HIF-PHD Inhibitor Research Articles

Effects of HIF-PHD inhibitors in kidney development.

Effects of HIF-PHD inhibitors in kidney development.

Abstract 5271: Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer

Abstract Purpose: Microsatellite-stable (MSS) colorectal cancer is resistant for immune therapy, necessitating the exploration of novel modalities for its conquest. Recent revelations have revealed the genetic upregulation of HIF-1α as a potential activator of CD8+ T-cells, enhancing an anti-tumor effect. Pseudohypoxia, characterized by normoxic upregulation of Hypoxia-Inducible Factor (HIF-1α), is known to be induced by iron chelators, including HIF-PHD inhibitors. In this study, we aimed to examine whether pseudohypoxia by HIF-PHD inhibitor can enhance immune response and elucidate the underlying mechanism. Method: Colon 26, SW480 and HT29 were used as murine and human MSS colorectal cancer model. Human and murine CD8+ T-cells were represented by Jurkat cells and isolated splenic CD8+ T-cells, respectively. HIF-PHD inhibitors Roxadustat and Vadadustat were used in the study, and a subcutaneous Colon 26 tumor model was established in BALB/c WT and nude mice. Hypoxic culture conditions were maintained for 48 hours at 2% O2. Results: Roxadustat and Vadadustat did not suppress the proliferation of cancer cells under conditions marked by HIF-1α upregulation. Roxadustat and Vadadustat suppress the colon 26 subcutaneous tumor growth in WT mice, the effect was cancelled in the nude mice. The treatment regimen was associated with an augmentation in the total number and effector-like subtypes of CD8+ T-cells (Tex) infiltrating the tumor. Combination therapy involving Roxadustat and PD-1 antibody synergistically inhibited tumor growth and was accompanied by an increase in PD-1lo, Ly108-, TIM-3+ CD8+ T-cells within the tumor microenvironment. Immunohistochemistry revealed that Interleukin-2 (IL-2) expression of murine spleen was stronger in Roxadustat than control group. Interestingly, HIF-PHD inhibitors induced the secretion of IL-2 from isolated CD3+ T-cells of murine spleens in ELISA, a phenomenon uniquely observed under pseudo-hypoxic conditions created by HIF-PH inhibitors, rather than actual hypoxia. This result indicates that pseudo-hypoxia may induce more robust hypoxic response than real hypoxia. Conclusion: The induction of pseudohypoxia by HIF-PHD inhibitors activates the tumor immune response for MSS colorectal cancer via induction of splenic IL-2. Citation Format: Toshiaki Ohara, Yuehua Chen, Yusuke Hamada, Seitaro Nishimura, Hotaka Kawai, Kazuhiro Noma, Hiroshi Tazawa, Toshiyoshi Fujiwara, Akihiro Matsukawa. Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5271.

Exploring the natural products chemical space through a molecular search to discover potential inhibitors that target the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD).

Introduction: Hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes are major therapeutic targets of anemia and ischemic/hypoxia diseases. To overcome safety issues, liver failure, and problems associated with on-/off-targets, natural products due to their novel and unique structures offer promising alternatives as drug targets. Methods: In the current study, the Marine Natural Products, North African, South African, East African, and North-East African chemical space was explored for HIF-PHD inhibitors discovery through molecular search, and the final hits were validated using molecular simulation and free energy calculation approaches. Results: Our results revealed that CMNPD13808 with a docking score of -8.690kcal/mol, CID15081178 with a docking score of -8.027kcal/mol, CID71496944 with a docking score of -8.48kcal/mol and CID11821407 with a docking score of -7.78kcal/mol possess stronger activity than the control N-[(4-hydroxy-8-iodoisoquinolin-3-yl)carbonyl]glycine, 4HG (-6.87kcal/mol). Interaction analysis revealed that the target compounds interact with Gln239, Tyr310, Tyr329, Arg383 and Trp389 residues, and chelate the active site iron in a bidentate manner in PHD2. Molecular simulation revealed that these target hits robustly block the PHD2 active site by demonstrating stable dynamics. Furthermore, the half-life of the Arg383 hydrogen bond with the target ligands, which is an important factor for PHD2 inhibition, remained almost constant in all the complexes during the simulation. Finally, the total binding free energy of each complex was calculated as CMNPD13808-PHD2 -72.91kcal/mol, CID15081178-PHD2 -65.55kcal/mol, CID71496944-PHD2 -68.47kcal/mol, and CID11821407-PHD2 -62.06kcal/mol, respectively. Conclusion: The results show the compounds possess good activity in contrast to the control drug (4HG) and need further in vitro and in vivo validation for possible usage as potential drugs against HIF-PHD2-associated diseases.

