EP0904102A1

EP0904102A1 - Milk of transgenic animals containing human alpha 1-antitrypsin and use of human alpha 1-antitrypsin to treat bile acid related diseases

Info

Publication number: EP0904102A1
Application number: EP97914726A
Authority: EP
Inventors: Joyce Carlson; Sabine-Marija Janciauskiene
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-03-21
Filing date: 1997-03-20
Publication date: 1999-03-31
Also published as: AU2186497A; SE9601091L; WO1997034628A1; SE9601091D0

Abstract

The use of human α1-antitrypsin as a foodstuff or as a medicament, utilizing its capacity to bind steroids and steroid-like substances, and transporting them in biological systems is described. Particularly the direct oral administration of the milk of transgenic animals containing abundant amounts (10-60 g/L) of human α1-AT to reinstate a defect intestinal synthesis or to complement the normal physiological biosynthesis of α1-AT is described. Such treatment will reduce the total body load of bile acids by increasing their gastrointestinal elimination. It is expected to be beneficial for bile acid related diseases such as all cholestatic liver diseases, and bile-reflux gastritis. Such treatment is expected to be particularly beneficial in cases of neonatal cholestasis, as newborns circulate large quantities of hydrophobic bile acids which cause liver injury and may contribute to injury of other tissues. It will be protective in cases where bile acids cause tissue injury such as vasculitis, glomerulonephritis, and inflammatory bowel disease. It will be beneficial against diarrhoea in intestinal bacterial overgrowth and bile acid malabsorption. Increased gastrointestinal elimination of the steroid structure may also reduce the total body load of cholesterol and thus be efficient in the treatment of hyperlipidemia.

Description

MILK OF TRANSGENIC ANIMALS CONTAINING HUMAN ALPHAi-ANTTTRYPSIN AND USE OF HUMAN ALPHA^ANTTTRYPSIN TO TREAT BILE ACID RELATED DISEASES

Background

The discovery of α^-antitrypsin (c -AT) deficiency some 30 years ago triggered intensive research in pro- tease-antiprotease interactions, protein synthesis and export, and has been a leader in understanding the mole¬ cular mechanisms of disease¹"⁴.The clear causal associa¬ tion of the deficiency with pulmonary emphysema has been supported by the demonstration of free elastase activity in bronchioalveolar lavage (BAL) fluid and the presence of elastase in complex with its inhibitor, ot]_-AT, after intravenous injections of purified αχ-AT. The protease- antiprotease imbalance theory has also helped to explain the development of emphysema in smokers with normal Pi M α^-AT phenotype. αi-A deficiency genotype Pi ZZ is one of the most common known genetic diseases with a frequen¬ cy in Sweden of 1 in 1600 and in northern Europe of about 1/2000. Half of these individuals develop pulmonary emphysema with a mean age at onset of 32 years for smokers and 51 years for non-smokers. PiMZ heterozygotes have considerably milder disease, but non-smoking Pi MZ individuals also experience a significant decrease in lungfunction compared with normal Pi MM controls which was verified in a prospective study from mean age 30 to 40 years. The specific therapeutic options of intravenous augmentation therapy, and inhalation augmentation therapy with plasma purified or recombinant human c -AT have been approved in the absence of customary case-controlled studies according to the orphan drug principles of the US FDA, and intensive efforts to develop somatic gene thera- py via retroviral vectors, adenoviral vectors, or other delivery systems are ongoing. When all else has failed, more than 100 Pi ZZ patients have now received orthoptic lung transplants, and thus far shown about 60% 2 year post-transplant survival which is similar to that of other lung transplant recipients.

In contrast to the lung story, numerous clinical and epidemiological observations since the first reports of neonatal cholestas and juvenile cirrhosis in α_]_-AT defi¬ ciency, delineation of intracellular protein aggregation, identification of specific cell-membrane receptors, and the creation of transgenic animal models for the disease have thus far failed to produce any specific therapeutic option for the devastating childhood disease which pro¬ gresses to death or liver transplantation for about 5-6% of Pi ZZ children. An indolent but relentless development of cholestatic liver cirrhosis occurs during adulthood in nearly half of Pi ZZ individuals with progress to hepato- cellular or cholangiocellular carcinoma in about 25% with a 2:1 male predominance.

