AU9514701A - Colostrinin, and uses thereof - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Ludwick Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences and Georgiades Biotech Limited Actual Inventor(s): Marin Janusz, Jozef Lisowski, Anna Dubowska-lnglot Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: COLOSTRININ, AND USES THEREOF Our Ref: 657783 POF Code: 1541/358588, 358596 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- COLOSTRININ. AND USES THEREOF The present application is a divisional application from Australian patent application number 45651/97, the entire disclosure of which is incorporated herein by reference.

The present invention relates to Colostrinin, and to its use as a medicament.

Colostrum is the thick, yellowish fluid produced by a mammalian mother's breasts during the first few days after childbirth. It is replaced by mature breast milk about four to five days after birth. Compared with mature breast milk, colostrum contains low sugar. However, colostrum is richer in lipids. protein, mineral salts, vitamins and immuno-globulin. It also contains various floating cells such as granular and stromal cells, neutrophils.

monocyte/macrophages and lymphocytes and includes growth factors, hormones and cytokines.

Various factors have been isolated and characterised from mammalian colostrum. In 1974, Janusz et al (FEBS Lett., 49, 276-279) .isolated a proline-rich polypeptide (PRP) from ovine colostrum. The contents of this reference are incorporated herein by reference. It has since been o discovered that mammals other than sheep have analogues of PRP as a component of their colostrum. PRP has since been called Colostrinin and is tentatively identified as a new class of cytokine.

Janusz et al in "Proline-Rich Polypeptide (PRP) an Immunorrodulatory Peptide from Ovine Colostrum" (Archivum Immunologiae et Therapiae Experimentalis, 1993, 41, 275-279) mentioned that PRP from ovine colostrum has immunotropic activity in mice. However, there was no suggestion in this document that the PRP would have any therapeutic effect on any other animal. It will be appreciated that the fact that a composition has a therapeut.c effect on mice cannot be taken to suggest that there will be a therapeutic effect on any other animal.

We have now-found that Colostrinin has a number of previously unknown therapeutic effects. More particularly, we have found that Colostrinin provides an immunotropic action and provides a psychotropic WO 98/14473 PCT/GB97/02721 -2action.

According to one aspect of the invention we provide Colostrinin for use as a medicament. The Colostrinin may be used as a medicament for non-rodent mammals; we have found that Colostrinin is especially useful as a medicament for the treatment of humans.

According to another aspect of the invention there is provided the use of Colostrinin in the manufacture of a medicament for treating disorders of the central nervous system or disorders of the immune system.

In one advantageous embodiment of the invention, the Colostrinin is for use in the treatment of disorders of the central nervous system, particularly chronic disorders of the central nervous system. The disorders of the central nervous system that may be treated with Colostrinin include neurological disorders and mental disorders.

Examples of neurological disorders that can, with advantage, be treated by Colostrinin include dementia, and also disorders that cause dementia, such as neurodegenerative disorders. Neurodegenerative disorders include, for example, senile dementia and motor neurone disease; Parkinson's disease is an example of a motor neurone disease that can be treated with Colostrinin. Colostrinin has been found particularly effective in the treatment 20 of the neurodegenerative disease known as Alzheimer's disease.

Examples of mental disorders that can, with advantage, be treated by Colostrinin include psychosis and neurosis. For example, the Colostrinin may be used to treat emotional disturbances, especially the emotional Sdisturbances of psychiatric patients in a state of depression the use of 25 Colostrin-n has been found to help the patient with an improvement in feelings of wellbe.ng and with mood stabilisation. The Colostrinin may also be used as an auxiliary withdrawal treatment for drug addicts, after a period of detoxificEtion, and in persons dependent on stimulants.

In another advantageous embodiment of the invention, the Colostrinin is for use in the treatment of disorders of the immune system, particularly chronic disorders of the immune system. Thus, we have found that Colostrinin can be used in the treatment of disease requiring immunomodulation. In particular, we have found that Colostrinin is useful for treating such disorders in non-rodent animals, including humans. Colostrinin is useful in the treatment of a variety of diseases with an immunological and infectious basis. For example, the Colostrinin can be used to treat chronic diseases with a bacterial and viral aetiology, and to treat acquired immunological deficiencies that have developed, for example, after chemotherapy or radiotherapy of neoplasms. The invention is particularly useful for treating chronic bacterial and viral infections requiring non-specific immunostimulation and immunocorrection.

In general, a chronic disorder is a disorder that has persisted for a long time, usually at least one week, more usually at least one month, and often at least 3 months or at least 6 months.

