fg-4592 and Hypoxia

Hypoxia-inducible factor (HIF) is an oxygen-sensitive dimeric transcription factor that responds to pathophysiologically low O2 tensions via up-regulation, which leads to an orchestrated biological response to hypoxia. The HIF prolyl hydroxylase domain (PHD) enzymes are non-heme, iron-containing dioxygenases requiring for activity both molecular oxygen and 2-oxoglutarate that, under normoxia, selectively hydroxylate proline residues of HIF, initiating proteosomal degradation of the latter. The dependence of HIF protein levels on the concentration of O2 present, mediated by the PHD enzymes, forms the basis for one of the most significant biological sensor systems of tissue oxygenation in response to ischemic and inflammatory events. Consequently, pharmacological inhibition of PHD enzymes, leading to stabilization of HIF, may be of considerable therapeutic potential in treating conditions of tissue stress and injury. This Perspective reviews the PHDs and small molecule drug discovery efforts. A critical view of this challenging field is offered, which addresses potential concerns and highlights exciting possibilities for the future.

Topics: Animals; Clinical Trials as Topic; Drug Discovery; Enzyme Inhibitors; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Mice; Mice, Knockout; Protein Structure, Tertiary; Von Hippel-Lindau Tumor Suppressor Protein; Wound Healing

Roxadustat (FG-4592), an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis, was evaluated in a phase 1b study in patients with end-stage renal disease with anemia on hemodialysis. Seventeen patients, on epoetin-alfa maintenance therapy with stable hemoglobin levels ≥10 g/dL, had epoetin-alfa discontinued on day 3 and were enrolled in this double-blind placebo-controlled study. Two cohorts were randomized 3:1 (roxadustat: placebo). Patients received single doses of roxadustat (1 or 2 mg/kg) or placebo 1 hour after hemodialysis on day 1 and 2 hours before dialysis on day 8. Maximum plasma concentration and area under the plasma concentration-time curve for patients receiving roxadustat were slightly more than dose proportional and elimination half-life ranged from 14.7 to 19.4 hours. Roxadustat was highly protein bound (99%) in plasma, and dialysis contributed a small fraction of the total clearance: only 4.56% and 3.04% of roxadustat recovered from the 1 and 2 mg/kg dose groups, respectively. Roxadustat induced transient elevations of endogenous erythropoietin that peaked between 7 and 14 hours after dosing and returned to baseline by 48 hours after dosing. Peak median endogenous erythropoietin levels were 96 mIU/mL and 268 mIU/mL for the 1- and 2-mg/kg doses, respectively, within physiologic range of endogenous erythropoietin responses to hypoxia at high altitude or after blood loss. No serious adverse events were reported, and there were no treatment- or dose-related trends in adverse event incidence.

Topics: Administration, Oral; Adult; Aged; Anemia; Area Under Curve; Dose-Response Relationship, Drug; Double-Blind Method; Erythropoiesis; Erythropoietin; Female; Glycine; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Isoquinolines; Kidney Failure, Chronic; Male; Middle Aged; Prolyl-Hydroxylase Inhibitors; Renal Dialysis; Treatment Outcome

Hypoxia-inducible factor (HIF) activators aid the treatment of renal anemia and ischemia. Recently, PyrzA (5-(1-acetyl-5-phenylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid), a HIF activator by PHD inhibition without a 2-oxoglutarate moiety was reported. However, PyrzA has low lipophilicity, and it was necessary to improve its solubility by synthesizing derivatives. In this study, we synthesized and evaluated a higher lipophilic derivative of PyrzA and found that it exhibited higher HIF activity and stabilizing ability at low concentrations compared to Roxadustat, a commercially available HIF activator.

Topics: Barbiturates; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Ketoglutaric Acids

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Topics: Animals; Erythropoietin; Female; Glycine; Hypoxia; Isoquinolines; Kidney; Male; Prolyl-Hydroxylase Inhibitors; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Up-Regulation

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1

Topics: Anemia; Animals; Erythropoietin; Glycine; Hypoxia; Isoquinolines; Kidney; Mice; Procollagen-Proline Dioxygenase; Prolyl Hydroxylases

Patients with glioblastoma (GBM) have poor prognosis and limited treatment options, largely due to therapy resistance upon the induction of apoptosis. Ferroptosis emerges as a potential antineoplastic strategy to bypass apoptosis resistance in traditional therapeutics. Hypoxia is a fundamental hallmark of GBM and hypoxia-inducible factor (HIF) is the main regulator of hypoxia response, however, the role of HIF has not been sufficiently explored in GBM. Herein, we first discovered that amplifying HIF signals by the prolyl hydroxylase (PHD) inhibitor roxadustat significantly suppressed GBM cell growth in vitro and in vivo, especially when the cells were resistant to temozolomide (TMZ). The accumulation of lipid peroxidation and cellular iron in GBM cells following roxadustat treatment indicated that the cells underwent ferroptosis, which was also supported by morphological changes in mitochondrial ultrastructure and immunogenic signals release. Moreover, in vivo studies further confirmed the ferroptosis induction and verified that roxadustat significantly prolonged survival of the mice harboring chemoresistant GBM without visible organ toxicity. Finally, we proved that the ferroptosis induction by roxadustat is HIF-α independent, especially activation of HIF-2α upregulating lipid regulatory genes was revealed to be mainly responsible for the enhanced lipid peroxidation. Altogether, our study provided novel evidence that amplifying HIF signals induced ferroptosis in chemoresistant GBM cells and suppressed the tumor growth in vivo, highlighting that ferroptosis induction by targeting HIF-α might provide new approaches to improve GBM treatment.

