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Title:
MISTLETOE EXTRACTS
Document Type and Number:
WIPO Patent Application WO/2001/060389
Kind Code:
A2
Abstract:
The invention provides a method for preventing the development of an autoimmune disease in a susceptible mammal by administering a mistletoe extract to the mammal. The invention further provides a method for prolonging acceptance of an engrafted tissue by administering a mistletoe extract to an engrafted tissue recipient.

Inventors:
DELOVITCH TERRY (CA)
ARREAZA GUILLERMO (CA)
Application Number:
PCT/CA2001/000193
Publication Date:
August 23, 2001
Filing Date:
February 19, 2001
Export Citation:
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Assignee:
ROBARTS JOHN P RES INST (CA)
DELOVITCH TERRY (CA)
ARREAZA GUILLERMO (CA)
International Classes:
A61K36/185; A61P37/00; (IPC1-7): A61K35/78; A61P37/00
Other References:
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1997 JURIN M ET AL: "Viscum album L. preparation Isorel modifies the immune response in normal and in tumour-bearing mice." Database accession no. PREV199799597866 XP002185842 & ANTI-CANCER DRUGS, vol. 8, no. SUPPL. 1, 1997, pages S27-S31, ISSN: 0959-4973
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1998 WERNER M ET AL: "Stimulation of T-cell locomotion in an in vitro assay by various Viscum album L. preparations (IscadorX)." Database accession no. PREV199900125556 XP002185843 & INTERNATIONAL JOURNAL OF IMMUNOTHERAPY, vol. 14, no. 3, 1998, pages 135-142, ISSN: 0255-9625
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1990 MUELLER E A ET AL: "A VISCUM-ALBUM OLIGOSACCHARIDE ACTIVATING HUMAN NATURAL CYTOTOXICITY IS AN INTERFERON GAMMA INDUCER" Database accession no. PREV199191063963 XP002185844 & CANCER IMMUNOLOGY IMMUNOTHERAPY, vol. 32, no. 4, 1990, pages 221-227, ISSN: 0340-7004
Attorney, Agent or Firm:
Rae, Patricia A. (Ontario M5G 1R7, CA)
Download PDF:
Claims:
CLAIMS :
1. A method for preventing the development of an autoimmune disease in a susceptible mammal comprising administering to the mammal an extract of a mistletoe plant.
2. The method of claim 1 wherein the autoimmune disease is autoimmune diabetes mellitus.
3. The method of claim 1 wherein the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, multiple sclerosis, lupus erythematosis and glomerulonephritis.
4. The method of any one of claims 1 to 3 wherein the mistletoe plant is of the species Viscum album.
5. The method of any one of claims 1 to 4 wherein the mammal is a human.
6. The method of any one of claims 1 to 5 wherein the extract is an aqueous extract.
7. The method of any one of claims 1 to 5 wherein the extract is selected from the group consisting of mistletoe extract Helixor M, mistletoe extract Helixor P, mistletoe extract Helixor A, mistletoe extract Iscador, mistletoe extract Isorel and mistletoe extract Plenesol.
8. The method of claim 7 wherein the extract is mistletoe extract Helixor M.
9. The method of any one of claims 1 to 5 wherein the extract contains at least one compound selected from the group consisting of a lectin, a viscotoxin, an alkaloid, a flavonoid, a lignin, an amine, a phenyl carboxylic acid or a polysaccharide.
10. A method for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising administering to the mammalian recipient an acceptanceprolonging amount of an extract of a mistletoe plant.
11. The method of claim 10 wherein the autoimmune disease is autoimmune diabetes mellitus.
12. The method of claim 10 wherein the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, multiple sclerosis, lupus erythematosis and glomerulonephritis.
13. The method of any one of claims 10 to 12 wherein the mistletoe plant is of the species Viscum album.
14. The method of any one of claims 10 to 13 wherein the mammal is a human.
