| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
BLI-489 targets bacterial beta-lactamases, a family of enzymes produced by Gram-negative and some Gram-positive bacteria that hydrolyze and inactivate beta-lactam antibiotics (penicillins, cephalosporins, carbapenems). Specifically, BLI-489 is active against class A beta-lactamases (including TEM, SHV, and CTX-M-type ESBLs), class C (AmpC beta-lactamases), and some class D beta-lactamases (e.g., OXA-type enzymes). The compound acts as a mechanism-based inhibitor, forming a stable covalent complex with the beta-lactamase active site serine residue, thereby preventing the enzyme from hydrolyzing the companion beta-lactam antibiotic (piperacillin). This irreversible inhibition restores the antibacterial activity of co-administered piperacillin against beta-lactamase-producing resistant bacteria. BLI-489 has minimal intrinsic antibacterial activity itself.
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| ln Vitro |
In vitro susceptibility testing using broth microdilution and checkerboard synergy assays demonstrates that the combination of piperacillin and BLI-489 is active against a broad range of beta-lactamase-producing Gram-negative bacteria, including clinical isolates expressing ESBLs, AmpC enzymes, and some class D beta-lactamases. The combination improves the antibacterial activity of piperacillin alone, reducing MIC values to clinically achievable levels. Compared to piperacillin-tazobactam (the current standard of care in some settings), the piperacillin-BLI-489 combination shows improved activity against strains expressing ESBLs and AmpC enzymes. In vitro time-kill assays confirm the bactericidal activity of the combination. BLI-489 alone has minimal intrinsic antibacterial activity. The compound has been evaluated in a mouse model of systemic infection.
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| ln Vivo |
A dose ratio of 8:1 was determined to be ideal for maintaining increased efficacy based on initial results with different ratios. When given in an 8:1 ratio, piperacillin-BLI-489 was effective against infections in mice brought on by bacteria expressing class A (which includes extended-spectrum beta-lactamases), class C (AmpC), and class D beta-lactamases[2].
The in vivo efficacy of piperacillin in combination with BLI-489 hydrate was evaluated in acute lethal systemic infection mouse models. In this study, mice were infected intraperitoneally with a lethal challenge of various beta-lactamase-producing Gram-negative pathogens, including ESBL-producing Escherichia coli, AmpC-producing Enterobacter cloacae, and class D beta-lactamase-producing Pseudomonas aeruginosa. Piperacillin and BLI-489 were co-administered intravenously or subcutaneously at an 8:1 ratio. The combination was efficacious, producing significant survival rates and reduction of bacterial burden compared to vehicle controls and compared to piperacillin alone. The effective doses of BLI-489 in the combination were consistent with its mechanism of beta-lactamase inhibition. The combination was well tolerated at efficacious doses. Despite promising preclinical data, the compound did not advance to Phase III clinical trials. |
| Enzyme Assay |
A typical non-cellular beta-lactamase inhibition assay for BLI-489 uses purified TEM-1 (class A), AmpC (class C), or OXA-10 (class D) enzymes in a spectrophotometric assay using nitrocefin as a chromogenic substrate. The reaction mixture (1 mL) contains 50 mM phosphate buffer (pH 7.0), enzyme (20-100 nM), varying concentrations of BLI-489 (0.001-10 uM), and 50 uM nitrocefin. The reaction is initiated by adding nitrocefin after pre-incubation of BLI-489 with the enzyme for 10 min at 30degC. Absorbance at 486 nm is monitored over 10 min using a UV-vis spectrophotometer. The IC₅0 value (concentration required to inhibit 50% of enzyme activity) is calculated. For irreversible inhibitors, the second-order inactivation rate constant (k2/Kᵢ, where k2 is the inactivation rate and Kᵢ is the inhibitor concentration at half-maximal inactivation) is determined by measuring the time-dependent loss of enzyme activity in the presence of varying inhibitor concentrations. Pre-steady-state kinetics are also performed by monitoring the reaction progress curve for the first few seconds after mixing.
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| Cell Assay |
In vitro combination susceptibility testing for BLI-489 with piperacillin is performed using the checkerboard broth microdilution method. Bacterial isolates (e.g., E. coli, K. pneumoniae, P. aeruginosa) are grown overnight in Mueller-Hinton broth (MHB) and diluted to approximately 5 × 10⁵ CFU/mL. In 96-well plates, piperacillin is serially diluted two-fold (0.25-512 mg/L) in MHB across the rows, while BLI-489 is serially diluted two-fold (0.125-64 mg/L) down the columns. The bacterial inoculum is added to each well, and plates are incubated at 35degC for 18-24 h. The MIC of piperacillin in the presence of increasing concentrations of BLI-489 is determined. The fractional inhibitory concentration (FIC) index is calculated as FIC index = (MIC piperacillin in combination/MIC piperacillin alone) + (MIC BLI-489 in combination/MIC BLI-489 alone). An FIC index ≤0.5 indicates synergy. Alternatively, time-kill assays are performed: bacterial cultures (~10⁶ CFU/mL) are treated with piperacillin (4× MIC alone), BLI-489 (fixed 4 mg/L), or the combination, and incubated at 37degC. Aliquots are removed at 0, 2, 4, 6, and 24 h, diluted, and plated on MHB agar for CFU enumeration. Synergy is defined as a ≥2 log10 reduction in CFU/mL by the combination compared to the most active single agent at 24 h.
