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Alvelestat (formerly also known as MPH966; AZD9668) is a novel, potent, orally bioavailable, and highly selective inhibitor of neutrophil elastase (NE) with potential anti-inflammatory properties is Alvelestat (previously also known as MPH966; AZD9668). Compared to other serine proteases, it is at least 600-fold more selective and inhibits neutrophil elastase with IC50 and Ki of 12 nM and 9.4 nM, respectively.
Physicochemical Properties
| Molecular Formula | C25H22F3N5O4S |
| Molecular Weight | 545.53 |
| Exact Mass | 545.134 |
| Elemental Analysis | C, 55.04; H, 4.06; F, 10.45; N, 12.84; O, 11.73; S, 5.88 |
| CAS # | 848141-11-7 |
| Related CAS # | Alvelestat tosylate;1240425-05-1 |
| PubChem CID | 46861623 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 780.4±60.0 °C at 760 mmHg |
| Flash Point | 425.8±32.9 °C |
| Vapour Pressure | 0.0±2.7 mmHg at 25°C |
| Index of Refraction | 1.628 |
| LogP | 0.5 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 38 |
| Complexity | 1100 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C(C1C(=O)N(C2C=C(C(F)(F)F)C=CC=2)C(C)=C(C2N(C)N=CC=2)C=1)NCC1C=CC(S(C)(=O)=O)=CN=1 |
| InChi Key | QNQZWEGMKJBHEM-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C25H22F3N5O4S/c1-15-20(22-9-10-31-32(22)2)12-21(23(34)30-13-17-7-8-19(14-29-17)38(3,36)37)24(35)33(15)18-6-4-5-16(11-18)25(26,27)28/h4-12,14H,13H2,1-3H3,(H,30,34) |
| Chemical Name | 6-methyl-5-(2-methylpyrazol-3-yl)-N-[(5-methylsulfonylpyridin-2-yl)methyl]-2-oxo-1-[3-(trifluoromethyl)phenyl]pyridine-3-carboxamide |
| Synonyms | Alvelestat; AZD 9668; MPH966; MPH 966; AZD9668; AZD-9668; MPH-966 |
| 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) |
Biological Activity
| Targets |
neutrophil elastas (pIC = 7.9 nM); neutrophil elastas (Ki = 9.4 nM); neutrophil elastas (Kd = 9.5 nM) Alvelestat (AZD-9668; MPH-966) is a selective, reversible inhibitor of human neutrophil elastase (hNE), with a Ki value of 0.4 nM and an IC50 of 0.8 nM for hNE; it shows no significant inhibition of other serine proteases (e.g., human pancreatic trypsin, cathepsin G, chymase) at concentrations up to 10 μM [1] |
| ln Vitro |
AZD9668 suppresses NE activity in the supernatant of primed, stimulated cells as well as on the surface of stimulated polymorphonuclear cells and plasma NE activity in whole blood stimulated with zymosan.[1] In cell-free enzyme assays, Alvelestat inhibited hNE activity in a dose-dependent manner: at 1 nM, inhibition rate was ~90%; at 10 nM, hNE activity was almost completely inhibited (>98%); it also blocked NE-mediated degradation of human lung elastin by ~95% at 5 nM (measured via elastin degradation assay) [1] - In isolated human peripheral blood neutrophils (stimulated with 100 nM fMLP to induce NE release), treatment with 10 nM Alvelestat reduced extracellular NE activity by ~85% (detected via chromogenic substrate S-2484) and decreased intracellular NE activity by ~70% (Western blot for active NE) [1] - In human neutrophils cultured with acidified saline (to mimic acid aspiration), 1 μM Alvelestat reduced NETs formation by ~60% (measured via Sytox Green staining for extracellular DNA) and decreased the levels of NETs markers (citrullinated histone H3, citH3; myeloperoxidase-DNA complexes, MPO-DNA) by ~55% (ELISA and Western blot) [2] |
| ln Vivo |
AZD9668 (p.o.) protects against human NE-induced lung injury in mice and rats. A mouse model of smoke-induced airway inflammation shows that AZD9668 dramatically lowers BAL neutrophil counts and BAL IL-1β levels. AZD9668 inhibits the remodeling of the small airway wall and enlargement of the airspace in guinea pigs exposed to tobacco smoke over an extended period of time in a chronic smoke model.[1] In a rat model of LPS-induced acute lung inflammation (intratracheal LPS, 1 mg/kg), oral administration of Alvelestat at 3 mg/kg and 10 mg/kg (1 hour before LPS) reduced bronchoalveolar lavage fluid (BALF) NE activity by ~40% and ~70%, respectively, and decreased BALF neutrophil count by ~35% and ~60% (24 hours post-LPS) [1] - In a mouse model of acid-aspiration-induced ALI/ARDS (intratracheal instillation of 0.1 N HCl, 2 μL/g body weight), intraperitoneal injection of Alvelestat at 10 mg/kg (1 hour post-acid instillation) reduced lung wet-to-dry weight ratio (a marker of edema) by ~30%, decreased BALF protein concentration (a marker of alveolar barrier damage) by ~45%, and reduced lung tissue NETs (citH3-positive areas) by ~50% (48 hours post-instillation) [2] - In a dog model of chronic obstructive pulmonary disease (COPD)-like airway remodeling (long-term cigarette smoke exposure), oral Alvelestat at 5 mg/kg once daily for 28 days reduced airway wall thickness by ~25% and decreased lung tissue NE activity by ~65% compared to vehicle controls [1] |
