WO1998012219A1 - Methionine, penicillamine and cysteine-analogue containing peptides having immunomodulating activity - Google Patents

Abstract

Physiologically active peptides of 8ormula (I): A-R1-R2-R3-(R4)x-B, wherein each variable has the meaning mentioned in the description at least one of R1, R2 and R3 being R8 and at most one of R1, R2 and R3 being cysteine; and R8 is an amino acid residue of formula (II) or formula (III), wherein each variable has the meaning described in the description, the entire peptide sequence containing 3 to 30 amino acid residues; or a homo- or heterodimers thereof. These peptides are absorbable by the epithelial cell lining in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.

Description

METHIONINE. PENICILLAMINE AND CYSTEINE-ANALOGUE CONTAINING PEPTIDES HAVING IMMUNOMODULATING ACTIVITY

Field of Invention

The present invention relates to non-antigen-specific immunomodulation, including both immunosuppression and immunostimulation. In particular, the invention relates to immunomodulatory peptides comprising amino acid residues which contain S, Se orTe, which peptides are capable of inducing an immunomodulatory response in mammals and thereby a therapeutic effect, and uses thereof.

Background of Invention

The immune system, when it is working properly, protects the individual from infection and from growth of cancers. In order to cany out these functions, it must be able to recognise and mount an attack against foreign antigens (including cancer-specific antigens), but not against self antigens present on normal cells throughout the body.

It is possible to stimulate the immune system in order to improve its level of protection. Vaccines, including single-protein antigens such as diptheria toxoid, are widely used to generate immunity against a specific antigen and thus a specific disease. Where general stimulation of the immune system is desired, this can sometimes be achieved with nonspecific agents such as adjuvants, interleukins, interferons, and colony stimulating factors.

Occasionally, the immune system loses its critical ability to distinguish self from non-self. The resulting immunological assault on the individual's own tissues can take the form of autoimmune disease: for example, systemic lupus erythrematosis, Type 1 diabetes, or rheumatoid arthritis. In such a case, or alternatively where the individual is the recipient of a transplanted organ or tissue, suppression rather than stimulation of the immune response is desirable.

Non-specific down-regulation of the immune response is typically achieved by treatment with corticosteroids, azathioprine, cyclosporine, tacrolimus (FK506), rapamycin, or mycophenolate mofetil. Certain immunoglobulins, including the monoclonal antibody OKT3, have also been used for this purpose. Suppression of immunity against a specific antigen, called "tolerance induction", may also be possible. Methods that have been used for inducing tolerance against a particular antigen include intravenous or repeated topical administration of the antigen in dilute form, treatment with a very high dose of the antigen, and oral administration of the antigen..

It has now been found that certain peptides containing amino acid residues containing S, Se and Te have activity as immunomodulators. A surprising property associated with the immunomodulatory activity is that the activity has been found to be immunoinhibitory or immunostimulatory in effect, on the basis of experiments described herein and furthermore, that the immunomodulatory activity has been shown to be indicative of having some therapeutic effect in the treatment of certain diseases, such as cancer and arthritis.

Furthermore, it has been found that when the administration to the epithelial cell lining is by way of oral administration, the administration of the peptides has been observed to correlate with a modulating effect on the growth of tumours.

A further surprising finding is that the oral presentation of "naked" peptides of the invention did not require the inclusion of added transport agents. Thus, the peptides of the invention do not need to be administered in association with transport agents such as delivery vehicles e.g. vesicular delivery systems which are designed to improve delivery to the mucosal epithelial cell lining of the gut. In addition, it has been found that the amount of peptide required to produce the therapeutic effect by oral delivery can be significantly lower than that required to produce a similar effect when the peptide is delivered systemically, eg by parenteral injection.

It is an object of the present invention to provide an effective means for treating disease using immunomodulatory peptides.

It is another object of the invention to provide immunomodulatory peptides which can be utilised in the treatment of disease.

These and other objects of the invention will become apparent from the following description and examples.

Prior Art

WO92/00995 and W094/15958 purport to describe cyclic short-chain peptides useful in modulating cell adhesion. The cyclising moiety may be a disulphide bridge between cysteine residues. Also the possibility of the use of penicillamine (Pen) or homocysteine (Hey) as linking residue is mentioned. In WO92/00995 a cyclised peptide comprising Ser Pro (Pen) NH₂ is mentioned.

