WO2000006546A1

WO2000006546A1 - Immunotherapeutic anti-cancer pharmaceutical compositions

Info

Publication number: WO2000006546A1
Application number: PCT/US1998/012198
Authority: WO
Inventors: Kevin J. Tracey; Minghuang Zhang
Original assignee: Picower Institute for Medical Research
Current assignee: Picower Institute for Medical Research
Priority date: 1998-07-30
Filing date: 1998-07-30
Publication date: 2000-02-10
Anticipated expiration: 2001-01-30
Also published as: AU8657398A

Abstract

There is disclosed a class of pharmaceutical compositions useful for the treatment of cancer, particularly solid tumors, and having a mechanism of action as spermine antagonists. The present pharmaceutical compositions are able to inhibit the inhibitory effect of spermine upon macrophage function, and thus prevent spermine-induced immunosuppression.

Description

IMMUNOTHERAPEUTIC ANTI-CANCER PHARMACEUTICAL COMPOSITIONS

Technical Field of the Invention

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.

Background of the Invention

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 6but not after 18 days of treatment with DFMO ( -(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 IFN or IFNβ. 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 larger 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, lmmun. 61:970- 974, 1993 and Buetler et al., Science 232:977-980, 1986). Another class of agents is the anti- inflammatory" cytokines, consisting of IL10 and TGF-β, which effectively suppress macrophage activation and pro-inflammatory cytokine synthesis (Gonzalez et al., Infect, lmmun. 61:970-974, 1993; Tsunawaki et al., Nature 334:260-262, 1988; Donnelly et al., J Immunol. 155:1420-1427, 1995; and Bogdan et 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 intacellular second messenger signaling agent (Blanchard et al., Infect. lmmun. 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 Szabe 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 and 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 the 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 TNFα. 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 "activation" 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. Summary of the Invention

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 "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R4, R₅, R , R₇, R₈ and R₉ are each independently H, Cι_-6 alkyl, Ci-β alkenyl, Cι-₆ alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-ιo alkyl, C_2-ιo alkenyl, and R10-R1 ι=Rιo; wherein Rio is independently

alkyl or C_2- alkenyl, and wherein Rπ is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises: H₂N-B-D-B-NH₂ II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched Cι.₆ alkyl, straight or branched C _-6 alkenyl, straight or branched Cι_-6 alkyl substituted with an amine moiety, and straight or branched C _-6 alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-CsN III wherein "B" and "D" are defined as in formula II.

Preferably, Ri is H; R₂ through Ro. is H or Cι_-3 alkyl. The preferred compounds of formula I are:

wherein the upper structure has a designation compound 38 and the bottom structure is called "214140".

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 "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R₄, R₅, R₆, R₇, Rg and R₉ are each independently H, Cι_-6 alkyl, C).₆ alkenyl, Cι.₆ alkoxy. or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-]0 alkyl, C₂_ι₀ alkenyl, and

wherein R_{] 0} is independently C_!- alkyl or C_2- alkenyl, and wherein- Rn is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises: H₂N-B-D-B-NH₂ II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched Ci-6 alkyl, straight or branched C_2-6 alkenyl, straight or branched Cι_-6 alkyl substituted with an amine moiety, and straight or branched C _-6 alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are defined as in formula II.

Preferably, Ri is H; R through R₉ is H or Cj.₃ alkyl. The preferred compounds of formula I are:

The preferred compounds of formula II are:

compound 91 and

compound 94. The preferred compound of formula III is:

compound 92. Brief Description of the Drawings

Figure 1 shows dose-related activity of compound Ca91 to reverse or override spermine-induced suppression of macrophage/monocyte activation, measured as a TNF response of cultured human monocytes in response to LPS challenge.

Figure 2 shows that cytotoxic activity of LPS-stimulated monocytes for co-cultured tumor cells is inhibited by spermine, and that this spermine-dependent effect is apparent at several different target celheffector cell ratios.

Figure 3 shows that cytotoxic activity of LPS-stimulated monocytes for co-cultured tumor cells is not inhibited by compound Ca38, and that at certain target celheffector cell ratios, cytotoxicity is enhanced.

Figure 4 shows that several of the immunomodulatory compounds (compound 38 of formula I, compounds 91 and 94 of formula II) with anti-cancer activity are not themselves directly cytotoxic for tumor cells at their effective immunostimulatory concentrations. Figure 5 shows that compound Ca38 is efficacious in overriding or reversing the spermine-induced suppression of macrophage activation and of macrophage effector functions in vivo, as determined in a carrageenan-induced footpad edema model.

Figure 6 shows that in vivo treatment with compound Ca91 increases intra-tumor TNF levels. Figure 7 shows that the inhibitory effects of compound Ca91 on growth of a representative solid tumor in vivo are negated by administration of anti-TNF antibodies, indicating that the anti-cancer effects of compound Ca91 are mediated, at least in part, through an effect on TNF levels in the tumor-bearing animal.