Hypoxia-Inducible Factor-Prolyl Hydroxyl Domain Inhibitors: From Theoretical Superiority to Clinical Noninferiority Compared with Current ESAs?

Anemia is a common complication of chronic kidney disease; it is mainly treated with erythropoiesis-stimulating agents (ESAs) and iron. Experimental studies extensively investigated the mechanisms involved in the body's response to hypoxia and led to the discovery of the hypoxia-inducible factor (HIF) pathway and the enzymes regulating its function. HIF-prolyl-hydroxyl domain (PHD) inhibitors are a new class of oral drugs developed to treat anemia in chronic kidney disease. By inhibiting the function of PHD enzymes, they mimic the exposure to moderate hypoxia and stimulate the production of endogenous erythropoietin and very likely increase iron availability. Some data also suggest that their efficacy and, consequently, dose needs are less influenced by inflammation than ESAs. Overall, data from phases 2 and 3 clinical development showed efficacy in anemia correction and maintenance for all of the class molecules compared with placebo (superiority) or erythropoiesis-stimulating agents (noninferiority). Three molecules, roxadustat, vadadustat, and daprodustat, underwent extensive clinical investigation to assess their safety on hard cardiovascular end points, mortality, and special interest events (including cancer and thrombosis). Aside from vadadustat in the nondialysis population, at the prespecified primary analyses, all three molecules met the noninferiority margin for the risk of major cardiovascular events compared with erythropoiesis-stimulating agents or placebo. The reason for this discrepancy is difficult to explain. Other safety signals came from secondary analyses of some of the other randomized clinical trials, including a higher incidence of thrombosis. A more extensive clinical experience with post-marketing data on hard safety issues is needed to define better when and how to use HIF-PHD inhibitors compared with already available ESAs.

Disturbance of Hypoxia Response and Its Implications in Kidney Diseases.

Significance: The disturbance of the hypoxia response system is closely related to human diseases, because it is essential for the maintenance of homeostasis. Given the significant role of the hypoxia response system in human health, therapeutic applications targeting prolyl hydroxylase-hypoxia-inducible factor (HIF) signaling have been attempted. Thus, systemically reviewing the hypoxia response-based therapeutic strategies is of great significance. Recent Advances: Disturbance of the hypoxia response is a characteristic feature of various diseases. Targeting the hypoxia response system is, thus, a promising therapeutic strategy. Interestingly, several compounds and drugs are currently under clinical trials, and some have already been approved for use in the treatment of certain human diseases. Critical Issues: We summarize the molecular mechanisms of the hypoxia response system and address the potential therapeutic implications in kidney diseases. Given that the effects of hypoxia response in kidney diseases are likely to depend on the pathological context, specific cell types, and the differences in the activation pattern of HIF isoforms, the precise application is critical for the treatment of kidney diseases. Although HIF-PHIs (HIF-PHD inhibitors) have been proven to be effective and well tolerated in chronic kidney disease patients with anemia, the potential on-target consequence of HIF activation and some outstanding questions warrant further consideration. Future Direction: The mechanism of the hypoxia response system disturbance remains unclear. Elucidation of the molecular mechanism of hypoxia response and its precise effects on kidney diseases warrants clarification. Considering the complexity of the hypoxia response system and multiple biological processes controlled by HIF signaling, the development of more specific inhibitors is highly warranted. Antioxid. Redox Signal. 37, 936-955.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and “branched-tail” oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors’ IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure–activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 μM concentrations matching the effect of 30 μM roxadustat and 500 μM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Whether Prolyl Hydroxylase Blocker-Roxadustat-In the Treatment of Anemia in Patients with Chronic Kidney Disease Is the Future?

