AU2525700A - Utilization of orosomucoid in order to produce a pharmaceutical preparation - Google Patents

Abstract

The invention relates to the utilization of orosomucoid in order to produce a pharmaceutical preparation for the treatment of acute pancreatitis.

Description

The Use of Orosomucoid for Producing a Pharmaceutical Preparation The invention relates to a new use of Orosomucoid. a,-acidic glycoprotein (AGP), also termed Orosomucoid, is a substance recovered from plasma which has a molecular weight of 40 000 Daltons and a carbohydrate content of between 30 and 50%. AGP consists of a single polypeptide chain with 183 amino acids and includes two disulfide bonds. Furthermore, it comprises five carbohydrate chains, all localized in the first half of the peptide chain. About 14% of these carbohydrate groups are neutral hexoses, 14% hexosamines, 11% sialic acid and 1% fructose. Depending on the source of the AGP preparation and on the recovery or characterization method used, respectively, AGP occurs in various forms which are due to the differences in the carbohydrate chains. The properties and the biological functions of Orosomucoid have been described in the reviews by Schmid (in "The Plasma Proteins Structure Function and Genetic Control", Vol. 1 (1975), Academic Press, Ed. Frank A. Putnam, 2 nd Edition, pp. 183-228) and Kremer et al. (Pharmacological Reviews 40 (1988), pp. 1-47). In the field of medicine, AGP so far has been considered as substantial carrier substance for mainly - 1basic medicaments in plasma (cf. Kremer et al.). Furthermore, a positive effect of Orosomucoid on inflammatory reactions could be demonstrated. Thus, Denko et al. (Agents and Actions 15, 5/6 (1984), 539 540) have described an antiinflammatory action of AGP in case of urate crystal inflammations in rats. Libert et al. (J. Exp. Med. 180 (1994), 1571-1575) have demonstrated that a similar indication is the prevention of septic shock in connection with the action of TNF-a or of lipopolysaccharides. In connection with this antiinflammatory action of Orosomucoid it could be demonstrated that also local injuries and remote injuries, such as remote lung injury following intestinal ischemia in rat can be treated with Orosomucoid (Williams et al., Am. J. Physiol. 273 (1997), G1031-1035). The use of Orosomucoid for the treatment of impaired circulation and microcirculation of the non inflammatory type, in particular for the treatment of hemorrhagic and/or hypovolemic shock, has been described in WO 98/40087. In principle it is differentiated between acute and chronic pancreatitis, and the causes of both acute and also of chronic pancreatitis may be very different ones; in about 20% of the cases the cause remains unknown. -2- Pancreatitis denotes an inflammation of the pancreas which involves an autolysis of this organ, which, depending on the severity, pathologically/anatomically involves interstitial edematous changes up to hemorrhagic pancreas necrosis. Acute pancreatitis occurs often with alcoholism, diseases of the biliary tract, papillary stenosis, postoperatively after surgical interventions in the abdominal cavity, hyperlipoproteinemias, hyperparathyroidism, ulcus ventriculi, mumps and after ingestion of glucocorticoids. Also immunological causes have been postulated, yet so far no convincing proof has been found that immunological factors could be made responsible for the development of acute or chronic pancreatitis in humans. Letality varies with the severity of pancreatitis between 15 and 20%, yet with severe forms it may be up to 80%. Beger et al. (World J. Surg. 21 (1997), pp. 130-135) identify edematous pancreatitis and necrotizing pancreatitis as the most frequent clinical manifestations. The multiple organic damage involved in acute pancreatitis is due to various inflammation mediators which are a consequence of the necrotic processes in the course of pancreatitis. Local and systemic septic complications may also occur. In general, it is assumed that these necrotizing processes -3of pancreatitis are caused by a premature activation of the pancreas enzymes. For such a premature activation, however, a whole number of protective mechanisms in the acinar cells of the pancreas must be overcome. Only later on processes, such as biliary calculi or dysfunctions of the sphincter oddi or of the duodenal papilla which cause an entry of biliary liquid or of duodenal liquids into the ductus pancreaticus, are essential (Lerch et al., Int. J. Pancreatol. 