S" 15 It is a feature of the present invention to use Colostrinin for improving the development of the-immune system of a new born child. It is a further feature of the invention to use Colostrinin to correct immunological deficiencies in a child. These uses of the Colostrinin may be particularly applicable to babies or children who have been deprived of colostrum. This 20 may occur, for example, in babies and children who were not breast fed from birth; the artificial feed that such babies and children would have been given does not contain Colostrinin.

According to another aspect of the invention, we provide the use of Colostrinin as a dietary supplement. Colostrinin is particularly 25 advantageously used as a dietary supplement for babies and young children to correct deficiencies in the development of their immune system. As noted above, such deficiencies would arise in babies and children who had not been breast fed from birth. The Colostrinin may also be used as a dietary supplement for adults who have been subjected to chemotherapy, or have suffered from cahexia, or weight loss due to chronic disease. In an aspect of the invention, we provide a dietary supplement comprising an orally ingestible combination of Colostrinin in combination with a physiologically acceptable carrier. The dietary supplement may be provided in liquid or solid form; the dietary supplement may suitably be provided in the form of a tablet.

In accordance with the invention, the Colostrinin may be administered prophylactically in order to help to prevent the development of disorders of the central nervous system and the immune system.

The Colostrinin used in the aspects of the invention described above may be ovine Colostrinin, or it may be non-ovine Colostrinin. Nonovine Colostrinin may be derived from the colostrum of, for example, humans, cows, horses, goats, pigs, yaks, llamas and asses. The colostrum will normally be present in the beestings of these animals for 1 to 4 days after parturition.

The term "Colostrinin", as used herein refers to a polypeptide which, in its natural form, is obtained from mammalian colostrum.

Colostrinin is sometimes known as "colostrinine", and has the following properties: it has a molecular weight in the range 16,000 to 26,000 Daltons; (ii) it is a dimer or trimer of sub-units each sub-unit having a 20 molecular weight in the range 5,000 to 10,000 Daltons, preferably 6,000 Daltons; :(iii) it contains proline, and the amount of proline is greater than the amount of any other single amino acid.

We have also found that the Colostrinin, and also the sub-units 25 making up the Colostrinin, are non-polar.

The molecular weight can be determined by electrophoresis in the presence of SDS; the presence of the dimer or trimer can be shown by the same technique. It can be shown that the bonds between the sub-units are noncovalent by electrophoresis in reduced and non-reduced conditions. The presence of proline can be established by conventional amino acid analysis.

The non-poiarity can be demonstrated by chromatography in non-polar conditions.

The Colostrinin used in the present invention may be ovine- Colostrinin or non-ovine Colostrinin. Ovine Colostrinin has a molecular weight of about 18,000 Daltons, is made up of three non-covalently linked sub-units each having a molecular weight of' about 6,000 lDaltons and includes about 22 wtX proline. The amino-acid composition is made up of the following number of residues per sub-unit: lysine 2, histidine 1, arginine 0, aspartic acid 2, threonine 4, serine 3, glutamic acid 6, proline 11, glycine 2. alanine 0, valine 5. methioriine 2, isoleucine 2. leucine 6, tyrosine 1, phenylalanine -3 and cysteint 0.

As noted above, in its natural form Colostrinin is derived from miammaliarn colostrum. Colostrinin can be derived from mammalian colostrum by removing the lipids and the majority of the proteins from the colostrum.

15 Broadly, C'o]ostrinin can be obtained from the beestings of, for example, large farrn animals using column chromatography techniques and other biochemical 0 o~etechniques, or can be obtained by genetic engineering techniques.

oo*o More particularly, Colostrinin may be isolated from mammalian colostrum. by using the following steps: 0 20 Removing lipids, for example by centrifugation; (iij loving proteins such as casein, for example, by 00006:loweringpH; separatin'g Colostrinin bound to irmutnoglobulin, for 250 example by: 0 25(a) Processing the whey formed after the removal of lipids and proteins by ion exchangce chr~omatography; and Eluting with phosphate buffered saline and collecting a fraction containing Colostrinin bound to iranunoglobulin, for example IgG2 in sheep; (iv) Separating Colostrinin from the immunoglobulin, for example by sieving chromatography; and Further purifying the Colostrinin, preferably by: De-salting the fraction below 30,000 Daltons molecular weight; and Introducing antibodies to immunoglobulins and thereby remove this class of proteins to obtain the final product.

Whilst the above definition relates to naturally occurring mammalian Colostrinin, the term Colostrinin as herein used also includes analogues and fragments thereof having substantially the same biological activity, and mammalian Colostrinin, analogues thereof and fragments thereof produced by recombinant DNA technology. Colostrinin as used herein also includes biologically active polypeptides of substantially the same 15 composition as natural Colostrinin, which have been made by polypeptide synthesis.

