male mice given inoculations of virus in the first region in a constant volume of 0.01 mI/gm of experiment are presented in Chart 1. Treatment body weight. The regimens of treatment are mdi was initiated on the 17th day following virus in cated with the individual experiments. oculation and continued daily for 5 days. The

@@ RESULTS -,SISIS SSS DRUG DRUG DRUG Hi@topathology.â€”Forhistological examination CON- CON- CON- CON virus-inoculated mice were picked at random from TROtS NETROPSIN TROLNSC-712 TROLMELPHALANTROt @@@ the population in each experiment. In the first ex â€”1â€¢(2) (4)â€˜ @2)(7) (I)I : (JO) â€˜ (2) (9) periment control virus-inoculated mice were sacri 180 ficed 17 days after virus inoculation, and complete 160 autopsies were performed. The tissues were fixed 140 in Zenker-formol solution, and the sections were (20 stained with hematoxylin and eosin. In agreement v, with previous observations (3, 8), histological ex @8O amination of the tissues revealed that at 17 days w

post-virus inoculation the normal splenic architec I- 40 ture of the virus-inoculated animals was almost -J @ 20 completely absent. Few or no Malpighian bodies :D were seen. The splenic tissue consisted predomi U) M2 625 (25(25 200300300 @J2@&4 @J90 nantly of large immature lymphoid cells with scant @@@@@ -I I S DRUG' DRUd DRUd â€˜ DRU cytoplasm and large vesicular nuclei, the nucleoli w NSC- CON- CYâ€”CON- yiN- CON- CON of which were large and prominent. Mitotic figures 30689 TROt TOXANTRc@CRISTINETRf@5-FU TROt were numerous. A few degenerating cells were (I) (I) (2) (I) (3) (I) (I) (I) (2) (6) (2) interspersed with both immature leukocytes and 4 4 I I I I I I 4 t erythrocytes. The lymph nodes were characterized mainly by hyperplasia of lymphoid follicles and dilation of sinusoids. With the exception of marked (20 erythrocytopoiesis in the liver, the other tissues (00 were not unusual. 80

S Netropsin ($-[4-(4-quanidinoacetamidino-1-methyl-2-pyr 60 rolecarboxamido)-1-methyl-2-pyrrolecarboxamidoj-propionam 40 ide) was obtained from Mr. John Davenport, Charles Pfizer and Co., Inc., Maywood, N.J. Vincristinesulfate (7) was ob 20 @ tamed from Eli Lilly and Co. The remaining compounds were 25 50 (00 00 25 50 00 00025050 (0 12525 50 obtained from the Cancer Chemotherapy National Service MG/KG Center through the following sources: Melphalan (p-di[2- chloroethyl]-amino-r@-phenylalanine),Chester Beatty Be CHART 1.â€”Influence of chemotherapeutic agents on the search Institute; cyclophosphamide (2H-l,3,2-oxazaphospho survival time of 7- to 8-week-old BALB/c male mice given in rine, 2[bis(2-chloroethyl)aminojtetrahydro-2-oxide) (Cytox oculationsof 0.2 ml. of a 10@' dilution of a stock virus prepa an), Mend Johnson and Co.; 5-fluorouracil(5-PU), Hoffman ration. Chemotherapy was initiated 17 days followingvirus LaRoche, Inc.; methylglyoxalbisguanylhydrazone diacetate inoculation and continued daily for 5 days (days 17 through trihydrate (NSC-30,689), Frederic A. French, Mt. Zion lice 21). There were ten animals per experimental group, except in pital, San Francisco, Calif.; p-anisaldehyde thiosemicarbazone the control group, which contained 30 animals. Figures in (NSC-712), Merck, Sharp and Dohme, Rahway, N.J.; 6- parentheses represent the number of mice surviving to 180 mercaptopurine (6-MP), Burroughs Welcome Company. days.