15. The method of any one of claims 10 to 14 wherein the extract is an aqueous extract.
16. The method of any one of claims 10 to 14 wherein the extract is selected from the group consisting of mistletoe extract Helixor M, mistletoe extract Helixor P, mistletoe extract Helixor A, mistletoe extract Iscador, mistletoe extract Isorel and mistletoe extract Plenesol.
17. The method of claim 16 wherein the extract is mistletoe extract Helixor M.
18. The method of ciny one of claims 10 to 14 wherein the extract contains at least one compounc selected from the group consisting of a lectin, a viscotoxin, an alkaloid, a flavonoid, a lignin, an amine, a phenyl carboxylic acid or a polysaccharide.
19. A pharmaceutical composition for preventing the development of an autoimmune disease in a susceptible mammal comprising an effective amount of an extract of a mistletoe plant.
20. The composition of claim 19 wherein the autoimmune disease is autoimmune diabetes mellitus.
21. The composition of claim 19 wherein the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, multiple sclerosis, lupus erythematosis and glomerulonephritis.
22. The composition of any one of claims 19 to 21 wherein the mistletoe plant is of the species Viscum album.
23. The composition of any one of claims 19 to 22 wherein the mammal is a human.
24. The composition of any one of claims 19 to 23 wherein the extract is an aqueous extract.
25. The composition of any one of claims 19 to 23 wherein the extract is selected from the group consisting of mistletoe extract Helixor M, mistletoe extract Helixor P, mistletoe extract Helixor A, mistletoe extract Iscador, mistletoe extract Isorel and mistletoe extract Plenesol.
26. The composition of claim 25 wherein the extract is mistletoe extract Helixor M.
27. The composition of any one of claims 19 to 23 wherein the extract contains at least one compound selected from the group consisting of a lectin, a viscotoxin, an alkaloid, a flavonoid, a lignin, an amine, a phenyl carboxylic acid or a polysaccharide.
28. A pharmaceutical composition for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising an effective amount of an extract of a mistletoe plant.
29. The composition of claim 28 wherein the autoimmune disease is autoimmune diabetes mellitus.
30. The composition of claim 28 wherein the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, multiple sclerosis, lupus erythematosis and glomerulonephritis.
31. \.
32. The composition of any one of claims 28 to 30 wherein the mistletoe plant is of the species Viscum album.
33. The composition of any one of claims 28 to 31 wherein the mammal is a human.
34. The composition of any one of claims 28 to 32 wherein the extract is an aqueous extract.
35. The composition of any one of claims 28 to 32 wherein the extract is selected from the group consisting of mistletoe extract Helixor M, mistletoe extract Helixor P, mistletoe extract Helixor A, mistletoe extract Iscador, mistletoe extract Isorel and mistletoe extract Plenesol.
36. The composition of claim 34 wherein the extract is mistletoe extract Helixor M.
37. The composition of any one of claims 28 to 32 wherein the extract contains at least one compound selected from the group consisting of a lectin, a viscotoxin, an alkaloid, a flavonoid, a lignin, an amine, a phenyl carboxylic acid or a polysaccharide.
38. Use of an extract of a mistletoe plant for the preparation of a pharmaceutical composition for preventing the development of an autoimmune disease in a mammal.
39. Use of an extract of a mistletoe plant for the preparation of a pharmaceutical composition for prolonging the acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant.
Description:
MISTLETOE EXTRACTS Field of the Invention The present invention relates to methods and compositions for preventing the development of autoimmune diseases in susceptible subjects.

