Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Ester doai
Reexamination Certificate
2001-08-31
2003-04-29
Krass, Frederick (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Ester doai
C514S362000, C514S363000, C514S364000, C514S381000, C514S520000, C514S826000
Reexamination Certificate
active
06555576
ABSTRACT:
SCOPE OF THE INVENTION
This invention covers compounds which preferentially inhibit, or bind, one form of a phosphodiesterase isozyme denominated 4 (PDE 4 hereafter) while exhibiting equal or, preferably less binding or inhibition for a second form of the enzyme and are thus useful for treating exercise induced bronchoconstriction in patients with asthma. These isoenzyme forms, believed to be different forms of non-interconvertible conformations of the same enzyme, are distinguished by their binding affinity for rolipram, an archtypical PDE 4 inhibitor. Rolipram binds with high affinity to a site of one form but with low affinity to the catalytic site of the other. Herein one form is denominated the high affinity rolipram binding site and the other form is identified as the low affinity rolipram binding site. A method for selectively treating exercise induced asthma (EIA) by inhibiting preferentially the low affinity form of the catalytic site in the PDE 4 isozyme is also disclosed. A method for treating (EIA) by administering a compound preferentially binding to the low affinity-binding site is also disclosed.
BACKGROUND OF THE INVENTION
Cyclic nucleotide phosphodiesterases (PDEs) represent a family of enzymes that hydrolyze the ubiquitous intracellular second messengers, adenosine 3′,5′-monophosphate (cAMP) and guanosine 3′,5′-monophosphate (cGMP) to their corresponding inactive 5′-monophosphate metabolites. At least nine distinct classes of PDE isozymes are believed to exist, each possessing unique physical and kinetic characteristics and each representing a product of a different gene family. These have been distinguished using the numerals 1 through 9.
The target enzyme in this invention is the PDE 4 isozyme in all its various forms and in the full domain of its distributions in all cells. It is a low K
m
(cAMP K
m
=1-5 &mgr;M) cAMP-selective enzyme that has little activity against cGMP (Km>100 &mgr;M). Members of this isozyme class have the interesting characteristics of existing in two or more non-interconvertible or slowly interconvertible forms that bind rolipram and other PDE 4 inhibitors with distinct rank order potencies. Thus the same gene product can exist in more than one catalytically active conformational state. Importantly, the relative proportions of the different binding forms may vary depending on the tissue cell type. For example, inflammatory cells may contain a relatively high proportion of the form that binds rolipram with a low affinity while brain and parietal cells may contain a relatively high proportion of the form that binds rolipram with a high affinity.
Of particular interest in this invention is the role this class of isozymes play in inflammation and airway smooth muscle. Studies indicate that it plays a prominent role in regulating cAMP in a wide variety of inflammatory cells (i.e., mast cells, basophils, eosinophils, neutrophils, and monocytes) and airway smooth muscle. The work of this invention is particularly applicable to inflammatory cells and airway smooth muscle; the isozyme type expressed in human monocytes is of particular interest. This is because cyclic AMP serves as a second messenger to inhibit chemotaxis and activation of inflammatory cells. In addition, cAMP mediates smooth airway muscle relaxation. This coupled with the major role of PDE 4 in metabolizing cAMP has provided the underpinnings for investigating PDE 4 inhibitors: by virtue of their ability to elevate cAMP content in leukocytes and airway smooth muscle, PDE 4 inhibitors may posses anti-inflammatory and bronchodilator activities.
Current PDE inhibitors used in treating inflammation and as bronchodilators, drugs like theophylline and pentoxyfyllin, inhibit PDE isozymes indiscriminately in all tissues. These compounds exhibit side effects, apparently because they non-selectively inhibit all or most PDE isozyme classes in all tissues. This is a consideration in assessing the therapeutic profile of these compounds. The targeted disease state may be effectively treated by such compounds, but unwanted secondary effects may be exhibited which, if they could be avoided or minimized, would increase the overall therapeutic effect of this approach to treating certain disease states. Taken collectively, this information suggests that the side effects associated with the use of standard non-selective PDE inhibitors might be reduced by targeting novel isozyme-selective inhibitors for the predominant PDE in the tissue or cell of interest. Although in theory isozyme-selective PDE inhibitors should represent an improvement over non-selective inhibitors, the selective inhibitors tested to date are not devoid of side effects produced as an extension of inhibiting the isozyme of interest in an inappropriate or not-targeted tissue. For example, clinical studies with the selective PDE 4 inhibitor rolipram, which was being developed as an antidepressant, indicate it has psychotropic activity and produces gastrointestinal effects, e.g., pyrosis, nausea and emesis. Indications are that side effects of denbufylline, another PDE 4 inhibitor targeted for the treatment of multi-infarct dementia, may include pyrosis, nausea and emesis as well. These side effects are thought to occur as a result of inhibiting PDE 4 in specific areas of the CNS and gastrointestinal system.
In 1986, Schneider and colleagues described the presence and characteristics of high affinity, stereoselective [
3
H]-rolipram binding sites in rat brain homogenates. Although it was assumed that these binding sites represented the catalytic site of the rat brain “non-calmodulin-dependent, cAMP phosphodiesterase” (i.e. PDE 4), a striking anomaly was apparent in the data. Under similar albeit not identical experimental conditions, data showed rolipram had a K
d
=1 nM, whereas it inhibited rat brain PDE 4 activity with a K
i
=1 &mgr;M. Thus, there was a 1000-fold difference in the affinity of rolipram for the binding site versus its affect on catalytic activity. Although comprehensive structure activity relationships (SARs) for PDE inhibition and competition for [
3
H]-rolipram binding were not established, the substantial difference in the potency of rolipram as a PDE 4 inhibitor compared with its potency at the binding site seemed to question the validity of the assumption that both activities were contained within the same molecular locus.
Because of this conundrum, several studies were initiated. One sought to determine whether rolipram's high affinity binding site existed on the same protein as the cAMP catalytic site. Another study sought to determine whether or not the SAR for inhibition of PDE 4 was the same as the SAR for competition with the high affinity rolipram binding site. A third study undertook to try and elucidate what biological significance, if any, there might be in these findings, particularly as it might relate to developing new drug therapies.
As data were collected from several assays, it became apparent that there are at least two binding forms on human monocyte recombinant PDE 4 (hPDE 4) at which inhibitors bind. One explanation for these observations is that hPDE 4 exists in two distinct forms. One binds the likes of rolipram and denbufylline with a high affinity while the other binds these compounds with a low affinity. Herein we distinguish these forms by referring to them as the high affinity rolipram binding form (HPDE 4) and the low affinity rolipram binding form (LPDE 4).
The importance of this finding lies in the discovery that compounds which potently compete for the high affinity rolipram binding form (HPDE 4) have more side effects or more intense side effects than those which more potently compete with the LPDE 4 (low affinity rolipram binding form). Further data indicate that compounds can be targeted to the low affinity binding form of PDE 4 and that this form is distinct from the binding form for which rolipram is a high affinity binder. Distinct SARs were found to exist for inhibitors acting at the high aff
Christensen, IV Siegfried B.
Nieman Richard
Torphy Theodore J.
Kanagy James M.
Kinzig Charles M.
Krass Frederick
SmithKline Beecham Corporation
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