Hypoxia-inducible Factor Prolyl Hydroxylase Inhibition Research Articles

Discovery of DS-1093a: An oral hypoxia-inducible factor prolyl hydroxylase inhibitor for the treatment of renal anemia

Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We discovered DS44470011 in our previous study, which showed potent in vitro activity and in vivo efficacy based on HIF-PHD inhibition. However, DS44470011 was also found to exert genotoxic effects. By converting the biphenyl structure, which is suspected to be the cause of this genotoxicity, to a 1-phenylpiperidine structure, we were able to avoid genotoxicity and further improve the in vitro activity and in vivo efficacy. Furthermore, through the optimization of pyrimidine derivatives, we discovered DS-1093a, which has a wide safety margin with potent in vitro activity and an optimal pharmacokinetic profile. DS-1093a achieved an increase in hemoglobin levels in an adenine-induced rat model of chronic kidney disease after its continuous administration for 4 days.

The Efficacy of Roxadustat in Multiple Myeloma Patients with Renal Insufficiency and Anemia: A Real-World Study

Background: Anemia and renal insufficiency are two common symptoms of multiple myeloma (MM). There are currently few treatment options for anemia in MM. Renal anemia appears to be one of the causes of anemia in MM. Roxadustat is an inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH) that has been proven effective to improve anemia conditions in patients with kidney disease. HIF-PH inhibition with Roxadustat may offer a novel approach to the management of anemia in MM patients with renal insufficiency. This is the first study exploring the efficacy of Roxadustat in multiple myeloma patients with renal insufficiency. Methods: The clinical data of MM patients with renal insufficiency (defined as serum creatinine (Scr) ≥177mol/L) and severe anemia (defined as Hb＜80g/L) in the First Affiliated Hospital, Zhejiang University School of Medicine were analyzed retrospectively in this study. Patients who were treated with Roxadustat complied the drug instructions. Roxadustat was administered three times a week (TIW) at a dose of 100 mg (in patients weighing 45 to 60 kg) or 120 mg (in patients weighing 60 kg) in hemodialysis patients, and 70 mg (in patients weighing 40 to 60 kg) or 100 mg (in patients weighing 60 kg) in patients not receiving hemodialysis. Mann-Whitney U, chi-square test and mixed-effect models for repeated measures (MMRM) were applied to the study. Results: Among a total of 98 MM patients with renal insufficiency (Scr≥177μmol/L) and severe anemia (Hb＜80g/L), 30 MM patients received Roxadustat treatment along with the anti-tumor treatment. The choice anti-tumor therapy was commonly consisted of combination therapy with a bortezomib, cyclophosphamide/thalidomide and dexamethasone or an equivalent regimen. 12 patients in Roxadustat group and 10 patients in control group (40.0% vs. 14.7%, P=0.006) underwent short-term or long-term hemodialysis at the start of the anti-tumor treatment. 25 patients in Roxadustat group and 59 patients in control group (83.3% vs. 86.8%, P=0.655) were newly-diagnosed MM (NDMM). The mean (standard deviation [SD]) baseline hemoglobin (Hb) levels for the Roxadustat and control groups were 62.73 (9.04) g/L and 68.03 (7.32) g/L, respectively. The mean (SD) baseline Scr levels for the Roxadustat and control groups were 441.93 (252.60) g/L and 342.53 (185.38) μmol/L, respectively. The median increase in Hb from baseline to week 4, week 8, week 12 was 5.50 versus 17.00 g/L (P＜0.001), 13.00 versus 30.50 g/L (P＜0.001), and 20.00 versus 41.00 g/L (P＜0.001) in patients from control group and Roxadustat group. Analyzing with mixed models, the interaction of time* Roxadustat was 6.77 g/L (95% CI=4.80-8.73, P＜0.001) in the whole cohort. The proportions of MM patients with Hb increases of ≥10 g/L, ≥20 g/L, and ≥30 g/L at week 12 were 39.7% (n=27) versus 70.0% (n=21) (P=0.006), 61.8% (n=42) versus 93.3% (n=28) (P=0.001), and 66.2% (n=45) versus 100% (n=30) (P＜0.001) in MM patients from control group and Roxadustat group. Roxadustat was well tolerated by MM patients with renal insufficiency and severe anemia. Metabolic acidosis (23.3% vs. 7.4%, P=0.026) and hyperkalemia (33.3% vs. 14.7%, P=0.035) occurred more frequently in the Roxadustat group than in control group. Although there was no significant difference, deep vein thrombosis occurred relatively more frequently in the Roxadustat group than in control group (16.7% vs. 7.4%, P=0.160). Conclusion: This is the first study exploring the efficacy of Roxadustat in multiple myeloma patients with renal insufficiency. According to this study, Roxadustat is well tolerated by MM patients with renal insufficiency and effectively improves their anemia conditions. Metabolic acidosis and hyperkalemia occurred more frequently in the Roxadustat group than in control group. The data indicated that Roxadustat could be proved to be a potential, effective therapy for anemia in MM patients with renal insufficiency.

