Inhibitor Of Inosine Monophosphate Dehydrogenase Research Articles

Mizoribine Synchronized Methotrexate Therapy should be Considered when Treating Rheumatoid Arthritis Patients with an Inadequate Response to Various Combination Therapies.

Objective The objective of this study was to confirm the efficacy of low-dose mizoribine (MZR), an inhibitor of inosine monophosphate dehydrogenase, as part of synchronized methotrexate (MTX) therapy for rheumatoid arthritis (RA) patients with an inadequate response to various combination therapies of MTX, other synthetic disease-modifying anti-rheumatic drugs (DMARDs) and biological DMARDs. Methods Low-dose MZR was administered to 56 uncontrolled RA patients being treated with MTX and various biological DMARDs. The observation period was 12 months, and the disease activity was evaluated based on the Disease Activity Score in 28 joints (DAS28)-ESR, Simplified Disease Activity Index (SDAI) and serum MMP-3 level. Results All of the disease activity indices were significantly improved within three months, and the serum MMP-3 levels were also significantly decreased around four months after starting low-dose MZR therapy. No patients experienced any adverse effects. Conclusion The present preliminary findings suggest that low-dose MZR therapy with MTX should be considered for the treatment of RA patients with an inadequate response to various combination therapies including MTX, other synthetic DMARDs and biological DMARDs or in whom increasing the dose of MTX is difficult for reasons such as adverse effects and complications.

Inhibition of Impdh As an Effective Treatment for MLL-Fusion Leukemia

Uncontrolled cell proliferation is a hallmark of cancer and requires adequate nucleotide biosynthesis. Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to XMP at the branch point of guanine nucleotide biosynthesis. Inhibition of IMPDH results in the depletion of guanine nucleotides, thereby suppressing the growth of cancers cells. Therefore, IMPDH inhibition constitutes a rational approach to treat cancers including acute myeloid leukemia (AML).We have developed an experimental system to model myeloid leukemogenesis using primary human cord blood (CB) cells. AML1-ETO is a leukemogenic fusion protein and promotes the self-renewal and long-term proliferation of CB cells in vitro. Another fusion protein MLL-AF9 immortalizes CB cells in vitro and produces human leukemia in immunodeficient mice. These engineered pre-leukemic and leukemic cells recapitulate many features of the clinical diseases and have been useful in testing potential anti-leukemic drugs. Using these human cell-based models, we assessed the effect of a specific IMPDH inhibitor, mycophenolic acid (MPA), on CB cells and those expressing AML1-ETO and MLL-AF9. MPA showed substantial growth-inhibitory effect against MLL-AF9-expressing CB cells, while it had only marginal effects on normal CB cells and AML1-ETO cells. Mechanistically, MPA treatment caused a cell cycle arrest at G0/G1-phase, triggered apoptosis, and induced upregulation of p53 and its downstream target genes including p21/CDKN1A in MLL-AF9 cells. The MPA-mediated changes in MLL-AF9 cells were almost fully reversed by the supplementation of guanosine, confirming that guanine nucleotide depletion underlies the effects of MPA. In contrast, knockdown of p53 or p21, or expression of a dominant-negative form of p53 (p53-DD), did not abrogate the growth-inhibitory effect of MPA on MLL-AF9 cells, indicating that MPA inhibits the leukemic growth largely through p53-independent mechanisms.To examine whether MPA has single-agent activity for MLL-fusion leukemia in vivo, we next established a mouse bone marrow transplant assay for MLL-AF9 leukemia. Bone marrow progenitors were transduced with MLL-AF9 and were transplanted into recipient mice. MLL-AF9-expressing bone marrow progenitors produced AML within 3 months. Leukemic cells were isolated from the spleens of moribund primary mice, and were transplanted into secondary recipient mice. These mice were treated either with injections of MPA (100 mg kg−1) every other day or with vehicle. MPA administration led to a delay in disease progression and significantly extended survival. We also tested the in vivo effect of another IMPDH inhibitor FF-10501-01 on MLL-AF9 leukemia. FF-10501-01 is a potent new competitive IMPDH inhibitor undergoing phase I clinical trials for patients with AML and high-risk myelodysplastic syndrome (MDS). As expected, FF-10501-01 also showed significant therapeutic value, providing survival benefit in a mouse MLL-AF9 leukemia model. Finally, we assessed the sensitivity of p53-deficient mouse MLL-AF9 leukemia cells to IMPDH inhibition. We generated p53-deficient leukemia cells by expressing MLL-AF9 into bone marrow progenitors derived from p53 knockout mice. Consistent with earlier results, the p53-deficient MLL-AF9 cells were still sensitive to MPA and FF-10501-01 both in vitro and in vivo, indicating that p53 activation is dispensable for the anti-leukemia effect of these IMPDH inhibitors.Taken together, these findings establish the inhibition of IMPDH as a promising therapy for MLL-fusion leukemia, including those with defective p53 signaling. DisclosuresGoyama:Fuji Film: Research Funding. Iwamura:FUJIFILM Corporation: Employment. Saito:Fuji Film: Employment.

