0192-0561/92 $5.00 + .00 Pergamon Press Ltd. © 1992 International Society for lmmunopharmacology.

Int. J. Immunopharmac., Vol. 14, No. 7, pp. 1259-1266, 1992. Printed in Great Britain.

P O T E N T I A T I O N OF I M M U N E R E S P O N S E S IN MICE BY A N E W INOSINE DERIVATIVE - - M E T H Y L INOSINE M O N O P H O S P H A T E (MIMP) MARINA SOSA, ANUTOSH R. SAHA, YULAI WANG, JORGE COTO, ALFREDO GINER-SOROLLA, ELBA M. HADDEN and JOHN W. HADDEN University of South Florida College of Medicine, Division of Immunopharmacology, Tampa, FL 33612, U.S.A. (Received 15 November 1991 and in revised form 31 March 1992)

Abstract - - Inosine 5'- methyl monophosphate (MIMP) is a new immunomodulator designed to improve upon the activity of other thymomimetic purines. In Balb/c mice, MIMP was assessed for toxicity and activity on immune responses. The lethal dose for half the mice (LOso)exceeded 500 mg/kg of body weight by both the parenteral and oral routes. At doses of 1 - 100 mg/kg, the mice showed no visible untoward effects. The antibody response of splenocytes to sheep erythrocytes (SRBC) was measured by IgM plaque-forming cells (PFC) in soft agar under optimal conditions of immunization and challenge. MIMP (1 - 100 mg/kg) was given by both the intraperitoneal and oral routes (gavage) at the time of SRBC injection and 4 days thereafter. The PFC response was found to be significantly augmented. The maximum effect (approximately 2 x) was observed at 50 and 100 mg/kg, via intraperitoneal (i.p.) and oral routes, respectively. Increases (maximally 1.5 x) in the responses of splenic lymphocytes to mitogen stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A) were observed under similar conditions of MIMP treatment. SRBCinduced delayed-hypersensitivity (DTH) was also measured under optimal conditions. By both i.p. and oral routes, enhancement of DTH response was produced by the lower doses of MIMP (0.01 - 1 mg/kg). Again, a second peak of optimum stimulation of DTH response was produced by 50 mg/kg of MIMP when administered by both routes. The effect was observed mainly on the sensitization rather than on the expression phase. MIMP qualifies as an effective immunopotentiator in normal mice.

Immunopotentiators, including drugs and biological substances, have been extensively employed in the prevention and treatment of different diseases (Hadden, 1987). One class of immunologically active compounds has been derived from purine structures such as inosine and hypoxanthine, e.g. isoprinosine and NP T 15392, respectively. Purine nucleosides, particularly inosine derivatives, generally share the capacity to mimic thymic hormone action (thymomimetic) to induce precursor T-cell differentiation and to potentiate the functional response of mature T-cells (Hadden, Cornaglia-Ferraris & Coffey, 1983; Hadden, 1985). It has been shown that inosine 5'-monophosphate augments mitogen-induced proliferation of T-lymphocytes in culture; however, this action is not apparent in vivo presumably due to the rapid catabolism of inosine 5'-monophosphate by 5'-nucleotidase (Y. Wang & J. W. Hadden, unpublished). In order to take advantage of this newly discovered property of inosine 5'-monophosphate, a

new purine nucleotide has been synthesized: inosine 5 '-methyl monophosphate (MIMP) (Hadden, GinerSorolla & Hadden, 1992). This synthesis was carried out with the assumption that methyl esterification of inosine 5'-monophosphate might result in the formation of a stable derivative resistant to hydrolysis by 5'-nucleotidase (confirmed by us; Hadden, Hadden, Wang, Sosa, Coffey & GinerSorolla, 1991). In previous reports, MIMP was found to have effects in vitro on T-lymphocyte greater than on B-lymphocyte responses (Hadden et al., 1991) and on T-cell differentiation (Touraine, Sanhadji, de Bouteiller & Hadden, 1991), and it can be classified as a thymomimetic drug (Hadden, 1985). The present study was designed to evaluate the effect of MIMP on in vivo immune responses. MIMP was assessed for its efficacy to modulate the expression of humoral (antibody-mediated) immunity by the plaque-forming cell (PFC) assay and cellular (T-lymphocyte-mediated) immunity by delayed-type hypersensitivity (DTH) response in

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Fig. 1. Inosine 5'-methyl monophosphate (MIMP). normal mice. It was also examined to determine whether administration of MIMP to mice could enhance mitogen-induced spleen cell proliferation. The results of this study confirm that enhancement of in vivo immune responses in mice by MIMP involves T-cells.

