HIF-PH/hypoxia-inducible factor prolyl hydroxylase

Vadadustat improves iron mobilization and stimulates the body's natural production of erythropoietin. Vadadustat raises reticulocytes, plasma EPO, and Hb levels in a dose-dependent manner and is well tolerated in both healthy volunteers and patients with chronic renal disease. The vadadustat-induced increases in plasma EPO levels followed a typical diurnal pattern, peaking before the subsequent dose and being similar in size to physiological increases at moderate elevations. By raising transferrin levels and decreasing hepcidin, vadadustat promotes iron homeostasis. When compared to conventional ESAs, once-daily oral vadadustat that has been titrated to raise and maintain Hb within the target range may offer a number of benefits [1]. It has been noted that Vadadustat has a half-life of roughly 4.5 hours. The mean hemoglobin levels of the patients rose from 9.91 g/dL at baseline to 10.54 g/dL on day 29. From 334.1 ng/mL at baseline to 271.7 ng/mL on day 29, ferritin levels dropped [2].

Current treatment of anemia in chronic kidney disease (CKD) with erythropoiesis-stimulating agents can lead to substantial hemoglobin oscillations above target range and high levels of circulating erythropoietin. Vadadustat (AKB-6548), a novel, titratable, oral hypoxia-inducible factor prolyl hydroxylase inhibitor induces endogenous erythropoietin synthesis and enhances iron mobilization. In this 20-week, double-blind, randomized, placebo-controlled, phase 2b study, we evaluated the efficacy and safety of once-daily vadadustat in patients with stages 3a to 5 non-dialysis-dependent CKD. The primary endpoint was the percentage of patients who, during the last 2 weeks of treatment, achieved or maintained either a mean hemoglobin level of 11.0 g/dl or more or a mean increase in hemoglobin of 1.2 g/dl or more over the predose average. Significantly, the primary endpoint was met in 54.9% of patients on vadadustat and 10.3% of patients on placebo. Significant increases in both reticulocytes and total iron-binding capacity and significant decreases in both serum hepcidin and ferritin levels were observed in patients on vadadustat compared with placebo. The overall incidence of adverse events was comparable between the 2 groups. Serious adverse events occurred in 23.9% and 15.3% of the vadadustat- and placebo-treated patients, respectively. Three deaths occurred in the vadadustat arm. Thus, this phase 2b study demonstrated that vadadustat raised and maintained hemoglobin levels in a predictable and controlled manner while enhancing iron mobilization in patients with nondialysis-dependent CKD[1].

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Wild type and ERFE knockout mice with and without CKD were treated with vadadustat or vehicle. In both wild type and ERFE knockout CKD models, vadadustat was similarly effective, as evidenced by normalized hemoglobin concentrations, increased expression of duodenal iron transporters, lower serum hepcidin levels, and decreased tissue iron concentrations. This is consistent with ERFE-independent increased iron mobilization. Vadadustat treatment also lowered serum urea nitrogen and creatinine concentrations and decreased expression of kidney fibrosis markers. Lastly, vadadustat affected fibroblast growth factor 23 (FGF23) profiles: in non-CKD mice, vadadustat increased plasma total FGF23 out of proportion to intact FGF23, consistent with the known effects of hypoxia-inducible factor-1α and erythropoietin on FGF23 production and metabolism. However, in the mice with CKD, vadadustat markedly decreased both total and intact FGF23, effects likely contributed to by the reduced loss of kidney function. Thus, in this CKD model, vadadustat ameliorated anemia independently of ERFE, improved kidney parameters, and decreased FGF23. How vadadustat affects CKD progression in humans warrants future studies.[3]

Analysis of BM-MSCs Cytokine Secretion by Antibody Array Proteome Profiler[4]
The relative changes in secretory activity of Vadadustat treated BM-MSCs compared to control cells were examined using the Proteome Profiler Human XL Cytokine Array. The Proteome Profiler membrane-based antibody array enables to simultaneously measure the relative level of 102 human cytokines in a single sample. For the purpose of this assay, BM-MSCs from 6 donors were grown in a standard growth medium on a 6-well plates until approximately 80% confluency was achieved. 24 h before the start of the experiment, all cells were primed with IFNγ (25 ng/mL). Next day, cells were washed and culture medium was replaced with OptiMEM Medium, no phenol red with reduced FBS content to 4% and supplemented with 1.0% penicillin–streptomycin with/without Vadadustat 40 μM. Cells of each population were treated in triplicate. After 24 h treatment cells supernatants were collected in an Eppendorf tube (1.5 mL), centrifuged at 4500 rpm for 5 min, transferred to new tubes, mixed and divided into 200 µL aliquots and frozen in −80 °C. Prior to the analysis, cell supernatants from 6 donors were thawed on ice and pooled. The analysis was performed according to the manufacturer’s instruction. Chemiluminescence of membranes was detected with ChemiDoc MP Imaging System and the integrated optical density of each spot was measured and quantified using Image Lab software.