In a further aspect of the present invention there is provided a method of treating disorders of the central nervous system or of the immune system using Colostrinin. The disorders that can, with advantage, be treated using the method according to the invention are described above. In a preferred embodiment the Colostrinin is administered to a patient for a first period at about 1 to 2 therapeutic units daily, followed by a second period when no Colostrinin is administered. The first period is preferably about 2 to 4 weeks, more preferably about 3 weeks; and the second period is preferably 25 about 2 to 5 weeks, more preferably about 4 weeks. This cycle is preferably repeated at least once, and is more preferably repeated more than once.

The therapeutic unit for use in methods of the invention is preferably in the range 25:to 1,000 micrograms of Colostrinin, most preferably to 100 micrograms.

The Colostrinin may be formulated for administration in any -7suitable form. For example, is may be formulated for oral, rectal or parenteral administration, More specifically, the Colostrinin may be formulated for administration by injection, or, preferably, in a form suitable for absorption through the mucosa of the oral/nasopharyngeal cavity, from the alimentary canal or any other mucosal surface. The oral formulations may be provided in a form for swallowing; or, preferably, in a form for dissolving in the saliva, whereby the formulation can be absorbed in the mucous membranes of the oral/nasopharyngeal cavity. The oral formulations may be in the form of a tablet for oral administration, lozenges a sweet-like tablet in a form suitable to be retained in the mouth and sucked), adhesive gels for rubbing into the gum. The Colostrinin may be formulated as an adhesive plaster or patch, which may be applied to the gums. The Colostrinin may also be formulated for application to mucous-membranes of the genito-urinary organs.

Whilst it would, of course, be possible to administer the 15 Colostrinin in the form of whole colostrum, this is not preferred, because whole colostrum has an unpleasant taste, and is difficult to store.

Colostrinin for use in the present invention may be obtained from any mammal, including human sources or animals such as cows, horses, goats, pigs, yaks, sheep, llamas or asses, camels etc.

20 Tests on Colostrinin were performed using ovine and human Colostrinin. Ovine Colostrinin is marketed under the trade mark ColostrininTM During tests on experimental animals it was found that Colostrinin is characterized by immunotropic action, both in vivo and in vitro, 25 based on the properties of modulation, development of differentiation and maturation, of thymocytes to active T cells and on the stimulation or inhibition of an immunological response, and on induction of the expression of various surface markers on the thymocytes. In intraperitoneal administration in mice, Colostrinin inhibits the development of haemolytic anaemia in mice of the NZB line, inhibit the growth of sarcoma 180 in mice, and in mice exposed to gamma radiation it protects the animals against radiation sickness.

Toxicological studies on mice showed, both after oral-and parenteral administration, a very low toxicity, as LD50 is above 1.25 g/kg of body weight. Colostrinin also exhibits capacity to stimulate the growth, maturation and differentiation of immunologically active cells both in humans and in experimertal animals. In cultures of lymphocytes of human peripheral blood (including cultures of lymphocytes isolated from the cord blood) Colostrinin is characterized in that it stimulates the production of cytokines, especially gamma interferon (IFN-y), tumour necrosis factor (TNF-a), interleukins (e.g.

IL-6 and IL-10) and various growth factors. The cytokines produced are determined quantitatively by known methods.

In natural conditions analogues of ovine Colostrinin but possessing the biochemical properties thereof are present in human colostrum and in the beestings of all mammals, and especially large farm animals such as 15 cows, horses, goats, pigs, yaks, sheep, llamas or asses, camels etc. for 1-4 days after parturition. The Colostrinin enters the body of the newborn animals during sucking and swallowing of its mother's colostrum. Owing to the low molecular weight it is possible for Colostrinin to act on the mucosa of the oral/nasopharyngeal cavity via cell receptors, and even as a result of ordinary 20 diffusion. Because the mucosa and epithelium of the upper part of the digestive tract contain receptors for certain immunomodulators, it has been shown in investigations that the Colostrinin can be administered in the form of tablets and sublingual tablets for sucking, tablets and capsules for swallowing, in the form of adhesive gels, and in the form of adhesive plasters for fastening 25 to the gums. Colostrinin can also be applied to the mucous membranes of genito-urinary organs.

