CB₂
Acute systemic LY2828360 administration suppresses mechanical and cold allodynia induced by paclitaxel in a dose-dependent manner in WT mice. Paclitaxel-evoked mechanical and cold hypersensitivities are time-dependently suppressed by LY2828360, and suppression of allodynia is sustained for at least 4.5 hours post-injection in comparison to drug pre-injection levels. The mechanical allodynia caused by paclitaxel has returned to the hypersensitive levels seen prior to the drug injection at 24 hours after the injection. By 72 hours after starting LY2828360 treatment, there was no longer any residual suppression of cold allodynia. In WT mice, LY2828360 has been shown to prevent tolerance to the antiallodynic effects of morphine over time, but not in CB₂KO mice. Chronic LY2828360 treatment suppresses paclitaxel-induced mechanical and cold allodynia in WT mice but not in CB₂KO mice previously render tolerant to morphine[1].

Acute systemic LY2828360 administration suppresses mechanical and cold allodynia induced by paclitaxel in a dose-dependent manner in WT mice. Paclitaxel-evoked mechanical and cold hypersensitivities are time-dependently suppressed by LY2828360, and suppression of allodynia is sustained for at least 4.5 hours post-injection in comparison to drug pre-injection levels. The mechanical allodynia caused by paclitaxel has returned to the hypersensitive levels seen prior to the drug injection at 24 hours after the injection. By 72 hours after starting LY2828360 treatment, there was no longer any residual suppression of cold allodynia. In WT mice, LY2828360 has been shown to prevent tolerance to the antiallodynic effects of morphine over time, but not in CB₂KO mice. Chronic LY2828360 treatment suppresses paclitaxel-induced mechanical and cold allodynia in WT mice but not in CB₂KO mice previously render tolerant to morphine[1].

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LY2828360 was characterized as a slowly acting, G protein-biased agonist at both mouse and human CB2 receptors.
In HEK293 cells stably expressing mouse CB2 receptors, 1 µM LY2828360 inhibited forskolin-stimulated cAMP accumulation, but this effect was only observed after 30 minutes of incubation, not at 5 minutes. This delayed inhibition was abolished by pertussis toxin (PTX) pretreatment, confirming Gi/o protein dependence.[1]
Full concentration-response experiments showed that LY2828360 potently and efficaciously inhibited cAMP accumulation at mouse CB2 with an EC50 of 13.6 nM after 30-minute incubation. The same assay at human CB2 yielded an EC50 of 16.7 nM after 35-minute incubation.[1]
LY2828360 (1 µM) increased phosphorylated ERK1/2 (pERK1/2) in HEK-mCB2 cells only at later time points (20, 30, and 40 minutes), not at early time points (5 and 10 minutes). This activation was completely abolished by PTX pretreatment, demonstrating G protein dependence.[1]
In a concentration-response experiment, LY2828360 increased pERK1/2 at mouse CB2 with an EC50 of 339 nM after 20-minute incubation. At human CB2, the EC50 was 33.5 nM after 30-minute incubation.[1]
In contrast to the balanced agonist CP55940, LY2828360 failed to recruit β-arrestin to mouse CB2 receptors in a 90-minute assay.[1]
LY2828360 was less potent and efficacious than CP55940 in inducing internalization of mouse CB2 receptors (Emax ~19% vs ~49% for CP55940). It failed to internalize human CB2 receptors.[1]
LY2828360 did not affect myo-inositol phosphate 1 (IP1) accumulation via mouse or human CB2 receptors, indicating no activation of Gq signaling.[1]

In WT mice, acute systemic administration of LY2828360 inhibited paclitaxel-induced mechanical and cold allodynia in a dose-dependent manner. LY2828360 produced a time-dependent inhibition of paclitaxel-induced mechanical and cold hypersensitivity, and the inhibition of allodynia was maintained for at least 4.5 hours post-injection relative to pre-drug injection levels. By 24 hours after injection, paclitaxel-induced mechanical allodynia had returned to pre-drug injection hypersensitivity levels. Residual suppression of cold allodynia was absent 72 hours after LY2828360 treatment. Chronic administration of LY2828360 previously blocked tolerance to the anti-allodynic effects of morphine in WT mice but not in CB2KO mice. Long-term LY2828360 treatment inhibited paclitaxel-induced mechanical and cold allodynia in WT mice but not in CB2KO mice that had previously developed tolerance to morphine [1].

