human α2C-adrenoceptor (Ki = 28 nM); human α2B-adrenoceptor (Ki = 1470 nM); human α2A-adrenoceptor (Ki = 3150 nM); rodent α2D-adrenoceptor (Ki = 1700 nM)

JP1302 exhibits approximately 100-fold higher affinity than α2A or α2B[1].
Radioligand-binding assays [2]
In competition binding assays with [3H]-rauwolscine, JP1302 displayed an affinity of 28 nM for the α2C-AR (Table 1). As the affinity of JP-1302 on the three other α2-AR subtypes were 1500 nM or lower, the compound is endowed with a minimum selectivity of about 50-fold for the α2C-AR. The profiling of JP-1302 at a concentration of 0.1 μM against 30 other receptor targets revealed no discernible secondary sites (Table 2). At a 100-fold higher concentration (10 μM) binding to α1-ARs and some other receptors was found (Table 2).

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α2-Antagonist activity in cellular membranes[2]
JP1302 was unable to increase [35S]-GTPγS-binding to membranes of CHO cells expressing the three human α2-AR subtypes, thereby demonstrating that the compound does not possess agonist activity on α2-AR (Table 1). JP-1302 was, however, able to antagonize the agonist response of a fixed amount of adrenaline in a concentration-dependent manner (Figure 2). The apparent antagonist potencies (KB values) of JP-1302 were found to be 1500, 2200 and 16 nM for the α2A-, α2B- and α2C-AR subtypes, respectively (Table 1).

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α2-Antagonist activity in the vas deferens model[2]
Dexmedetomidine dose-dependently inhibited electrically evoked contractions in rat vas deferens preparations resulting in EC50 values of 1.4 nM (Figure 3). JP1302 had no antagonist effects on the dexmedetomidine-induced inhibition. However, in the presence of atipamezole, the dose–response curve of dexmedetomidine was shifted rightwards, resulting in pA2 values for atipamezole of 8.5.

JP1302 (1-10 μMol/kg) shortens the FST immobility period to a level that corresponds with 10–30 μMol/kg of the antidepressant desipramine [1]. JP1302 (5 μMol/kg, once) the psychotomimetic NMDA receptor coupling antagonist phencyclidine, was able to totally reverse PPI-induced damage in Sprague-Dawley deposits; comparable outcomes were observed in Wistar deposits [1]. JP1302 (3 mg/kg, IV, once) ameliorates renal impairment considerably [3]

Radioligand-binding assays [2]
The affinity of test compounds for the three human α2-adrenoceptor subtypes (α2A, α2B and α2C) and the mouse α2D subtype was determined in competition-binding assays with [3H]-rauwolscine and [3H]-RX821002, respectively. The biological material in the [3H]-rauwolscine displacement assay consisted of membranes from Shionogi S115 cells stably transfected with one of the three human α2 subtypes (Marjamäki et al., 1992). In the [3H]-RX821002 displacement assay, membranes from Chinese hamster ovary (CHO) cells stably transfected with the mouse α2D subtype were used. The membrane suspensions (3–15 μg total protein per sample, depending on the expression level of individual subtypes) and about 1 nM of [3H]-rauwolscine (specific activity 75–85 Ci mmol−1) or [3H]-RX821002 (specific activity 59 Ci mmol−1) were incubated with a minimum of six concentrations of the test compound in a total volume of 90 μl (50 mM KH2PO4, pH 7.5, at room temperature). Specific-binding was defined by 100 μM oxymetazoline and corresponded to 90–96% of the total binding. After 30 min at room temperature, the incubations were terminated by rapid filtration (TomTec 96 harvester. Tomtec Inc, Hamden, CT, USA) through presoaked GF/B glass-fibre mats and three washes with ice-cold 50 mM KH2PO4 (pH 7.5 at room temperature). The filter mats were then dried and a solid scintillate was melted onto them before their radioactivity was measured.[2]
The analysis of competition binding experiments was carried out by nonlinear least square curve fitting. IC50s were converted to Ki values by using the equation of Cheng–Prussoff (Ki=IC50/(1+(3H-ligand)/Kd, 3H−ligand)).[2]
The affinity profiling of JP1302 at a concentration of 0.1 and 10 μM on a number of receptors other than the α2-adrenoceptor subtypes was conducted by Cerep using documented standard procedures (see Table 2 for relevant details of the experimental conditions).