15 (3) (1994), pp. 159-170). Because of the many possible causes of a pancreatitis, also numerous invasive and chemical therapies have been proposed, some of which are also applied in combination. These therapies include the excretory relaxation of the organ by a food and liquid restriction period, aspiration of gastric secretion and inhibition of pancreas secretion by azetazolamide, calcitonin, glucagon, cimetidine, hymecromon, or surgical measures, such as laparotomy (opening the abscess and necrectomy, choledochus drainage, partial or (sub)total pancreas resection, sanitation of the biliary ducts (papillotomy) and general therapies, such as antibiotics (because of the invasion of intestinal bacteria into the inflamed pancreas), shock therapy (because of the high liquid and plasma losses), alleviation of pain and administration of corticoids. -4- However, Tenner et al. (World J. Surg. 21 (1997), pp. 143-148) could find out that the "no oral intake" (NPO)-treatment (i.e. the food and liquid restriction period) as well as the treatment with anticholinergic substances and H 2 blocking agents could neither ease the pain involved in this disease, nor shorten the lengths of hospital stays. It could also be demonstrated that treatment with somatostatin or octreotide, aprotinin (Trasylol) and other protease inhibitors, as well as Gabexatmesilate, atropin, glucagon or calcitonin, in clinical tests were not effective and thus cannot really be recommended for the therapy of pancreatitis. Treatment with antibiotics, such as Ciprofloxacin, Ofloxacin and Imipenem, on the other hand, significantly reduced pancreatic sepsis and also significantly reduced the mortality caused by gram negative sepsis and secondary infection. These effects, however, are exclusively due to the antibacterial action of the antibiotics and not to fighting the actual causes of pancreatitis. Since there are no suitable chemical-medical treatment methods, the care of pancreatitis patients in the intensive care unit with measures, such as rigorous hydration, electrolyt monitoring, oxygen supply, early complete parenteral feeding, in combination with -5surgical measures, such as removal of biliary calculi and/or aspiration of the necrotic foci by a targeted percutaneous thin-needle aspiration (GPA) (in each case after a check by means of endoscopic retrograde cholangiopancreatography), optionally in combination with antibiotics, at present constitutes the only effective therapy for pancreatitis (cf. Tenner et al. (1997)). Therefore, there is a high demand for a medical chemical treatment of pancreatitis, which may be used alone or in combination with the medical intensive-care treatment methods for the therapy of pancreatitis, in particular of acute pancreatitis. It is thus the object of the present invention to provide a medical-chemical treatment method which may be used alone or in combination with known healing methods for pancreatitis. According to the invention, this object is achieved by using Orosomucoid for the production of a pharmaceutical preparation for the treatment of acute pancreatitis. Surprisingly, it has been shown that Orosomucoid in acknowledged pancreatitis animal models, as described in Lerch et al. (1994) and in Fedderson et al. (Int. J. Pancreatol. 8 (1991), pp. 323-331), exhibited an amazing effectiveness. In the animal model -6of edematous pancreatitis, a markedly reduced edema formation and vacuolisation could be attained by administering Orosomucoid. In the animal model of the hemorrhagic/necrotic pancreatitis it could be shown by the present invention that with the administration of Orosomucoid, the necrotic processes, in particular autolysis, could efficiently be counteracted. This is a completely new medical indication for Orosomucoid. As has been mentioned, the field of application of the present invention is the treatment of acute pancreatitis, in particular if it is an edematous or necrotizing pancreatitis. It has also been shown that according to the invention a pancreatitis-caused organic injury of other organs, such as, e.g., the lungs, can effectively be fought by the Orosomucoid pancreatitis treatment according to the invention. In particular, the ARDS (adult respiratory distress syndrome) which is known to occur in connection with pancreatitis, can be fought. ARDS often is a life-threatening complication in pancreatitis patients, the cause thereof and the connection between pancreatitis and ARDS still being largely unclear. There is, however, an animal model (Feddersen et al. (1991)), with which these conditions can be examined. Therefore, according to the invention, -7it is also possible to efficiently treat a pancreatitis-caused injury of the lungs, possibly of the kidney and of the liver. Also in connection with a pancreatitis-caused sepsis, Orosomucoid is provided according to the invention, and also a combination thereof with antibiotics is suggested. The mode of preparing Orosomucoid has been known (e.g. from WO 95/07703); as the source for human Orosomucoid, preferably human plasma or a plasma fraction, e.g. a COHN fraction, such as COHN IV or COHN V, is used. According to a preferred embodiment of the present invention, a pharmaceutical preparation comprising plasmatic Orosomucoid is provided for the treatment of acute pancreatitis. Alternatively, the Orosomucoid used according to the invention may also be recombinantly prepared (Merritt et al., Gene 66 (1998), pp. 97-106); and in the recombinant preparation, the glycosylation, in particular the sialylation level of Orosomucoid, may be varied, cf., e.g., Williams et al. (1997). According to the