It has been clearly demonstrated that Pi ZZ α^-AT, which differs from the normal Pi M α^-AT by either a single amino acid change (342 Glu->Lys) or by this change in combination with a neutral substitution (213 Val->Ala) aggregates within the endoplasmic reticulum of hepatocy¬ tes causing a secretory defect and a deficiency of the circulating inhibitor. This aggregation may be due to a spontaneous concentration- and temperature dependent polymerization of the Pi Z protein, although a more complex mechanism is suspected ⁵. There is generally a preponderance of globular PAS-positive material corres¬ ponding to immunoreactive α]_-AT accumulated in periportal cells, as well as presence of periportal inflammation, seen on microscopy of liver tissue from affected patients. ct_-AT deficiency may also systematically involve other organs than lungs and liver, as summarized in ref. 4. Numerous reports verify the prevalence of glomerulone- phritis, in particular membranoproliferative forms (MPGN) in Pi Z children, adolescents, and adults. (The glomeruli can be seen to be "clogged" by deposits of αι_-AT using lmmunperoxidase staining, Joyce Carlson unpublished results.) This association has also been seen in pros¬ pective studies of Pi ZZ children with cholestatic liver disease. MPGN may be the cause of severe hypertension m the post liver transplantation period for several Pi ZZ children. Recent reports suggest an association with vasculitis and antmuclear cytoplasmatic antibodies (c-ANCA) such as found in Wegener's granulomatosis. Such antibodies have been shown to react with proteinase 3, one of the target enzymes of α]_-AT, although the exact causal mechanism is not yet clear. The Mayo Clinic has found a significant overrepresentation of Pi ZZ patients among those with fibromuscular dysplasia. Reports on a theoretical association with aortic aneurysms conflict, but arterial aneurysms have complicated liver transplan¬ tation in some pediatric cases. An increased frequency of the Pi MZ phenotype is also seen among patients with rheumatoid arthritis.

Studies in transgenic mice in which each mouse con- tains about 20 copies of the human Pi Z gene per cell have been performed to determine if the aggregates of Pi Z α^-AT in liver cells cause liver disease or if disease is caused by the lack of protease-inhibitory capacity due to the deficiency. The mice were expected to develop liver disease only if the mtracellular inclusions were responsible because they continued to produce their own endogenous "mouse inhibitors" and therefore have no cir¬ culatory deficiency.

Mice produced in the laboratory of Savio Woo⁶'⁷ developed significant intracellular aggregates and signs of liver inflammation but, although large numbers reached an age well past 2 years, and autopsies were performed on essentially every mouse, with liver histology evaluated by Joyce Carlson and a specialist in human liver patho- logy (Professor Milton Finegold, Texas Children's

Hospital, Houston, Texas and/or Professor Francesco Callea, Children's Hospital G. Gaslini Institue, Genova, Italy) , no case of typical liver cirrhosis or hepato- cellular hepatoma could be found. Furthermore, studies performed on the mice in Woo' s laboratory and in other laboratories⁷ clearly demonstrated biosynthesis of human ct]_-AT in many organs, including the antrum of the stomach, the small intestine, and the kidneys. Insofar as such studies have been performed, these findings have also been reproduced in humans. Specifically, Perlino et al.⁸ have demonstrated expression with alternative usage of 5' -first exons in macrophages and monocytes compared to liver cells. We (Arne Egesten and Joyce Carlson, un¬ published results) have found expression with alternative splicing in eosinophils and Joyce Carlson has demonstrat¬ ed alternative splicing in the mucosa of the distal ileum. David Perlmutter has shown expression of the human α_]_-AT gene in human intestinal epithelial cells in cul¬ ture⁹. Human biosynthesis in other organs such as the exocrine (and possibly endocrine) pancreas, the antrum of the stomach, the proximal renal tubulus, cartilage, mucus glands of the bronchial epithelium, and sweat glands, has also been suggested or verified by us or by other authors as cited in ref.6.