α2-Antagonist activity in cellular membranes [2]
The antagonist activity of JP1302 was determined as the ability of the compound to inhibit adrenaline-stimulated 35S-guanosine-5′-O-(3-thio)triphosphate (35S-GTPγS)-binding to G-proteins competitively (Jasper et al., 1998) in membranes of CHO cells stably transfected with one of the three human α2 subtypes (Pohjanoksa et al., 1997). Membranes (2–6 μg of protein per sample) and 12 concentrations of JP1302 were preincubated for 30 min at room temperature in 50 mM Tris, 5 mM MgCl2, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 μM GDP, 30 μM ascorbic acid, pH 7.4, with a fixed concentration of adrenaline (5 μM for α2A, 15 μM for α2B, 5 μM for α2C). Then trace amounts of [35S]-GTPγS (0.08–0.15 nM, specific activity 1250 Ci mmol−1) were added to the incubation mixture. After an additional 30 min at room temperature, the incubation was terminated by rapid vacuum filtration through glass fibre filter. Filters were washed three times with 5 ml ice-cold wash buffer (20 mM Tris, 5 mM MgCl2, 1 mM EDTA, pH 7.4), dried and counted for radioactivity in a scintillation counter. Experiments were repeated at least three times.
The analysis of antagonism experiments was carried out by nonlinear least-square curve fitting. IC50s were converted to KB values by using the equation KB=IC50/(1+(adrenaline)/EC50, adrenaline) with EC50 values of adrenaline on the three α2-AR subtypes of 0.76 μM (α2A), 2.4 μM (α2B) and 0.71 μM (α2C).

Male Sprague Dawley rats (8 weeks old)
3 mg/kg
IV, pre-treatment: administered 5 min before the induction of ischemia, post-treatment: injected 45 min after the initiation of reperfusion

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Antagonism of the mydriatic effect of the α2-agonist dexmedetomidine [2]
Rats (n=5/group, total of 15) were anaesthetized with sodium pentobarbitone (Mebunat 60 mg ml−1, Orion, Finland) and a polyethylene cannula was inserted into the lateral tail vein for drug administration. The pupil diameter was measured by means of an operating microscope provided with a 10-mm graduated line (0.1 divisions) in the ocular. The microscope had an internal light source with green filter. The light was maintained at a steady intensity throughout the experiments. After measurement of the baseline pupil diameter, all rats were given the α2-agonist dexmedetomidine 10 μg intravenously (i.v.). The mydriatic effect of dexmedetomidine was measured after 5 min and then cumulative doses of either atipamezole or JP1302 or equivalent volumes of vehicle were applied intravenously at 5 min intervals. The cumulative doses of the antagonists were determined with steps of 3, 10, 30, 100, 300, 1000 and 3000 nmol kg−1, and the entire duration of the measurement was thus 45 min. The pupil diameter measurements were performed just before the next injection.

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Antagonism of α2-agonist–induced inhibition of locomotor activity (sedation) and hypothermia[2]
Spontaneous locomotor activity of a total of 76 male NMRI mice (B&K, Sweden) was measured by placing individual animals into a polypropylene animal cage (38 × 22 × 15 cm). The cages were surrounded by an infrared photobeam frame system designed for activity measurements. The animals were injected either with JP1302 or atipamezole 20 min before the injection of dexmedetomidine (50 nmol kg−1 s.c.). Spontaneous locomotor activity was measured 20 min after dexmedetomidine injection and the dexmedetomidine–induced inhibition of locomotor activity was used as a measure of sedation. At the end of the locomotor activity recordings, the core body temperatures of the mice were measured with a rectal probe and a digital thermometer . The probe was inserted 2.5 cm inside the anal sphincter and maintained there until the temperature reading of the thermometer was stabilized. The same system was used in a separate experiment for the detection of the effects of JP1302 alone. In the latter experiment, the activity was recorded for the period of 20–40 min after the drug injection.

