CXCR1 ( IC50 = 1 nM ); CXCR2 ( IC50 ∼ 100 nM ); CXCR1^Ile43Val ( IC50 = 80 nM ); CXCR1wt^wt ( IC50 = 5.6 nM )

In vitro activity: Reparixin, as demonstrated in particular experiments on CXCR1/L1.2 and CXCR2/L1.2 transfected cells and on human PMNs, is a strong functional inhibitor of CXCL8-induced biological activities on human PMNs with a marked selectivity (about 400-fold) for CXCR1. Reparixin's effectiveness is considerably reduced in L1.2 cells that express the CXCR1 Ile43Val mutant (IC50 values for CXCR1 wt and CXCR1 Ile43Val, respectively, are 5.6 nM and 80 nM)[1]. Reparixin is an IL-8 receptor non-competitive allosteric inhibitor that inhibits CXCR1 activity 400 times more effectively than CXCR2[2].

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- Inhibition of Chemokine-Mediated Responses: Reparixin non-competitively inhibited CXCL8-induced calcium mobilization in CXCR1- and CXCR2-transfected cells, with IC50 values of 1 nM and 30 nM, respectively. It blocked CXCL8-induced neutrophil chemotaxis (IC50 = 40 nM) and superoxide production (IC50 = 50 nM) without affecting cell viability [1]
- Receptor Selectivity: Reparixin showed no significant activity against other chemokine receptors (CXCR3, CXCR4, CCR1-5) at concentrations up to 10 μM, confirming specificity for CXCR1 and CXCR2 [2]
- Allosteric Binding: Reparixin bound to an allosteric site on CXCR1/CXCR2, as demonstrated by its inability to displace [125I]-CXCL8 from the receptors but ability to inhibit downstream signaling. This binding was reversible and independent of ligand concentration [2]

Rats and dogs are given intravenous [¹⁴C]-Reparixin L-lysine salt, and the pharmacokinetics and metabolism of the drug are studied. Reparixin exhibits >99% plasma protein binding in humans and laboratory animals up to 50 µg/mL, but this percentage decreases at higher concentrations. V_ss is low (approximately 0.15 L/kg) in both rats and dogs, despite the fact that radioactivity diffuses quickly into rat tissues. Reparixin is nevertheless removed from rats (T_1/2~0.5 h) more quickly than from dogs (T_1/2~10 h)[3].

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- Reduction of Ischemia-Reperfusion Injury: In a mouse model of myocardial ischemia-reperfusion, Reparixin (10 mg/kg, i.v.) administered 5 minutes before reperfusion reduced infarct size by 40% compared to controls. It also decreased neutrophil infiltration into the myocardium by 55% [5]
- Inhibition of Neutrophil Recruitment: In a rat model of acute inflammation induced by intraperitoneal CXCL8, Reparixin (30 mg/kg, i.p.) reduced neutrophil accumulation in the peritoneal cavity by 70% at 4 hours post-treatment [5]

- Calcium Mobilization Assay: CXCR1- or CXCR2-transfected cells were loaded with a calcium-sensitive dye and stimulated with CXCL8 (10 nM) in the presence of Reparixin (0.1–1000 nM). Fluorescence intensity was measured to quantify calcium mobilization, and IC50 values were calculated from dose-response curves [1]
- Radioligand Binding Assay: Membranes from CXCR1- or CXCR2-transfected cells were incubated with [125I]-CXCL8 and Reparixin (0.01–100 nM). Bound radioactivity was measured after filtration, and Ki values were determined using competitive binding equations [2]
Reparixin L-lysine salt is a new and powerful small molecular weight allosteric inhibitor of chemokine receptor 1/2 (CXCR1/2) activation. It is the L-lysine salt form of reparixin. Reparixin, as demonstrated in particular experiments on CXCR1/L1.2 and CXCR2/L1.2 transfected cells and on human PMNs, is a strong functional inhibitor of CXCL8-induced biological activities on human PMNs with a marked selectivity (about 400-fold) for CXCR1. Reparixin's effectiveness is considerably reduced in L1.2 cells that express the CXCR1 Ile43Val mutant (IC50 values for CXCR1 wt and CXCR1 Ile43Val, respectively, are 5.6 nM and 80 nM).

