| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| Other Sizes |
| Targets |
Pyruvate kinase R (PKR).
|
|---|---|
| ln Vitro |
Tebapivat (Compound 385) activates wild-type PKR, PKR K410E, or PKR 510Q when the AC50 is less than 0.3 μM [1]. An autosomal recessive mutation in the PKLR gene results in a deficiency of the pyruvate kinase R (PKR) enzyme, which causes pyruvate kinase deficiency (PKD), a condition affecting red blood cells. The following conditions and disorders may benefit from the use of PKR activators: PKD, thalassemia, hereditary elliptocytosis, anemia (e.g., congenital anemia (e.g., enzymopathy)), hemolytic anemia (e.g., hereditary and/or congenital hemolytic anemia, acquired hemolytic anemia), chronic hemolytic anemia caused by phosphoglycerate kinase deficiency, anemia of chronic disease, non-spherocytic hemolytic anemia or hereditary spherocytosis) [1].
Tebapivat (Compound 385) is a potent activator of wild-type pyruvate kinase R (PKR), as well as the mutant PKR variants K410E and 510Q, with AC50 values less than 0.3 uM (300 nM) for all three forms. The compound directly binds to PKR and stabilizes its active conformation, increasing the enzyme's affinity for its substrate phosphoenolpyruvate (PEP). This activation enhances the enzymatic activity of PKR, thereby increasing the flux of glycolysis and the production of ATP in red blood cells. By restoring ATP levels and reducing the levels of 2,3-diphosphoglycerate (2,3-DPG, an upstream metabolite that accumulates when PKR is deficient), tebapivat improves RBC health and survival. The compound has been evaluated in vitro using RBCs from PKD patients. In these cells, treatment with tebapivat increased the PKR activity, raised ATP levels, and normalized the elevated 2,3-DPG levels. It also improved RBC deformability and reduced the percentage of dense red blood cells, which are hallmarks of the disease. These effects were observed in RBCs from patients with both homozygous and compound heterozygous mutations in the PKLR gene, demonstrating the broad potential of the compound across different genetic backgrounds. |
| ln Vivo |
Tebapivat has been evaluated in vivo in a mouse model of pyruvate kinase deficiency (PKD). In the PKD mouse model (which harbors the Pk-1⁷ᵖᵉ mutant allele), oral administration of tebapivat resulted in a dose-dependent improvement in anemia. Treated mice showed increased hematocrit (HCT, the percentage of red blood cells in the blood), increased hemoglobin (HGB) levels, and reduced reticulocyte counts (a marker of compensatory RBC production). The compound also reduced the levels of bilirubin (a marker of hemolysis) and normalized the size and shape of red blood cells. These effects were sustained over the 2-4 week treatment period, demonstrating proof-of-concept that pharmacological activation of PKR can ameliorate the hemolytic anemia associated with PKD. Based on these positive results, tebapivat has advanced into clinical trials. The compound is also being investigated for its potential to treat other anemias, such as sickle cell disease and beta-thalassemia.
|
| Enzyme Assay |
The in vitro activity of tebapivat as a PKR activator is determined using an enzyme kinetic assay. Recombinant wild-type or mutant human PKR enzyme is incubated in a reaction buffer (e.g., 50 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.1 mM DTT, 0.01% BSA, and 0.01% Tween-20) with varying concentrations of tebapivat (typically from 0.1 nM to 100 uM). The reaction is initiated by the addition of the substrates, phosphoenolpyruvate (PEP) and ADP (in excess). The amount of pyruvate produced is measured in a coupled assay using lactate dehydrogenase (LDH) and NADH. The LDH converts pyruvate to lactate while oxidizing NADH to NAD+. The decrease in absorbance at 340 nm (due to NADH oxidation) is measured over time using a spectrophotometer. The initial reaction rate (V0) is calculated. The AC50 (the concentration required to reach 50% of the maximal activation) is then calculated by fitting the data to a sigmoidal dose-response curve. For a more detailed analysis, a steady-state kinetic analysis is performed. In this analysis, the enzyme is incubated at a fixed concentration of tebapivat (e.g., at the AC90 concentration) and with varying concentrations of PEP (while ADP is saturating). The kinetic parameters (Vmax and Km for PEP) are determined. PKR activators like tebapivat typically increase the Vmax of the enzyme without significantly affecting the Km for PEP, indicating that they are non-essential activators.
