IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Results of a Phase 1/2a dose-escalation study of FF-10501-01, an IMPDH inhibitor, in patients with acute myeloid leukemia or myelodysplastic syndromes.

FF-10501-01 potently inhibits inosine-5-monophosphate dehydrogenase (IMPDH), inducing anti-proliferative and pro-apoptotic effects in acute myeloid leukemia (AML) human cell lines resistant to hypomethylating agents. In this Phase 1/2a study, Phase 1 enrolled 38 patients with relapsed/refractory AML (n = 28) or myelodysplastic syndromes (MDS/CMML, n = 10) to receive FF-10501 oral doses 50-500 mg/m2 BID for 14 or 21 days out of each 28-day cycle. Fifteen additional patients with HMA-resistant MDS/CMML (Phase 2a) were treated at 400 mg/m2 BID for 21 days. Most Phase 1 adverse events were disease-related and low-grade. 3 of 19 (16%) evaluable AML patients achieved partial remission (31, 7, and 5 months). 2 of 20 (10%) evaluable MDS/CMML patients (Phase 1 and 2a) attained marrow complete remission, one continuing treatment for 17 months. While FF-10501-01 demonstrated clinical activity and target inhibition in heavily pretreated patients with AML and MDS/CMML, increased mucositis events led to Phase 2a closure (ClinTrials.gov#NCT02193958).

FF-10502, an Antimetabolite with Novel Activity on Dormant Cells, Is Superior to Gemcitabine for Targeting Pancreatic Cancer Cells.

In this paper, we report that 1-(2-deoxy-2-fluoro-4-thio-ß-d-arabinofuranosyl) cytosine (FF-10502), a pyrimidine nucleoside antimetabolite with a chemical structure similar to gemcitabine, shows beneficial anticancer activity via a novel mechanism of action on dormant cells. The growth inhibition of pancreatic cancer cell lines by FF-10502 (IC50, 60-330 nM) was moderately weaker than that by gemcitabine in vitro. In contrast, an in vivo orthotopic implantation model in mice with established human pancreatic cancer cell line, SUIT-2, revealed no mortality with FF-10502 intravenous treatment, which was related to regression of implanted tumor and little metastasis, whereas 75% of the mice treated with gemcitabine died by day 128. Two in vivo patient-derived xenograft models with gemcitabine-resistant pancreatic cancer cells also demonstrated complete tumor growth suppression with FF-10502, but only partial inhibition with gemcitabine. We also investigated the mechanism of action of FF-10502 by using dormant cancer cells, which are reportedly involved in the development of resistance to chemotherapy. In vitro serum starvation-induced dormant SUIT-2 cells developed resistance to gemcitabine even in combination with DNA damage inducers (DDIs; H2O2, cisplatin, and temozolomide). Interestingly, FF-10502 in combination with DDIs significantly induced concentration-dependent cell death in accordance with enhanced DNA damage. FF-10502 was far more potent than gemcitabine in inhibiting DNA polymerase ß, which may explain the difference in dormant cell injury, although further investigations for direct evidences are necessary. In conclusion, our study demonstrated the beneficial antitumor effects of FF-10502 in clinically relevant in vivo models, and suggests the importance of preventing DNA repair unlike gemcitabine.

Molecular targeting of inosine-5'-monophosphate dehydrogenase by FF-10501 promotes erythropoiesis via ROS/MAPK pathway.

One of the major symptoms of myelodysplastic syndromes (MDS) is severe cytopenia. Despite cytokine therapies, such as erythropoiesis-stimulating agents, many patients still require blood transfusions, and the development of new therapeutic approaches is needed. In this work, we studied the effects of the inosine-5'-monophosphate (IMP) dehydrogenase (IMPDH) inhibitor FF-10501 on erythropoiesis of human hematopoietic cells. Differentiation of K562 chronic myeloid leukemia cells to an erythroid lineage was promoted by FF-10501 in a dose-dependent manner. Interestingly, we found that metabolic conversion of IMP to hypoxanthine leads to elevation of reactive oxygen species (ROS). The differentiative effects of FF-10501 were abolished by the ROS scavenger dimethylthiourea or the p38 MAPK inhibitor SB203580. Furthermore, FF-10501 promoted erythropoiesis from CD34+ hematopoietic stem/progenitor cells, accompanied with ROS accumulation, while high-dose FF-10501 mainly showed cytotoxic effects. These findings denote the potential of IMPDH inhibition therapy with FF-10501 in amelioration of anemia in MDS patients.

