Comparison of the immunosuppressive properties of two related dimethanesulfonates

Chem-Btol lnteracttons

363

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

C O M P A R I S O N OF T H E I M M U N O S U P P R E S S I V E P R O P E R T I E S OF T W O RELATED DIMETHANESULFONATES

aS VADLAMUDI, av S WARAVDEKAR ANDbA GOLDIN aMtcrobtologwal Assocmtes, Inc and bNattonal Cancer Institute, National Institutes of Health, Bethesda, Md 20014 ( U S A )

(Received December 14th, 1970) (Accepted January 25th, 1971)

SUMMARY Studies were conducted to determme the effect of treatment with two related dxmethanesulfonates, 1-propanol-3,3'-lminodl-dlmethanesulfonate (ester) hydrochlonde(NSC-102,627) and 1-propanol-3,Y (methyl-lmmo) dl-dlmethanesulfonate hydrochloride (NSC-84,641), on immune suppression m mice. Administration of sheep erythrocytes (antigen) following daily treatment with 23-65 mg/kg of NSC-84,641, or a single treatment with 65-108 mg/kg of this drug, resulted in suppression of hemagglutlnln (HA) synthesis. In contrast, treatment with equivalent doses ofNSC-102,627 prior to admlmstration of antigen had no effect on H A synthems. However, both the dimethanesulfonates, NSC-84,641 and NSC-102,627, suppressed H A synthesis when administered subsequent to antigenic challenge In the test system that measures the degree of chlmerlsm, as evidenced by the presence of allogenelc y-globulins m BALB/c mice, NSC-84,641 was found to be more effective than NSC-102,627 These results indicate that at equivalent doses NSC-84,641 is more lmmunosuppresstve than NSC-102,627, and it appears that NSC-84,641, like cyclophosphamlde (NSC-26,271), not only damages the prlmmve cells which evolve into plasmablasts but also inhibits the phase of antibody synthesis in which plasmablasts predominate.

Abbreviations HA, hemagglutlnln, l p, mtraperltoneally, NSC-84,641, l-propanol-3,3'-(methyllmlno)dl-dlmethanesulfonate hydrochlonde, NSC-102,627, 1-propanol-3,3'-lmlnodl-dlmethanesulfonate (ester) hydrochlorlde, SRBC, sheep erythrocytes Chem-Btol Interactions, 3 (1971) 363-370

364

s VADLAMUDI, V S. WARAVDEKAR, A GOLDIN

INTRODUCTION

Two new dtmethanesulfonxyl derivatives of amlnoglycols, synthesized by EL-MERZABANI AND SAKURAI, were found to be active against a wide spectrum of tumors in rats, mice and dogs l'z. Their activity against leukemia L1210 in mice has been reported z'3 It has also been demonstrated that on a daily treatment schedule (days 1-6) a greater therapeutic effect was observed with NSC-102,627 than with its analogue NSC-84,641 or with cyclophosphamide (NSC-26,271; ref. 4). Also, NSC-102,627 was less toxic to the stem cells (colony-forming units) than was NSC-84,641 in nonleukemlc mice Since an immune response to an antigenic stimulus involves cell division, cellular dlfferentlat~on and protein synthesis, it might be anticipated that these agents may have some influence on antibody formation The current studies were conducted to compare the lmmunosuppressant action of these two drugs. Determination was made of the effect of single or daily treatments with these two drugs prior to, or after, antigenic stimulation on primary antibody formation and on themductlon of tolerance to allogenelc spleen cells in the estabhshment of chimeras. MATERIALS AND METHODS