EXPERIMENTAL PROCEDURES

Animals and reagents. Balb/c female mice were obtained from Harlan Co. (Indianapolis, IN). For all the experiments, 8 - 12-week-old Balb/c female mice were selected. All the animals were allowed to recover in our animal facility for at least 1 week before the beginning of the experiment. Inosine 5'-methyl monophosphate (MIMP) was synthesized and purified in our laboratory by Dr Alfredo Giner-Sorolla (Fig. 1). Its composition was confirmed by elementary analysis and its structure by nuclear magnetic resonance (melting point, 145°C). MIMP stock solution at concentrations from 10 to 1000 tag/ml was tested for the presence of ep_dotoxin. At 1000 lag/ml the endotoxin value was < 0.1 ng/ml compared with the standard. Agar Noble and penicillin- streptomycin sulfate were purchased from Difco Laboratories (Detroit, MI) and Gibco Laboratories Life Technologies, Inc. (Grand Island, NY), respectively. RPMI 1640 medium, Eagle's minimum essential medium (MEM), phosphatebuffered saline (PBS), DEAEdextran and concanavalin A (Con A) were obtained from Sigma Chemical Company (St. Louis, MO). Phytohemagglutinnin ( P H A ) w a s purchased from Burroughs Wellcome (Research Triangle Park, NC). Guinea-pig complement and sheep red blood cells were obtained from Diamedix Corporation (Miami, FL). Fetal calf serum was obtained from Hyclone Sterile Systems (Logan, UT).

Antibody-forming cell assay. The animals were immunized with 1 × l0 ~ sheep red blood cells (SRBC) by i.p injection. MIMP was dissolved in water and administered in the dose range of 0 . 1 - 1 0 0 mg/kg in 0.2 ml by the i.p. route and orally, by garage (p.o.) on the day of immunization or on the day of immunization plus 4 consecutive days. After 4 days of immunization eight to nine animals per each group were sacrificed and the spleen cells were examined for the number of antiSRBC IgM plaque-forming cells by the hemolytic plaque assay according to the Jerne's method (Jerne & Nordin, 1963). In this assay, 0.2 ml of a 5 × 10~'/ ml spleen cell suspension was mixed with 2 ml of 0.7% agar medium, 0.1% DEAE-dextran and 0.96% of MEM. The mixture was immediately poured into the Petri dish and the plates were incubated for 2 h at 37°C in a humid atmosphere of 5% CO2 in air. The plates were flooded with 2 ml of diluted (l : 20) guinea-pig complement. After 1 h incubation, hemolytic plaques were developed and counted using the magnification of a colony counter. Results are presented as PFC/106 spleen cells. For both the i.p. and oral routes, experiments were done twice. Data from seven to nine animals in each dose group are presented as mean + standard error of the mean (S.E.M.) and were analyzed with Student's t-test. Delayed-type hypersensitivity (DTH). The estimation of the DTH response to SRBC was carried out following the methods described previously (Lagrange, Mackaness & Miller, 1974) with slight modificationl Mice were immunized by intraperitoneal administration of SRBC (1 × 10~) . After 4 days, each mouse was challenged by injecting the left hind footpad subcutaneously with 1 × 10~ SRBC in 50 lal of phosphate-buffered saline (PBS). Vehicle (50 lal PBS) was injected subcutaneously into the right hind footpad as a control. MIMP was administered both i.p. and orally by gavage either at the time of immunization or of elicitation. After 24 h of challenge, footpad swelling was measured as the increase in footpad thickness (left minus right) using an engineer's caliper. For both i.p. and oral routes, experiments were carried out two to three times. Data from an average of eight animals in each dose group are presented. The results are expressed as increase in footpad thickness in 0.1 mm units. Mitogen stimulation. Spleens from each group of mice were aseptically removed, and minced in RPMI 1640 culture medium supplemented with 100 units/ ml of penicillin and 100 lag/ml streptomycin. The culture was performed in flat bottomed 96-well microculture plates (Costar, Cambridge, MA) in a 0.2 ml volume containing 1 × l0 s splenocytes,

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Fig, 2. Effect of MIMP on anti-SRBC PFC response when given orally by gavage (p.o.) to 8-week-old female Balb/c mice. MIMP was administered simultaneously with immunization of mice with 1 x 108 SRBC. PFC were enumerated after 4 days. Results represent mean _+ S.E.M. from two experiments with eight to nine animals in each dose group. Significantly different from the control: *P< 0.05, **P< 0.005.

