The present invention provides a class of pharmaceutical compositions useful for the treatment of cancer, particularly solid tumors, and having an immuno-modulating mechanism of action as spermine antagonists. The present pharmaceutical compositions are able to override the inhibitory effect of spermine upon tumor killing, and thus prevent spermine-induced immunosuppression.
Anti-cancer therapy has usually followed the model wherein a cytotoxic agent is administered that kills rapidly dividing cells, which are cancer cells and some rapidly dividing host cells, such as bone marrow cells and gut epithelial cells. Thus, conventional cytotoxic cancer therapies have been limited by their side effects targeting those tissues or organs having rapidly dividing cells, (e.g., bone marrow suppression and mucositis). Advances in cancer therapies have been made to better schedule the dosing of cytotoxic agents in creative combinations and to administer growth factors that are designed to rescue host rapidly dividing cells. such as blood cell growth factors G-CSF, EPO and others.
Other approaches to cancer therapy have been to better target the cytotoxic agent to localize to the site of the tumor by antibodies, creative dosing apparatus and by a severe form of treatment, bone marrow transplantation wherein all of the rapidly dividing cells are killed and host cells needed for patient survival are replaced.
The approach of augmenting the host""s immune response to a growing tumor or the presence of a tumor has been a desired approach form many years, but it has achieved limited success. For example, interleukin-2 (IL-2) is available for limited kinds of cancers but this immune activator has experienced many severe and life-threatening side effects. Other cytokines and interleukins are also being tried. yet there remains a strong need for effective and less toxic immunotherapeutic agents for cancer therapy.
Spermine and Macrophages
Under certain conditions, macrophages undergo differentiation to become capable of phagocytosis. The involvement of polyamines (of which spermine is one) in functional aspects of macrophages has been studies mainly with respect to malignant processes and treatment with inhibitors of polyamine biosynthesis. Macrophage-mediated tumorocidal activity directed against B16 melanoma cells is transiently augmented after 6 but not after 18 days of treatment with DFMO (xcex1-(difluoromethyl)ornithine, an inactivator of ornithine decarboxylase (ODC); Bowlin et al., Cancer Res. 46:5494-5498, 1986). Treatment with Cornebacterium parvum enhanced the DFMO effect in vivo (Bowlin et al., Cancer Immunol. Immunother. 20:214-218, 1985) and reduced polyamine levels in macrophages, but had no effect on tumoricidal macrophage activation that can be promoted by other agents, such as IFNxcex1 or IFNxcex2.
During the early immune response to infection or injury, macrophages synthesize pro-inflammatory cytokines which orchestrate the inflammatory reaction. Relatively small amounts of these cytokines produced locally in tissues benefit the host by activating antimicrobial pathways and stimulating tissue repair. On the other hand, if the inflammatory stimulus triggers an uncontrolled release of large amounts of cytokines, the resulting cytokine cascade mediates the development of lethal shock and tissue injury (Tracey et al., Science 234:470-474, 1986; Tracey et al., Nature 330:662-664, 1987; and Tracey in Remnick and Friedland, eds., Tumor Necrosis Factor, Marcel Dekker, Inc., 1996). This potentially disastrous scenario is normally prevented by endogenous counter-regulatory mechanisms that have evolved to inhibit cytokine over-production. One class of endogenous cytokine synthesis inhibitors are the glucocorticoid hormones, which are produced during a stress response, and suppress immune activation and cytokine synthesis (Gonzalez et al., Infect. Immun. 61:970-974, 1993 and Buetler et al., Science 232:977-979, 1986). Another class of agents is the anti-inflammatory cytokines, consisting of IL 10 and TGF-xcex2, which effectively suppress macrophage activation and pro-inflammatory cytokine synthesis (Gonzalez et al., Infect. Immun. 61:970-974, 1993; Tsunawaki et al., Nature 334:260-262, 1988; Donnelly et al., J. Immunol. 155:1420-1427, 1995; and Szabxc3x3et al., Br. J. Pharmacol. 113:757-766, 1994). Lastly, prostaglandin E2, which accumulates at sites of inflammation, can also suppress TNF synthesis by increasing intracellular cAMP (Lehmmann et al., J. Immunol. 141:587-591,1988; and Sinha et al., Eur. J. Immunol. 25:147-153,1995). Together, these endogenous molecular mediators are supposed to counter-regulate or dampen the inflammatory response, and to prevent overabundant production of potentially injurious pro-inflammatory cytokines.
