PDE4 ( IC50 = 3-240 nM)

The activity of immunopurified PDE4B and PDE4D was also inhibited by the PDE4-selective inhibitor Rolipram, with IC50 values of 130 nM and 240 nM, respectively. Conversely, immunopurified PDE4A's activity was inhibited by Rolipram, albeit at a much lower IC50 of about 3 nM. Rolipram increases cAMP response element binding protein (CREB) phosphorylation in U937 cells in a dose-dependent manner, suggesting the presence of components with high and low affinity (IC50 ~1 nM and IC50 ~120 nM, respectively). With a 290 nM IC50, rolipram dose-dependently and simply and monotonically inhibits the phosphorylation of p38 MAPK when stimulated by IFN-γ [1]. All four PDE4 isoforms are inhibited by the selective PDE4 inhibitor rolipram. Rolipram inhibits the production of TNF induced by LPS in a maximal/submaximal manner and in a dose-dependent manner (IC50 of 25.9 nM). At a dosage of 2 μM, inhibitory effects were noted in J774 cells [2].

Rolipram appears to have decreased the TNF mRNA and protein expression that LPS produced in WT mice's peritoneal macrophages (PM) (74% and 63% suppression of TNF mRNA and TNF protein, respectively). In line with previous findings, LPS-induced TNF production was higher in MKP-1 (-/-) mice's PM than in WT mice's PM. It's interesting to note that Rolipram's suppression of TNF mRNA and protein expression was much reduced and did not reach statistical significance in the PM of MKP-1 (-/-) mice [2]. In trained helpless rats, repeated intraperitoneal injections of Rolipram (1.25 mg/kg) can decrease the number of unsuccessful escape attempts [3].

Immunoprecipitation and PDE assays [1]
Selective immunoprecipitation of the enzymes of the four PDE4 classes was performed as described previously. As dis cussed before, sufficient antiserum was used to ensure that all isoenzymes of the target PDE4 subclass were selectively immunoprecipitated; these were then subjected to the PDE assay. PDE assays were done by a modification of the twostep Thompson and Appleman method. Determinations of total cellular PDE activity and the amounts of PDE3 and PDE4 components were performed with fresh cell lysates. As described before, determination of the total PDE3 and PDE4 activities was done using 1 µM cAMP as substrate and 10 µM of either the PDE3 selective inhibitor, cilostimide, or the PDE4 selective inhibitor, rolipram.

J774 murine macrophages were cultured at 37°C in 5% CO2 atmosphere in DMEM supplemented with glutamax-1 containing 10% heat-inactivated FBS, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin and 250 ng·mL−1 amphotericin B. For experiments, cells were seeded on 24-well plates at a density of 2 × 105 cells per well. Cell monolayers were grown for 72 h before the experiments were started. Rolipram, IBMX and BIRB 796 were dissolved in dimethyl sulfoxide (DMSO), and 8-Br-cAMP in HBSS. LPS (10 ng·mL−1) or the compounds of interest at concentrations indicated or the solvent (DMSO, 0.1% v/v) were added to the cells in fresh culture medium containing 10% FBS and the supplements. Cells were further incubated for the time indicated. The effect of LPS and the tested chemicals on cell viability was evaluated by Cell Proliferation Kit II (XTT). Neither LPS nor the other chemicals used in the experiments were observed to evoke cytotoxicity. [2]

Carrageenan-induced paw oedema [2]
C57BL/6 mice (20–25 g) were divided into groups of six mice and treated with 200 μL of PBS or rolipram (100 mg·kg−1 in PBS) by an i.p. injection 2 h before applying carrageenan. Before the administration of carrageenan, the mice were anaesthetized by i.p. injection of 0.5 mg·kg−1 of medetomidine (Domitor® 1 mg·mL−1) and 75 mg·kg−1 of ketamine (Ketalar® 10 mg·mL−1). The mice received a 30 μL i.d. injection of carrageenan (1.5%, dissolved in normal saline) in one hind paw. The contralateral paw received 30 μL of saline and it was used as a control. Paw volume was measured before and 3 h after the carrageenan injection with a plethysmometer. Oedema is expressed as a change in paw volume over time. After the experiments, the anaesthetized animals were killed by cervical dislocation.

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Mice were weighed and each one was placed in an individual cage. To introduce the mouse to sucrose solution and to obtain baseline data on sucrose consumption, mice were given a bottle of 2% sucrose. Twenty-four hours later, the bottles were removed and weighed to measure liquid intake. The water bottles were then replaced. Sucrose intake was measured again for a 1-h period. On the basis of body weight and sucrose intake (during the 24- and 1-h period), mice were assigned to either experimental or control groups (n=12 in each group). Body weight, in addition to sucrose consumption, was used to separate animals in an effort to minimize future changes in sucrose intake caused by differences in body size. Experimental animals were exposed to 6 weeks of chronic mild stress. Antidepressant-treated animals received a daily dose per os [po] of rolipram starting from the beginning of the 3rd week up to the end of the 6th week of CMS. The control animals were left undisturbed during the 6 week-period, except for scheduled daily po administration of distilled water in the last 3 weeks simulating the test group of treated animals, in addition to cleaning, feeding and weighing procedures. [3]

Drug administration and forced swimming test [3]
Where indicated, mice were given per os with a once daily dose of either distilled water (control group), rolipram (0.1 mg/kg/day) dissolved in distilled water in the last 3 weeks of exposure to CMS. The injected volume did not exceed 20 ml/kg body weight. This dose was selected by a pilot study that was done before the start of the experimental study and denoted the presence of changes by its administration.