range of individual deaths in the untreated con of NSC-712 tested. Nondiseased drug-treated ani trols (Chart 1), from time of virus inoculation, was mals showed tolerance to the highest dose level of from 37 to 178 days. The median survival time NSC-712 tested, indicating that higher dose levels (MST) for the group was 78 days. might have been more effective. All the drug Meiphalan (3.2 mg/kg) and 5-FU (25 mg/kg) treated, infected mice surviving for longer than exerted the most extensive therapeutic effect, pro 180 days showed signs of the virus-induced disease ducing an approximately twofold increase in MST. as evidenced grossly by enlarged spleens. Treatment with higher dose levels of the drug In the second experiment BALB/c female mice produced earlier deaths, indicative of drug toxic were treated with one dose level of Melphalan, ity. Netropsin, at dose levels of 3.12, 6.25, and 6-MP, and Vincristine (Chart 2). Melphalan or ______6-MP treatment was initiated on the 38th day VINCRISTINE following virus inoculation and continued daily for CONTROLS MELPHALAN 6-NP (I) (I) (4) 5 days. It had been planned to treat with Yin cristine daily for 5 days. However, four of the nine @ (80- t @ U) mice in the group died 1 day after the first injec .@ tion, and treatment was discontinued (Chart 2). (60- @ Li â€”.-. In this experiment, despite the delay in adminis

5- tering the drug (3 weeks prior to the median time (40 - @ -J of death of controls) as compared with the first experiment (8@weeks prior to the median time of (20 - @ -.- death of controls) (Chart 1), effective therapy was U)

z â€¢ obtained with Meiphalan. Treatment with Mel @ .@ K@O- â€¢ phalan (3.2 mg/kg, Chart 1, and 5.4 mg/kg, Chart Li 2) resulted in a twofold increase in survival time. @ 80- â€¢â€¢ In both experiments, treatment with 0.5 mg/kg .@ Vincristine produced early deaths. Treatment with -J @ .4 60- S 6-MP (Chart 2) resulted in .a 2k-fold increase in 0 S. S. â€¢!â€¢@â€¢survival time. All the drug-treated, infected mice 0 40- z â€¢â€¢ â€¢ â€¢â€¢â€¢â€¢.surviving for 179 days (Chart 2) showed signs of the virus-induced disease as evidenced grossly by @ 20 - enlarged spleens and microscopically by the pres ence of pathologic numbers of lymphoblasts, bands,

V 54 60 0@5 myelocytes, and metamyelocytes in smears of tail

. MG/KG . blood.

CHART 2.â€”Influence of chemotherapeutic agents on the It was of interest to determine whether a cot'- survival time of 7- to 8-week-old BALB/c female mice given relation occurred between spleen reduction and in inoculations of 0.2 ml. of a 10'' dilution of a stock virus crease in survival time as a result of drug treat preparation. Melphalan or 6-MP treatment was initiated on ment. The dosage level of each drug which pro the 38th day following virus inoculation and continued daily duced the greater increase in survival time (Charts for 5 days. Vincristine was administered on day 38 only. There were nine animals per experimental group. Figures in paren 1 and 2) and its effect on splenomegaly is presented theses represent the number of mice surviving to 179 days. in Table 1. Treatment with Netropsin, Melphalan, Vincris 12.5 mg/kg, produced a 27-, 67-, and 28-day in tine, and 5-FU reduced splenomegaly with a con crease in MST, respectively. Netropsin therapy comitant increase in the lifespan of the animals resulted in a greater number of mice which sur (Table 1, Exp. 1). Treatment with Melphalan and vived for more than 180 days (eight of 30) than 6-MP (Table 1, Exp. 2) also reduced splenomegaly, did treatment with other compounds. Cytoxan at with a concomitant increase in the survival time 2.5, 50, and 100 mg/kg produced a 40-, 6-, and of the animals. Vincristine (0.5 mg/kg) reduced 30-day increase in MST, respectively. The lowest, splenomegaly but produced early deaths which and only nontoxic, dose of Vincristine (0.25 mg/ were attributed to drug toxicity. kg) resulted in a 32-day increase in MST. Although Male BALB/c control mice had a MST of 78 treatment with NSC-30,689 failed to substantially days (Chart 1). In contrast, female mice of the increase the MST of the mice, there was a moder same strain had a MST of 60 days (Chart 2). This ate increase in the percentage of mice surviving to difference is in agreement with the observation of 180 days. A minimal increase in MST also oc Rauscher3 that female BALB/c mice are more

curred following treatment with the highest dose 3 F. J. Rauscher, personal communication.