No benefit of HIF prolyl hydroxylase inhibition for hypertensive renal damage in renovascular hypertensive rats.

Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified "preconditioning" approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

Hypoxia-inducible factor stabilisation-related lncRNAs in retinopathy of prematurity

Long non-coding RNAs (lncRNAs) play an important role in the response to many diseases. The previous study reported the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, retinopathy of prematurity (ROP) model) by hypoxia-inducible factor (HIF) stabilisation via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarateanalog dimethyloxalylglycine (DMOG). However, there is little understanding of how those genes are regulated. In the present study, 6918 known lncRNAs and 3654 novel lncRNAs were obtained, and a series of differentially expressed lncRNAs (DELncRNAs) were also identified. By cis- and trans-regulation analyses, the target genes of DELncRNAs were predicted. Functional analysis demonstrated that multiple genes were involved in the MAPK signalling pathway, adipocytokine signalling pathway was regulated by the DELncRNAs. By HIF-pathway analysis, two lncRNAs Gm12758 and Gm15283 were found that can regulate the HIF-pathway by targeting the Vegfa, Pgk1, Pfkl, Eno1, Eno1b and Aldoa genes. In conclusion, the present study provided a series of lncRNAs for further understanding and protecting the extremely premature infant from oxygen toxicity.

FC 073REGIONAL EFFICACY AND SAFETY RESULTS OF ROXADUSTAT COMPARED WITH PLACEBO OR DARBEPOETIN ALFA IN NON-DIALYSIS-DEPENDENT (NDD) CHRONIC KIDNEY DISEASE (CKD) PATIENTS WITH ANAEMIA

