Sickle hemoglobin (HbS) polymerization
Osivelotor targets hemoglobin S (HbS), the abnormal hemoglobin variant that causes sickle cell disease. It is an allosteric regulator that binds reversibly to the N-terminal valine of the alpha-globin chain. This binding stabilizes the oxygenated, relaxed (R-state) conformation of hemoglobin, increasing its oxygen affinity. The increased oxygen affinity reduces the proportion of deoxygenated hemoglobin S (deoxy-HbS), which is the form that polymerizes and leads to red blood cell sickling. By inhibiting HbS polymerization, Osivelotor reduces the formation of sickled red blood cells, improves RBC deformability, and ameliorates the pathophysiology of SCD, including hemolysis, vaso-occlusion, and chronic inflammation. Unlike earlier HbS modulators, Osivelotor is potent, selective, and orally effective. It does not target the heme iron or interfere with oxygen binding to the heme; it stabilizes the hemoglobin tetramer.

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

---

In vitro, Osivelotor has been characterized using biochemical and biophysical assays. In a hemoglobin oxygen affinity assay, Osivelotor shifts the oxygen dissociation curve (ODC) to the left, indicating increased oxygen affinity. The P50 (partial pressure of oxygen at which hemoglobin is 50% saturated) is decreased in a concentration-dependent manner. The compound shows an EC50 for increasing oxygen affinity in the low micromolar range. In a HbS polymerization assay, Osivelotor inhibits the formation of deoxy-HbS polymers at concentrations consistent with its effects on oxygen affinity. The concentration required for 50% inhibition (IC50) of polymerization is typically in the sub-micromolar to low micromolar range. In whole blood from SCD patients, treatment with Osivelotor (1-10 uM) reduces the percentage of sickled red blood cells under hypoxic conditions and improves red blood cell deformability (measured by ektacytometry). The compound also reduces hemolysis markers (e.g., lactate dehydrogenase, LDH, and plasma-free hemoglobin) in vitro.

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]

---

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]

---

In vivo, Osivelotor has demonstrated efficacy in murine models of sickle cell disease, such as the Townes SCD mouse model (which expresses human HbS). In these models, oral administration of Osivelotor (20-150 mg/kg, once daily) increases hemoglobin oxygen affinity, inhibits HbS polymerization, and prevents red blood cell sickling. The compound reduces biomarkers of hemolysis (e.g., decreased LDH, bilirubin, and reticulocyte counts) and improves red blood cell survival (reduced % phosphatidylserine-exposing RBCs). Osivelotor also ameliorates vaso-occlusion, as evidenced by reduced plasma levels of inflammatory cytokines (e.g., IL-6, TNF-alpha) and decreased organ pathology (e.g., reduced splenomegaly, improved kidney function). In the Townes model, chronic treatment with Osivelotor improves overall RBC health and reduces the characteristic pathophysiology of SCD, including pulmonary thrombosis and renal injury. The compound is orally bioavailable and achieves adequate plasma exposures in mice, rats, and dogs. Osivelotor is in clinical development and has shown promising results in early-phase human trials for SCD.

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

---

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

---

A non-cellular (cell-free) protocol for evaluating the effect of Osivelotor on hemoglobin oxygen affinity uses purified human hemoglobin S or whole blood lysate. Hemoglobin S is purified from red blood cells of SCD patients or expressed recombinantly. The oxygen equilibrium curve (OEC) is measured using a Hemox Analyzer or a tonometer coupled with a spectrophotometer. In a typical Hemox Analyzer experiment, 5 uL of Osivelotor solution (in DMSO) is added to 5 mL of hemoglobin solution (0.1-1 mg/mL) in Tris buffer (pH 7.4, 37degC) to achieve final concentrations of 0.1-100 uM. The sample is oxygenated at 160 mmHg for 10 minutes, then deoxygenated by bubbling with nitrogen at a slow rate. The oxygen saturation is recorded against the partial pressure of oxygen (pO2). The P50 (pO2 at 50% saturation) is calculated from the curve. A leftward shift (decrease in P50) indicates increased oxygen affinity. Alternatively, a high-throughput oxygen affinity assay can be performed in a 96-well plate using an oxygen-sensitive probe (e.g., MitoXpress-Xtra). For HbS polymerization inhibition, a turbidity assay is used: deoxy-HbS (1-2 mM) in phosphate buffer (pH 7.4, 25degC) is prepared, and Osivelotor (0.01-100 uM) is added. Polymerization is initiated by raising the temperature to 30degC or by adding a reducing agent. The increase in turbidity at 700 nm is monitored over time, and the lag time and final turbidity are measured. The IC50 for inhibiting polymerization is determined.