Scientific studies aiming to elucidate the biochemical activity induced by Colostrinin also revealed the existence of similar biological activity when using human colostrum, collected from women in the period of 1-7 days after parturition. The method used for isolating Colostrinin of human PCTIGB97/02721 -9origin was analogous to the methods of obtaining it from the beestings of farm animals, and especially from sheep beestings. It was found that the colostrum secreted by the mammary gland of women from 1-7 days after parturition contains the polypeptide Colostrinin, the amount of which depends on lactation and is optimum between 2-3 days, when about 300 mg of it is detected in 1 litre of colostrum. This quantity of human Colostrinin is within the range seen in ovine colostrum. It was demonstrated that human Colostrinin has many biological properties similar to, for example, the sheep analogue. It was found that Colostrinin originating from human colostrum stimulates the lymphocytes present in the colostrum, and cord blood lymphocytes, to secrete cytokines, including mainly interferon (IFN-y), tumour necrosis factor TNF-e, interleukins (IL-10 and IL-6) and other cytokines. These cytokines are involved in humoral and cellular immune reactions.

These biological activities of the Colostrinin both of human and 15 of animal origin were determined by the method known from a Polish patent (PL 170592 B and from European patent no. EP-B-0609225, pt.: Testing Immunology.

In studies on human volunteers it was shown that after administration of tablets for sucking, containing at least 25 pg of Colostrinin S 20 at doses of at least one daily, for a period of about 3 weeks, no adverse reactions were observed; on the other hand it brings about a state of hyporeactivity to induction of interferon IFN-y and partially also of tumour necrosis factor TNF-c. After discontinuing administration of the Colostrinin, the state of hyporeactivity returns to normal. The reactions obtained are the 25 result of involvement of cytokines produced by Thl and Th2 lymphocytes and by helper cells.

During clinical studies it was shown in addition that Colostrinin has an immunomodulatory effect and an effect on the central nervous system, which can be utilized prophylactically and therapeutically in the following diseases, e.g. for preventing occurrence of, progression of and for remission of Alzheimer's disease, in senile dementia of some other origin and in the treatment of chronic diseases with an immunologic and infectious basis.

Instead of Colostrinin, it is possible to use a nonapeptide designated NP having the following composition and amino acid sequence: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro. NP can be obtained from Colostrinin by digesting with chymotrypsin and isolation by column chromatography, or by means of chemical synthesis. NP is an example of a useful fragment of Colostrinin. It has been found to have similar biological effects to Colostrinin, and, in particular, is useful for treating the chronic immune and central nervous system disorders described above. es-ialy Alzheimer's disease. NP may be used to treat any of disord" -d above. It may also be used in a dietary supplement. I' .a in the same way as Colostrinin. NP is useful for ,nals, especially humans.

In oraei me invention may be more fully understood, reference will now be made to the accompanying Examples by way of illustration only.

20 Example I In ovine Colostrinin with molecular weight of 18 000 Daltons, constructed from three non-covalently linked subunits with molecular weight of 6 000 Daltons, each sub-unit had the following amino-acid composition: I I-

S

*5 S S

S

S S S.

*SS.

Amino Acid No. of residues per subunit Lysine 2 Histidine 1 Arg inine0 Aspartic acid Threonine4 [Serine I'l Glutarni: acid 16 Pro line I I1 SlV cine Al anine 0 Valine is Methionine 2) Isoleucine 6 Tyrosine Ph envl a.an in 3 Cyste ine I0 The Colostrinin was obtained from sheep beestings, taken after parturition up to 24 hours after commencement of lactation. The material was centrifuged at 35 000 x g in order to remove fats and part of the casein. The Ig fraction obtained was applied to a DEAE-Cellulose column, in order to isolate 25 irnmunoglobulin LgG2 cornplexed with the polypeptide Colostrinin, and upon elution fromn the column the Colostrinin was separated from the lgG2_ by means of column chromatography on Sephadex 0- 100 and was chromatographed again on Sephadex G-75. Then a fraction of this polypeptide was applied to a column of Sepharose conjugated with anti-bodies 55 S S

S.

S. S S S against sheep Ig02 to remove traces Of IgG2 contamination. The preparation obtained was desalinated by means of Sephadex G-25 and lyophilized, after which it was stored at a temperature of W4C or The Colostrinin thus obtained has been designated by the trade mark Colcstrinir.rtM.

In polyacrylamide gel electrophoresis in the presence of SDS. a strong band was observed, corresponding to molecular weight of 5 800 Daltons, and two weak bands corresponding to molecular weight of 12 400 Daltonis and 18 200 Daltons.

Examole II The noriapeptide NP having the compositioni and amino acid sequence V al- Glu-S er-Tyr-Va1- Pro -Leu-Ph e-Pro was obtained from the Colostriflin made in Example 1.