invention, preferably an Orosomucoid having a physiological sialylation level of approximately 11% is used. This level is enabled by producing the Orosomucoid preparation from pooled human plasma. -8- Preferably, the pharmaceutical Orosomucoid preparation according to the invention may further comprise albumin. As a liquid preparation, the pancreatitis treatment preparation according to the invention preferably has an aluminum content of less than 100 pg/l, preferably less than 10 g/1l, in particular less than 200 ng/l. Suitable methods of producing aluminum-reduced, storage-stable pharmaceutical preparations have, e.g., been cited in WO 98/12225 or in EP 0 484 464. According to the invention, the Orosomucoid preparation may be applied in any manner, in particular i.v., s.c., i.m., as well as locally, intravenous application being particularly preferred. The dose applied will depend on the severity of the patient's condition, a daily individual dose of from 0.05 to 1 g/kg body weight being a preferred standard quantity which preferably is used as a start according to the invention. Preferably, a stabilizer is admixed to the pharmaceutical preparation before it is used according to the invention, in particular sodium caprylate and, optionally, tensides, in addition to albumin, so as to increase its storage stability and its stability during a thermal treatment, respectively. -9- It is also suitable to treat the pharmaceutical preparation for inactivation and depletion, respectively, of viruses, in particular by at least one physical treatment, such as thermal treatment and/or filtration. A number of physical, chemical or physico chemical methods are known for the inactivation of viruses, such as, e.g., a thermal treatment, e.g. pasteurizing, or according to EP 0 159 311 A or EP 0 637 451 A, a hydrolysis treatment according to EP 0 247 998 A or a radiation treatment or a treatment with organic solvents and/or tensides, e.g. according to EP 0 131 740 A. Further suitable virus inactivation steps during the production of the preparations according to the invention have been described in EP 0 506 651 A or in WO 94/13329 A. The invention will now be described in more detail by way of the following examples and the drawing figures, to which, however, it shall not be restricted. Fig. 1 shows the effect of Orosomucoid in the GDOC pancreatitis animal model, and Fig. 2 shows the effect of Orosomucoid in the rat caerulein pancreatitis model. Example : E x a m p 1 e 1 Hemorrhagic-necrotic - 10 pancreatitis in rats Following laparotomy, the Ductus pancreaticus of fasted male Wistar rats (> 220 g) is cannulated under Ketamin/Xylazin anaesthesia (i.m.); the Ductus choledochus is clamped off. The retrograde application of glycodesoxycholic acid (GDOC; 4 mg/0.5 ml/kg for 2 min, subsequently the Ductus is rinsed free with 0.2 ml of isotonic saline) is effected into the Ductus pancreaticus. Two minutes after application, the Ductus pancreaticus catheter is removed again and the clamp of the Ductus choledochus is opened. Subsequently, the laparotomy wound is closed again. Via a V. jugularis, a permanent catheter is implanted which allows for a continuous infusion on the freely movable, concsious animal. Treatment regime with Orosomucoid: 1"t Series: (24 h substance application, after which the animals are sacrificed) Treatment group: GDOC into D. pancreaticus; immediately thereafter a bolus of 600 mg/10.75 ml/kg + infusion of 600 mg/kg/h Orosomucoid for 24 h (i.v.); negative control group: 0.5 ml/kg isotonic saline into the D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h NaCl for 24 h (i.v.), positive control group: GDOC into D. pancreaticus; - 11 bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h NaCl for 24 h (i.v.) placebo group: GDOC into D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h of placebo formulation for 24 h (i.v.); 2 nd Series: (6 h substance application, after which the animals were sacrificed) Treatment group: GDOC into D. pancreaticus; immediately thereafter, a bolus of 600 mg/10.75 ml/kg + infusion of 600 mg/kg/h Orosomucoid for 6 h (i.v.); negative control group: 0.5 ml/kg isotonic saline into D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h NaCl for 6 h (i.v.); positive control group: GDOC into D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h NaCl for 6 h (i.v.); placebo group: GDOC into D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h placebo formuation for 6 h (i.v.) 3 rd Series: (6 h substance application, the animals being sacrificed after 24 h) Treatment group: GDOC into D. pancreaticus; immediately thereafter, a bolus of 600 mg/10.75 ml/kg + infusion of 200 mg/10.75 ml/kg/h Orosomucoid for 6 h (i.v.); - 12 negative control group: 0.5 ml/kg isotonic saline into the D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h NaCl for 6 h (i.v.); positive control group: GDOC into D. pancreaticus; bolus of 10.75 ml/kg + infusion of 10.75 ml/kg/h NaCl for 6 h (i.v.); placebo group: GDOC into D. pancreaticus; bolus of 10.75 ml/kg + infusion of placebo formulation