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In a paclitaxel-induced neuropathic pain model in wild-type (WT) mice, acute systemic administration of LY2828360 suppressed mechanical and cold allodynia in a dose-dependent manner. A dose of 3 mg/kg i.p. fully reversed allodynia, normalizing responses to pre-paclitaxel baseline levels. The antiallodynic effect lasted for at least 4.5 hours post-injection.[1]
Chronic administration of LY2828360 (3 mg/kg/day i.p. for 12 days) suppressed paclitaxel-induced neuropathic pain in WT mice without producing tolerance to its own antiallodynic effects over the treatment period.[1]
Prior chronic treatment with LY2828360 (3 mg/kg/day i.p. for 12 days) prevented the subsequent development of tolerance to the antiallodynic effects of morphine (10 mg/kg/day i.p.) in paclitaxel-treated WT mice. This effect was absent in CB2 knockout (KO) mice.[1]
Coadministration of a submaximal dose of LY2828360 (0.1 mg/kg/day i.p.) with morphine (10 mg/kg/day i.p.) for 12 days blocked the development of morphine tolerance in paclitaxel-treated WT mice, but not in CB2KO mice.[1]
Chronic LY2828360 treatment (3 mg/kg/day i.p. for 12 days) showed sustained antiallodynic efficacy in paclitaxel-treated WT mice that had been previously rendered tolerant to morphine. This efficacy was absent in morphine-tolerant CB2KO mice.[1]
In paclitaxel-treated WT mice with a history of LY2828360 pretreatment followed by morphine, there was a trend toward fewer naloxone-precipitated withdrawal jumps compared to CB2KO mice with the same treatment history. Similarly, coadministration of LY2828360 with morphine trended to reduce naloxone-precipitated jumps in WT versus CB2KO mice.[1]

For the cAMP accumulation assay, HEK293 cells stably expressing mouse or human CB2 receptors were used. Cells were resuspended in stimulation buffer and incubated. They were then transferred to a 384-well plate and stimulated with drugs in the presence of forskolin for defined times (e.g., 5 or 30 minutes). Cells were lysed, and cAMP levels were quantified using a time-resolved fluorescence resonance energy transfer (TR-FRET) kit according to the manufacturer's instructions. Plates were read on a compatible plate reader.[1]
For the phosphorylated ERK1/2 (pERK1/2) detection assay, HEK293 cells stably expressing CB2 receptors were seeded on poly-D-lysine coated plates and serum-starved. Cells were challenged with drugs for indicated times, then quickly fixed. After fixation and permeabilization, cells were incubated with a blocking buffer, followed by an anti-phospho-ERK1/2 primary antibody overnight. After washing, a fluorescent dye-conjugated secondary antibody was added. The plates were washed again, dried, and scanned using an infrared imaging scanner to quantify pERK1/2 levels.[1]
For the IP1 accumulation assay, HEK293 cells expressing mouse CB2 receptors were detached and resuspended in stimulation buffer containing lithium chloride. Cells were incubated and then transferred to a 384-well plate, followed by stimulation with drugs. Cells were then lysed, and IP1 levels were quantified using a homogeneous time-resolved fluorescence (HTRF) assay kit according to the manufacturer's protocol. Plates were read in HTRF mode on a plate reader.[1]

For the arrestin recruitment assay, a Chinese hamster ovary (CHO) cell line engineered to express a mouse CB2 receptor-β-aractosin enzyme complementation system was used. Cells were thawed, grown, and maintained in appropriate media. For the assay, cells were treated with compounds. Upon receptor activation and arrestin recruitment, an active β-galactosidase enzyme is reconstituted, acting on a substrate to produce luminescence, which was detected.[1]
For the receptor internalization assay, HEK293 cells stably expressing hemagglutinin-tagged CB2 receptors were grown to confluence. Cells were treated with drugs for 90 minutes at 37°C, then fixed. After blocking, cells were incubated with an anti-hemagglutinin primary antibody, followed by a fluorescent dye-conjugated secondary antibody. The plate was imaged using an infrared scanner to measure surface receptor remaining, indicating the extent of internalization.[1]