WO96/06108 purports to describe cyclic peptides that inhibit the binding between VLA-4 receptor expressed on inflammatory leukocytes and the fibronectin CS-1 peptide expressed on endothelial cells. There is mentioned a cyclised peptide of the formula:

R-Xaa-Z,-Asp-Phe-Y₂-Xaa-NH₂

with at least one Xaa being oxidized cysteine and the other being oxidized cysteine, homocysteine, or penicillamine such that the two Xaa's together form a disulphide bond. Y₂ may be absent, Pro, Pro-Ser or Pro-Ser-Thr. WO96/22106 purports to describe compounds which inhibit CD8 mediated T-cell activation and have a molecular surface that corresponds to the molecular surface of human CD at specified amino acids. Among conformationally restricted peptides described, decapeptides including -R₂₈-R₂₉-R₃₀- wherein R_2g is glutamine or asparagine, R₂₉ is arginine and R₃₀ is cysteine or penicillamine are mentioned.

EP-A-0359338 purports to describe pentapeptides of the formula R^a-R^b-R^c-R^d-(R^e)_n-R^f having good activity in inhibition of stem cell proliferation particularly for use in the treatment of immune diseases. The R grouping may be a substituted cysteine, e.g. benzyl cysteine.

EP-A-0425212 purports to describe cyclic anti-aggregatory short-chain peptides. The cyclising moiety may be a disulphide bridge. The linking bridges are generally formed through cysteine residues but the possibility of penicillamine or 3-phenyl cysteine is mentioned.

EP-A-0535730 purports to describe inhibitors for farnesyl protein transferase. N-(2)-R- amino-3-mercaptopropyl-isoleucyl-isoleucyl-homocysteine lactone is mentioned.

WO92/ 14751 purports to describe peptides active against herpes virus infection of the general formula I-A-B-C-D-E-F-G-H-II in which H may, inter alia, be Met(O) or Cys and II may be Cys, an amide group, a substituted amide group, an ester group or absent. The peptides may be linear or cyclic.

EP-A-0249169 and EP-A-0288965 purport to describe novel peptides. Amongst the peptides mentioned is H-Arg-Pro-Cys(StBu)-Arg-Phe-OtBu. WO96/01318 purports to describe polypeptides, other than human interleukin 10, having specified characteristics and which are interleukin 10 agonists. Certain of the peptides described contain the amino acid sequence Thr Met Lys.

JP-A2-08151396 purports to describe HLA-oligopeptides and immunomodulating agents containing them. Among the peptide sequences identified is sequence 55 comprising Ala Pro Met Phe.

Statement of Invention

In a first aspect of the present invention, there is provided a physiologically active peptide of formula (I):

A-Rι-R₂-R3-(R4)χ-B (I)

wherein each A is independently selected from H, a protecting group e.g. ethyl, trityl (Tit), allyl, or t-butyl, or at least one amino acid residue independently selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains;

Rι is a residue of an amino acid selected from glycine (Gly), proline (Pro), aspartic acid (Asp), arginine (Arg), alanine (Ala), isoleucine (He), tryptophan (Trp), serine (Ser), cysteine (Cys), glutamic acid (Glu), asparagine (Asn), and R₈,

R₂ is a residue of an amino acid selected from cysteine (Cys), proline (Pro), isoleucine (He), alanine (Ala), tyrosine (Tyr), threonine (Thr), arginine (Arg), pipecolic acid (Pec), and Rg, R₃ is a residue of an amino acid selected from cysteine (Cys) and R₈, and

at least one of R>, R₂ and R₃ being R₈ and at most one of R,, R₂ and R₃ being cysteine; and

R₈ is an amino acid residue of formula (II) or formula (III) Formula (II):

Formula (III):

wherein each of R₅ and R^ is independently selected from H, alkyl and alkoxy, suitably of 1 to 6, generally 1 to 4, carbon atoms, e.g. methyl or t-butyl, aryl e.g. phenyl,

R₇ is SOH, SO₂H, SO₃H, SR,, SeR<, or TeRo (wherein R₉ is H, alkyl and alkoxy, suitably of 1 to 6, generally 1 to 4, carbon atoms, e.g. methyl or t-butyl, aryl, e.g. phenyl, SR_I0, SOR₁₀ or SO₂R₁₀, and R₁₀ is H, alkyl or alkoxy, suitably of 1 to 6, generally 1 to 4, carbon atoms e.g. methyl or t-butyl, aryl e.g. phenyl),

n is selected from 0, 1 , 2, 3 and 4, and

m is selected from 0, 1, 2, 3 and 4; provided that, when R₈ is an amino acid residue of formula (II), when n is 0 and R₇ is SH, R₅ and R_$ are not both H;

