Cannabinoid receptor

BAY 59-3074 [3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butane-sulfonate] is a structurally novel cannabinoid CB1/CB2 receptor partial agonist with analgesic properties. [1]

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CP in the presence of both inverse agonists (hereafter SR) almost completely inhibits the aggregation of intracellular sAβPPβf and p-Tau, increases ΔΨm, decreases oxidation of DJ-1Cys106-SH residue, and blocks the activation of c-Jun, p53, PUMA, and caspase-3 independently of CB1Rs signaling in mutant ChLNs. CP also inhibits the generation of reactive oxygen species partially dependent on CB1Rs. Although CP reduced extracellular Aβ42, it was unable to reverse the Ca2+ influx dysregulation as a response to acetylcholine stimuli in mutant ChLNs. Exposure to anti-Aβ antibody 6E10 (1:300) in the absence or presence of SR plus CP completely recovered transient [Ca2+]i signal as a response to acetylcholine in mutant ChLNs.[2]

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CP55940 restores the mitochondrial membrane potential (ΔΨm) and blunts generation of ROS in PSEN1 E280A ChLNs in an independent concentration fashion.[2]
CP55940 restores ΔΨm in a CB1 receptor-independent manner but diminishes ROS production partially dependent on CB1 receptors in PSEN1 E280A ChLNs.[2]
CP55940 reduces intracellular sAβPPβf aggregation in a CB1Rs-independent fashion but it reduces oxidized DJ-1 partially dependent of CB1Rs in PSEN1 E280A ChLNs.[2]
CP55940 blocks apoptosis in a CB1Rs-independent manner in PSEN1 E280A ChLNs.[2]
CP55940 inhibits tau phosphorylation in a CB1Rs-independent fashion PSEN1 E280A ChLNs.[2]
CP55940 reduces the levels of eAβ42 protein fragment independent of CB1Rs in PSEN 1 E280A ChLNs.[2]
CP55940 does not recover Ca2+ dysregulation in PSEN1 E280A ChLNs.[2]

The present study was performed to confirm its receptor binding profile in a highly sensitive in vivo assay. Rats (n=10) learned to discriminate BAY 59-3074 (0.5 mg/kg, p.o., t-1 h) from vehicle in a fixed-ratio: 10, food-reinforced two-lever procedure after a median number of 28 training sessions. BAY 59-3074 generalized dose-dependently (ED(50): 0.081 mg/kg, p.o.) and the cue was detectable between 0.25 and 4 h after administration. The selective cannabinoid CB1 receptor antagonist SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] blocked the discriminative effects of BAY 59-3074 (ID50: 1.79 mg/kg, i.p.). Complete generalization was also obtained after i.p. administration of BAY 59-3074 (ED50 value: 0.41 mg/kg), and the reference cannabinoids BAY 38-7271 [(-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate, 0.011 mg/kg], CP 55940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol, 0.013 mg/kg], HU-210 [(-)-11-OH-Delta8-tetrahydrocannabinol dimethylheptyl, 0.022 mg/kg], WIN 55,212-2 [(R)-4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo [3,2,1-ij] quinolin-6-one, 0.41 mg/kg] and (-)-Delta9-tetrahydrocannabinol (0.41 mg/kg). Non-cannabinoids with analgesic properties, such as morphine, amitriptyline, carbamazepine, gabapentin and baclofen, did not generalize to the cue. It is concluded that the discriminative stimulus effects of BAY 59-3074 are specifically mediated by cannabinoid CB1 receptor activation.[1]

Receptor Binding Assays. [3]
1. CB1 Assay. CB1 receptor affinities were determined using membrane preparations of Chinese hamster ovary (CHO) cells in which the human cannabinoid CB1 receptor is stably transfected19 in conjunction with [3H]CP 55940 as radioligand. After incubation of a freshly prepared cell membrane preparation with the [3H]-radioligand, with or without addition of test compound, separation of bound and free ligand was performed by filtration over glassfiber filters. Radioactivity on the filter was measured by liquid scintillation counting. The IC50 values from at least three independent measurements were combined and converted to Ki values using the assumptions of Cheng and Prusoff.

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CB2 Assay. [3]
CB2 receptor affinities were determined using membrane preparations of Chinese hamster ovary (CHO) cells in which the human cannabinoid CB2 receptor is stably transfected in conjunction with [3H]CP-55,940 as radioligand. After incubation of a freshly prepared cell membrane preparation with the [3H]-radioligand, with or without addition of test compound, separation of bound and free ligand was performed by filtration over glassfiber filters. Radioactivity on the filter was measured by liquid scintillation counting. The IC50 values from at least two independent measurements were combined and converted to Ki values using the assumptions of Cheng and Prusoff.

