L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition
Introduction
Cyclic adenosine monophosphate (cAMP) is a potent second messenger with a variety of physiological and pathophysiological manifestations [1]. A few of the parameters which cAMP can modulate are smooth muscle contraction, inflammatory cell activity, mediator release, neurochemical release, and cellular adhesion. Adenylate cyclases, of which there are presently 10 human isoforms, catalyze the formation of cAMP from 5′AMP [2], [3], [4], [5]. Since even short spikes of cAMP elevation within a cell can cause activation of several signaling pathways, the synthesis and metabolism of cAMP is tightly controlled [6]. The enzymes that metabolize cAMP to 5′AMP are termed phosphodiesterases. The phosphodiesterase gene family is compromised of 11 members of which 6 of these genes can metabolize specifically either cAMP or cGMP while the remaining 5 genes can metabolize both cAMP and cGMP [7].
Elevations of cAMP through β2 adrenergic stimulation have been utilized extensively for the treatment of asthma and COPD [8]. The primary clinical utility for β2 adrenergic agonists has been to cause bronchodilatation in patients with bronchial hyper-reactivity [9]. The present clinical data suggest that elevation of cAMP through β2 adrenergic stimulation does not affect the inflammatory component of asthma or COPD [10]. In preclinical paradigms and in clinical trials, significant data have been generated that demonstrate prolonged elevation of cAMP through inhibition of PDE4 results in significant anti-inflammatory effects. Roflumilast, one of the most advanced, potent and specific PDE4 inhibitor, has been shown to decrease parenchymal lung damage and infiltration of lung macrophages in a 7-month model in mice chronically exposed to cigarette smoke [9], [11]. Also, genetic mouse deletions of either PDE4B or PDE4D (two of the four orthologues of the PDE4 gene family) results in either absence of LPS induced TNFα formation from monocytes or decreased hyper-responsiveness to antigen challenge in a mouse OVA-model of airway resistance, respectively [12], [13]. The most compelling data for PDE4 inhibitors is the clinical data generated with roflumilast and cilomilast. Roflumilast in a 24-week daily dosing regime of either 0.25 or 0.5 mg/day in COPD patients resulted in a statistically significant improvement in postbronchodilator FEV1[14]. In a clear indication of anti-inflammatory activity with PDE4 inhibitors, cilomilast dosed at 15 mg/BID for 12 weeks in COPD patients resulted in a 48 and 47% reduction in CD8+ and CD68+ cells, respectively, in lung biopsies from the inhibitor treated patients [15]. These data with cilomilast are the only published data that demonstrate decreases in airway inflammatory cells in lung tissues from COPD patients. Of the latter two compounds, only roflumilast has also demonstrated efficacy in asthma, with statistically significant improvement in FEV1's during the late asthmatic response in a 10-day study of allergen challenged asthmatics [16].
One of the major development hurdles for PDE4 inhibitors has been toxicity and specifically, gastrointestinal intolerance that is manifested as emesis and diarrhea. With the use of genetically manipulated mice, the data suggest that a majority of the anti-inflammatory effect of PDE4 inhibition is through PDE4B and PDE4D. The PDE4B null mice have a significantly decreased TNFα production in response to LPS and have decreased lung inflammation in an LPS challenged model of asthma [17]. The PDE4D null mice have a decreased α2-adrenoreceptor mediated anesthesia that is a correlate of emesis and therefore identifies PDE4D as the major isoform in the brain responsible for emesis [18]. Subtype selective PDE4 inhibitors have been a major challenge throughout the development process and recent crystallography data demonstrates that the first shell of the PDE4 active site is completely conserved between PDE4B and D [19].
Another significant indication for PDE4 inhibitors is the treatment of cognitive dysfunction. Disorders in which cognitive deficits are inherent include Alzheimer's disease, Parkinson's disease, vascular dementia, schizophrenia and stroke. Alterations in brain levels of cAMP have been linked to increases in long term potentiation (LTP) that appears critical for signaling pathways involved in cognition. Both studies with weak PDE4 selective inhibitors and PDE4D null mice have demonstrated significant increases in LTP and/or learning behavior in several assays [20], [21], [22]. The present study details effects observed with L-454,560 in the Morris watermaze in rodents and, moreover, determines a therapeutic window between memory enhancement and potential emesis.
Thus, this study describes the development of a potent and selective PDE4 inhibitor, L-454,560. This compound is a competitive and reversible PDE4 inhibitor that preferentially inhibits the hydrolysis of cAMP by PDE4A, 4B and 4D with comparable IC50 values ranging from 0.5 to 1.6 nM. It is efficacious in an ovalbumin-challenged guinea pig model of airway hyper-reactivity with an ED50 of 0.03 mg/kg. L-454,560 also inhibits significantly the late asthmatic response in ascaris sensitized and challenged sheep with 95% of the LAR response inhibited at a 0.5 mg/kg IV dose. The overall profile of L-454,560 is significantly more potent than cilomilast and equivalent in human whole blood assays to the advanced clinical candidate, roflumilast. Finally, L-454,560 appears to have utility in the treatment of cognitive dysfunction by enhancing memory processes at a dose significantly lower than that found to induce conditioned taste aversion.
Section snippets
Reagents
Analytical grade HEPES, EDTA, cAMP, ultra grade MgCl2, KCl, PBS, IBMX, mepyramine, Al(OH)3, carbachol, lithium chloride and ovalbumin reagents were purchased from Sigma/Aldrich (St. Louis, MO). [3H]-cAMP, [3H]-cGMP, and sepharose beads were purchased from GE Healthcare (Piscataway, NJ). Recombinant baculovirus proteins were prepared by the Biotechnology Research Institute (Montreal, Quebec). All products for quantitative PCR were purchased from Applied Biosystems (Foster City, CA). L-454,560
PDE4 inhibitor potency and selectivity
A number of splicing variants exist for each of the four PDE4 isoforms. In order to remove the variabilities in enzyme activity and inhibitor sensitivity due to their divergent N-terminal sequences, PDE4AQT (PDE4A248), PDE4BQT, PDE4CQT and PDE4DQT enzymes, comprising the common region of all variants of each isoform were utilized to test PDE4 inhibitors. Functionally, the PDE4QT enzymes mimic the fully activated and PKA-phosphorylated PDE4 enzymes [24], [31]. Table 1 summarizes the potency of
Discussion and conclusions
This study demonstrates that L-454,560 is a potent, selective and reversible inhibitor of PDE4. L-454,560 is a low nM inhibitor of all four of the PDE4 enzyme isoforms, and the compound is shifted about 300-fold (IC50 = 160 nM) with respect to the TNFα surrogate of inhibition in human whole blood. Based on the PDE enzymes tested, L-454,560 is at least 132-fold selective for inhibition of PDE4 over the other eight PDE gene families tested. L-454,560 is unique amongst the three compounds profiled in
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