In patients with chronic kidney disease (CKD), anemia develops gradually, which is primarily due to an inadequate synthesis of erythropoietin by the kidneys, as well as to iron disorders in the body, blood loss, shortened erythrocyte survival and inflammation. The currently accepted treatment employs iron, vitamin B12, folic acid supplementation and the use of erythropoiesis stimulants, which are administered only parenterally. Research is currently underway on the new erythropoiesis drugs that can be orally administered, i.e., hypoxia-inducible factor-propyl hydroxylase inhibitor (HIF-PHI) inhibitors which temporarily block propyl hydroxylase [PHD] catalysis and promote a transient increase in the expression of genes regulated by HIF, including kidney and liver erythropoietin [EPO]. Roxadustat is the first oral drug in this class and a potent HIF-PHD inhibitor, exerted to treat anemia in patients with CKD. In phase 1, 2 and 3 studies with CKD-affected patients, roxadustat was more effective to stimulate erythropoiesis for anemia correction than previously used drugs. Roxadustat can be orally given, unlike other erythropoiesis drugs with parenteral administration only, which grants roxadustat a considerable advantage. Our paper presents the results of studies with roxadustat applied for the treatment of anemia in CKD patients with or without dialysis. We are currently not yet able to know the exact role of roxadustat in the treatment of anemia in patients with CKD, but time will tell. It is possible that roxadustat has benefits an iron metabolism and cardiovascular risk.

Effects of molidustat, a HIF-PHD inhibitor, on sodium dynamics and distribution in hypertensive subtotally nephrectomized rats

Effects of molidustat, a HIF-PHD inhibitor, on sodium dynamics and distribution in hypertensive subtotally nephrectomized rats

Effects of post-renal anemia treatment with the HIF-PHD inhibitor molidustat on adenine-induced renal anemia and kidney disease in mice

The kidneys are the major organs for erythropoietin (EPO) production in adults, and thus, kidney damage results in reduced EPO levels and anemia. Inhibitors of Hypoxia-inducible factor-prolyl hydroxylase domain-containing protein (HIF-PHD) are awaited as new therapeutic options for renal anemia. It can be predicted that most patients who receive HIF-PHD inhibitors have renal dysfunction as a cause of anemia. Therefore, in the present study, we investigated the effects of the HIF-PHD inhibitor molidustat on anemia and renal dysfunction when initiated after the onset of renal anemia. Male C57BL/6J mice received adenine orally to induce nephropathy. After the onset of nephropathy, the mice were treated with either vehicle or molidustat. After 4 weeks of administration, vehicle-treated mice displayed significant anemia, and molidustat ameliorated this anemia. Vehicle-treated mice exhibited reduced creatinine clearance and body weight, increased blood urea nitrogen levels, histopathological changes, immune cell infiltration, and dehydration. Molidustat reversed immune cell infiltration, dehydration, and renal fibrosis without improving renal functional parameters. In conclusion, molidustat treatment initiated after the onset of nephropathy and renal anemia reversed anemia in mice. Molidustat improved some parameters of renal abnormality, but it did not restore renal function.

Click Chemistry-Based Discovery of [3-Hydroxy-5-(1 H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia-Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of Anemia.

As a gene associated with anemia, the erythropoiesis gene is physiologically expressed under hypoxia regulated by †hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study, we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time, and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assays. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin-induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.

新型PHD抑制剂类口服抗贫血药物分子的计算机辅助设计

目的：以目标靶蛋白为研究对象设计一系列可口服的抗贫血类化学药物分子结构。方法：本文针对肾性和肿瘤相关性贫血设计口服小分子治疗药物。贫血，特别是糖尿病性贫血治疗领域是一个非常有前景的市场。HIF-PHD抑制剂作为新型治疗贫血药物，可从注射用促红细胞生成药物, 尤其是透析前慢性肾病患者中获取较大的市场份额。更重要的是，由于这类药物可作为对现有促红细胞生成药物治疗无效或因故不能使用、或因价格太贵而未使用人群的一种新的、有效的替代治疗选择，故也具显著拓展贫血治疗市场规模的潜力。通过对已知有活性的化合物的结构特性以及与靶蛋白的作用机制为基础进行研究，设计合成出治疗效果更好的、副作用更小的潜在治疗贫血的口服药物，在贫血治疗领域必将占有一席之地。采用计算机辅助药物设计将理论计算和实际试验相结合，通过计算机的模拟、计算和预算药物与受体生物大分子之间的关系，设计和优化脯氨酸羟化酶抑制剂类先导化合物。采用体外模拟试验进行理论验证，以期对发现新的具有更大的潜在活性药物提供较大的应用价值。结果：设计出一系列以吲哚为母核结构的化合物分子，通过分子对接从理论上证实其中一些分子化合物为潜在的活性化合物。结论：为新型口服抗贫血化学药物的开发奠定了一定的理论基础。 A series of small molecule compound structures oral administration anti-anemia drugs were de-signed based on target protein. Small molecule drug designed in this project is targeted at renal and cancer related anemia. Anemia treatment field, especially diabetic anemia, is a very promising market. As a new kind of anemia inhibitor, HIF-PHD inhibitors could obtain a larger market share from erythropoietic drugs for injection, especially pre-dialysis patients with chronic kidney diseases. More importantly, this kind of drugs could be used as a new, effective replacement therapy for those patients that could not be treated by the existing erythropoietic drugs for that the drugs are invalid for them, or cannot be used for them for some other reasons, or too expensive for them to afford to. So, the potential drugs with better therapeutic effects, less adverse effects, and oral administration developed on the basis of structural characteristic of known active compound and its mechanism of action with target proteins, are doomed to hold a place in the field of anemia treatment. By com-bining theoretical calculation and practical test together via computer aided drug design, with the computer’s simulation, calculation and estimation on the relationship between drugs and biological receptor molecules, this project starts from designing and optimizing the leading compounds of proline hydroxylase inhibitors, then carrying out In Vitro experiments to verify the theoretical ac-tivity, which is of great application value for discovering greater potential active agents. Result: A series of compounds with indole as the parent nucleus structure were designed and some molecular compounds were theoretically confirmed by molecular docking as potential reactive compounds. Conclusion: It has laid a theoretical foundation for the development of new oral anti-anemia drugs.

