GPR55

O-1602 causes non-CB1/CB2-mediated vasodilatation in vitro. The mechanism of action mediating the actions of O-1602 is still under debate. Several reports have found that O-1602 activates GPR55, a seven-transmembrane G protein-coupled receptor, and have suggested that GPR55 might be a novel type of cannabinoid receptor. However, the pharmacology of GPR55 has often provided conflicting results, probably because of the fact that the activity of GPR55 ligands is influenced by the functional assay employed.[2]

---

O‐1602 significantly increased proliferation rates of hNSCs as compared to vehicle control. [3]

Central and peripheral administration of O-1602 acutely stimulates food intake, and chronically increases adiposity. The hyperphagic action of O-1602 is mediated by the downregulation of mRNA and protein levels of the anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript. The effects on fat mass are independent of food intake, and involve a decrease in the expression of lipolytic enzymes such as hormone sensitive lipase and adipose triglyceride lipase in white adipose tissue. Consistently, in vitro data showed that O-1602 increased the levels of intracellular calcium and lipid accumulation in adipocytes. Finally, we injected O-1602 in GPR55 −/− mice and found that O-1602 was able to induce feeding behaviour in GPR55-deficient mice. Conclusions: These findings show that O-1602 modulates food intake and adiposity independently of GPR55 receptor. Thus atypical cannabinoids may represent a novel class of molecules involved in energy balance.[2]

---

GPR55 agonist O-1602 inhibited METH-induced anxiety- and depressive-like behaviors; GPR55 activation by O-1602 prevented hippocampal neurogenesis impairment; GPR55 activation by O-1602 prevented microglial activation and reduce neuroinflammation. Intraperitoneal O-1602 administration prevented anxiety and depression caused by chronic social defeat. In line with previous research, GPR55 was inhibited in our study, and activating GPR55 by O-1602 alleviated METH-induced anxiety- and depression-like behavior; these findings demonstrate the involvement of the endocannabinoid system in METH-induced negative emotion.[1]

---

Continuous administration of O‐1602 into the hippocampus via a cannula connected to an osmotic pump resulted in increased Ki67+ cells within the dentate gyrus. O‐1602 increased immature neuron generation, as assessed by DCX+ and BrdU+ cells, as compared to vehicle‐treated animals. GPR55−/− animals displayed reduced rates of proliferation and neurogenesis within the hippocampus while O‐1602 had no effect as compared to vehicle controls.[3]

Effect of O‐1602 on Intracellular Calcium [(Ca2+)i] Mobilization and Differentiation in 3T3-L1 Adipocytes[2]
3T3-L1 cells were differentiated into adipocytes as previously described [28]. Briefly, 3T3-L1 cells were cultured onto 0.1 mg/ml poly-l-lysine coated, 25-mm round cover slips in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 4 mM glutamine and 1% antibiotic–antimicotic solution. At 100% confluence (day 0), cells were incubated in DMEM containing 10% FBS, 0.5 mM isobutylmethylxanthine, 0.25 µM dexamethasone and 10 µg/ml insulin for 72 h (day 3). Afterwards, the culture medium was replaced by DMEM with 10% FBS and 10 µg/ml insulin for an additional 72-h period (day 6) and was then exchanged by DMEM without insulin until days 10–12, when cells were processed for (Ca2+)i measurements. Differentiated 3T3-L1 cells were loaded with 2.5 µM Fura-2AM and 0.02% Pluronic F127 in phenol red-free DMEM containing 20 mM NaHCO3 (pH 7.4) for 30 min at 37°C. Cover slips bearing the cells were then washed with phenol red-free DMEM, mounted in Sykes–Moore chambers and placed on the temperature-controlled stage of a Nikon Eclipse TE2000-E fluorescence microscope fitted with a Plan-Fluor 40× oil immersion objective. Cells were then sequentially epi-illuminated at 340- and 380-nm for 100 ms every 5 s for 8–10 min. Image acquisition was controlled using Metafluor PC software and fluorescence emission was captured at 505/510 nm before (basal line) and after addition of 100 µM O-1602 using a back thinned-charge coupled device cooled digital camera running in one-bit mode. Changes in (Ca2+)i were recorded as ratio of the corresponding excitation wavelengths (F340/F380).

---

Effect of O‐1602 on Adipocyte Differentiation of 3T3-L1 Cells[2]
The 3T3-L1 adipocyte differentiation procedure was carried out in the presence or absence of O-1602 (10 µM) during 10 days. During this period, treatment was renewed every 2 days. At day 10, cells were fixed in paraformaldehyde (4%) for 15 min, thoroughly washed in 60% isopropanol and incubated in Oil Red O solution (Sigma, St. Louis, MO, USA) for 30 min. After washing out the excess of dye with H2O2, plates were placed in a bright-field microscope and snapshots of random fields were captured using a 10× objective. Afterwards, Oil Red O was eluted by incubating cells with 100% isopropanol for 10 min and optical density measured at 500 nm to estimate dye incorporation. In another set of differentiated 3T3-L1 cells, we isolated RNA and measured the gene expression of master regulators of adipocyte differentiation as described below.

