kappa 1 opioid receptor/κ Opioid Receptor/KOR

The selective kappa agonist U-50488 was recently discovered and characterized. In this study, the receptor binding properties of [3H]U-69593, an analog of U-50488, were characterized. [3H]U-69593 binds with high affinity (3 nM) to membranes prepared from guinea pig, mouse and rat brain. The number of kappa binding sites comprise only 13%, 9% and 4% of the total opioid sites, respectively. The benzmorphans, dynorphin, and compounds structurally related to U-50488 have high affinity for this kappa site. [1]

U-69593 (0.16 mg/kg; sc) reduces behavioral sensitization associated with addictive states [2]. In the lateral striatum, U-69593 (1, 10, 25 nmol/μL; microinjection) potentiates U-69593 (0.32 mg/kg; sc) and immediately lowers amphetamine-induced behavior, as well as calcium-induced diabetic fluid dopamine and abdominal glutamate [4].

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The effects of treatment with the selective kappa-opioid receptor agonist U-69593 upon cocaine-induced changes in locomotor activity and stereotypy were examined in rats. U-69593 (0.16 mg/kg s.c.) administered either acutely or chronically attenuated both the motor stimulant effect and stereotypy produced by an acute injection of cocaine (20 mg/kg i.p.). Daily cocaine treatment resulted in sensitization to both effects of cocaine. In contrast, no such sensitized responses were seen in animals which had received U-69593 either prior to or in conjunction with daily cocaine treatment. These data demonstrate that activation of kappa-opioid receptors attenuates the acute and chronic effects of cocaine on locomotor activity and stereotypy. Given the inhibitory effects ascribed to both exogenous and endogenous kappa-opioid agonists upon dopamine release in the mesolimbic dopaminergic system, it is suggested that this action may underlie the observed effects of U-69593 on cocaine-induced changes in locomotor activity and stereotypy. [2]

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The present report investigated the contributions of the ventromedial prefrontal cortex to the control of spontaneous alternation/working memory and anxiety-related behaviour. In Experiment 1, we examined the effects of microinjections of the selective kappa(1) receptor agonist, U-69,593, in the infralimbic cortex (IL) of CD-1 mice on several ethologically-derived anxiety indices in the elevated plus-maze (EPM) and defensive/withdrawal (D/W) anxiety in the open field, as well as on memory in the EPM transfer-latency (T-L) test and implicit spontaneous alternation memory (SAP) in the Y-maze. In week 1, pretreatment with one injection of vehicle, 1, 10 or 25 nmol/1.0 microliter U-69593 in the IL dose-dependently prolonged T-L and produced a dose-dependent anxiolytic behavioural profile in the first EPM trial. Following a 24-h delay, the same mice were given a drug-free second trial in the EPM tests of T-L memory and anxiety. Whereas T-L memory was not disturbed, small but detectable carry-over effects were observed in trial-2 EPM behaviour relative to vehicle-treated animals. In week 2, the same groups of mice were again pretreated with one injection of the same doses of U-69593 in the IL and given a D/W test in an open field, followed immediately by an 8-min SAP trial in the Y-maze. The smallest U-69,593 dose was anxiolytic in the D/W test, and SAP/working memory was dose-dependently enhanced in the Y-maze. In Experiment 2, we evaluated whether 0.5 microliter volume microinjections would produce comparable behavioural and carry-over effects in the IL of three new groups of CD-1 mice, in the event that the 1.0 microl volume injections used in Experiment 1 diffused beyond the IL and therefore may have confounded some effects. Experiment 2 procedures were carried out in the same manner as in Experiment 1, except the animals were tested in reverse order. Thus in week 1, SAP memory was tested in the Y-maze followed by D/W anxiety in the open field for half of the animals in each group, and the other half was tested in reverse order. In week 2, T/L memory and anxiety were tested in the EPM in 2 trials as described in Experiment 1. Pretreatment with one injection of vehicle, 10 or 25 nmol/0.5 microliter U-69,593 in the IL reduced D/W anxiety and enhanced SAP memory regardless of testing order in week 1. In week 2, the same groups of mice were again pretreated with one injection of the same doses of U-69,593 in 0.5 microliter volumes in the IL and tested in the EPM. In a similar fashion to Experiment 1, U-69,593 dose-dependently prolonged T/L and produced an anxiolytic behavioural profile in the first EPM trial. Following a 24-h delay, T/L recall memory was again not significantly influenced, but a robust anxiolytic behavioural profile was observed in the second drug-free anxiety trial in the EPM relative to vehicle-treated animals. Results are discussed relative to a) injection volumes and testing order, b) the possible influence kappa receptors may exert on neurochemical responsivity to anxiety-provoking environments in the IL area of the mPFC, c) the possibility that kappa-mediated anxiolysis from the IL in CD-1 mice results from interactions with neurochemical systems involved in the blunting of incoming anxiety-provoking information, d) evidence that SAP memory may be an implicit subtype of working memory, and e) the possibility that IL implicit working memory processes may modulate the induction and expression of anxiety-related behaviour. [3]