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Fig. 3. Effect of MIMP on anti-SRBC PFC response when administered i.p. to 8-week-old female Balb/c mice. MIMP was administered simultaneously with immunization of mice with 1 x 108 SRBC. PFC were enumerated after 4 days. Results represent mean _+ S.E.M. from two experiments with eight to nine animals in each dose group. Significantly different from the control: *P< 0.05. 0.5 ~ g / m l o f P H A or C o n A a n d 10°70 heat-inactivated fetal calf serum. C u l t u r e plates were i n c u b a t e d at 37°C in a h u m i d i f i e d a t m o s p h e r e o f 5o70 CO2 for 72 h. P r o l i f e r a t i o n was assessed by pulsing the lymphocytes with 0.5 ~Ci" 3H-thymidine (New E n g l a n d Nuclear, Boston, M A ) for the last 18 h. The

cells were harvested o n glass fiber filter p a p e r ( W h a t m a n 934-AH, W h a t m a n L a b o r a t o r y P r o d ucts, Inc., Clifton, N J) using a multiple a u t o m a t i c sample harvester ( C a m b r i d g e Technology, Inc.) a n d 3H-thymidine i n c o r p o r a t i o n was d e t e r m i n e d by a scintillation counter.

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Fig. 4. Effect of MIMP on anti-SRBC PFC response when administered by i.p. route at the time of immunization with SRBC and daily for 4 days thereafter. PFC in the spleen cells were measured at the end of the 5 days. Results represent mean _+ S.E.M. from two experiments with eight to nine animals in each dose group. Significantly different from the control: *P< 0.02, **P< 0.01, ***P< 0.005.

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Fig. 5. Effect of MIMP on anti-SRBC PFC response when administered by p.o. route at the time of immunization with SRBC and daily for 4 days thereafter. PFC in the spleen cells were measured at the end of the 5 days. Results represent mean + S.E.M. from two experiments with eight to nine animals in each dose group. Significantly different from the control: *P< 0.005, **P< 0.001. RESULTS

Effect o f M I M P on antibody response M I M P was given by b o t h oral and intraperitoneal routes at four different doses: 1, 10, 50 and

100 m g / k g on the same day o f i m m u n i z a t i o n with SRBC. The n u m b e r o f IgM a n t i b o d y - f o r m i n g cells in the spleen was determined 4 days after immunization. As s h o w n in Figs 2 and 3, b o t h oral and intraperitoneal

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MIMP (mglKg) Fig. 6. Effect of MIMP on 24 h DTH response when given orally by gavage (p.o.) to 8-week-old Balb/c female mice, on the day of immunization. Mice were immunized with 1 x 10 7 SRBC and after 4 days were challenged with 1 x 108 SRBC in the footpad. Results represent mean _+ S.E.M. from two experiments with three to five animals in each dose group. Significantly different from the control: *P< 0.05, **P< 0.0005. administration of MIMP augmented significantly the humoral response in mice assessed by anti-SRBC PFC response. Approximately, a two-fold maximum increase was obtained by both routes. In the case of intraperitoneal administration, the 50 mg/kg dose was found to be the most effective dose, and 100 mg/kg by oral route was found to produce the maximum increase. In separate experiments, when MIMP was administered for 5 consecutive days including the day of immunization, similar profiles of augmentation of PFC response were found by both the i.p. and the oral routes (Figs 4 and 5). In both cases greater effects of MIMP at low doses were observed by the i.p. route.

Effect o f M I M P on delayed-type hypersensitivity to SRBC As depicted in Figs 6 and 7, cellular immune function in mice determined by the D T H response was augmented significantly by MIMP administration by both the oral and intraperitoneal routes on the day of immunization. With oral administration (Fig. 6), the DTH response was enhanced by doses from 0.1 to 50 mg/kg. With higher doses, substantial enhancement was observed at 50 mg/kg dose by both routes. In the case of intraperitoneal administration (Fig. 7), significant enhancement of the DTH response appeared from the lowest dose of 0.01 to 50 mg/kg, with a statistically insignificant dip at the dose of 10 mg/kg. When MIMP was administered

intraperitoneally for 4 consecutive days from the day of immunization, the same profile of enhancement in the DTH response was found (data not shown). MIMP administered on the day of challenge did not augment the DTH response (Fig. 8). These data indicate that MIMP promotes cellular immunity, presumably through an action on the afferent limb of the immune response, possibly on T-helper cells.

