Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-11-12
2002-10-01
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S017400, C514S019300, C530S331000, C530S332000
Reexamination Certificate
active
06458760
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a method for treating tissue damage in a mammal caused by ischemia, such as from a stroke or from myocardial ischemia, utilizing a peptidyl diazomethyl ketone.
BACKGROUND OF THE INVENTION
An ischemia is a deficiency of blood flow to a body part, due to functional constriction or actual obstruction of a blood vessel. Ischemia may occur in various parts of the body of an animal, but it has the most dire consequences when it occurs in the brain, in the heart or in the bowels.
For example, a stroke occurs when there is an interruption of blood flow to an area of the brain. It is usually caused by an embolism or clot that blocks a significant artery in the brain. Blood flow to this area is blocked, causing the affected tissue to become hypoxic. Upon resolution of the cause of the stroke, at least partial blood flow is restored to the affected tissue of the animal. These two processes, lack of blood flow and then restoration of blood flow both produce effects that damage the affected brain tissue.
Among the mechanism of damage initiated by stroke is the activation of calcium sensitive proteases due to the influx of calcium ions into the hypoxic tissue.
At least two classes of proteases, the calpains and the cathepsins, are believed to be activated during cerebral ischemia. Both enzymes are cysteine proteases, the former being located in the cytoplasm, while the latter is located in the lysosome of the cells. Brain tissue contains the cathepsins B, L, S, and H, of which the first two predominate. Moreover, at physiological pH, cathepsin L is less stable than cathepsin B.
Both the calpains and cathepsins play a role in the basal recycling of neural cytoskeletal components. These enzymes are part of a series of enzymes which includes phospholipases, phosphatases, certain kinases and proteases, and which participate in this recycling process.
Cathepsins have been studied under conditions of cardiac ischemia and are believed to play a role in tissue damage by their release from the lysosome into the cytoplasm, where they are able to contribute to cellular injury by the breakdown of cellular structural components. Increased intracellular calcium and acidic pH are believed to be factors leading to lysosomal membrane instability and the activation of these proteases during cardiac ischemia. The same phenomena of calcium influx and acidosis occurs following brain ischemia, and these are believed to cause further cellular damage.
Because tissue perfusion is required for the distribution of pharmaceuticals to target tissues, it is difficult to intervene pharmacologically in the stroke process to prevent the damage caused by hypoxia that occurs during stroke. Thus, the objective of medical research in this area is to reduce the amount of time that the tissue is hypoxic and simultaneously therewith to develop therapies to prevent the activation of the destructive processes that ensues upon restoration of blood flow. In this regard, attention has been focused on the calpains and cathepsins and inhibitors of these two protease classes have been proposed for treating patients afflicted with brain ischemia, myocardial ischemia, and other diseases.
A number of investigators have indeed suggested that calpain inhibition could reduce stroke damage. These cytosolic neutral cysteine proteases are thought to trigger release of the lysosomal proteases. Reduction in tissue damage resulting therefrom has been observed in some experimental systems. However, these results are often complicated by the use of calpain inhibitors that also inhibit a number of other proteases.
Recently, U.S. Pat. No. 4,518,825 to Rasnik, discloses that &agr;-amino fluoroketone peptide derivatives inhibit various proteases, including serine and cysteine proteases, including, but not limited to cathepsins B, H, C, G, R; elastase; trypsin; plasma kallikrein; glandular kallikrein; plasmin; plasminogen activator; and the like. These compounds, however, exhibit non-specific binding to serine proteases, as well as other non-cathepsin cysteine proteases.
Krantz, et al. in U.S. Pat. No. 5,055,451 disclose aryloxy and arylacyloxy methylketones as thiol protease inhibitors, including inhibitors of Cathepsin B. Krantz, et al. recognized that there is a need for potent and specific thiol protease inhibitors and in particular a need for chemically stable inhibitors that minimize the likelihood of non-specific reactions with plasma or cellular constituents. Unfortunately, the aryloxy and arylacyloxy compounds displayed unacceptable toxicity in large animals.
PCT Application WO 96/21655 and U.S. Pat. No. 5,691,368 to Peet disclose oxazolidine inhibitors of calpain and/or cathepsin B; it is alleged that the compounds are useful in the treatment of patients afflicted with acute or chronic neurodegenerative disorders.
EPO Application 525,420 to Ando, et al. disclose peptidyl aminomethylketones that exhibit reversible inhibition of calpains and cathepsins. They allege that these compounds are clinically useful in the treatment of various diseases, including stroke.
It is to be noted that in each of these references, the compounds disclosed therein inhibit a number of enzymes, including but not limited to both cathepsins and calpains. None of the references disclose inhibitors that are specific for only one type of cysteine protease. However, as recognized by both Rasnik and Krantz, et al. described hereinabove, for a drug in this field to be effective, it must not only interact with one type of cysteine protease, but, in addition, there must also be a lack of interaction with other cysteine proteases to prevent unwanted side effects.
The present, inventors, however have found such a system. In particular, they have found that peptidyl diazomethyl ketones, and in particular, N-terminus amino protected diazomethyl ketones such as benzyloxy-carbonyl peptidyl diazomethyl ketones, are capable of specifically inhibiting cathepsins significantly better than calpains.
Benzyloxycarbonyl peptidyl diazomethyl ketones, however, are not new compounds, but have been described in earlier publications by Shaw, et al. For example, Shaw, et al. in
Arch. Biochem. Biophys
, 1983, 222, 424-429 reveal a number of benzyloxycarbonyl-Phe-X-CHN
2
derivatives, wherein X is one of several amino acids or derivatized amino acids and their effect on the in vitro activity of bovine spleen cathepsins. However, these compounds were not considered viable clinical candidates for treating damaged tissues because, as indicated by Powers in U.S. Pat. No. 5,610,297, these compounds are thought, inter alia, to be poorly membrane permeant and to have low specificity.
But, the present inventors have surprisingly found that these compounds are not poorly membrane permeants and do not have low specificity.
SUMMARY OF THE PRESENT INVENTION
Accordingly, the present invention is directed to treating tissue damage in a patient caused by ischemia comprising administering to said patient a therapeutically effective amount of a compound which is an inhibitor of at least one of cathepsin B or cathepsin L, wherein the inhibition of cathepsin B or L is significantly greater than that of calpain, said compound being a peptidyl diazomethyl ketone and more preferably a N-terminus amino protected peptidyl diazomethyl ketone.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a method for treating tissue damage in a mammal resulting from ischemia. The compounds useful for this purpose are small lipophilic molecules which are specific inhibitors of cathepsin B or cathepsin L or both. They may inhibit cathepsin B, but not cathepsin L or vice versa or they may inhibit both enzymes. However, it is a significantly poorer inhibitor of calpain relative to either cathepsin B or cathepsin L.
Another characteristic of the present invention is that the compounds utilized are peptidyl diazomethyl ketones. These compounds are preferably di or tri-peptides. In addition, they have a molecular weight ranging from about 450 to about 1000 dal
Anagli John
Seyfried Donald M.
Henry Ford Health System
Low Christopher S. F.
Mohamed Abdel A.
Scully Scott Murphy & Presser
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