50 rng of the Colostrinin is digested by means of 10 activity units of the pro-,eolytic enzyme chymotrypsin, for 20 hours at a temperature of Isol.ation from the product of digestion was carried out by means of at least one cycle of column chromatography using Sephadex G-10. The preparaticn obtained was lyophilised, and was then stored at a temperature of 20 +I4'C or -20*C. The isolated nonapeptide wvas isolated by means of determination of the N-terminal amino acid.

Example III Dosage unit in the form of a tablet for sucking, with the 00:~ 25 composition- Active ins,,redient: Colostrinin® polypeptide 0.0001 g obtained according to Example I Stabilizer- Albumin, free from 0.000135 g i !mpurities. mainly I.P.S.

-13- Lubricant/binder: Magnesium stearate 0.003 g Carrier: Mannitol 0.15 g (0.1497 g) The above components were suspended in 0.0001 M NaCI.

Tablets for sucking are primarily intended for treating early and lare stages of dementia, including Alzheimer's disease, and various stages of senile dementia, for treating chronic bacterial and viral infections, requiring nonspecific immuncstimulation and immunocorrection and acquired immunologic deficiencies caused by various agents. In addition it is used in emotional disturbances especially in states of depression in psychiatric patients to improve the general feeling of well being and for mood stabilization, and in auxiliary withdrawal treatment of drug addicts, after a period of detoxification and in persons dependent on stimulants. ColostrininTM can also be used in the 15 newborn and in young children for correcting nutritional deficiencies connected with artificial feeding, Example IV Preparation in the form of gel for application to mucous 20 membranes with the composition: Active ingredient: Colostrinin T M polypeptide obtained according to Example I 25 Stabilizer Albumin Gel carrier: Orabase-Plain® containing pectin, gelatin, sodium salt of carboxymethylcellulose and hydrocarbon gel 0.0003 g of the polypeptide Colostrinin

M

and 0.0015 g of -14albumin were used per 1 ml of gel carrier, The dosage unit thus formulated is primarily intended for cyclic treatment of bacterial and viral infections of the oral cavity and upper respiratory tract.

Example V Preparation intravenous injections.

Active ingredient: in the form of intramuscular, subcutaneous or ColostrininT M polypeptide obtained according to Example I Stabilizer: Carrier: Human albumin, free from impurities Sterile, none pyrogenic water 9* 0.0003 g of ColostrininTM polypeptide, 0.0015 g of albumin and, as antibacterial agent, 0.00:1% of Merthiolate, i.e. sodium salt of ethylmercurithiosalicylic acid, were used per 1 ml ampoule. The dosage unit thus obtained is primarily intended for cyclic treatment of bacterial and viral 20 infections.

Example VI Preparation inlthe form of intramuscular, subcutaneous or intravenous injections.

Active ingredient: Stabilizer: Carrier: NP obtained according to Example 11 fIuman albumin, free from impurities Sterile, none pyrogenic water Sterile, none pyrogenic water 0.0003 g of NP, 0.0015 g of albumin and, as antibacterial agent, 0.001% of Merthiolate, i.e. sodium salt of ethylmercurithiosalicylic acid, were used per 1 ml ampoule. The dosage unit thus obtained is primarily intended for cyclic treatment of bacterial and viral infections.

Example VII Induction. of cytokines by the polypeptide Colostrinin

T

M in vitro on blood taken from healthy and sick volunteers taking the pharmaceutical in the form stated in Example III, in cyclic treatment, is carried out as follows.

Whole blood, containing 10 units per 1 ml of heparin without preservatives is diluted at 1:10 ratio in RPMI 1640 culture medium.

Incubation is conducted in the same culture medium without inducers (negative control), or in the presence of 1-100 ig/ml of the polypeptide Colostrinin T M or in the presence of 2 ug/ml blood of phytohaemagglutinin 15 (PHA) and 2 gg/ml blood of lipopolysaccharide (LPS), at a temperature of 37°C, in an atmosphere of 5% carbon dioxide, for 20 hours. Samples with mixture of PHA and LPS are the positive control, as they can stimulate the maximum quantities of cytokines. The test results are presented in the Tables 1 and 2. They show that ColostrininTM at concentrations of 1-100 tg/ml 20 stimulates the production of cytokines in a dose-dependent fashion. Relative S"to the negative control (without inducers) these results are statistically significant (p 0.0001). Patients with Alzheimer's disease exhibit diminished capacity for production of IFN and to a lesser extent also TNF.

Table I Induction of cytokines by ColostrininTM1 (series A 1993) from sheep in cultures of lymphocytes present in whole blood of healthy volunteers or of patients with Alzheimer's disease.