Phase 1 Results of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in a Phase 1 clinical study in advanced AML and MDS, including HMA failures.Previous preclinical studies demonstrated potent anti-proliferative and apoptotic effects of FF-10501-01 on AML cell lines, including HMA-resistant derivatives, through inhibition of de novo guanine nucleotide synthesis. Therefore, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ≥ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. A total of 29 patients, 15M and 14F (23 AML, 6 MDS) have been treated in 7 dose cohorts (50 - 500 mg/m2 PO BID) for 14 days on and 14 days off, and 400 mg/m2 for 21 days on and 7 days off, each 28-day cycle. Median (range) values: age 75 yrs (59 - 88); baseline bone marrow blast counts for AML 34% (12 - 82), for MDS 10% (5 - 16), and overall 30% (5 - 82); and prior treatment regimens 2 (1 - 7). All patients relapsed from, or progressed on, prior HMAs.At baseline, mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A, NOTCH1, JAK2, IDH2, PTPN11, KRA, TP53, RUNX1, EZH2 and/or MDM2 were present in 13 of 29 (45%) of patients.Atrial fibrillation (Gr 2) was reported in 2 subjects at a dose of 500 mg/m2 BID. This met the definition of dose-limiting toxicity (DLT) and no further enrollment was made at this dose level. The maximally tolerated dose (MTD) was declared at 1 dose level lower, 400 mg/m2 BID, and this cohort was expanded to 6 subjects. No DLTs have been observed in N=7 total subjects treated at 400 mg/m2 BID x 14 days. FF-10501-01 has been very well tolerated through 24 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related thrombocytopenia, neutropenia and bone marrow aplasia (all Gr 4) were reported in 1 patient at 200 mg/m2 BID.The median number of FF-10501-01 cycles received to date is 2 (range 1 - 24). Partial remissions have occurred in 2 AML patients (50 and 100 mg/m2 BID) after 3 cycles, lasting for 5 and 24 cycles, respectively, with the higher dose patient still on study after 24 cycles. A total of 8/23 (34.8%) AML patients, including the 2 PRs, have attained stable disease (SD) control with no disease progression over 3 - 24 cycles. Three AML patients remain on study through 3, 23 and 24 cycles, respectively.A bone marrow complete response was achieved in 1 MDS patient treated at 400 mg/m2 BID after 1 cycle. Although the bone marrow blast counts have increased since, this patient remains stable and is still on therapy through 14 cycles. Three of 6 MDS patients (50%), including the marrow CR, attained SD control with no disease progression over 3, 14 and 14 cycles, and 2 remain on study through 3 and 14 cycles, respectively.FF-10501-01 was rapidly absorbed with mean Tmax of 2.74 hours and mean t1/2 of 4.05 hours. Drug exposure (AUC0-24 and AUCcourse) increased with dose in a near linear manner.Potent suppression of circulating xanthine monophosphate (XMP), a marker of IMPDH activity, has been observed following FF-10501-01 administration on Day 1 of Cycles 1 and 2 at dose levels of 50 mg/m2 BID and above.FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS in patients who have failed or progressed on HMAs and with one or more baseline mutations in pathways known to be affected in AML and MDS. Preclinical activity was seen in multiple leukemia cell lines, including HMA-resistant derivatives. In a Phase 1 trial, clinical activity with a marrow CR, PRs, long-term disease stabilization (≥ 5 cycles) and a highly tolerable safety profile were observed. The Phase 2a expansion phase of the study is soon to begin. DisclosuresDiNardo:Agios: Research Funding; Daiichi Sankyo: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Abbvie: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Daver:BMS: Research Funding; Kiromic: Research Funding; Pfizer: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Ariad: Research Funding; Karyopharm: Honoraria, Research Funding; Sunesis: Consultancy, Research Funding. Denton:Westat Corporation: Employment. Smith:Westat Corporation: Employment. Tiefenwerth:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

