National Toxicology Program

Immunotoxicity of Morphine Sulfate in Female B6C3F1 Mice
(CAS No. 64-31-3)

NTP Report Number IMM92044

ABSTRACT

Morphine sulfate represents the prototype opiate antagonist which binds to the opiate receptor and produces biological effects on several systems of the body. Its actions as an analgesic and tolerance to this effect is well documented.¹ Other systems of the body affected include the autonomic nervous system,^2,3 cardiovascular and pulmonary systems,^4,5,6 hepatobiliary system^7,8,9,10 and the immune system. ^{11,12,13,14,15,16}

The studies reported here represent an holistic investigation of morphine's effect on the immune system. Since heroin works through the same mechanism as morphine and also has been implicated in immune suppressive activity, these studies will help in understanding the relationship between drugs of abuse such as heroin and immune suppression.

Morphine sulfate was nominated for investigation by the National Institute of Drug Abuse based on several diverse studies that indicated that it is immunosuppressive.

Morphine sulfate was administered by slow release pellet. In a range-finding study, a time course for immunosuppression was performed using the IgM spleen antibody-forming cell response as the indicator of immunosuppression. The peak time for immunosuppression occurred when the antigen was administered 24 hours after implant. In these same studies, complete tolerence to the morphine action occurred by day 5 after implantation. Morphine levels reaching 1610 ng/ml were obtained within 24 hours of pellet implant. A request was made to the National Institute of Drug Abuse for 3 pellet dose levels of morphine. As a part of the range-finding investigation, a dose-response study was performed which measured morphine levels and corticosterone levels as well as the IgM spleen antibody-forming cell response. The pellet sizes used were 8, 25 and 75 mg pellets. The studies showed that 24 hours after implant morphine levels were 904 ng/ml, while mice implanted with 25 and 75 mg dose p! ellets had mean blood levels of 1299 and 1610 ng/ml. The bioavailability of morphine in these pellets was not linear and the release on corticosterone was very similar for all three pellet sizes.

Executive Summary Table 1 (ES-1) shows a summary of the standard toxicology studies. The primary objective of the toxicology studies was to characterize the toxicological profile produced by morphine. The implantation of morphine did not result in any mortality. Implantation of morphine pellets which produced blood levels as high as 1600 ng/ml caused changes in body weight and body weight gain, changes in hematological parameters and in serum chemistries.

Morphine implantation also increased the blood levels of corticosterone which is believed to have an effect on some of the parameters measured. Body weights of mice implanted with morphine have up to a 10% decrease in body weight. Changes in hematology, as manifested in an increase in erythroid elements, may be related to hemoconcentration, while the decrease in leukocyte numbers may be related to the cortiscosterone-induced apoptosis of lymphocyte and the activation of the sympathetic nervous system which may increase neutrophil output of the bone marrow. Elevation of SGPT, serum albumin and total protein are indicative of liver damage.

Executive Summary Table 2 (ES-2) summarizes the immunology studies of mice treated with morphine sulfate. Morphine sulfate, administered by a slow release subcutaneously implanted pellet to female B6C3F1 mice, is associated with several changes in immune function. The changes in immune function are also associated with elevation of corticosterone levels and may be the major mediator of the immune changes. Spleen and thymus weights and spleen cellularity are decreased by as much as 42%. Spleen cell differentials showed a 50.4% decrease in the percent of Ig+ cells and an increase in the percent of Thy 1.2+ (26.6%), CD4+ (17.3%) and CD8+ (27.3%) cells. Because of the decrease in spleen cellularity, there was an overall decrease of the B cells and T cells in the spleen. The IgM AFC response to the T-dependent antigen sRBC was decreased 57% when expressed as specific activity, i.e. AFC/10⁶ spleen cells, and 72% when expressed on a per spleen basi! s. The spleen proliferative response to T cell and B cell specific mitogens or to allogeneic cells (MLR) were not markedly altered as was the abililty of the T cells to produce cytotoxic T cells (CTL). Innate immunity was markedly altered as seen in suppression of macrophage activity, NK cell activity and decreased serum C3 levels. The decreased levels of C3 are not believed to be sufficient to be responsible for the decrease in macrophage function. Kupffer cell phagocytosis was suppressed by 51.4%, NK activity by 68.4% and C3 levels by 47.4%. The suppression of NK activity is reversed by naltrexone and RU486 indicating that this action is mediated through the opiate receptor elevating the levels of corticosterone which is the final effector hormone.¹⁷ The suppression of Kupffer cell function is reversed by naltrexone and only partially reversed by RU486, indicating that the action is mediated through the opiate receptor and but not completely t! hrough the elevation of corticosterone.¹⁸

Executive Summary Table 3 (ES-3) summarizes the host resistance studies in mice treated with morphine sulfate. Implantation of morphine sulfate pellets was associated with decreased resistance in the tumor model, B16F10 melanoma, but increased host resistance to the bacterial pathogenic models, Listeria monocytogenes and Streptococcus pneumoniae. The increase in resistance is not understood and in-depth studies will be needed. In a preliminary study, serum from morphine sulfate-implanted mice did not inhibit the growth of the bacteria in vitro.

The conclusion from these investigations is that morphine sulfate, administered by pellet implant, is associated with elevations in corticosterone that may in part be responsible for effects on selected aspects of the immune system. The most sensitive immune parameters include the antibody response to sRBC, Kupffer cell phagocytosis and NK cell activity. Statistically significant effects were seen at the lowest dose levels of morphine sulfate administered.

REFERENCES

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¹¹ Bayer, B.M., Daussin, S., Hernandez, M. and Irvin, L. Morphine inhibition of lymphocyte activity is mediated by an opioid dependent mechanism. Neuropharmacol. 29:369-374, 1990.

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¹³ Perz-Castrillion, J. Perez-Arellano, J., Garcia-Palomo, J., Jimenez-Lopez, A. and DeCastro, S. Opioids depresses in vitro human monocyte chemotaxis. Immunopharmacology 23:57-61, 1992.

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¹⁵ Tubaro, E., Borelli, G., Croce, C. Cavallo, G., and Santiangeli, C. Effect of morphine on resistance to infection. J. Infect. Dis., 148:656-666, 1983.

¹⁶ Pruett, S.B., Han, Y.C. and Fuchs, B.A. Morphine Suppresses Primary Humoral Immune Response by a Predominantly Indirect Mechanism. J. Pharmacol. and Exp. Ther., 262:923-928, 1992.

¹⁷ Frier, D. and Fuchs, B.A. A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity. J Pharmacol Exp Ther. 1994 Sep;270(3):1127-33.

¹⁸ LeVier, D. G., Brown, R.D., McCay, J.A. Fuchs, B.A., Harris, L.S. and Munson, A.E. Hepatic and splenic phagocytosis in female B6C3F1 mice implanted with morphine sulfate pellets. J Pharmacol Exp Ther. 1993 Oct;267(1):357-63.

Report Date: December, 1992

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