HA studies The Jmmunosuppressant action of these two drugs was measured by their capablhty to depress H A synthesis CDF1 mice (DBA/2 male × BALB/c female) F1, 10-12 weeks old, were treated with the two dlmethanesulfonates or cyclophosphamlde. The drugs (NSC-102,627, NSC-84,641 and NSC-26,271)* were &ssolved in saline and the p H for each drug was adjusted to 4 5 with N a O H solution The drugs were injected i.p at 0 01 ml/g body weight. Fresh sheep erythrocytes (SRBC) in Alsevere's solution were washed twice in phosphate buffered saline and the cell concentration was adjusted to 30 ~ (approx. 108/ml) The SRBC suspension (0 25 ml/mouse) was injected l p at the designated intervals, either before or after treatment as indicated. In order to determine the degree of an anamnestic response, mice were injected for a second time with SRBC. Mice from various groups were bled by the retro-orbltal method at designated intervals Sera from each experimental group of animals were pooled for use in the H A tests The H A tests were performed by the method described by STAVITSKYs using disposable mtcroplates The HA titers were read 18-24 h after incubation at --4 ° and the results were expressed as reciprocal tlters. Tolerance to allogeneie spleen cells T h e lmmunosuppressive capability of the two dlmethanesulfonates was also determined by measuring the degree of chlmerism which can occur In mice following treatment with the two drugs In this test BALB/c mice, 8-10 weeks old, were treated * The two methanesulfonates were supplied to the Cancer Chemotherapy National Service Center by Dr Y SAKURA~,Tokyo Cancer Institute and Yoshltoml Company, Tokyo (Japan)

Chem-Biol. Interactzons, 3 (1971) 363-370

365

IMMUNE SUPPRESSION BY DIMETHANESULFONATES

p with the agents and then injected intravenously 4 h later w]th spleen cells (107/0.2 ml) from C D F : mice. Sera collected from groups of mice treated with a series of doses of the two d:methanesulfonates were employed in the lmmunodlffusion studies 6 The presence of DBA/2-y-globuhn in BALB/c mice was examined employing DBA/I antisera prepared by the method described by LIEBERMANAND DRAY7 and GLYNN et al 8 RESULTS

Effect on H A synthesis o f multiple treatments with two related dtmethanesulfonates admmtstered prior to antigenic stimulus In these experiments mice were injected daily with the dlmethanesulfonates for 6 days. 24 h after the last treatment they were injected with SRBC and then bled on days 5, 12 and 19. Theresults are summarized m Fig. 1. The values for the reciprocal

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Fzg 1 HA tlters of sera from mice pretreated with NSC-102,627 and NSC-84,641. Six CDF1 mace per group, SRBC reJected 1p on day 0 Drug treatment xp on days --6 to --1, (survivors)/6 day 60, HA tlter of sera collected on days 5 (O), 12 (O) and 19 (A) H A tlters of sera of mice treated wlth NSC-102,627 (5-65 mg/kg) were the same as the controls injected with SRBC alone, but not pretreated In the sera of mlce treated with NSC-84,641 m the range 14-39 mg/kg, a significant reduction m H A tlters was observed on days 12 and 19. Although by day 26 these values had recovered m the mice treated with 14 and 23 mg/kg of the drug, the H A tlters m the sera of the mice treated with 39 mg/kg of the drug remained suppressed. When tested with SRBC on day 70, these rmce had also regained their capability to form antibody. A dose of 65 mg/kg of NSC-84,641 was found to be lethal to the mice. A second experiment was conducted to determine the effect of a booster mjecUon of SRBC (Fig. 2). In this experiment, following pretreatment wxth the drugs and admlmstratlon of SRBC on day 0, the ammals were bled on day 5 and injected with