Autophagy suppression sensitizes glioma cells to IMP dehydrogenase inhibition-induced apoptotic death

We investigated the role of autophagy, a process of controlled self-digestion, in the in vitro anticancer action of the inosine monophosphate dehydrogenase (IMPDH) inhibitor ribavirin. Ribavirin-triggered oxidative stress, caspase activation, and apoptotic death in U251 human glioma cells were associated with the induction of autophagy, as confirmed by intracellular acidification, appearance of autophagic vesicles, conversion of microtubule associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, and degradation of autophagic target p62/sequestosome 1. Ribavirin downregulated the activity of autophagy-inhibiting mammalian target of rapamycin complex 1 (mTORC1), as indicated by a decrease in phosphorylation of the mTORC1 substrate ribosomal p70S6 kinase and reduction of the mTORC1-activating Src/Akt signaling. Guanosine supplementation inhibited, while IMPDH inhibitor tiazofurin mimicked ribavirin-mediated autophagy induction, suggesting the involvement of IMPDH blockade in the observed effect. Autophagy suppression by ammonium chloride, bafilomycin A1, or RNA interference-mediated knockdown of LC3 sensitized glioma cells to ribavirin-induced apoptosis. Ribavirin also induced cytoprotective autophagy associated with Akt/mTORC1 inhibition in C6 rat glioma cells. Our data demonstrate that ribavirin-triggered Akt/mTORC1-dependent autophagy counteracts apoptotic death of glioma cells, indicating autophagy suppression as a plausible therapeutic strategy for sensitization of cancer cells to IMPDH inhibition.

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads againstMycobacterium tuberculosis

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

Mycophenolic Acid-Induced Developmental Defects in Zebrafish Embryos.

With the increasing use of mycophenolic acid (MPA) in solid organ transplantation, some clinical studies indicate that it is also a human teratogen. However, it is unknown by which mechanism MPA acts as a teratogen. Mycophenolic acid was a selective blocker of de novo purine synthesis, and its immunosuppressive effect is mediated by the inhibition of inosine monophosphate dehydrogenase, which could be a target for MPA-induced toxicity as well. The aim of our study was to examine the direct influence of MPA exposure on zebrafish (Danio rerio) embryos. Morphological defects including tail curvature and severe pericardial edema in zebrafish embryos caused by MPA (3.7-11.1 µmol/L) were found in a dose-dependent manner. The teratogenic index (25% lethal concentration value (LC25)/no observed adverse effect level ratio) was 16, which indicated MPA as a teratogen. Quantitative polymerase chain reaction analysis revealed that the expression level of impdh1b and impdh2 was significantly reduced by MPA treatment at 8 µmol/L (equals to LC25 level). All the toxic effects could be partially reversed by the addition of 33.3 µmol/L guanosine. Our results indicated that MPA impairs the development of zebrafish embryos via inhibition of impdh activity, which subsequently caused a guanosine nucleotide depletion in vivo.

