METHOD FOR USING PROTEASE INHIBITORS TO TREAT AND/OR PREVENT PANCREATITIS

Abstract: The present invention relates to a method for preventing and/or treating acute pancreatitis. The method involves the administration of a protease inhibitor using a variety of protocols. The preferred protease inhibitor is pancreatic secretory trypsin inhibitor (PSTI). The desired level of inhibitor needed in the pancreas would be the amount sufficient to block activity of any activated trypsin present inside the pancreas.

Description: 1 METHOD FOR USING PROTEASE INHIBITORS TO TREAT AND/OR PREVENT PANCREATITIS BACKGROUND OF.THE INVENTION The exocrine pancreas is an organ specialized for the synthesis of zymogens of digestive enzymes. In the nor¬ mal course of events, these zymogens are activated only after they leave the pancreas to perform their digestive functions in the duodenum and small intestine. Thus, self-digestion of the pancreas is not normally a common condition. However, when premature activation of these zymogens occurs self- digestion of the pancreas results, causing a state commonly referred to as pancreatitis. The etiology leading to acute pancreatitis is unknown, but there is strong correlations between the initiation of that disease and biliary tract disease and chronic alcoholism. In addition to acute pancreatitis caused by disease and chronic alcoholism, severe acute pancreatitis is a dreaded complication of certain interventional proce¬ dures. These interventional procedures include endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic surgery. Acute pancreatitis is a life-threatening condition for which 161,000 people were hospitalized in the U.S. in 1985. Mortality rates of up to 20% are reported. There is currently no efficacious treatment for acute pancreatitis. Macroscopically, acute pancreatitis is character¬ ized by edema, hemorrhage, and necrosis of the pancreas, and necrosis of adipose tissue adjacent to the pancreas. Histologically, an inflammatory reaction is another typical feature. Although the exocrine pancreas is an organ special¬ ized for the synthesis of digestive enzymes, self-digestion of the pancreas, as noted above, is not a common condition. In the normal course of events, several protective mechanisms exist that prevent self-digestion of the pancreas from occur¬ ring. Among these factors are that: (A) the proteolytic enzymes (trypsin, chy otrypsin, carboxypeptidase, elastase) and phospholipase A, which, in their active forms, can destroy tissues, are synthesized as inactive zymogens and are not activated until they reach the intestines; (B) the zymogens are stored in specialized granules which are bounded by a phospholipid membrane and are thereby isolated from other components of the cell which might cause their prema¬ ture activation; (C) pancreatic tissue and pancreatic juice contain a protein, herein referred to as pancreatic secretory trypsin inhibitor (PSTI) which inactivates trypsin and there¬ by prevents prematurely-activated trypsin from activating all the other zymogens. The onset of pancreatitis indicates that all of these protective screens have broken down as a result of some direct or indirect insult to the pancreas. As noted above, a<_ute pancreatitis strikes at least about 150,000 people per year in this country. In addition, the reported frequency of pancreatitis after the interven¬ tional procedures discused herein approaches 1-2%. Moreover, the popularity of these interventional procedures is increas¬ ing both for diagnostic purposes and as an adjunct to gall stone removal. Thus, it is predicted that the rate of acute pancreatisis will continue to rise. The present inventors believe that interventionally-induced pancreatitis has been shown to be characterized by a pronounced release of trypsin, as is se¬ vere pancreatitis of other causes. The PSTI, described above and which is produced in all acinar cells of the pancreas, is secreted in parallel with trypsinogen and thought to protect the pancreas from the effects of accidential activation of trypsinogen. The amount of PSTI normally present in the pan¬ creas corresponds, however, only to about 2% of the Trypsinogen present. Thus, it is believed that a major trypsinogen activation caused by the diseaε = and/or interven¬ tional procedures could easily overwhelm the local inhibitor barrier. Active trypsin would then activate all the other pancreatic zymogens with acute pancreatitis as a consequence. The present inventors have found a method to pre¬ vent and/or treat acute pancreatitis. Since the maximum amount of trypsin possibly present in the pancreas corre¬ sponds to the amount of trypsinogen which is present and SUBSTITUTESHEET could thus be activated, the maximum amount of protease in¬ hibitor required to prevent pancreatitis is that necessary to inhibit the trypsinogen were it to become activated. There¬ fore, this method