Comprehensive studies of structure-function rela¬ tionships for the entire family of serine protease inhi- bitors (serpins) of which a_-hT is the most studied member, have clearly demonstrated homologous structure for all members of the group and a drastic change in structure after interaction of each serpin with its tar¬ get protease⁵. The new structure exposes a hydrophobic pentapeptide on the surface of the molecule, and this peptide is recognized by a specific serpin-enzyme-complex (SEC) receptor in the cell membranes of hepatocytes and monocyte derived cells. Other serpins have been shown to have specific transport functions: corticosteroid binding globulin for cortisol, thyroxin binding globulin for thyroxin; and antithrombin III for heparin, and may release their transported ligand upon cleavage at the active site by a serine protease. No transport function has been thus far identified for ct]_-AT.

It is therefore of extreme interest that we have now demonstrated hydrophobic binding of cholesterol and bile salts to α]_-AT^10"13. We have shown that incubation of bile salts with α^-AT results in the formation of stable complexes. Lithocholic acid appears to have at least 3- fold greater affinity for ct -AT than for albumin, suggesting that this binding is biologically significant (Sabina Janciauskiene and Joyce Carlson, unpublished) . The binding of lithocholic acid to α^-AT is accompanied by a conformational change which permits or induces polymerization. We have demonstrated the spontaneous formation of insoluble fibrils when α^-AT has been in- cubated with the hydrophobic bile salt lithocholic acid. This polymeric complex is furthermore inactive as an in¬ hibitor of α^-AT' s preferred target enzyme, neutrophile elastase. Another interesting observation made when puri¬ fying α^-AT from a PiZZ patient with gemfibrozil treat- ment for hyperlipidemia was that gemfibrozil also formed a stable complex with PiZ α^-AT, ablating its capacity to inhibit elastase¹³. α^-AT or proteolytic peptides derived from α_-AT have previously been identified in the bile. We have now demonstrated that after addition of purified α -AT to bile, the α^-AT is converted into a polymer, and also loses its immunoreactivity, possibly due to incorporation into micellar structures. This behavior cannot be observ¬ ed for another of the serpins, antichymotrypsin. Other unpublished observations include the disappearance of measurable aldosterone in the plasma of a patient with hyperaldosteronism upon the addition of a molar excess of native αχ-AT (Sabina Janciauskiene, unpublished) .

Together, several experiments indicate that c -AT has three mutually exclusive functions: 1) α]_-AT inhibits the proteolytic effect of target serine proteases. 2) c -AT appears to form complexes with a number of steroids and steroid-like hydrophobic com¬ pounds, and functions as a transport vehicle for these substances in analogy to corticosteroid binding globulin. Similarity in structure with sulfate transporters (PROSITE databasePDOC00870) suggests that α_]_-AT may also be active in the metabolic conjugation or transformation of such substances. Transport may be directed to specific cells via the SEC receptor, for further detoxification or metabolism. 3) Native αι_-AT is capable of complexing free immunoglobulin lambda light chains ¹⁴ and αi-antichymo- trypsin binds β-amyloid peptide¹⁵. Peptides may be insert¬ ed into the beta sheet of ct_-AT, occupying the position normally available for sheet 4A of α^-AT after its inter¬ action with its target enzyme (fig. I)⁵.

Fig. 1. Shows the crystalline structure of human αχ~ AT (a) and chicken ovalbumin (b) . αι_-AT was crystallized after cleavage by trypsin at the active site Pl-Pl' . The strand s4A is the active site loop (cfr b) , which upon cleavage is inserted into the β-sheeted plate A⁵. The hydrophobic core in which steroid-like substances are transported is marked ^"*^" .

Whereas presence of a steroid in the hydrophobic core of α_-AT appears to inhibit the binding of .such peptides, each of these two functions independently block α^-AT's enzyme inhibitory function. Presumably, incorpo¬ ration of hydrophobic substances according to 2) and insertion of a peptide as in 3) limit the flexibility of the native protease inhibitory active site loop, causing steric hindrance for the "docking" of target proteases.