R₄ is a residue of an amino acid selected from glycine (Gly), phenylalanine (Phe), valine (Val), isoleucine (He), lysine (Lys), proline (Pro), tryptophan (Trp), tyrosine (Tyr), glutamic acid (Glu), leucine (Leu) and methionine (Met);

x is 0 or 1 ;

with the provisos that

(i) when R₃ is other than penicillamine (Pen) or methionine (Met) (i.e. it is not a residue R₈ of formula II in which n is 0, R₇ is SH and R₅ and R₍. are both methyl or in which n is 1 , R₇ is SCH₃ and R₅ and R^ are both H), and R, is serine (Ser) and R₂ is threonine (Thre) or R, and R₂ are both isoleucine (He), x is 1 ;

(ii) when R₃ is penicillamine (Pen), and R, is serine (Ser) and R₂ is proline (Pro) or threonine (Thr), or R, is asparagine (Asn) and R₂ is arginine (Arg), x is 1 ;

(iii) when the or each R₈ is methionine, x is 1 , and

R_l is a residue of an amino acid selected from glycine (Gly), proline (Pro), aspartic acid (Asp), arginine (Arg), alanine (Ala), tryptophan (Trp), serine (Ser), methionine (Met), cysteine (Cys), and glutamic acid (Glu),

R₂ is a residue of an amino acid selected from cysteine (Cys), proline (Pro), isoleucine (He), methionine (Met), pipecolic acid (Pec), threonine (Thr), and arginine (Arg), and

R₃ is a residue of an amino acid selected from cysteine (Cys) and methionine (Met), and R is a residue of an amino acid selected from phenylalanine (Phe), valine (Val), isoleucine (He), proline (Pro), tryptophan (Trp), tyrosine (Tyr), glutamic acid (Glu), methionine (Met), and lysine (Lys);

except that, when R₃ is methionine and R, is alanine (Ala), R, is not phenylalanine (Phe), or R₂ is threonine (Thr), - is not lysine (Lys);

each B is independently selected from the group consisting of OH, NH₂, an oxygen or a nitrogen carrying a protecting group, such as ethyl, trityl (Trt), allyl or t-butyl, or at least one amino acid residue selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains; provided that when x is 0, B is OH, NH₂, or an oxygen or a nitrogen carrying a protecting group;

the entire peptide sequence containing 3 to 30 amino acid residues; or

a homo- or heterodimer thereof.

Amino acid residues of A and B independently selected from amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, and sulphur containing side chains. Suitable amino acids may be independently selected from the groups comprising naturally and non- naturally occurring amino acid residues. Examples of naturally occurring amino acid residues include isoleucine (He), leucine (Leu), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), lysine (Lys), phenyl alanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), methionine (Met), valine (Val) and histidine (His). Naturally, the skilled addressee will appreciate that naturally occurring amino acid residues means those amino acid residues which are found in peptides and/or proteins of living organisms. The skilled addressee will also appreciate that such naturally occurring amino acid residues may be present in peptides of the invention in chemically modified forms eg including added protecting groups such as ethyl, trityl (Trt), allyl, t-butyl and the like. Naturally, the skilled addressee will appreciate that any protecting group(s) which may be present on the peptides of the invention should be such so as not to substantially interfere with the immunomodulatory properties thereof and hence therapeutic effect thereof.

Peptides of the invention can be made synthetically, for example by chemical means, or through the use of recombinant DNA technology. Alternatively, peptides according to the invention can be isolated from polypeptides or problems or the like.

Amino acid residues R₈ of formula (II) or formula (III) in which R₇ is SH, SOH, SO₂H, S0₃H, SeH or TeH in particular and cysteine residues within formula (I) are capable of reacting to form intermolecular links with the formation of dimers or intramolecular bridges with the formation of cyclised monomers.

The amino acid units making up the peptides according to the invention may be in L- and D- form. Generally the amino acid units will be in L- form. For example a methionine or penicillamine residue in D- form may be used.

