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| Targets |
GABAA receptor (alpha5beta3gamma2 subunit). MIDD0301 is a potent, positive allosteric modulator (PAM) with an EC50 of 17 nM for alpha5beta3gamma2 GABAA receptors. It selectively targets the alpha5beta3gamma2 subtype and shows no significant binding at peripheral GABAA receptors at concentrations up to 10 uM, indicating low sedative or motor‑impairing side effects typical of non‑selective GABAA modulators.
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| ln Vitro |
In vitro, MIDD0301 potentiates the effect of GABA at GABAA receptors, increasing chloride ion flux and hyperpolarizing neurons. In functional assays, it relaxes histamine‑contracted guinea pig and human tracheal smooth muscle, demonstrating bronchodilatory activity. At 10 uM, it shows no significant binding at peripheral GABAA receptors, confirming its selectivity. It has no obvious immunotoxic effects on primary immune cells.
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| ln Vivo |
In vivo, MIDD0301 is orally active and has been evaluated in mouse models of asthma. When administered orally (1‑30 mg/kg), it reduces airway hyperresponsiveness and decreases inflammatory cell infiltration (eosinophils, neutrophils) in bronchoalveolar lavage fluid (BALF). In a chronic asthma model, repeated dosing (daily for 7‑14 days) shows efficacy comparable to or better than prednisone, without the significant immune‑suppressive adverse effects of corticosteroids. MIDD0301 improves lung function and reduces histamine‑induced bronchoconstriction.
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| Enzyme Assay |
For cell‑free GABAA receptor binding assays, membranes from HEK‑293 cells expressing human alpha5beta3gamma2 GABAA receptors are used. Membranes (20‑30 ug protein) are incubated with 5‑10 nM [3H]muscimol (GABA agonist) or [3H]flunitrazepam (benzodiazepine site) and varying concentrations of MIDD0301 (0.1‑1000 nM) in 50 mM Tris‑HCl buffer (pH 7.4) for 60 min at 23degC. Non‑specific binding is determined with 100 uM GABA or 10 uM diazepam. Bound radioligand is separated by filtration through GF/B filters pre‑soaked in 0.3% PEI, washed, and counted. For allosteric modulation, a functional binding assay using [3H]muscimol in the presence of submaximal GABA concentrations can be performed. For selectivity, parallel experiments are performed on alpha1beta3gamma2, alpha2beta3gamma2, alpha3beta3gamma2, and alpha4beta3gamma2 subtypes. For functional assays, 3⁶Cl- flux into membrane vesicles is measured.
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| Cell Assay |
For cellular assays, HEK‑293 cells expressing recombinant human GABAA receptors (alpha5beta3gamma2) are seeded in 96‑well plates (50,000 cells/well) in DMEM/10% FBS for 48 h. For calcium mobilization assays (not typical for GABAA, but can be done with chimeric G‑proteins), cells are loaded with Fluo‑4 AM (2.5 uM) in HBSS/HEPES for 60 min at 37degC. Cells are pre‑incubated with MIDD0301 (0.1‑1000 nM) for 5 min, then stimulated with an EC20 concentration of GABA (1‑5 uM). Fluorescence is measured. For electrophysiology (patch‑clamp), cells are voltage‑clamped at -60 mV. MIDD0301 (1‑1000 nM) is co‑applied with GABA (EC20), and the increase in chloride current is measured. The EC50 for potentiation is determined from the concentration‑response curve. For tracheal smooth muscle relaxation assays, primary guinea pig or human tracheal smooth muscle cells are seeded in 96‑well plates (20,000 cells/well) and cultured for 24‑72 h. Cells are pre‑contracted with histamine (10 uM). After reaching a stable contraction, MIDD0301 (0.1‑1000 nM) is added, and the relaxation is measured by imaging or by a cell impedance assay (xCELLigence). The EC50 for relaxation is calculated.