These observations offer a new interpretation to the pathogenesis of liver disease (and its complications) in α_]_-AT deficiency. The function of α^-AT synthesized in the small intestine is clearly not only to inactivate excess pancreatic proteolytic enzymes (this occurs pri- marily in the proximal small intestine) , but also to bind toxic hydrophobic bile acids produced from the beneficial hydrophilic bile acids by bacterial deconjugation and oxidation in the more distal gut. Possibly it also parti- cipates in inactivation/elimination of such toxic sub¬ stances as endotoxins. Complex formation with α]_-AT and subsequent polymerization decreases the solubility of both substances, increasing their fecal excretion. In more dilute solutions, it may facilitate metabolic transforma- tion of the hydrophobic bile acids to less toxic products in the gut, in intestinal cells, or after enterohepatic recirculation, in the liver.

In Pi Z α]_-AT deficiency, intestinal as well as hepatic secretion of α^-AT is reduced. Therefore there is a decreased fecal excretion of toxic hydrophobic sub¬ stances, decreased metabolic transformation to non-toxic products, and a greatly increased delivery of toxic hydrophobic substances to the liver. Whereas hydrophilic bile acids such as ursodeoxycholic acid have been shown to increase bile flow, the hydrophobic lithocholic acid is hepatotoxic. Hydrophobic bile acids readily cross membranes and have been shown to disrupt the endoplasmic reticulum membrane in liver cells. Binding to Pi Z α_x-AT in the endoplasmic reticulum induces polymerization and simultaneoulsy prevents c^-AT from performing its other beneficial functions. The increasing total body burden of hydrophobic bile acids in liver disease results in the formation of complexes with circulating o^-AT, inactiva¬ tion of its protease inhibitor function, and possibly spontaneous polymerization in blood vessels. The end result may be vasculitis and glomerulonephritis.

This hypothesis is compatible with the increased prevalence of liver disease in males with α -AT defi¬ ciency, as androgens are known to compete with hydro- phobic bile acids for the same "detoxifying" enzymes¹⁶. It may explain the beneficial effect of breast-feeding in some cases of neonatal cholestasis¹⁷, due to the low con- centration of α₁-AT in the milk of the heterozygous mother. It is also compatible with decreased intestinal absorption and delivery of the steroid-like fat soluble vitamins D, E and K to the liver in cholestasis, as hydrophobic bile acids compete with these vitamins for the binding site in ct]_-AT . Deficiencies of vitamins D and K result in osteomalacia and hemorrhage, common complications of cholestatic liver disease. Vitamin E deficiency and excess hydrophobic bile acids exert a combined oxidative stress on the hepatocyte, which is further potentiated by the accumulation of iron and copper in cholestatic disease¹⁸.

Assuming that the intestinal biosynthesis of αχ-AT is essential for the maintenance of a normal bile acid pool, then even those PiZZ patients who have received liver transplants will successively develop problems from an increased circulatory load of hydrophobic bile acids. Intestinal bile acids transported via lymph vessels to the sytemic vasculature, will presumably be complexed by circulating Pi M α^-AT provided by the liver transplant, and be transported either to the liver or directly to the kidney. This is highly compatible with the observation of hypertension after liver transplantation in children, suggested to be due to MPGN¹⁹. The etiology of this di- sease may well be due to circulating bile acids probably in complex with α^-AT.

These observations offer new therapeutic options for liver disease in α]_-AT deficiency , as well as choles¬ tatic conditions in general. Invention