Preconditioning of Human BM-MSCs with Vadadustat[4]
In the presented study “pharmacological” hypoxia was achieved by culturing cells with the selective PHDs inhibitor, Vadadustat (AKB-6548). Based on preliminary data (Western blot analysis of HIF-1α stabilization and the MTT test) we decided to select the Vadadustat concentration of 40 μM for further studies. Vadadustat was dissolved and stored in −80 °C as 5 mM stock solution in DMSO according to the manufacturer instruction. Notably, no more than 0.8% (v/v) of DMSO was finally present in the culture medium, which did not cause any noticeable cytotoxic effect (MTT analysis presented in Supplementary Figure S1). The control group of MSCs was incubated with the same dose (0.8% v/v) of DMSO alone.

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Mixed Lymphocyte Reaction (MLR) Assay[4]
For the purpose of MLR assay human PBMCs were isolated from buffy coats from 6 healthy blood donors. The assay was performed in three independent sets of experiments on two donors each. Supernatants from 6 populations of IFNγ (25 ng/mL) primed BM-MSCs treated for 24 h with/without Vadadustat 40 μM were used to determine the effect of Vadadustat pretreatment on immunomodulatory activity of MSCs secretome. In this study, half of the isolated PBMCs were inactivated for 90 min with γ-irradiation. Next, 1 × 105 both responder (active) and irradiated (stimulatory) PBMCs were seeded into wells of 96-well plates in a combination of auto- (AAir, BBir) and allo- (ABir, BAir) stimulation. Cells were maintained in RPMI-1640 supplemented with 10% FBS and antibiotic–antimycotic solution (1% penicillin-streptomycin; 0.5% amphotericin B). The MLR assay were performed using 96-well plates. In the part of the wells where the direct effect of Vadadustat on auto- and allostimulated PBMCs as well as its effect on the interaction between MSCs and PBMCs were studied, 40 μM Vadadustat was added to the experimental wells daily as a stock solution. Control wells were treated daily with equivalent volumes of DMSO. In the remaining wells, in which the indirect effect of Vadadustat pre-conditioning on the interaction between MSCs and PBMCs was studied, a 1:1 mixture of RPMI-1640 growth medium and supernatants from 24 h cell culture of control or Vadadustat preconditioned MSCs was added once at the beginning of the experiment. Plates were then cultured for 5 days at 37 °C in a humidified atmosphere with 5% CO2. After 5 days of cell culture, PBMCs were pulsed with 1 μCi/well of 3H-thymidine (113 Ci/nmol, NEN) for the last 18 h of incubation and then harvested with an automated cell harvester. The 3H-thymidine incorporation into cells was measured based on the level of radioactivity reported as ‘Corrected Counts per Minute’ (CCPM) using a scintillation counter. All treatments were performed in triplicate.

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Transwell Migration Assay[4]
The effect of 40 μM Vadadustat preconditioning on the chemotactic properties of the BM-MSCs secretome was investigated using a “96 Well Cell Migration Assay” reagent kit from Cultrex®, which utilize a simplified design of a Boyden chamber with polyethylene terephthalate (PET) membrane with pores of 8 μm size. For the migration test, we used monocyte-enriched PBMCs (n = 4) suspended in RPMI containing 0.5% human serum at a density of 4 × 106/mL. 50 μL of cell suspension from each donor were applied to the upper chambers of the plate (2 × 104 cells per well), each in duplicate. Quantities of 150 µL per well of growth medium (RPMI with 0.5% human serum) or freshly thawed, pooled supernatants from cultures of 7 MSCs populations were applied to the bottom chambers of the plate. Each of three treatments: growth medium alone, supernatants from control MSCs and MSCs preconditioned with Vadadustat was applied in duplicate. Plates were then incubated under standard conditions (37 °C, 5% CO2) for 48 h. After incubation, the upper chambers were carefully aspirated and the cells that migrated to the bottom compartments of the plate were detached using a cell dissociation solution with calcein acetomethylester (calcein-AM). Afterwards, plates were incubated at 37 °C for 30 min. During this time, cells internalized calcein-AM, and cellular esterases then cleaved it into free calcein. Released calcein possess strong fluorescence, that was used to estimate the number of migrated cells. After incubation, plates were disassembled and bottom chambers were fluorescently read at 485 nm excitation and 520 nm emission on Perkin Elmer Victor X4 plate reader. The degree of cell migration was assessed by comparing fluorescence in the wells with MSCs culture supernatants to fluorescence in wells with growth medium alone, and expressed as the ratio of migrating cells.