Abstract Background and Aims Roxadustat regulates erythropoiesis and iron metabolism through hypoxia-inducible factor prolyl hydroxylase inhibition. This regional analysis evaluated efficacy and safety for roxadustat versus placebo/darbepoetin alfa (DA) in NDD CKD patients with anaemia. Method Results from three, double-blind phase 3 studies comparing roxadustat to placebo (ALPS, ANDES, OLYMPUS) in patients with anaemia and stage 3-5 NDD CKD were pooled and evaluated alongside results of an open-label study comparing roxadustat to DA (DOLOMITES) in the same population. Efficacy outcomes were compared in three regions (Europe, US, and other) in placebo-controlled studies (PCS) and two regions (Western Europe/Israel and Central/Eastern Europe) in the DA-controlled study (DCS). The primary efficacy endpoint in Europe was haemoglobin (Hb) response, defined as Hb ≥11.0 g/dL that changed from baseline (CFB) by ≥1.0 g/dL in patients with Hb >8.0 g/dL or as CFB ≥2.0 g/dL in patients with Hb ≤8.0 g/dL. The secondary efficacy endpoint was Hb CFB to Weeks 28-36 without rescue therapy (intravenous iron, red blood cell [RBC] transfusion, or erythropoiesis-stimulating agent [ESA] for PCS; RBC transfusion or ESA [roxadustat only] for DCS). Incidence of treatment-emergent adverse events (TEAEs) was summarised descriptively in two regions in PCS (Europe and non-Europe) and two regions in DCS (Western Europe/Israel and Central/Eastern Europe). Results A total of 4886 patients were randomised (2709 roxadustat; 1884 placebo; 293 DA). A significantly greater proportion of patients in all regions who received roxadustat had a Hb response without rescue therapy vs placebo (Europe: 77.9% vs 16.5%, difference of proportion [DOP] 61.4%, 95% CI: 56.5-66.2; US: 75.4% vs 8.3%, DOP 67.2%, 95% CI: 62.7-71.6; and other: 83.4% vs 5.5%, DOP 78.0%, 95% CI: 75.4-80.5) and a numerically greater proportion in both regions vs DA (Western Europe/Israel: 93.9% vs 83.5%, DOP 10.4%, 95% CI: 1.2-19.6; Central/Eastern Europe: 86.5% vs 75.4%, DOP 10.9%, 95% CI: 3.6-18.3). Mean Hb CFB was significantly greater with roxadustat vs placebo in Europe (2.01 vs 0.37 g/dL, LSMD 1.70, 95% CI: 1.55-1.84), US (1.80 vs 0.21 g/dL, LSMD 1.67, 95% CI: 1.53-1.82), and other (1.80 vs 0.06 g/dL, LSMD 1.77, 95% CI: 1.64-1.89) (all P<0.0001) and similar vs DA in Western Europe/Israel (1.64 vs 1.75 g/dL, LSMD -0.03, 95% CI: -0.30 to 0.23, P=0.80) and Central/Eastern Europe (1.94 vs 1.88 g/dL, LSMD 0.03, 95% CI: -0.14 to 0.21, P=0.72). The range in incidence for select TEAEs (arteriovenous fistula thrombosis, deep vein thrombosis, nausea, and seizure) was: 0.3-8.4% for roxadustat and 0.2-4.3% for placebo in Europe; 0.8-10.8% for roxadustat and 0.1-7.0% for placebo in non-Europe; 0-12.1% for roxadustat and 0-11.8% for DA in Western Europe/Israel; 0.4-6.3% for roxadustat and 0-3.4% for DA in Central/Eastern Europe. Conclusion In patients with anaemia and stage 3-5 NDD CKD, roxadustat was more effective than placebo for correcting Hb and generating Hb response without rescue therapy in Europe, the US, and other countries. It was similarly effective as DA for correcting Hb and generating a Hb response without rescue therapy in Western Europe/Israel and Central/Eastern Europe. The incidence of select TEAEs was comparable and relatively low in all regions.

Neuroprotective Effect of HIF Prolyl Hydroxylase Inhibition in an In Vitro Hypoxia Model.

A novel potent analog of the branched tail oxyquinoline group of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, neuradapt, has been studied in two treatment regimes in an in vitro hypoxia model on murine primary hippocampal cultures. Neuradapt activates the expression of HIF1 and HIF2 target genes and shows no toxicity up to 20 μM, which is more than an order of magnitude higher than its biologically active concentration. Cell viability, functional activity, and network connectivity between the elements of neuronal networks have been studied using a pairwise correlation analysis of the intracellular calcium fluctuations in the individual cells. An immediate treatment with 1 μM and 15 μM neuradapt right at the onset of hypoxia not only protects from the death, but also maintains the spontaneous calcium activity in nervous cells at the level of the intact cultures. A similar neuroprotective effect in the post-treatment scenario is observed for 15 μM, but not for 1 μM neuradapt. Network connectivity is better preserved with immediate treatment using 1 μM neuradapt than with 15 μM, which is still beneficial. Post-treatment with neuradapt did not restore the network connectivity despite the observation that neuradapt significantly increased cell viability at 1 μM and functional activity at 15 μM. The preservation of cell viability and functional activity makes neuradapt promising for further studies in a post-treatment scenario, since it can be combined with other drugs and treatments restoring the network connectivity of functionally competent cells.

Rank Order of Small Molecule Induced Hypoxiamimesis to Prevent Retinopathy of Prematurity.