Combination therapy with corticosteroid and mycophenolate mofetil in a case of refractory cardiac sarcoidosis

Management of cardiac sarcoidosis (CS) can be challenging. The first-line therapy for this condition is corticosteroids, but other immunosuppressive agents are sometimes co-administered to reduce the dosage of corticosteroid and to thereby avoid steroid-induced adverse effects or to increase its therapeutic efficacy. Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid, an inhibitor of inosine monophosphate dehydrogenase that acts more selectively on T and B lymphocytes when compared with azathioprine. A 40-year-old man was diagnosed with CS after presenting with ventricular fibrillation. His left ventricular ejection fraction was severely reduced (30%), and cardiac positron emission tomography (PET) showed abnormal uptake of 18F-fluorodeoxyglucose. A cardioverter-defibrillator was implanted and prednisolone (30mg/day) was administered. He was re-admitted with recurrent sustained ventricular tachycardia and a positive PET finding despite a 5-month course of prednisolone, and MMF (1000mg/day) was administered. Six months later, he had not required re-hospitalization for heart failure or arrhythmia. We conclude that combination therapy with MMF and corticosteroids is useful for refractory CS.<Learning objective: Management of cardiac sarcoidosis (CS) can be challenging. Although some immunosuppressive agents are co-administered to reduce the dosage of corticosteroids or to intensify the effect of corticosteroids, the optimal combination regimen has not yet been established. This case report shows that combination therapy with corticosteroid and mycophenolate mofetil was useful for CS that was refractory to corticosteroid monotherapy.>

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication.

Current methods of the analysis of immunosuppressive agents in clinical materials: A review

More than 100000 solid organ transplantations are performed every year worldwide. Calcineurin (cyclosporine A, tacrolimus), serine/threonine kinase (sirolimus, everolimus) and inosine monophosphate dehydrogenase inhibitor (mycophenolate mofetil), are the most common drugs used as immunosuppressive agents after solid organ transplantation. Immunosuppressive therapy, although necessary after transplantation, is associated with many adverse consequences, including the formation of secondary metabolites of drugs and the induction of their side effects. Calcineurin inhibitors are associated with nephrotoxicity, cardiotoxicity and neurotoxicity; moreover, they increase the risk of many diseases after transplantation. The review presents a study of the movement of drugs in the body, including the processes of absorption, distribution, localisation in tissues, biotransformation and excretion, and also their accompanying side effects. Therefore, there is a necessity to monitor immunosuppressants, especially because these drugs are characterised by narrow therapeutic ranges. Their incorrect concentrations in a patient’s blood could result in transplant rejection or in the accumulation of toxic effects. Immunosuppressive pharmaceuticals are macrolide lactones, peptides, and high molecular weight molecules that can be metabolised to several metabolites. Therefore the two main analytical methods used for their determination are high performance liquid chromatography with various detection methods and immunoassay methods. Despite the rapid development of new analytical methods of analysing immunosuppressive agents, the application of the latest generation of detectors and increasing sensitivity of such methods, there is still a great demand for the development of highly selective, sensitive, specific, rapid and relatively simple methods of immunosuppressive drugs analysis.

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.

Therapeutic Drug Monitoring of Mycophenolic Acid.

Mycophenolic acid (MPA) is an immunosuppressant requiring therapeutic drug monitoring. Although immunoassays are commercially available, there is significant positive bias using this approach when compared to high-performance liquid chromatography or LC combined with mass spectrometry (LC/MS) or tandem mass spectrometry (LC/MS/MS). Positive bias is due to variable cross-reactivity of MPA acyl glucuronide with antibodies traditionally used in immunoassay formats. As can be expected, the magnitude of bias varies considerably. MPA strongly binds albumin and, as a result, disproportionate increases in free MPA occur in patients with uremia, hypoalbuminemia, and hepatic dysfunction. As such, monitoring free MPA poses additional challenges. Because MPA inhibits inosine monophosphate dehydrogenase, monitoring this enzyme may provide an alternative approach.