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Dissolved in 100% PEG at an appropriate concentration; 1 mL/kg; i.v. injection

Male Hartley guinea pigs

mouse LD oral >300 mg/kg Biological and Pharmaceutical Bulletin., 17(498), 1994 [PMID:8069256]

[1]. Action of rolipram on specific PDE4 cAMP phosphodiesterase isoforms and on the phosphorylation of cAMP-response-element-binding protein (CREB) and p38 mitogen-activated protein (MAP) kinase in U937 monocyticcells. Biochem J. 2000 Apr 15;347(Pt 2):571-8.

[2]. Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase-1. Br J Pharmacol. 2013 Aug;169(7):1525-36.

[3]. Effect of rolipram, a phosphodiesterase enzyme type-4 inhibitor, on γ-amino butyric acid content of the frontal cortex in mice exposed to chronic mild stress. J Pharmacol Pharmacother. 2012 Apr;3(2):132-7.

Rolipram is a member of the lclass of pyrrolidin-2-ones that is pyrrolidin-2-one bearing a 3-(cyclopentyloxy)-4-methoxyphenyl substituent at the 4-position. It is a type IV-specific phosphodiesterase (PDE4) inhibitor. It has a role as an antidepressant and an EC 3.1.4.* (phosphoric diester hydrolase) inhibitor.
A phosphodiesterase inhibitor with antidepressant properties.
A phosphodiesterase 4 inhibitor with antidepressant properties.

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U937 monocytic cells are shown here to express a range of PDE4, cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes, PDE4A4, PDE4D5 and PDE4D3, plus the short isoenzyme, PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A, PDE4B and PDE4D activities were 0.63+/-0.09, 8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor, rolipram, inhibited immunopurified PDE4B and PDE4D activities similarly, with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast, rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis, it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes.[1]

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Background and purpose: 3',5'-Cyclic nucleotide PDE4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5'AMP, down-regulating cAMP signalling in cells. MAPK phosphatase-1 (MKP-1) is an endogenous p38 MAPK signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and whether MKP-1 is involved in the anti-inflammatory effects of rolipram. Experimental approach: The effect of rolipram on TNF production was investigated in J774 mouse macrophage cell line and in primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(-/-) mice. We also investigated the effect of rolipram on carrageenan-induced paw inflammation in WT and MKP-1(-/-) mice. Key results: MKP-1 expression was enhanced by rolipram, by a non-selective PDE inhibitor IBMX and by a cAMP analogue 8-Br-cAMP in J774 cells and in PM. Enhanced MKP-1 mRNA expression by rolipram was reversed by a PKA inhibitor. Rolipram, IBMX and 8-Br-cAMP also inhibited TNF production in activated macrophages. Accordingly, rolipram inhibited TNF production in PMs from WT mice but, interestingly, not in PMs from MKP-1(-/-) mice. Furthermore, rolipram attenuated carrageenan-induced paw inflammation in WT but not in MKP-1(-/-) mice. Conclusions and implications: PDE4 inhibitor rolipram was found to enhance the expression of MKP-1, and MKP-1 mediated, at least partly, the anti-inflammatory effects of PDE4 inhibition. The results suggest that compounds that enhance MKP-1 expression and/or MKP-1 activity hold potential as novel anti-inflammatory drugs.[2]

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Objectives: To investigate the alterations in GABA levels by rolipram in the model of depression. Materials and methods: The alteration of GABA content by rolipram as a phosphodiesterase enzyme type-4 inhibitor in the frontal cortex (FCx; as a brain region crucial for the control of emotion and cognition) obtained from male mice exposed to chronic mild stress (CMS)-induced anhedonia (the loss of pleasure or lack of sensitivity to pleasure stimuli) was recorded. Results: The results demonstrated the reversal of CMS-induced anhedonia after 3 weeks per os of rolipram in a dose of 0.1 mg/kg/day dissolved in distilled water. Furthermore, rolipram showed a significant reduction in duration of immobility in long-term behavioral changes recorded by the FST. Additionally, there was a significant increase in the GABA content of the FCx of rolipram-treated mice exposed to CMS-induced anhedonia. Conclusions: The present study suggested that GABA levels may be decreased in an animal model of depression and its reversal together with the behaviour improvement by rolipram could support the hypothesis that modification in GABAergic activity has a role in mood disorders. These effects may complement the antidepressant effect of rolipram that is originally mediated via inhibition of phosphodiesterase enzyme type-4 [PDE4] that increases cyclic adenosine monophosphate signalling the pharmacotherapy of depression.[3]

C16H21NO3

275.34

275.152

C, 69.79; H, 7.69; N, 5.09; O, 17.43

61413-54-5

(R)-(-)-Rolipram;85416-75-7;(S)-(+)-Rolipram;85416-73-5

5092

White to off-white solid powder

1.2±0.1 g/cm3

472.7±45.0 °C at 760 mmHg

127-133ºC

239.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.552

1.43

1

3

4

20

341

0

HJORMJIFDVBMOB-UHFFFAOYSA-N

InChI=1S/C16H21NO3/c1-19-14-7-6-11(12-9-16(18)17-10-12)8-15(14)20-13-4-2-3-5-13/h6-8,12-13H,2-5,9-10H2,1H3,(H,17,18)

4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one

ZK-62711; SB 95952; SB95952; rolipram; 61413-54-5; (+/-)-Rolipram; (R,S)-rolipram; ZK 62711; Rolipramum [Latin]; 4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one; Rolipramum; SB-95952; ME-3167; ZK-62711; ME3167; ZK62711; ME 3167; ZK 62711

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol:10 mg/L

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