Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Phosphorus containing other than solely as part of an...
Reexamination Certificate
1996-10-08
2002-04-09
Weber, Jon P. (Department: 1651)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Phosphorus containing other than solely as part of an...
C514S143000, C568S008000, C435S197000
Reexamination Certificate
active
06369045
ABSTRACT:
The present invention relates to the use of inhibitors of an enzyme in the therapy, in particular in the treatment of atherosclerosis. The present invention also relates to the isolation and purification of the enzyme, to isolated nucleic acids encoding the enzyme, to recombinant host cells transformed with DNA encoding the enzyme, to the use of the enzyme in diagnosing a patient's susceptibility to atherosclerosis, and to the use of the enzyme in identifying compounds which are potentially useful for the treatment of atherosclerosis.
Lipoprotein Associated Phospholipase A
2
(Lp-PLA
2
), also previously known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase). During the conversion of LDL to its oxidised form, Lp-PLA
2
is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-phosphatidylcholine and an oxidatively modified fatty acid. Both of these products of Lp-PLA
2
action are potent chemoattractants for circulating monocytes. As such, this enzyme is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, causing the characteristic ‘fatty streak’ associated with the early stages of atherosclerosis. Inhibition of the Lp-PLA
2
enzyme would therefore be expected to stop the build up of this fatty streak (by inhibition of the formation of lysophosphatidylcholine), and so be useful in the treatment of atherosclerosis. In addition, it is proposed that Lp-PLA
2
plays a direct role in LDL oxidation. This is due to the poly unsaturated fatty acid-derived lipid peroxide products of Lp-PLA
2
action contributing to and enhancing the overall oxidative process. In keeping with this idea, Lp-PLA
2
inhibitors inhibit LDL oxidation. Lp-PLA
2
inhibitors may therefore have a general application in any disorder that involves lipid peroxidation in conjunction with the enzyme activity, for example in addition to conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion injury and acute and chronic inflammation.
The present invention therefore provides in a first aspect an inhibitor of the enzyme lipoprotein associated Lp-PLA
2
for use in therapy, in particular in the treatment of atherosclerosis. Suitable compounds able to inhibit the Lp-PLA
2
enzyme are known in the art and include for example, the following compounds of structure (1):
in which
R is C
1-6
alkylCONR
2
;
R
2
is hydrogen or C
1-6
alkyl;
X is oxygen, sulphur or —O(CO)—;
R
1
is C
8-20
alkyl;
Z is N(R
3
)
2
,
⊕
N(R
3
)
3
, SR
3
,
⊕
S(R
3
)
2
, in which each group R
3
is the same or different and is C
1-6
alkyl, OR
2
, C
1-4
alkanoyl, imidazolyl or N-methylimidazolyl
Suitably R
2
is hydrogen or C
1-6
alkyl; preferably R
2
is hydrogen.
Suitably X is oxygen, sulphur or —O(CO)—; preferably X is oxygen
Suitably R
1
is C
8-20
alkyl; preferably R
1
is C
16-18
alkyl
Suitably Z is N(R
3
)
2
,
⊕
N(R
3
)
3
, SR
3
,
⊕
S(R
3
)
2
, in which each group R
3
is the same or different and is C
1-6
alkyl, OR
2
, C
1-4
alkanoyl, imidazolyl or N-methylimidazolyl; preferably Z is SR
3
in which R
3
is methyl or OR
2
in which R
2
is hydrogen
The compounds of structure (I) can be prepared by processes known to those skilled in the art for example as described in J Chem Soc Chem Comm.,1993, 70-72; J Org Chem, 1983, 48, 1197 and Chem Phys Lipids, 1984,35,29-37 or procedures analogous thereto.
When used in therapy, the compounds of structure (I) are formulated in accordance with standard pharmaceutical practice.
The compounds of structure (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and, lozenges.
A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
A typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
Preferably the composition is in unit dose form such as a tablet or capsule.
Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of a compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base.
The daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day. Suitably the compounds will be administered for a period of continuous therapy.
The enzyme, lipoprotein associated Lp-PLA
2
has not hitherto been available in isolated purified form. The present invention therefore provides in a further aspect, the enzyme lipoprotein associated Lp-PLA
2
in purified form. By purified form is meant at least 80%, more preferably 90%, still more preferably 95% and most preferably 99% pure with respect to other protein contaminants.
The enzyme Lp-PLA
2
may be characterised by one or more partial peptide sequences selected from SEQ ID NOs:1, 2, 3, 4, 10 and 11 or by the partial peptide sequence comprising residues 271 to 441 or consisting of residues 1 to 441 of SEQ ID NO:9. The enzyme Lp-PLA
2
may further or alternatively characterised by its molecular weight found to be 45 kDa, at least 45 kDa, 45-47 kDa, 46-47 kDa or 45-50 kDa.
The invention also provides fragments of the enzyme having Lp-PLA
2
activity.
The enzyme can be isolated and purified using the methods hereafter described. Once isolated, the protein sequence of the enzyme can be obtained using standard techniques. In identifying said sequence, a number of protein fragments have been identified, each of which comprises part of the whole sequence of the enzyme. These sequences are themselves novel and form a further aspect of the invention.
This invention also provides isolated nucleic acid molecules encoding the enzyme, including mRNAs, DNAs, cDNAs as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In particular, the invention provides an isolated nucleic acid molecule consisting of bases 1 to 1361 or 38 to 1361 or comprising the sequence correspond
Hickey Deirdre Mary Bernadette
Macphee Colin Houston
Tew David Graham
Han William T.
King William T.
Kinzig Charles M.
SmithKline Beecham p.l.c.
Weber Jon P.
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