A peptide of the invention may take the form of a dimer consisting of two like or two dissimilar peptide monomers linked to each other by way of covalent bonds (e.g. bonds between S, SO, S0₂, SO₃, Se, Te). In an aspect of the invention, the peptide monomers may be linked one to the other through bonds between at least one R₈ and/or cysteine amino acid residue of the first monomer and at least one of R₈ amino acid residue of a second monomer. Dimers of the present invention may be in parallel form, i.e. two peptide monomers aligned parallel one to the other such that both the peptide monomers are readable in one direction eg from the N- terminal to C-terminal direction. The peptide monomers making up the dimer may or may not be the same length. Preferably, the peptide monomers are the same length and the N- and C- terminal amino acid residues of one monomer are located adjacent to the N- and C- terminal amino acid residues of the second peptide monomer. Alternatively, a dimer of the present invention may be in anti -parallel form. That is to say, a first monomer read from the N-terminal amino acid residue to the C- terminal amino acid residue is aligned against a second monomer which is read from the C- terminal amino acid residue to the N-terminal amino acid residue ie in the opposite direction to that of the first peptide monomer; the two peptide monomers being linked through covalent bonds as described above for parallel or anti-parallel dimers of the invention.

Where the two peptide monomers forming a peptide dimer of the invention are dissimilar one to the other, the dimer is referred to as an heterodimer. A heterodimer can be in parallel or anti-parallel form. Thus, a heterodimer can be composed of two peptide monomers of the same length, differing, for example, in the substitution of an amino acid residue having the L- form to an amino acid residue having the D- form. Alternatively, the lengths of the peptide monomers making up the dimer may be different. In a heterodimer, at least one of the monomers is of formula (I) and generally both are of formula (I).

Preferably, the first and second peptide monomers making up a dimer of the present invention are the same.

Peptides of the invention also include monomers which can be linear or cyclic e.g. wherein at least two contiguously aligned or spaced apart R₈ and R₈ or cysteine amino acid residues are linked through bridges between S, SO, SO₂, SO₃, Se, Te. Preferably, the monomers are linear.

Administration of peptides of the invention by way of, for example, oral administration, intra-tracheal, nasal or parenteral administration gives rise to a measurable modulated immune response, as indicated in the examples herein. "Epithelial cell lining" is defined as being the cell lining and associated cells thereto which covers the internal and external surfaces of the body, including the lining of vessels and other small cavities. For the purposes of the present invention the epithelial cell lining is regarded as being at least one cell layer in depth and as many as several cell layers deep. Cells included within the ambit of "epithelial cell lining" also includes those cells and specialised lymphoid tissues which are located in or associated with the said epithelial cell lining and which influence the immune response such as T- lymphocytes, B- lymphocytes, enterocytes, NK-cells, monocytes, dendritic cells and cells comprising mucosal associated lymphoid tissue (MALT), such as Peyer's patches and the like. Thus, the skilled addressee will appreciate that so-called migratory cells, such as T- and B- lymphocytes which can be regarded as transient resident cells of the epithelial cell lining as defined above are included within the ambit of the definition of epithelial cell lining. The peptides of the invention may be absorbed by the epithelial cell lining in a passive or active sense. For example, the peptides may be absorbed on the cell surface, or actively or passively taken up by cells located on the lumen surface side of the epithelial cell lining , or they may pass inbetween cells located on the lumen surface side of the epithelial cell lining and are taken up by cells located deeper in the epithelial cell lining eg T-lymphocytes or Peyer's patches. The skilled addressee will also appreciate that "absorption" as defined herein also includes the situation wherein peptides of the invention initiate an immune response by interacting with cell surface receptors found in or on the membranes of certain specialised cells located in the epithelial cell lining, such as on enterocytes, and intra-epithelial lymphocytes, without physically penetrating the epithelial cell lining. Thus, the skilled addressee will understand that peptides of the invention may interact with, bind to, pass through or penetrate the epithelial cell lining.

The peptides of the invention are preferably administered by oral, nasal, or intra-tracheal administration in oral, nasal or intra-tracheal dosage forms. It has been found that the amount of a peptide of the invention required to produce a given therapeutic effect when orally administered can be significantly lower than that required to produce the same effect via other types of administration, such as parenteral administration. In a further aspect of the invention there is provided an oral dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the gastrointestinal tract in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.