---

To investigate the effects of GPR55 activation on hNSC proliferation, cells were plated on laminin‐coated 6‐well plates. Cells were allowed to adhere overnight and then treated with LPI (1 μM), the endogenous ligand for GPR55, or synthetic agonists, O‐1602 (1 μM) or ML184 (1 μM), in a reduced growth factor media (5% growth factor). Reduced growth factor medium was utilized to better mimic a less proliferative phenotype while still maintaining a ‘stemness’ state. Analysis by flow cytometry showed no significant reduction of nestin+ or Sox2+ populations after 48 h (data not shown). Cells treated with the selective GPR55 antagonist ML193 (5 μM) were pretreated for 30 min prior to addition of agonist. Vehicle‐treated cells received 0.1% DMSO in 5% growth factor media. For differentiation studies, cells were treated with either vehicle, ML184 (1 μM), ML193 (5 μM), or a combination of ML184 (1 μM) and ML193 (5 μM) in ReNcell medium that did not contain growth factors.[3]

Researchers experimented to observe the effect of GPR55 activation by systematic administration of O-1602, a GPR55 agonist. The dosage and route of administration refer to previous literature. Mice were randomly divided into the Vehicle group, METH group, and O-1602+METH group. After a 7-day adaptation period, mice from the METH group and O-1602+METH group were intraperitoneally injected with 10 mg/kg of METH once daily for 14 consecutive days, while mice from the control group received an equivalent volume of saline. During the 14 days of METH treatment, mice received an additional intraperitoneal injection of 10 mg/kg of O-1602 30 min before each METH injection in the O-1602+METH group, while in Vehicle and METH group mice received an equivalent volume of saline. Then a 2-day withdrawal was performed after METH treatment. On the next day, the behavioral tests were conducted for 4 consecutive days during the light cycle.[1]

---

Metabolic Effects of Acute O-1602 Injection[2]
For the study of acute peripheral effects, rats received a single i.p. injection of vehicle [dimethyl sulfoxide (DMSO) 5%] or O-1602 at a dose of 2, 20 and 200 µg/kg of body weight. For the study of acute central effects, rats received a single intracerebroventricular (i.c.v.) injection of vehicle (DMSO 5%), or O-1602 at a dose of 0.02, 0.1, 0.5, 1, 5 and 10 µg/rat. In both experiments, food intake was measured after 1, 2, 4, 6 and 24 h of O-1602 injection. To know the precise amount of food ingested by each rat, we weighted the food in the same order as the rats were injected with either vehicle or O-1602. Moreover, we also weighted the spillage of food.

---

Metabolic Effects of Subchronic O-1602 Infusion[2]
To assess the metabolic effects of O-1602, we subchronically infused this compound during a 1-week period at the peripheral and central level. At peripheral level, we injected daily O-1602 at a dose of 0.1, 0.5 and 1 mg/kg of body weight for 7 days (i.p. injection). For the subchronic central infusion of O-1602, rats were anaesthetized and chronic i.c.v. cannulae were implanted in the lateral ventricle stereotaxically using the following coordinates: 1.3 mm posterior to bregma, 1.9 mm lateral to the midsagittal suture and to a depth of 3.5 mm as described previously. A catheter tube was connected from the brain infusion cannula to the osmotic minipump flow moderator (model 2001D or 2ML2). A subcutaneous pocket on the dorsal surface was created using blunt dissection and the osmotic minipump was inserted. The incision was closed with sutures, and rats were kept warm until fully recovered. Rats were then infused with either vehicle or O-1602 (1, 10 and 100 µg/day) for 7 days. Food intake, body weight, body composition and adipocyte metabolism were assessed after peripheral and central administration of O-1602. Body composition was measured in rats infused using nuclear magnetic resonance (NMR) imaging Measurements were performed before surgery and prior to sacrifice following the treatment period.

---

Metabolic Effects of Acute O-1602 Injection on GPR55-Deficient Mice[2]
To test if the orexigenic effects of O-1602 were specifically mediated by GPR55, mice lacking GPR55 received a single i.p. injection of vehicle (DMSO 50%), or O-1602 at a dose of 200 µg/kg of body weight and 2 µg/kg of body weight, respectively. Food intake was measured after 1, 2, 4, 6 and 24 h of O-1602 injection.