An Impdh Inhibitor, FF-10501 Possesses Activating Effects on Myeloid Differentiation As Well As Anti-Proliferation

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders, characterized by peripheral blood cytopenia and risk of leukemic progression. The treatment strategy is subdivided into anti-cancer therapy and the improvement of refractory cytopenia. The therapeutic options are still limited while the number of patients is increasing with growth of the aging population. Interestingly, some of the cytotoxic agents such as alkylating agents, antimetabolites, and antitumor antibiotics have been reported the potency of promoting the differentiation of leukemic cell-lines in cultures. FF-10501, the inhibitor of inosine-5-monophosphate dehydrogenase (IMPDH), which interferes with the synthesis of nucleic acids, is one of candidates for a novel agent for MDS treatment, and now phase I clinical trials for high-risk MDS patients are running in the United States (FF10501US101) and Japan (FF10501JP101). In this study, we evaluated the effects of this agent on myeloid differentiation using human hematopoietic cells. Human samples were collected after informed consent with protocols approved by the Investigational Review Board of Osaka University Hospital.First, FF-10501 inhibited the proliferation of 9 cell-lines of hematological malignancy, including K562, HL-60 and MOLM-13. The erythroid differentiation of K562 human leukemic cells and F36P MDS cells was promoted in the same condition. The induction of differentiation to either granulocytes or monocytes in acute myeloid leukemia cells, MOLM-13 and HL-60 was also observed. These effects were dose-dependent. To elucidate the detailed mechanisms of the FF-10501-induced differentiation, we studied the alternative pathway activated after the IMPDH inhibition. As expected, the production of GMP, GDP and GTP was suppressed while that of ADP was not altered. The accumulation of inosine-5-monophosphate (IMP) was recognized. Among the metabolic changes induced by FF-10501 treatment, we found the elevation in the concentration of inosine and hypoxanthine. Because it is known that reactive oxygen species (ROS) are generated during the degradation of IMP, we examined whether the imbalance of ROS would be associated with the differentiation effect. When ROS scavenger, dimethylthiourea was added into cultures with FF-10501, the erythroid differentiation of K562 and the granulocytic differentiation of MOLM-13 were attenuated. Moreover, the inhibition of p38 also cancelled the effects. These results indicate that overproduction of ROS and the consequent activation of p38 contribute to the promotion of myeloid differentiation of hematological tumor cells by FF-10501.To apply our findings for clinical settings, we examined the effects of FF-10501 using human mononuclear blood cells. When bone marrow cells derived from healthy donors and MDS patients were cultured in colony-forming unit assays, the number of recovered colonies decreased in a dose-dependent manner. Although high dose of FF-10501 (≥ 40 μM) showed strong cytotoxicity, we found that the intermediate dose of FF-10501 promoted generation of erythroid cells from CD34+ hematopoietic stem/progenitor cells. After 3 week-cultures with 4 μM of FF-10501 under erythroid condition (20 ng/ml IL-3, 10 ng/ml Flt3-ligand, 50 ng/ml thrombopoietin, 20 ng/ml stem cell factor and 10 ng/ml erythropoietin) on MS5 stromal cells, the development of glycophorin A+ cells were significantly promoted. Regarding ROS accumulation, we examined cultured cells under the erythroid condition with a fluorescent sensor, Red CC-1. As a result, FF-10501 increased the ROS production.In summary, FF-10501 showed enough anti-proliferative effects to consider the clinical use. Furthermore, in this study we showed that FF-10501 improves normal erythropoiesis. The effect was cancelled by the inhibition of ROS accumulation or MAPK signaling. Our results suggest that FF-10501 could ameliorate myelosuppression during MDS treatment by maintaining myelopoiesis, and the low-dose therapy for low-risk MDS patients might be effective for the improving refractory anemia. DisclosuresMurase:FUJIFILM Corporation: Employment. Komatsu:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Anti-Leukemia Effect of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures.Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with <100 µM FF-10501-01. Decreased intracellular concentrations of guanine nucleotides were observed in MOLM13 and SKM1 cell lines treated with 3 to 30 µM of FF-10501-01 for 24 hours. Proliferation was partially rescued after 72 hours of treatment with 3 µM guanosine and FF-10501-01 in MOLM-13, HL60 cells and their HMA-resistant derivatives. No treatment synergy was observed with the combination of FF-10501-01 with HMAs in MOLM-14 and HL-60 or their HMA-resistant cell lines.In summary, FF-10501-01 produced potent anti-proliferative and apoptotic effects on AML cell lines through inhibition of de novo guanine nucleotide synthesis.In view of these pre-clinical findings, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ³ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. Sixteen patients (12 AML, 4 MDS) have been enrolled in 5 dose cohorts (50 - 400 mg/m2 PO BID) for 14 days on/14 days off each 28-day cycle, including 8 M and 8 F. Median (range) values: age 75.3 yrs (59.1 - 88.6); bone marrow blasts for AML patients 40.5% (12 - 71), for MDS patients 10% (6 - 13), or 30% overall (6 - 71); and prior treatment regimens 2.5 (1 - 6). All patients relapsed from, or progressed on, prior HMAs. Mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A and/or MDM2 were present in 4/16 (25%) patients.The median number of FF-10501-01 cycles received to date is 1.5 (range 1 - 10). No DLTs or drug-related serious adverse events (AEs) have been observed and FF-10501-01 has been very well tolerated through 5 - 10 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related Gr 4 prolonged thrombocytopenia and Gr 4 prolonged neutropenia were reported in one patient at 200 mg/m2 BID.Two partial responses (PRs) have been achieved in 1 patient each at 50 and 100 mg/m2 BID after 3 cycles, 7 (50%) patients demonstrated long-term stable disease over 2 - 10 cycles, and 4 patients have remained on study drug through 5 - 10 cycles and are still ongoing. Updated safety and efficacy data, including PK/PD, will be presented at the meeting.FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS. Preclinical activity was seen in multiple leukemia cell lines. In a Phase 1 trial, clinical activity with PRs, prolonged disease stabilization and a highly tolerable safety profile were observed. The Phase 2 expansion phase will be initiated soon. DisclosuresDiNardo:Novartis: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Smith:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutic, Inc.: Employment. Madden:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