Chem-Btol Interactions, 3 (1971) 363-370

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Fig. 2. Effect of the two related dlmethanesulfonates and cyclophosphamlde on the HA tlters of mice post-treated with the drugs. Six CDF1 mxce per group, SRBC injected I p on days 0 and 5 Drug treatment I p on days --6 and --1, (surwvors)/6 day 30 HA titers of sera collected on days 5 (O) and 12 (A) a b o o s t e r dose o f SRBC. These mice were bled again o n d a y 12, 1 e 7 days following the b o o s t e r rejection o f SRBC. F o r NSC-102,627 the H A tlters before the m j e c t m n o f the b o o s t e r were again the same as the controls. However, after the second injection o f SRBC, a m o d e r a t e d r o p was observed in mice receiving 39 a n d 65 m g / k g o f NSC-102,627. F o r NSC-84,641, as in the first experiment, a m a r k e d reduction in the H A tlter was observed at the high doses (23-65 mg/kg) before the a d m i n i s t r a t i o n o f the booster. A similar r e d u c t i o n was n o t e d following the a d m t m s t r a t l o n o f the b o o s t e r ; the H A txter was sharply reduced with 39 a n d 65 m g / k g The depression o f H A titer following t r e a t m e n t with NSC-84,641 (65 mg/kg) or c y c l o p h o s p h a m i d e was similar following one reJection or a b o o s t e r rejection with S R B C Thus, these results indicate t h a t NSC-84,641 has l m m u n o s u p p r e s s t v e p r o p e r t i e s similar to cyclophosphamide.

Effect on H A synthesis of a single treatment with two related dtmethanesulfonates admimstered prior to anttgentc stimulus The effects on H A synthesis o f a single t r e a t m e n t with the two related d i m e t h a n e sulfonates, or c y c l o p h o s p h a m i d e , a d m i n i s t e r e d before antigenic stimulus, are summarIzed in Fig. 3. Mice were treated at doses ranging f r o m 23 to 180 m g / k g with the two dlmethanesulfonates, or 250 m g / k g o f c y c l o p h o s p h a m l d e , one d a y p r i o r to the a d m i n i s t r a t i o n o f S R B C Sera f r o m the d r u g - t r e a t e d and u n t r e a t e d mice were collected on days 5, 14, 21 a n d 28 after S R B C rejection The H A titers o f sera from the u n t r e a t e d c o n t r o l mice were 512, 512, 64 a n d 32 at the respective intervals. In the mice treated with NSC-102,627, the H A titers o f the sera were similar to those

Chem-Btol Interactions, 3 (1971) 363-370

367

I M M U N E S U P P R E S S I O N BY D I M E T H A N E S U L F O N A T E S

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Fig 3 Comparlsonoftheeffectofaslnglepretreatmentwlthtwodlmethanesulfonates (NSC-102,627 and NSC-84,641) and cyclophosphamlde on the HA synthesis in mice Six CDF 1 mice per group, SRBC rejected 1p on day 0 Drug treatment i p on day --1, (survivors)/6 day 60 Sera collected on days 5 (O), 14 (A), 21 (0) and 28 (A) of the untreated controls, indicating that at these doses the drug was not able to suppress H A synthesis In contrast, w~th NSC-84,641 the H A tlters of sera collected from mice on day 5 were almost completely suppressed at 65-180 mg/kg of the drug. The tlters recovered at later intervals (14, 21 and 28 days) in mice treated at doses ranging from 23 to 108 mg/kg. At 180 mg/kg the values remained close to zero. In mice treated with a dose of 250 mg/kg of cyclophosphamlde, the H A ttters on days 5 and 14 were also markedly suppressed.

Effect on HA synthesis of a smgle treatment with two related dtmethanesulfonates admmtstered subsequent to antigenic stimulus In this study (Fig 4) mice were injected with SRBC on day 0, given a single treatment with the drugs on day 2, and then bled on day 7. Following this bleeding the mice were rejected with another dose of SRBC on day 7 and were bled on days 14, 23 and 30, respectively Doses of 300 mg/kg of NSC-102,627 and 180 or 300 mg/kg of NSC-84,641 were lethal. The H A tlters of sera from mice treated with 180 mg/kg of NSC-102,627 and bled on day 7 were reduced, whereas those bled on days 14, 23 and 30 were almost similar to the controls In the case of NSC-84,641 the H A titers on days 7 and 14 were markedly reduced following treatment with 108 mg/kg of the drug. The H A tlters of mice bled on days 23 and 30 were similar to the untreated controls at 23-108 mg/kg of the drug In contrast to these observations, cyclophosphamxde, at 250 mg/kg, completely suppressed the H A titers at all intervals. These results indicate that NSC-102,627 is Immunosuppressive If given after antigenic treatment but not before. NSC-84,641 and cyclophosphamxde were both lmmunosuppressive when given before or after antigenic stimulus. When no prior treatment was given with the drug, a second injection with SRBC resulted in an anamnestlc response of H A tlter. This anamnestlc response was less ewdent in the groups receiving prior