In a further aspect of the invention there is provided an oral dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the gastrointestinal tract in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease wherein the amount of the at least one orally administered peptide needed to induce an observable level of modulated immune response in a mammal is less than the amount of the same at least one peptide administered parenterally and needed to achieve a similar observable level of modulated immune response in the said mammal.

In a further aspect of the invention there is provided a nasal dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the nasal passages in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.

In a further aspect of the invention there is provided a nasal dosage form comprising at least one immunomodulatory peptide according to the invention, the peptide being absorbable by the epithelial cell lining of the nasal passages in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease wherein the amount of the nasally administered peptide needed to induce an observable level of modulated immune response in a mammal is less than the amount of the same peptide administered parenterally and needed to achieve a similar observable level of modulated immune response in the said mammal. In a further aspect of the invention there is provided an intra-tracheal dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the lung in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease.

In a further aspect of the invention there is provided an intra-tracheal dosage form comprising at least one immunomodulatory peptide according to the invention, the at least one peptide being absorbable by the epithelial cell lining of the lung in a mammal resulting in a modulated immune response and thereby a therapeutic effect against disease wherein the amount of the at least one intra-tracheally administered peptide needed to induce an observable level of modulated immune response in a mammal is less than the amount of the same at least one peptide administered parenterally and needed to achieve a similar observable level of modulated immune response in the said mammal.

Peptides of the invention contain 3 to 30 amino acid residues in the or each sequence of formula (I). Generally, the or each peptide sequence is from 4 amino acid residues up to 30 amino acid residues in length. Preferably, the or each peptide sequence is from 4 amino acid residues to about 20 amino acid residues in length. More preferably, the or each peptide sequence is from 4 to 15 amino acids in length (e.g. 4 to 10 or 4 to 9), and, most preferably, from 4 to 7 amino acids in length. For example, the peptide sequences can be 4, 5, 6, 7, or 8 amino acid residues in length, with or without protecting groups.

The peptides of the invention may or may not be associated with transport agents as defined herein. Preferably, the peptides of the invention are administered in a "naked" form ie free from added transport agents. Added transport agents are those with which the peptides of the invention are intentionally placed in contact or in association either before, during or immediately after administration and which may serve to improve absorption and/or improve the stability of the peptide. Thus, in one preferment there is provided a physiologically active peptide according to the invention free from added transport agents.

When x is 0, B is OH, NH₂ or an oxygen or a nitrogen carrying a protecting group.

When R₈ is an amino acid residue of formula (II), when n is 0 and R₇ is SH, R₅ and R₆ are not both H.

Suitable R₈ groups of formula (II) include those wherein R₅ and R₆ are both H, n is 0 and R₇ is S-alkyl, S-O-alkyl or S-O₂-aIkyI (wherein the alkyl group contains 1 to 6, e.g. 1 to 4, carbon atoms) e.g. homocysteine (Hey); and those wherein R₅ and Rg are both H, n is 0 and R₇ is SCH₃ (Cys (Me)), SOCH-, (Cys(Me,O)), SO₂CH₃ (Cys(Me,0₂) or S-t-butyl (Cys(tBu)), wherein R₅ and Rg are both H, n is 1 and R₇ is SCH₃ (Met), SOCH-, (Met,O) or S0₂CH₃ (Met,O₂), wherein one of R₅ and R₆ is hydrogen and the other is methyl, n is 0 and R₇ is SH, or wherein R₅ and R_€ are both methyl, n is 0 and R₇ is SH (Pen),

Suitable R₈ groups of formula (III) include that when m is 1, n is 0 and R₇ is SH.

Within formula (I), when x is 0, R| to R₃ may be residues of the amino acids selected from the following:

R,; selected from Gly, Pro, Asp, Arg, Ala, He, Trp, Ser, R₂ selected from Cys, Pro, He, Pec, R₈, R₃ selected from Cys, R_g;

at least one of R₂ and R₃ being R₈.

For example R, to R₃ may be residues of amino acids as follows:

R, selected from Gly, Pro, Ser, R₂ selected from Cys, Pro, R₈, R₃ selected from Cys, R_g;

at least one of R₂ and R₃ being R₈.

Within formula (I), when x is 1, R. to R₄ may be residues of the amino acids selected from the following:

R, selected from Gly, Pro, Asp, Arg, Ala, He, Trp, Ser, R₂ selected from Cys, Pro, He, Pec, R₈, R₃ selected from Cys, R₈, R₄ selected from Gly, Phe, Val, He, Pro, Leu;

at least one of R₂ and R₃ being R₈.