Figure 8 shows that compound 38 is effective to delay, inhibit or prevent the establishment of solid tumors from experimentally introduced tumor cell suspensions. The effect in this model of tumor establishment (or metastasis) is dose-sensitive.

Figure 9 shows growth curves of solid tumors in animals treated daily with compound Ca91. Tumors were established from experimental engraftment of tumor cell suspensions before initiation of treatment. Administration of compound Ca91 was efficacious in slowing or preventing further tumor growth, and this activity was dose-sensitive.

Detailed Description of the Invention

wherein "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R , R₅, R₆, R7, Rs and R₉ are each independently H, Cι_-6 alkyl, Cι.₆ alkenyl, Cι- alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-ιo alkyl, C_2-ι₀ alkenyl, and Rι₀-Rn=Rιo; wherein Rio is independently C_{1 -4} alkyl or C_2-4 alkenyl, and wherein Rπ is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises:

wherein "B" is a linker moiety independently selected from the group consisting of straight or branched Cι_6 alkyl, straight or branched C₂-₆ alkenyl, straight or branched C_1-6 alkyl substituted with an amine moiety, and straight or branched C₂.₆ alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are defined as in formula II. Preferably, Ri is H; R₂ through R₉ is H or .₃ alkyl. The preferred compounds of formula I are:

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 "A" is independently -CH -, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R , R₅, R₆, R7, Rs and R are each independently H, Cι_₆ alkyl, Cι_-6 alkenyl, Cι_-6 alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-ιo alkyl, C_2-ιo alkenyl, and Rι₀-Rn=Rιo; wherein Rio is independently Cι_₄ alkyl or C₂-₄ alkenyl, and wherein Rπ is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises:

H2N-B-D-B-NH2 II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched Cι_₆ alkyl, straight or branched C_2-6 alkenyl, straight or branched Cι_-6 alkyl substituted with an amine moiety, and straight or branched C₂. alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are independently defined as in formula II.

Preferably, Ri is H; R₂ through R₉ is H or C_1-3 alkyl. The preferred compounds of formula I are:

The preferred compounds of formula II are:

compound 91 and

compound 94. The preferred compound of formula III is:

compound 92.

Each of the foregoing preferred compounds are available commercially as research reagents (Aldrich). Compound 38, for example, has industrial applications in plastics and synthetic textiles but no pharmaceutical uses.

Spermine

The pro-inflammatory cytokines TNF, IL-1, IL-6, MlP-lα and MlP-l β play an important role in stimulating the early stages of acute inflammation, including recruitment and activation of inflammatory cells, stimulation of endothelial activation and direct cytotoxicity.

These "inflammatory" cytokines play an important role for recovery from infection or injury, however, normal counter-regulatory mechanisms are also critical to the success of an immune response, because inappropriate or excessive production of pro-inflammatory cytokines can lead to shock or tissue injury. Counter-regulatory mechanism has focused upon the cytokine inhibitory roles of the glucocorticoid hormones, the anti-inflammatory cytokines, such as TGF- β and IL-10, and prostaglandin PGE₂. Specifically, local production of proinflammatory cytokines mediates a host response to inflammation, infection and injury whereas overexpression of these mediators can injure or kill the host. Spermine plays an important counter-regulatory role for pro-inflammatory cytokine production and that an excessive counter-regulatory response, mediated by excessive spermine production, can be a mechanism protecting tumors against host defense mechanisms. Thus, spermine is an important point of therapeutic intervention for immunotherapy of cancers, particular solid tumors, by inhibiting an excessive counter-regulatory response and allowing endogenous immune and inflammatory mechanisms to take place. Moreover, local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carageenan.

Spermine can effectively inhibit cytokine synthesis in serum-free conditions and in the presence of the polyamine oxidase inhibitor aminoguanidine. Oxidative metabolism of spermine is not required for the counter-regulatory activity of spermine on cytokine synthesis. The spermine concentrations are readily achievable in vivo as high spermine concentrations have been reported in tumors and in patients infected with bacteria, mycobacteria and viruses (Kurihara et al., Neurosurg. 32:372-375, 1993; Susuki et al., Experimentia 40:838-839, 1984; Seiler et al., Biochem J. 225:219-226, 1985; and Cipolla et al., Eur. Urol. 24:124-131, 1993). The counter-regulatory activity of spermine also appears to act via a different cellular mechanism from that of the glucocorticoids. Others have shown that LPS stimulation of monocytes activates spermine uptake via a protein kinase C (PKC)-dependent mechanism (Khan et al., J. Cell. Physiol. 157:493-501, 1993; and Ding et al., J. Biol. Chem. 264:3924- 3929, 1989). Net spermine uptake occurs following LPS stimulation in human PBMCs (peripheral blood mononuclear cells). Immunotherapeutic Compounds