Nicotinamide Adenine Dinucleotide Based Therapeutics, Update.

About 500 NAD (P)-dependent enzymes in the cell use NAD (P) as a cofactor or a substrate. This family of broadly diversified enzymes is crucial for maintaining homeostasis of all living organisms. The NAD binding domain of these enzymes is conserved and it was believed that NAD mimics would not be of therapeutic value due to lack of selectivity. Consequently, only mycophenolic acid which selectively binds at the cofactor pocket of NAD-dependent IMP-dehydrogenase (IMPDH) has been approved as an immunosuppressant. Recently, it became clear that the NAD (P)-binding domain was structurally much more diversified than anticipated and numerous highly potent and selective inhibitors of NAD (P) dependent enzymes have been reported. It is likely, that as in the case of protein kinases inhibitors, inhibitors of NAD (P)-dependent enzymes would find soon their way to the clinic. In this review, recent developments of selective inhibitors of NAD-dependent human IMPDH, as well as inhibitors of IMPDHs from parasites, and from bacterial sources are reported. Therapies against Cryptosporidium parvum and the development of new antibiotics that are on the horizon will be discussed. New inhibitors of bacterial NAD-ligases, NAD-kinases, NMN-adenylyl transferases, as well as phosphoribosyl transferases are also described. Although none of these compounds has yet to be approved, the progress in revealing and understanding crucial factors that might allow for designing more potent and efficient drug candidates is enormous and highly encouraging.

Neural crest requires Impdh2 for development of the enteric nervous system, great vessels, and craniofacial skeleton

Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5′ monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.

