Good cop, bad cop: Polyamines play both sides in host immunity and viral replication.

Viruses rely on host cells for energy and synthesis machinery required for genome replication and particle assembly. Due to the dependence of viruses on host cells, viruses have evolved multiple mechanisms by which they can induce metabolic changes in the host cell to suit their specific requirements. The host immune response also involves metabolic changes to be able to react to viral insult. Polyamines are small ubiquitously expressed polycations, and their metabolism is critical for viral replication and an adequate host immune response. This is due to the variety of functions that polyamines have, ranging from condensing DNA to enhancing the translation of polyproline-containing proteins through the hypusination of eIF5A. Here, we review the diverse mechanisms by which viruses exploit polyamines, as well as the mechanisms by which immune cells utilize polyamines for their functions. Furthermore, we highlight potential avenues for further study of the host-virus interface.

Polyamine homeostasis-based strategies for cancer: The role of combination regimens.

Spermine, spermidine and putrescine polyamines are naturally occurring ubiquitous positively charged amines and are essential metabolites for biological functions in our life. These compounds play a crucial role in many cell processes, including cellular proliferation, growth, and differentiation. Intracellular levels of polyamines depend on their biosynthesis, transport and degradation. Polyamine levels are high in cancer cells, which leads to the promotion of tumor growth, invasion and metastasis. Targeting polyamine metabolism as an anticancer strategy is considerably rational. Due to compensatory mechanisms, a single strategy does not achieve satisfactory clinical effects when using a single agent. Combination regimens are more clinically promising for cancer chemoprevention because they work synergistically with causing little or no adverse effects due to each individual agent being used at lower doses. Moreover, bioactive substances have advantages over single chemical agents because they can affect multiple targets. In this review, we discuss anticancer strategies targeting polyamine metabolism and describe how combination treatments and effective natural active ingredients are promising therapies. The existing research suggests that polyamine metabolic enzymes are important therapeutic targets and that combination therapies can be more effective than monotherapies based on polyamine depletion.

Inhibition of Polyamine Biosynthesis Using Difluoromethylornithine Acts as a Potent Immune Modulator and Displays Therapeutic Synergy With PD-1-blockade.

Polyamines are known to play a significant role in cancer progression and treatment using difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, has shown some clinical promise. It is interesting to note that, while DFMO is directly cytostatic in vitro, recent work has suggested that it achieves its antitumor efficacy in vivo by enhancing adaptive antitumor immune responses. On the basis of these data, we hypothesized that DFMO might act as an immune sensitizer to increase tumor responsiveness to checkpoint blockade. To test this hypothesis, we treated tumors with DFMO, in either the presence or absence of additional PD-1 blockade, and subsequently analyzed their immunological and therapeutic responses. Our data demonstrates that treatment with DFMO significantly enhances both the viability and activation status of intratumoral CD8+ T cells, most likely through an indirect mechanism. When combined with PD-1 blockade, this increased viability resulted in unique proinflammatory cytokine profiles and transcriptomes within the tumor microenvironment and improved therapeutic outcomes. Taken together, these data suggest that DFMO might represent a potential immunomodulatory agent that can enhance current PD-1-based checkpoint therapies.

Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Antitumor Efficacy of PD-1 Blockade.

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade (ICB), responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of cotreatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit antitumor immunity, and limit the efficacy of ICB. In this study, we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow-derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the antitumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti-PD-1 monotherapy, increasing tumor-specific cytotoxic T cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that cotreatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in patients with cancer with resistant tumors.

Inhibitors of tri- and tetra- polyamine oxidation, but not diamine oxidation, impair the initial stages of wound-induced suberization.