Preclinical activity of FF-10501-01, a novel inosine-5'-monophosphate dehydrogenase inhibitor, in acute myeloid leukemia.

BACKGROUND: FF-10501-01 is a selective inosine monophosphate dehydrogenase (IMPDH) inhibitor that has shown activity in cancer cell lines. We studied whether FF-10501-01 is effective in targeting a variety of hypomethylating agent (HMA)-sensitive and -resistant acute myelogenous leukemia (AML) cell lines. METHODS: We treated multiple cell lines (including HMA-resistant cells) with FF-10501-01 and analyzed proliferation, apoptosis, and cell cycle status. We also assessed HMA-FF-10501-01 combinations and the ability of extracellular guanosine to rescue cell proliferation in FF-10501-01-treated cells. We performed high-performance liquid chromatography (HPLC) to study guanine nucleotide levels in treated and untreated cells. Finally, we studied the effects of FF-10501-01 in fresh peripheral blood cells taken from AML patients. RESULTS: FF-10501-01 showed a strong dose-dependent effect on proliferation and induced apoptosis at approximately 30µM. The effects of FF-10501-01 treatment on cell cycle status were variable, with no statistically significant trends. Guanosine rescued proliferation in FF-10501-01-treated cells, and HPLC results showed significant decreases in phosphorylated guanosine levels in MOLM13 cells. FF-10501-01 effectively reduced proliferation at concentrations of 300µM and above in 3 primary AML samples. CONCLUSIONS: FF-10501-01 effectively induces AML cell death and reduces AML peripheral blood cell proliferation by targeting guanine nucleotide biosynthesis regardless of HMA resistance status.

Lack of cross-resistance to FF-10501, an inhibitor of inosine-5'-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell lines.

Resistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine-resistant cells derived from SKM-1 and MOLM-13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth-inhibitory effect of azacitidine, expression levels of uridine-cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross-resistance of greater than 10-fold (shift in IC50 value) was observed in azacitidine-resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors FF-10501 and mycophenolate mofetil (cross-resistance to 5-fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth-inhibitory activities in the azacitidine-resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF-10501 to its active form, FF-10501 ribosylmonophosphate [FF-10501RMP]), FF-10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF-10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.

Involvement of central cannabinoid CB2 receptor in reducing mechanical allodynia in a mouse model of neuropathic pain.

We sought to examine the involvement of central cannabinoid CB2 receptor activation in modulating mechanical allodynia in a mouse model of neuropathic pain. JWH133 was demonstrated to be a selective cannabinoid CB2 receptor agonist in mice, reducing forskolin-stimulated cAMP production in CHO cells expressing mouse cannabinoid CB2 and cannabinoid CB1 receptors with EC50 values of 63 nM and 2500 nM, respectively. Intrathecal administration of JWH133 (50 and 100 nmol/mouse) significantly reversed partial sciatic nerve ligation-induced mechanical allodynia in mice at 0.5 h after administration. In contrast, systemic (intraperitoneal) or local (injected to the dorsal surface of the hindpaw) administration of JWH133 (100 nmol/mouse) was ineffective. Furthermore, the analgesic effects of intrathecal JWH133 (100 nmol/mouse) were absent in cannabinoid CB2 receptor knockout mice. These results suggest that the activation of central, but not peripheral, cannabinoid CB2 receptors play an important role in reducing mechanical allodynia in a mouse model of neuropathic pain.

Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors.

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through two main pathways, immunoglobulin E-dependent and -independent activation. In the latter, mast cells are activated by a diverse range of basic molecules, including peptides and amines such as substance P, neuropeptide Y, and compound 48/80. These secretagogues are thought to activate the G proteins in mast cells through a receptor-independent mechanism. Here, we report that the basic molecules activate G proteins through the Mas-related gene (Mrg) receptors on mast cells, leading to mast cell degranulation. We suggest that one of the Mrg receptors, MrgX2, has an important role in regulating inflammatory responses to non-immunological activation of human mast cells.

Involvement of cannabinoid CB(2) receptor-mediated response and efficacy of cannabinoid CB(2) receptor inverse agonist, JTE-907, in cutaneous inflammation in mice.