Modulation of viral infection in plants by exogenous guanosine

To evaluate the role of purines in antiviral treatments in plants, ribavirin (RB) and tiazofurin (TZ) were applied in combination with guanosine (GS) or adenosine (AS) in in vitro grapevine or tobacco explants infected by Grapevine leafroll associated virus 3 (GLRaV-3) and Cucumber mosaic virus (CMV), respectively. Using a microelectrochemical (trans-plasma membrane electron transport, t-PMET) technique, in vivo assay of free reduced nicotinamide adenine dinucleotide (NADH) was also carried out to estimate the inosine monophosphate dehydrogenase inhibition caused by drugs. Antiviral effectiveness of TZ, evaluated as virus-free explants or virus copies, was significantly hindered by GS in both species, while AS did not interfere with the drugs. GS, but not AS, slightly hindered the antiviral effectiveness of RB. With regard to NADH tests, t-PMET inhibition caused by RB and TZ was dose dependent and the interference of drugs with the NAD+/NADH conversion was confirmed by NADH content. Findings indicate that exogenous GS up to 0.50 mM replenished the GS pool depleted by drugs, contrasting antiviral action. At higher doses of GS, the TZ antiviral action was completely inhibited and exogenous GS caused a feedback that reduced t-PMET activity. The reversal was partially against RB, suggesting that the reduction of the GS pool contributed to the antiviral activity of RB, but it was not the only cause of antiviral effectiveness.

Inosine 5'-monophosphate dehydrogenase inhibitors as antimicrobial agents: recent progress and future perspectives.

Inosine 5'-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an important target for cancer, bacterial, parasitic and viral infections and autoimmune disorders. Several classes of IMPDH inhibitors are known in the literature. The current review succinctly summarizes the progress made in the design and development of IMPDH inhibitors as antimicrobial agents in last five years or so. The focus is on the inhibitor and enzyme structural features responsible for imparting selectivity for the microbial over the host enzyme. Future perspectives clearly outline the inhibitor design opportunities available in this area to address the present challenges of drug resistance and re-emergence of newer and deadly strains of microbes, posing a serious threat to public.

Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture.

The use of mycophenolate mofetil in experimental encapsulating peritoneal sclerosis.

Encapsulated peritoneal sclerosis (EPS) is a rare complication of long-term peritoneal dialysis usually associated with the inadequacy and early termination of dialysis modality. Adequate treatment of peritoneal fibrosis has not been achieved by medical intervention so far. Mycophenolate mofetil (MMF), which inhibits inosine monophosphate dehydrogenase reversibly and highly selectively, is the most widely used drug for maintenance immunosupression in renal transplantation. Recent studies have shown that MMF has also antifibrotic effects. In this study, we evaluated the effects of MMF on EPS model in rats based on antifibrotic effects. Twenty-four Wistar albino rat have been randomly divided into four groups. Group I (control group) received isotonic saline intraperitoneally (i.p) 2 ml/day for (0-3rd weeks). Group II (chlorhexidine (CG) group) received CG 2 ml/day i.p. for (0-3rd weeks). Group III (chlorhexidine + MMF group) received CG (2 ml/day) i.p. for (0-3rd weeks) plus MMF 30 mg/kg/day peroral (4th-6th weeks). Group IV (resting group) received CG 2 ml/day) i.p. (0-3rd weeks) plus peritoneal resting without any treatment (4th-6th weeks) At the end of the sixth weeks, all of the rats were killed. All of the groups were analyzed in terms of peritoneal thickness, degree of inflammation, vasculopathy, neovascularization and fibrosis. Also, the parietal peritoneal tissue samples were evaluated for matrix metalloproteinase 2 (MMP-2) by using the immunohistochemical analysis. When the CG group was compared with the MMF group, the medication resulted in a statistically significant reduction in peritoneal thickness, inflammation and fibrosis score (53.23 ± 16.24 vs. 17.22 ± 3.62, 1 ± 1.225 vs. 1 ± 0, 1.6 ± 0.548 vs. 0.2 ± 0.447, respectively, all p < 0.05). In the resting group, no beneficial effects on morphological abnormality of the peritoneum were observed as compared with MMF group. However, according to immunohistochemical analysis of the expression of MMP-2 on peritoneal samples, the highest expression of MMP-2 was observed in the MMF group. MMF was effective for the treatment of encapsulating peritoneal fibrosis in our rat model. Most recently, MMF may be first choice for EPS due to antifibrotic effect.

High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication.

Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.