The present compounds that exhibit immunotherapeutic activity have been described structurally according to three formulae. The compounds exhibit cancer immunotherapeutic pharmacologic activity by inhibiting spermine-induced counter-regulatory activity that tumor cells induce to protect themselves from a host defense immune response. Therefore, the inventive pharmaceutical compositions stimulate an immune response of the host against tumor cells by inhibiting an immune-inhibitory response, or a double negative makes a positive. Tumors, particularly solid tumors, are typically found to be unaffected by macrophages capable of participating in a vigorous host immune response against the tumor tissue. However, for the most part, these macrophages are not active (i.e., are dormant) against the invading tumor tissue. Therefore, it appears that the tumor cells are able to counter-regulate or suppress macrophage immune functions likely by influencing an endogenous counter- regulatory mechanism, which in view of data showing high spermine concentrations in solid tumor tissue, is mediated by spermine. Therefore, it is desirable to inhibit the spermine counter-regulatory effect for therapeutic benefit. Compound Synthesis Compound 38 is N,N'-bis(2,2,6,6-tetramethyl-4-piperidyl) hexamethylenediamine.

This compound has been used as an ultraviolet stabilizer for polymeric materials. One process to synthesize compound 38 is described in Maiz et al. Chemical Industry v. 47 (Catal. Org. React), 369-371, Marcel Dekker, 1992. Briefly, the synthetic process involves a reductive alkylation of 2,2,6,6-tetramethyl-4-piperdone with hexamethylenediamine. Crude N,N'- bis(2,2,6,6-tetramethyl-4-piperidyl) hexamethylenediamine can be synthesized with 1047 parts of 2,2,6,-tetramethyl-4-piperidone and 373 parts of hexamethylenediamine, and 2260 parts of methanol and 5.5 parts of platinum-on-carbon catalyst charged into an autoclave. The temperature of this mixture is gradually elevated from 30 °C. The mixture is subject to hydrogenation at a hydro pressure of 5 kg/cm2 while the temperature is maintained at 60-70 °C. It takes about 8 hours under the foregoing reaction conditions to complete the hydrogenation process. The product is filtered at about 50 °C to remove the catalyst. The filtrate is then subject to distillation to remove solvent and water formed as a by-product during the reaction. This process obtains about 1325 parts of crude N,N'-bis(2,2,6,6-tetramethyl-4- piperidyl) hexamethylenediamine having a purity of 92.9%, and an unreacted 2,2,6,6- tetramethylpiperidone content of about 0.8%. The product can be further purified by, for example crystalization. Briefly, about 100 parts of crude N,N'-bis(2,2,6,6-tetramethyl-4- piperidyl) hexamethylenediamine is added to 80 parts of acetone and the mixture heated to boiling to dissolve the N,N'-bis(2,2,6,6-tetramethyl-4-piperidyl) hexamethylenediamine. The vessel containing this mixture is cooled from the outside walls. Crystals begin to precipitate at about 29 °C. Cooling is continued until the product reaches a temperature of about 5-10 °C. The crystals formed are separated by filtration, washed with pure acetone, and then dried to obtain about 79.6 parts of pure N,N'-bis(2,2,6,6-tetramethyl-4-piperidyl) hexamethylenediamine having a purity of about 99.5% and good long term stability. Pharmaceutical Formulations Because of their pharmacological properties, the compounds of the formulae I-III can be used especially as immunotherapeutic agents to treat patients suffering from cancer. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well-known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Example 1 : Screening assay for compounds active in overriding the spermine-dependent inhibition of macrophage activation in vitro.

This example illustrates the in vitro activity of spermine to inhibit the macrophage/monocyte cytokine response to a standardized challenge with bacterial (E. coli) endotoxin (lipopolysaccharide, LPS), and further illustrates the use of a screening assay for compounds that reverse or override such spermine-dependent inhibition of macrophage activation and identifies compounds active in this assay.

Human monocytes were isolated and cultured from aliquots of fresh, EDTA-treated human blood from healthy donors (Long Island Blood Services; Melville, NY, USA) using standard techniques. Briefly, blood cells were fractionated to obtain peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation through Ficoll-Paque (Pharmacia; Upsalla, Sweden). Buffy coat cells were collected, suspended in RPMI 1640 medium (Gibco BRL; Grand Island, NY, USA) supplemented with 10% heat-inactivated human serum, 0.1% L-glutamine and 0.01% gentamycin, and cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air. PBMCs were distributed into 96-well microtiter culture plates at 5 X 10⁵ cells per well for cytokine assay and spermine uptake studies. Nonadherent cells were removed by aspiration after overnight culture, and adherent cells (i.e., human monocytes) were then used in experimental procedures.

