Kappa opioid receptor

HSK21542 is a Peripherally-Restricted Kappa Opioid Receptor Agonist[2]
To unravel the pharmacological profiles of HSK21542 at KOR, [3H]diprenorphine binding assay was performed to investigate the inhibitory effects of HSK21542 on [3H]diprenorphine competition binding and determine the binding kinetics of unlabeled HSK21542. U69593, a positive control, obviously prevented [3H]diprenorphine binding to KOR with an IC50 value of 14.72 nM (95% CI: 9.08–22.38 nM). As anticipated, HSK21542 significantly inhibited [3H]diprenorphine binding to KOR with an IC50 value of 0.54 nM (95% CI: 0.38–0.75 nM), while CR845 had an IC50 value of 1.16 nM (95% CI: 0.85–1.57 nM, Figure 2A). The results of the binding kinetics study revealed that HSK21542 and CR845 bound to KOR with K d values of 0.068 nM (95% CI: 0.028–0.092 nM) and 0.23 nM (95% CI: 0.17–0.26 nM), respectively (Figures 2B,C). Meanwhile, HSK21542 had a t 1/2 value of 90.6 min (95% CI: 53.6–292.7 min), which was found to be longer than that of CR845 (42.0 min, 95% CI: 28.6–79.4 min). On the other hand, HSK21542 significantly inhibited forskolin-induced cAMP accumulation in HEK-293 cells that stably expressed human κ opioid receptor with an EC50 value of 2.41 pM (95% CI: 1.43–4.67 pM), which was 12.4-fold and 747-fold lower than those of CR845 and U69593, respectively (Figure 2D).

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To investigate the specificity, the in vitro profile of HSK21542 was observed against a broad panel of receptors, ion channels, transporters and enzymes, including MOR and DOR. At a concentration of 10 μM, HSK21542 was shown to bind to cannabinoid CB1 receptor with an inhibitory rate of 47%, and no obvious activity was observed at the remaining 85 targets (Supplementary Table S1).

In animal models of pain, HSK21542 significantly inhibited acetic acid-, hindpaw incision- or chronic constriction injury-induced pain-related behaviors, and the efficacy was comparable to CR845 at 15 min post-dosing. HSK21542 had a long-lasting analgesic potency with a median effective dose of 1.48 mg/kg at 24 h post-drug in writhing test. Meanwhile, the antinociceptive activity of HSK21542 was effectively reversed by a KOR antagonist nor-binaltorphimine. In addition, HSK21542 had powerful antipruritic activities in compound 48/80-induced itch model. On the other hand, HSK21542 had a weak ability to produce central antinociceptive effects in a hot-plate test and fewer effects on the locomotor activity of mice. HSK21542 didn't affect the respiratory rate of mice. Therefore, HSK21542 might be a safe and effective KOR agonist and promising candidate for treating pain and pruritus.[2]
HSK21542 Causes Potent Antinociceptive Effects. HSK21542 Produces Significant Antiallodynic Effects. HSK21542 Attenuates Compound 48/80-Induced Itch. HSK21542 Showed Fewer CNS Side Effects.

In vitro SafetyScreen Panel: In vitro off-target pharmacological activities of Anrikefon (HSK21542) were evaluated on 86 targets using a SafetyScreen panel (target selectivity panel) and the corresponding methods could be found at https://www.eurofinsdiscoveryservices.com/.

[3H]Diprenorphine Binding Assay: HEK-293 cells (ATCC) were maintained in Eagle’s Minimum Essential Medium with 10% FBS, and incubated at 37°C in humidified air containing 5% CO2. HEK-293 cells that stably express human κ opioid receptor were established in our laboratory and used in this assay. The cell membranes were prepared in 50 mM Tris-HCl buffer (pH 7.4). An equivalent of 30 μg of membranes was incubated with compounds and 0.6 nM [ 3 H]diprenorphine (an opioid antagonist) at 25°C for 60 min (inhibitory effect) or the multiple time points (binding kinetics). Nonspecific binding was estimated in the presence of 10 μM naloxone. The bound and free fractions were separated by vacuum filtration through a GF/B filter that was pretreated with 0.3% polyetherimide. The filters were washed with ice-cold buffer and then were counted to specifically determine the bound radioligand (Olianas et al., 2006). The percentage inhibition of [3H]diprenorphine binding was calculated as follows: inhibition rate (%) = (CPMtotal−CPMcompound)/(CPMtotal−CPMnon-specific) × 100, where CPMtotal = total [3H]diprenorphine bound (membrane +0.6 nM [3H]diprenorphine) and CPMnon-specific = non-specific [3H]diprenorphine bound (membrane +0.6 nM [3H]diprenorphine + 10 μM naloxone). For the unlabeled compounds, the association/dissociation constants were calculated by fitting the data using equations as described by Motulsky and Mahan (Motulsky and Mahan, 1984).[2]

For in vivo experiments, all the test compounds (e.g. Anrikefon (HSK21542)) were solubilized in normal saline, and intravenously administered with a volume of 10 μL/g, except for morphine and nor-binaltorphimine (subcutaneously) when the animals were not under anesthesia and were awake. All other reagents used were of analytical grade unless otherwise stated.