---

Intrahippocampal injections[3]
Animals were anaesthetized with isoflourane and placed in a stereotaxic frame. Isoflurane was vaporized and a concentration of 4–5% at induction and maintenance at 1.5–2%. Stereotactic coordinates were calculated from Bregma (in mm) −2.0 anterio‐posterior, +1.5 medial‐lateral, −1.5 dorso‐ventral. Vehicle [artificial cerebral spinal fluid (ACSF, Tocris Bioscience), 0.05% EtOH] or O‐1602 (O‐1602 diluted in 100% EtOH, then further diluted in ACSF with a final concentration of no more than 0.05% EtOH) was injected via a stainless‐steel cannula (Alzet Durect, Cupertino, CA, USA; brain infusion kit 3) connected through a polyvinyl tube to an osmotic mini‐pump (Alzet Durect, model 1002). Pumps were primed in sterile saline overnight at 37°C to ensure infusion would begin upon implantation. The infusion period was a continuous 14 days. Infusion doses were set to 4 μg·kg−1·day−1. O‐1602 was chosen for treatment in vivo due to ML184 being a piperazine and structurally similar to another benzoylpiperazine GPR55 agonist from GlaxoSmithKline (GSK494581A), which is active at human GPR55 but not rodent GPR55 (Brown et al., 2011).
For in vivo experiments, master stock solutions of O‐1602 were prepared in 100% ethanol (EtOH). All master stock solutions were aliquoted and stored at −20°C.

[1]. GPR55 activation improves anxiety- and depression-like behaviors of mice during methamphetamine withdrawal.Heliyon, 10 (2024), e30462.

[2]. The atypical cannabinoid O-1602 stimulates food intake and adiposity in rats. Diabetes, Obesity and Metabolism. March 2012, Volume14, Issue3.

[3]. IAVPGEVA: Orally Available DPP4-Targeting Soy Glycinin Derived Octapeptide with Therapeutic Potential in Nonalcoholic Steatohepatitis.Journal of Agricultural and Food Chemistry. 2024, 72, 13, 7167–7178.

Methamphetamine is a potent and highly addictive neurotoxic psychostimulant that triggers a spectrum of adverse emotional responses during withdrawal. G-protein coupled receptor 55 (GPR55), a novel endocannabinoid receptor, is closely associated with mood regulation. Herein, we developed a murine model of methamphetamine-induced anxiety- and depressive-like behavior during abstinence which showed a decreased GPR55 expression in the hippocampus. Activation of GPR55 mitigated these behavioral symptoms, concomitantly ameliorating impairments in hippocampal neurogenesis and reducing neuroinflammation. These findings underscore the pivotal role of GPR55 in mediating the neuropsychological consequences of methamphetamine withdrawal, potentially via mechanisms involving the modulation of hippocampal neurogenesis and inflammation.[1]

---

Aims: Cannabinoids are known to control energy homeostasis. Atypical cannabinoids produce pharmacological effects via unidentified targets. We sought to investigate whether the atypical cannabinoid O-1602 controls food intake and body weight. Methods: The rats were injected acutely or subchronically with O-1602, and the expression of several factors involved in adipocyte metabolism was assessed by real-time polymerase chain reaction. In vivo findings were corroborated with in vitro studies incubating 3T3-L1 adipocytes with O-1602, and measuring intracellular calcium and lipid accumulation. Finally, as some reports suggest that O-1602 is an agonist of the putative cannabinoid receptor GPR55, we tested it in mice lacking GPR55.[2]

---

Background and Purpose The cannabinoid system exerts functional regulation of neural stem cell (NSC) proliferation and adult neurogenesis, yet not all effects of cannabinoid‐like compounds seen can be attributed to the cannabinoid 1 (CB1) or CB2 receptor. The recently de‐orphaned GPR55 has been shown to be activated by numerous cannabinoid ligands suggesting that GPR55 is a third cannabinoid receptor. Here, we examined the role of GPR55 activation in NSC proliferation and early adult neurogenesis. Experimental Approach The effects of GPR55 agonists (LPI, O‐1602, ML184) on human (h) NSC proliferation in vitro were assessed by flow cytometry. Human NSC differentiation was determined by flow cytometry, qPCR and immunohistochemistry. Immature neuron formation in the hippocampus of C57BL/6 and GPR55−/− mice was evaluated by immunohistochemistry.[3]

C17H22O2

258.35538

258.161

C, 79.03; H, 8.58; O, 12.38

317321-41-8

45073499

White to light yellow ointment

1.1±0.1 g/cm3

401.6±45.0 °C at 760 mmHg

185.2±23.3 °C

0.0±1.0 mmHg at 25°C

1.565

4.91

2

2

2

19

372

2

CC1=C[C@H]([C@@H](CC1)C(=C)C)C2=C(C=C(C=C2C)O)O

KDZOUSULXZNDJH-LSDHHAIUSA-N

InChI=1S/C17H22O2/c1-10(2)14-6-5-11(3)7-15(14)17-12(4)8-13(18)9-16(17)19/h7-9,14-15,18-19H,1,5-6H2,2-4H3/t14-,15+/m0/s1

5-methyl-4-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]benzene-1,3-diol

O-1602; 317321-41-8; 5-METHYL-4-[(1R,6R)-3-METHYL-6-(1-METHYLETHENYL)-2-CYCLOHEXEN-1-YL]-1,3-BENZENEDIOL; O 1602; 5-methyl-4-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]benzene-1,3-diol; CHEMBL3402654; O1602; 59F4R2N5N5;

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)

DMSO : ~100 mg/mL (~387.06 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to 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 (9.68 mM) (saturation unknown) 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.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (9.68 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)