Matrix metalloproteinase 12 (MMP-12)
MMP-12 (macrophage elastase). Aderamastat is a potent, selective, and orally active MMP-12 inhibitor with >90-fold selectivity over MMP-2 and 200-300-fold selectivity over other MMPs.

Aderamastat is a highly selective oral MMP-12 inhibitor targeting inflammatory and fibrotic diseases. MMP-12 plays a role in asthma pathophysiology and is associated with disease severity.

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In vitro, Aderamastat is a highly selective and potent MMP-12 inhibitor. It shows approximately 90-fold selectivity over MMP-2 and 200-300-fold selectivity over other MMP family members. The compound effectively inhibits the enzymatic activity of MMP-12, which plays a role in asthma pathophysiology and is associated with disease severity.

Aderamastat showed sustained anti-inflammatory effects and attenuated allergen-induced histopathology in a house dust mite (HDM) mouse model. FP‐025 in a dose‐dependent manner significantly attenuated AHR, BAL inflammatory cell numbers and lung pathohistology in a mouse model of persistent HDM‐allergic asthma. FP‐025 markedly reduced MMP‐12 expression in airway epithelial cells and levels of MMP‐12 in lung parenchyma and airways, indicating that the enhanced expression in allergic asthma depends on an autocrine process. FP‐025 did not negatively affect anti‐viral responses, contributing to its safety profile. These findings warrant interventions with FP‐025 in allergic asthma, also as FP‐025 showed safety, tolerability, and good pharmacokinetics characteristic in a randomized, placebo‐controlled, single, and multiple ascending dose study in healthy subjects.

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In vivo, Aderamastat showed sustained anti-inflammatory effects and attenuated allergen-induced histopathology in a house dust mite (HDM) mouse model of allergic asthma. It is an orally active compound in development for respiratory conditions, including chronic inflammatory airway diseases and pulmonary fibrosis. Positive primary outcomes from a Phase 2a clinical trial were reported.

MMP-12 quantification[2]
Levels of MMP-12 in BAL and lung homogenates were measured by ELISA (MMP-12, LSBio, LS-F2819) according to manufacturers’ specifications.

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For non-cellular assays, recombinant human MMP-12 is incubated with a fluorogenic FRET peptide substrate in a standard MMP assay buffer (e.g., 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl). Aderamastat is added at varying concentrations (0.1-1000 nM). The increase in fluorescence is monitored (excitation 325 nm, emission 393 nm). The IC50 value is calculated from the dose-response curve. Selectivity is assessed by testing the compound against a panel of other MMPs.

Sampling and primary cell isolation[2]
Bronchoalveolar lavage (BAL) was collected as previously described. Briefly, mice were cannulated through the trachea and the lavage was performed by instilling and retrieving 1 mL of sterile NaCl 0.9% into the right lung. BAL recovered was centrifuged, the supernatant was stored, whereas the pellet was immediately suspended in sterile PBS. Cell counts were then determined for each BAL sample and the cellular subset phenotypes were determined by FACS analysis. To remove the intravascular pool of cells, lungs were perfused with 5 ml of sterile NaCl 0.9% via the pulmonary circulation. The left lungs (not lavaged) were removed and minced using iridectomy scissors, thereafter enzymatically digested and grinded, as previously described2,3. Briefly, to prepare a lung homogenate, lungs were incubated for 1 hour at 37C with Collagenase type III (200 U/mL Worthington Biochemical, Lakewood, NJ) and DNAse I (100 U/mL; grade II from bovine pancreas, Sigma) in RPMI 1640. Pulmonary cell suspensions were obtained by grinding the tissue through 70-mm nylon sieves, and red blood cells were lysed with ammonium chloride buffer. Cell counts were then performed before staining for flow cytometric analysis. The cell suspension was lysed with Greenberger buffer (75 mmol/L NaCl, 7.5 mmol/L Tris, 0.5 mmol/L MgCl2, 0.5 mmol/L CaCl2, 0.5% Triton X-100, 4 mg/mL 4-[2-aminoethyl] benzenesulfonyl fluoride, 50 mg/mL Na2-EDTA, 10 ng/mL Pepstatin A, and 10 ng/mL leupeptin, pH 7.4) for 30 minutes on ice, followed by centrifugation at 680g for 10 minutes. Supernatants were stored at -80°C until further use. Cell counts were then performed before staining for flow cytometry analysis. Supernatants were stored at -80°C until further use.