Full methods are detailed in the Supplementary Material. Six-week-old male EKO mice and WT littermates were placed on 8-week diets that did or did not contain 0.2% adenine, which induces CKD. To assess the treatment effects of vadadustat in the setting of CKD, for the last 3 weeks of the diets, the mice were treated daily with vadadustat solution, dosed at 75 mg/kg/d via oral gavage, or vehicle solution. Mice were killed at 14 weeks of age. We obtained whole blood, serum, plasma, liver, spleen,...[3]

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Effects of vadadustat on erythropoietic parameters in WT and EKO mice with and without CKD...[3]
To assess the efficacy of vadadustat in the presence or absence of ERFE and CKD, we evaluated the effects of 3 weeks of daily vadadustat treatment in WT and EKO mice with and without adenine diet-induced CKD. In the non-CKD model, in which the mice were not anemic, vadadustat induced similar increases in hemoglobin concentrations from pretreatment values in the WT and EKO groups (Supplementary Figure S1). In the CKD model, as expected, both WT and EKO groups developed moderate anemia...[3]

Absorption, Distribution and Excretion
Following single and repeated doses, vadadustat is rapidly absorbed. The Tmax of vadadustat ranges between 2 and 3 hr. No significant accumulation was detected in healthy subjects given repeated doses of vadadustat. Compared to fasted conditions, the administration of a 450 mg vadadustat tablet with a standard high-fat meal decreased the Cmax and AUC by 27% and 6%, respectively. Vadadustat may be taken with or without food. The mean blood-to-plasma ratio of vadadustat went from 0.50 to 0.55, suggesting that the sequestration of vadadustat into red blood cells is minimal.
In healthy subjects given a single dose of 650 mg of radiolabelled vadadustat, 85.9% of the dose was recovered, with 58.9% appearing in urine and 26.9% in feces. Small amounts of the unchanged form of vadadustat are excreted in urine and feces (<1% and 9%, respectively).
The apparent volume of distribution of vadadustat in patients with chronic kidney disease is 11.6 L.
In healthy subjects given a single dose of 300 mg of vadadustat, the apparent total body clearance ranged from 1.7 L/h to 1.9 L/h. In patients with chronic kidney disease, the clearance of vadadustat is 0.8 L/h.

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Metabolism / Metabolites
Vadadustat is mainly metabolized by UDP-glucuronosyltransferase (UGT) enzymes, forming O-glucuronide conjugates via direct glucuronidation. The metabolism of vadadustat via cytochrome P450s (CYPs) is minimal. The major metabolite of vadadustat is vadadustat-O-glucuronide, which has 15% of the AUC of plasma radioactivity, and it is catalyzed by multiple UGT enzymes, including UGT1A1, UGT1A7, UGT1A8 and UGT1A9. Vadadustat acyl glucuronide is a minor metabolite with 0.047% of the total radioactivity in plasma. None of the vadadustat metabolites are active.

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Biological Half-Life
In patients with dialysis-dependent chronic kidney disease, the half-life of vadadustat was 9.2 hours.

Protein Binding
The protein binding of vadadustat is greater than or equal to 99.5% in human plasma.

[1]. Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in nondialysis-dependent chronic kidney disease. Kidney Int. 2016 Nov;90(5):1115-1122.

[2]. Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors: A Potential New Treatment for Anemia in Patients With CKD. Am J Kidney Dis. 2017 Feb 24. pii: S0272-6386(17)30110-5.

[3]. Amelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone. Kidney Int. 2021 Jul;100(1):79-89.

[4]. Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells. Cells. 2020 Nov 1;9(11):2396..