in a volume of 10.75 ml/kg/h for 6 h (i.v.) When the tests are over, the animals are anesthetized again and exsanguinated after cardiopuncture. After the abdominal cavity has been opened, aszites liquid is recovered and its volume determined, and also pancreatic tissue is removed for a histological examination. Amylase, lipases and lactate dehydrogenase are photometrically determined from the plasma. - 13 - Results: Orosomucoid exhibited positive effects in all the treatment groups; in Fig. 1, a survey of the results of the negative and positive control groups as well as of the treatment groups is given. The results of the placebo groups are discussed in the text. 1. Celiocentesis specimen: Animals of the positive control group form a markedly larger amount of aszites fluid as compared to the negative control group. GDOC animals which had been treated with Orosomucoid exhibit smaller amounts than the positive control group. 2. Plasma parameters: 2.1 Amylase: Animals of the negative control group have plasma amylase levels in the range of 8000 U/1. In animals treated with GDOC + isotonic saline (positive control group), the amylase levels rise within 24 h to the 2- to 3-fold. Treatment with Orosomucoid can clearly reduce the increase of amylase, the placebo formulation had no positive effect. 2.2. Plasmalipases: the lipase levels of positive control animals were more than twice as high than those of animals of the negative control group. GDOC animals which received infusions of Orosomucoid exhibit lower lipase levels. GDOC animals treated with - 14 placebo formulation partially exhibit higher lipase levels than the positive control group. 2.3 Plasma lactate dehydrogenase (LDH): Animals of the negative control group have LDH-levels in the range of approximately 300 U/1. In the positive control group, the levels are clearly higher. Orosomucoid treated GDOC animals have lower LDH levels than the animals of the positive control group. In GDOC animals which received placebo formulation infusions, the LDH levels clearly rise. 3. Histology: In the histologic examination of pancreatic tissue, the animals of the positive control group showed clear indications of an acute hemorrhagic necrotic pancreatitis. Thus, massive necroses of acinar cells, edemas, hemorrhages as well as clear infiltrations with inflammation cells could be observed. Animals in which isotonic saline had been administered into the D. pancreaticus + isotonic saline had been administered intravenously (negative control group) showed individual edematizing and low-grade hemorrhages in the pancreatic tissue, yet never necroses. Treatment of GDOC- animals with Orosomucoid (treatment group) could clearly mitigate the development of hemorrhagic-necrotic pancreatitis. In GDOC-animals with infused placebo formulations (placebo - 15 group), however, large amounts of necrotic pancreas acinar cells could be found. E x a m p 1 e 2 : In conscious rats, edematous pancreatitis was induced by infusion of 10 pg/kg h~ 1 Caerulein i.v. for 6 h. As parameter, the amylase increase in plasma and the water content of the pancreas were assayed, and also a histologic examination was carried out. In the present examinations, Orosomucoid was applied at 600 mg/kg i.v. as a bolus prior to Caerulein infusion, as well as at 200 mg/kg h~ 1 in parallel with the Caerulein infusion. The results are illustrated in Fig. 2. Caerulein dramatically increased the plasma amylase activity (from 3600 U/1 on negative controls to 26700 U/l). An administration of Orosomucoid lowered this level significantly to 18000 U/1. The placebo formulation did not have this effect (24800 U/l). What was interesting was that Orosomucoid in this model did not have any effect on the pancreas edema. The water content rose from 69% in negative controls to 91.6% in Caerulein treated animals, and this effect was influenced neither by Orosomucoid (90.6% nor by the placebo formulation (90.6%). In this histologic examination of pancreatic tissue, animals of the positive control group - 16 - (Caerulein infusion + isotonic saline) showed clear signs of an acute edematous pancreatitis. Thus, the massive development of an interstitial edema as well as a high-grade vacuolization of the acinar cells could be observed. Animals which had only been infused with isotonic saline (negative control group) did not exhibit pathologically changed pancreas tissue in any case. Caerulein-pre-treated animals to which additionally Orosomucoid had been administered (treatment group) exhibited reduced edema formation in the histologic picture. The Caerulein-caused vacuolization of the cells of the exocrine pancreas is markedly reduced by an Orosomucoid treatment. In animals which had been given placebo formulation in addition to the Caerulein infusion, again massive edema formation as well as extremely numerous vacuoles in the pancreas acinar cells could be found. Thus, for the first time a favorable influence on an experimental pancreatitis by Orosomucoid could be demonstrated, which is a completely new finding. - 17 -