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In Vitro Pharmacology. [3]
Measurement of Arachidonic Acid Release. CB1 receptor antagonism was assessed with the human CB1 receptor cloned in Chinese hamster ovary (CHO) cells. CHO cells were grown in a Dulbecco's modified Eagle's medium (DMEM) culture medium, supplemented with 10% heat-inactivated fetal calf serum. Medium was aspirated and replaced by DMEM, without fetal calf serum, but containing [3H]-arachidonic acid and incubated overnight in a cell culture stove (5% CO2/95% air; 37 °C; water-saturated atmosphere). During this period [3H]-arachidonic acid was incorporated in membrane phospholipids. On the test day, medium was aspirated and cells were washed three times using 0.5 mL of DMEM, containing 0.2% bovine serum albumin (BSA). Stimulation of the CB1 receptor by WIN 55,212-2 led to activation of PLA2 followed by release of [3H]-arachidonic acid into the medium. This WIN 55,212-2-induced release was concentration dependently antagonized by CB1 receptor antagonists. The CB1 antagonistic potencies of the test compounds were expressed as pA2 values.

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P-Glycoprotein Assay. [3]
The capability of the human MDR1 P-glycoprotein pump to translocate compounds over a cellular monolayer of PK1 LLC MDR cells was assessed. The transport method essentially described in the literature31 was used. Compounds were added at the start of the experiment at 1 μg/mL to one side of the cellular layer. The bottom to top transport was measured as well as the top to bottom transport. The P-glycoprotein (Pgp) factor was expressed as the ratio of the bottom to top transport and top to bottom transport. The membrane passage was expressed as the mean percentage of compound transported from bottom to top and from top to bottom at 3 h after adding the compound. Compound detection was performed using a LC/MS method.

Analysis of cells [2]
Assay protocol [2]
The methodology for both WT and PSEN1 E280A ChLNs cell culture assays was the same. Initial CP 55940 screening was performed at least twice in triplicate between 10 nM and 1μM. Subsequently, CP 55940 (1μM) was established as an optimal concentration for further experiments. ChLNs were divided in four groups: 1) untreated; 2) treated with SR141716 (CB1 receptor inverse agonist) and SR144528 (CB2 receptor inverse agonist) at 1μM final concentration each (hereafter SR); 3) CP55940 (or CP); 4) RS + CP55940 (also named as SR + CP cocktail). To block eAβ42, WT and mutant ChLNs were incubated four days with anti-Aβ42 antibody 6E10 (1:300 in RCm) after differentiation in the presence or absence of CP or CP + RS.

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Wild type (WT) and PSEN1 ChLNs were exposed to CP (1μM) only or in the presence of the CB1 and CB2 receptors (CB1Rs, CB2Rs) inverse agonist SR141716 (1μM) and SR144528 (1μM) respectively, for 24 h. Untreated or treated neurons were assessed for biochemical and functional analysis[2].

In Vivo Pharmacology. 1. CP 55940 Induced Hypotension in Rat. Male normotensive rats (225−300 g; Harlan, Horst, The Netherlands) were anaesthetized with pentobarbital (80 mg/kg ip). Blood pressure was measured, via a cannula inserted into the left carotid artery, by means of a Spectramed DTX-plus pressure transducer (Spectramed B. V., Bilthoven, The Netherlands). After amplification by a Nihon Kohden Carrier amplifier (Type AP-621G; Nihon Kohden B. V., Amsterdam, The Netherlands), the blood pressure signal was registered on a personal computer (Compaq Deskpro 386s), by means of a Po-Ne-Mah data-acquisition program (Po-Ne-Mah Inc., Storrs, USA). Heart rate was derived from the pulsatile pressure signal. All compounds were administered orally as a microsuspension in 1% methylcellulose 30 min before induction of the anesthesia which was 60 min prior to administration of the CB1 receptor agonist CP-55,940. The injection volume was 10 mL kg-1. After haemodynamic stabilization the CB1 receptor agonist CP-55,940 (0.1 mg kg-1 i.v.) was administered and the hypotensive effect22 established. Typical blood pressure after administration of CP-55,940 was approximately 60% as compared to vehicle treated animals.[3]

[1]. Eur J Pharmacol.2004 Nov 28;505(1-3):127-33.
[2]. J Alzheimers Dis. 2021; 82(Suppl 1): S359–S378.
[3]. J Med Chem. 2004 Jan 29;47(3):627-43.

series of novel 3,4-diarylpyrazoline compounds were synthesized and their activities against human CB1 and CB2 receptors were evaluated. These 3,4-diarylpyrazoline compounds exhibited potent in vitro CB1 receptor antagonistic activity and, overall, high selectivity for both CB1 and CB2 receptor subtypes. Some key compounds showed significant in vivo pharmacological activity in CB1 receptor agonist-induced hypertension and CB2 receptor agonist-induced hypothermia models after oral administration. The absolute configuration of its enantiomer 80 (SLV319) at the C4 position was determined to be 4S by chiral separation of racemate 67 combined with crystallization and X-ray diffraction analysis. Bioanalytical studies showed that candidate drug 80 had a high central nervous system/plasma concentration ratio. Molecular modeling studies showed that compound 80 has a relatively close three-dimensional structural overlap with the known CB(1) receptor antagonist rimonaban (SR141716A). Further analysis of X-ray diffraction data of 80 revealed the presence of intramolecular hydrogen bonds, a finding also confirmed by computational methods. Computational models and X-ray diffraction data indicated that the intramolecular hydrogen bonding pattern of inactive compound 6 differs from that of 80. Furthermore, X-ray diffraction studies of 6 showed that its intermolecular packing is more compact than that of 80, which may be one of the reasons for its poor in vivo absorption. Replacing the amidine group -NH(2) with the -NHCH(3) group proved to be a key change to improve the oral bioavailability of this series of compounds, ultimately identifying compound 80. [3]