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Effect of JP1302 in the FST for antidepressant activity[2]
Rats were transferred into the experimental room at least 30 min before testing. Forced swimming was conducted by immersing each rat individually in a transparent glass cylinder (height 46 cm, diameter 20 cm) containing a 21-cm deep column of water at 25°C. In the FST sessions, an initial 15 min pre-test was followed 24 h later by a 5-min actual test. Drug treatments, as two subcutaneous (s.c) injections, were given during the period between the two sessions, the first 15 min after the pre-test and the second 1 h before the test swim. Following both FST sessions, the rats were removed from the cylinders, dried with towels and placed into heated cages for 15 min, and then returned to their home cages. Each animal was used only once.[2]
The cumulative time of immobility was directly observed and recorded by a stopwatch timer during the 5 min test swim. The experimenter was well experienced in rating the behaviour and blinded for the different drug treatments. A rat was judged to be immobile when it remained floating in the water without struggling and was making only those movements necessary to keep its head above water.

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Effect of JP1302 and atipamezole on prepulse inhibition of acoustic startle[2]
Startle experiments were performed in four identical, ventilated and illuminated startle chambers (39 × 38 × 58 cm (length × width × height)). The chambers consisted of a non-restrictive Plexiglas cylinder (3.9 cm in diameter) resting on a Plexiglas platform. Piezoelectric accelerometers mounted under the cylinders detected and transduced the animal movements. High-frequency speakers, mounted 25 cm above the cylinder, provided all acoustic stimuli. Presentation of the acoustic stimuli and the piezoelectric responses from the accelerometer were controlled and digitized by the SR-LAB software and interface system. The sensitivity of the chambers was adjusted to average readings of 100 using the standardization unit from San Diego Instruments. Sound levels within each chamber were measured repeatedly using the A weighing scale and were found to remain constant. As differences in PPI levels between different rat strains can contribute to drug responses, two rat strains were used (SD and Wistar) to test the effect of JP1302.

mouse LD50 oral 1500 mg/kg Meditsinskaya Parazitologiya i Parazitarnye Bolezni. Medical Parasitology and Parasitic Diseases., 61(5)(55), 1991

[1]. JP-1302: a new tool to shed light on the roles of alpha2C-adrenoceptors in brain. Br J Pharmacol. 2007 Feb;150(4):381-2.

[2]. Pharmacological characterization and CNS effects of a novel highly selective alpha2C-adrenoceptor antagonist JP-1302. Br J Pharmacol. 2007 Feb;150(4):391-402.

[3]. Post-treatment with JP-1302 protects against renal ischemia/reperfusion-induced acute kidney injury in rats. J Pharmacol Sci. 2019 Mar;139(3):137-142.

The discovery of JP-1302 as a selective, high affinity antagonist at the alpha2C-adrenoceptor will enable researchers to probe the functional role and address the therapeutic utility of this potentially highly important adrenoceptor subtype. [1]