FG-4497, a Pharmacological Stabilizer of HIF-1α Protein, Synergistically Enhances Hematopoietic Stem Cells (HSC) Mobilization in Response to G-CSF and Plerixafor

AbstractAbstract 216Up to 5% allogeneic healthy donors and up to 40–60% of chemotherapy-treated patients in autologous setting, fail to reach minimal threshold of 2×106 blood CD34+cells/kg in response to G-CSF, precluding transplantation. Plerixafor, a small inhibitor of the chemokine receptor CXCR4, used for 4 days in combination with G-CSF enables this minimal threshold to be reached in up to 60% patients who previously failed to mobilise in response to G-CSF alone. However, the remaining 40% of patients who failed to mobilise in response to G-CSF alone, still fail to mobilize adequately with G-CSF + Plerixafor. In an attempt to further boost HSC mobilization in response to combinations of G-CSF and Plerixafor, we have investigated the role of the hypoxia-sensing pathway in HSC mobilization.HIF-1α (Hypoxia-inducible factor-1α) controls HSC proliferation and self-renewal in poorly perfused hypoxic bone marrow (BM) niches where very quiescent HSC with highest self-renewal potential reside. When O2 concentration is above 2% in the cell microenvironment, HIF-1α protein is rapidly hydroxylated on Pro residues by prolyl hydroxylases PHD1-3. This recruits the E3 ubiquitin ligase VHL, which targets HIF-1α to rapid proteasomal degradation. When O2concentration is below 2% (hypoxia), PHDs are inactive; HIF-1α protein is stabilized, associates with its β subunit ARNT, translocates to the nucleus and activates of transcription and hypoxia-responsive genes.In this study, we have investigated the effect of pharmacological stabilization of HIF-1α protein on HSC mobilization in mice using the HIF-PHD inhibitor FG-4497. We report that FG-4497 treatment stabilizes HIF-1α protein in mouse BM. We find that FG-4497 synergizes with G-CSF and Plerixafor to enhance HSC mobilization. C57/Bl6 mice were in 4 treatment groups: (G) 250μg/kg/day G-CSF alone for 2 days; (GF) G-CSF for 2 days + 20mg/kg/day FG-4497 for 3 days; (GP) G-CSF for 2 days together with16mg/kg Plerixafor 1 hour prior harvest; (GPF) G-CSF together with Plerixafor and FG-4497 with same dosing as above. Mobilization of colony-forming cells (CFC), phenotypic Lin-CD41-Sca1+Kit+CD48-CD150+ HSC, and functional HSC in long-term competitive transplantation assays were measured. Mice in the GF group (G-CSF + FG-4497) mobilized CFC to the blood 4-fold and phenotypic HSC 3-fold more than mice mobilized with G-CSF alone (p<0.005), whereas FG-4497 alone had a poor mobilizing effect. This demonstrates synergy between G-CSF and PHD inhibition. Expectedly, Plerixafor enhanced mobilization of CFC 10-fold and phenotypic HSC 2-fold in response to G-CSF (p<0.005). Most interestingly, addition of FG-4497 boosted 4-fold mobilization of CFC and phenotypic HSC in response to G-CSF+Plerixafor (p<0.005). This was confirmed in competitive repopulation assays following transplantation of 20μL mobilized blood in competition with 2×105BM cells from congenic donors. CD45.2/CD45.1 chimerism showed that combination of G-CSF+Plerixafor+FG-4497 mobilized competitive repopulating HSC 6-fold more than G-CSF+Plerixafor (p<0.005), the best mobilizing cocktail used in the clinic currently.To determine which cell types drive HSC mobilization in a HIF-1α-dependant manner, we crossed HIF1αflox/floxmice with osxCre (HIF-1α gene deletion in osteoprogenitors), LysMCre (deletion in myeloid cells), or with SclCreER mice (tamoxifen-induced deletion in HSC). While studies in LysMCre and SclCreER mice are ongoing, we find that deletion of HIF-1α gene in osteoprogenitors (osxCre mice) decreased 2.5-fold the number of CFU/mL blood following 2 and 3 days treatment with G-CSF. This suggests that HIF-1α in osteoprogenitors and their osteoblastic progenies is necessary for optimal mobilization in response to G-CSF.In conclusion, our data highlight the importance of HIF-1α in HSC mobilization and provide a novel therapeutic strategy for increasing HSC mobilization above levels obtained with combinations of G-CSF and Plerixafor. Thus PHD inhibitors could be useful agents in patients who still fail to mobilize in response to G-CSF and plerixafor. Disclosures:Walkinshaw:Fibrogen Inc.: Employment, Equity Ownership.