Spermine is a ubiquitous biogenic amine that is positively charged at physiological pH. Spermine is a ubiquitous natural polyamine that has been implicated as an inhibitor of some immune responses, including human neutrophil locomotion, T cell activity and NO production in murine macrophages. Pathological conditions, such a major injury or cancer, result in massive impairment of immunological reactivity with clinical consequences of high susceptibility towards serious infection and tumor escape from the immune system. Spermine has been widely studied for its biological roles in regulating DNA synthesis and cellular proliferation, modulation of ion channel function, and as a intracellular second messenger signaling agent (Blanchard et al., Infect. Immun. 55:433-437, 1987). Spermine has been implicated as an inhibitor of an immune response. For example, spermine prevents the synthesis of nitric oxide synthase and NO production in macrophages activated by bacterial endotoxin (Southan et al., Biochem. Biophys. Res. Comm. 203:1638-1644, 1944; and Kaczmarek, et al., Cancer Res. 52:1891-1894, 1992), down-regulates human neutrophil locomotion (Ferrante, Immunol. 54:785-790, 1985), and is immunosuppressive to T cells (Quan et al., Am. J. Reprod Immunol. 22:64-69, 1990). Increased spermine levels have been measured in tissues following injury, inflammation, and infection, derived, in part, from a release of intracellular spermine from dying and injured cells. Several theories have been proposed that the accumulation of spermine an the products of its oxidative metabolism via polyamine oxidase mediate anti-inflammatory activity found in inflammatory exudates, human pregnancy serum, plasma from arthritic rats, and human rheumatoid synovial fluid (Ferrante, Immunol. 54:785-790, 1985; Hempel et al., Nature 225:32-35, 1983; Lewis et al., Biochem. Pharmacol. 25:1435, 1976; Persellin, Arthritis Rheum. 15:144, 1972; Rinandi, Indian J. Med. Res. 44:144, 1956 and Robinson and Robson Br. J. Pharmacol. 23:420, 1964). In addition, high spermine concentrations have been found in solid tumor tissue, leading to a supposition that spermine concentrations are secreted by tumor cells to act as an immunoprotectant mechanism against the host defense system.
One of the early cytokines discovered was tumor necrosis factor (TNF) and, as implied by its name, this cytokine was thought to be an immune effector that could lyse and kill tumor cells. Macrophages are terminally differentiated immune effector cells that, when activated, can lyse tumor cells. The macrophage lytic activity is mediated, in part, by secreting the cytokine TNFxcex1. Curiously, the anti-tumor activity of macrophages is somehow suppressed during tumor growth.
The role of a macrophage, or a mononuclear phagocyte in immunology has long been recognized. Macrophages act by phagocytosis and intracellular disposal. It is a goal of cancer immunotherapy to activate macrophages, since activated macrophages have been shown to lyse tumor cells under both in vitro and in vivo conditions. Macrophages have a continuous function for removal of senescent or damaged red blood cells from the circulation, but this function is constitutive and does not require activation. By contrast, macrophages require activation to perform infrequent functions, such as participation in a host defense against cancer. By xe2x80x9cactivationxe2x80x9d it is generally meant in the literature that an activated macrophage may mean any change in behavior of the macrophage, such as increased adherence, altered motility, increased enzymatic activity, or increased phagocytosis. There are many mechanisms that have been studied that can activate macrophages and include, for example, bacterial endotoxin, and cytokines such as TNF, GM-CSF, IL-2, IL-1 and others. Subsequent direct tumor cell lysis occurs both by direct macrophage-tumor cell contact and the release of a plethora of cytotoxic molecules from the activated macrophages (e.g., H2O2, NO, IL-1, TNF, and collagenases). The importance of direct contact of the macrophage to the tumor cell requires that the macrophage be located within or in proximity to tumor cell tissue. If there are substances secreted by tumor cells that deactivate or prevent macrophage activation, the ability to deactivate the deactivators (a double negative makes a positive) represents an important therapeutic advance for immunotherapeutic treatment of cancer.