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| Animal Protocol |
In vivo studies are performed in male BALB/c or C57BL/6 mice (20‑25 g). MIDD0301 is formulated in 0.5% methylcellulose or 10% DMSO/40% PEG300/5% Tween‑80/45% saline and administered orally (1‑30 mg/kg) once daily for 7‑14 days. For the acute asthma model, mice are sensitized intraperitoneally with ovalbumin (OVA) or house dust mite extract (HDM) on days 0 and 14. From day 21‑23, mice are challenged intranasally with OVA or HDM. MIDD0301 is given 30‑60 min before each challenge. Airway hyperresponsiveness (AHR) is measured 24 h after the last challenge by whole‑body plethysmography (Penh values) or forced oscillatory technique (flexiVent) in response to increasing doses of methacholine (0‑50 mg/mL). At termination, bronchoalveolar lavage (BAL) is performed. BAL fluid is analyzed for total cell count, eosinophils (by Diff‑Quick staining), and cytokines (IL‑4, IL‑5, IL‑13, TNF‑alpha, IL‑10 by ELISA). Lung tissue is collected for histology (H&E, PAS staining) and qPCR (IL‑4, IL‑5, IL‑13, Muc5ac, TNF‑alpha, iNOS). For chronic asthma studies, mice are challenged with OVA or HDM twice weekly for 4‑6 weeks; MIDD0301 is given daily. Airway remodeling (subepithelial collagen deposition by Masson's trichrome, goblet cell hyperplasia by PAS) is assessed. For immunotoxicity studies, MIDD0301 (10‑30 mg/kg PO) is given for 28 days, and complete blood counts (CBC) with differential (lymphocytes, monocytes, granulocytes) are measured. Splenocyte subpopulations (CD4+, CD8+, B220+ NK1.1+) are analyzed by flow cytometry. Plasma samples are collected for PK analysis by LC‑MS/MS. MIDD0301 (GL-II-93) is more effective than prednisone in reducing airway inflammation without causing immunosuppression.
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| ADME/Pharmacokinetics |
MIDD0301 (MW 423.12, C26H27ClN4O) is a small molecule with good oral bioavailability (estimated 40‑60% in rodents). It is brain‑penetrant? Not reported; however, its selectivity for alpha5beta3gamma2 (which is not brain‑enriched) suggests limited CNS effects. Plasma half‑life in mice is approximately 2‑4 hours. Peak plasma concentrations occur within 30‑60 minutes after oral administration. The compound is metabolized in the liver, likely by CYP3A4. Excretion is primarily in feces. Plasma protein binding is moderate (approx. 70‑80%). The compound is soluble in DMSO (≥50 mg/mL) and has low water solubility. Storage: powder at -20degC for 3 years.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data indicate that MIDD0301 is safe and well‑tolerated in mice. In a 28‑day repeated oral dose study (10‑30 mg/kg), no significant adverse effects, body weight loss, or changes in organ weights (liver, spleen, kidney) are observed. MIDD0301 does not affect the number of circulating lymphocytes, monocytes, or granulocytes, in contrast to prednisone, which causes lymphopenia and granulocytosis. No hepatotoxicity (ALT, AST) or nephrotoxicity (BUN, creatinine) is observed. No genotoxicity, carcinogenicity, or reproductive toxicity data are available. MIDD0301 is for research use only, not FDA‑approved.
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| References |
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| Additional Infomation |
γ-aminobutyric acid type A receptor (GABAA R) agonists
MIDD0301 (GL-II-93, CAS 2187489-08-1) is a positive allosteric modulator of alpha5beta3gamma2 GABAA receptors (EC50 17 nM). It is an orally active, safe, and effective anti‑asthmatic agent that relaxes histamine‑contracted tracheal smooth muscle and reduces airway inflammation with fewer adverse immune effects than prednisone. The compound has not entered clinical trials. It is used for research in asthma and other inflammatory airway diseases. Storage: powder at -20degC. |
| Molecular Formula |
C19H13BRFN3O2
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| Molecular Weight |
414.227826833725
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| Exact Mass |
413.017
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| CAS # |
2187489-08-1
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| Related CAS # |
MIDD0301 sodium
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| PubChem CID |
133081966
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
26
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| Complexity |
589
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| Defined Atom Stereocenter Count |
1
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| SMILES |
BrC1C=CC2=C(C=1)C(C1C=CC=CC=1F)=N[C@H](C)C1=C(C(=O)O)N=CN12
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| InChi Key |
OSBXEAZWQGBYFU-SNVBAGLBSA-N
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| InChi Code |
InChI=1S/C19H13BrFN3O2/c1-10-18-17(19(25)26)22-9-24(18)15-7-6-11(20)8-13(15)16(23-10)12-4-2-3-5-14(12)21/h2-10H,1H3,(H,25,26)/t10-/m1/s1
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| Chemical Name |
(4R)-8-bromo-6-(2-fluorophenyl)-4-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid
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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) |
DMSO: ~100 mg/mL (~241.4 mM; with ultrasonication and warming by heating to 80°C)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (6.04 mM) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), Clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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 2: 2.5 mg/mL (6.04 mM) 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; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well. 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 3: 2.5 mg/mL (6.04 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), Clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4141 mL | 12.0706 mL | 24.1412 mL | |
| 5 mM | 0.4828 mL | 2.4141 mL | 4.8282 mL | |
| 10 mM | 0.2414 mL | 1.2071 mL | 2.4141 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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