Background of the Invention In the description which follows, references are made to certain literature citations which are listed at the end of the specification and all of which are incorporated herein by reference.

The mistletoe order of plant parasites encompasses numerous different families and species that demonstrate similar properties. The mistletoes comprise a polyphyletic group of nearly 2000 species of predominantly tropical, dicotyledonous semi-parasitic plants within six families in the order Santalales. Mistletoes make up about 50% of all parasitic angiosperms, and represent the largest and most diverse group of aerial parasites. The six families comprising the Santales order are Loranthaceae, Misodendraceae, Olacaceae, Piliaceae, Santalaceane and Viscaceane.

In temperate regions, mistletoes grow on trees such as apple, cherry, oak, ash, hawthorn and lime.

Santales Mistletoe species are dependent on their host trees for water and minerals, which are drawn from tree fluids, but are capable of synthesising their own carbohydrates through photosynthesis. Chemical compounds found in extracts of mistletoe plants may have been synthesised either by the host tree or by the mistletoe plant. Extracts of mistletoe plants typically contain compounds such as lectins, viscotoxins, alkaloids, flavonoids, lignins, amines, phenyl carboxylic acids and polysaccharides (1-4). Such compounds have been demonstrated to activate various different components of the immune system, including B and T cells, granulocytes, NK cells, monocytes, macrophages and other inflammatory white blood cells.

Extracts of mistletoe plants have been shown to have a number of biological activities in mammals, and mistletoes such as Viscum album L, the

European white-berry mistletoe, have been used in a number of traditional herbal remedies (5). The most active components of these extracts appear to be lectins and viscotoxins (6).

Recent investigations have shown that an extract of V. album can induce apoptosis of cultured tumour cells and of lymphocytes, specifically CD8+ cytotoxic T cells, can stimulate the immune system and can protect DNA against chemotherapy and radiation damage (7-9).

Mistletoe extracts have therefore been used to upregulate the human immune system. Furthermore, mistletoe extracts can also mediate release of several pro-inflammatory cytokines within humans, including IL-1, IL-6, TNF-a and IFN-Y. These pro-inflammatory effects of mistletoe extract have been utilized in the treatment of a variety of cancerous tumors in humans (3, 10, 11).

Mistletoe extracts from, for example, African mistletoe, Loranthus bequrensis, and from V. album have also been shown to have antidiabetic effects in mammals, both stimulation of insulin release and possibly insulin- independent lowering of blood glucose (12, 13).

Type I diabetes has an autoimmune pathogenesis with a multifactorial etiology. T cell infiltration of the pancreatic islets of Langerhans and the progressive T cell-mediated destruction of insulin-producing beta cells heralds the onset of autoimmune diabetes (14, 15). Nonobese diabetic (NOD) mice spontaneously develop a form of type 1 diabetes that shares many immunologic and pathologic features of the human disease. Diabetes occurs in these mice as a result of the interaction of genetic factors with the environment (14, 15).

Type I or autoimmune diabetes develops gradually in both humans and NOD mice and it is desirable to find ways of intervening during this development process to prevent progression to actual diabetes.

Prior to the work of the present inventors, it was not realised that mistletoe extracts could be used to modulate the autoimmune response leading to Type I diabetes and to prevent the development of the disease.

Summary of the Invention The inventors have snown that extracts of mistletoe plants are effective to prevent the development of autoimmune diseases such as autoimmune diabetes. Mistletoe extracts may also be used to suppress rejection and prolong the acceptance of transplanted or engrafted tissue in a mammalian recipient.

In accordance with one embodiment, the invention provides a method for preventing the development of an autoimmune disease in a susceptible mammal comprising administering to the mammal an extract of a mistletoe plant.

In accordance with a further embodiment, the invention provides a method for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising administering to the mammalian recipient an acceptance-prolonging amount of an extract of a mistletoe plant.

In accordance with a further embodiment, the invention provides a pharmaceutical composition for preventing the development of an autoimmune disease in a susceptible mammal comprising an effective amount of an extract of a mistletoe plant.

In accordance with a further embodiment, the invention provides a pharmaceutical composition for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising an effective amount of an extract of a mistletoe plant.

In accordance with a further embodiment, the invention provides for the use of an extract of a mistletoe plant for the preparation of a pharmaceutical composition for preventing the development of an autoimmune disease in a mammal.

In accordance with a further embodiment, the invention provides for the use of an extract of a mistletoe plant for the preparation of a pharmaceutical composition for prolonging the acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant.