The present invention is based upon the discovery of a group of compounds having such activity, wherein the tumor-secreted deactivating substance is spermine.
The present invention provides a pharmaceutical composition for cancer immunotherapy treatment comprising a compound selected from formula I and a pharmaceutically acceptable carrier, wherein formula I comprises: 
wherein xe2x80x9cAxe2x80x9d is independently xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94, phenyl, or pyrimidinyl;
wherein R1,R2, R3, R4, R5, R6, R7, R8, and R9 are each independently H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkoxy, or phenyl;
wherein xe2x80x9cXxe2x80x9d is a linker moiety selected from the group consisting of C2-10 alkyl, C2-10 alkenyl, and R10-R11=R10; wherein R10 is independently C1-4 alkyl or C2-4 alkenyl, and wherein R11 is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and xe2x80x94Oxe2x80x94;
wherein formula II comprises:
H2Nxe2x80x94Bxe2x80x94Dxe2x80x94Bxe2x80x94NH2xe2x80x83xe2x80x83II
wherein xe2x80x9cBxe2x80x9d is a linker moiety independently selected from the group consisting of straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkyl substituted with an amine moiety, and straight or branched C2-6 alkenyl substituted with an amine moiety;
wherein xe2x80x9cDxe2x80x9d is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, xe2x80x94CHxe2x80x94CH2xe2x80x94NH2, xe2x80x94CHxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2, xe2x80x94CHxe2x80x94NH2, and piprazinyl;
wherein formula III comprises:
Nxe2x89xa1Cxe2x80x94Bxe2x80x94Dxe2x80x94Bxe2x80x94Dxe2x80x94Bxe2x80x94Cxe2x89xa1Nxe2x80x83xe2x80x83III
wherein xe2x80x9cBxe2x80x9d and xe2x80x9cDxe2x80x9d are defined as in formula II.
Preferably, R1 is H; R2 through R9 is H or C1-3 alkyl. The preferred compounds of formula I are: 
wherein the upper structure has a designation compound 38 and the bottom structure is called xe2x80x9c214140xe2x80x9d.
The preferred compounds of formula II are: 
compound 91 and 
compound 94. The preferred compound of formula III is: 
compound 92.
The present invention further provides a method for treating a patient with cancer, comprising administering an effective amount of a compound selected from the group consisting of formula I, formula II and formula III, wherein formula I comprises: 
wherein xe2x80x9cAxe2x80x9d is independently xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94, phenyl, or pyrimidnyl;
wherein R1, R2, R3, R4, R5, R6, R7, R8 and R9 are each independently H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkoxy, or phenyl;
wherein xe2x80x9cXxe2x80x9d is a linker moiety selected from the group consisting of C2-10 alkyl, C2-10 alkenyl, and R10-R11xe2x95x90R10; wherein R10 is independently C1-4 alkyl or C2-4 alkenyl, and wherein R11 is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and xe2x80x94Oxe2x80x94;
wherein formula II comprises:
H2Nxe2x80x94Bxe2x80x94Dxe2x80x94Bxe2x80x94NH2xe2x80x83xe2x80x83II
wherein xe2x80x9cBxe2x80x9d is a linker moiety independently selected from the group consisting of straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkyl substituted with an amine moiety, and straight or branched C2-6 alkenyl substituted with an amine moiety;
wherein xe2x80x9cDxe2x80x9d is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, xe2x80x94CHxe2x80x94CH2xe2x80x94NH2, xe2x80x94CHxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2, xe2x80x94CHxe2x80x94NH2, and piprazinyl;
wherein formula III comprises:
Nxe2x89xa1Cxe2x80x94Bxe2x80x94Dxe2x80x94Bxe2x80x94Dxe2x80x94Bxe2x80x94Cxe2x89xa1Nxe2x80x83xe2x80x83III
wherein xe2x80x9cBxe2x80x9d and xe2x80x9cDxe2x80x9d are defined as in formula II.
Preferably, R1 is H; R2 through R9 is H or C1-3 alkyl. The preferred compounds of formula I are: 
wherein the upper structure has a designation compound 38 and the bottom structure is called xe2x80x9c214140xe2x80x9d.
The preferred compounds of formula II are: 
compound 91 and 
compound 94. The preferred compound of formula III is: 
compound 92.