An in vitro cellular protocol for evaluating Osivelotor‘s effect on red blood cell sickling uses human SCD blood samples obtained from patients with informed consent. Whole blood (5 mL) is collected in heparinized tubes from SCD patients in steady state. Red blood cells (RBCs) are washed three times with PBS and resuspended in HBSS (Hanks' Balanced Salt Solution) to a hematocrit of 10%. Osivelotor is dissolved in DMSO to prepare a 10 mM stock and diluted in HBSS to various concentrations (0.1-100 uM). RBCs (100 uL) are incubated with 100 uL of Osivelotor solution for 1-4 hours at 37degC. To induce sickling, the RBCs are deoxygenated by adding sodium metabisulfite (0.2% w/v) or by bubbling nitrogen gas (95% N2, 5% CO2) for 30-60 minutes. Aliquots are fixed with 0.5% glutaraldehyde in PBS, and the percentage of sickled RBCs (typical crescent, holly-leaf, or elongated forms) is counted under a light microscope (counting at least 500 RBCs per sample). The percentage of sickling is calculated, and the EC50 for inhibition of sickling is determined. Alternatively, RBC deformability is measured by ektacytometry (e.g., using a Lorrca instrument) under increasing shear stress. RBCs are treated with Osivelotor, then deoxygenated, and elongation index (EI) is measured. Increased EI indicates improved deformability. The hemolysis assay: treated RBCs are deoxygenated, and the supernatant is collected. Lactate dehydrogenase (LDH) activity and plasma-free hemoglobin are quantified using commercial kits.

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.

---

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.

---

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.

---

An in vivo animal protocol for evaluating the efficacy of Osivelotor uses the Townes sickle cell disease (SCD) mouse model (HBA^Tg(HBA) HBB^tm1Tow HBB^Tg(HBG1, HBB). Both male and female mice (6-12 weeks old, 20-30 g) are used. Mice are randomized into treatment groups (n=8-12 per group). Osivelotor is formulated in a suitable vehicle, such as 0.5% methylcellulose (MC) or 10% DMSO/40% PEG300/5% Tween-80/45% saline. The compound is administered orally once daily at doses of 20, 50, 100, or 150 mg/kg for 2-4 weeks. The vehicle control group receives an equivalent volume of vehicle. A positive control group may receive hydroxyurea (100 mg/kg/day) or another HbS modulator. During the treatment period, body weight, food consumption, and clinical signs are monitored. Blood samples are collected from the tail vein or retro-orbital sinus at various time points (e.g., baseline, days 7, 14, 21, 28) for measurement of complete blood count (CBC), reticulocyte count, bilirubin, LDH, and blood urea nitrogen (BUN). At the end of the study, mice are euthanized, and tissues (spleen, liver, kidneys, lungs) are harvested, weighed, and processed for histology (H&E staining, Perls‘ Prussian blue for iron, and picrosirius red for fibrosis). Hemoglobin oxygen affinity is measured by a Hemox Analyzer using blood samples. Red blood cell survival may be assessed by biotinylation or other methods. Plasma inflammatory cytokines (TNF-alpha, IL-6) are measured by ELISA.

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]

---

Osivelotor (GBT-601) has been evaluated in preclinical and clinical pharmacokinetic (PK) studies. In animal studies, oral administration results in rapid absorption with a Tmax of 1-2 hours. The terminal elimination half-life (t½) is approximately 4-8 hours in mice and dogs. The compound has a moderate volume of distribution and is highly protein-bound (>99% in plasma). Metabolism occurs primarily via glucuronidation (UGT1A9) and to a lesser extent via CYP3A4. The compound is eliminated primarily in the bile, with some renal excretion. In Phase 1 clinical trials in healthy volunteers and SCD patients, Osivelotor demonstrated dose-proportional pharmacokinetics, with a Tmax of 1-3 hours and a t½ of 8-12 hours, supporting once-daily dosing. The compound shows a low potential for drug-drug interactions and does not significantly prolong the QTc interval. The long-acting properties of Osivelotor allow for once-daily oral administration. Detailed clinical PK data are available in regulatory filings and clinical trial publications.

In preclinical toxicology studies, Osivelotor (GBT-601) has been generally well-tolerated. In repeat-dose toxicity studies in rodents and dogs, the no-observed-adverse-effect level (NOAEL) was at least 10-20 times higher than the projected therapeutic human exposure. Adverse effects observed at high doses included mild gastrointestinal disturbances, slight body weight loss, and minimal changes in liver enzyme levels (ALT, AST). No significant genotoxicity was observed in the Ames test or micronucleus assay. Chronic toxicology studies and carcinogenicity studies have not been fully completed or published. In clinical trials, the most common adverse events reported were headache, nausea, diarrhea, and fatigue, which were mostly mild to moderate in severity. No serious adverse events or dose-limiting toxicities have been reported at the studied doses. Osivelotor is not approved for clinical use as of 2026, but it is under active development. Standard laboratory precautions should be used when handling the compound.

[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]

---

Osivelotor (GBT-601, GBT-021601) is an orally bioavailable, next-generation allosteric modulator of hemoglobin S (HbS) for the potential treatment of sickle cell disease (SCD). It is a potent second-generation HbS polymerization inhibitor that achieves higher hemoglobin modification at lower doses, reducing pill burden. By stabilizing the oxygenated conformation of HbS, it increases oxygen affinity, inhibits HbS polymerization, and prevents RBC sickling. As of mid-2026, Osivelotor is in clinical development (Phase 2/3 trials) and has not yet received regulatory approval from the FDA, EMA, or other agencies. It is not a research tool; it is an investigational drug. However, it is available for research use in the context of preclinical and clinical studies. The compound should be handled with standard laboratory safety precautions and stored at -20degC.

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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