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.

Multiple indirect pieces of evidence support the hypothesis that activation of protein kinase C (PKC) and elevated intracytoplasmic Ca++ levels are essential for T cell activation and proliferation in response to physiological stimuli, i.e., MHC class II restricted antigen presentation. We validated this hypothesis using Ro 32-0432, a highly potent, cell-penetrating, and selective PKC inhibitor. Ro 32-0432 inhibited interleukin-2 (IL-2) secretion, IL-2 receptor expression, and proliferation of peripheral blood human T cells stimulated with phorbol ester in combination with phytohemagglutinin or anti-CD3 antibody, but did not inhibit IL-2-induced proliferation in cells expressing the IL-2 receptor. Ro 32-0432 inhibited the proliferation of influenza peptide antigen HA 307-319-specific human T cell clones (HA27) after exposure to antigen pulsed autopresenting cells. Oral administration of Ro 32-0432 inhibited subsequent edema induced by phorbol ester in rats, indicating that the compound has systemic efficacy in inhibiting PKC-driven responses. Ro 32-0432 also inhibits more physiologically driven T cell responses, such as host antigraft response and secondary claw swelling in adjuvant-induced arthritis. These data suggest that PKC plays a key role in T cell activation and that selective oral administration of highly bioavailable PKC inhibitors can effectively prevent T cell-driven chronic inflammatory responses in vivo. Inhibition of protein kinase C (PKC) is an important mechanism for preventing T cell activation, and such compounds may have important therapeutic applications for chronic inflammation and autoimmune diseases. [1]

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G protein-coupled receptor kinase 5 (GPK5) is thought to mediate multiple signal transduction cascades that are associated with the occurrence of nicotine withdrawal syndrome. Therefore, this study investigated the effects of the GPK5 inhibitor Ro 32-0432 on the development of nicotine dependence and withdrawal symptoms in a subchronic nicotine mouse model. Our experimental protocol consisted of subcutaneous injection of nicotine (2.5 mg/kg) four times a day for seven consecutive days. To induce nicotine withdrawal, mecaramine (3 mg/kg) was administered intraperitoneally one hour after the last nicotine injection on the test day (day 8). Behavioral observation was conducted immediately 30 minutes after mecaramine treatment. The severity of withdrawal syndrome was quantified by a comprehensive withdrawal severity score, jump frequency, and nicotine-induced hyperalgesia measured by the tail-flick test. Withdrawal syndrome-related anxiety was assessed by the results of the elevated cross maze test. Ro 32-0432 dose-dependently reduced mecaramine-induced nicotine withdrawal syndrome in mice. It is concluded that Ro 32-0432 may attenuate the spread of nicotine dependence and alleviate withdrawal symptoms through a mechanism related to G protein-coupled receptor kinase 5 activation. [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.)