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In vivo Brain/Plasma Distribution of Anrikefon (HSK21542)in Rats[2]
SD rats (half male and half female) were intravenously given a single dose of 0.3 mg/kg Anrikefon (HSK21542). The samples were collected at 0.083, 0.5, 1.5 and 4 h after dosing. The rats were anesthetized with isoflurane and then sacrificed by taking blood from the abdominal aorta. The whole brains were rapidly removed from the crania. The plasma (∼100 μL) was separated from the blood by centrifugating at 2000 ×g for 10 min at 4°C. The brains were rinsed with ice-cold normal saline, blotted dry, weighted and placed into a plastic tube. For 1.0 g of brain sample, 4 ml of acetonitrile-ultrapure water solution (1:4, v/v) was added to the tube. The brain samples were then homogenized for 120 s at 50 Hz and ultrasound was performed for 5 min. The plasma and the brain samples were analyzed using a LC-MS/MS assay as detailed in supplementary materials.

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LC-MS/MS Assay[2]
The plasma or brain homogenate was ice thawed. After 30 μL of plasma or brain homogenate was transferred into a centrifuge tube, 50 μL of internal standard (D4-HSK21542, 50 ng/ml) and 120 μL of acetonitrile were added. The mixture was vortexed for 10 min and centrifuged at 2000 ×g for 10 min at 4°C. The collected supernatant (150 μL) was placed in a 96-well plate and dried under nitrogen. The residue was reconstituted with 150 μL of ultrapure water and vortexed for 10 min. The resulting solution was then analyzed to determine the concentrations of Anrikefon (HSK21542) on a LC-MS/MS system, which consisted of a DGU-20A5R degasser, a LC-30AD pump, a SIL-30AC autosampler, a CTO-20A column oven (Shimadzu, Japan) and an AB Sciex Triple Quad 5500 mass spectrometer (Sciex, Canada). The LC system was coupled to mass spectrometer by using an electro-spray ionization (ESI) source (Yang et al., 2011; Dong et al., 2018). Chromatographic separation was performed on a reverse phase column (Venusil ASB C18, 4.6 mm × 50 mm) under a ternary gradient elution. The temperatures of autosampler and column were maintained at 4 and 40°C, respectively. The mobile phase A consisted of 0.3% formic acid in 2 mM acetic acid solution and the mobile phase B consisted of 0.2% formic acid in acetonitrile. The flow rate was held constant (0.7 ml/min) and the injection volume was set to 20 μL. Quantification was conducted in positive ion mode. The MRM transition of m/z 704.4→295.2 was used to quantify HSK21542.

Furthermore, it was extremely hard for HSK21542 to penetrate into the brain tissues with a brain/plasma concentration ratio of 0.001 (Supplementary Figure S2).

[1]. WHO Drug Information, Vol. 36, No. 2, 2022. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO.

[2]. Antinociceptive and Antipruritic Effects of HSK21542, a Peripherally-Restricted Kappa Opioid Receptor Agonist, in Animal Models of Pain and Itch. Front Pharmacol . 2021 Nov 16:12:773204..

Kappa opioid receptor (KOR) agonists have been promising therapeutic candidates, owing to their potential for relieving pain and treating intractable pruritus. Although lacking morphine-like central nervous system (CNS) effects, KOR agonists do elicit sedation, dysphoria and diuresis which seriously impede their development. Peripherally-restricted KOR agonists have a poor ability to penetrate into the CNS system, so that CNS-related adverse effects can be ameliorated or even abolished. However, the only approved peripherally-restricted KOR agonist CR845 remains some frequent CNS adverse events. In the present study, we aim to address pharmacological profiles of HSK21542, with an expectation to provide a safe and effective alternative for patients who are suffering from pain and pruritus. The in vitro experimental results showed that HSK21542 was a selective and potent KOR agonist with higher potency than CR845, and had a brain/plasma concentration ratio of 0.001, indicating its peripheral selectivity. [2]

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In conclusion, the in vitro findings revealed that HSK21542 is a selective KOR agonist with a higher potency than CR845. The brain/plasma distribution study showed that HSK21542 has an extremely poor ability to penetrate into the CNS system. The in vivo pharmacological activities supported the translational potential of HSK21542 as a safe and effective analgesic and antipruritic candidate. Generally, HSK21542 has the ability to avoid adverse CNS effects that are associated with centrally penetrating KOR agonists and MOR agonist, and might provide an effective alternative for treating patients with pain or pruritus.[2]

C41H61N7O7

763.97

763.4632

2584931-05-3

Anrikefon;2269511-95-5

168265997

DPhe-DPhe-DLeu-DLys-1-(2,7-diazaspiro[3.5]non-2-yl)ethanone

White to off-white solid powder

HHHGOGUNHJGVTN-BBOJDXSOSA-N

InChI=1S/C39H57N7O5.C2H4O2/c1-27(2)22-33(36(49)42-32(16-10-11-19-40)38(51)45-20-17-39(18-21-45)25-46(26-39)28(3)47)44-37(50)34(24-30-14-8-5-9-15-30)43-35(48)31(41)23-29-12-6-4-7-13-29;1-2(3)4/h4-9,12-15,27,31-34H,10-11,16-26,40-41H2,1-3H3,(H,42,49)(H,43,48)(H,44,50);1H3,(H,3,4)/t31-,32-,33-,34-;/m1./s1

acetic acid;(2R)-N-[(2R)-1-(2-acetyl-2,7-diazaspiro[3.5]nonan-7-yl)-6-amino-1-oxohexan-2-yl]-2-[[(2R)-2-[[(2R)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanamide

Anrikefon; Anrikefon [INN]; 22SCY98BXV; 2269511-95-5; UNII-22SCY98BXV; 1-(7-(D-Phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2,7-diazaspiro(3.5)non-2-yl)ethanone; 1-(7-(D-Phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)- 2,7-diazaspiro(3.5)nonan-2-yl)ethan-1-one; Ethanone, 1-(7-(D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl)-2,7-diazaspiro(3.5)non-2-yl)-;

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.)