susceptible to the virus-induced disease and sue Moloney virus and in decreasing the weight of cumb earlier than BALB/c males. thymus, liver, and spleen. NSC-30,689 and 5-FU caused a moderate increase in survival time (2). DISCUSSION In the current study, inhibition of splenomegaly In the current study, eight drugs were tested appeared to accompany prolongation of the life against primary virus-induced leukemia in mice. span of Rauseher virus-induced leukemic mice. With each drug, treatment was initiated when Whether the reduction in splenomegaly, by drug mice showed definite evidence of leukemia. In the therapy, had any effect on the leukemic process first experiment drug therapy was withheld until has not, as yet, been elucidated. Sugiura (11) 84 weeks prior to the median day of death of con tested the effectiveness of various drugs in mice trols. Netropsin, Meiphalan, and 5-FU were effec inoculated with the Friend virus and showed that

TABLE 1 EFFECT OF CHEMOTHERAPY ON SPLENOMEGALY AND SURVIVAL TIME OF BALB/c MICE GIvEN INOCULATIONS OF RAUSCHER Vmus

sp. PALP.SCOREtMEDIAN

Exp. SURVIVAL DOSE TREAT NO.GROUPNO.DRUGDAILY TOTAI417 @4 31 38 43 83 TIMR (Mo/KG)No. MENTS*Av. (DAY8)SuRvlvoas/ (days)11

2 Netropsin 2+ 3+ 3+ 4+ 4+ 4+ 146 4/10 3 NSC-712 300 5 2+ 4+ 4+ 4+ 4+ 4+ 93 0/10 4 Melphalan 3.2 5 2+ Â± 2+ 3+ 3+ 4+ 155 2/10 5 NSC-30,689 50 5 2+ 4+ 4+ 4+ 4+ 4+ 95 1/10 6 Cytoxan â€˜25 5 2+ 1+ 4+ 4+ 4+ 4+ 118 3/10 7 Vincristine 0.25 .5 2+ 1+ 3+ 3+ 4+ 4+ 112 2/10 2/1021 8None 5-FU6.25 255 .52+ 2+4+ 2+4+ 4+4+ 4+4+ 4+4+ 4+78 1540/30

(days)537760

2 Melphalan 4+ 2+ 2+ 2+ 3+ 4+ 113 1/9 3 6-MP 60 5 4+ 3+ 2+ 2+4+ 4+4+ 4+ 156 4/9 4None Vincristine5.4 0 .505 1384@46490/94+4+4+ 1+4+ 3+4+ 441/9

* No. of treatments from day 17 for Experiment 1, and from day 38 for Experiment 2. t Av. sp. paip. score = Averagespleenpalpation score;the figures represent the average spleen palpation of the survivors of the group. Enlargement based on an arbitrary scale ranging from 1+ to 4+. Average spleen weight, based on palpations obtained from a separate study, were: Â±(0.20gm. or less), 1+ (0.3 gm.), 2+ (0.6gm.), 3+ (1.0 gm.), and 4+ (2.0â€”4.0gm.). @ The number of survivors when the experiment was terminated at 180days and 179days for Experiments I and 2, respec tively.

tive in producing a twofold increase in survival Netropsin, 6-MP, and 5-FU were capable of sup time of leukemic animals. Cytoxan and Vincristine pressing splenomegaly. Mirand (6) demonstrated were moderately effective; NSC-712 and NSC that, in animals given inoculations of the Friend 30,689 were minimally effective. virus, 6-MP caused significant decreases in splenic In the second experiment, in which drug therapy uptake of Fe59 and in spleen weight, and prolonged was withheld until 3 weeks prior to the median the survival time of the animals. 5-FU was effec day of death of controls, Melphalan and 6-MP tive in decreasing splenic uptake of Fe59 and produced at least a twofold increase in survival diminishing spleen weight but did not increase the time of leukemic animals. A similar effect was survival time. obtained with 6-MP in leukemic mice in a pre As a result of drug therapy, leukemic animals vious study (3). displayed some loss of body weight. The dosage A previous report from this laboratory (2) level of each drug that produced the greater in showed that Cytoxan was also effective in increas crease in survival time (Netropsin, 6.25 mg/kg; ing the survival time of mice inoculated with the Melphalan, 3.2 mg/kg; Cytoxan, 25 mg/kg; Vin

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. Studies with the Murine Leukemogenic Rauscher Virus: II. Chemotherapy of Virus-induced Lymphoid Leukemia

Michael A. Chirigos, Frank J. Rauscher, Ismail A. Kamel, et al.

Cancer Res 1963;23:1646-1650.

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