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The effects of a kappa-opioid receptor agonist on acute amphetamine-induced behavioral activation and dialysate levels of dopamine and glutamate in the ventral striatum were investigated. Amphetamine (2.5 mg/kg i.p.) evoked a substantial increase in rearing, sniffing, and hole-poking behavior as well as dopamine and glutamate levels in the ventral striatum of awake rats. U-69593 (0.32 mg/kg s.c.) significantly decreased the amphetamine-evoked increase in behavior and dopamine and glutamate levels in the ventral striatum. Reverse dialysis of the selective kappa-opioid receptor antagonist, nor-binaltorphimine, into the ventral striatum antagonized the effects of U-69593 on amphetamine-induced behavior and dopamine and glutamate levels. Reverse dialysis of low calcium (0.1 mM) into the ventral striatum decreased basal dopamine, but not glutamate, dialysate levels by 91% 45 min after initiation of perfusion. Strikingly, 0.1 mM calcium perfusion significantly reduced the 2.5 mg/kg amphetamine-evoked increase in dopamine and glutamate levels in the ventral striatum, distinguishing a calcium-dependent and a calcium-independent component of release. U-69593 did not alter the calcium-independent component of amphetamine-evoked dopamine and glutamate levels. These data are consistent with the view that a transsynaptic mechanism augments the increase in dopamine and glutamate levels in the ventral striatum evoked by a moderately high dose of amphetamine and that stimulation of kappa-opioid receptors suppresses the calcium-dependent component of amphetamine's effects. [4]

Animal/Disease Models: Spontaneous memory alternation [3]. Rat[2]
Doses: 0.16 mg/kg
Route of Administration: Sc (acute injection of cocaine (20 mg/kg ip))
Experimental Results: Attenuation of acute and chronic effects of cocaine on locomotor activity and stereotypes.

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Animal/Disease Models: CD-1 mice [3]
Doses: 1, 10, 25 nmol/μL
Route of Administration: Microinjection (infralimbic cortex (IL)), once weekly, 2 weeks
Experimental Results: Dose-dependent prolonged metastasis latency (TL) ) and produced a dose-dependent anxiolytic behavioral profile, with small but detectable residual effects observed in mice after 24 hrs (hrs (hours)). At week 2, U-69593 dose-dependently prolonged T/L and produced an anxiolytic profile in the first EPM (elevated plus maze) test, but a robust anxiolytic profile was observed.

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Animal/Disease Models: 280-350 g, male Wistar rats [4]
Doses: 0.32 mg/kg
Route of Administration: subcutaneous injection (amphetamine (2.5 mg/kg ip) 15 minutes later)
Experimental Results: Dramatically diminished Amphetamine Amphetamine stimulates an increase in dialysate dopa

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U-69593 was dissolved using a 45% cyclodextrin solution to concentrations of 1, 10 and 25 nmol/μl. Vehicle was a 45% cyclodextrin/distilled H2O solution. Cyclodextrin is a cyclic sucrose array that increases drug solubility. Because of poor solubility in saline, U-69593 is often injected in suspension. Cyclodextrin is inert and excreted rapidly. The use of cyclodextrin allowed for more precise microinjections and avoided cannula blockage.

Intracerebral drug administration [3]
Following a 2-week recovery period, mice were transferred from the main holding area to the laboratory and left undisturbed for 1 h prior to drug administration. The mice were randomly assigned to one of a vehicle, 1, 10 or 25 nmol U-69593 treatment condition. Each mouse was lightly restrained and a 30 gauge injection cannula was inserted into the guide cannula, the injection cannula connected using polyethylene tubing to a 5 μl Hamilton microsyringe. Each drug concentration was infused in a 1 μl volume over a 30-s period. The injection cannula remained in place for an additional 30 s to allow for drug diffusion.

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In experiment 1, the effect of U-69593 or reduced calcium on basal dopamine and glutamate levels was investigated. In one group of rats, after the collection of baseline samples, U-69593 (0.32 mg/kg s.c.) was injected followed 15 min later by saline. In a second group of rats, following the collection of baseline samples, a 12% propylene glycol (vehicle) solution was injected followed 15 min later by saline. In a third group of rats, the calcium concentration in the perfusate was reduced from 1.2 mM to 0.1 mM for the remainder of the experiment. [4]
In experiment 2, the effect of U-69593 on amphetamineevoked dopamine and glutamate levels was investigated. After three baseline dialysis samples were collected, rats received an injection of vehicle (1 ml/kg s.c.) or U-69593 (0.32 mg/kg s.c.) followed 15 min later by an injection of amphetamine (2.5 mg/kg i.p.). In an additional group of rats, nor-binaltorphimine (nBNI ; 100 nM) was infused through the dialysis probe just before the injection of U-69593 (0.32 mg/kg s.c.), which was followed 15 min later by an injection of amphetamine (2.5 mg/kg i.p.). Samples were collected for 2 h after amphetamine while the behavior of the rats was rated (see below). [4]
In experiment 3, the effect of lowered calcium on amphetamine-evoked dopamine and glutamate levels was investigated in the presence or absence of U-69593. In one group of rats, after baseline samples were collected, the calcium concentration in the ACSF was reduced to 0.1 mM. Fifteen minutes later, amphetamine (2.5 mg/kg i.p.) was injected. In a separate group of rats, U-69593 (0.32 mg/kg s.c.) administration and reduced calcium infusion were initiated 15 min before the amphetamine injection. Samples were collected for 2 h after amphetamine was administered. [4]
U-69593 was suspended in vehicle (12% propylene glycol), whereas nBNI was dissolved in ACSF. Doses were calculated as the salt.

