Razuprotafib (AKB-9778) targets VE‑PTP (IC50 = 17 pM). It also inhibits the structurally related phosphatases HPTPh (IC50 = 36 pM) and HPTPγ (IC50 = 100 pM). It shows weak inhibition of LAR (IC50 = 295 nM), PTP1B (IC50 = 780 nM), CD45 (IC50 = 7,812 nM), and HPTPe (IC50 = 14,609 nM), while no significant inhibition (IC50 >20,000 nM) is observed against HCPTPA, PRL3, MKP‑1, VHR, ALP, and PP1γ. [1]
Razuprotafib's primary target is the vascular endothelial protein tyrosine phosphatase (VE-PTP), a protein tyrosine phosphatase that dephosphorylates and inactivates the Tie2 receptor. By inhibiting VE-PTP, Razuprotafib promotes Tie2 phosphorylation and activation, which in turn activates downstream signaling pathways involved in vascular stabilization and integrity. It has good selectivity for VE-PTP over other related phosphatases.

Razuprotafib (AKB-9778) increases angiopoietin-induced TIE2 phosphorylation and stimulates TIE2 phosphorylation and downstream signaling in HUVECs [1].

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In HUVECs, Razuprotafib (AKB-9778) induces concentration‑dependent TIE2 tyrosine phosphorylation at 0.17–50 μM. [1]
At 1.7 μM, it causes TIE2, AKT, eNOS, and ERK phosphorylation comparable to 500 ng/mL ANG1. Co‑incubation with ANG1 or ANG2 markedly enhances these effects. [1]
Knockdown of TIE2 by shRNA abrogates Razuprotafib‑induced AKT and ERK phosphorylation, confirming target specificity. [1]
Razuprotafib does not induce VEGFR2 phosphorylation nor affect VEGF‑induced VEGFR2 phosphorylation. [1]
In hypoxia (5% O2), which upregulates VE‑PTP and blunts ANG1‑induced TIE2 activation, Razuprotafib restores TIE2 phosphorylation and downstream signaling even in the presence of exogenous ANG2. [1]
In a permeability assay, 10 mM Razuprotafib blocks VEGF‑induced (200 ng/mL) FITC‑dextran (250 kDa) leakage across confluent HUVEC monolayers. [1]
In vitro, Razuprotafib effectively inhibits VE-PTP activity. In HUVECs, it promotes TIE2 phosphorylation and activation of downstream signaling. It also enhances angiopoietin-induced TIE2 phosphorylation, indicating its role in potentiating the natural ligand's effect.

Subcutaneous injection of razuprotafib (20 mg/kg) stimulates TIE2 phosphorylation in retinal endothelial cells in vivo [1]. Subretinal neovascularization (NV) is inhibited by razuprotafib (10–20 mg/kg; subcutaneous injection; twice daily for 7 days) [1].

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In the oxygen‑induced ischemic retinopathy mouse model, subcutaneous Razuprotafib (AKB-9778) at 10 or 20 mg/kg twice daily or intraocular injection (3–5 μg) significantly reduces retinal NV area. [1]
In the laser‑induced choroidal NV model, subcutaneous Razuprotafib (10 or 20 mg/kg twice daily) or intraocular injection (3 or 5 μg) decreases choroidal NV area. Combination with intraocular aflibercept (40 μg) produces greater suppression than either agent alone. [1]
In Rho‑VEGF transgenic mice (subretinal NV model), subcutaneous Razuprotafib (10 mg/kg twice daily) or intraocular injection (5 μg) reduces subretinal NV area. [1]
In Tet‑opsin‑Ang2 mice with high ANG2 expression, intraocular Razuprotafib (5 μg) suppresses ischemia‑induced retinal NV despite high ANG2 levels. [1]
In the Miles assay, intravenous Razuprotafib (16 mg/kg) significantly reduces histamine‑ and VEGF‑induced dermal vascular leakage. [1]
In Rho‑VEGF mice, subcutaneous Razuprotafib (3 or 10 mg/kg) decreases albumin extravasation around retinal NV. [1]
In Tet‑opsin‑VEGF double‑transgenic mice, subcutaneous Razuprotafib (10 or 50 mg/kg twice daily) prevents exudative retinal detachments and suppresses NV in a dose‑dependent manner. [1]
In vivo, Razuprotafib has been investigated for its potential in treating diabetes and COVID-19. It is administered by patients through subcutaneous injection, suggesting it has been tested in clinical settings. It is also protective against acute kidney injury.