The invention describes the use of human α_-anti- trypsin as a foodstuff or as a medicament, utilizing its capacity to bind hydrophobic substances and steroids and steroid-like substances, and transporting such substances in biological systems. It does not exclude the use of a_- antitrypsin for the transport of other substances after demonstration of specific binding to such substances. In particular it describes the direct oral administra tion of the milk of transgenic animals containing abundant amounts of human ct]_-AT. Such treatment will reinstate a normal physiological function, i.e. to reduce the total body load of bile acids by increasing their gastrointes¬ tinal elimination. It is expected to be beneficial for bile acid related diseases such as all cholestatic liver diseases, and bile-reflux gastritis. Such treatment is expected to be particularly beneficial in cases of neonatal cholestasis, as newborns circulate large quanti¬ ties of hydrophobic bile acids which cause liver injury and may contribute to injury of other tissues. It will be protective in cases where bile acids cause tissue injury such as vasculitis, glomerulonephritis, and inflammatory bowel disease. It will be beneficial against diarrhoea in intestinal bacterial overgrowth and bile acid malabsorp- tion. Increased gastrointestinal elimination of the steroid structure may also reduce the total body load of cholesterol and thus be efficient in the treatment of hyperlipidemia.

The use of such a foodstuff or medicament is readily accessible to experimental investigation as well as to clinical trials with treatment modalities that should be non-toxic, and may become relatively inexpensive. This possiblitiy is a radical contrast to the expense, uncer¬ tainties and risks involved in either somatic gene thera¬ py²⁰ or liver transplantation. In particular we consider the current production 10-40 g/L of recombinant human a^- AT in the milk of transgenic sheep²¹. This treatment alone or in combination with a supplement of hydrophilic bile acids to improve the qualitative characteristics of bile²² or with a supplement of specific nutriments such as fat soluble vitamins A, D, E and K, may aid in the resolution of neonatal cholestasis. Subsequent longterm supplements, possibly in the form of yoghurt, ice cream, or entero- capsules, should be beneficial to prevent the slow, sub- clinical development of liver disease in Pi ZZ indivi¬ duals, and to prevent vascular complications in liver transplanted Pi ZZ patients. Availability of a reasonable therapeutic alternative should also increase motivation for early diagnosis of the condition, its longterm follow up and treatment.

Other interesting observations and conclusions may be drawn in parallel to the above. Hydrophobic bile acids should be highly detrimental in other cholestatic liver diseases, producing a vicious circle with increased hepatocellular injury due to the "bad" quality of the bile. Such diseases include intrahepatic biliary atresia, primary biliary cirrhosis, sclerosing cholangitis, and possibly secondary injury due to the cholestatic compo- nent of other metabolic diseases such as hemochromatosis and Wilson's Disease. All of these conditions should also be alleviated by the same treatment, regardless of access to or inavailability of a primary causal therapy. Such treatment may even have a beneficial effect on the pro- gress of all forms of cirrhosis, including alcoholic liver cirrhosis, in which abnormal bile acid pools have developed secondary to liver disease. The delivery of such a foodstuff or medicament supplemented with the fat soluble vitamins could provide a targetted delivery system using the SEC receptor and replenish deficiencies secondary to the liver disease, as well as protect against oxidative stress. This treament could also be effective in the liver disease associated with cystic fibrosis, and might alleviate diarrhea caused by bacte- rial overgrowth.

Pretreatment of animals with a combination of antithrombin III and α^-antitrypsin has been shown to prevent the symptoms of adult respiratory distress syndrome (ARDS) which reproducibly occurs upon the infusion of endotoxin into blood vessels of these ani¬ mals²³. The combination of these two serpins has had a much greater effect than corresponding concentrations of either of the serpins alone, suggesting synergism. The delivery of a foodstuff or medicament containing α^-anti- trypsin could protect against the intestinal absorption of free endotoxin. The "beefy red" mucosa of the stomach lining follow¬ ing surgery for ulcers or cancer is thought to be due to bile reflux. Such bile reflux has also been implicated as the cause of "post gastrectomy" cancer. Part of the injury in both small and large intestine in patients with Crohn' s disease and ulcerative colitis is thought to be due to the excess presence of bile acids. Even in such cases, the ingestion of αχ-AT rich milk should provide local protection of the mucosal cells. By reducing the total body load of hydrophobic bile acids, it would be expected to decrease the risk for glomerulonephritis and other systemic complications of cholestasis. Similarly, the structural similarites between several major liver carcinogens with the bile acids and with steroids is of interest in conjunction with the high frequency of hepatocellular carcinoma in Pi ZZ men. Pi M α^-AT is expected to have a protective effect in the elimination of such carcinogens.