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Background: Alzheimer's disease (AD) is characterized by structural damage, death, and dysfunction of cholinergic neurons (ChNs) due to intracellular amyloid β protein (Aβ) aggregation, extracellular neuroinflammatory plaques, and hyperphosphorylation of tau protein (p-Tau). Objective: To evaluate the effects of synthetic cannabinoid CP 55940 (CP) on PSEN1 E280A cholinergic-like neurons (PSEN1 ChLNs), a natural model of familial AD. Methods: Wild-type (WT) and PSEN1 ChLNs were exposed to CP alone (1 μM) or to CP in the presence of the inverse agonists of CB1 and CB2 receptors (CB1R, CB2R), SR141716 (1 μM) and SR144528 (1 μM), respectively, for 24 h. Biochemical and functional analyses were performed on untreated and treated neurons. Results: In the presence of both inverse agonists (hereinafter referred to as SR), CP almost completely inhibited the accumulation of intracellular sAβPPβf and p-Tau, increased mitochondrial membrane potential (ΔΨm), reduced the oxidation of DJ-1Cys106-SH residues, and blocked the activation of c-Jun, p53, PUMA, and caspase-3 in mutant ChLNs, with this effect independent of the CB1R signaling pathway. CP also inhibited the production of reactive oxygen species, with this effect partially dependent on CB1R. Although CP reduced extracellular Aβ42 levels, it could not reverse the Ca2+ inward disregard induced by acetylcholine stimulation in mutant ChLNs. Treatment of mutant ChLNs with anti-Aβ antibody 6E10 (1:300) in the presence or absence of SR and CP completely restored the transient [Ca2+]i signaling induced by acetylcholine stimulation. Conclusion: In summary, our results suggest that the combined use of cannabinoids, CB1R inverse agonists, and anti-Aβ antibodies may be a promising treatment for familial Alzheimer's disease (AD). [2]

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BAY 59-3074 [3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate] is a novel cannabinoid CB1/CB2 receptor partial agonist with analgesic effects. This study aimed to verify its receptor binding properties through highly sensitive in vivo experiments. Rats (n=10) were trained to distinguish between BAY 59-3074 (0.5 mg/kg, orally, t-1 h) and the solvent control after an average of 28 training sessions in a fixed-ratio, food-fortified two-bar experiment. The generalization effect of BAY 59-3074 was dose-dependent (ED50: 0.081 mg/kg, orally), and the signal was detectable from 0.25 to 4 hours after administration. The selective cannabinoid CB1 receptor antagonist SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] blocked the discrimination of BAY 59-3074 (ID50: 1.79 mg/kg, intraperitoneal injection). Complete generalization was achieved after intraperitoneal injection of BAY 59-3074 (ED50: 0.41 mg/kg), as well as after reference cannabinoids BAY 38-7271 [(-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate, 0.011 mg/kg], CP 55940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol, 0.013 mg/kg], HU-210 [(-)-11-OH-Δ8-tetrahydrocannabinol dimethylheptyl, 0.022 mg/kg], and WIN 55,212-2. [(R)-4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthylcarbonyl)-6H-pyrrolo[3,2,1-ij]quinoline-6-one, 0.41 mg/kg] and (-)-Δ9-tetrahydrocannabinol (0.41 mg/kg). Non-cannabinoid drugs with analgesic effects, such as morphine, amitriptyline, carbamazepine, gabapentin, and baclofen, did not generalize to this clue. It is concluded that the discriminative stimulant effect of BAY 59-3074 is specifically mediated by cannabinoid CB1 receptor activation. [1]

C24H40O3

376.57

376.297

C, 76.55; H, 10.71; O, 12.75

83002-04-4

104895

Typically exists as solid at room temperature

1.0±0.1 g/cm3

494.4±45.0 °C at 760 mmHg

209.2±23.3 °C

0.0±1.3 mmHg at 25°C

1.526

5.97

3

3

10

27

408

3

CCCCCCC(C)(C)C1=CC(=C(C=C1)[C@@H]2C[C@@H](CC[C@H]2CCCO)O)O

YNZFFALZMRAPHQ-SYYKKAFVSA-N

InChI=1S/C24H40O3/c1-4-5-6-7-14-24(2,3)19-11-13-21(23(27)16-19)22-17-20(26)12-10-18(22)9-8-15-25/h11,13,16,18,20,22,25-27H,4-10,12,14-15,17H2,1-3H3/t18-,20-,22-/m1/s1

2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol

CP55940; CP-55940; 83003-12-7; 83002-04-4; 2-[(1r,2r,5r)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol; CP55,940; CP-55,940; 8YX8JK1BQG;

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.)