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| Animal Protocol |
In vivo animal studies for BLI-489 hydrate were conducted using acute lethal systemic infection models in female Swiss-Webster or BALB/c mice (6-8 weeks old, 18-22 g). Mice were challenged intraperitoneally with bacterial inocula of ~1-5 × 10⁶ CFU/mouse of beta-lactamase-producing pathogens (e.g., ESBL-producing E. coli, AmpC-producing Enterobacter cloacae) suspended in 5% hog gastric mucin to ensure lethality. One hour post-challenge, mice were treated subcutaneously or intravenously with a fixed ratio of piperacillin:BLI-489 (typically 8:1 weight ratio) at doses of 10-200 mg/kg for piperacillin and 1.25-25 mg/kg for BLI-489. Dosing was administered twice daily for 2-5 days. Efficacy was assessed by survival monitoring (Kaplan-Meier analysis) over 7 days and/or by organ bacterial burden quantification (spleen, liver, kidneys) at necropsy. The PD₅0 (the BLI-489 dose required to achieve 50% survival or 50% reduction of bacterial burden) was calculated. Pharmacokinetic studies of piperacillin and BLI-489 in mice were also performed to support PK/PD correlation. All animal procedures must be approved by the institutional animal care and use committee.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of BLI-489 have been evaluated in animal models (mice) as part of its preclinical development. BLI-489 is formulated for intravenous or subcutaneous administration. Following subcutaneous administration in mice, BLI-489 is rapidly absorbed, with maximum plasma concentrations (Cmax) achieved within 0.5-1 h (Tmax). The compound has a moderately short elimination half-life (t1/2) of approximately 1-2 h in mice. The volume of distribution (Vd) is consistent with distribution into extracellular fluid. BLI-489 is primarily eliminated unchanged by renal excretion, as it does not undergo extensive metabolic transformation. The area under the plasma concentration-time curve (AUC) is dose-proportional over the tested therapeutic range. In combination with piperacillin, no significant pharmacokinetic drug-drug interaction between the two agents was observed, allowing for straightforward PK/PD modeling for the combination. Clinical pharmacokinetic data in humans were likely evaluated during the discontinued clinical development program, but specific parameters are not publicly available.
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| Toxicity/Toxicokinetics |
Toxicological data for BLI-489 hydrate have been evaluated in preclinical animal models, though specific findings are not extensively reported in the public literature due to the discontinued development program. Standard acute and subchronic toxicity studies would have been conducted as part of the preclinical safety package. The compound is generally well tolerated at efficacious doses in mouse models, with no significant adverse effects reported at therapeutic doses (e.g., BLI-489 up to 25 mg/kg in combination with piperacillin). The safety profile is consistent with the mechanism of action involving beta-lactamase inhibition without inhibition of human serine hydrolase enzymes. Off-target activity against human enzymes is expected to be minimal due to the specificity for bacterial beta-lactamases. No mutagenicity or genotoxicity has been reported. Reproductive and developmental toxicity studies have not been publicly disclosed. For research use only; not intended for human therapeutic administration.
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| References |
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| Additional Infomation |
BLI-489 hydrate is not approved for human therapeutic use. Clinical development by Wyeth was discontinued following evaluation of clinical data, and the compound is now only available for research purposes. Its mechanism of action involves irreversible inhibition of class A, C, and some class D beta-lactamases, thereby restoring the antibacterial activity of companion beta-lactam antibiotics (piperacillin). The combination of piperacillin and BLI-489 showed improved efficacy compared to piperacillin-tazobactam against ESBL- and AmpC-producing strains, which is a significant unmet medical need due to increasing antibiotic resistance. No Phase III clinical trials have been completed. The compound remains a valuable tool for studying beta-lactamase inhibition and for use in in vitro and in vivo models of bacterial resistance. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C13H12N3NAO5S
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| Molecular Weight |
345.306252479553
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| Exact Mass |
345.039
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| CAS # |
2580120-08-5
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| PubChem CID |
117072614
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| Appearance |
Light yellow to yellow solid powder
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
23
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| Complexity |
582
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1COCC2=NC(=CN21)/C=C/3\[C@@H]4N(C3=O)C(=CS4)C(=O)[O-].O.[Na+]
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| InChi Key |
FOAVDIXLMXLMAH-UVEQJXRBSA-M
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| InChi Code |
InChI=1S/C13H11N3O4S.Na.H2O/c17-11-8(12-16(11)9(6-21-12)13(18)19)3-7-4-15-1-2-20-5-10(15)14-7;;/h3-4,6,12H,1-2,5H2,(H,18,19);;1H2/q;+1;/p-1/b8-3-;;/t12-;;/m1../s1
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| Chemical Name |
sodium;(5R,6Z)-6-(6,8-dihydro-5H-imidazo[2,1-c][1,4]oxazin-2-ylmethylidene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate;hydrate
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | 2.8959 mL | 14.4797 mL | 28.9595 mL | |
| 5 mM | 0.5792 mL | 2.8959 mL | 5.7919 mL | |
| 10 mM | 0.2896 mL | 1.4480 mL | 2.8959 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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