Claims (13)

1. The use of Orosomucoid for producing a pharmaceutical preparation for the treatment of acute pancreatitis.

2. The use according to claim 1, characterized in that the pancreatitis is an edematous pancreatitis.

3. The use according to claim 1, characterized in that the pancreatitis is a necrotizing pancreatitis.

4. The use according to any one of claims 1 to 3, characterized in that an organ injury caused by pancreatitis is treated.

5. The use according to claim 4, characterized in that an injury of the lungs caused by pancreatitis is treated.

6. The use according to any one of claims 1 to 5, characterized in that a sepsis caused by pancreatitis is treated.

7. The use according to any one of claims 1 to 6, characterized in that the pharmaceutical preparation - 18 - comprises a plasmatic Orosomucoid.

8. The use according to any one of claims 1 to 6, characterized in that the pharmaceutical preparation comprises an Orosomucoid encoded by a recombinant nucleic acid.

9. The use according to claim 7 or 8, characterized in that the Orosomucoid has a physiological sialylation level.

10. The use according to any one of claims 1 to 9, characterized in that the pharmaceutical preparation further comprises albumin.

11. The use according to any one of claims 1 to 10, characterized in that the pharmaceutical preparation is characterized by an aluminum content of less than 100 pg/l.

12. The use according to any one of claims 1 to 11, characterized in that the pharmaceutical preparation is intravenously administered.

13. The use according to claim 12, characterized in that the dose is 0.05 - 1 g/kg as a daily single dose. - 19 - DA/fm - 20 -