Sickle hemoglobin (HbS) polymerization

GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD.

This report details the discovery path of GBT021601 (osivelotor) (16), a novel, small molecule, sickle hemoglobin (HbS) polymerization inhibitor. Following a streamlined testing funnel with cassette dosing in rat pharmacokinetic (PK) studies, we identified this next-generation HbS polymerization inhibitor, which had improved PK properties compared with the first-in-class drug, voxelotor (1). GBT021601 has ∼4.8-fold greater exposure and a ∼3.5-fold longer half-life in rats compared with voxelotor. In a murine model of sickle cell disease (SCD), GBT021601 treatment resulted in an increase in hemoglobin oxygen affinity, a reduction in sickling of red blood cells (RBCs), and an increase in both RBC half-life and hemoglobin levels not seen with voxelotor preclinically. The improved half-life and exposure appear to translate to similar levels of HbS occupancy at lower doses than voxelotor, thus reducing treatment burden. GBT021601 is being investigated in a phase 2/3 clinical trial for the treatment of patients with SCD (NCT05431088).[2]

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The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.[3]

Modified oxygen dissociation assay [3]
The ability of GBT021601-modified HbS to release O2 was evaluated using a modified oxygen dissociation assay.

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In vitro HbS polymerization[3]
The in vitro HbS polymerization reaction, described in the Supplemental Information, was modified from a previously described method.

PK measurements [3]
PK analysis of GBT021601 was conducted in mice (C57BL/6 and SS), Sprague–Dawley rats, Beagle dogs and cynomolgus monkeys following intravenous (IV, 1 mg/kg) and oral (PO, 2 or 10 mg/kg) administration. Blood and plasma were se- rially collected from each animal up to 96–336 h post-dose and analysed for GBT021601 concentration using liquid chromatography with tandem mass spectrometry.

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Murine model of SCD [3]
Studies with ~8- to 12-week-old male knock-in Townes mice (B6; 129-Hbb tm2(HBG1,HBB*)Tow /Hbb tm3(HBG1,HBB)Tow Hba tm1(HBA)Tow/J) with an HbSS genotype (homozygous for Hba tm1(HBA)Tow and homozygous for Hbb tm2(HBG1,HBB*)Tow) (Jackson Laboratory) were performed under the oversight of the Institutional Animal Care and Use Committee. GBT021601 (20, 40, 75 or 150 mg/kg QD) or vehicle only was administered via oral gavage to SS mice as a repeat dose for 21 days. At the end of the study, whole blood was collected (~3 h [Cmax] or 24 h [Cmin] after the last dose) either from the tail vein or by cardiac puncture under anaesthesia (using iso- flurane in 100% O2) for PK and PD analyses.

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Tissue oxygenation and tolerance to hypoxia in SS mice [3]
GBT021601 150 mg/kg QD was administered to SS mice via oral gavage for 21 days before subjecting the mice to any pro- cedure. Blood gases and hypoxia tolerance measurements were taken ~2.5 to 3.5 h after the final dose of GBT021601. Cardiac output, blood gases, total Hb and lactate were meas- ured in normoxia (21% fraction of inspired O2 [FiO2]) and hypoxia (10% FiO2) as described previously. 8 O2 delivery (DO2) and O2 consumption (VO2) were calculated from the cardiac output, total Hb, and arterial (SaO2) and venous O2 saturation (SvO2) as described previously. 8 Tolerance to hy- poxia was determined as described previously.

In summary, in order to find a next-generation HbS polymerization inhibitor with a longer half-life (t1/2), we conducted a structure-activity relationship (SAR) study on the initial lead compound GBT1580 (2). Guided by p50-shifted oxygen extractable concentration (OEC) measurements and t1/2 and exposure data in rats, we identified GBT021601 (16). GBT021601 has desirable physicochemical properties, including high water solubility and high permeability, and exhibits consistent pharmacokinetic (PK) characteristics across different animal species, showing significant efficacy in a sickle cell disease (SCD) mouse model (18). Consistent with its preclinical PK characteristics, GBT021601 has a very long t1/2 in both SCD patients (10 days) and healthy volunteers (30 days) (22). In contrast, voxelotor has t1/2 of 2 days in SCD patients and 2.5–3.5 days in healthy volunteers. (23) In a phase II/III study, adult patients with sickle cell disease who received GBT021601 at daily doses of 100 mg and 150 mg for 12 weeks showed an average increase in hemoglobin of 2.67 g/dL and 3.17 g/dL, respectively. In addition, clinical markers of hemolysis decreased at 6 and 12 weeks of treatment. (24) Currently, a phase II/III clinical trial of GBT021601 for the treatment of adult patients with sickle cell disease is underway. [2]

[1]. WHO Drug Information-World Health Organization (WHO).

[2]. Discovery of Osivelotor (GBT021601): A Potent, Next-Generation Sickle Hemoglobin Polymerization Inhibitor. ACS Med Chem Lett. 2025 Jul 8;16(8):1526-1532.

[3]. GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model. Br J Haematol. 2023 Jul;202(1):173-183.

In summary, GBT021601 significantly improved erythrocyte health and restored multiple hematological parameters, including hemoglobin, to normal in a sickle cell disease mouse model, demonstrating its potential to provide optimal therapeutic benefits to patients with sickle cell disease by effectively inhibiting HbS polymerization, reducing erythrocyte sickling, and maintaining peripheral tissue oxygen delivery. Further clinical studies are needed to fully assess the potential benefits/risks of GBT021601 in patients with sickle cell disease. [3]

C20H22N2O6

386.398485660553

386.147

C, 62.17; H, 5.74; N, 7.25; O, 24.84

2417955-18-9

146567655

White to light yellow solid powder

0.9

2

7

7

28

519

1

C1COC[C@H](N1C(=O)C2=C(N=CC=C2)CCO)COC3=CC=CC(=C3C=O)O

NIWBSQAKKNNWBT-AWEZNQCLSA-N

InChI=1S/C20H22N2O6/c23-9-6-17-15(3-2-7-21-17)20(26)22-8-10-27-12-14(22)13-28-19-5-1-4-18(25)16(19)11-24/h1-5,7,11,14,23,25H,6,8-10,12-13H2/t14-/m0/s1

2-hydroxy-6-[[(3S)-4-[2-(2-hydroxyethyl)pyridine-3-carbonyl]morpholin-3-yl]methoxy]benzaldehyde

Osivelotor; 2417955-18-9; GBT-601; GBT021601; GBT-021601; GBT601; PF-07940367; UK749B4S16;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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 (258.80 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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