One of the most common symptoms of advanced renal disease is anemia, caused primarily by the inability of the kidney to respond to anemic conditions with a corresponding increase in [erythropoietin] (EPO) production. The treatment of anemia associated with chronic kidney disease (CKD) has traditionally involved the administration of exogenous erythropoiesis-stimulating agents (ESAs), such as [darbepoetin alfa], to counter the decrease in endogenous EPO production. While efficacious, the overuse of ESAs has been associated with cardiovascular complications, progression of CKD, and increases in overall mortality. A relatively new and alternative treatment option for patients with anemia associated with CKD is the use of small molecule inhibitors of hypoxia-inducible factor prolyl-hydroxylase (HIF-PH). These agents inhibit prolyl-hydroxylase domain oxygen sensors, mimicking hypoxic conditions and activating hypoxia-inducible factors. These transcription factors serve a multitude of roles, including the stimulation of erythropoiesis. Vadadustat is an orally administered inhibitor of HIF-PH with a safety and efficacy profile non-inferior to [darbepoetin alfa] for the treatment of anemia in patients with CKD undergoing dialysis. It was first approved in Japan, and in April 2023, it was approved by the EMA for the treatment of symptomatic anemia associated with CKD in adults on chronic maintenance dialysis. Vadadustat is currently awaiting a regulatory decision by the FDA. Vadadustat did not meet the prespecified noninferiority criterion for cardiovascular safety in patients with non-dialysis-dependent CKD.
Vadadustat is an orally bioavailable, hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor (HIF-PHI), with potential anti-anemic and anti-inflammatory activities. Upon administration, vadadustat binds to and inhibits HIF-PH, an enzyme responsible for the degradation of transcription factors in the HIF family under normal oxygen conditions. This prevents HIF breakdown and promotes HIF activity. Increased HIF activity leads to an increase in endogenous erythropoietin production, thereby enhancing erythropoiesis. It also reduces the expression of the peptide hormone hepcidin, improves iron availability, and boosts hemoglobin (Hb) levels. HIF regulates the expression of genes in response to reduced oxygen levels, including genes required for erythropoiesis and iron metabolism. In addition, HIF 1-alpha (HIF1A) may play a role in reducing inflammation during acute lung injury (ALI) through HIF-dependent control of glucose metabolism in the alveolar epithelium.

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Drug Indication
Vadadustat is indicated for the treatment of symptomatic anemia associated with chronic kidney disease (CKD) in adults on chronic maintenance dialysis.
Vafseo is indicated for the treatment of symptomatic anaemia associated with chronic kidney disease (CKD) in adults on chronic maintenance dialysis.
Treatment of anaemia due to chronic disorders

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Mechanism of Action
Hypoxia-inducible factors (HIFs) are transcription factors responsible for cellular survival under hypoxic conditions. They regulate a number of processes including angiogenesis, cell growth and differentiation, various metabolic processes, and erythropoiesis. Under normoxic conditions, HIFs are degraded via hydroxylation by prolyl-hydroxylase dioxygenases. Vadadustat is an inhibitor of HIF-prolyl-hydroxylases (HIF-PHI), that facilitates increased HIF activity in the absence of hypoxic conditions. The increased levels of HIF prompted by vadadustat stimulate endogenous erythropoietin production, increasing iron mobilization and contributing to the gradual rise of hemoglobin levels and the correction of iron metabolism. In patients with anemia of chronic kidney disease, in whom normal erythropoiesis is dysfunctional, this leads to the correction of anemia.

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Pharmacodynamics
The use of vadadustat was compared to darbepoetin alfa for the treatment of anemia in adult patients with dialysis-dependent chronic kidney disease. Vadadustat was non-inferior to darbepoetin alfa and met the primary hemoglobin level endpoint. In healthy subjects given 600 mg to 1200 mg of vadadustat, the use of this drug was not associated with clinically significant QTc prolongation. Compared to darbepoetin alfa, patients with dialysis-dependent chronic kidney disease treated with vadadustat have similar risks for death, myocardial infarction and stroke. The use of vadadustat may also lead to the development of thromboembolic events, hepatic impairment, hepatotoxicity, convulsions, as well as an increase in blood pressure.

C14H11CLN2O4

306.7011

306.041

C, 54.83; H, 3.62; Cl, 11.56; N, 9.13; O, 20.87

1000025-07-9

23634441

White to off-white solid powder

2.312

3

5

4

21

393

0

JGRXMPYUTJLTKT-UHFFFAOYSA-N

InChI=1S/C14H11ClN2O4/c15-10-3-1-2-8(4-10)9-5-11(18)13(16-6-9)14(21)17-7-12(19)20/h1-6,18H,7H2,(H,17,21)(H,19,20)

(5-(3-chlorophenyl)-3-hydroxypicolinoyl)glycine

AKB-6548; PG1016548; B-506; AKB 6548; AKB-6548; Vafseo; PG-1016548; Vadadustat [USAN]; B506; PG1016548; PG 1016548; B506; AKB6548; PG-1016548;Vadadustat

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~326.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)