Here we rank order small molecule inhibitors of hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) using severity of oxygen induced retinopathy (OIR) as an outcome measure. Dose response analyses in cell cultures of hepatoma (Hep3B), retinal Müller cells (MIO-M1) and primary retinal endothelial cells were conducted to evaluate potency by comparing dose to HIF-1,2 protein levels by western blotting. In vivo dose response was determined using the luciferase-transgene HIF reporter (luc-ODD). Each compound was placed in rank order by their ability to reduce neovascularization and capillary drop out in the OIR mouse model. An Epas1 KO confined to retinal Müller cells was used to determine whether successful protection by HIF stabilization requires HIF-2. Two candidate small molecules can prevent OIR by stabilizing HIF-1 to prevent oxygen induced growth attenuation and vascular obliteration. Müller cell HIF-2, the mediator of pathologic retinal angiogenesis, is not required for protection. The lack of dependence on Müller cell HIF-2 predicts that inhibition of HIF PHD will not drive pathological angiogenesis.

Acidosis induces antimicrobial peptide expression and resistance to uropathogenic E. coli infection in kidney collecting duct cells via HIF-1α.

Acute pyelonephritis is frequently associated with metabolic acidosis. We previously reported that metabolic acidosis stimulates expression of hypoxia-inducible factor (HIF)-1α-induced target genes such as stromal derived factor-1 and cathelicidin, an antimicrobial peptide. Since the collecting duct (CD) plays a pivotal role in regulating acid-base homeostasis and is the first nephron segment encountered by an ascending microbial infection, we examined the contribution of HIF-1α to innate immune responses elicited by acid loading of an M-1 immortalized mouse CD cell line. Acid loading of confluent M-1 cells was achieved by culture in pH 6.8 medium supplemented with 5-(N-ethyl-N-isopropyl)-amiloride to block Na+/H+ exchange activity for 24 h. Acid loading induced antimicrobial peptide [cathelicidin and β-defensin (Defb2 and Defb26)] mRNA expression and M-1 cell resistance to uropathogenic Escherichia coli infection to an extent similar to that obtained by inhibition of HIF prolyl hydroxylases, which promote HIF-1α protein degradation. The effect of acid loading on M-1 cell resistance to uropathogenic E. coli infection was reduced by inhibition of HIF-1α (PX-478), and, in combination with prolyl hydroxylase inhibitors, acidosis did not confer additional resistance. Thus, metabolic stress of acidosis triggers HIF-1α-dependent innate immune responses in CD (M-1) cells. Whether pharmacological stabilization of HIF prevents or ameliorates pyelonephritis in vivo warrants further investigation.

Reactive Oxygen Species Formation in the Brain at Different Oxygen Levels: The Role of Hypoxia Inducible Factors.

Hypoxia inducible factor (HIF) is the master oxygen sensor within cells and is central to the regulation of cell responses to varying oxygen levels. HIF activation during hypoxia ensures optimum ATP production and cell integrity, and is associated both directly and indirectly with reactive oxygen species (ROS) formation. HIF activation can either reduce ROS formation by suppressing the function of mitochondrial tricarboxylic acid cycle (TCA cycle), or increase ROS formation via NADPH oxidase (NOX), a target gene of HIF pathway. ROS is an unavoidable consequence of aerobic metabolism. In normal conditions (i.e., physioxia), ROS is produced at minimal levels and acts as a signaling molecule subject to the dedicated balance between ROS production and scavenging. Changes in oxygen concentrations affect ROS formation. When ROS levels exceed defense mechanisms, ROS causes oxidative stress. Increased ROS levels can also be a contributing factor to HIF stabilization during hypoxia and reoxygenation. In this review, we systemically review HIF activation and ROS formation in the brain during hypoxia and hypoxia/reoxygenation. We will then explore the literature describing how changes in HIF levels might provide pharmacological targets for effective ischaemic stroke treatment. HIF accumulation in the brain via HIF prolyl hydroxylase (PHD) inhibition is proposed as an effective therapy for ischaemia stroke due to its antioxidation and anti-inflammatory properties in addition to HIF pro-survival signaling. PHD is a key regulator of HIF levels in cells. Pharmacological inhibition of PHD increases HIF levels in normoxia (i.e., at 20.9% O2 level). Preconditioning with HIF PHD inhibitors show a neuroprotective effect in both in vitro and in vivo ischaemia stroke models, but post-stroke treatment with PHD inhibitors remains debatable. HIF PHD inhibition during reperfusion can reduce ROS formation and activate a number of cellular survival pathways. Given agents targeting individual molecules in the ischaemic cascade (e.g., antioxidants) fail to be translated in the clinic setting, thus far, HIF pathway targeting and thereby impacting entire physiological networks is a promising drug target for reducing the adverse effects of ischaemic stroke.