ultimately protects the pancreas from the adverse effects of trypsinogen activation by increasing the local protease inhibitor concentration in the pancreas to a level capable of inhibiting the maximum amount of potential trypsin. Thus, in one embodiment, the present inventors have discovered a method of using PSTI, preferably recombinant human PSTI, to act as a prophylactic drug to prevent pancreatitis induced by an internventional procedure. In another embodiment, the present inventors have discovered a method of using PSTI and other protease inhibitors to treat acute pancreatitis. Detailed Description of the Preferred Embodiments Reference will now be made to the presently pre¬ ferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the invention. As noted above, the present invention relates to a method of preventing and/or treating acute pancreatitis. This method involves, in part, the intraductal administration of a protease inhibitor. In a second embodiment, this inven¬ tion involves administration, through a variety of methods, of pancreatic secretory trypsin inhibitor (PSTI). The preferred protease inhibitor of the present invention is PSTI. This PSTI, to the extent it is equivalent to PSTI isolatable from a human pancreas, is referred to herein as human PSTI or HPSTI. Human PSTI has been previous¬ ly described and methods for its isolation and recombinant- DNA methods for its production have been disclosed in U.S. Patent Application Serial No. 924,991, filed October 30, 1986 by Tadahiko Kohno et al. , entitled "Human Pancreatic Secreto¬ ry Trypsin Inhibitors Produced by Recombinant DNA Methods and Processes of Same." This United States patent application is specifically incorporated herein by reference. SUBSTITUTESHEET The PSTI of the present invention is preferably produced according to the method of Kohno et al. disclosed in U.S. Patent Application Serial No. 924,991 referred to above using the modifications set forth in Example 1. Thus, the presently preferred PSTI is human PSTI made by recombinant- DNA methods. This HPSTI is also referred to herein as "rHPSTI" or "rPSTI." The protease inhibitor, preferably PSTI, is admin¬ istered at a dosage sufficient to result in a level of protease inhibitor in the pancreas capable of protecting the pancreas from autodigestion by trypsin and other proteases. In one embodiment where in the protease inhibitor is PSTI, the PSTI is administered at a dosage sufficient to cause a PSTI concentration in the pancreas sufficient to inhibit any trypsin produced by conversion of trypsinogen. Thus, the level of PSTI desired in the pancreas is that sufficient to block activity of any trypsinogen that had become activated inside the pancreas. Likewise, should a protease inhibitor other than PSTI be deemed useful, the maximum level of inhib¬ itor needed in the pancreas would be an amount sufficient to block activity of any related protease zymogens, particularly trypsin, that had become activated inside the pancreas. For an intraductal mode of administration, it is presently preferred that the amount of protease inhibitor that is administered by at least about a 2 to 1 molar ratio of inhibitor to trypsinogen present in the pancreas. The inventors presently believe that there is approximately one- half gram of trypsinogen in a human pancreas at any one time. Therefore, in a preferred embodiment of the present inven¬ tion, approximately 125 to approximately 250 mg PSTI would be administered. For an intraperitoneal or intravenous dose, approximately 1 to 20 grams of PSTI per dose would be admin¬ istered with repeated doses contemplated. Further refinements of the calculations necessary to determine the appropriate dosage for treatment involving each of the above-mentioned formulations are routinely made by those of ordinary skill in the art and are within the S UBS TITUTESHEET ambit of tasks routinely performed by them without undue experimentation, especially in light of the dosage informa¬ tion and assays disclosed herein. These dosages may be ascertained through use of the established assays for determining dosages utilized in connection with appropriate dose-response data. The PSTI of the present invention may by supplied dry as a lyopholized powder or as a frozen solution or in liquid form. To the extent the PSTI is supplied in a liquid form, it is suggested that it be stored at a pH 4-6. For the PSTI which is stored in a liquid form, the PSTI is admixed with a carrier. The preferred carrier is a saline solution, but is is contemplated that other pharmaceutically-acceptable liquid carriers may also be used. It is to be noted that the PSTI will be administered in a formulation that has a physiological pH. Thus, to the extent that the PSTI has been provided in a liquid form at pH 4-6, the pH is to be adjusted to a physiological pH prior to administration. The preferred modes of administration of