A screen for inducers of p21waf1/cip1 identifies HIF prolyl hydroxylase inhibitors as neuroprotective agents with antitumor properties

Preventing neuronal death is a priority for treating neurological diseases. However, therapies that inhibit pathological neuron loss could promote tumorigenesis by preventing the physiological death of cancerous cells. To avert this, we targeted the transcriptional upregulation of p21waf1/cip1 (p21), an endogenous tumor suppressor with neuroprotective and pro-regenerative activity. We identified potential p21 indcuers by screening a FDA-approved drug and natural product small molecule library against hippocampal HT22 cells stably expressing a luciferase reporter driven by the proximal 60bp of the p21 promoter, and tested them for neuroprotection from glutathione depletion mediated oxidative stress, and cytotoxicity to cancer cell lines (DLD-1, Neuro-2A, SH-SY5Y, NGP, CHLA15, CHP212, and SK-N-SH) in vitro. Of the p21 inducers identified, only ciclopirox, a hypoxia-inducible factor prolyl-4-hydroxylase (HIF–PHD) inhibitor, simultaneously protected neurons from glutathione depletion and decreased cancer cell proliferation at concentrations that were not basally toxic to neurons. We found that other structurally distinct HIF–PHD inhibitors (desferrioxamine, 3,4-dihydroxybenzoate, and dimethyloxalyl glycine) also protected neurons at concentrations that killed cancer cells. HIF–PHD inhibitors stabilize HIF transcription factors, mediating genetic adaptation to hypoxia. While augmenting HIF stability is believed to promote tumorigenesis, we found that chronic HIF–PHD inhibition killed cancer cells, suggesting a protumorigenic role for these enzymes. Moreover, our findings suggest that PHD inhibitors can be used to treat neurological disease without significant concern for cell-autonomous tumor promotion.

Oxygen-Independent Stabilization of the Oxygen-Labile Transcription Factor HIF-1α with Dimethyloxalyl Glycine or FG-4497 Increases Hematopoietic Stem Cell Quiescence In Vivo and Mobilization in Response to G-CSF