Effect o f M I M P on spleen cell response to mitogens It was confirmed in this study that both PHA- and Con A-induced mitogenic responses of spleen cells are stimulated by oral administration of MIMP (Fig. 9). Splenocytes were obtained from mice which were administered MIMP orally at doses of 1 - 100 mg/kg for five times in 1 week. Under these conditions basal thymidine incorporation was not affected (data not shown). The results show that stimulation of both T-cell mitogen responses appeared at 10 m g / k g and above. The maximum enhancement of mitogen response was achieved with 100 mg/kg and it was approximately 1.48 and 1.35 fold for Con A and PHA, respectively.

DISCUSSION

The results presented here demonstrate that MIMP is able to influence the development of cellmediated and humoral immune responses. As for

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Fig. 8. Effect of MIMP on 24 h DTH response when administered by i.p. route on the day of challenge. 8-week-old Balb/c female mice were immunized with SRBC 4 days before challenge. Results represent mean _+ S.E.M. from four to five animals. m a n y other i m m u n o m o d u l a t o r s , the dosage and timing o f M I M P a d m i n i s t r a t i o n are o f critical significance. Toxicity studies showed that at the doses utilized in this study, M I M P was active b o t h parenterally and orally, and was apparently nontoxic. The acute toxicity (LDs0) o f m e t h y l - I M P was greater than 500 m g / k g o f b o d y weight intraperitoneally, and greater t h a n 5000 m g / k g o f b o d y

weight orally. It showed no obvious toxic effect in experimental animals even at doses o f 100 and 200 m g / k g . Since we f o u n d previously in our laboratory that M I M P , unlike I M P , is resistant to hydrolysis by 5 ' - n u c l e o t i d a s e , the effect o f M I M P on in vivo h u m o r a l and cellular i m m u n e responses was undertaken in the present study. The anti-SRBC hemolytic

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Fig. 9. Effect of MIMP on mitogenic response of mouse spleen cells. Splenocytes were obtained from mice which were administered MIMP p.o. five times in 1 week and incubated with Con A (0.5 ~g/ml) and PHA (0.5 ~g/ml) for 48 h. Cultures were pulsed with tritiated thymidine for last 18 h. (11) PHA, (El) Con A. Results represent mean _+ S.E.M. of count per minutes (counts/min) from culture of splenocytes from three to four animals. Significantly different from the control group: *P< 0.05; **P< 0.005. plaque assay was chosen as it is a well-known macrophage and T-cell-induced response that is expressed through the B-cell and thus involves the participation of a complete range of i m m u n o c o m petent cells. The foregoing data show that M I M P at a single intraperitoneal dose of 50 m g / k g can induce a m a x i m u m two-fold increase o f the P F C response to SRBC in Balb/c mice. On the other hand, by the oral route, a single administration of 100 m g / k g induced the same magnitude of augmentation. When M I M P was administered intraperitoneally for 5 consecutive days, a significant response was found with all the doses of M I M P (1 - 100 m g / k g i.p.). On the contrary, a single i.p. administration showed a m a x i m u m two fold augmentation only at the dose of 50 m g / k g . This reveals that consecutive administrations of M I M P increase its activity range. By both i.p. and oral routes, administration for 5 consecutive days shows a significant response at a dose as low as 1 mg/kg. In the D T H experiments, in comparison with the oral route, a lower dose of M I M P by the i.p. route produced a significant effect. An i.p. dose of M I M P as low as 0.01 m g / k g produced a significant 1.5 fold enhancement in the D T H response. On the contrary, the same dose was found to be inactive by the oral route. In general, a 10 fold higher dose was required orally than by the i.p. route to produce a comparable level of potentiation of the D T H response. This is