Comparative systems pharmacology of HIF stabilization in the prevention of retinopathy of prematurity

Retinopathy of prematurity (ROP) causes 100,000 new cases of childhood blindness each year. ROP is initiated by oxygen supplementation necessary to prevent neonatal death. We used organ systems pharmacology to define the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, ROP model) by hypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarate analog dimethyloxalylglycine (DMOG). Although both molecules conferred a protective phenotype, gene expression analysis by RNA sequencing found that Roxadustat can prevent OIR by two pathways: direct retinal HIF stabilization and induction of aerobic glycolysis or indirect hepatic HIF-1 stabilization and increased serum angiokines. As predicted by pathway analysis, Roxadustat rescued the hepatic HIF-1 knockout mouse from retinal oxygen toxicity, whereas DMOG could not. The simplicity of systemic treatment that targets both the liver and the eye provides a rationale for protecting the severely premature infant from oxygen toxicity.

Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibition Attenuates Chronic Stress Induced Intestinal Barrier Dysfunction

This is a non-final version of an article published in final form in the journal Medicine and Science in Sports and Exercise, 47: 5(S) and can be found at: http://dx.doi.org/10.1249/01.mss.0000466118.29837.67

HIF prolyl hydroxylase inhibition prior to transient focal cerebral ischaemia is neuroprotective in mice.

This study investigated the effects of 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8- to 12-week-old C57/B6 mice were subjected to 45min of middle cerebral artery occlusion (MCAO) either immediately or 24h after receiving IOX3. Mice receiving IOX3 at 20mg/kg 24h prior to the MCAO had better neuroscores and smaller blood-brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up-regulated HIF-1α, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up-regulated HIF-1α and delocalized ZO-1. Pre-treating IOX3 on RBE4 cells 24h before oxygen-glucose deprivation had a protective effect on endothelial barrier preservation with ZO-1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia. We show that IOX3, a selective small molecule (280.66Da) HIF prolyl hydroxylase inhibitor, could up-regulate HIF-1α and increase erythropoietin expression in mice. We further demonstrate that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of blood-brain barrier (BBB) protection, while immediate application is detrimental both in vivo and in vitro. These findings provide new insights into the role of HIF stabilization in ischaemic stroke.

Prolyl hydroxylase inhibition during hyperoxia prevents oxygen-induced retinopathy in the rat 50/10 model.

To study the effect of systemic hypoxia-inducible factor prolyl hydroxylase inhibition (HIF PHDi) in the rat 50/10 oxygen-induced retinopathy (OIR) model. Oxygen-induced retinopathy was created with the rat 50/10 OIR model. OIR animals received intraperitoneal injections of dimethyloxalylglycine (DMOG, 200 μg/g), an antagonist of α-ketoglutarate cofactor and inhibitor for HIF PHD, on postnatal day (P)3, P5, and P7. Control animals received intraperitoneal injections of PBS. On P14 and P21, animals were humanely killed and the effect on vascular obliteration, tortuosity, and neovascularization quantified. To analyze HIF and erythropoietin, rats at P5 were injected with DMOG (200 μg/g). Western blot or ELISA measured the levels of HIF-1 and Epo protein. Epo mRNA was measured by quantitative PCR. Alternating hyperoxia and hypoxia in untreated rats led to peripheral vascular obliteration on day P14 and P21. Rats that were treated with systemic DMOG by intraperitoneal injections had 3 times less ischemia and greater peripheral vascularity (P = 0.001) than control animals treated with PBS injections. Neovascularization similarly decreased by a factor of 3 (P = 0.0002). Intraperitoneal DMOG administration increased the levels of HIF and Epo in the liver and brain. Serum Epo also increased 6-fold (P = 0.0016). Systemic DMOG had no adverse effect on growth of rats treated with oxygen. One of the many controversies in the study of retinopathy of prematurity is whether hyperoxia or alternating hyperoxia and hypoxia creates the disease phenotype in humans. We have previously demonstrated that PHDi prevents OIR in mice exposed to 5 days of sustained 75% oxygen followed by 5 days of 21% oxygen. The 50/10 rat experiments demonstrate that PHDi is also effective in a 24-hour alternating hyperoxia-hypoxia model. The rat OIR model further validates the therapeutic value of HIF PHDi to prevent retinopathy of prematurity because it reduces oxygen-induced vascular obliteration and retinovascular growth attenuation in prolonged and/or alternating hyperoxia.

Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibition: Robust New Target or Another Big Bust for Stroke Therapeutics?

A major challenge in developing stroke therapeutics that augment adaptive pathways to stress has been to identify targets that can activate compensatory programs without inducing or adding to the stress of injury. In this regard, hypoxia-inducible factor prolyl hydroxylases (HIF PHDs) are central gatekeepers of posttranscriptional and transcriptional adaptation to hypoxia, oxidative stress, and excitotoxicity. Indeed, some of the known salutary effects of putative 'antioxidant' iron chelators in ischemic and hemorrhagic stroke may derive from their abilities to inhibit this family of iron, 2-oxoglutarate, and oxygen-dependent enzymes. Evidence from a number of laboratories supports the notion that HIF PHD inhibition can improve histological and functional outcomes in ischemic and hemorrhagic stroke models. In this review, we discuss this evidence and highlight important gaps in our understanding that render HIF PHD inhibition a promising but not yet preclinically validated target for protection and repair after stroke.

Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

SummaryThe Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

HIF prolyl hydroxylase inhibitors for anemia

Introduction: The hypoxia-inducible factor (HIF) system mediates the body's response to hypoxia, locally, inducing angiogenesis and a shift to anaerobic metabolism, and systemically, increasing red cell mass in anemia. HIF prolyl hydroxylases (HIF-PH) modify HIF, decreasing its activity. Increasing HIF activity through inhibition of HIF-PH may provide an alternative treatment for anemia and may protect against damage related to ischemia–reperfusion.Areas covered: The review discusses the basic science underpinnings of the HIF system and the clinical effects of the HIF system and its pharmacologic manipulation.Expert opinion: Manipulation of the HIF system may improve outcomes in anemia by bypassing the effective iron deficiency found in anemia of chronic disease and by increasing red cell mass without supraphysiologic increases in erythropoietin. HIF-PH may also find a clinical use in the prevention of ischemia–reperfusion damage in strokes, cardiac ischemia, ischemic renal failure, etc.

HIF prolyl hydroxylase inhibition increases cell viability and potentiates dopamine release in dopaminergic cells

Hypoxia-inducible factor (HIF) controls the expression of genes that adapts the cellular condition to accommodate oxidative stress. The potential beneficial effect of HIF up-regulation in ischemia has recently gained interest substantiated by the known HIF-regulation of erythropoietin and other hypoxia accommodating genes. So far the perspectives for HIF up-regulation has been focused on anemia and ischemia related diseases but little information is available about the relevance of HIF biology for neurodegenerative disease like Parkinson's disease. We therefore sought out to characterize the effect of HIF-up-regulation on survival and dopamine homeostasis in dopaminergic cells. We used a low molecular weight HIF prolyl hydroxylase (HPH) inhibitor and lentiviral based shRNA knockdown of HPH subtypes as molecular tools to increase HIF protein level and downstream HIF-regulated genes. We show that HIF induction results in protection against oxidative stress in cellular models based on PC12 cells and LUHMES cells. In addition, HPH inhibition elevates tyrosine hydroxylase expression and activity, which causes increased dopamine synthesis and release in both PC12 cells and a primary rat ventral mesencephalic cell culture. All together these findings suggest that prolyl hydroxylases may represent novel targets for therapeutic intervention in disorders characterized by dopamine homeostasis dysregulation like Parkinson's disease.