Chem-Btol Interactions, 3 (1971) 363-370

368

s. VADLAMUDI, V, S. WA.RAVDEKAR, A. GOLDIN

chemotherapy In mtce pretreated wtth NSC-84,641, although an anamnestm response was observed at doses of 5-23 mg/kg, at higher doses there was actually an m h i b m o n of H A tlter. {SURVIVORS} /6 DAY 30 (6} 1024-

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related d l m e t h a n e s u l f o n a t e s (NSC-102,627 l r n m u n e response m mine Sxx C D F 1 mice t r e a t m e n t 1 p on day 2 only, (survivors)/6 a n d 30 (ll0

Tolerance to allogenetc spleen cells The results of mmro-lmmunodlffUSlOn tests performed with sera from BALB/c mice treated with the two related dlmethanesulfonates and DBA/1 antisera are surnmanzed in Table I DBA/1 antlsera were placed in the center well and the test sera (2-fold dilutions) in the peripheral wells A posmve preclpmn reaction reflects the presence of DBA/2-y-globuhns m BALB/c mice The reciprocal ttters represent the degree of chimensm (allogenelc ~-globulms) produced m BALB/c mine after treatment with the drugs. In mtce treated with 180 or 108 mg/kg of NSC-102,627, sera collected on day 7 showed a positive preclpmn hne at a reciprocal dilution of 32. In sera collected on days 14 and 28 only a weak reaction, or no reaction, was observed over the whole dose range (23-180 mg/kg). In contrast, the sera collected on day 7 from mine treated with NSC-84,641 showed reciprocal precipmn ttters of 64 at all dose levels of the drug Significant levels of allogenelc 7-globuhns persisted until day 28. These results indicate that a more extensive persistent chtmerlc state was established in mice treated with NSC-84,641 than m mice treated with NSC-102,627.

Chem-Bwl lnteracttons, 3 (1971) 363-370

369

IMMUNE SUPPRESSION BY DIMETHANESULFONATES

TABLE

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MICRO-IMMUNODIFFUSION TEST WITH SERA FROM B A L B / c DIMETHANESULFONATES AND D B A / 1 ANTISERAh

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a 6 BALB/c male mice per group transplanted intravenously with 107/ml spleen cells from CDF 1 mice 4 h after admxmstratmn of the drug. b DBA/1 antlsera produced against Proteus mtrabths by the method described by LIEBERMANAND DRAY 7.

DISCUSSION A number of chemical agents which exhibit antlleukemlc activity have been shown to interfere to some degree with the immunological response of the host. Since it has been demonstrated that the two structurally related dlmethanesulfonates, NSC-102,627 and NSC-84,641, exert antlleukemlc activity m a m m a l test systems2-% comparison of their lmmunosuppressive properties was undertaken. The data presented here show that the dlmethanesulfonate NSC-84,641 was approximately as lmmunosuppresswe as cyclophosphamlde. It exerted stronglmmunosuppressive action when administered prior to or subsequent to antlgemc antigen stimulus, and had a marked capacity for estabhshment and persistence of a clumerxc state. Recently, MAKINODANet aL 9 placed lmmunosuppressive drugs into three classes depending upon whether they are active (a) prior to (Class I), (b) following (Class II) or (c) at all mtervals (Class III) relatwe to antlgemc stimulus. Accordingly, NSC84,641 may be placed m the same class as cyclophosphamxde. The current results with cyclophospharmde are in agreement with those of OKADA et aL to and FRISCH AND DAVIES11 that this drug suppressed H A synthesis when given before or after antigemc stimulation. NSC-102,627 was only mildly lmmunosuppressive. It showed no activity when administered prior to antigenic stimulus, but moderate action when admmlstered following antigen. It had only mild and transient actwlty in the reduction of a chimeric state. NSC-102,627 would appear to more closely resemble nitrogen mustard than busulphan 12. Thus, it is indicated that the two dlmethanesulfonates, hke cyclophosphamlde, may inhlblt cell division and interfere with antibody synthesis. However, only the Chem.-Biol. lnteractlons, 3 (1971) 363-370

