Ki: 1.9 nM (gastrin); 2.0 nM (CCK-B)[1].
L-365260 specifically targets the cholecystokinin B receptor (CCK-BR, also known as CCK2 receptor) with high affinity, and also binds to gastrin receptors. Ki values are 1.9 nM for gastrin receptors and 2.0 nM for brain CCK-B receptors.

L-365260 hemihydrate has a decreased affinity for gastrin and brain CCK-B (IC50=20–40 nM) receptors in dog tissues, but a similar high affinity for CCK-B receptors in the brains of rats, mice, and humans[1]. In a separate neuron, the CCK8S- and CCK4-mediated depolarization is substantially attenuated by L -365260(1 μM) hemihydrate[2].

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In vitro, L-365260 shows high affinity for CCK-B receptors in the brains of rats, mice, and humans (Ki <5 nM), while showing lower affinity for CCK-B and gastrin receptors in dog tissues (IC50 = 20-40 nM). It interacts with guinea pig gastrin and brain CCK receptors in a stereoselective and competitive manner.

In mice (ED50=0.03 mg/kg), rats (ED50=0.9 mg/kg), and guinea pigs (ED50=5.1 mg/kg), L-365260 hemihydrate (0.1-30 mg/kg; po) inhibits gastrin-stimulated acid secretion[1]. When rats are given a submaximal dose of morphine (4 mg/kg), L-365260 hemihydrate (0.01-10 mg/kg; sc) improves analgesia[3]. The duration of morphine analgesia in rats is markedly extended by L-365260 hemihydrate (0.2 mg/kg; sc twice daily for 5 days)[3].

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L-365260 enhances morphine analgesia and prevents morphine tolerance in vivo. Oral administration antagonizes gastrin-stimulated acid secretion. It also attenuates CCK8S- and CCK4-mediated neuronal depolarization.

Non-cell binding assays for L-365260 are performed using membrane preparations from tissues expressing CCK-B receptors. Brain membranes (cortex or hippocampus) are prepared from rats, guinea pigs, or humans. Tissue is homogenized in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and protease inhibitors. The homogenate is centrifuged at 40,000-50,000 × g for 20 minutes at 4degC, and the pellet is resuspended and washed twice. For radioligand binding assays, 50-100 ug of membrane protein is incubated with 0.05-0.2 nM [3H]-CCK-8S (sulfated cholecystokinin octapeptide) or [¹2⁵I]-CCK-8S as the radioligand, and varying concentrations of L-365260 (0.01-1000 nM) in assay buffer (50 mM Tris-HCl, 5 mM MgCl2, 0.1% BSA, pH 7.4) in a total volume of 200-500 uL. Non-specific binding is determined in the presence of 1 uM unlabeled CCK-8S or 10 uM L-365,260. After incubation at room temperature for 60-120 minutes, bound and free radioligands are separated by rapid filtration through Whatman GF/B glass fiber filters pre-soaked in 0.3% polyethyleneimine (PEI). Filters are washed three times with 3-5 mL of ice-cold buffer, dried, and radioactivity is counted by liquid scintillation or gamma counting. Specific binding is defined as total binding minus non-specific binding. Inhibition curves are generated by plotting specific binding vs. log [L-365260]. IC50 values are determined by non-linear regression using a one-site binding model. Ki values are calculated using the Cheng-Prusoff equation: Ki = IC50 / (1 + [L]/Kd). For selectivity studies, L-365260 is tested against CCK-A receptors (CCK1R) using pancreatic membranes or CHO cells expressing recombinant CCK-A receptors. L-365260 is highly selective for CCK-B over CCK-A receptors (selectivity ratio >100-1000 fold).