The mechanisms regulating, and modulating potato wound-healing processes are of great importance in reducing tuber infections, reducing shrinkage and maintaining quality and nutritional value for growers and consumers. Wound-induced changes in tuber polyamine metabolism have been linked to the modulation of wound healing (WH) and in possibly providing the crucial amount of H2O2 required for suberization processes. In this investigation we determined the effect of inhibition of specific steps within the pathway of polyamine metabolism on polyamine content and the initial accumulation of suberin polyphenolics (SPP) during WH. The accumulation of SPP represents a critical part of the beginning or inchoate phase of tuber WH during closing-layer formation because it serves as a barrier to bacterial infection and is a requisite for the accumulation of suberin polyaliphatics which provide the barrier to fungal infection. Results showed that the inhibitor treatments that caused changes in polyamine content generally did not influence wound-induced accumulation of SPP. Such lack of correlation was found for inhibitors involved in metabolism and oxidation of putrescine (arginine decarboxylase, ornithine decarboxylase, and diamine oxidase). However, accumulation of SPP was dramatically reduced by treatment with guazatine, a potent inhibitor of polyamine oxidase (PAO), and methylglyoxal-bis(guanylhydrazone), a putative inhibitor of S-adenosylmethione decarboxylase which may also cross-react to inhibit PAO. The mode of action of these inhibitors is presumed to be blockage of essential H2O2 production within the WH cell wall. These results are of great importance in understanding the mechanisms modulating WH and ultimately controlling related infections and associated postharvest losses.

An oral form of methylglyoxal-bis-guanylhydrazone reduces monocyte activation and traffic to the dorsal root ganglia in a primate model of HIV-peripheral neuropathy.

Peripheral neuropathy (PN) is a major comorbidity of HIV infection that is caused in part by chronic immune activation. HIV-PN is associated with infiltration of monocytes/macrophages to the dorsal root ganglia (DRG) causing neuronal loss and formation of Nageotte nodules. Here, we used an oral form of methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine biosynthesis inhibitor, to specifically reduce activation of myeloid cells. MGBG is selectively taken up by monocyte/macrophages in vitro and inhibits HIV p24 expression and DNA viral integration in macrophages. Here, MGBG was administered to nine SIV-infected, CD8-depleted rhesus macaques at 21 days post-infection (dpi). An additional nine SIV-infected, CD8-depleted rhesus macaques were used as untreated controls. Cell traffic to tissues was measured by in vivo BrdU pulse labeling. MGBG treatment significantly diminished DRG histopathology and reduced the number of CD68+ and CD163+ macrophages in DRG tissue. The number of recently trafficked BrdU+ cells in the DRG was significantly reduced with MGBG treatment. Despite diminished DRG pathology, intraepidermal nerve fiber density (IENFD) did not recover after treatment with MGBG. These data suggest that MGBG alleviated DRG pathology and inflammation.

Biochemical evaluation of the anticancer potential of the polyamine-based nanocarrier Nano11047.

Synthesizing polycationic polymers directly from existing drugs overcomes the drug-loading limitations often associated with pharmacologically inert nanocarriers. We recently described nanocarriers formed from a first-generation polyamine analogue, bis(ethyl)norspermine (BENSpm), that could simultaneously target polyamine metabolism while delivering therapeutic nucleic acids. In the current study, we describe the synthesis and evaluation of self-immolative nanocarriers derived from the second-generation polyamine analogue PG-11047. Polyamines are absolutely essential for proliferation and their metabolism is frequently dysregulated in cancer. Through its effects on polyamine metabolism, PG-11047 effectively inhibits tumor growth in cancer cell lines of multiple origins as well as in human tumor mouse xenografts. Promising clinical trials have been completed verifying the safety and tolerance of this rotationally restricted polyamine analogue. We therefore used PG-11047 as the basis for Nano11047, a biodegradable, prodrug nanocarrier capable of targeting polyamine metabolism. Following exposure of lung cancer cell lines to Nano11047, uptake and intracellular degradation into the parent compound PG-11047 was observed. The release of PG-11047 highly induced the polyamine catabolic enzyme activities of spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX). By contrast, the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis and a putative oncogene, was decreased. Consequently, intracellular levels of the natural polyamines were depleted concurrent with tumor cell growth inhibition. This availability of Nano11047 as a novel drug form and potential nucleic acid delivery vector will potentially benefit and encourage future clinical studies.