the protease inhibitor of the present invention vary. To the extent a protease inhibitor other than PSTI is to be adminis¬ tered, it is administered intraductally into the pancreas. To the extent that protease inhibitor is PSTI , PSTI may be administered intraductally, intravenously, or in- traperitoneally. As noted previously, the incidence of pancreatitis occurring as result of interventional procedures is rising. Thus, a preferred embodiment of the present invention in¬ volves the use of protease inhibitor, particularly PSTI, in¬ traductally as a prophylactic measure to prevent pancreatitis during these procedures. In this embodiment, it is possible to dissolve lyophilyzed protease inhibitor and to administer the protease inhibitor intraductally immediately prior to the interventional procedure. In an alternate embodiment, a protease inhibitor, preferably PSTI, supplied as a liquid may be injected intraductally prior to the interventional procedure. SUBSTITUTESHEET In alternate embodiments, lyophilized or liquid protease inhibitor may be admixed with a liquid to be used in the internventional procedure, for example admixed with a ra- diopaque dye, and administered to a patient with the inter¬ ventional procedure liquid. Moreover, the protease inhibitor may be administered at the conclusion of the procedure. When administering the protease inhibitor in¬ traductally, it is important to monitor carefully the pres¬ sure being used to administer the protease inhibitor to avoid rupture of the duct or pancreas. Such maximum pressures have been established by those of routine skill in the art and are known, especially to those who commonly employ contrast mate¬ rials. In an alternate embodiment, it is also possible to administer the therapeutic composition containing PSTI, as noted above, intraductally, intraperitoneally, or as an in¬ travenous solution. To the extent PSTI is administered in¬ traperitoneally, a preferred system for administration is to add the PSTI to intraperitoneal dialysis fluid. It should also be noted that the PSTI formulations described herein may be used for veterinary applications, with the dosage ranges being the same as those specified above humans. It is understood that the application of teachings of the present invention to a specific problem or environment will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein. Exam¬ ples of the products of the present invention and representa¬ tive processes for their preparation and use appear in the following examples. EXAMPLES Example 1 - Preparation of Recombinant Human PSTI Recombinant human PSTI (rPSTI) was prepared gener¬ ally according to the method of Kohno et al. described in U.S. Patent Application Serial No. 924,991. Specifically, the rPSTI used in the fc.lowing examples was prepared from a E. coli strain carrying a plasmid with a synthetic gene for SUBSTITUTESHEET human PSTI joined to DNA encoding the ompA leader sequence as described by J. Ghrayed et al. in EMBO J. 1984; 1JJ:3:2437-2442, specifically incorporated herein by refer¬ ence. Human PSTI was purified from the culture medium by a trypsin affigel-10 column and an SPC25 or S-Sepharose column. These two column chromatography steps yielded a preparation of human PSTI that was better than 95% pure and had an a ino acid analysis, an N-terminal sequence, a tryptic peptide map, and inhibitory properties that were identical to those of na¬ tive human PSTI. The purified human PSTI was depyrogenated with detoxigel and sterilized using 0.45 urn filter prior to use in the following examples. A second, preferred embodiment for making HPSTI uses similar procedures and an HPSTI production strain called JDC110. THis is E. co ' _i JM109 with a plasmid carrying an ompA-HPSTI fusion from SGE45, driven by the tac promoter in vector pT3XI-2. Translation of ompA-HPSTI is coupled to the T7 gene 10 start with a translation coupler sequence known as TC4. The sequence of the DNA between the tac promoter and the ompA-HPSTI gene is given below. The vector pT3XI-2, was derived from pKK223-3 (Pharmacia), which was in turn derived from pBR322. The modifications that were made to pKK223-3 to generate pT3XI-2 include the following: reconstruction of the complete tetracycline resistance gene from pBR332; mutagenesis of the tet gene to remove the Hindlll , Sail and BamHI sites while still retaining tetracylcline resistance; and insertion of the lad gene from E. coli into the Pvull site. Example 2 - Effect on Human PSTI on Taurocholate-Induced Murine Pancreatitis Pancreatitis was induced after laparotomy under general anesthesia as described previously by W.H. Marks et al. in Scand. J. Gastroenterol 1984; 1.9:552-560, specifically incorporated herein by reference, in 70 rats. This procedure generally involves injecting 0.4 ml of 4% sodium taurocholate in phospate buffered saline (pH 7.4) into the common bile duct, temporary