Monocyte cultures were activated with 100 ng/ml sonicated LPS (Sigma; St. Louis, MO, USA), and activation was measured by assaying for the elicited cytokine response; typically, tumor necrosis factor (TNF) levels in culture supernatants were measured by a standard TNF-specific enzyme-linked immunosorbent assay (ELISA). In some experiments, LPS challenge was supplemented with 25 U/ml recombinant human interferon-γ (IFN- γ; Boehringer Mannheim; Mannheim, Germany); results with LPS alone challenge and LPS/ IFN- γ challenge were comparable. To treat cultures with spermine, spermine (Sigma) was added to culture wells, typically 30-60 min before LPS challenge. Fresh spermine stock solutions were prepared before each experiment, by dissolving spermine in sterile-filtered deionized water to a concentration of 51.2 mM or 35 mM; further dilution in RPMI 1640 followed by addition to cultured cells gave the final concentrations indicated (range 1-1000 μM). Control cultures (not spermine-treated) received an equal volume of unadulterated medium. Standard lactate dehydrogenase assays indicated that spermine at the final concentrations indicated did not affect cell viability significantly. LPS was prepared as a 400 μg/ml stock solution by sonication for 10 min, then further diluted with RPMI 1640 and by addition to cultures to provide the indicated final concentrations.

Fifty compounds with some molecular resemblance to naturally occurring polyamines (e.g., spermine) were screened for activity in reversing the spermine-dependent suppression of the tumor necrosis factor (TNF) response of monocyte cultures challenged with LPS. Briefly, dilutions of test compounds were pre-incubated at various concentrations with monocyte cultures 30-60 min before the addition of spermine (35 μM), which was introduced 30-60 min before LPS challenge. Culture supernatants were collected 4 hours after LPS challenge and assayed for TNF by ELISA. Control incubations showed that the TNF response of monocyte cultures to LPS challenge was inhibited by pre-treatment with spermine in the micromolar range (50%) inhibitory concentration was 20 ± 15 μM). The screening assay identified several compounds active in reversing the spermine-mediated suppression of monocyte/macrophage activation. Identifying internal reference numbers, structural and molecular formulae, formula weights, and the 50% effective concentrations (EC₅o; that is, the concentration of test compound which restored about 50% of the uninhibited TNF response seen in monocyte cultures not treated with spermine) of four compounds particularly efficacious in reversing the spermine-dependent inhibition of monocyte activation are presented in Table I, which collates the results of many separate experiments. Results of a typical experiment with compound 91, showing the dose-dependent reversal of spermine-mediated inhibition of the monocyte TNF response to LPS challenge is shown in Figure 1. Compound 21 and compound 11 (2-(2- aminoethylamino)-5-nitropyridine) also showed dose-dependent activity in this "spermine overriding" assay. The activity of the compounds of the present invention to reverse or override the spermine-mediated inhibition of monocyte/macrophage activation in vitro is predictive of their utility as therapeutic compounds in vivo for the same purpose (i.e., releasing monocytes/macrophages from spermine-mediated inhibition of activation or effector functions).

Compounds Able To Override Spermine (35 μM) Suppression of Monocyte TNF Synthesis Completely

No. Structure FW EC50

PI-Ca91

PI-Ca92

C₉H₂₃N3 173.3 7.9±3.2 μM

PI-Ca38

The activity of these compounds to reverse spermine suppression of macrophage activation and effector functions must be attributed to antagonism of the spermine effect since TNF synthesis and release in cultured human monocytes/macrophages was not stimulated by treatment with these compounds alone (that is, in the absence of LPS challenge; TNF levels in monocyte cultures prepared as above were undetectable by ELISA in control-, Ca91- and Ca38-treated cultures; TNF = 8000 ± 820 pg/ml in LPS-stimulated monocyte culture supernatants). Toxicity of the compounds was also tested in mice; the LD₅₀ for compound 91 , for example, was estimated to be 300-400 mg/kg. The mechanism by which the compounds of the present invention antagonize the macrophage suppressive activity of spermine was further investigated by measuring spermine accumulation in LPS-stimulated human PBMC cultures in the presence or absence of compound 91. Briefly, PBMC cultures were pre-cultured with LPS (100 ng/ml) for two hours, then treated with test compound (or medium as control) for 30-60 min, and then supplemented with ¹⁴C-spermine as tracer. After 30 min, cells were flushed of unincorporated radiolabel, chased with cold spermine, and cell-associated radioactivity was measured by standard methods using a microtiter plate-based detection system. Treatment with compound 91 caused a concentration-dependent decrease in cell-associated spermine (measured as ¹⁴C-associated counts from cultures incubated with labeled spermine at 37°C corrected for binding at 4°C), indicating that Ca91 interferes with cellular accumulation of spermine. The 50% inhibitory concentration (IC₅₀) of compound 91 for spermine uptake was 8 ± 3 μM, and this activity significantly correlated with the effect of Ca91 in reversing spermine-dependent suppression of LPS-elicited TNF synthesis in monocyte/macrophage cultures (R = 0.99). To address the specificity of this activity, the effect of treatment with compound 91 on cell accumulation of structurally related polyamines (i.e., spermidine and putrescine) versus other metabolites (i.e., glutamine and aspartic acid) was determined in similar label-uptake experiments. IC o values for Ca91 in inhibiting cellular accumulation of spermidine (about 10 μM) and putrescine (about 7.5 μM), which share a common polyamine uptake system with spermine, were comparable to the potency of the compound in inhibiting spermine transport. In contrast, compound 91 did not interfere with the cellular accumulation of glutamine nor aspartic acid, indicating that the activity of Ca91 in antagonizing cellular accumulation of polyamines is specific. Taken together, these results are predictive of the activity of compounds of the present invention to alleviate spermine-dependent suppression of monocyte/macrophage activation and effector functions by inhibiting cellular spermine accumulation.