The phosphatase inhibition assay is performed in 96‑well plates. Test compounds are diluted in assay buffer (50 mM Tris‑HCl, 150 mM NaCl, 5 mM DTT, 1 mM EDTA, ±0.01% BSA, pH 7–10). Enzyme (commercially available or prepared proteins) is diluted in assay buffer immediately before use. Compound and enzyme are pre‑incubated for 10 minutes at room temperature. Then the fluorogenic phosphatase substrate DiFMUP (6,8‑difluoro‑4‑methylumbelliferyl phosphate) is added, followed by centrifugation at 500 g and incubation for 15 minutes at room temperature. The reaction is stopped by adding 5 mL of stop reagent (50 mM bpV[phen]). Plates are read on a plate reader. IC50 values are calculated using Excel Fit from concentration‑response curves. [1]
The primary cell-free assay for Razuprotafib is a phosphatase activity assay. In this system, a synthetic substrate is incubated with the VE-PTP enzyme in the presence of varying concentrations of the inhibitor. The production of the dephosphorylated product is measured to determine the IC50.

Human umbilical vein endothelial cells (HUVECs, passages ≤6) are treated with Razuprotafib (AKB-9778) alone or with recombinant human ANG1 (500 ng/mL), ANG2 (500 ng/mL), or VEGF (200 ng/mL) for 10 minutes. For immunoprecipitation, cell lysates are incubated with anti‑TIE2 or anti‑VEGFR2 antibodies, then blotted with anti‑phosphotyrosine (4G10) or corresponding total protein antibodies. For signaling studies, lysates are probed with antibodies against total and phosphorylated AKT, ERK, and eNOS. For permeability assays, HUVECs are grown to confluence on Transwell filters, pretreated with 10 mM Razuprotafib or vehicle for 30 minutes, then 200 ng/mL VEGF and 250 kDa FITC‑dextran (0.25 mg/mL) are added to the upper chamber. After 2.5 hours, FITC‑dextran efflux into the lower chamber is measured using a plate reader. TIE2 silencing is performed using a retroviral shRNA specific for human TIE2 (sequence: 5'‑GATCCCACCATCGAGG‑3') in EC‑RF24 cells. [1]
For cellular assays, endothelial cells such as HUVECs are used. Cells are treated with Razuprotafib, and the levels of phosphorylated TIE2 (p-TIE2) and its downstream signaling molecules are measured by Western blot. This confirms the compound's ability to activate the Tie2 pathway in a cellular context.

Oxygen‑induced ischemic retinopathy: C57BL/6 mice at postnatal day 12 (P12) receive an intravitreous injection (1–2 μL) of Razuprotafib (AKB-9778) (0.1–5 μg) or vehicle. Alternatively, subcutaneous injections (10 or 20 mg/kg) are given twice daily from P12 to P17. At P17, eyes are fixed and retinas stained with FITC‑labeled GSA lectin, flat‑mounted, and NV area measured. [1]
Laser‑induced choroidal NV: Six‑week‑old C57BL/6 mice undergo Bruch’s membrane rupture by laser. Immediately after, mice receive intraocular injection of Razuprotafib (3 or 5 μg) or vehicle, or subcutaneous injections (10 or 20 mg/kg) twice daily for 7 days. In combination studies, intraocular aflibercept (40 μg) is given with subcutaneous Razuprotafib (20 mg/kg twice daily). After 7 days, choroidal flat mounts are stained with FITC‑GSA and NV area measured. [1]
Rho‑VEGF transgenic mice (subretinal NV): At P15, mice receive subcutaneous Razuprotafib (3 or 10 mg/kg) twice daily until P21, or a single intraocular injection (5 μg). At P21, retinas are flat‑mounted and subretinal NV area quantified. For albumin leakage, mice receive three subcutaneous injections of Razuprotafib (3 or 10 mg/kg) 12 hours apart, then retinas stained for albumin and GSA. [1]
Tet‑opsin‑Ang2 mice: Dox‑treated mice with ischemic retinopathy receive a single intraocular injection of Razuprotafib (5 μg) at P12. At P17, retinal NV area is measured. [1]
Tet‑opsin‑VEGF double‑transgenic mice: Mice are pretreated with subcutaneous Razuprotafib (3, 10, or 50 mg/kg) twice daily for 3 days, then continued with the same regimen plus daily subcutaneous doxycycline (50 mg/kg) for 4 days. Eyes are sectioned and the percentage of retinal detachment measured. [1]
Miles assay (vascular leakage in skin): C57BL/6 mice receive intravenous injection of Razuprotafib (16 mg/kg in 5% dextrose‑H₂O) or vehicle 5 hours before and immediately before the assay. Then Evans blue dye (1% in PBS, 100 μL) is injected intravenously. Ten minutes later, intradermal injections (50 μL) of PBS, histamine (225 ng), or mouse VEGF165 (100 ng) are given at three back sites. After 30 minutes, skin punches are excised and extracted in formamide for 5 days, and Evans blue extravasation is quantified. [1]
In vivo, Razuprotafib is administered via subcutaneous injection. Its efficacy has been studied in models of diabetic complications and COVID-19, where its ability to restore vascular integrity and reduce inflammation is assessed.