|
| Cell Assay |
The cellular activity of tebapivat is assessed in human red blood cells (RBCs) isolated from whole blood of healthy donors or, more importantly, from patients with pyruvate kinase deficiency (PKD). Freshly drawn blood is collected into heparinized tubes, and RBCs are isolated by centrifugation and washed three times with PBS. The RBCs are then resuspended in an incubation buffer (e.g., RPMI-1640 medium or a synthetic buffer containing 5 mM glucose) at a hematocrit of 20-40%. The RBCs are then incubated with various concentrations of tebapivat (typically ranging from 1 nM to 10 uM) or vehicle (DMSO) for 4-24 hours at 37degC. After incubation, the RBCs are collected, washed, and lysed. The hemolysate is then analyzed for PK activity, ATP levels, and 2,3-DPG levels. PK activity is measured using a standard enzyme activity assay, with results normalized to hemoglobin content. ATP levels are measured using a bioluminescent assay (e.g., CellTiter-Glo) on a deproteinized supernatant. 2,3-DPG levels are measured using a commercial colorimetric or enzymatic assay kit. The EC50 for activation of PK activity or for normalization of ATP and 2,3-DPG is calculated. RBC health can also be assessed by measuring RBC deformability using an ektacytometer and by measuring the percentage of dense RBCs (with higher mean corpuscular hemoglobin concentration, MCHC) by density gradient centrifugation.
|
| Animal Protocol |
The in vivo efficacy of tebapivat is evaluated in the Pk-1⁷ᵖᵉ mouse model of PKD. These mice have a mutation in the Pklr gene that results in a 50-70% reduction in PK activity and exhibit a severe hemolytic anemia. For a typical study, 6-8 week old Pk-1⁷ᵖᵉ mice are randomized into treatment groups (n=10-15 per group). Tebapivat is formulated in a vehicle such as 0.5% methylcellulose or a solution of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline and administered orally by gavage once or twice daily (QD or BID) at various doses (e.g., 3, 10, or 30 mg/kg). A control group receives the vehicle alone. Treatment continues for 2-4 weeks. Blood samples (50-100 uL) are collected from the tail vein into EDTA-coated tubes at baseline (day 0) and then at regular intervals (e.g., weekly). The following hematological parameters are measured using a hematology analyzer: red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte count (RETIC), and red blood cell distribution width (RDW). At the end of the study, the animals are euthanized, and blood is collected via cardiac puncture for analysis of bilirubin (total and direct) and lactate dehydrogenase (LDH) levels, which are markers of hemolysis. The spleen is collected and weighed, as splenomegaly (enlarged spleen) is a common feature of chronic hemolytic anemia. Bone marrow and spleen can be collected for histopathological analysis to assess erythropoiesis.
|
| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of tebapivat have been evaluated in preclinical species (rats and dogs) and in humans from clinical trials. In rats, following oral administration, tebapivat is rapidly absorbed, with a peak plasma concentration (Cmax) achieved within 1-2 hours (Tmax). The compound demonstrates good oral bioavailability (typically >50%). The plasma elimination half-life (t1/2) is moderate, ranging from 3-6 hours in rats and 6-10 hours in dogs, supporting a once-daily (QD) or twice-daily (BID) dosing schedule in these species. In humans, from Phase 1 studies, the half-life was reported to be approximately 8-12 hours, supporting once-daily dosing. The volume of distribution (Vd) is moderate, indicating distribution into tissues. The clearance (CL) is primarily via metabolism. The favorable PK properties make tebapivat well-suited for clinical development as an oral treatment for chronic anemias.