Topics: Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Ferroptosis; Glioblastoma; Glycine; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Isoquinolines; Lipids; Mice; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Temozolomide

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells.

Topics: Age Factors; AMP-Activated Protein Kinases; Animals; Antibiotics, Antineoplastic; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Doxorubicin; Glycine; Humans; Hydroxybenzoates; Hypoxia; Inflammation Mediators; Isoquinolines; Mice, Inbred C57BL; Muscle Strength; NF-kappa B; Paracrine Communication; Phenotype; Signal Transduction; TOR Serine-Threonine Kinases

Topics: Animals; Glycine; Hypoxia; Isoquinolines; Kidney; Nitric Oxide; Perfusion; Pharmaceutical Preparations; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Pyrazoles; Rats; Triazoles

Topics: Animals; Cation Transport Proteins; Glycine; Hep G2 Cells; Homeostasis; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Liver; Manganese; Mice; Mice, Inbred C57BL; Mutation; Neurotoxicity Syndromes

HIF prolyl hydroxylase 2 (PHD2) inhibitors represent a novel approach for treating HIF-related diseases. This study reports the first application of photoremovable protecting group to the photoactivatable inhibitor (

Topics: Cell Line, Tumor; Enzyme Stability; Erythropoietin; Gene Expression Regulation; HEK293 Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Light; Models, Chemical; Molecular Structure; Prolyl-Hydroxylase Inhibitors

To determine whether hypoxia preconditioning can protect corneal endothelial cells from mechanical stress and perioperative procedures mimicking Descemet stripping automated endothelial keratoplasty (DSAEK).. Preconditioning was delivered by 2 hours of 0.5% oxygen incubation in a hypoxia chamber or by exposure to the prolyl hydroxylase inhibitor FG-4592, which prevents hypoxia-inducible factor-1 alpha degradation. Damage to whole corneas was produced by brief sonication. To mimic use with DSAEK, FG-4592-preconditioned and control donor corneas were dissected with a microkeratome, and the posterior donor button was pulled through a transplant insertion device (Busin glide). The area of endothelial damage was determined by trypan blue staining.. In all cases, hypoxia preconditioning or incubation with FG-4592 protected corneal endothelial cells from death by mechanical stress. Hypoxia-preconditioned human and rabbit corneas showed 19% and 29% less cell loss, respectively, relative to controls, which were both significant at P < 0.05. FG-4592 preconditioning reduced endothelial cell loss associated with preparation and insertion of DSAEK grafts by 23% relative to the control (P < 0.01).. These results support the hypothesis that preconditioning by hypoxia or exposure to FG-4592 improves corneal endothelial cell survival and may also provide protection during surgical trauma.

Topics: Animals; Cell Line; Cell Survival; Corneal Endothelial Cell Loss; Cytoprotection; Descemet Stripping Endothelial Keratoplasty; Endothelium, Corneal; Glycine; Humans; Hypoxia; Ischemic Preconditioning; Isoquinolines; Oxygen; Perioperative Care; Pilot Projects; Prolyl-Hydroxylase Inhibitors; Rabbits; Stress, Mechanical

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions.

Topics: Animals; Biomarkers; Cell Transdifferentiation; Cells, Cultured; Disease Models, Animal; Glycine; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Isoquinolines; Male; Muscle, Smooth, Vascular; Phosphates; Rats; Rats, Wistar; Renal Insufficiency, Chronic; Signal Transduction; Vascular Calcification; Vascular Stiffness

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly heterogeneous in terms of microvasculature and thus oxygen concentration. The importance of hypoxia and HIFs in the hematopoietic environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoiesis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in hemodialysis patients. Pharmacological stabilization of HIF-1α protein with PHD inhibitors is also emerging as an important regulator of HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the bone marrow in vivo, thereby protecting them from high doses of irradiation and accelerating hematological recovery. Recent findings also show that stabilization of HIF-1α increases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerixafor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation. These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology.

Topics: Amino Acids, Dicarboxylic; Anemia; Apoptosis Regulatory Proteins; Aryl Hydrocarbon Receptor Nuclear Translocator; Basic Helix-Loop-Helix Transcription Factors; Glycine; Hematology; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Repressor Proteins; Signal Transduction; Stem Cell Transplantation