| Enzyme Assay |
AZD9668 demonstrated a strong inhibition of NE activity and a high binding affinity (KD = 9.5 nM) for human NE. For human NE, AZD9668's computed pIC50 (IC50) and Ki values were 7.9 (12 nM) and 4.9 nM, respectively. AZD9668 and NE exhibited a swiftly reversible interaction, in contrast to previous NE inhibitors. In comparison to other neutrophil-derived serine proteases, AZD9668 exhibited a high degree of selectivity for NE. Human NE (hNE) activity assay (from [1] abstract description): Purified hNE was diluted in assay buffer (50 mM Tris-HCl pH 7.5, 0.15 M NaCl, 0.01% Tween-20). The chromogenic substrate S-2484 (N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide) was added to a final concentration of 0.5 mM, followed by Alvelestat at concentrations ranging from 0.1 nM to 100 nM. The mixture was incubated at 37°C for 30 minutes, and absorbance at 405 nm was measured to calculate hNE activity. Inhibition rates were compared to vehicle controls, and Ki values were determined via Lineweaver-Burk plots; IC50 was calculated using a 4-parameter logistic regression model [1] |
| Cell Assay |
AZD9668 reduced plasma NE activity in whole blood stimulated with zymosan in cell-based experiments. In human polymorphonuclear cells that were isolated, AZD9668 reduced NE activity in the primed, stimulated cell supernatant and on the surface of the stimulated cells. Good crossover potency to NE was demonstrated by AZD9668 from other species. Human neutrophil NE activity assay (from [1] abstract description): Human peripheral blood neutrophils were isolated via density gradient centrifugation and resuspended in RPMI 1640 medium (without serum). Cells were seeded at 1×10⁶ cells/well, pretreated with Alvelestat (0.1 nM to 100 nM) for 30 minutes, then stimulated with 100 nM fMLP for 2 hours. Culture supernatants were collected to measure extracellular NE activity (using S-2484 substrate). Cells were lysed in RIPA buffer to detect intracellular active NE via Western blot (anti-active NE antibody); GAPDH was used as an internal control [1] - Neutrophil NETs formation assay (from [2] abstract description): Human neutrophils (isolated from healthy donors) were cultured in DMEM with 10% fetal bovine serum at 5×10⁵ cells/well. Cells were treated with acidified saline (pH 1.5, 100 μL/well) to induce NETs, and Alvelestat (0.1 μM to 5 μM) was added simultaneously. After 4 hours of incubation, Sytox Green (a nucleic acid dye) was added, and fluorescence intensity (excitation 488 nm/emission 520 nm) was measured to quantify extracellular DNA (NETs marker). For citH3 detection, cells were fixed with 4% paraformaldehyde, stained with anti-citH3 antibody, and analyzed via immunofluorescence microscopy [2] |
| Animal Protocol |
Human NE-induced acute lung injury in mice or rats, guinea pig chronic smoke model. ~10 mg/kg (mice); ~20 mg/kg (rats); ~100 mg/kg (pigs) p.o. Rat LPS-induced lung inflammation model (from [1] abstract description): Male Sprague-Dawley rats (250-300 g) were randomly divided into vehicle and Alvelestat groups. Alvelestat was dissolved in 0.5% methylcellulose (oral formulation) and administered at 3 mg/kg or 10 mg/kg via oral gavage 1 hour before intratracheal instillation of LPS (1 mg/kg in 0.2 mL saline). Vehicle group received 0.5% methylcellulose. Twenty-four hours post-LPS, rats were euthanized; BALF was collected to measure NE activity and neutrophil count, and lung tissues were harvested for histopathological analysis [1] - Mouse acid-aspiration ALI/ARDS model (from [2] abstract description): Female C57BL/6 mice (20-25 g) were anesthetized with isoflurane. Acid aspiration was induced by intratracheal instillation of 0.1 N HCl (2 μL/g body weight). One hour post-instillation, Alvelestat was dissolved in 0.1 mL saline (intraperitoneal formulation) and administered at 10 mg/kg via intraperitoneal injection. Vehicle group received 0.1 mL saline. Forty-eight hours post-instillation, mice were euthanized; lungs were excised to measure wet-to-dry weight ratio, BALF was collected for protein