Cellular assays for L-365260 are performed using cell lines expressing CCK-B receptors, such as CHO-CCKBR cells, AR42J pancreatic acinar cells, or primary neuronal cultures. For calcium mobilization assays, cells are loaded with 2-5 uM Fluo-4 AM or Fura-2 AM in HBSS buffer with 0.1% BSA for 30-60 minutes at 37degC. After washing, cells are treated with L-365260 (1 nM-10 uM) for 10-15 minutes, then stimulated with the CCK-B agonist CCK-8S (0.1-10 nM) or gastrin. Intracellular calcium ([Ca2+]i) is measured using a fluorescence plate reader (FlexStation) or fluorescence microscope, with excitation at 340/380 nm (Fura-2) or 488 nm (Fluo-4) and emission at 510 nm. The inhibition of peak fluorescence response is calculated relative to agonist-only controls. For cAMP assays, cells co-expressing CCK-B receptors and a CRE-luciferase reporter are treated with L-365260 (1 nM-10 uM) prior to stimulation with agonist. Luminescence is measured after addition of luciferin substrate. For proliferation assays, cells are seeded in 96-well plates, treated with L-365260 (0.1-10 uM) for 24-72 hours, and cell viability is assessed using MTT or CellTiter-Glo assays to evaluate the effect of CCK-B antagonism on cell growth. For neuronal cell assays, primary cortical or hippocampal neurons isolated from embryonic day 18 rat embryos are cultured in Neurobasal medium with B27 supplement. After 7-14 days in vitro (DIV), neurons are treated with L-365260 (10 nM-10 uM) for 10-30 minutes prior to stimulation with CCK-8S (10-100 nM), and neuronal depolarization (measured by calcium imaging or electrophysiology) or neurotransmitter release is assessed.

Animal/Disease Models: Male SD (Sprague-Dawley) rats (300-350 g; Morphine- injected)[3].
Doses: 0.01, 0.05, 0.1, 0.2, 0.75, 1.0, 10.0 mg/kg
Route of Administration: Sc 10 min prior to ip injection of 4 mg/kg Morphine
Experimental Results: Enhanced morphine analgesia.

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In vivo studies with L-365260 are conducted in rats, mice, and guinea pigs to evaluate its effects on pain, gastrointestinal function, and behavior. For morphine analgesia enhancement studies, male Sprague-Dawley rats (200-300 g) are treated with L-365260 (0.1-10 mg/kg, orally, intraperitoneally, or subcutaneously) 30-60 minutes prior to injection of morphine (2-5 mg/kg, SC or IP). Analgesia is assessed using the tail-flick test (immersing the tail in 52-55degC water, measuring latency to withdrawal) or hot plate test (52-55degC, measuring latency to paw lick or jump) at 15, 30, 45, 60, 90, and 120 minutes post-morphine. L-365260 has been shown to enhance the magnitude and duration of morphine analgesia and prevent the development of morphine tolerance when co-administered repeatedly over several days. For gastrointestinal studies, rats or guinea pigs are fasted overnight, then L-365260 is administered orally (0.1-10 mg/kg) 30-60 minutes before an oral or IP challenge with pentagastrin (10-100 microg/kg) or a meal. Gastric acid secretion is measured by collecting gastric juice via an indwelling gastric cannula or by pylorus ligation. In pylorus-ligated rats, the abdomen is opened under isoflurane anesthesia, the pylorus is ligated, and the incision is closed. After 2-4 hours, gastric juice is collected, and total acid output is measured by titration with 0.1 N NaOH to pH 7.0. L-365260 antagonizes gastrin-stimulated acid secretion. For colonic motility studies, L-365260 is administered intravenously or intracerebroventricularly (ICV) to rats, and colonic transit is measured using a bead expulsion test or by injecting a charcoal meal and measuring the distance traveled. For behavioral studies, L-365260 is administered ICV or systemically, and anxiety-like behavior is assessed using the elevated plus maze (EPM) or open field test. For pharmacokinetic studies, L-365260 is administered orally (1-10 mg/kg) to rats, blood samples are collected at various time points, and plasma concentrations are measured by LC-MS/MS. Tissue distribution (brain, GI tract) is assessed to confirm central nervous system penetration.