370

s VADLAMUDI, V S WARAVDEKAR, A GOLDIN

&methanesulfonate

NSC-84,641

resembles cyclophospham~de

m t h a t at m a y a l s o

damage the plasmablasts and thereby lnhlbxt the lnductmn phase of antibody synthests ACKNOWLEDGMENTS T h e a u t h o r s a r e g r a t e f u l t o M e s s r s M PADARATHSINGH, B KRISHNA, W VIEIRA and M

SPEEL f o r t h e i r t e c h n i c a l a s s i s t a n c e . T h e s e e x p e r i m e n t s w e r e s u p p o r t e d m p a r t

by Contract PH43-68-1283 from Chemotherapy, Instxtutes of Health, Bethesda, Maryland

Nattonal Cancer Institute, National

20014.

REFERENCES 1

M. M EL-MERZABANIAND Y SAKURAI, Inhlbmon of tumor growth by new sulfonlc acid esters of ammoglycols, Gann, 56 (1965) 575-587 2 M M EL-MERZABANI AND Y SAKURAI, Antlcancer activities of methanesulfomc acid esters of armnoglycols against mouse leukemia L-1210, Gann, 58 (1967) 199-201 3 S VADLAMUDI,M PADARATHSINGH,M SPEEL, V S WARAVDEKARand A GOLDIN, Comparison of antlleukemlc (L1210) and lmmunosuppresslve properties of two dlmethanesulfonates, Federatlon Proc, 28 (1969) 264 4 S VADLAMUDI, M PADARATHSINGH,V S WARAVDEKARAND A GOLDII~, Effect of treatment with two related &methanesulfonates on the hfespan and spleen and bone marrow cells of leukemic and nonleukemlc mice, Chemotherapy, m the press 5 A B STAVITSKY, Mlcromethods for the study of proteins and antibodies, I Procedure and general application of hemagglutlnatlon and hemagglutlnatlon inhibition reaction with tannic acid and protein treated red blood cells, J Immunol, 72 (1954) 360-367 6 O OUCHTERLONY, Diffusion-In-gel methods for immunological analysis 11, in P KALLOS AND B H WAKSMAN (Eds), Progress tn Allergy, Vol I l, Karger, Basel, 1962, pp 30-154 7 R LIEBERMANAND S DRAY, Five allehc genes of the ISA locus which control gammaglobuhn allotype speclfiCltles m mice, J Immunol, 93 (1964) 584-594 8 J P GLYNN, A FEFER AND B L HALPERN, Cyclophosphamlde-lnduced chlmerlsm, Cancer Res, 28 (1968) 41-43 9 T MAKINODAN, G W SANTOS AND R P QUINN, lmmunosuppressJve drugs, Pharmacol Rev, 22 (1970) 189-247 10 Y OKADA, T YAGURA, Y MATSUOKA AND ~" YAMAMURA, Immunologic unresponsiveness reduced by antlcarcmogenlc agents and steroid hormone, J Btochem (Tokyo), 55 (1964) 352-354 11 A W FRISCH AND G H DAVIES, Inhibition of hemagglutmm synthesis by cytoxan, Cancer Res, 25 (1965) 745-751 12 M C BERENBAUM, Effects of carcinogens on immune processes, Brtt Med Bull, 20 (1964) 159-163 Chem -Btol Interactions, 3 (1971) 363-370