PKCα 9.3 nM (IC50) PKC-βI 28 nM (IC50) PKC-βII 30 nM (IC50) PKCγ 36.5 nM (IC50) PKCε 108.3 nM (IC50) G protein-coupled receptor kinase 5 (GRK5)

(S)-Ro 32-0432 suppresses the release of interleukin-2 (IL-2) and the expression of IL-2 receptors in peripheral human T-cells stimulated with phorbol ester in combination with phytohemagglutin or anti-CD3. However, it has no effect on the proliferation of IL-2-induced cells that have already been stimulated to express IL-2 receptors. The influenza peptide antigen HA 307-319-specific human T-cell clone (HA27) is also blocked by (S)-Ro 32-0432 from proliferating after being exposed to antigen-pulsed autologous presentation cells. HA27 proliferation is inhibited by (S)-Ro 32-0432, with an IC50 of 0.15 μM[1].

Treatment with (S)-Ro 32-0432 (10–50 mg/kg; oral dose; once; female AHH/R rats) prevents edema produced by phorbol ester in rats later on, indicating the compound's systemic effectiveness in suppressing PKC-driven reactions. Ro 32-0432 also inhibits the induction of more physiologically driven T-cell responses, such as host versus graft responses and subsequent paw edema in adjuvant-induced arthritis[1].

The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon[Biochem J. 1993 Sep 1;294 ( Pt 2)(Pt 2):335-7].

Several lines of circumstantial evidence support the assumption that protein kinase C (PKC) activation together with elevated levels of cytosolic Ca++ are necessary for T-cell activation and proliferation in response to a physiological stimulus, i.e., MHC class II restricted antigen presentation. By using a potent, cell-permeable and selective inhibitor of PKC, Ro 32-0432, we have tested this hypothesis. Ro 32-0432 inhibits interleukin-2 (IL-2) secretion, IL-2 receptor expression in, and proliferation of, peripheral human T-cells stimulated with phorbol ester together with phytohemagglutin or anti-CD3, but does not inhibit IL-2 induced proliferation in cells already stimulated to express IL-2 receptors. Proliferation of the influenza peptide antigen HA 307-319-specific human T-cell clone (HA27) after exposure to antigen-pulsed autologous presenting cells was also inhibited by Ro 32-0432[2].

Animal/Disease Models: Female AHH/R rats (200-250 g) induced with phorbol ester[1]
Doses: 10 mg/kg, 30 mg/kg, 50 mg/kg
Route of Administration: Oral administration; once
Experimental Results: Inhibited subsequent phorbol ester-induced edema in rats.

[1]. Ro 32-0432, a Selective and Orally Active Inhibitor of Protein Kinase C Prevents T-cell Activation. J Pharmacol Exp Ther. 1994 Feb;268(2):922-9.

[2]. Ro 32-0432 Attenuates Mecamylamine-Precipitated Nicotine Withdrawal Syndrome in Mice. Naunyn Schmiedebergs Arch Pharmacol. 2013 Mar;386(3):197-204.

Several lines of circumstantial evidence support the assumption that protein kinase C (PKC) activation together with elevated levels of cytosolic Ca++ are necessary for T-cell activation and proliferation in response to a physiological stimulus, i.e., MHC class II restricted antigen presentation. By using a potent, cell-permeable and selective inhibitor of PKC, Ro 32-0432, we have tested this hypothesis. Ro 32-0432 inhibits interleukin-2 (IL-2) secretion, IL-2 receptor expression in, and proliferation of, peripheral human T-cells stimulated with phorbol ester together with phytohemagglutin or anti-CD3, but does not inhibit IL-2 induced proliferation in cells already stimulated to express IL-2 receptors. Proliferation of the influenza peptide antigen HA 307-319-specific human T-cell clone (HA27) after exposure to antigen-pulsed autologous presenting cells was also inhibited by Ro 32-0432. Oral administration of Ro 32-0432 inhibited subsequent phorbol ester-induced edema in rats demonstrating the systemic efficacy of the compound to inhibit PKC-driven responses. Induction of more physiologically T-cell driven responses such as host vs. graft responses and the secondary paw swelling in adjuvant-induced arthritis were also inhibited by Ro 32-0432. These data demonstrate the crucial role for PKC in T-cell activation and that selective p.o. bioavailable PKC inhibitors are efficacious in preventing T-cell driven chronic inflammatory responses in vivo. Inhibition of PKC represents an important mechanistic approach to prevent T-cell activation and compounds of this class may have important therapeutic applicability to chronic inflammatory and autoimmune diseases.[1]

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G protein-coupled receptor kinase 5 is noted to mediate a number of signal transduction cascades involved in the causation of nicotine withdrawal syndrome. Therefore, the present study investigated the effect of Ro 32-0432, a G protein-coupled receptor kinase 5 inhibitor, on propagation of nicotine dependence and resultant withdrawal signs in subchronic nicotine mouse model. Our experimental protocol consisted of administration of nicotine, (2.5 mg/kg, subcutaneously), four times daily for 7 days. In order to precipitate nicotine withdrawal, mice were given one injection of mecamylamine (3 mg/kg, intraperitoneally) 1 h after the last nicotine injection on the test day (day 8). Behavioral observations were made for a period of 30 min immediately after mecamylamine treatment. Withdrawal syndrome was quantitated in terms of a composite withdrawal severity score, jumping frequency, nicotine-induced hyperalgesia by tail flick method, and withdrawal syndrome-related anxiety was assessed by elevated plus maze test results. Ro 32-0432 dose dependently attenuated mecamylamine-induced nicotine withdrawal syndrome in mice. It is concluded that Ro 32-0432 attenuates the propagation of nicotine dependence and reduce withdrawal signs possibly by G protein-coupled receptor kinase 5 activation-linked mechanisms.[2]

C28H29CLN4O2

488.197

C, 68.77; H, 5.98; Cl, 7.25; N, 11.46; O, 6.54

1781828-85-0

70346044

Typically exists as solid at room temperature

2

3

4

35

869

1

CN1C=C(C2=CC=CC=C21)C3=C(C(=O)NC3=O)C4=C5C[C@H](CCN5C6=CC=CC=C64)CN(C)C.Cl

HSPRASOZRZDELU-LMOVPXPDSA-N

InChI=1S/C28H28N4O2.ClH/c1-30(2)15-17-12-13-32-22-11-7-5-9-19(22)24(23(32)14-17)26-25(27(33)29-28(26)34)20-16-31(3)21-10-6-4-8-18(20)21;/h4-11,16-17H,12-15H2,1-3H3,(H,29,33,34);1H/t17-;/m0./s1

3-[(8S)-8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]-4-(1-methylindol-3-yl)pyrrole-2,5-dione;hydrochloride

Ro 32-0432 hydrochloride; 1781828-85-0; Ro 32-0432 (hydrochloride); 3-[(8S)-8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]-4-(1-methylindol-3-yl)pyrrole-2,5-dione;hydrochloride; SCHEMBL8321924; Ro 32-0432 HCl;

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.)