At present there are two principally different options for the general treatment of cholestasis and/or gallstone disease (caused by aggregation and precipita¬ tion of lithocholic and other bile acids with cholesterol in the gall bladder) . One is the oral administration of the hydrophilic bile acids ursodeoxycholic acid and chenodeoxy cholic acid as stated above. Whereas this treatment has been shown to increase the volume of bile produced, and decrease the concentration of lithocholic acid in the bile, it does not decrease the amount of lithocholic acid reaching the liver²⁴. The other concep¬ tual approach has been to form insoluble complexes with anion exchange resins in the intestine. Since such resins have here been shown to form complexes with Pi Z αχ-AT ¹³ which ablate its protease inhibitory capacity in plasma, they must be considered as unsuitable in patients with the Pi Z allele.

The suggested treatment using direct ingestion of milk containing a high concentration of cq-AT (see patents WO 92/11358, WO 90/05188 and WO 88/00239 for the method of producing the milk) is built upon the new con¬ cept that biosynthesis of ctι_-AT in the intestine plays an essential role in the normal metabolism and circulation of bile acids, and that it is a defect in this physio- logical function which is central in the development of liver disease in α^-AT deficiency. Such treatment would provide both proteinase inhibition and bile acid and/or steroid transport in a physiological fashion, and would be expected to produce fewer - if any - side effects. It is different from these patents in that it does not re¬ quire the purification of α^-AT for intravenous injec¬ tion, although the use of purified α^-AT is not excluded from this invention (see below) . The suggested use of milk containing human α^-AT clearly differs from the use of milk of transgenic animals containing human fibrinogen (WO 95/23868) or the use of milk with a low concentration of phenylalanine due to genetic modification of the α-lactalbumin gene for treatment of hyperphenylalaninuria and PKU (WO95/02692) . This suggested use of α^-AT as a transport vehicle for steroid-like substances is completely different from the documented therapeutic use of human αι_-AT purified from plasma (Prolastin) as a protease inhibitor, in the treatment of emphysema^25-28. It differs from its use for the prophylaxis or treatment of mast cell implicated disease by local application to dermatitis as in patents USP 5134119, 5217951 and 5346886, and does not conflict with JP 04074133A in which α^-AT is an example of a serine protease inhibitor which may be combined with calcitonin (to prevent proteolytic degradation of calci¬ tonin) and bile acid salts (to enhance percutaneous absorption) in an "easily administered percutaneous absorption composition". Neither does it conflict with patent WO 9407525 in which α^-AT may be administered as an example of a "serine protease inhibitor of viral replication" m which administration is preferably by infusion and α -AT is intended as an antiviral drug. Nonetheless, use of α]_-AT purified from the milk of transgenic animals is not excluded from this invention.

Numerous other documents mention human αχ-AT, in¬ cluding patents for gene therapy (W093/18794, WO93/19660, WO94/04696), fusion constructs of DNA producing hybrids between α_-AT and other proteins (WO92/025539) , methods for analysis (JP 06181798), use of mutated α_]_-AT with increased thermal stability (W094/26781) , decreased oxidative potential, etc. None of these patents deals with the α_-AT' s affinity for bile acids, the treatment of liver disease, the enterohepatic circulation or gastrointestinal elimination of bile acids.

References

1. Carlson J, Sifers R, Woo SLC. Molecular defects in an inheritable liver disease: α_]_-antitrypsin deficiency. in Arias I et al., eds, The Liver: Biology and

Pathobiology, Second Edition. New York, Raven Press, Ltd. , 1988, pp 1161-8.

2. Eriksson S and Carlson J. α_-Antitrypsin Deficiency and Related Disorders" Chapter 20.3 in Mclntyre N,

Benhamou J-P, Bircher J, Rizzetto M and Rodes J, eds. et al. eds. OXFORD Textbook of Clinical Hepatology, Oxford University Press, 1991, 958-66.