Efficacy and safety of eflornithine (CPP-1X)/sulindac combination therapy versus each as monotherapy in patients with familial adenomatous polyposis (FAP): design and rationale of a randomized, double-blind, Phase III trial.

BACKGROUND: Molecular studies suggest inhibition of colorectal mucosal polyamines (PAs) may be a promising approach to prevent colorectal cancer (CRC). Inhibition of ornithine decarboxylase (ODC) using low-dose eflornithine (DFMO, CPP-1X), combined with maximal PA export using low-dose sulindac, results in greatly reduced levels of normal mucosal PAs. In a clinical trial, this combination (compared with placebo) reduced the 3-year incidence of subsequent high-risk adenomas by >90 %. Familial Adenomatous Polyposis (FAP) is characterized by marked up-regulation of ODC in normal intestinal epithelial and adenoma tissue, and therefore PA reduction might be a potential strategy to control progression of FAP-related intestinal polyposis. CPP FAP-310, a randomized, double-blind, Phase III trial was designed to examine the safety and efficacy of sulindac and DFMO (alone or in combination) for preventing a clinically relevant FAP-related progression event in individuals with FAP. METHODS: Eligible adults with FAP will be randomized to: CPP-1X 750 mg and sulindac 150 mg, CPP-1X placebo and sulindac 150 mg, or CPP-1X 750 mg and sulindac placebo once daily for 24 months. Patients will be stratified based on time-to-event prognosis into one of the three treatment arms: best (ie, longest time to first FAP-related event [rectal/pouch polyposis]), intermediate (duodenal polyposis) and worst (pre-colectomy). Stage-specific, "delayed time to" FAP-related events are the primary endpoints. Change in polyp burden (upper and/or lower intestine) is a key secondary endpoint. DISCUSSION: The trial is ongoing. As of February 1, 2016, 214 individuals have been screened; 138 eligible subjects have been randomized to three treatment groups at 15 North American sites and 6 European sites. By disease strata, 26, 80 and 32 patients are included for assessment of polyp burden in the rectum/pouch, duodenal polyposis and pre-colectomy groups, respectively. Median age is 40 years; 59 % are men. The most common reasons for screening failure include minimal polyp burden (n = 22), withdrawal of consent (n = 9) and extensive polyposis requiring immediate surgical intervention (n = 9). Enrollment is ongoing. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov ( NCT01483144 ; November 21, 2011) and the EU Clinical Trials Register( EudraCT 2012-000427-41 ; May 15, 2014).

Polyamine Antagonist Therapies Inhibit Neuroblastoma Initiation and Progression.

PURPOSE: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. EXPERIMENTAL DESIGN: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. RESULTS: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. CONCLUSIONS: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391-404. ©2016 AACR.

Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect.

Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines. Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer.

Polyamine metabolism is sensitive to glycolysis inhibition in human neuroblastoma cells.

Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors.

Polyamine-blocking therapy reverses immunosuppression in the tumor microenvironment.

Correcting T-cell immunosuppression may unleash powerful antitumor responses; however, knowledge about the mechanisms and modifiers that may be targeted to improve therapy remains incomplete. Here, we report that polyamine elevation in cancer, a common metabolic aberration in aggressive lesions, contributes significantly to tumor immunosuppression and that a polyamine depletion strategy can exert antitumor effects that may also promote immunity. A polyamine-blocking therapy (PBT) that combines the well-characterized ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) with AMXT 1501, a novel inhibitor of the polyamine transport system, blocked tumor growth in immunocompetent mice but not in athymic nude mice lacking T cells. PBT had little effect on the proliferation of epithelial tumor cells, but it increased the number of apoptotic cells. Analysis of CD45(+) tumor immune infiltrates revealed that PBT decreased levels of Gr-1(+)CD11b(+) myeloid suppressor cells and increased CD3(+) T cells. Strikingly, in a model of neoadjuvant therapy, mice administered with PBT one week before surgical resection of engrafted mammary tumors exhibited resistance to subsequent tumor rechallenge. Collectively, our results indicate that therapies targeting polyamine metabolism do not act exclusively as antiproliferative agents, but also act strongly to prevent immune escape by the tumor. PBT may offer a general approach to heighten immune responses in cancer.