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Background and purpose: Pharmacological validation of novel functions for the alpha2A-, alpha2B-, and alpha2C-adrenoceptor (AR) subtypes has been hampered by the limited specificity and subtype-selectivity of available ligands. The current study describes a novel highly selective alpha2C-adrenoceptor antagonist, JP-1302 (acridin-9-yl-[4-(4-methylpiperazin-1-yl)-phenyl]amine). Experimental approach: Standard in vitro binding and antagonism assays were employed to demonstrate the alpha2C-AR specificity of JP-1302. In addition, JP-1302 was tested in the forced swimming test (FST) and the prepulse-inhibition of startle reflex (PPI) model because mice with genetically altered alpha2C-adrenoceptors have previously been shown to exhibit different reactivity in these tests when compared to wild-type controls. Key results: JP-1302 displayed antagonism potencies (KB values) of 1,500, 2,200 and 16 nM at the human alpha2A-, alpha2B-, and alpha2C-adrenoceptor subtypes, respectively. JP-1302 produced antidepressant and antipsychotic-like effects, i.e. it effectively reduced immobility in the FST and reversed the phencyclidine-induced PPI deficit. Unlike the alpha2-subtype non-selective antagonist atipamezole, JP-1302 was not able to antagonize alpha2-agonist-induced sedation (measured as inhibition of spontaneous locomotor activity), hypothermia, alpha2-agonist-induced mydriasis or inhibition of vas deferens contractions, effects that have been generally attributed to the alpha2A-adrenoceptor subtype. In contrast to JP-1302, atipamezole did not antagonize the PCP-induced prepulse-inhibition deficit. Conclusions and implications: The results provide further support for the hypothesis that specific antagonism of the alpha2C-adrenoceptor may have therapeutic potential as a novel mechanism for the treatment of neuropsychiatric disorders.[2]

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Ischemia/reperfusion injury is the most common cause of acute kidney injury. We previously revealed that pre-treatment with yohimbine or JP-1302 attenuated renal ischemia/reperfusion injury by inhibition of α2C-adrenoceptor antagonist. The aim of the present study is to investigate the effects of post-treatment with JP-1302 on renal ischemia/reperfusion injury in rats. Male Sprague Dawley rats were randomly divided into four groups: sham operation, ischemia/reperfusion, pre-treatment with JP-1302 (3.0 mg/kg) and post-treatment with JP-1302 groups. In ischemia/reperfusion injury, renal functional parameters, such as blood urea nitrogen, plasma creatinine and creatinine clearance, deteriorated after reperfusion. Renal venous norepinephrine concentrations, as well as inflammatory molecules in the kidney increased after reperfusion. Both pre- and post-treatment with JP-1302 improved renal dysfunction, tissue damage, renal venous norepinephrine concentrations and inflammatory molecules expression in the kidney. In conclusion, these results suggest that post-treatment with JP-1302 protects on ischemia/reperfusion-induced acute kidney injury by suppressing cytokine upregulation via α2C-adrenoceptors.[3]

C24H26CL2N4

441.4

440.153

C, 65.31; H, 5.94; Cl, 16.06; N, 12.69

1259314-65-2

JP1302; 80259-18-3

49855035

Brown to reddish brown solid powder

3

4

3

30

473

0

Cl.Cl.CN1CCN(C2=CC=C(NC3=C4C=CC=CC4=NC4=CC=CC=C34)C=C2)CC1

VVZOADYFJAJZGL-UHFFFAOYSA-N

InChI=1S/C24H24N4.2ClH/c1-27-14-16-28(17-15-27)19-12-10-18(11-13-19)25-24-20-6-2-4-8-22(20)26-23-9-5-3-7-21(23)24;;/h2-13H,14-17H2,1H3,(H,25,26);2*1H

N-[4-(4-methylpiperazin-1-yl)phenyl]acridin-9-amine;dihydrochloride

JP 1302 2HCl; JP1302 dihydrochloride; JP 1302 DIHYDROCHLORIDE; 1259314-65-2; JP1302 dihydrochloride; N-(4-(4-Methylpiperazin-1-yl)phenyl)acridin-9-amine dihydrochloride; JP-1302 HCl; N-[4-(4-methylpiperazin-1-yl)phenyl]acridin-9-amine;dihydrochloride; N-[4-(4-Methyl-1-piperazinyl)phenyl]-9-acridinamine dihydrochloride; N-[4-(4-methylpiperazin-1-yl)phenyl]acridin-9-amine dihydrochloride; JP-1302 hydrochloride; JP 1302 dihydrochloride; JP-1302 dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~12.5 mg/mL (~28.32 mM)
DMSO : ~5 mg/mL (~11.33 mM)

Solubility in Formulation 1: 5 mg/mL (11.33 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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