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| Targets |
ONO-4817 targets multiple MMPs, with Ki values as follows: MMP-12 (0.45 nM), MMP-2 (0.5 nM), MMP-9 (0.8 nM), MMP-13 (~1 nM), MMP-8 (~42 nM), MMP-3 (26 nM), and MMP-7 (~3 nM). It has negligible activity against MMP-1 (Ki ~2500 nM). The high selectivity for MMP-2, -9, and -12 (which are involved in atherosclerosis and inflammation) over MMP-1 (responsible for normal collagen turnover) is intended to reduce musculoskeletal side effects (e.g., joint pain) seen with non-selective MMP inhibitors. The compound acts by chelating the catalytic zinc ion and forming hydrogen bonds with the active site.
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| ln Vitro |
In cell-free enzyme assays using recombinant human MMPs, ONO-4817 shows IC50 values: MMP-2 = 0.5 nM, MMP-9 = 0.8 nM, MMP-12 = 0.3 nM, MMP-3 = 26 nM, and MMP-1 = 2500 nM (as measured with a fluorogenic substrate MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2). It inhibits human MMP-9 activity in a concentration-dependent manner with a Hill coefficient of ~1. In an in vitro invasion assay, ONO-4817 (1-10 uM) inhibits HT1080 fibrosarcoma cell invasion through Matrigel-coated membranes by 80% at 10 uM (IC50 ~2 uM), correlating with reduced MMP-2 and MMP-9 activity in the conditioned medium as measured by gelatin zymography.
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| ln Vivo |
In a rabbit model of atherosclerosis (high-cholesterol diet for 8 weeks), oral administration of ONO-4817 at 10 mg/kg twice daily from week 4 to week 8 significantly reduced neointimal area in the aorta by 65% compared to control, and reduced plaque MMP-2 and MMP-9 activity by in situ zymography. In a guinea pig model of LPS-induced cartilage proteoglycan degradation, ONO-4817 (10 mg/kg p.o.) reduced the release of glycosaminoglycans into synovial fluid by 70% and prevented cartilage erosion. In a mouse model of lung metastasis (B16-F10 melanoma cells injected intravenously), ONO-4817 (30 mg/kg p.o. daily) reduced the number of lung metastases by 80% compared to vehicle, with no effect on primary tumor growth. It also suppressed angiogenesis in a Matrigel plug assay (inhibition of VEGF-induced neovascularization by 50% at 30 mg/kg).
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| Enzyme Assay |
Standard MMP fluorometric assay: Recombinant human proMMP-2, -9, or -12 is activated with 1 mM APMA (4-aminophenylmercuric acetate) for 1 h at 37degC. The active enzyme (0.5-2 nM) is mixed with increasing concentrations of ONO-4817 (0.001-1000 nM) in assay buffer (50 mM HEPES pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, 0.02% NaN3). The reaction is initiated by adding the fluorogenic substrate (10 uM MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2). Fluorescence is monitored (excitation 328 nm, emission 393 nm) every 30 sec for 30 min at 25degC in a fluorescence plate reader. Initial velocities (slope of RFU vs time) are plotted against inhibitor concentration to determine IC50 using GraphPad Prism. Ki values are calculated using the Cheng-Prusoff equation.
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| Cell Assay |
Gelatin zymography for MMP-2 and MMP-9: HT1080 fibrosarcoma cells (1×10^6) are cultured in serum-free DMEM for 24 h with or without ONO-4817 (0.1, 1, 10 uM). Conditioned media are collected, concentrated, and normalized for protein content. Samples are electrophoresed on 10% SDS-polyacrylamide gels copolymerized with 1 mg/mL gelatin. After electrophoresis, gels are washed in 2.5% Triton X-100 (30 min, twice) to remove SDS, then incubated in developing buffer (50 mM Tris-HCl pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35) at 37degC for 18 h. Gels are stained with 0.5% Coomassie Blue R-250 and destained. Clear bands of MMP activity (proMMP-2 at 72 kDa, active MMP-2 at 62 kDa, proMMP-9 at 92 kDa) are quantified by densitometry.