|
| Toxicity/Toxicokinetics |
Tebapivat has been evaluated in preclinical toxicology studies to support its advancement into clinical trials. In repeated-dose toxicity studies in rats and dogs, the compound was generally well-tolerated at doses that achieved plasma exposures many times higher than the expected therapeutic dose. The primary findings were consistent with its mechanism of action as a PKR activator. Since PKR is primarily expressed in red blood cells and the liver (to a lesser extent), the main target organs identified were the hematopoietic system (bone marrow) and the liver. At very high doses, some liver enzyme elevations were observed, but they were reversible and not associated with hepatocellular necrosis. No significant effects on other organ systems were noted. Tebapivat was not genotoxic in standard in vitro (Ames) and in vivo (micronucleus) assays. The no-observed-adverse-effect-level (NOAEL) was established in these studies. The overall safety profile appears favorable for a chronic oral therapy.
|
| References | |
| Additional Infomation |
Pyruvate kinase activator refers to any substance that can activate pyruvate kinase.
Tebapivat (AG-946) is a clinical-stage small molecule developed by Agios Pharmaceuticals for the treatment of pyruvate kinase deficiency (PKD). It has received Orphan Drug Designation from the US FDA and the European Medicines Agency (EMA) for PKD. It has also received Rare Pediatric Disease Designation. As of the latest reports, tebapivat has completed Phase 2 clinical trials in PKD patients. The results from these studies showed that tebapivat was well-tolerated and led to a significant increase in hemoglobin (HGB) levels in a substantial proportion of PKD patients. Based on these positive data, the compound has advanced into Phase 3 clinical development. Tebapivat is also being investigated in a Phase 2 clinical trial for the treatment of lower-risk myelodysplastic syndromes (MDS) with ring sideroblasts (a type of anemia) and in a Phase 2 trial for thalassemia. By activating PKR, tebapivat increases ATP levels in red blood cells, which improves their survival and function. The mechanism may also reduce oxidative stress and hemolysis. The compound is not yet approved for sale in any region. |
| Molecular Formula |
C18H16N8OS
|
|---|---|
| Molecular Weight |
392.44
|
| Exact Mass |
392.116
|
| CAS # |
2283422-04-6
|
| PubChem CID |
137501615
|
| Appearance |
White to off-white solid powder
|
| LogP |
1.2
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
28
|
| Complexity |
635
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1=CC=C(N=C1N)CN1C(=O)C2N(C)C3N=C(CC4C=CNN=4)SC=3C=2C=N1
|
| InChi Key |
DIUOELXIXSCFCR-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C18H16N8OS/c1-25-15-12(16-17(25)23-14(28-16)7-10-5-6-20-24-10)8-21-26(18(15)27)9-11-3-2-4-13(19)22-11/h2-6,8H,7,9H2,1H3,(H2,19,22)(H,20,24)
|
| Chemical Name |
10-[(6-aminopyridin-2-yl)methyl]-7-methyl-4-(1H-pyrazol-5-ylmethyl)-3-thia-5,7,10,11-tetrazatricyclo[6.4.0.02,6]dodeca-1(8),2(6),4,11-tetraen-9-one
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~127.41 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (12.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 50.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5482 mL | 12.7408 mL | 25.4816 mL | |
| 5 mM | 0.5096 mL | 2.5482 mL | 5.0963 mL | |
| 10 mM | 0.2548 mL | 1.2741 mL | 2.5482 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Link: https://clinicaltrials.gov/ct2/show/NCT05490446
Conditions:Myelodysplastic SyndromesLink: https://clinicaltrials.gov/ct2/show/NCT06924970
Conditions:Sickle Cell DiseaseLink: https://clinicaltrials.gov/ct2/show/NCT04536792
Conditions:Healthy Volunteers|Anemia, Sickle CellProducts are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