Example 2: Screening assay for compounds active in overriding the spermine-dependent inhibition of macrophage-mediated tumor cell killing in vitro. Test compounds were further screened for activity in reversing the spermine-dependent inhibition of the cytotoxic activity of cultured monocytes against co-cultured tumor cells. In this in vitro assay that is predictive of enhancement of monocyte-dependent tumorolytic activity in vivo, monocyte effector cells are co-cultured with cancer target cells (in this case, G361 human melanoma cell line), the co-cultures are treated with LPS to activate the monocytes, and the activation-dependent cytotoxicity mediated by the monocytes against the tumor cell targets is measured. In this model, the addition of spermine to the co-cultures inhibits the tumor cell killing of the monocytes, and test compounds may be screened for activity in reversing or overriding this spermine-dependent inhibition of tumor cell killing activity by activated monocyte/macrophages. The activity of compounds of the present invention in this predictive screening assay has been determined. The follow data use the foregoing screen assay procedure. G361 tumor cell cultures were split and incubated overnight in the presence of 20 μCi/ml ³H-thymidine to label tumor target cells. Labeled tumor cells were rinsed to remove unincorporated radiolabel, and labeled cells were freed from culture plates by trypsin treatment, suspended in Dulbecco's-modified PBS, rinsed and counted. Tumor target cell suspensions were prepared in 2X serial dilutions in RPMI 1640 and 200 μl aliquots were added to cultures of 3 X 10⁵ human monocytes (prepared from human blood samples as described above, except that the PBMC fraction was cultured overnight in the presence of 2 ng/ml human MCSF) to provide effector celhtarget cell ratios of 2:1, 4:1, 8:1, 16:1, 32:1 and 64:1. After incubation for 2 hours, such co-cultures were treated with control medium or test compounds (to provide the indicated final concentration). 30-60 min later, spermine (final concentration 50-100 μM) or control medium was added to the co-cultures, and 30-60 min later LPS (final concentration, 100 ng/ml) was added to activate the monocytes. In the absence of spermine or test compounds, this standard method provides robust monocyte-dependent tumor cell killing that is conveniently assayed by measuring the release of radiolabel into the culture supernatants and establishing an index of "percent cytotoxicity," where 100% is determined by intentionally lysing all tumor target cells. Experimentally treated co-cultures were incubated for 40-48 hours and death of tumor target cells was determined by measuring the appearance ³H in the culture supernatants and computing the corresponding percent cytotoxicity. Control cultures of tumor cells alone were assayed to determine the spontaneous release of ³H from labeled tumor cell cultures (defined as 0% cytotoxicity). Figure 2 shows that spermine interferes with the cytotoxicity of LPS -activated monocytes for cancer target cells at several different target: effector cell ratios. In separate^" replications of this experiment, TNF levels in co-culture supernatants were assayed in a TNF- specific ELISA (as described above), which showed that TNF levels in spermine-treated, LPS- challenged co-cultures were unchanged or significantly lower than TNF levels in control cultures challenged with LPS but not treated with spermine. Figure 3 shows that compound 38, in contrast to spermine, did not interfere with macrophage/monocyte tumor cell killing, and at some target: effector ratios significantly enhanced the cytotoxicity of LPS -activated monocytes for co-cultured melanoma target cells. In separate experiments, compounds 38, 91 and 94 were shown not to be cytotoxic for cultured melanoma cells (B16 cell line) over a concentration range of 0.1 to 100 μM which corresponds to the range of concentrations over which the compounds showed immunostimulatory effects (see Figure 4). Cell viability was assessed by a standard MTT-based assay after 24 hours of incubation in the presence of test compounds; in additional experiments compound 91 showed no significant toxicity for B16 cells after five days at a concentration of 100 μM (cell counts: 2 X 10⁴ cells/well on day 0; 123.5 ± 5 X 10⁴ cells/well on day 5 for compound 91 -treated cultures; 111.4 ± 4 X 10⁴ cells/well on day 5 for untreated control cultures). Taken together, these experiments indicate that the compounds of the instant invention enhance the cytotoxicity of LPS-activated monocytes towards cancer cells in vitro, which is predictive of a similarly beneficial anti- cancer effect in vivo.