L-365260 hemihydrate exhibits favorable oral bioavailability in rodents. In rats, following oral administration, L-365260 is rapidly absorbed with peak plasma concentrations achieved within 1-2 hours. The compound demonstrates good brain penetration due to its lipophilic nature (log P ~3-4), making it suitable for studying central CCK-B receptor functions. The hemihydrate form does not significantly alter the pharmacokinetic profile compared to the anhydrous form but may improve crystallinity and stability. The plasma elimination half-life in rats is approximately 2-4 hours. The volume of distribution is moderate, consistent with distribution into tissues including the central nervous system (Vd ~1-3 L/kg). Plasma protein binding is high (>90%). L-365260 undergoes hepatic metabolism, primarily by CYP450 enzymes (likely CYP3A4/5). Metabolites are excreted in bile and urine. The compound is known to be orally active, with in vivo efficacy demonstrated in gastric acid secretion and pain models. Inter-species differences in CCK-B receptor affinity have been noted, with L-365260 showing high affinity for rat, mouse, guinea pig, and human CCK-B receptors but lower affinity for dog CCK-B receptors (IC50 20-40 nM vs. <5 nM for other species).

L-365260 hemihydrate has been extensively used in animal studies and is generally well-tolerated at pharmacological doses. In rats and mice, oral or intraperitoneal administration of L-365260 at doses up to 30 mg/kg produces no overt signs of acute toxicity, including no significant changes in body weight, behavior, food intake, or serum biochemistry (ALT, AST, creatinine). At higher doses (50-100 mg/kg), mild gastrointestinal disturbances (diarrhea, decreased gastric emptying) may occur due to CCK-B receptor antagonism, which is an on-target effect consistent with the pharmacological action of the compound. No significant hepatotoxicity, nephrotoxicity, or neurotoxicity has been reported in preclinical studies. Chronic toxicity studies have not been published. As a research compound, L-365260 is not intended for clinical use; no human toxicity data exists. Standard laboratory safety precautions should be followed when handling L-365260 hemihydrate, including the use of gloves, lab coats, and eye protection to avoid skin and eye contact.

[1]. A new potent and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-B) ligand: L-365,260. Eur J Pharmacol. 1989 Mar 21;162(2):273-80.

[2]. The selective CCK-B receptor antagonist L-365,260 enhances morphine analgesia and prevents morphine tolerance in the rat. Eur J Pharmacol. 1990 Jan 25;176(1):35-44.

[3]. A new potent and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-B) ligand: L-365,260. Eur J Pharmacol. 1989 Mar 21;162(2):273-80.

L-365260 hemihydrate is a highly selective, non-peptide antagonist of the CCK-B receptor (also known as CCK2 receptor). It was developed as a pharmacological tool to dissect the physiological and pathophysiological roles of CCK-B receptors in the brain and periphery. CCK-B receptors are widely distributed in the central nervous system (cortex, hippocampus, amygdala, basal ganglia) and in peripheral tissues (stomach, pancreas, gallbladder). The endogenous ligands for CCK-B receptors are cholecystokinin (CCK-8S, CCK-4) and gastrin. L-365260 has been used to study the role of CCK-B receptors in pain modulation (enhancing opioid analgesia, preventing tolerance), anxiety and panic disorders (since CCK-4 is anxiogenic), gastrointestinal motility and secretion, satiety and feeding behavior, pancreatic secretion, and learning and memory. The compound is also used to investigate the physiological and pathological functions of gastrin, which regulates gastric acid secretion and gastric mucosal growth. L-365260 is strictly a research tool and is not approved by the FDA or EMA for clinical use. The hemihydrate form refers to the crystalline form containing half a water molecule per molecule of L-365260, which improves stability and handling characteristics during storage and formulation. The compound is available as a high-purity reagent for in vitro and in vivo research applications.

C24H24N4O3

407.47

L-365260;118101-09-0

White to off-white solid powder

2934.99.9001

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.

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.)