Abstract 2334The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIF). At O2 concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O2 < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo.In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals.C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p<0.001, 5 mice/group). Conversely, DMOG decreased the proportion of HSC in phase G1 from 31±5% to 14±5% (p<0.001). Similar enhancement of quiescence was observed in less primitive cells such as L-K+S+48+ cells and L-K+S- myeloid progenitors. In mice given BrdU in their drinking water for the last 3 days of the experiment, a 18 day DMOG treatment reduced 3-fold the proportion of L-K+S+48- HSC that incorporated BrdU and halved the proportion of BrdU+ L-K+S+48+ progenitors. Shorter DMOG treatments (6 days or 12 days) did not alter hematopoietic stem and progenitor cell (HSPC) cycling or BrdU incorporation. In contrast, mice treated with 20mg/kg/day FG-4497 had significantly increased proportion of HSPC in G0 phase after only 6 days of treatment, likely due to the more lasting effect of FG-4497 on HIF-1α stabilization. Proportion of HSPC that incorporated BrdU was also significantly decreased. As HIF-PHD inhibitors also increase erythropoietin (EPO) expression by stabilizing HIF in the kidney, we injected a parallel cohort of mice with EPO daily for 18 days. Despite a very strong increase in red cells and hemoglobin in the blood, EPO had no effect of HSPC cycling and BrdU incorporation. Therefore the effects of HIF-PHD inhibitors on HSPC cycling are not an indirect effect of increased EPO.Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery.Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p<0.05). Without G-CSF, circulating CFC were less than 10 / mL blood. Similarly, FG-4497 pre-treatment increased the number of CFC mobilized into the spleen 4-fold compared to G-CSF alone (p<0.01).In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation. Disclosures:Walkinshaw:Fibrogen Inc.: Employment, Equity Ownership.

Chronic Inhibition of Hypoxia-inducible Factor Prolyl 4-hydroxylase Improves Ventricular Performance, Remodeling, and Vascularity After Myocardial Infarction in the Rat

Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI). GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group). Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.

HIF Prolyl Hydroxylase Inhibitors Prevent Neuronal Death Induced by Mitochondrial Toxins: Therapeutic Implications for Huntington's Disease and Alzheimer's Disease

Mitochondrial dysfunction is a central feature of a number of acute and chronic neurodegenerative conditions, but clinically approved therapeutic interventions are only just emerging. Here we demonstrate the potential clinical utility of low molecular weight inhibitors of the hypoxia inducible factor prolyl-4-hydroxylases (HIF PHDs) in preventing mitochondrial toxin-induced cell death in mouse striatal neurons that express a "knock-in" mutant Huntingtin allele. Protection from 3-nitropropionic acid (3-NP, a complex II inhibitor)-induced toxicity by HIF PHD inhibition occurs without rescue of succinate dehydrogenase activity. Although HIF-1alpha mRNA is dramatically induced by mutant huntingtin, HIF-1alpha depletion by short interfering RNAs (siRNA) does not affect steady-state viability or protection from 3-NP-induced death by HIF PHD inhibitors in these cells. Moreover, 3-NP-induced complex II inhibition in control or mutant striatal neurons does not lead to activation of HIF-dependent transcription. HIF PHD inhibition also protects cortical neurons from 3-NP-induced cytotoxicity. Protection of cortical neurons by HIF PHD inhibition correlates with enhanced VEGF but not PGC-1alpha gene expression. Together, these findings suggest that HIF PHD inhibitors are promising candidates for preventing cell death in conditions such as Huntington's disease and Alzheimer's disease that are associated with metabolic stress in the central nervous system.

Selective Inhibition of Hypoxia-Inducible Factor (HIF) Prolyl-Hydroxylase 1 Mediates Neuroprotection against Normoxic Oxidative Death via HIF- and CREB-Independent Pathways

Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress. Iron chelators are well established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton chemistry and the formation of highly reactive hydroxyl radicals. Here, we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1alpha and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1alpha and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2alpha levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1alpha and CREB; and that HIF-2alpha, not HIF-1alpha, regulates susceptibility to normoxic oxidative neuronal death.

Non-hypoxic Stabilization of Hypoxia-Inducible Factor Alpha (HIF-α): Relevance in Neural Progenitor/Stem Cells

Hypoxia-inducible factor-1 (HIF-1) plays an important role in neural progenitor cell (NPC) propagation and dopaminergic differentiation. In the presence of oxygen and iron, hypoxia-inducible factor 1 alpha (HIF-1alpha) is rapidly degraded via the prolyl hydroxylase (PHD)/VHL pathway. In addition to hypoxia, various non-hypoxic stimuli can stabilize HIF-1alpha in NPCs and influence the transcription of HIF-regulated genes. Here, we investigate various hypoxia mimetics: deferoxamine (DFO), ciclopirox olamine (CPX), dimethyloxallyl glycine (DMOG), a novel HIF-PHD inhibitor (FG-4497) and cobalt chloride (CoCl(2)) with respect to their ability to enhance in vitro proliferation, neurogenesis and dopaminergic differentiation of human fetal mesencephalic NPCs (hmNPCs) in ambient oxygen (21%). Although able to stabilize HIF-1alpha, iron chelators (DFO and CPX) and DMOG were toxic to hmNPCs. CoCl(2) was beneficial only towards neuronal and dopaminergic differentiation, while FG-4497 enhanced proliferation, neurogenesis and dopaminergic differentiation of hmNPCs. Both CoCl(2) and FG-4497 were protective to human dopaminergic neurons. Finally, exposure to hyperbaric oxygen (HBO) also stabilized HIF-1alpha in hmNPCs and induced neurogenesis in vitro. These findings suggest that several HIF stabilizing agents or conditions can rescue impaired neurons and promote neurogenesis in vitro.