Summary of the Drawings Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein : Figure 1 shows the effect of administration of a mistletoe extract on the development of diabetes in non-obese diabetic mice treated from age 3 weeks to 18 weeks ; Figure 2 shows the effect of administration of a mistletoe extract on the development of diabetes in NOD mice treated from 12 to 27 weeks of age ; Figure 3 shows the effect of administration of a mistletoe extract on the development of diabetes in NOD mice treated from 18 to 25 weeks of age ; Figure 4 shows the severity of insulitis in treated and control NOD mice.

Detailed Description of the Invention The present invention provides a method for preventing the development of an autoimmune disease in a susceptible mammal comprising administering to the mammal an extract of a mistletoe plant.

The inventors have shown that the development of autoimmune diabetes in NOD mice can be prevented by treating the mice, before appearance of diabetes, with a mistletoe extract. This treatment was effective even when begun as late as 18 weeks of age, by which time the autoimmune process leading to diabetes in NOD mice is well under way and diabetes is imminent.

Without wishing to be limited to a particular mechanism for this observed therapeutic effect, it is proposed that the effect of mistletoe extract treatment is to modulate the balance between Th1 and Th2 T cells in the treated animal or human, leading to a reduction in the process of destructive insulitis which results in the specific destruction of the insulin-producing pancreatic beta cells. Example 3 shows the reduced insulitis seen after treatment of NOD mice with mistletoe extract.

Many other autoimmune diseases are believed to develop by a similar mechanism to that involved in the development of autoimmune diabetes, with

autoreactive T cells recognising different autoantigens characteristic of each disease.

Mistletoe extracts may therefore be used to prevent the development of autoimmune diseases including, but not limited to psoriasis, rheumatoid arthritis, multiple sclerosis, lupus erythematosis and glomerulonephritis.

The invention also provides a method for prolonging acceptance of an engrafted tissue in a mammalian recipient of a tissue transplant comprising administering to the mammalian recipient an acceptance-prolonging amount of an extract of a mistletoe plant.

When a tissue or organ is transplanted into a mammalian recipient, the immune system of the recipient mounts an immune response to antigens in the transplanted tissue or organ, leading to rejection of the transplanted tissue or organ. It is therefore necessary to treat the recipient with an immunosuppressive agent prior to transplantation and often continuously thereafter.

The invention provides a new immunosuppressive therapy in the form of a mistletoe extract to promote and prolong acceptance of a tissue or organ transplant in a mammal.

A"mistletoe plantas used herein means a plant of any species of mistletoe within the order Santalales. A mistletoe plant of the species V. album is preferred.

An"extract of a mistletoe plant", as used herein, means an aqueous extract of a mistletoe plant, either of the whole plant or of a part of the plant such as leaves or berries. The extract may be made, for example, by macerating the mistletoe plant or plant parts in water or phosphate buffered saline followed by removal of solid plant material from the extract, for example by centrifugation.

The extract preferably contains at least one compound selected from the group consisting of a lectin, a viscotoxin, an alkaloid, a flavonoid, a lignin, an amine, a phenyl carboxylic acid or a polysaccharide.

Suitable mistletoe extracts for use in the methods of the invention include those previously described for cancer treatment. Such mistletoe

extracts may be known by different names in different parts of the world ; for example V. album extracts are produced by Helixor Heilmettel GmbH, Rosenfeld, Germany and are supplied in Europe under the names Helixor M, Helixor P and Helixor A, and V. album extracts are supplied by Weleda in North America under the names Iscador or Iscar (11). Further examples of mistletoe extracts are Plenesol and Isorel (10).

These and similar mistletoe extracts may be used in the methods and pharmaceutical compositions of the invention.

For use in the methods and pharmaceutical compositions of the invention, a mistletoe extract may be administered to a human patient without further formulation or may be mixed, modified or introduced into other compositions to develop pharmaceutically acceptable vehicles for delivery into human patients.