evident when the potentiating effect of 0.01, 0.5, 1.0 and 5.0 m g / k g M I M P administered intraperitoneally are compared with the impact of 10 fold higher doses given by the oral route. As was mentioned earlier, the dose required to produce acute toxicity was also 10 times higher by the oral than by the i.p. route. This 10 fold difference between the i.p. and oral dose of M I M P required to yield both D T H potentiation and acute toxicity (LDs0) may be attributed to differences in the rate of absorption or metabolism of M I M P by the two routes. It was found further that the stimulating effect on D T H was observed mainly on the sensitization rather than on the expression phase. It is known that three different subsets of immunocompetent cells are involved in delayed-type hypersensitivity; these sets consist of specifically antigen-reactive T-cell, a suppressive regulatory cell, and the macrophage (Morikawa, Baba, H a r a d a & Mitsuo!~a, 1977). Also, indirect interactions mediated by ly~nphokin's or monokines can occur. It may be suggested ~hat '~he target subset of immunocompetent cells !nvolved in the enhancement of the D T H response by M I M P depends on the time of its administration. To understand the effect of M I M P on lymphocytes proliferation under in vivo conditions, spleen cells of MIMP-administered mice were cultured with T-cell mitogens. The present study confirmed that

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b o t h the P H A a n d C o n A responses o f spleen lymphocytes were stimulated by oral a d m i n i s t r a t i o n o f M I M P , for five times in 1 week. In a parallel experiment, spleen cells o f M I M P - a d m i n i s t e r e d (i, p.) mice f r o m the D T H experiment were cultured with P H A a n d C o n A. It is o f interest to note t h a t the p a t t e r n o f mitogenic response was parallel to the D T H response o f the same a n i m a l s (data n o t shown). In the present study, M I M P was f o u n d to be a

p o t e n t stimulant for T-cell-dependent a n t i b o d y responses, T - l y m p h o c y t e proliferative responses, a n d cellular i m m u n i t y as m e a s u r e d by the D T H response. T h e in vivo results in mice indicate t h a t MIMP possesses immunopotentiating activity characteristic o f a t h y m o m i m e t i c drug. Acknowledgement - - This work was supported by a grant-

in-aid from Genta, Inc., San Diego, California.

REFERENCES

HADDEN, J. W., (1985). Thymomimetic drugs. In Serono Symposium on Immunopharmacology (eds Miescher, P. A., Bolis, L. and Ghione, M.), Vol. 23, pp. 183 - 192. Raven Press, New York. HADDEN, J. W. (1987). The immunopharmacology of the immunotherapy of cancer, infection and autoimmunity. Fund. clin. Pharmac., 1, 2 8 3 - 332. HADDEN, J. W., CORNAGLIA-FERRARIS,P. E. & COFFEY, R. G. (1983). Purine analogs as immunomodulators. In Progress in Immunology V (eds Yamamura, Y. and Tada, T.), pp. 1393- 1408. Academic Press, Japan. HADDEN, J. W., GINER-SOROLLA, A. & HADDEN, E. M. (1992). Methyl Inosine Monophosphate (MIMP), a new purine immunomodulator for HIV infection. Int. J. Immunopharmac., 14, 5 5 5 - 563. HADDEN, J. W., HADDEN, E. M., WANG, Y., SOSA, M., COFFEY, R. G. & GINER-SOROLLA, A. (1991). Methyl Inosine Monophosphate (MIMP) - - a new purine immunomodulator. 5th International Conference on Immunopharmacology, Tampa, Florida. Abstract No. 107. Int. J. Immunopharmac., 13, 761. JERNE, N. K. & NORDIN, A. A. (1963). Plaque formation in agar by single antibody producing cell. Science, 140, 405. LAGRANGE, P. H., MACKANESS, G. B. & MILLER, T. (1974). Potentiation of T cell-mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. exp. Med., 139, 1529- 1539, MORIKAWA, S., BABA, M., HARADA, T. & MITSUOKA, A. (1977). Studies on delayed hypersensitivity in mice III. Evidence for suppressive regulatory T-cell population in delayed hypersensitivity. J. exp. Med., 145, 237-248. TOURAINE, J.-L., SANHADJI, K., BOUTE1LLER, O. DE & HADDEN, J. W. (1991). In vitro effects of inosine 5'-methyl monophosphate (MelMP) on human prothymocyte differentiation. 5th International Conference on Immunopharmacology, Tampa, Florida. Abstract No. 109. Int. J. lmmunopharmac., 13, 761.

Potentiation of immune responses in mice by a new inosine derivative--methyl inosine monophosphate (MIMP).

Inosine 5'-methyl monophosphate (MIMP) is a new immunomodulator designed to improve upon the activity of other thymomimetic purines. In Balb/c mice, M...
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