For example R. to R^. may be residues of amino acids as follows:

R, selected from Gly, Pro, Ser, R₂ selected from Cys, Pro, R₈₎ R₃ selected from Cys, R₈,

R, selected from Gly, Phe, He, Pro;

at least one of R₂ and R₃ being R₈.

For example when R₈ is methionine (Met) (i.e. n is 1, R₇ is SCH₃ and R₅ and R are both H) R. to R₄ may be residues of amino acids as follows:

R₍ selected from Gly, Asp, Arg, Ala, Trp, Ser, R₂ selected from Pro, He, Pec, R₃ Met, R₄ selected from Phe, He, Pro; e.g.

R, selected from Gly, Ser, R₂ Pro, R₃ Met,

R₄ selected from Phe, He, Pro.

Preferred peptides, including cyclised monomers and dimers, according to the invention include those wherein R. to R, are residues of the amino acids selected from the following:

R, selected from Gly, Pro, Asp, Arg, Ala, He, Ser, Glu, R₈,

R₂ selected from Cys, Pro, He, Pec, R₈,

R₃ selected from Cys, R₈,

R₄ selected from Gly, Phe, Val, He, Pro, Lys, Leu;

at least one of R», R₂ and R₃ being R₈;

and R₈ being Met, Pen, homocysteine (Hey), a residue of the formula (II) wherein R₅ and R are both H, n is 0 and R₇ is SCH₃ (Cys(Me)), SOCH₃ (Cys(Me,O)), or SO₂CH₃ (Cys(Me,0₂)), wherein R₅ and R^ are both H, n is 1 and R₇ is SOCH-, (Met,O) or SO₂CH₃ (Met,0₂), or wherein one of R₅ and R^ is H and the other is methyl, n is 0 and R₇ is SH or a residue of formula (III) wherein m is 1, n is 0 and R₇ is SH.

Particularly preferred such peptides are those wherein R, to R, are residues of amino acids as follows:

R, selected from Gly, Pro, Ser, Asp, Ala, Glu, R₈, R₂ selected from Cys, Pro, He, R₈, R₃ selected from Cys, R₈, R₄ selected from Gly, Phe, He, Pro, Lys, Met, at least one of R,, R₂ and R₃ being R₈ and R₈ being Pen or Met.

Preferred peptides wherein R_g is methionine (Met) include those wherein R_{ to R, are residues of amino acids as follows:

R, selected from Gly, Pro, Asp, Ala, Ser, Met, Glu, R₂ selected from Pro, Met, He, R₃ Met, R₄ selected from Phe, Val, He, Pro, Tyr, Met, Lys.

Particularly preferred such peptides include those wherein R. to R, are residues of amino acids as follows:

R. selected from Gly, Asp, Ser, Met, Glu, R selected from Pro, He, R₃ Met, _t selected from Phe, He, Met, Lys.

Preferred peptides, including cyclised monomers and dimers, wherein R₈ is penicillamine (Pen) include those wherein R_! to R,, are residues of the amino acids selected from the following:

R_[ selected from Gly, Pro, Asp, Arg, He, Pen, R₂ selected from Cys, Pro, He, Pen, Pec, R₃ selected from Cys, Pen, R, selected from Gly, He, Pro, Lys, Leu;

at least one of R,, R₂ and R₃ being Pen. Particularly preferred such peptides including those wherein R, to R, are residues of amino acids as follows:

R[ selected from Gly, Pro, Pen, R₂ selected from Cys, Pro, He, Pen, R₃ selected from Cys, Pen, R₄ selected from Gly, He, Pro, Lys;

at least one of R., R₂ and R₃ being Pen.

Also amongst preferred peptides, including cyclised monomers and dimers, include those wherein R. to R₄ are residues of the amino acids selected from the following:

R, selected from Gly, He, Ser, R₂ selected from Pro, R_g R₃ selected from Cys, R₈ R₄ selected from Phe, He, Trp, Leu;

at least one of R₂ and R₃ being R₈ and R₈ being homocysteine (Hey), a residue of the formula (II) wherein R₅ and Rg are both H, n is 0 and R₇ is SCH₃ (Cys(Me)), SOCH₃ (Cys(Met,O)), or SO₂CH₃ (Cys(Me,O₂)), wherein R₅ and R<. are both H, n is 1 and R₇ is SOCH₃ (Met,O) or SO₂CH₃ (Met,O₂), or wherein one of R₅ and R^ is H and the other is methyl, n is 0 and R₇ is SH or a residue of formula (III) wherein m is 1, n is 0 and R₇ is SH.

