Annette Robichaud

Deletion of phosphodiesterase 4D in mice shortens α2-adrenoceptor–mediated anesthesia, a behavioral correlate of emesis

A combination of pharmacological and genetic approaches was used to determine the role of type 4 cAMP-specific cyclic nucleotide phosphodiesterase 4 (PDE4) in reversing alpha(2)-adrenoceptor-mediated anesthesia, a behavioral correlate of emesis in non-vomiting species. Among the family-specific PDE inhibitors, PDE4 inhibitors reduced the duration of xylazine/ketamine-induced anesthesia in mice, with no effect on pentobarbital-induced anesthesia. The rank order of the PDE4 inhibitors tested was 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (PMNPQ) > (R)-rolipram > (S)-rolipram >> (R)-N-[4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl]N-ethylurea (CT-2450). The specific roles of PDE4B and PDE4D in this model were studied using mice deficient in either subtype. PDE4D-deficient mice, but not PDE4B-deficient mice, had a shorter sleeping time than their wild-type littermates under xylazine/ketamine-induced anesthesia, but not under that induced with pentobarbital. Concomitantly, rolipram-sensitive PDE activity in the brain stem was decreased only in PDE4D-deficient mice compared with their wild-type littermates. While PMNPQ significantly reduced the xylazine/ketamine-induced anesthesia period in wild-type mice and in PDE4B-null mice, it had no effect in PDE4D-deficient mice. These findings strongly support the hypothesis that inhibition of PDE4D is pivotal to the anesthesia-reversing effect of PMNPQ and is likely responsible for emesis induced by PDE4 inhibitors.

The next generation of PDE4 inhibitors

A number of highly potent PDE4 inhibitors are being developed for the treatment of asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, multiple sclerosis and Crohns disease. Cilomilast (Ariflo, SB 207499, SmithKline Beecham), the most advanced member of the class in Phase III clinical trials, was reported to have a limited therapeutic window. Other inhibitors with improved profiles in preclinical models are entering into (or are in) clinical trials. The recent developments in understanding PDE4 catalysis, inhibitor binding and their emetic response should facilitate the design of the next generation of PDE4 inhibitors.

PDE4 inhibitors induce emesis in ferrets via a noradrenergic pathway

The objective of this work was to assess the role of alpha(2)-adrenoceptors in emesis induced by inhibitors of type 4 phosphodiesterase (PDE4) in ferrets. Pre-treatment with yohimbine, MK-912 or MK-467 (alpha(2)-adrenoceptor antagonists) caused sudden and unexpected vomiting. In contrast, clonidine (alpha(2)-adrenoceptor agonist) did not induce emesis at doses ranging from 62.5-250 microg/kg s.c. At the dose of 250 microg/kg, clonidine also provided protection against emesis induced by the PDE4 inhibitors, PMNPQ (i.e. 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline, CT-2450 and R-rolipram. It was postulated that PDE4 inhibitors trigger emesis by mimicking the pharmacological actions of alpha(2)-adrenoceptor antagonists. This hypothesis was strengthened by the demonstration that PDE4 inhibitors can reverse the hypnotic effect of an alpha(2)-adrenoceptor mediated anaesthetic regimen in rats and ferrets. Similar to alpha(2)-adrenoceptor antagonists, PMNPQ, R-rolipram and S-rolipram dose-dependently decreased the duration of anaesthesia in rats injected with the combination xylazine/ketamine. While subcutaneous injections of CT-2450 (3-30 mg/kg) were without effect, a central infusion (6 microg i.c.v.) decreased the duration of anaesthesia. These studies suggest that the ferret is an appropriate model to study emesis induced by PDE4 inhibitors and that these compounds trigger the emetic reflex via a noradrenergic pathway, mimicking the pharmacological actions of a pre-synaptic alpha(2)-adrenoceptor inhibition.

Assessing the emetic potential of PDE4 inhibitors in rats

Type 4 phosphodiesterase (PDE4) inhibitors mimic the pharmacological actions of alpha2‐adrenoceptor antagonists. This has been postulated as the mechanism by which PDE4 inhibitors induce emesis and was also demonstrated by their ability to reverse xylazine/ketamine‐induced anaesthesia. We further characterized this latter effect since it appears to reflect the emetic potential of PDE4 inhibitors. Selective inhibitors of PDE 1, 2, 3, 4 and 5 were studied in rats, on the duration of anaesthesia induced by the combination of xylazine (10u2003mgu2003kg−1, i.m.) and ketamine (10u2003mgu2003kg−1, i.m.) PMNPQ (i.e. 6‐(4‐pyridylmethyl)‐8‐(3‐nitrophenyl)quinoline) u2003–u2003 PDE4 inhibitor: 0.01u2003–u20033u2003mgu2003kg−1), like MK‐912 (alpha2‐adrenoceptor antagonist: 0.01u2003–u20033u2003mgu2003kg−1), dose‐dependently reduced the duration of anaesthesia. In contrast, vinpocetine (PDE1 inhibitor), EHNA (PDE2 inhibitor), milrinone (PDE3 inhibitor) and zaprinast (PDE5 inhibitor) had no significant effect at the doses tested (1u2003–u200310u2003mgu2003kg−1). Analysis of plasma and cerebrospinal fluid (CSF) of treated animals confirmed the absorption and distribution to the brain of the inactive inhibitors. Neither MK‐912 (3u2003mgu2003kg−1) nor PMNPQ (0.1u2003–u20031u2003mgu2003kg−1) altered the duration of anaesthesia induced via a non‐alpha2‐adrenoceptor pathway (sodium pentobarbitone 50u2003mgu2003kg−1, i.p.) Central NK1 receptors are involved in PDE4 inhibitor‐induced emesis. Consistently, [sar9, Met(O2)11]‐substance P (NK1 receptor agonist, 6u2003μgu2003i.c.v.) reduced the duration of anaesthesia induced by xylazine/ketamine. In summary, this model is functionally coupled to PDE4, specific to alpha2‐adrenoceptors and relevant to PDE4 inhibitor‐induced emesis. It therefore provides a novel way of evaluating the emetic potential of PDE4 inhibitors in rats.

Localization of phosphodiesterase-4 isoforms in the medulla and nodose ganglion of the squirrel monkey

Pre-clinical and clinical studies are currently underway to evaluate the potential of phosphodiesterase-4 (PDE4) inhibitors for the treatment of chronic obstructive pulmonary disease and other inflammatory conditions of the airways. The most common side effect associated with this class of compounds is emesis. The squirrel monkey provides a model for evaluating the efficacy of PDE4 inhibitors and their emetic potential. The distribution of three PDE4 isoforms (A, C and D) has been investigated in the squirrel monkey medulla and nodose ganglion to determine which isoform(s) could be responsible for the emetic adverse effects. The distribution of PDE4 isoforms was delineated using immunohistochemistry with antibodies specific for PDE4A, PDE4C and PDE4D and by in situ hybridization with isoform-selective riboprobes. PDE4A was present in the medulla where expression was mostly restricted to glial cells and the vasculature. PDE4C was not detected in either the medulla or nodose ganglion. Finally, the PDE4D isoform was localized to neurons in the nodose ganglion and found through many structures of medulla including the area postrema, neurons of the nucleus tractus solitarius and locus coeruleus. These data are consistent with a role for PDE4D in the emetic response.

Substituted furans as inhibitors of the PDE4 enzyme

The synthesis and in vitro activity of a series of substituted furans as a novel structural class of PDE4 inhibitors is described. Comparison of emetic threshold with known PDE4 inhibitors is presented.