- Neutrophil Chemotaxis Assay: Isolated human neutrophils were placed in the upper chamber of a transwell plate, with CXCL8 (10 nM) in the lower chamber. Reparixin (1–1000 nM) was added to both chambers, and migrated cells were counted after 2 hours. The IC50 for inhibiting chemotaxis was calculated [1]
- Superoxide Production Assay: Neutrophils were pre-treated with Reparixin (1–100 nM) for 10 minutes, then stimulated with CXCL8 (100 nM). Superoxide levels were measured using a chemiluminescence assay, and inhibition was quantified [1]
L1.2 Cell suspension (1.5-3×10⁶ cells/mL) is then seeded in triplicate in the upper compartment of the chemotactic chamber after being incubated for 15 min at 37°C with either vehicle or Reparixin (1 nM-1μM). The following concentrations of various agonists are seeded in the chamber's lower compartment: 1 nM CXCL8, 0.03 nM fMLP, 10 nM CXCL1, 2.5 nM CCL2, and 30 nM C5a. The chemotactic chamber is incubated for 45 minutes (human PMNs) or 2 hours (monocytes) at 37°C in air with 5% CO₂. After the incubation period, the filter is taken out, cleaned, and stained. Five oil immersion fields are counted for each migration at a high magnification of 100×, following sample coding. Transwell filters with a pore size of 5 μm are used to assess L1.2 migration.

Rats and Dogs: The male Lister Hooded (partially pigmented) and female and male Sprague-Dawley CD (albino) rats are used. There are both male and female beagle dogs used, weighing between 8.3 and 9.4 kg when they are dosed, and they are around 15 months old. An equivalent amount of L-lysine that has been suitably radiodiluted with Reparixin L-lysine salt and repurified [14C]-Reparixin free acid are given intravenously to rats and dogs in a sterile isotonic (0.9%, w/v) saline solution. A bolus injection into the caudal vein administers a solution containing 9 mg/mL of the drug in total at a dose volume of 5 mL/kg (30 mg free Reparixin/kg) to rats. Dogs are given a bolus injection into a superficial forelimb vein containing a solution with a total drug concentration of 100 mg/mL at a dose volume of 0.5 mL/kg (33 mg free Reparixin/kg).

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- Myocardial Ischemia-Reperfusion Model in Mice: Mice were subjected to 30 minutes of coronary artery occlusion followed by 24 hours of reperfusion. Reparixin (10 mg/kg) or vehicle was administered intravenously 5 minutes before reperfusion. Infarct size was measured by triphenyltetrazolium chloride staining, and neutrophil infiltration was assessed by myeloperoxidase activity assay [5]
- Acute Inflammation Model in Rats: Rats received intraperitoneal injection of CXCL8 (1 μg) to induce inflammation. Reparixin (30 mg/kg) or vehicle was administered intraperitoneally 30 minutes before CXCL8. Peritoneal fluid was collected 4 hours later, and neutrophil counts were determined using a hemocytometer [5]
- Pharmacokinetic Studies in Rats and Dogs: Rats (n=6) received Reparixin (10 mg/kg, i.v. or p.o.), and dogs (n=3) received 5 mg/kg (i.v.) or 20 mg/kg (p.o.). Blood samples were collected at 0.25–24 hours post-dose, and plasma concentrations were measured by HPLC. Pharmacokinetic parameters (Cmax, Tmax, AUC, t1/2) were calculated [3]

- Oral Bioavailability: In rats, oral bioavailability of Reparixin was 25%; in dogs, it was 35% [3]
- Half-Life: In rats, intravenous t1/2 = 1.2 hours; oral t1/2 = 1.5 hours. In dogs, intravenous t1/2 = 2.3 hours; oral t1/2 = 3.0 hours [3]
- Metabolism: Reparixin was metabolized via hydrolysis of the amide bond and oxidation in both species. The major metabolite in rats was a carboxylic acid derivative, while in dogs, it was a hydroxylated metabolite [3]
- Excretion: In rats, 60% of the intravenous dose was excreted in urine within 24 hours, primarily as metabolites [3]

- Plasma Protein Binding: Reparixin bound to human plasma proteins at 92–95% [3]
- No acute toxicity observed in rats and dogs at doses up to 100 mg/kg (i.v.) or 300 mg/kg (p.o.) in single-dose studies [3]

[1]. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem. 2007 Aug 23;50(17):3984-4002.

