Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
1999-04-08
2004-08-17
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S193100, C424S543000, C424S001210, C424S010400
Reexamination Certificate
active
06776990
ABSTRACT:
FIELD OF THE INVENTION
The present invention includes methods and compositions for the treatment of acute pancreatitis. In a preferred embodiment the invention concerns the use of agents to reduce or prevent the secretion of pancreatic digestive enzymes within the pancreas. Such agents are targeted to pancreatic cells, and serve to prevent the exocytotic fusion of vesicles containing these enzymes with the plasma membrane. The invention is also concerned with methods of treating a mammal suffering from pancreatitis through the administration of such agents.
BACKGROUND OF THE INVENTION
Pancreatitis is a serious medical condition involving an inflammation of the pancreas. In acute or chronic pancreatitis the inflammation manifests itself in the release and activation of pancreatic enzymes within the organ itself, leading to autodigestion. In many cases of acute pancreatitis, the condition can lead to death.
In normal mammals, the pancreas, a large gland similar in structure to the salivary gland, is responsible for the production and secretion of digestive enzymes, which digest ingested food, and bicarbonate for the neutralization of the acidic chyme produced in the stomach. The pancreas contains acinar cells, responsible for enzyme production, and ductal cells, which secrete large amounts of sodium bicarbonate solution. The combined secretion product is termed “pancreatic juice”; this liquid flows through the pancreatic duct past the sphincter of Oddi into the duodenum. The secretion of pancreatic juice is stimulated by the presence of chyme in the upper portions of the small intestine, and the precise composition of pancreatic juice appears to be influenced by the types of compounds (carbohydrate, lipid, protein, and/or nucleic acid) in the chyme.
The constituents of pancreatic juice includes proteases (trypsin, chymotrypsin, carboxypolypeptidase), nucleases (RNAse and DNAse), pancreatic amylase, and lipases (pancreatic lipase, cholesterol esterase and phospholipase). Many of these enzymes, including the proteases, are initially synthesized by the acinar cells in an inactive form as zymogens: thus trypsin is synthesized as trypsinogen, chymotrypsin as chymotypsinogen, and carboxypolypeptidase as procarboxypolypeptidase. These enzymes are activated according to a cascade, wherein, in the first step, trypsin is activated through proteolytic cleavage by the enzyme enterokinase. Trypsinogen can also be autoactivated by trypsin; thus one activation has begun, the activation process can proceed rapidly. Trypsin, in turn, activates both chymotypsinogen and procarboxypolypeptidase to form their active protease counterparts.
The enzymes are normally activated only when they enter the intestinal mucosa in order to prevent autodigestion of the pancreas. In order to prevent premature activation, the acinar cells also co-secrete a trypsin inhibitor that normally prevents activation of the proteolytic enzymes within the secretory cells and in the ducts of the pancreas. Inhibition of trypsin activity also prevents activation of the other proteases.
Pancreatitis can occur when an excess amount of trypsin saturates the supply of trypsin inhibitor. This, in turn, can be caused by underproduction of trypsin inhibitor, or the overabundance of trypsin within the cells or ducts of the pancreas. In the latter case, pancreatic trauma or blockage of a duct can lead to localized overabundance of trypsin; under acute conditions large amounts of pancreatic zymogen secretion can pool in the damaged areas of the pancreas. If even a small amount of free trypsin is available activation of all the zymogenic proteases rapidly occurs, and can lead to digestion of the pancreas (acute pancreatitis) and in particularly severe cases to the patient's death.
Pancreatic secretion is normally regulated by both hormonal and nervous mechanisms. When the gastric phase of stomach secretion occurs, parasympathetic nerve impulses are relayed to the pancreas, which initially results in acetylcholine release, followed by secretion of enzymes into the pancreatic acini for temporary storage.
When acid chyme thereafter enters the small intestine, the mucosal cells of the upper intestine release a hormone called secretin. In humans, secretin is a 27 amino acid (3400 Dalton) polypeptide initially produced as the inactive form prosecretin, which is then activated by proteolytic cleavage. Secretin is then absorbed into the blood. Secretin causes the pancreas to secrete large quantities of a fluid containing bicarbonate ion. Secretin does not stimulate the acinar cells, which produce the digestive enzymes. The bicarbonate fluid serves to neutralize the chyme and to provide a slightly alkaline optimal environment for the enzymes.
Another peptide hormone, cholecystokinin (CCK) is released by the mucosal cells in response to the presence of food in the upper intestine. As described in further detail below, human CCK is synthesized as a protoprotein of 115 amino acids. Active CCK forms are quickly taken into the blood through the digestive tract, and normally stimulate the secretion of enzymes by the acinar cells. However, stimulation of the CCK receptor by the CCK analogs cerulein and CCK-octapeptide (CCK-8) appears to lead to a worsening of morbidity and mortality in mammals in whom pancreatitis is induced. See Tani et al.,
Pancreas
5:284-290 (1990).
As indicated above, the digestive enzymes are synthesized as zymogens; proto-enzyme synthesis occurs in the rough endoplasmic reticulum of the acinar cells. The zymogens are then packaged within vesicles having a single lipid bilayer membrane. The zymogens are packed within the vesicles so densely that they appear as quasi-crystalline structures when observed under light microscopy and the zymogen granules are electron-dense when observed under the electron microscope. The vesicles are localized within the cytoplasm of the acinar cells. Secretion of zymogens by the acinar cells occurs through vesicle docking and subsequent fusion with the plasma membrane, resulting in the liberation of the contents into the extracellular milieu.
Nerve cells appear to secrete neurotransmitters and other intercellular signaling factors through a mechanism of membrane fusion that is shared with other cell types, see e.g., Rizo & Sudhof,
Nature Struct. Biol.
5:839-842 (October 1998), hereby incorporated by reference herein, including the pancreatic acinar cells.
Although the Applicants do not wish to be bound by theory, it is believed that a vesicle first contacts the intracellular surface of the cellular membrane in a reaction called docking. Following the docking step the membrane fuses with and becomes part of the plasma membrane through a series of steps that currently remain relatively uncharacterized, but which clearly involve certain vesicle and membrane-associated proteins, as has been illustrated using neural models.
In neurons, neurotransmitters are packaged within synaptic vesicles, formed within the cytoplasm, then transported to the inner plasma membrane where the vesicles dock and fuse with the plasma membrane. Recent studies of nerve cells employing clostridial neurotoxins as probes of membrane fusion have revealed that fusion of synaptic vesicles with the cell membrane in nerve cells depends upon the presence of specific proteins that are associated with either the vesicle or the target membrane. See id. These proteins have been termed SNAREs. As discussed in further detail below, a protein alternatively termed synaptobrevin or VAMP (vesicle-associated membrane protein) is a vesicle-associated SNARE (v-SNARE). There are at least two isoforms of synaptobrevin; these two isoforms are differentially expressed in the mammalian central nervous system, and are selectively associated with synaptic vesicles in neurons and secretory organelles in neuroendocrine cells. The target membrane-associated SNAREs (t-SNARES) include syntaxin and SNAP-25. Following docking, the VAMP protein forms a core complex with syntaxin and SNAP-25; the formation of the core complex appears to be an essential step t
Aoki Kei Roger
Sachs George
Allergan Inc.
Baran Robert J.
Fisher Carlos A.
Nolan Patrick J.
Voet Martin A.
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