obstructed proximally with a vascular clip. S UB S T IT UTESHEET Doses of 50 mg/kg body weight of PSTI were infused in- traperitoneally 15 minutes and 2 hours after the taurocholate injection and 25 mg/kg was infused 4, 6 and 24 hours in 40 rats. A second treatment group of 30 rats was infused in¬ traductally with 40 mg/kg body weight of PSTI in 0.2 ml of saline immediately before taurocholate infusion. As controls, one group of 30 rats was infused with taurocholate and treated with intraperitoneal administration of PBS vehicles. A second group of 30 rats was infused with vehicles both intraductally and intraperitoneall . Survival of the animals was observed daily to 4 days. Fifteen animals from each treatment group were fol¬ lowed for one month. The survival rate in the control group of rats receiving intraductal taurocholate was 13%. In con¬ trast, the survival rate in the group receiving in¬ traperitoneal PSTI in addition to taurocholate was 63% and survival rate in the group of animals receiving PSTI as a single dose into the pancreatic duct was 80% as shown in Table 1 below. The 15 animals of each treatment group fol¬ lowed beyond the 4 days all survived a one month observation. The control group receiving only one vehicle showed a surviv¬ al rate of 93%. TABLE 1 Experimental Pancreatitis in Rats -- Effects of hPSTI on Survival Rate Survival (number) Survival 24h 48h 72h 96h Rate (%) Intraductal adm. (N=30) 24 24 24 24 80 Intraperitoneal adm. (N=40) 26 25 25 25 63 Controls Saline intraperit (N=30) 4 4 4 13 Saline intraperit (N=30) 28 28 28 28 93 Example 3 - Effect of Human PSTI on Bile-Induced Canine Pancreatitis Pancreatitis was induced as described in Example 2 SUBSTITUTESHEET by infusion of 0.5 ml/kg body weight of autologous bile into the main pancreatic duct after laparotomy according ' to the method described by K. Ohlsson and A. Eddeland in Gastroenterology 1975; 6_9_:668-674, specifically incorporated herein by reference. In one group of 5 dogs, 20 mg/kg body weight of human PSTI in 2.0 ml of saline was injected immediately before the bile. Both infusions were directed only into the horizontal lobe of the pancreas. As a control, another group of 5 dogs was treated with vehicle by the same method before they received bile intraductally. Both groups of animals were kept under general anesthesia with respirator-assisted ventilation for 24 hours. Ringer's solution was given in¬ travenously at a rate of 100 ml per hour. Polyethylene cath¬ eters were inserted in one femoral vein for blood sampling and into one femoral artery for monitoring of arterial blood pressure. A Swan-Ganz catheter (Model 93-1325F, obtained from American Edwards Lab, Irvine, California) was inserted through the other femoral vein and forwarded in the pulmonary artery under pressure control. The Swan-Ganz catheter was used to measure cardiac output (CO) using thermodilution. Measurements were performed using a cardiac output computer (Model 9520 Edwards Lab) . Samples of venous blood and peritoneal exudate were drawn at regular intervals. The samples were centrifuged for one hour and the supernatants were then frozen in small ali- quots at -70°C. The pancreas of animals that died during the observation period were immediately removed and formalin- fixed. The same procedure was used at autopsy after 24 hours for those animals surviving the observation period. The survival rate of the control group under gen¬ eral anesthesia was only 40% and the two dogs alive at the end of the observation period were in bad physical condition with dropping blood pressure that reached about 70 mm hg at 24 hours. In the control group, there was only a decrease in the cardiac output to around 60% after 12 hours. In con¬ trast, in the PSTI-treated group under anesthesia, all SUBSTITUTESHEET animals survived the 24 hour observation period with blood pressure above 100 mm Hg and showed good urine production. The cardiac output in this group did not show any significant changes. In the treatment group under general anesthesia, the peritoneal exudates showed a decreasing blood-staining with time. The pancreatic glands were edematous with scat¬ tered bleeding areas, but this was less pronounced than in the control groups. Fat necrosis was difficult to find in this group. The peritoneal exudates of the control animals gen¬ erally showed high levels of kallikrein/trypsin-like activity on gel filtration. It was possible to inhibit about 90% of the activity with human PSTI. In contrast, peritoneal exudates of the PSTI-teated animals showed very low levels of protease activity. A similar situation was seen when the kallikrein/trypsin-like activity in blood was measured. This was considerably higher in the control animals than in the PSTI-treated animals, especially 10 or more hours after the insult. Both PSTI-treated and control groups showed a sharp increase in the plasma level of