Blood Inducers I Dose Cytokines (unit/mi SD) donors (Ag/mi) IFN TNF Colostrinin~m 100 344 ±254 670 =b560 Healthy toOtinfT 1050.!:59 316 -:371 voluntee-?s PHA LPS 2 -2 171 ±i162 521 :±447 Control 9 921 19± 26 Colostrinin~' 100 21 ±14 249 :t 187 Patients 'with Colo strinin TM 10 15 9 182 ±158 Alzheimer's disease PHA +LPS 2+2 117 ±76 397 252 Control 2 ±3 18 26 The group of healthy volunteers (22 people) was heterogeneous 20 (age range 20-64 years). The group of patients with Alzheimner's disease people) was also heterogeneous (age 63 7.5 years). Despite the high standard tion SD) the differences between the controls without induccrs -id ColostrininTM1 were statistically significant (p 0.00 1).

S

Table 2 Examples of stimulation of the production of interferon (IFN) by ColostrininTN( (series A 1993) from sheep in experiments using whole blood of healthy volunteers and patients with various diseases.

Blood donors Induce,- Dose (pglrrl) Titres of IFN determined by (diagnosis)------ Antivirus ELISA IFN-y biotest Pg/mI units/mi Colostrinin-,Ii 100 300 2920 Z.B. healthy Colostrinin--11 10 300 3402 young Colcstrin-inTM 1 100 1413 soldier PHA LPS 2 +2 200 3308 Control <324 Co~cstrininT11l 100. 600 3941 C.O. healthy ColostrininTM 10 200 3778 young Colostrininrm 1100 29 soldier PHA LI'S 2 +2 600 4631 Control 5 M. J. ColostrininThi 100 40 400 Alzheimer's Colostrinin~m 10 20 427 disease PHA LI'S 2 +2 300 3757 Control 3 46 S.E. Colostrinin'rh" 100 6 29 schizo- Colostrinin~m 10 6 29 Phrenia PHA LI'S 2 +2 30 243 Control -3 19

F.W,

breast cancer Colostrinjn Thl Colostrinin

T

M

PHA +LPS Control 100 10 2+2 523 307 2833 150 NOTE: the biotest for presence of IFN measures the levels of various types of interferons. whereas the ELISA test only determines the level of immunoactive IFN-y.

Example V111 Cord blood obtained from the Gynaecological-Obstetric Department of the Specialist District Hospital in Wroclaw. Induction of cytoki nes by ColostrininTM in vitro in lymphocytes of cord blood taken 4-6 hour-s after parturition is effected In the following way.

The lymphocytes are isolated using a Ficoll-Paque® gradient containing .5.7 g of the component Ficoll 400 and 9.0 g of the component Diatrizoate Sodium per 100 ml. The lymphocytes isolated are suspended in RPMI-1640 culture medium at density of 2 x 106 lymphocyte cells per 1 ml of culture medium. ColostrininMN at concentration of 1-20 jig/ml or phytohaernagglutinin at concentration of 10 jig/ml is added to the lymphocyte suspension. The culture is incubated for 20 hours at a temperature of 3)7*C.

Then the level of cytokines in the culture fluids is determined by biological methods. A typical example is given in Table ColostrininT," at concentration of 1-100 pig/ml has capacity for stimulation of cytokines (lFN and TNF) similar to that exhibited by the classical IFN-y inducer phytobaemagglutinin.

-19a a Table 3 Induction of cytokines by ColostrininTNI (series A 1996) in a culture of lymphocytes isolated from cord blood (CBL).

N Inducer Dose (pg/nil) Cytokines (unit/rn! SD) IFN TNF 32ColestrinT 20 89 79 59--41 39 ColostrininTM 10 78 80 3 7 i7ColostrininTN' 1 3 8 ±66 16--617 PHA 10 75 66 S3 -_69 control 3 3 4 -N number of CBL samples investigated 25 In the StudEnt t test the probability of significance of the difference Coos trinin TM1 "absent", both for IFN and TNF, is p 0.0001.

Because the cord blood is rich in immature stem cells, which are capable of reproduction of haemopoietic cells arid of various immunologically-active cells, the result obtained shows that ColostrininTM greatly accelerates the maturation of stem cells. The results obtained show that it is possible to use ColostrininT1! for treating various types of inrnune deficiencies and for stimulating the haemopoietic system, e.g. after injuries, infections, ;hemoherapy and radiotherapy. In biomedical studies, substances of natural origin with similar action are very seldom encountered.