Mistletoe extracts may be administered orally or parenterally, either by intravenous, intraperitoneal or subcutaneous injection, or by inhalation, transdermal application or rectal administration. The formulation of pharmaceutically acceptable vehicles for these various modes of administration are known to those of skill in the art and are described in standard texts on pharmaceutical formulation (16).

In order to identify a subject susceptible to an autoimmune disease and therefore a candidate for treatment by the method of the invention for preventing development of an autoimmune disease, various well known tests for diagnostic markers of susceptibility to autoimmune diseases may be employed. These diagnostic tests are known to those skilled in the art. For example, first degree relatives of patients with autoimmune diabetes are already screened for susceptibility to the disease by tests which look for, for example, antibodies to insulin or to glutamic acid dehydroxylase (17, 18).

Subjects found to be potential victims of autoimmune diabetes by such tests may be treated by the method described herein.

Similar diagnostic tests are available to detect susceptibility to other autoimmune diseases, looking for diagnostic markers characteristic of those diseases, for example anti-myelin antibodies in multiple sclerosis and anti-

collagen antibodies in rheumatoid arthritis. These tests may be employed to identify candidates for the treatment of the invention.

Subjects about to undergo a tissue or organ transplant may be treated by the method of the invention either alone or in combination with other immunosuppressive therapies.

The appropriate dosage of mistletoe extract administered in accordance with the methods of the invention may vary depending, for example, on the age, sex and weight of the patient. The dosage and dosing regimen may have to be adjusted, depending on the response of the patient, to provide the optimum therapeutic response. Determination of an appropriate effective dosage is within the skill of the ordinary physician.

In another embodiment of the invention, a mistletoe extract is administered to human subjects to prevent or decrease the recruitment of immune cells into human tissues. Administration of mistletoe extract may therefore be used to prevent or treat conditions associated with abnormal or deregulated immune cell recruitment into tissue sites of human disease.

In another embodiment of the invention, a mistletoe extract is administered to human subjects to prevent or decrease effects mediated by the release of cytokines within the human body. The term"cytokine"refers to all human cytokines that bind extracellular receptors upon the cell surface and thereby modulate cell function, including but not limited to IL-1, IL-4, IL-6, TNF-a and IFN-Y. It is envisioned that mistletoe extracts interfere with the in vivo role of mammalian cytokines. Therefore, administration of mistletoe extract may be used to prevent or treat conditions mediated through abnormal production or release of one or more cytokines within the human body.

EXAMPLES The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Methods of chemistry, molecular biology, protein and peptide biochemistry or immunology referred to but not explicitly described in this

disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.

Materials HELIXORT was obtained from Helixor Heilmittel GmbH, Rosenfeld, Germany.

HELIXOR is available in three different types, derived from three different subspecies of Viscum album which grow on different host trees : HELIXOR A from a mistletoe which grows on fir trees (abietis) ; HELIXOR M which grows on apple trees (genus malus) and HELIXOR P which grows on pine trees (genus pinus).

Each HELIXOR preparation is a sterile-filtered, unfermented extract of fresh Viscum album plants. It is provided as a 5% isotonic solution (50 mg of fresh plant per mi extract).

Toxicological studies of HELIXOR in animals and humans have demonstrated minimal toxicity with a LD50 in mice >500 mg/kg equivalent to about 700 times the clinical dose (Investigator brochure, HELIXOR Heilmittel GmbH & Co.).

Mistletoe extracts have been widely used for human adjuvant cancer therapy in various European countries, and their experimental use has been approved by the HPB (Health Protection Branch) of Canada and the FDA (Federal Drug Agency) in USA as an Investigational New Drug (IND) in clinical trials for patients with cancer.