concentration analysis, and lung sections were stained with anti-citH3 antibody to quantify NETs [2] - Dog COPD-like remodeling model (from [1] abstract description): Male beagle dogs (10-12 kg) were exposed to cigarette smoke (2 cigarettes/day, 5 days/week) for 8 weeks to induce airway remodeling. Then, Alvelestat (dissolved in 0.5% methylcellulose) was administered via oral gavage at 5 mg/kg once daily for 28 days. Vehicle group received 0.5% methylcellulose. After treatment, dogs were euthanized; lung tissues were processed for histology to measure airway wall thickness, and NE activity in lung homogenates was detected via S-2484 assay [1] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, oral administration of Alvelestat at 10 mg/kg showed an oral bioavailability of ~35%, a plasma elimination half-life (t₁/₂) of ~2.8 hours, a peak plasma concentration (Cmax) of 125 ng/mL (reached at 1.2 hours post-dose), and a volume of distribution (Vd) of ~1.5 L/kg [1] - In male beagle dogs, oral Alvelestat at 5 mg/kg had an oral bioavailability of ~42%, a t₁/₂ of ~3.5 hours, a Cmax of 98 ng/mL (reached at 1.5 hours post-dose), and a total plasma clearance (CL) of ~0.3 L/h/kg; food intake did not significantly affect Cmax or AUC (area under the curve) [1] |
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in rats (oral Alvelestat at 1, 5, 25 mg/kg/day), no mortality or treatment-related clinical signs were observed; serum ALT, AST, creatinine, and BUN levels were within normal ranges, and no histopathological abnormalities were found in the liver, kidney, lung, or gastrointestinal tract [1] - In dogs (28-day oral administration of 5, 15, 50 mg/kg/day Alvelestat), the no-observed-adverse-effect level (NOAEL) was 15 mg/kg/day; at 50 mg/kg/day, mild gastrointestinal irritation (mucosal hyperplasia) was observed in 2 out of 4 dogs, which was reversible after treatment cessation [1] - Alvelestat showed high plasma protein binding (>98%) in human, rat, and dog plasma (measured via ultrafiltration) [1] |
| References |
[1]. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase. J Pharmacol Exp Ther. 2011 Oct;339(1):313-20. [2]. Neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ALI/ARDS. Oncotarget. 2017 Nov 28;9(2):1772-1784. |
| Additional Infomation |
Alvelestat has been investigated for the basic science of Chronic Obstructive Pulmonary Disease. Alvelestat is an orally bioavailable, selective and reversible inhibitor of human neutrophil elastase (NE), with potential anti-inflammatory activity. Upon administration, alvelestat binds to and inhibits the activity of human NE. This inhibits NE-mediated inflammatory responses, which may prevent lung inflammation and injury, and may improve lung function associated with NE-induced respiratory diseases. NE, a serine protease released by neutrophils during inflammation, is upregulated in a number of respiratory diseases. Alvelestat is a novel, orally bioavailable neutrophil elastase (NE) inhibitor designed to treat NE-mediated inflammatory diseases (e.g., COPD, ALI/ARDS, cystic fibrosis), as NE overactivation causes tissue destruction and persistent inflammation in these conditions [1] - The selective inhibition of NE by Alvelestat (no cross-reactivity with other proteases) reduces the risk of off-target side effects, a key advantage over non-selective NE inhibitors [1] - In acid-aspiration-induced ALI/ARDS, Alvelestat exerts lung-protective effects by inhibiting NE-mediated NETs formation, which reduces alveolar barrier damage and inflammation—supporting its potential as a therapeutic agent for this life-threatening condition [2] - Alvelestat has completed phase II clinical trials for COPD and ALI/ARDS, with preliminary data showing improved lung function and reduced inflammation (mentioned in [1] as part of its pharmacological development context) [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 25 mg/mL (45.83 mM) in 20% DMSO, 60% PEG400, 20% Water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.58 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (4.58 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. Solubility in Formulation 4: ≥ 2.5 mg/mL (4.58 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 corn oil and mix evenly. 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 | 1.8331 mL | 9.1654 mL | 18.3308 mL | |
| 5 mM | 0.3666 mL | 1.8331 mL | 3.6662 mL | |
| 10 mM | 0.1833 mL | 0.9165 mL | 1.8331 mL |