Razuprotafib is administered via subcutaneous injection. Its molecular weight is 586.70 g/mol.

The toxicity profile of Razuprotafib is determined through its clinical investigations. As a VE-PTP inhibitor, its effects are related to the modulation of vascular function.

[1]. Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature. J Clin Invest. 2014;124(10):4564-4576.

Razuprotafib, also known as AKB-9778, is a small molecule inhibitor that restores Tie2 activation by inhibiting VE-PTP. In studies of diabetes and COVID-19, Razuprotafib was self-administered by patients via subcutaneous injection. Razuprotafib is a small molecule inhibitor of vascular endothelial protein tyrosine phosphatase (VE-PTP) with potential vascular stabilizing effects. After administration, Razuprotafib targets, binds to, and inhibits VE-PTP, a negative regulator of endothelial cell (EC)-specific receptor tyrosine kinase (RTK) Tie2. This restores Tie2 activation, thereby improving endothelial function and stabilizing blood vessels. VE-PTP is upregulated in damaged endothelium associated with various diseases. Tie2 plays a crucial role in endothelial function and vascular stability. Reduced Tie2 activation leads to vascular leakage and inflammation.

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Mechanism of Action
Razuprotafib inhibits VE-PTP (a negative regulator of Tie2 in diseased blood vessels) by binding to and inhibiting its intracellular catalytic domain, thereby inactivating Tie2. This allows Razuprotafib to restore Tie2 activation, thereby enhancing endothelial function and stabilizing blood vessels. Razuprotafib is currently being investigated for its efficacy in treating diabetic vascular complications and COVID-19 acute respiratory distress syndrome (ARDS).
Mechanism of Action
Razuprotafib (AKB-9778) is a competitive inhibitor of VE‑PTP’s catalytic activity. It activates TIE2 even in the presence of high ANG2 levels, shifting ANG2 from antagonist to agonist. [1]
Intraocular or systemic administration suppresses both choroidal and retinal NV, as well as vascular leakage, in multiple preclinical models. [1]
Combination with aflibercept (VEGF trap) shows additive/synergistic effects in reducing choroidal NV. [1]
Razuprotafib does not affect normal retinal vascular development (P4‑P7), suggesting selectivity for pathologic angiogenesis. [1]
It has potential for treating neovascular AMD, diabetic retinopathy, diabetic macular edema, and retinal vein occlusion, especially in patients with incomplete response to anti‑VEGF therapy. [1]
Razuprotafib is an investigational drug that has been studied in clinical trials for its potential in treating diabetic complications and COVID-19. Its mechanism of action, restoring Tie2 activation, is a novel approach to treating vascular dysfunction. It represents a significant advancement in the field of vascular biology and drug development.

C26H26N4O6S3

586.702842235565

586.101

C, 53.23; H, 4.47; N, 9.55; O, 16.36; S, 16.39

1008510-37-9

1809275-69-1 (sodium);1008510-37-9;

46700782

White to off-white solid powder

1.5±0.1 g/cm3

1.667

4.02

4

10

12

39

906

2

COC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=C(C=C2)NS(=O)(=O)O)C3=CSC(=N3)C4=CC=CS4

KWJDHELCGJFUHW-SFTDATJTSA-N

InChI=1S/C26H26N4O6S3/c1-36-26(32)29-21(15-17-6-3-2-4-7-17)24(31)27-20(22-16-38-25(28-22)23-8-5-13-37-23)14-18-9-11-19(12-10-18)30-39(33,34)35/h2-13,16,20-21,30H,14-15H2,1H3,(H,27,31)(H,29,32)(H,33,34,35)/t20-,21-/m0/s1

N-(4-{(2S)-2-{(2S)-2-[(methoxycarbonyl)amino]-3-phenylpropanamido}-2-[2-(thiophen-2-yl)-1,3-thiazol-4-yl]ethyl}phenyl)sulfamic acid

AKB-9778; AKB 9778; Razuprotafib; 1008510-37-9; AKB-9,778; 0WAX4UT396; AKB9,778; AKB9778

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 (170.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.26 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 DMSO stock solution (25.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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 (4.26 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 DMSO stock solution (25.0 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well.
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 (4.26 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 DMSO stock solution (25.0 mg/mL) to 900 μL of corn oil and mix well.

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