Example 3: Screening assay for compounds active in overriding the spermine-dependent inhibition of macrophage effector functions in vivo. This example shows that compounds of the instant invention can override the macrophage-suppressive effects of spermine in vivo. Carrageenan-induced edema of the rat footpad is a well-accepted model of inflammation, in which monocytes/macrophages play a pivotal role. Intra-footpad administration of carrageenan causes monocyte/macrophage activation, leading to pro-inflammatory cytokine release (including, importantly, TNF) which is associated with inflammatory cell infiltration, edema and paw swelling. To provide a mouse footpad assay for macrophage effector functions in vivo (measured as elicited inflammation and edema), female C3H/HeN mice (20-25 g; Jackson Laboratories, Bar Harbor, ME, USA) were used in experimental groups of five animals. Carrageenan (Sigma) was prepared at a concentration of 1.0% in PBS at 37°C three or more days before use. 50 μl doses of 0.2% carrageenan were injected into the plantar surface of the left hind paw of animals in the control group, and spermine (at the indicated doses) was co-administered in 50 μl PBS in three separate spermine-treatment groups. The right hind paw was injected with the same volume of PBS as the matched control in each animal. At between 20-28 hours after footpad injection, the thickness of the left versus the right hind paw was measured by an investigator blinded as to the treatment group. The difference in thickness (in mm) between the left versus right paws was taken as the inflammatory index. This assay is indicative of the activity of the test compound to override or reverse the inhibitory effects of spermine on macrophage activation and macrophage-mediated cytokine responses in vivo, and therapeutically beneficial activity of compounds of the present invention is identified thereby. As summarized in Figure 5, spermine is effective to suppress macrophage activation and effector functions (measured as footpad swelling) in this in vivo model. Thus, spermine is effective to suppress monocyte/macrophage activation by an inflammatory stimulus in vivo, and compound 38 was active in overriding or reversing this spermine effect; that is, the normal degree of inflammatory footpad swelling was observed in rats treated with both spermine and compound 38.

Example 4: Anti-cancer activity in vivo.

This example shows the effectiveness of the compounds of the present invention to enhance intra-tumor TNF levels, to cause necrosis in established tumors, and to significantly inhibit the growth of experimentally introduced tumors in vivo.

B16 Fl melanoma cells were proliferated under standard culture conditions and harvested at 80% confluency for administration to C57B1/6J female mice. Mice received 1 X 10⁵ melanoma cells subcutaneously in the left dorsal flank and tumors were allowed to establish for 7-8 days, at which time tumor-bearing mice were randomized into treatment groups (five animals per group). Animals received 1.0 mg/kg compound 91 (or PBS vehicle only as control) in a single i.p. injection daily for one week. In some experiments, tumor volume was estimated from orthogonal linear measurements of tumor diameter made with calipers. For analysis of intra-tumor TNF levels, tumors were harvested at day 15, lysed (in 50 mM Tris-HCl, 0.5% NP-40, 150 mM NaCl, 5 mm EDTA, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 0.02% sodium azide, pH 7.4) and assayed for TNF protein by ELISA. TNF levels were normalized per gram of tumor tissue lysed.

In Figure 6, each bar represents the intra-tumor TNF level in a tumor from either the untreated control group or the compound 91 -treated animals (TNF levels could not be determined in one animal treated with compound 91 due to small tumor size). The mean TNF level in the compound 91 -treated group was significantly higher than the mean TNF level in the untreated group; evidence that compound 91 was efficacious in reversing the inhibitory effects of tumor-associated spermine and restoring the TNF secretion of tumor-associated macrophages.

In parallel experiments, mice were similarly inoculated with B16 Fl melanoma cells, and tumors were harvested into 10% formalin for histological analysis at day 15 after one week of daily administration of compound 91 (1.0 mg/kg/day) or vehicle (as control). Tumors from compound 91 -treated animals showed extensive central necrosis of the solid tumor; melanomas from untreated animals did not show this zone of necrosis. In addition, numerous histiocytes and lymphocytes were noted at the periphery of tumors from compound 91 -treated animals; this histological appearance is typical of tumors from animals treated with TNF and other immune-activating therapies where macrophage activation and elevated TNF levels play a critical role in mediating tumor responses. There was no such intense cellular reaction at the margin of tumors harvested from control-treated animals.