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For cell-based assays, primary human alveolar macrophages or monocyte-derived macrophages are treated with Aderamastat (1-100 nM) and then stimulated with LPS or other activators to induce MMP-12 expression. The activity of MMP-12 in the cell culture supernatant is measured using the FRET substrate described above. The ability of the compound to inhibit the enzymatic activity of MMP-12 secreted into the medium can be assessed.

Male 8-week-old C57BL/6J mice purchased from Charles River Laboratories (France) were housed under specific pathogen-free conditions and maintained on a 12-hour light – dark cycle with access to food and water ad libitum. The sensitization schedule to elicit a severe and persistent allergic inflammation of the airways was based on a well-characterized model. In short, groups of C57BL/6J mice were randomly assigned to be treated for 5 days a week for five consecutive weeks with either the aeroallergen house dust mite (HDM) or NaCl through intranasal (i.n.) administration. Lyophilized HDM extract was resuspended in sterile PBS at a concentration of 5 mg (protein)/ml, and 25 µg (5 µl) protein per dose per mouse was administered i.n. in a final volume of 20 µl (in NaCl) in mice lightly anesthetized with isofluorane. The H3N2 influenza A virus HK X31 stocks and viral titers were obtained by infecting Madin–Darby canine kidney (MDCK) as described before. The viral stock suspension was diluted in NaCl and animals (lightly anesthetized with isofluorane) received i.n. 20 TCID50 in 50 µl or not. The MMP-12 inhibitor FP-025 , was dissolved in sterile NaCl 0.5% methyl cellulose containing 0.2% Tween-80 and administered orally, daily, for 7 consecutive days, at a dose of 10 mg/kg or 30 mg/kg or 100 mg/kg in a volume of 50 µl per mouse. Prednisone was diluted in sterile NaCl (5 mg/kg) in 100 µl and administered daily via the intraperitoneal (i.p) route [2].

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For animal studies, Aderamastat is administered orally to mice in an allergen challenge model, such as the house dust mite (HDM)-induced allergic airway inflammation model. Doses are typically in the range of 1-10 mg/kg, given once or twice daily. Efficacy is assessed by measuring the levels of inflammatory cells (eosinophils) in the bronchoalveolar lavage fluid (BALF), by quantifying cytokines and chemokines, and by histopathological analysis of lung tissue.

Aderamastat (FP-025) is an orally active small molecule. A bioanalytical method (LC-MS/MS) has been developed and validated for the quantification of Aderamastat in human plasma to support pharmacokinetic studies. The compound is intended to be orally bioavailable, with pharmacokinetic properties suitable for once-daily or twice-daily dosing.

In a completed Phase 2a clinical trial for allergic asthma, Aderamastat was reported to have positive outcomes and was likely well-tolerated, as the safety findings were not highlighted as a reason for termination. However, specific safety data from this trial have not been published. As an experimental compound, it would have been subjected to standard preclinical toxicology studies (safety pharmacology, genotoxicity, repeat-dose toxicity) to support clinical development.

[1]. Safe administration of MMP-12 inhibitor. Patent. WO2022261624 A1.

Aderamastat (FP-025) has been developed by Foresee Pharmaceuticals. The company completed a Phase 2a proof-of-concept clinical trial in the Netherlands for allergic asthma in 2023 with positive results. The drug was being investigated for its potential to treat rare immuno-fibrotic diseases. It is not approved by any regulatory agency and is intended for research use only. As of 2024, clinical development may have been discontinued, but it remains a valuable research tool.

394.099

877176-23-3

67177374

White to light yellow solid powder

2

5

6

28

567

0

CC1=CC(=CC=C1)COC2=CC=C(C=C2)SC3=C(OC=C3)C4C(=O)NC(=O)N4

CXEKSVVDMIAMLF-UHFFFAOYSA-N

InChI=1S/C21H18N2O4S/c1-13-3-2-4-14(11-13)12-27-15-5-7-16(8-6-15)28-17-9-10-26-19(17)18-20(24)23-21(25)22-18/h2-11,18H,12H2,1H3,(H2,22,23,24,25)

5-[3-[4-[(3-methylphenyl)methoxy]phenyl]sulfanylfuran-2-yl]imidazolidine-2,4-dione

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)