Polyamine transport inhibitors: design, synthesis, and combination therapies with difluoromethylornithine.

The development of polyamine transport inhibitors (PTIs), in combination with the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO), provides a method to target cancers with high polyamine requirements. The DFMO+PTI combination therapy results in sustained intracellular polyamine depletion and cell death. A series of substituted benzene derivatives were evaluated for their ability to inhibit the import of spermidine in DFMO-treated Chinese hamster ovary (CHO) and L3.6pl human pancreatic cancer cells. Several design features were discovered which strongly influenced PTI potency, sensitivity to amine oxidases, and cytotoxicity. These included changes in (a) the number of polyamine chains appended to the ring system, (b) the polyamine sequence, (c) the attachment linkage of the polyamine to the aryl core, and (d) the presence of a terminal N-methyl group. Of the series tested, the optimal design was N(1),N(1'),N(1″)-(benzene-1,3,5-triyltris(methylene))tris(N(4)-(4-(methylamino)butyl)butane-1,4-diamine, 6b, which contained three N-methylhomospermidine motifs. This PTI exhibited decreased sensitivity to amine oxidases and low toxicity as well as high potency (EC50 = 1.4 µM) in inhibiting the uptake of spermidine (1 µM) in DFMO-treated L3.6pl human pancreatic cancer cells.

Polyamine inhibitors for treatment of feline oral squamous cell carcinoma: a proof-of-concept study.

This study assessed proof-of-concept for use of polyamine inhibitor 2-diluoromethylornithine (DFMO) as a treatment for oral squamous cell carcinoma (SCC) in client-owned cats. Polyamine levels in tumor tissue and normal oral mucosa were quantified before and after treatment. DFMO was administered orally to 14 client-owned cats with histologically confirmed oral SCC. Patients were monitored for gastrointestinal, dermatologic, auditory, hematological, and biochemical abnormalities. Total polyamine levels in tumor tissue decreased after treatment, as did the specific polyamine putrescine in both tumor tissue and normal mucosa. Ototoxicity was observed in 5 of 6 cats receiving pre- and post-treatment brainstem auditory evoked potential tests. Subclinical thrombocytopenia was observed in 6 of 14 cats. One cat showed mild post-anesthetic tremors that resolved without treatment. Oral administration of DFMO at doses used in this study resulted in significantly decreased tumor polyamine levels without life-threatening clinical or hematological toxicities. Further studies are warranted to explore pathophysiology of polyamine biochemistry and use of polyamine inhibitors in treatment of cats with oral SCC.

Mathematical modelling of polyamine metabolism in bloodstream-form Trypanosoma brucei: an application to drug target identification.

We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.

Leishmaniasis: current status of available drugs and new potential drug targets.

The control of Leishmania infection relies primarily on chemotherapy till date. Resistance to pentavalent antimonials, which have been the recommended drugs to treat cutaneous and visceral leishmaniasis, is now widespread in Indian subcontinents. New drug formulations like amphotericin B, its lipid formulations, and miltefosine have shown great efficacy to treat leishmaniasis but their high cost and therapeutic complications limit their usefulness. In addition, irregular and inappropriate uses of these second line drugs in endemic regions like state of Bihar, India threaten resistance development in the parasite. In context to the limited drug options and unavailability of either preventive or prophylactic candidates, there is a pressing need to develop true antileishmanial drugs to reduce the disease burden of this debilitating endemic disease. Notwithstanding significant progress of leishmanial research during last few decades, identification and characterization of novel drugs and drug targets are far from satisfactory. This review will initially describe current drug regimens and later will provide an overview on few important biochemical and enzymatic machineries that could be utilized as putative drug targets for generation of true antileishmanial drugs.