To induce neuropathic pain, adult male C57BL/6J wild-type (WT) or CB2 knockout (KO) mice received paclitaxel (4 mg/kg, i.p.) on four alternate days (cumulative dose 16 mg/kg). Control mice received a cremophor-based vehicle. Allodynia was assessed after neuropathy was established.[1]
For acute efficacy and time-course studies, single doses of LY2828360 (dissolved in a vehicle containing DMSO, a surfactant, ethanol, and saline) were administered intraperitoneally (i.p.) to paclitaxel-treated mice. Mechanical allodynia was assessed using an electronic von Frey anesthesiometer, and cold allodynia was assessed by applying an acetone bubble to the hind paw and measuring the time spent attending to the paw.[1]
For chronic treatment studies, drugs were administered once daily for 12 consecutive days. LY2828360 was administered at 3 mg/kg/day i.p. Morphine was administered at 10 mg/kg/day i.p. In coadministration studies, morphine (10 mg/kg/day) and LY2828360 (0.1 mg/kg/day) were injected together i.p. for 12 days. Behavioral tests were performed on specified days during and after treatment periods.[1]
For withdrawal assessment, mice that had received chronic morphine (with or without LY2828360) were challenged with vehicle followed 30 minutes later by naloxone (5 mg/kg, i.p.). Mice were video-recorded for 30 minutes, and jumps were scored as a measure of opioid withdrawal.[1]

Literature indicates that LY2828360 showed good central nervous system (CNS) penetration and potent oral activity in a preclinical model of joint pain. [1]

In a phase II clinical trial for osteoarthritis pain, no specific risks or discomfort were observed in patients taking LY2828360 (up to 80 mg for 4 weeks). [1]

[1]. Slowly Signaling G Protein-Biased CB2 Cannabinoid Receptor Agonist LY2828360 Suppresses Neuropathic Pain with Sustained Efficacy and Attenuates Morphine Tolerance and Dependence. Mol Pharmacol. 2018 Feb;93(2):49-62.

LY2828360 is being investigated in the clinical trial NCT01319929 (LY2828360 for the treatment of knee pain in patients with osteoarthritis). LY2828360 is a potent and selective CB2 receptor agonist. In clinical trials for knee pain in patients with osteoarthritis, LY2828360 has shown poor efficacy, but it has shown improvement in an exploratory pain model in this trial. [1] LY2828360 is characterized by slow and G protein-biased signaling, activating Gi/o-mediated pathways (inhibiting cAMP, activating ERK1/2), but does not recruit β-arrestin or activate Gq signaling pathways. [1] This study suggests that LY2828360 may be a first-line treatment for chemotherapy-induced neuropathic pain with long-lasting efficacy and no tolerance. It may also prolong the effective duration of opioid analgesia while reducing opioid tolerance and dependence. [1]

C22H27CLN6O

426.95

426.193

C, 61.89; H, 6.37; Cl, 8.30; N, 19.68; O, 3.75

1231220-79-3

46833780

White to off-white solid powder

1.4±0.1 g/cm3

541.8±60.0 °C at 760 mmHg

281.5±32.9 °C

0.0±1.4 mmHg at 25°C

1.701

1.6

0

6

3

30

570

0

ClC1C=CC=CC=1C1=NC2C(=NC(C)=NC=2N1C1CCOCC1)N1CCN(C)CC1

UCMNDPDJRSEZPL-UHFFFAOYSA-N

InChI=1S/C22H27ClN6O/c1-15-24-21(28-11-9-27(2)10-12-28)19-22(25-15)29(16-7-13-30-14-8-16)20(26-19)17-5-3-4-6-18(17)23/h3-6,16H,7-14H2,1-2H3

8-(2-chlorophenyl)-2-methyl-6-(4-methylpiperazin-1-yl)-9-(oxan-4-yl)purine

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.87 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.87 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.87 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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