Tumors from additional Ca91 -treated and control-treated animals were collected into formalin and processed for in situ hybridization specific for TNF mRNA. The in situ hybridization analysis was performed by a commercial vendor (Molecular Histology Laboratory, Inc.; Gaithersburg, MD, USA), and the results are presented in Table 2 (below): Table 2. TNF expression in B16 melanoma tumors is localized in mononuclear cells (relative hybridization intensity)

These in situ hybridization results show that the increase in intra-tumor TNF levels seen with compound treatment is attributable to increased TNF production by tumor-associated mononuclear cells. This is predictive of the compounds' therapeutic activity to promote tumor-associated monocyte/macrophage activation and enhance tumor-associated monocyte/macrophage effector functions to result in an anti-cancer therapeutic benefit.

To test whether the immunomodulatory anti-cancer activity of compounds of the present invention depended on the restoration of monocyte/macrophage TNF responses within the tumor, the activity of anti-TNF antibodies to reverse the anti -tumor activity of the instant compounds was tested. Mice were given B16 Fl melanoma grafts as described above, and animals with size-matched tumors were randomized into treatment groups of five to receive intraperitoneal injections on days 7, 9 and 1 1 as follows: PBS group, 100 μl PBS vehicle only; Anti-TNF group, 2.0 mg anti-TNF IgG raised in rabbits/mouse; PI-Ca91, 2.0 mg/kg compound 91 ; +Rab IgG group, 2.0 mg/kg compound 91 plus 2.0 mg control rabbit IgG/mouse; +Anti- TNF group, 2.0 mg/kg compound 91 plus 2.0 mg anti-TNF IgG raised in rabbit/mouse. Tumor sizes were measured on day 13 and the average of such tumor sizes for each group are shown in Figure 7. Treatment with compound 91 significantly inhibited tumor growth (P<0.05), and co-treatment with anti-TNF antibodies (but not with control IgG) prevented such compound 91 -mediated inhibition. In that anti-TNF antibody treatment did not itself significantly affect tumor growth, this suggests that the anti-cancer effects of compound 91 are mediated, at least in part, by the alleviation of suppressed monocyte/macrophage anti-tumor activity that is mediated by the naturally occurring elevation of spermine levels within the tumor.

To determine the in vivo anti-tumor therapeutic activity of the compounds of the present invention, mice were studied in a melanoma engraftment model in which test compound was present from the time of subcutaneous implantation of the tumor cells. Such assays are predictive, among other things, of the therapeutic activity of the test compound against melastases; that is, the activity of the compound to inhibit or prevent establishment of a new tumor by "founder" tumor cells. 16 Fl tumor cells were cultured and isolated as described above for transfer into mice. Stock solutions of compound 38 were prepared to provide final concentrations of 0 μM. 10 μM and 100 μM when mixed with melanoma cell suspensions. Approximately 10^' cells in 50 μl vehicle treated without or with compound 8 at the indicated concentrations were then implanted by subcutaneous injection in the dorsal flank of subject mice numbering five per experimental group. Group 1 received melanoma cells but no lest compound; Group 2 received melanoma cells plus 10 μM compound 38, and received additional treatment with compound 38 in the same amount on days 3 and 6; Group 3 received melanoma cells plus 100 μM compound 38 with additional compound 38 treatment in the same amount on days 3 and 6. The additional treatments with compound 38 on days 3 and 6 were by peri-tumoral subcutaneous injection. As illustrated in Figure 8, compound 38 was efficacious in delaying or preventing the establishment of macroscopically apparent tumors following experimental transfer of tumor cells, and this "anti-melastatic" activity was dose-dependent. In further experiments to test whether compounds of the instant invention could slow the growth or cause the regression of established lumors, animals were given B16 Fl melanoma cells as above, and lumors were allowed to become established for 7-8 days, at which time animals were matched according to tumor size and then distributed in control and treatment groups. Animals were treated daily for eight days and tumor size was estimated from caliper measurements every other day (compound 91 was administered i.p. at 0.5 or 2.0 mg/kg/day; controls received vehicle alone). Tumor-bearing animals ucated with compound 91 showed significantly reduced Lumor growth, as illustrated in Figure 9. Al 2.0 mg/kg, treatment with compound 91 completely inhibited tumor growth in 40-60% of treated animals; the other animals showed partial inhibition of tumor growth. These in vivo treatment experiments in mice (together with the other working examples) are predictive of beneficial anti-cancer activity in other animal species, including human.