Involvement of cannabinoid CB(2) receptor and effect of cannabinoid CB(2) receptor antagonist/inverse agonists on cutaneous inflammation were investigated. Mice ears topically exposed to an ether-linked analogue of 2-arachidonoylglycerol (2-AG-E) or selective cannabinoid CB(2) receptor agonist, {4-[4-(1,1-dimethylheptyl)-2,6-dimethoxy-phenyl]-6.6-dimethyl-bicyclo[3.1.1]hept-2-en-2-yl}-methanol (HU-308), had early and late ear swelling (0--24 h and 1--8 days after exposure, respectively). Both types of responses induced by 2-AG-E were significantly suppressed by oral administration of cannabinoid CB(2) receptor antagonist/inverse agonists, [N-(benzo[1,3]dioxol-5-ylmethyl)-7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxamide] (JTE-907) and {N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2 yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide}} (SR 144528). In contrast, JTE-907 did not affect arachidonic acid-induced swelling. Orally administered JTE-907 (0.1-10 mg/kg) and SR 144528 (1 mg/kg) also produced significant inhibition of dinitrofluorobenzene-induced ear swelling, with increased cannabinoid CB(2) receptor mRNA expression observed in the inflamed ear. These results suggest that cannabinoid CB(2) receptor is partially involved in local inflammatory responses and cannabinoid CB(2) receptor antagonist/inverse agonist has beneficial effects on ear swelling.

JTP-27536 [(+)-1,3-dihydroxy-2-hydroxymethylpropyl-2-ammonium 2-[(R)-3-cyclo-hexyl-1-phenylpropyl]-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carboxylate monohydrate], a novel inhibitor of immunoglobulins and interleukin-5 with anti-inflammatory properties in mouse allergic dermatitis model.

We report a novel synthetic compound JTP-27536 [(+)-1,3-dihydroxy-2-hydroxymethylpropyl-2-ammonium 2-[(R)-3-cyclohexyl-1-phenylpropyl]-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carboxylate monohydrate] as an inhibitor of immunoglobulins (Igs) and interleukin (IL)-5 production in vitro and in vivo. JTP-27536 inhibited IgE production in mouse and human B cells with IC50 values of 2.5 and 2.1 microM, respectively, and the inhibition was stronger than that on IgG1 and IgM production (IC50 > 10 microM). JTP-27536 also inhibited IL-5 production in mouse splenocytes and human peripheral blood mononuclear cells with IC50 values of 3.3 and 1.3 microM, respectively, without affecting mouse interferon (IFN)-gamma, IL-2, IL-4, IL-10, or human IL-4 production. In contrast, prednisolone not only inhibited mouse IgE production but also mouse IFN-gamma, IL-2, IL-4, and IL-10 and human IL-4 and IL-5 production in vitro. The effect of suplatast tosilate, a Th2 cytokine inhibitor, on antibody and cytokine production was less potent than that of JTP-27536. In vivo animal experiments using dinitrophenylated ascaris-sensitized mice and 2,4,6-trinitro-1-chrolobenzene-induced chronic dermatitis mice showed that JTP-27536 was more potent than suplatast tosilate and comparable with prednisolone in inhibiting ear swelling, antigen-specific IgE and IL-5 production, and cell infiltrations into the inflamed tissue. These results indicate that JTP-27536 is an inhibitor of Igs, in particular IgE, and of IL-5, which has antiallergic properties in mouse dermatitis model, and suggest that an inhibitor of Igs and IL-5 like JTP-27536 may be useful as a drug for the treatment of allergic diseases.

Comparative study of glucocorticoids, cyclosporine A, and JTE-607 [(-)-Ethyl-N[3,5-dichloro-2-hydroxy-4-[2-(4-methylpiperazin-1-yl)ethoxy]benzoyl]-L-phenylalaninate dihydrochloride] in a mouse septic shock model.

Actions of glucocorticoids, cyclosporine A, and JTE-607 [(-)-ethyl-N-[3,5-dichloro-2-hydroxy-4-[2-(4-methylpiperazin-1-yl)ethoxy]benzoyl]-L-phenylalaninate dihydrochloride], a proinflammatory cytokine inhibitor that does not inhibit interleukin (IL)-2 or interferon-gamma, were compared in a mouse septic shock model induced by cecal ligation and puncture (CLP). CLP caused elevation of macrophage inflammatory protein (MIP)-2 in lung, and MIP-2 and IL-6 in plasma and peritoneal fluid, reaching a peak 4 to 8 h after CLP. Myeloperoxidase (MPO) activity in lung increased and reached a peak 8 to 12 h after CLP. Acute treatment (subcutaneous injections 1 h before and 2 h after CLP) of mice with JTE-607 and methylprednisolone showed significant inhibition of elevated cytokine levels and MPO activity, plus increased survival rate. Similar treatment with cyclosporine A and prednisolone was ineffective. Chronic treatment (subcutaneous injection for seven consecutive days before CLP) of mice with JTE-607 also showed an inhibitory effect on cytokine production, MPO activity and mortality. In contrast, chronic treatment with cyclosporine A and prednisolone did not inhibit cytokine production or MPO activity, but rather exacerbated mortality. These results indicate that JTE-607 has protective effect on mouse mortality induced by CLP, correlating with inhibition of proinflammatory cytokines, whereas the immunosuppressants cyclosporine A and prednisolone do not. This suggests that JTE-607, a multiple cytokine inhibitor that does not cause adverse immunosuppression, is useful for treatment of septic shock.

