P2X receptor

Antinociceptive effects of TNP-ATP [1]
The novel P2X receptor antagonist, TNP-ATP inhibited potently agonist-stimulated calcium flux in 1321N1 cells expressing either the rat P2X3 or P2X2/3 receptors (Table 1). As has been shown for the human P2X3 receptor (Virginio et al., 1998), sequential removal of the terminal phosphate groups significantly reduces antagonist potency at both P2X3 and P2X2/3 receptors. In particular, TNP-AMP shows little inhibitory activity at rat P2X3 receptors at concentrations up to 30 μM.

The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat [1].

Activation of rat P2X3 and P2X2/3 receptors in vitro [1]
The recombinant rat P2X3 and rat P2X2/3 receptor cDNAs were identical to the previously published sequences used in the in vitro characterization of the pharmacology of the rat homomeric and heteromeric P2X3 receptors (Bianchi et al., 1999). 1321N1 human astrocytoma cells stably expressing rat P2X3, or rat P2X2/3 receptors were constructed using standard lipid-mediated transfection methods. All cell lines were maintained in D-MEM containing 10% FBS and antibiotics as follows: 300 μg ml−1 G418 for rat P2X3 containing cells; and 75 μg ml−1 hygromycin and 150 μg ml−1 G418 for rat P2X2/3 containing cells. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. P2X receptor function was determined on the basis of agonist-mediated increases in cytosolic Ca2+ concentration as previously described (Bianchi et al., 1999). BzATP (10 μM) and α,β-meATP (10 μM) were used to activate rat P2X3 and P2X2/3 receptors, respectively. Briefly, a fluorescent Ca2+ chelating dye (Fluo-4) was used as an indicator of the relative levels of intracellular Ca2+ in a 96-well format using a Fluorescence Imaging Plate Reader (FLIPR). Cells were grown to confluence in 96-well black-walled tissue culture plates and loaded with the acetoxymethylester (AM) form of Fluo-4 (1 μM) in D-PBS for 1 – 2 h at 23°C. Fluorescence data was collected at 1 – 5 s intervals throughout each experimental run. Concentration response data were analysed using a four-parameter logistic Hill equation in GraphPad Prism.

Nociceptive testing [1]
Nociceptive responses were assessed using procedures previously described for the formalin test of chemically-induced persistent pain (Abbott et al., 1995; Tjosen et al., 1992). Experimentally naive animals were placed in individual plexiglass cages and allowed 30 min to acclimatize to the testing environment. Following this period, animals received subcutaneous injections of either a formalin solution (1, 2.5, 5%), different doses of BzATP alone, or in combination with TNP-ATP or cibacron blue, into the dorsal surface of the right hind paw using an insulin gauge (29G1/2) needle. The volume of injection was 50 μl for all treatments. Saline (0.9%) was used as drug vehicle for all experiments. For co-administration studies, all test compounds were combined and administered in a single injection. To assess acute nociception, animals were observed immediately following drug injections and the number of flinch behaviours (paw withdrawals) was recorded over a 1-min period. Additional observations were conducted at sequential 5-min intervals during the first 15 – 20 min following drug injections (Phase I, acute phase of the formalin test). For some experiments, observations began 30 min after formalin injection and continued for 20 min thereafter (Phase II, persistent phase of the formalin test). For each individual experiment, six rats were used in separate experimental and control groups. Mean cumulative flinch responses were analysed by analysis of variance and post-hoc comparisons were conducted using Fisher's least significant difference test (GB-STAT, Dynamics Microsystems, Inc., Silver Spring, MD, U.S.A.). Statistical significance was determined at P<0.05.

[1]. Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue. Br J Pharmacol. 2001 Jan;132(1):259-69.

Since the potent P2X receptor antagonist, TNP-ATP may also be rapidly degraded in vivo (Lewis et al., 1997), the potential antinociceptive effects of TNP-ATP were assessed following peripheral intradermal administration into the rat hindpaw in an attempt to maximize the local concentration of TNP-ATP at a relevant site of action. The present data demonstrate that TNP-ATP, administered locally, can effectively attenuate nociception produced by either exogenously administered P2X receptor agonist (BzATP and α,βmeATP) or formalin. The antinociceptive effects of TNP-ATP appear to be pharmacologically specific since the less active analogue, TNP-AMP, was ineffective in reducing the nociceptive effects of either BzATP (1000 nmol paw−1) or 5% formalin. These antinociceptive effects of intradermal TNP-ATP extend other recent data, demonstrating that the i.t. administration of TNP-ATP blocks acute thermal hyperalgesia produced by i.t. administration of P2X receptor agonists (Tsuda et al., 1999b) and reduces acute nociception produced by intradermally administered formalin and capsaicin (Tsuda et al., 1999b). The ability of TNP-ATP to attenuate dose-dependently acute nociception produced by either BzATP or formalin was similar. Additionally, the antinociceptive effects of peripherally administered TNP-ATP were also evident during the persistent portion (Phase II) of the formalin test. This latter result contrasts with a recent report indicating that i.t. administration of TNP-ATP attenuates Phase I, but not Phase II, nociception in a murine formalin test regardless of whether TNP-ATP administration occurred before or after formalin administration (Tsuda et al., 1999b). However, another more stable P2 receptor antagonist, PPADS, was equally effective in reducing nociception in both phases of the formalin test following i.t. administration (Tsuda et al., 1999b), further supporting the idea that TNP-ATP may have a short half-life in vivo. The ability of peripherally administered TNP-ATP to attenuate formalin-induced persistent nociception in the present study may also have resulted from the use of significantly higher doses as compared to the doses used for i.t. administration (Tsuda et al., 1999b). Taken together, these results suggest that activation of both peripheral and spinal P2X receptors may contribute to the expression of nociception following tissue injury. Further, the ability of intradermal TNP-ATP to attenuate paw flinching in the formalin model indicates that activation of peripheral P2X receptors contributes to nociception in both the acute afferent barrage and persistent central sensitization phases of this model (Coderre & Melzack, 1992). [1]

C40H77N12O19P3

1123.04

1122.46

C, 42.78; H, 6.91; N, 14.97; O, 27.07; P, 8.27

61368-63-6

Orange to red liquid

1097.7ºC at 760 mmHg

617.7ºC

6.652

CCN(CC)CC.CCN(CC)CC.CCN(CC)CC.CCN(CC)CC.O=N([O-])C1C=C(N(=O)[O-])C2(O[C@@H]3[C@@H]([C@@H](COP(=O)(O)OP(=O)(O)OP(O)(O)=O)O[C@@H]3N4C5C(N=C4)=C(N)N=CN=5)O2)C(N(=O)[O-])C=1

XTSNRUFLZVGKOU-NRBYJGOSSA-O

InChI=1S/C16H16N8O19P3.4C6H15N/c17-13-10-14(19-4-18-13)21(5-20-10)15-12-11(7(39-15)3-38-45(34,35)43-46(36,37)42-44(31,32)33)40-16(41-12)8(23(27)28)1-6(22(25)26)2-9(16)24(29)30;4*1-4-7(5-2)6-3/h1-2,4-5,7,11-12,15H,3H2,(H6-,17,18,19,25,26,31,32,33,34,35,36,37);4*4-6H2,1-3H3/q-1;;;;/p+1/t7-,11-,12-,15-;;;;/m1..../s1

2',3'-O-(2,4,6-Trinitrophenyl)adenosine-5'-triphosphate tetra(triethylammonium) salt

TNP ATP; TNPATP; TNP-ATP

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.)