The polyamine inhibitor alpha-difluoromethylornithine modulates hippocampus-dependent function after single and combined injuries.

Exposure to uncontrolled irradiation in a radiologic terrorism scenario, a natural disaster or a nuclear battlefield, will likely be concomitantly superimposed on other types of injury, such as trauma. In the central nervous system, radiation combined injury (RCI) involving irradiation and traumatic brain injury may have a multifaceted character. This may entail cellular and molecular changes that are associated with cognitive performance, including changes in neurogenesis and the expression of the plasticity-related immediate early gene Arc. Because traumatic stimuli initiate a characteristic early increase in polyamine metabolism, we hypothesized that treatment with the polyamine inhibitor alpha-difluoromethylornithine (DFMO) would reduce the adverse effects of single or combined injury on hippocampus structure and function. Hippocampal dependent cognitive impairments were quantified with the Morris water maze and showed that DFMO effectively reversed cognitive impairments after all injuries, particularly traumatic brain injury. Similar results were seen with respect to the expression of Arc protein, but not neurogenesis. Given that polyamines have been found to modulate inflammatory responses in the brain we also assessed the numbers of total and newly born activated microglia, and found reduced numbers of newly born cells. While the mechanisms responsible for the improvement in cognition after DFMO treatment are not yet clear, the present study provides new and compelling data regarding the potential use of DFMO as a potential countermeasure against the adverse effects of single or combined injury.

Methotrexate--how does it really work?

Methotrexate remains a cornerstone in the treatment of rheumatoid arthritis and other rheumatic diseases. Folate antagonism is known to contribute to the antiproliferative effects that are important in the action of methotrexate against malignant diseases, but concomitant administration of folic or folinic acid does not diminish the anti-inflammatory potential of this agent, which suggests that other mechanisms of action might be operative. Although no single mechanism is sufficient to account for all the anti-inflammatory activities of methotrexate, the release of adenosine from cells has been demonstrated both in vitro and in vivo. Methotrexate might also confer anti-inflammatory properties through the inhibition of polyamines. The biological effects on inflammation associated with adenosine release have provided insight into how methotrexate exerts its effects against inflammatory diseases and at the same time causes some of its well-known adverse effects. These activities contribute to the complex and multifaceted mechanisms that make methotrexate efficacious in the treatment of inflammatory disorders.

Lipophilic lysine-spermine conjugates are potent polyamine transport inhibitors for use in combination with a polyamine biosynthesis inhibitor.

Cancer cells can overcome the ability of polyamine biosynthesis inhibitors to completely deplete their internal polyamines by the importation of polyamines from external sources. This paper discusses the development of a group of lipophilic polyamine analogues that potently inhibit the cellular polyamine uptake system and greatly increase the effectiveness of polyamine depletion when used in combination with DFMO, a well-studied polyamine biosynthesis inhibitor. The attachment of a length-optimized C(16) lipophilic substituent to the epsilon-nitrogen atom of an earlier lead compound, D-Lys-Spm (5), has produced an analogue, D-Lys(C(16)acyl)-Spm (11) with several orders of magnitude more potent cell growth inhibition on a variety of cultured cancer cell types including breast (MDA-MB-231), prostate (PC-3), melanoma (A375), and ovarian (SK-OV-3), among others. These results are discussed in the context of a possible membrane-catalyzed interaction with the extracellular polyamine transport apparatus. The resulting novel two-drug combination therapy targeting cellular polyamine metabolism has shown exceptional efficacy against cutaneous squamous cell carcinomas (SCC) in a transgenic ornithine decarboxylase (ODC) mouse model of skin cancer. A majority (88%) of large, aggressive SCCs exhibited complete or nearly complete remission to this combination therapy, whereas responses to each agent alone were poor. The availability of a potent polyamine transport inhibitor allows, for the first time, for a real test of the hypothesis that starving cells of polyamines will lead to objective clinical response.