Example 1 Female NOD mice were treated with HELIXOR M extract at different times during the development of the inflammatory process (insulitis) that leads to islet beta cell damage and diabetes. In the first group of mice (n=20), treatment was begun at 3 weeks of age, before the onset of insulitis, and was continued for 15 weeks until the mice were 18 weeks of age. These mice received an amount of Helixor M extract corresponding to 0. 5 mg plant tissue extracted, intraperitoneally (ip) twice a week. An age-matched control group

of female NOD mice (n=20) was treated with the vehicle (PBS) according to the same schedule. Mice were screened twice weekly for the presence of hyperglycemia (>11. 1 mmol/L) starting at age 8 weeks. Diabetes was diagnosed when mice were hyperglycemic for two consecutive readings.

Mice were sacrificed at 30 weeks of age, at which time the incidence of diabetes in the control group was 90%. The NOD mice treated with the HELIXOR M extract were significantly protected from diabetes, as 95% of mice remained non-diabetic at 30 weeks of age. The results are shown in Figure 1.

Example 2 A second group of female NOD mice (n=10) of >12 weeks of age (time of onset of destructive insulitis) was treated ip with the same dose (corresponding to 0. 5 mg plant tissue) of HELIXOR M extract twice weekly for the same period of time (15 weeks). Controls received PBS. Mice were screened as described in Example 1. This treatment resulted in a lower amount of protection, as 40% of the mice treated with extract were diabetic at 30 weeks of age, compared to an incidence of 80% diabetes in the vehicle- treated control group of mice (Fig. 2).

The experiment was repeated on a new group of mice with administration of twice the dose of extract (corresponding to 1 mg plant tissue) ip twice weekly per mouse, from age 18 weeks to 25 weeks. Mice were screened for diabetes as described in Example 1. It was found that mice as old as 18 weeks of age or older at the start of treatment (stage of imminent diabetes) were protected from diabetes (Fig. 3). 90% of the mice treated with the extract were non-diabetic at 44 weeks of age whereas 100% of the control group were diabetic by that time.

Example 3 As shown in Figure 4, the effect of the mistletoe extract in female NOD mice was to significantly lower the proportion of islets affected by invasive and destructive insulitis (from ? % to ? %). All of the mice appeared healthy at the

time of euthanasia and no toxicity or abnormal histology was observed in the organs of mice treated with mistletoe extract.

The severity of insulitis was examined by standard histological techniques in NOD mice treated with Helixor M or PBS from 3 to 18 weeks of age and sacrificed at 25 weeks of age."Invasive insulitis"means lymphocytic infiltrate penetrating islet capsules with reduction of insulin staining."Non- invasive insulitis"means peri-vascular or peri-islet lymphocytic infiltration with normal insulin staining.

The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

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4. Stirpe et al., (1982), J. Biol. Chem., v. 257, pp. 13271-13277.

5."Mistletoe", in Herbal Medicines : A Guide for Health-Care Professionals ; Eds. Newall, C. A. et al., (1996), Pharmaceutical Press, London, U. K., at pp. 193-196.

6. Bussing, A., (1997), Anti-Cancer Drugs (Suppl.), 8 : S65.

7. Bussing, A. et al., (1996), Cancer Letters, v. 95, pp. 59-72.

8. Bussing, A., (1996), Apoptosis, v. 1, pp. 25-32.

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10. Zarkovic, N. et al., (1997), Anti-Cancer Drugs (Suppl.), 8 : S17-S22.

11. Kuttan, G. et al., (1988), Cancer Lett., v. 41, pp. 307-314.

12. Gray, A. M. et al. (1999), J. Endocrinol., v. 160, pp. 409-414.

13. Obatomi, D. K. et al., (1997), Med. Sci. Res., v. 25, pp. 651-654.

14. Delovitch, T. L. et al., (1997), Immunity, v. 7, pp. 727-738.

15. Tisch, R. et al., (1996), Cell, v. 85, pp. 291-297.

16. See, for example, Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA, U. S. A., 1985).

17. Verge et al., (1998), Diabetes, v. 47, pp. 1857-1866.

18. Gottlieb et al., (1998), Ann. Rev. Med., v. 49, pp. 391-405.