HIF prolyl hydroxylase inhibition augments dopamine release in the rat brain in vivo

The transcription factor hypoxia-inducible factor (HIF) is essential for the activation of several genes that promote the survival of cells exposed to oxidative stress. Expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme in the dopamine (DA) synthesis, is one of the genes that are positively regulated by HIF. Accordingly, HIF induction results in elevated DA release in various cell lines in vitro. HIF prolyl hydroxylase (HPH) is critically involved in the negative regulation of HIF levels. We investigated the in vivo effects of the HPH inhibitor FG0041 on brain DA function in rats by microdialysis in freely moving rats, locomotor activity, and Western blot analysis. Administration of FG0041 (10 mg/kg i.p.), as an acute (single injection), or as subchronic (once daily for 6 days) treatment and cobalt chloride (CoCl2) (60 mg/kg s.c.) potentiated potassium (K+) induced increases in extracellular levels of DA levels in the rat striatum. The increase in extracellular DA of freely moving rats was sought in relationship to locomotor activity in rats. A significant increase in locomotor activity was observed in FG0041-treated rats compared with vehicle on a cocaine challenge. In support of these findings, protein levels of TH in the rat brain stem were increased after treatment with FG0041. These data indicate that FG0041 augments DA function in the rat brain. Inhibition of HPH enhances DA function by increasing DA release, which has implications for the use of HIF induction in the treatment of neurodegenerative diseases.

Hypoxia and gastrointestinal disease

The gastrointestinal mucosa is a richly perfused vascular bed directly juxtaposed with the anaerobic and nonsterile lumen of the gut. As such, intestinal epithelial cells, which line the mucosa, experience a uniquely steep physiologic oxygen gradient in comparison with other cells of the body. Inflammation associated with a loss of epithelial barrier function and unregulated exposure of the mucosal immune system to luminal antigens leads to inflammatory bowel disease (IBD), a relatively common disorder with severe morbidity and a limited therapeutic repertoire. During IBD, increased tissue metabolism and vasculitis renders the chronically inflamed mucosa and particularly the epithelium hypoxic, giving rise to the activation of the hypoxia-responsive transcription factor hypoxia-inducible factor (HIF). Recent studies utilizing conditional intestinal epithelial hif1a-null mice have revealed a protective role for epithelial HIF-1alpha in murine models of IBD. Such protection occurs, at least in part, through HIF-dependent induction of barrier-protective genes in the epithelium. More recently, studies employing pharmacologic activation of HIF via inhibition of HIF prolyl hydroxylases revealed a profoundly protective effect of these agents in murine models of colitis. In this paper, we review this pathway in detail and examine the therapeutic potential for targeting HIF hydroxylases in intestinal mucosal inflammatory disease.

Mucosal Protection by Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibition

A number of recent studies have implicated tissue hypoxia in both acute and chronic inflammatory diseases, particularly as they relate to mucosal surfaces lined by epithelial cells. In this context, a protective role for the transcriptional regulator hypoxia-inducible factor (HIF) was shown through conditional deletion of epithelial HIF-1alpha in a murine model of colitis. Here, we hypothesized that pharmacologic activation of HIF would similarly provide a protective adaptation to murine colitic disease. For these purposes, we used a novel prolyl hydroxylase (PHD) inhibitor (FG-4497) that readily stabilizes HIF-1alpha and subsequently drives the expression downstream of HIF target genes (eg, erythropoietin). Our results show that the FG-4497-mediated induction of HIF-1alpha provides an overall beneficial influence on clinical symptoms [weight loss, colon length, tissue tumor necrosis factor-alpha (TNFalpha)] in murine trinitrobenzene sulfonic acid (TNBS) colitis, most likely because of their barrier protective function and wound healing during severe tissue hypoxia at the site of inflammation. Taken together these findings emphasize the role of epithelial HIF-1alpha during inflammatory diseases in the colon and may provide the basis for a therapeutic use of PHD inhibitors in inflammatory mucosal disease.