Induction of megakaryocytopoiesis and thrombocytopoiesis by JTZ-132, a novel small molecule with thrombopoietin mimetic activities.

We report in this paper that a novel small molecule, JTZ-132, induced growth and differentiation of megakaryocytic progenitor cells and improved thrombocytopenia in myelosuppressed mice. JTZ-132 stimulated proliferation of UT-7/TPO cells, a cell line highly sensitive to thrombopoietin (TPO), and exhibited full efficacy comparable to TPO with an approximate EC(50) (median effective concentration) value of 0.43 microM, whereas little proliferation was observed in a TPO-insensitive cell line, UT-7/EPO, and human carcinoma cell line, HCT116. Signal transduction studies revealed that JTZ-132 induced tyrosine phosphorylation of c-Mpl, Janus kinase-2 (JAK2), and signal transducers and activators of transcription 5 (STAT5) in UT-7/TPO cells as well as TPO. JTZ-132 increased the number of megakaryocyte-specific marker, CD61(+) and CD41(+), cells in cultures of mouse and human bone marrow cells, respectively, and the colony-forming unit megakaryocytes in mouse bone marrow cells. In vivo experiments in x-ray irradiation- or busulfan injection-induced myelosuppressed mice demonstrated that subcutaneously injected JTZ-132 at 30 mg/kg showed significantly higher platelet number at nadir and accelerated platelet recovery without affecting white blood cell number. These data suggest that JTZ-132 is a novel stimulator of megakaryocytopoiesis and thrombocytopoiesis in vitro and in vivo with TPO mimetic activities and that it is useful for the treatment of thrombocytopenia.

Prior burn insult induces lethal acute lung injury in endotoxemic mice: effects of cytokine inhibition.

Many patients who experience surgical stress, including burn injury, become susceptible to severe sepsis and septic organ dysfunction, which remains the primary contributor to morbidity and mortality in burn patients. In the present study, we developed a murine model of burn-primed sublethal endotoxemia by producing a 15% BSA full-thickness burn on the dorsum of BALB/c mice under ether anesthesia and administering 10 mg/kg of LPS intravenously on day 11 to model endotoxemia. The prior burn injury in this model induced two-peaked production of cytokines, TNF-alpha, and macrophage inflammatory protein-2 at 2 and 12 h after LPS administration, and it was associated with increased mortality. We also assessed the effect of pharmacological modulation with cytokine synthesis inhibitors prednisolone and JTE-607 and found that pretreatment with them attenuated later cytokine production and decreased mortality after LPS administration. We speculate that the prior burn injury primed the mice for the secondary insult via cytokine production. These results also suggested that an anticytokine strategy might serve as a novel prophylactic therapy for septic organ dysfunction in burn-primed patients.

Neutralization of IFN-inducible protein 10/CXCL10 exacerbates experimental autoimmune encephalomyelitis.

We examined the effect of a monoclonal antibody (mAb) against interferon (IFN)-inducible protein 10 (IP-10)/CXCL10 on the development of experimental autoimmune encephalomyelitis (EAE) in rats induced by injecting xenogeneic brain homogenates into footpads. Treatment with neutralizing mAb against CXCL10 exacerbated EAE with increased infiltrating CD4+ cells in the central nervous system. Furthermore, the exacerbation by the mAb treatment was accompanied by less enlarged draining popliteal lymph nodes (LN) in parallel with cell number compared with those of EAE rats treated with control mAb, whereas other lymphoid organs such as the spleen and thymus were not significantly different between rats treated with anti-CXCL10 and the control mAb. Induction of gene expression of CXCL9/Mig and CXCL10 and their receptor CXCR3 was confirmed in the draining LN in EAE rats. Induction of the third CXCR3 ligand, CXCL11/I-TAC was not seen in the draining LN, whereas all three CXCR3 ligands and CXCR3 itself were markedly detected in the spinal cords following the development of EAE. These findings suggest that CXCL10 produced in the LN plays a specific inhibitory role in the development of Th1-mediated diseases such as EAE by holding sensitized and activated Th1s expressing CXCR3 in the draining LN.