Claims

We claim:

1. A pharmaceutical composition for cancer immunotherapy treatment comprising a compound selected from formula I, formula II or formula III and a pharmaceutically acceptable carrier, (a) wherein formula I comprises:

wherein "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein R\, R₂, R₃, R<ι, R₅, Rό, R7, Rs and R₉ are each independently H, Cι_-6 alkyl, Cι_-6 alkenyl, Cι_-6 alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C₂_ι₀ alkyl, C₂.ι₀ alkenyl, and

wherein Rι₀ is independently C_]- alkyl or C_2-4 alkenyl, and wherein Rn is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-;

(b) wherein formula II comprises:

H₂N-B-D-B-NH2 II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched Cι_-6 alkyl, straight or branched C_2- alkenyl, straight or branched C_1-6 alkyl substituted with an amine moiety, and straight or branched C_2-6 alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; and

(c) wherein formula III comprises:

N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are defined as in formula II.

2. The pharmaceutical composition of claim 1 wherein Ri is H.

3. The pharmaceutical composition of claim 1 wherein R through R₉ is H or Cι_-3 alkyl.

The pharmaceutical composition of claim 1 wherein the compound of formula

5. The pharmaceutical composition of claim 1 wherein the compound of formula

6. The pharmaceutical composition of claim 1 wherein the compound of formula

7. 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 "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R , R₅, ό, R₇, Rs and R₉ are each independently H, Cι_-6 alkyl, Cι- alkenyl, Cι_-6 alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-10 alkyl, C_2-10 alkenyl, and Rιo-Rπ=Rιo; wherein Rι₀ is independently C_M alkyl or C_2-4 alkenyl, and wherein Rπ is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises: H₂N-B-D-B-NH₂ II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched C_1-6 alkyl, straight or branched C_2-6 alkenyl, straight or branched Cj.₆ alkyl substituted with an amine moiety, and straight or branched C_2-6 alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are independently defined as in formula II.

8. The method of claim 7 wherein R] is H.

9. The method of claim 7 wherein R₂ through R is H or Cι_-3 alkyl.

10. The method of claim 7 wherein the compound of formula II is

11. The method of claim 7 wherein the compound of formula II is

12. The method of claim 7 wherein the compound of formula III is

13. A method for treating cancer by inhibiting spermine uptake in macrophages, comprising administering a compound that inhibits spermine uptake in macrophages.

14. The method of claim 13 wherein the compound is selected from the group consisting of formula I, formula II and formula III, wherein formula I comprises:

wherein "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R , R5, Rό, R7, Rs and R₉ are each independently H, Cι_-6 alkyl, Cι- alkenyl, Cι.₆ alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-ι₀ alkyl, C_2-ι₀ alkenyl, and Rι₀-Rπ=Rιo; wherein R₁₀ is independently C_1-4 alkyl or C_2-4 alkenyl, and wherein Rπ is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises: H₂N-B-D-B-NH2 II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched Cι_-6 alkyl, straight or branched C .₆ alkenyl, straight or branched Cι_-6 alkyl substituted with an amine moiety, and straight or branched C_2- alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are independently defined as in formula II.

15. A method for increasing tumor necrosis factor (TNF) secretion in tumor- associated mononuclear cells, comprising administering a compound that increases TNF secretion to a solid tumor, wherein tumor-associated mononuclear cells are macrophages and monocytes located within a solid tumor mass but are not cancer cells.

16. The method of claim 15 wherein the compound selected from the group consisting of formula I, formula II and formula III, wherein formula I comprises:

wherein "A" is independently -CH₂-, -O-, -NH-, -CO-, phenyl, or pyrimidnyl; wherein Ri, R₂, R₃, R₄, R₅, Re, R7, Rs and R are each independently H, C_1-6 alkyl, Cι.₆ alkenyl, Cι_-6 alkoxy, or phenyl; wherein "X" is a linker moiety selected from the group consisting of C_2-ι₀ alkyl, C_2-ι₀ alkenyl, and

wherein Rio is independently C_M alkyl or C_2- alkenyl, and wherein Rπ is selected from the group consisting of oxo, phenyl, toluenyl, pyrimidinyl, amino, and -O-; wherein formula II comprises: H2N-B-D-B-NH2 II wherein "B" is a linker moiety independently selected from the group consisting of straight or branched C_]-6 alkyl, straight or branched C_2-6 alkenyl, straight or branched C_1-6 alkyl substituted with an amine moiety, and straight or branched C₂. alkenyl substituted with an amine moiety; wherein "D" is a nitrogen-containing moiety selected from the group consisting of pyrimidinyl, piperidyl, pyridinyl, -CH-CH₂-NH₂, -CH-CH₂-CH₂-NH₂, -CH-NH₂, and piprazinyl; wherein formula III comprises: N≡C-B-D-B-D-B-C≡N III wherein "B" and "D" are independently defined as in formula II.