[1]. [3H]U-69593 a highly selective ligand for the opioid kappa receptor. Eur J Pharmacol. 1985 Feb 26;109(2):281-4.

[2]. The kappa-opioid receptor agonist U-69593 attenuates cocaine-induced behavioral sensitization in the rat. Brain Res. 1993 Jul 9;616(1-2):335-8.

[3]. U-69,593 microinjection in the infralimbic cortex reduces anxiety and enhances spontaneous alternation memory in mice. Brain Res. 2000 Feb 21;856(1-2):259-80.

[4]. The kappa-opioid agonist, U-69593, decreases acute amphetamine-evoked behaviors and calcium-dependent dialysate levels of dopamine and glutamate in the ventral striatum. J Neurochem. 1999 Sep;73(3):1066-74.

U-69593 is a monocarboxylic acid amide obtained by formal condensation between the carboxy group of phenylacetic acid and the secodary amino group of (5R,7S,8S)-N-methyl-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-amine. It has a role as a kappa-opioid receptor agonist, an anti-inflammatory agent and a diuretic. It is an oxaspiro compound, a N-alkylpyrrolidine, an organic heterobicyclic compound and a monocarboxylic acid amide.

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The present series of experiments sought to determine whether U-69593 could not only replicate the aforementioned anxiolytic effects observed among CD-1 mice from the vmPFC in the elevated plus-maze, but also influence associative learning in the elevated plus-maze transfer-latency test in trial-1 (drug condition). Following a 24-h delay, the possibility that pretrial-1 U-69593 could influence transfer-latency memory and memory of the aversiveness of the elevated plus-maze in trial-2 (no drug condition) was evaluated. In week 2, we evaluated whether a second pretrial U-69593 microinjection in the IL could influence defensive/withdrawal anxiety and spontaneous alternation performance memory in the Y-maze in the same CD-1 mice. In Experiment 2 we evaluated whether a 50% smaller drug injection volume (0.5 relative to 1.0 μl) in the IL cortex could comparably influence the aforementioned behaviours in 3 new groups of CD-1 mice, but in reverse order testing.[3]

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In this study, the κ-opioid receptor agonist, U-69593, significantly decreased amphetamine-induced behavior and the ability of 2.5 mg/kg amphetamine to induce dialysate dopamine and glutamate levels in the ventral striatum. These effects of U-69593 were antagonized by intraaccumbal perfusion of the selective κ-opioid antagonist, nBNI, through the microdialysis probe. ACSF containing 0.1 mM calcium reduced amphetamine’s ability to increase dopamine and glutamate dialysate levels in the ventral striatum. Furthermore, U-69593 did not affect the calcium-independent component of amphetamine-evoked dopamine and glutamate levels. [4]

C22H32N2O2-HCL

356.246

C, 74.12; H, 9.05; N, 7.86; O, 8.98

96744-75-1

1313288-99-1;96744-75-1;U69593 HCl; U-69593 Maleate;

105104

White to off-white solid powder

1.14g/cm3

518.719ºC at 760 mmHg

267.512ºC

1.581

3.191

0

3

4

26

485

3

CN([C@H]1CC[C@@]2(CCCO2)C[C@@H]1N3CCCC3)C(=O)CC4=CC=CC=C4

PGZRDDYTKFZSFR-ONTIZHBOSA-N

InChI=1S/C22H32N2O2/c1-23(21(25)16-18-8-3-2-4-9-18)19-10-12-22(11-7-15-26-22)17-20(19)24-13-5-6-14-24/h2-4,8-9,19-20H,5-7,10-17H2,1H3/t19-,20-,22-/m0/s1

(+)-N-methyl-2-phenyl-N-[(5R,7S,8S)-7-pyrrolidin-1-yl-1-oxaspiro[4.5]decan-8-yl]acetamide

U-69593; U069593; 96744-75-1; U69,593; U 69,593; U-69,593; n-methyl-2-phenyl-n-[(5r,7s,8s)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide; N-methyl-2-phenyl-N-[(5R,7S,8S)-7-pyrrolidin-1-yl-1-oxaspiro[4.5]decan-8-yl]acetamide; J5S4K6TKTG; U 69593

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.)