Example IX The method of treatment of disorders of the central nervous a.

a a system was investigated on a group of volunteer patients in the early and moderate stages of Alzheimer's disease. The dosage units were administered in the form of tablets for sucking, between meals, containing 0.00015 g of Colostrinin T defined in Examples I and Ill. Firstly, I tablet was used daily for a period of 3 weeks, then therapy was interrupted for 2-4 weeks and treatment was repeated, administering 2 tablets daily for 3 weeks. It was found that ColostrininT

M

treatment induced a state ofhyporeactivity or tolerance. This is manifested by an inability to synthesise IFN and also tumour ne:rosis factor TNF-a. This phenomenon permits quantitative measurements of the action of active agent.

After cessation of administration of these drugs the state of tolerance reverses spontaneously. The state of temporary tolerance to the ColostrininT" is a result of the involvement of cytokines produced by Thi and Th2 lymphocytes and helper cells such as monocytes, macrophages, dendritic 15 cells, and endothelial cells and their products. As a result, improvement of contact and uplift of mood were observed in patients with Alzheimer's disease.

Fig. 1 illustrates the appearance and spontaneous disappearance of a state of hyporeactivity (partial tolerance) to induction of gamma interferon (IFN-y) in a female patient with Alzhcimer's disease, who 20 received Colostrinin T M in 100-p.g tablets every other day for three weeks. This was followed by a 3-week pause in treatment (during the pause, the tolerance to the inducer returns to normal). Blood samples for investigating stimulation of IFN-y by ColostrininTM and control inducers (PHA 10 gg/ml) were taken every week. The method of performing the tests for induction of cytokines and their cruantitative determination were described in previous sections.

The results of determinations of levels of induced IFN-y showed that hyporeactivity appears as early as during the first week of taking ColostrininT

M

L (100 jg/tablet every other day) and is maximum in the third week of treatment. Reversal of the state of tolerance to induction of IFN-y occurred spontaneously in a period of 3 weeks of a pause in treatment in -21the sixth week after commencement of treatment). Moreover, this chart shows that hyporeactivity that is "specific" with respect to sheep Colostrinin (OvCal) is still maintained in the sixth week of observation, whereas hyporeactivity to PHA had disappeared completely.

aml.

The method of treatment of disorders of the central nervous system was investigated on a group of volunteer patients in the early stages of Alzheimer's disease. The dosage units wcre'administered in the form of tablets for sucking, between meals, containing 0.00015 g of NP defined in Example II. Firstly. 1 tablet was used daily for a period of 3 weeks, then therapy was interrupted for 3 weeks and treatment was repeated, administering 2 tablets daily for 3 weeks. It was found that NP treatment induced a state of .o hyporeactivity or tolerance. This is manifested by an inability to synthesise 15 IFN and also tumour necrosis factor TNF-a. This phenomenon permits quantitative measurements of the action of active agent.

After cessation of administration of these drugs the state of tolerance reverses spontaneously. The state of temporary tolerance to the NP Sis a result of the involvement of cytokines produced by Th and Th2 20 lymphocytes and helper cells such as monocytes, macrophages, dendritic cells, and endothelial cells and their products. As a result, improvement of contact and uplift of mood were observed in patients with AIzheimer's disease.

o*

Claims (38)

1. Colostrinin when used as a medicament for humans.

2. Colostrinin when used in the treatment of chronic disorders of the central nervous system.

3. Colostrinin according to claim 2, when used in the treatment of neurological disorders and/or mental disorders.

4. Colostrinin according to claim 2, when used in the treatment of dementia and/or neurodegenerative diseases.

5. Colostrinin according to claim 2, when used in the treatment of 15 Alzheimer's disease and/or motor neurone disease.

6. Colostrinin according to claim 2, when used in the treatment of psychosis and/or neurosis. 20 7. Colostrinin when used in the treatment of chronic disorders of the immune system in humans.

8. Colostrinin according to claim 7, when used in the treatment of diseases with a bacterial and viral aetiology, and/or for use in the treatment of 25 acquired immunological deficiencies.

9. Colostrinin according to claim 7 or 8 when used in the treatment of chronic bacterial and/or viral infections.

10. Colostrinin according to any preceding claim, wherein said Colostrinin is derived from a non-ovine source.

11. The use of Colostrinin in the manufacture of a medicament for the treatment of chronic disorders of the central nervous system. W:~niceasop%45851.doc 23

12. The use according to claim 11, for the treatment of neurological disorders and/or mental disorders.

13. The use according to claim 12, for the treatment of dementia and/or neurodegenerative diseases.

14. The use according to claim 12, for the treatment of for use of Alzheimer's disease and/or motor neurone disease. The use according to claim 12, for the treatment of psychosis and/or neurosis.