immunoreactive anionic trypsin immediately after bile injection into the pancreatic duct. However, the level of anionic trypsin fell continuous¬ ly in the treated animals during the 24 hours after the in¬ sult whereas it increased substantially in the control groups during the period from 6 hours after insult until death. Example 4 In a long term study, a group of 8 dogs received 20 mg/kg body weight in 2.0 ml of saline of PSTI mixed with bile (0.5 ml/kg) immediately before injection into the main pancreatic duct. As a control, a group of 6 dogs were in¬ fused with bile mixed only with 2.0 ml of vehicles. The mix¬ ture was administred into the entire pancreas. After closure of the abdomen, the dogs were allowed to wake up and were taken off the respirator and extubated. Ringer's solution SUBSTITUTESHEET (100 ml/h) was given intravenously until the dogs removed the catheters. Blood samples were drawn through catheters in the external jugular veins during the same period at regular time intervals. No intraperitoneal catheter was used. Dogs surviving 48 hours were killed after 48 hours, 2, 4 and 6 weeks. Pancreatic tissues were recovered as described in the previous sample at autopsy. None of the animals in the control groups survived 48 hours, whereas all of the animals in the PSTI-treated group were alive at 48 hours. See Table 2. On autopsy, all of the animals of the control groups showed signs of severe hemorrhagic pancreatitis with a rich, dark red-brown peritoneal exudate and edema, and larger bleedings in the gland and tissue necrosis. There was fat necrosis on the surface of the gland and in the peripancreatic tissue. Pleural exudate was present and the lungs were edematous. TABLE 2 Experimental Pancreatitis in Poos — Effects of hPSTI on Survival Rate Survival (number) Survival 24h 28h Rate (%) Intraductal adm. 20MG/kg 8 8 100 body weight (N=8) Controls (N=6) 2 0 0 Histological examination of secretions from the glands from the control animals after haematoxylin staining showed signs of extensive proteolytic digestion with acinar necrosis in large areas of the glands. No immunoreactive trypsin was demonstrable in these parts. However, preserved acinar cells containing immunoreactive trypsin were fre¬ quently observed in the border zones of the lobules. Pro¬ nounced infiltration of inflammatory cells was observed. In the PSTI-treated animals autopsied after 24 or 48 hours, a finding of interstitial edema with the lobes spread apart was dominant. Some hypoxic vacuolation of acinar epitheilum was also seen. Acinar cell necrosis was hard to find in these SUBSTITUTESHEET glands and immunoreactive trypsin was present in an otherwise normal staining of the acinar cells. It will be apparent to those skilled in the art that various modifications and variations can be made to the processes and products of the present invention. Thus, it is intended that the present invention cover these modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. SUBSTITUTESHEET

Claims: WHAT IS CLAIMED IS 1. A method for treating pancreatitis comprising intraductal administering a protease inhibitor at a level and for a time sufficient to inhibit any active trypsin in the pancreas. 2. The method of claim 1 wherein said protease is PSTI. 3. A method for treating pancreatitis comprising administration of PSTI at a level and for a time sufficient to inhibit any active trypsin in the pancreas. 4. The method of claim 3 wherein said PSTI is administered intraperitoneally. 5. The method of claim 3 wherein said PSTI is administered intravenously. 6. A method for preventing pancreatitis during interventional procedures comprising the intraductal adminis¬ tration of a protease inhibitor at a level and for a time sufficient to inhibit any trypsin activated in the pancreas as a result of said interventional procedure. 7. The method of claim 6 wherein said protease inhibitor is administered prior to said interventional proce¬ dure. 8. The method of claim 6 wherein said protease inhibitor is administered after said interventional proce¬ dure. 9. The method of claim 6 wherein said protease inhibitor is administered during said interventional proce¬ dure. 10. The method of claim 6 wherein said in- ternventional procedure is endoscopic surgery. 11. The method of claim 6 wherein said interven¬ tional procedure is retrograde cholangiopancreatography. 12. The method of claim 6 wherein said protease inhibitor is PSTI. 13. The method of claim 12 wherein said PSTI is administered in an amount in a 2:1 molar ratio with the amount of trypsinogen present in the pancreas. SUBSTITUTESHEET 14. The method of claim 12 wherein about 125 to about 250 mg of PSTI is administered. SUBSTITUTESHEET

Inventors: OHLSSON KJELL, THOMPSON ROBERT C, OHLSSON, KJELL, THOMPSON, ROBERT, C.
Applicants: SYNERGEN INC, SYNERGEN, INC.
Application year: 1989
Main classification: C12P21
IPC codes: A61K38, A61P1