3. Triger D, Carlson J and Millward-Sadler GH. α^-

Antitrypsin Deficiency and Liver Disease in Millward- Sdler GH, Wright R and Arthur MJP, eds. Wright's Liver and Biliary disease, London, W.B. Saunders Company Ltd., 1992, pp 1170-1188.

4. Carlson J. Diagnosis, Clinical course and therapy of alphal-antitrypsin deificiency in Schmid, et al. Acute and Chronic Liver Diseases: Molecular Biology and Clinics. Lancaster, Kluwer academic publishers, In Press.

5. Stein PE and Carrell RW. What do dysfunctional serpins tell us about molecular mobility and disease? Structural Biology 1995; 2: 96-113.

Carlson J, Rogers B, Sifers R, et al.. Multiple tissues express α^-antitrypsin in transgenic mice and man. J Clin Invest 1988;82:26-36. 7. Carlson J, Rogers B, Sifers R, et al.. Accumulation of PiZ α -antιtrypsm causes liver damage in transgenic mice. J Clin Invest 1989;83:1183-1190.

8. Perlino E, Cortes R, Ciliberto G. The human αl-anti- trypsin gene is transcribed from two different pro¬ moters in macrophages and monocytes. EMBO J 1987; 2767-2771.

9. Perlmutter DH et al. The αl-antitrypsm gene is expressed in a human intestinal epithelial cell line. J Biol Chem 1989; 264:9485-90.

10. Janciauskiene S and Eriksson S. In vitro complex formation between cholesterol and α -protemase inhibitor. FEBS Lett 1992;316:269-72.

11. Janciauskiene S and Eriksson S. Conformational changes of the αχ-proteinase inhibitor affecting its cholesterol binding ability. FEBS Lett 1993;323:236- 38.

12. Janciauskiene S and Eriksson S. The interaction of hydrophobic bile acids with the αχ-proteinase inhibitor. FEBS Lett 1994;343:141-145.

13. Janciauskiene S and Eriksson S. An interaction between Gemfibrozil and alphal-antitrypsm. J Int Med 1994; 236: 357-360.

14.Eriksson S, Janciauskiene S, Merlini G. The putative role of alpha-1-antitrypsιn in the disaggregation of amyloid lambda fibrils. J Int Med 1995; 237: 143-9. 15.Eriksson S, Janciauskiene S, Lannfelt L. αl-

Antichymotrypsm regulates Alzheimer b-amyloid peptide fibril formation. Proc Natl Acad Sci 1995;92:2313-7.

16. Radominska A, Treat S, Little J. Bile acid metabolism and the pathophysiology of cholestasis. Sem In Liver Dis 1993; 13: 219-234.

17.Uldall IN, Dixon M, Newman AP, Wright JA, James B, Block KI. Liver idsease in alpha-1-antitrypsin deficiency. A retrospective analysis of the influence of early breast versus bottle feeding. JAMA 1985;253:2679-82.

18. Pittschieler, K. Liver disease and heterozygous alphal-antitrypsin deficiency Acta Paediatr Scand 1991;80:323-7.

19. Noble-Jamieson-G; Barnes-ND; Thiru-S; Mowat-AP. Severe hypertension after liver transplantation in alpha 1 antitrypsin deficiency. Arch-Dis-Child. 1990 Nov; 65(11) : 1217-21.

20.Knoell DL, Wewers MD. Clinical implications of gene therapy for alphal-antitrypsin deficiency. Chest 1995;107:535-45.

21.Wilmut I, Archibald Al, McClenaghan M, Simons JP, Whitelaw CB, Clark AJ. Production of pharmaceutical proteins in milk. Experientia. 1991; 47 (9) : 905-12.

22. Redens-TB, Leach-WJ, Bogdanoff-DA, Emerson-TE.

Synergistic protection from lung damage by combining antithrombin-III and alpha 1-proteinase inhibitor in the E. coli endotoxemic sheep pulmonary dysfunction model. Circ-Shock. 1988 Sep; 26(1) : 15-26

23.Reichen J. Pharmacologic treatment of cholestasis. Sem in Liver Dis 1993; 13: 302-15.