Generally in formula (I) x is 1.

Peptides according to the invention may have two of R₈ and Cys aligned contiguous one to the other as follows:

A-R.-Rg-Rg-R^B A-R_rR₈-Cys-R₄-B

A-R_rCys-R₈-R₄-B

(wherein A, R_t, R and B are as defined above), or separated by no more than one amino acid as follows:

A-R_g-R₂-Cys-R₄-B

A-Cys-R₂-R_g-R₄-B

(wherein A, R₂, R₄ and B are as defined above).

When R₈ is penicillamine (Pen) (i.e. n is 0, R₇ is SH and R₅ and Rg are both methyl) peptides according to the invention may have two of Pen and Cys aligned contiguous one to the other as follows:

A-R,-Pen-Pen-R₄-B

A-RpPen-Cys-Pvt-B

A-R,-Cys-Pen-R₄-B

(wherein A, R_b R₄ and B are as defined above), or separated by no more than one amino acid as follows:

A-Pen-R₂-Pen-R₄-B A-Pen-R₂-Cys-R₄-B

A-Cys-R₂-Pen-R₄-B

(wherein A, R₂, R and B are as defined above).

Peptides according to the invention may have two of Met and Cys aligned contiguous one to the other as follows:

A-R,-Met-Met-R₄-B

A-R.-Met-Cys-R^B

A-R_rCys-Met-R₄-B

(wherein A, R,, ₄ and B are as defined above), or separated by no more than one amino acid as follows:

A-Met-R₂-Met-R₄-B

A-Met-R₂-Cys-R,-B

A-Cys-R₂-Met-R₄-B

(wherein A, R₂, R₄ and B are as defined above).

Peptides according to the invention include the peptides:

wherein R8 is a residue of formula

• Leu Leu Phe Gly Pro (R,R)R8 He • Leu Leu Phe Gly Pro (S,S)R8 He

• Leu Leu Phe Gly Pro (R,S)R8 He

• Leu Leu Phe Gly Pro (S,R)R8 He

. Leu Leu Phe Gly Pro (R,R)R8 He (ox, dimer)

. Leu Leu Phe Gly Pro (R,S)R8 He (ox, dimer) . Leu Leu Phe Gly Pro (S,S)R8 He (ox, dimer)

. Leu Leu Phe Gly Pro (S,R)R8 He (ox, dimer)

• Leu Leu Tyr Ser Pro (R,R)R8 Phe

• Leu Leu Tyr Ser Pro (R,S)R8 Phe

• Leu Leu Tyr Ser Pro (S,S)R8 Phe . Leu Leu Tyr Ser Pro (S,R)R8 Phe

• Leu Leu Tyr Ser Pro (R,R)R8 Phe (ox, dimer) . Leu Leu Tyr Ser Pro (R,S)R8 Phe (ox, dimer) . Leu Leu Tyr Ser Pro (S,S)R8 Phe (ox, dimer) . Leu Leu Tyr Ser Pro (S,R)R8 Phe (ox, dimer)

wherein R8 is a homocysteine (Hey) residue

• Leu Leu Phe Gly Pro Hey He

• Leu Leu Tyr Ser Pro Hey Phe • Leu Leu Phe Asp Pro Hey He

• Gly Pro Cys Hey Pro Gly

• Ala Pro Hey Hey Val Pro

• Pro Asp Hey Cys He Pro • Leu Leu Phe Gly Pro Hey He Leu Asn Arg

• Leu Leu Phe Gly Pro Hey He

Leu Leu Phe Gly Pro Cys He • Phe Hey Leu Gly Pro Hey Pro

Val Val Phe Ala Pro Hey Tyr

Phe Hey He Hey Lys

Met Leu Phe Ser Pro Hey Trp (ox, dimer)