Small Molecule Activation of Adaptive Gene Expression

A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2-oxoglutarate-dependent dioxygenases, known as the HIF prolyl 4-hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF-1 in some of the salutary effects of iron chelation-induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development of HIF activators that do not bind iron a high priority. Here we report the development of a high throughput screen to develop novel HIF activators and/or PHD inhibitors for therapeutic use in the central nervous system (CNS). We show that tilorone, a low-molecular weight, antiviral, immunomodulatory agent is the most effective activator of the HIF pathway in a neuronal line. We also show that tilorone enhances HIF protein levels and increases the expression of downstream target genes independent of iron chelation and HIF PHD inhibition in vitro. We further demonstrate that tilorone can activate an HIF-regulated reporter gene in the CNS. These studies confirm that tilorone can penetrate the blood-brain barrier to activate HIF in the CNS. As expected from these findings, we show that tilorone provides effective prophylaxis against permanent ischemic stroke and traumatic spinal cord injury in male rodents. Altogether these findings identify tilorone as a novel and potent modulator of HIF-mediated gene expression in neurons with neuroprotective properties.

Detection of Hypoxia-Inducible Gene Manipulation.

Erythropoietin (Epo) gene expression is controlled by hypoxia-inducible factor-1 (HIF-1); it is positively regulated through the HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. Drugs that inhibit GATA or activate HIF-1 might increase the production of Epo and restore hemoglobin concentration. Epo gene doping and the illicit use of HIF-PHD inhibitor (FG-2216) and GATA inhibitor (K-11706) or HIF-1 activators may be used as new doping practices to increase the number of red blood cells. The broad scope of this research is to develop a system for detecting illegal hypoxia-inducible gene manipulation. We performed hematologic analyses, a treadmill exercise test, microarray and real-time RT-PCR (reverse transcriptase-polymerase chain reaction) to examine the effects of K-11706 and FG-2216 on mice, and compared these effects with those induced by recombinant human Epo (rhEpo) as a positive control. Oral administration of K-11706 for 5 and 8 days, FG-2216 for 5 days, and 14-day intra-peritoneal injection of rhEpo on alternate days significantly increased Epo and hemoglobin concentrations, hematocrit, and endurance performance, respectively, compared with the control. Transgenic lines of mice were generated to evaluate erythropoiesis in living mice. Firefly luciferase reporter line generated with the human β-globin locus control region (Hbb-LCR) is referred as Hbb-LCR-Luc; this mouse model provides a sensitive, noninvasive, and real-time method to monitor erythropoiesis in vivo in response to the administration of K-11706 and FG-2216. The oral administration of K-11706 and FG-2216 for 5 days significantly accumulated Hbb-LCR-Luc activity in the spleen. DNA chip technology was used to investigate the molecular mechanisms responsible for hypoxia-inducible gene manipulation. Total RNA was prepared from the bone marrow cells of mice treated with K-11706 for 28 days, FG-2216 for 5 days, and rhEpo for 14 days, and in untreated mice. The genes that appeared to exhibit a difference in the expression level (increase more or decrease less than five times for K-11706 and rhEpo, two times for FG-2216) were 251 genes for K-11706, 343 genes for FG-2216, and 142 genes for rhEpo. Twenty-eight genes showed a significant difference between the 3 groups, and 4 genes showed it with K-11706 and FG-2216. Among them, real-time RT-PCR revealed that FG-2216 significantly decreased the expression levels of FOS, OSM, IL8rb, Ms4a8a, and Arl5c genes to 0.58, 0.54, 0.56, 0.46, and 0.53-fold compared with the control, respectively. A panel of several candidate genes to detect hypoxia-inducible gene manipulation by real-time RT-PCR will provide a new detection system.