24. Poupon R, Chretien Y, Poupon RE. Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis? Lancet 1987; 1:834-36.

25. Guidelines for the approach to the patient with severe hereditary αχ-antitrypsin deficiency. Am Rev Respir Dis.1989;140:1494-7.

26.Wewers M, Casolaro M, Sellers S, Swayze S, McPhaul K, Wittes J, Crystal RG. Replacement therapy for alpha-1- antitrypsin dificiency associated with emphysema. N Engl J Med. 1987;316:1055-61.

27.Hubbard RC, Sellers S, Czerski D, Stephens L, Crystal RG. Biochemical efficacy and safety of monthly augmentation therapy for αχ-antitrypsin deficiency . JAMA.1988;260:1259-64.

28.Hubbard RC, McElvaney NG, Sellers SE, Healy JT,

Czerski DB, Crystal RG. Recombinant-DNA produced αι_- antitrypsin administered by aerosol augments lower respiratory tract antineutrophil elastase defenses in individuals with αχ-antitrypsin deficiency. J Clin Invest. 1989;84:1349-54.

Claims

1. Milk of dairy animals, which animals have been genetically modified by recombinant DNA techniques to produce and in their milk secrete human α-_L-antitrypsin, for use in the treatment of diseases.

2. Milk of dairy animals, which animals have been genetically modified by recombinant DNA techniques to produce and in their milk secrete human Oi-antitrypsin, for use in the treatment of bile acid related diseases.

3. Milk of dairy animals, which animals have been genetically modified by recombinant DNA techniques to produce and in their milk secrete human o^-antitrypsin, for the transport of hydrophobic substances, steroids or steroid-like substances in biological systems.

4. Milk according to claims 1-3, which milk has been processed to another dairy product, such as yoghurt, cultures with lactobacillus, ice cream, cheese, butter, dried milk powders, etc.

5. Use of human α -antitrypsin or its derivatives for the manufacture of a foodstuff or medicament for the treatment of bile acid related diseases and/or transport of hydrophobic substances and/or steroids or steroid-like substances in biological systems.

6. Use according to claim 5 in which said αy-anti- trypsin is purified from human plasma.

7. Use according to claim 5 in which said α^anti- trypsin is prepared by recombinant DNA techniques.

8. Use according to claim 5 in which said αχ-anti- trypsin is produced by recombinant DNA techniques in the milk of dairy animals.

9. Use according to claim 8 in which proteins from said milk have been concentrated and packaged for speci- fie intestinal delivery (enterocapsles) .

10. Use according to claim 8 in which said α -anti- trypsin has been modified to increase its affinity for specific hydrophobic and/or steroids or steroid-like substances.

11. Use according to claim 8 in which said milk has been modified by the addition of a specific beneficial steroid-like substance.

12. Use according to claim 11 in which said steroid- like substance is vitamin A, vitamin D, vitamin E and/or vitamin K.

13. Use according to claim 5, 8 or 10 in which said substance is cholesterol or its derivatives.

14. Use according to claim 5, 8 or 10 in which said substance is lithocholic acid or another hydrophobic bile acid.

15. Use according to claim 5, 8 or 10 in which said substance is a toxic substance.

16. Use according to claim 15 in which said toxic substance is a polycyclic carcinogen.

17. Use according to calim 15 in which said toxic substance is endotoxin.

18. Use according to claim 5, 8 or 10 in which said disease is neonatal cholestasis.

19. Use according to claim 5, 8 or 10 in which said disease is juvenile cirrhosis.

20. Use according to claim 5, 8 or 10 in which said disease is any liver disease.

21. Use according to claim 5, 8 or 10 in which said disease is bile-reflux gastritis.

22. Use according to claim 5, 8 or 10 in which said disease is glomerulonephritis.

23. Use according to claim 5, 8 or 10 in which said disease is vasculitis.

24. Use according to claim 5, 8 or 10 in which said disease is inflammatory bowel disease.

25. Use according to claim 5, 8 or 10 in whicn said disease is diarrhoea.

26. Use according to claim 5, 8 or 10 in which said disease is hyperlipidemia.