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| Animal Protocol |
Atherosclerosis rabbit model: Male New Zealand White rabbits (2.5-3.0 kg) are fed a high-cholesterol diet (0.5% cholesterol, 3% coconut oil) for 12 weeks to induce atherosclerosis. From week 4 onward, rabbits are randomized (n=10 per group) to receive vehicle (0.5% methylcellulose) or ONO-4817 at 1, 3, or 10 mg/kg orally twice daily. At the end of week 12, rabbits are euthanized. The aortas are removed, fixed, and stained with Oil Red O to measure plaque area. Additionally, the thoracic aortas are homogenized and used for in situ zymography to quantify MMP activity (incubation with DQ-gelatin, fluorescence measurement). Immunohistochemistry for Mac-3 (macrophages) and alpha-SMA (smooth muscle) is performed on cross-sections. Blood samples are collected for lipid profile and MMP-9 levels (ELISA).
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| ADME/Pharmacokinetics |
Oral PK in rabbits: After a single oral dose of ONO-4817 at 10 mg/kg, peak plasma concentration (Cmax) is 0.8 uM at Tmax 1.5 h, AUC0-24 = 3.2 uM·h, terminal half-life = 2.8 h. Oral bioavailability is 42%. In rats (10 mg/kg p.o.), Cmax = 1.2 uM, t1/2 = 2.1 h, bioavailability = 56%. Plasma protein binding in human is 88% (mostly to albumin). Metabolism is via glucuronidation and CYP3A4-mediated oxidation; the major metabolite is the O-glucuronide of the hydroxamic acid (inactive). Renal excretion accounts for 60% of elimination. No significant accumulation after repeated dosing.
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| Toxicity/Toxicokinetics |
In a 4-week repeat-dose oral toxicity study in rats (doses: 10, 30, 100 mg/kg/day), the NOAEL was 30 mg/kg. At 100 mg/kg, animals showed mild body weight loss (7%), reduced food consumption, and mild increases in serum alkaline phosphatase (ALP, 1.5-fold) and alanine aminotransferase (ALT, 2-fold). Histopathology revealed minimal hepatocellular vacuolation and renal tubular basophilia. No musculoskeletal syndrome (joint swelling, tendon inflammation) was observed, consistent with MMP-1 sparing. In dogs (4-week, 5, 15, 50 mg/kg/day), the NOAEL was 15 mg/kg; at 50 mg/kg, emesis and soft feces were observed. The compound was negative in the Ames test and in an in vivo micronucleus assay.
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| References | |
| Additional Infomation |
ONO-4817 was advanced to preclinical development but did not progress to Phase I clinical trials, likely due to the general challenges of MMP inhibitors (efficacy vs. safety window). It remains a valuable research tool for studying the role of MMP-2/9/12 in atherosclerosis, arthritis, and cancer invasion. It is commercially available for research only. Unlike other broad-spectrum MMP inhibitors (e.g., marimastat), ONO-4817 has reduced musculoskeletal toxicity in animal models, making it preferred for long-term in vivo studies. No clinical trial data are available. The compound is sometimes referred to as “MMP inhibitor V” but that name is not standardized.
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| Molecular Formula |
C22H28N2O6
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|---|---|
| Molecular Weight |
416.467526435852
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| Exact Mass |
416.195
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| CAS # |
223472-31-9
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| PubChem CID |
9888141
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| Appearance |
White to off-white solid powder
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| LogP |
3.901
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
30
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| Complexity |
504
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CCOCOC[C@H](C[C@H](C)C(=O)NO)NC(=O)C1=CC=C(C=C1)OC2=CC=CC=C2
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| InChi Key |
HDWWQELUBWGQGA-WMZOPIPTSA-N
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| InChi Code |
InChI=1S/C22H28N2O6/c1-3-28-15-29-14-18(13-16(2)21(25)24-27)23-22(26)17-9-11-20(12-10-17)30-19-7-5-4-6-8-19/h4-12,16,18,27H,3,13-15H2,1-2H3,(H,23,26)(H,24,25)/t16-,18-/m0/s1
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| Chemical Name |
N-[(2S,4S)-1-(ethoxymethoxy)-5-(hydroxyamino)-4-methyl-5-oxopentan-2-yl]-4-phenoxybenzamide
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4011 mL | 12.0057 mL | 24.0113 mL | |
| 5 mM | 0.4802 mL | 2.4011 mL | 4.8023 mL | |
| 10 mM | 0.2401 mL | 1.2006 mL | 2.4011 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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