16. The use of Colostrinin in the manufacture of a medicament for the 15 treatment of chronic disorders of the immune system in humans.

17. The use according to claim 16, for the treatment of diseases with a bacterial and viral aetiology, and/or for use in the treatment of acquired immunological deficiencies.

18. The use according to claim 16 or 17, for the treatment of bacterial and/or viral infections.

19. The use according to any one of claims 16 to 18, wherein said 25 Colostrinin is non-ovine Colostrinin. A method of treating disorders of the immune system comprising administering a predetermined amount of a composition containing Colostrinin to a patient for a predetermined period of time.

21. A method of treating disorders of the central nervous system comprising administering a predetermined amount of a composition containing Colostrinin to a patient for a predetermined period of time. W:anice\spec\45651.doc

22. A method according to claim 20 or claim 21, wherein the patient is a human patient.

23. A method according to any one of claims 20 to 22, wherein the Colostrinin is non-ovine Colostrinin.

24. A method according to any one of claims 20 to 23, comprising wherein said predetermined amount of Colostrinin is in the range of about 25 to 2000 micrograms. A method according to claim 24, comprising a cycle of administering 25 to 2000 micrograms of Colostrinin each day to a patient for a first period, followed by a second period when no Colostrinin is administered.

26. A method according to claim 25, wherein the first period is in the Srange of about 2 to 4 weeks, and the second period is in the range of about 2 to 5 weeks. a

27. A method according to claim 25 or 26, wherein the cycle is 20 repeated at least once.

28. A pharmaceutical composition in a form suitable for oral administration comprising a preselected amount of Colostrinin in combination with a physiologically acceptable carrier.

29. A composition according to claim 28, wherein the Colostrinin is non-ovine Colostrinin A composition according to claim 28 or 29, in a form suitable for absorption through the mucosa of the oral/nasopharyngeal cavity and/or in a form suitable for absorption in the alimentary canal.

31. A composition according to any one of claims 28 to 30, in the form of a tablet, lozenge, gel, patch or plaster. w:\ianiceru pe45651.doc 25

32. A composition according to any one of claims 28 to 31, comprising to 1000 micrograms of Colostrinin.

33. A composition according to any one of claims 28 to 32, wherein the Colostrinin is provided in isolated form.

34. A composition according to any one of claims 28 to 31 comprising to 100 micrograms of Colostrinin. The use of Colostrinin in isolated form as a dietary supplement.

36. The use of Colostrinin in isolated form as a dietary supplement for babies, small children, adults who have been subjected to chemotherapy and/or 15 adults who have suffered from cahexia, or weight loss due to chronic disease.

37. A dietary supplement comprising an orally ingestible combination of Colostrinin in combination with a physiologically acceptable carrier. 20 38. Colostrinin for use in the stimulation and/or modulation of the immune system of mammals.

39. Colostrinin for use in the stimulation and/or modulation of the immune system of humans. **25 The use of Colostrinin in the manufacture of a medicament for use in the stimulation and/or modulation of the immune system of mammals.

41. The use of Colostrinin in the manufacture of a medicament for use in the stimulation and/or modulation of the immune system of humans.

42. Colostrinin for use as a prophylactic medicament. w:'jnices pecus551.doc 26

43. Colostrinin for use as a prophylactic medicament for humans, to prevent or inhibit the development of Alzheimer's disease.

44. The use of Colostrinin in the manufacture of a prophylactic medicament for humans, to prevent or inhibit the development of Alzheimer's disease. The use of a nanopeptide having the composition and amino acid sequence Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro in the manufacture of a medicament for treating chronic disorders of the immune system in humans.

46. The use of a nanopeptide having the composition and amino acid sequence Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro in the manufacture of a medicament for treating chronic disorders of the central nervous system. 0

47. A pharmaceutical composition comprising a nanopeptide having the composition and amino acid sequence Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe- Pro in combination with a physiologically acceptable carrier. 20 48. A method of making a pharmaceutical composition comprising combining a nanopeptide having the composition and amino acid sequence Val- Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro with a physiologically acceptable carrier, and forming said mixture into a form in which it can be administered to a patient. i 25 DATED: 19 February, 2001 PHILLIPS ORMONDE FITZPATRICK Attorneys for: LUDWICK HIRSZFELD INSTITUTE OF IMMUNOLOGY AND EXPERIMENTAL THERAPY POISH ACADEMY OF SCIENCES AND GEORGIADES BIOTECH LIMITED D44d4^ J4J4^ W\janiceaspc045851.doc