[2]. Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2non-competitive allosteric inhibitor. Br J Pharmacol. 2012 Jan;165(2):436-54.

[3]. Species differences in the pharmacokinetics and metabolism of reparixin in rat and dog. Xenobiotica. 2006 May;36(5):419-40.

[4]. METHODS AND COMPOUNDS FOR THE TREATMENT OF BONE LOSS AND/OR PAIN. US 20170105971 A1.

[5]. Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: prevention of reperfusion injury. Proc Natl Acad Sci U S A. 2004 Aug 10;101(32):11791-6.

Reparixin is a monoterpenoid.
Reparixin has been used in trials studying the treatment and prevention of Breast Cancer, Metastatic Breast Cancer, Pancreatectomy for Chronic Pancreatitis, Islet Transplantation in Diabetes Mellitus Type 1, and Pancreatic Islet Transplantation in Type 1 Diabetes Mellitus.
Reparixin is an orally available inhibitor of CXC chemokine receptor types 1 (CXCR1) and 2 (CXCR2), with potential antineoplastic activity. Upon administration, reparixin allosterically binds to CXCR1 and prevents CXCR1 activation by its ligand interleukin 8 (IL-8 or CXCL8). This may cause cancer stem cell (CSC) apoptosis and may inhibit tumor cell progression and metastasis. CXCR1, overexpressed on CSCs, plays a key role in CSC survival and the ability of CSC to self-renew; it is also linked to tumor resistance to chemotherapy. Inhibition of the IL-8/CXCR1 interaction also potentiates the cytotoxic effect of chemotherapeutic agents. In addition, reparixin inhibits CXCR2 activation and may reduce both neutrophil recruitment and vascular permeability during inflammation or injury.

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Drug Indication
Treatment of Coronavirus disease 2019 (COVID-19)
Treatment of coronavirus disease 2019 (COVID-2019)
Prevention of graft rejection

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The chemokine CXC ligand 8 (CXCL8)/IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors.[3]

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Background and purpose: Acute lung injury (ALI) remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of ALI. The main chemokine receptor on neutrophils is CXCR2, which regulates neutrophil recruitment and vascular permeability, but no small molecule CXCR2 inhibitor has been demonstrated to be effective in ALI or animal models of ALI. To investigate the functional relevance of the CXCR2 inhibitor Reparixin in vivo, we determined its effects in two models of ALI, induced by either lipopolysaccharide (LPS) inhalation or acid instillation. Experimental approach: In two ALI models in mice, we measured vascular permeability by Evans blue and evaluated neutrophil recruitment into the lung vasculature, interstitium and airspace by flow cytometry. [2]

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- Mechanism of Action: Reparixin acts as a non-competitive allosteric inhibitor of CXCR1 and CXCR2, blocking downstream signaling (calcium mobilization, chemotaxis) without displacing CXCL8 from the receptors. This prevents neutrophil recruitment and activation in inflammatory conditions [1,2,5]
- Therapeutic Potential: Reparixin is being investigated for treating inflammatory disorders, including ischemia-reperfusion injury, acute lung injury, and rheumatoid arthritis, due to its ability to reduce neutrophil-mediated tissue damage [5]

C₂₀H₃₅N₃O₅S

429.57

429.23

C, 53.97; H, 8.03; N, 8.58; O, 22.87; S, 6.55

266359-93-7

Reparixin; 266359-83-5

9932389

Off-white to light yellow solid powder

4.913

4

7

10

29

495

2

S(C([H])([H])[H])(N([H])C([C@]([H])(C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)(=O)=O.O([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])N([H])[H])=O

JEJFWWFZAQBZMJ-GVKMLHTLSA-N

InChI=1S/C14H21NO3S.C6H14N2O2/c1-10(2)9-12-5-7-13(8-6-12)11(3)14(16)15-19(4,17)18;7-4-2-1-3-5(8)6(9)10/h5-8,10-11H,9H2,1-4H3,(H,15,16);5H,1-4,7-8H2,(H,9,10)/t11-;5-/m10/s1

(2S)-2,6-diaminohexanoic acid;(2R)-2-[4-(2-methylpropyl)phenyl]-N-methylsulfonylpropanamide

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, 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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 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 (5.82 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 (5.82 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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Solubility in Formulation 4: 40 mg/mL (93.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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