Pro Gly Hey Cys Gly Pro

Pro Gly Hey Hey Gly Pro

Met Leu Phe Ser Pro Hey Trp

wherein R8 is a residue of Cys(Me) of the formula

OH

NH,

• Leu Leu Phe Gly Pro Cys(Me) He

• Leu Leu Tyr Ser Pro Cys(Me) Phe • Pro Gly Cys(Me) Cys(Me) Gly Pro

• Leu Ala Phe Glu Pro Cys(Me) Met

wherein R8 is a residue of Cys(Me,O) of the formula

• Leu Leu Phe Gly Pro Cys(Me,O) He

• Gly Pro Cys(Me,O) Cys(Me,O) Pro Gly • Leu Leu Tyr Ser Pro Cys(Me,O) Phe

• Leu Leu Phe Arg Pro Cys(Me,O) He

wherein R8 is a residue of Cys(Me,0₂) of the formula

^^S.. T OH

O NH,

• Leu Leu Phe Gly Pro Cys(Me,O₂) He

• Leu Leu Tyr Ser Pro Cys(Me,O₂) Phe

• Leu Leu Phe Trp Pro Cys(Me,O₂) He • Ala Pro Cys(Me,O₂) Cys(Me,O₂) Val Pro

wherein R8 is a residue of Cys(tBu) of the formula

• Leu Leu Phe Gly Pro Cys(tBu) He

• Leu Leu Tyr Ser Pro Cys(tBu) Phe

• Leu Leu Phe Gly Pec Cys(tBu) He

• He Cys Tyr Cys(tBu) Glu

wherein R8 is a residue of Met(O) of the formula

• Leu Leu Phe Gly Pro Met(O) He

• Leu Leu Tyr Ser Pro Met(O) Phe • Leu Leu Phe Ala Pro Met(O) He

• Val He Met(O) Cys Leu Thr

wherein R8 is a residue of Met(O₂) of the formula

Leu Leu Phe Gly Pro Met(O₂) He

• Leu Leu Tyr Ser Pro Met(O₂) Phe

• Leu Leu Phe Ala Pro Met(O₂) He . Val He Met(O₂) Met(O₂) Leu Thr

wherein R8 is a residue of Aaa of the formula

• Leu Leu Phe Gly Pro Aaa He

• Leu Leu Tyr Ser Pro Aaa Phe

• Pro Gly Aaa Aaa Gly Pro

• Leu Leu Phe Gly Pro Aaa He (ox, dimer)

wherein R8 is a residue of Aaa of the formula

• Leu Leu Phe Gly Pro Aaa He

• Leu Leu Tyr Ser Pro Aaa Phe • Pro Gly Aaa Aaa Gly Pro

• Leu Leu Phe Gly Pro Aaa He (ox, dimer)

wherein R8 is a residue of Aaa of the formula

• Leu Leu Phe Gly Pro Aaa He

• Leu Leu Tyr Ser Pro Aaa Phe

• Pro Gly Aaa Aaa Gly Pro • Leu Leu Phe Gly Pro Aaa He (ox, dimer)

wherein R₈ is a residue of Seleno-cysteine (denoted by Aaa below)

• Leu Leu Tyr Ser Pro Aaa Phe (ox, dimer) • Leu Leu Phe Gly Pro Aaa He (ox, dimer)

ox, dimer denotes dimer formed by oxidation.

Preferred peptides according to the invention include those of the formulae:

Val Val Phe Ala Pro Met Tyr Leu Leu Tyr Ser Pro Met Phe Ala Pro Met Met Val Pro Leu Leu Phe Asp Pro Met He Leu Leu Phe Gly Pro met He Leu Leu Tyr Ser Pro met Phe Pro Gly met met Gly Pro Leu Ala Phe Glu Pro met Met Phe Met He Met Lys

wherein met denotes the residue of D-methionine; and

Leu Leu Phe Gly Pro Pen He Phe Pen He Cys Lys Phe Pen He Pen Lys Leu Leu Phe Asp Pro Pen He Leu Leu Phe Arg Pro Pen He Leu Leu Phe Gly Pec Pen He (Leu Leu Phe Gly Pro pen Ile)₂ Pro Gly Cys Pen Gly Pro (ox)

Pro Gly Pe un Cys Gly Pro (ox)

Phe Cys Le uu Gly Pro Pen Pro (ox)

Pro Gly Pen Cys Gly Pro Pro Gly Pen Pen Gly Pro Gly Pro Pen Cys Pro Gly Val He Pen Pen Leu Thr Pro Gly Pen(Amc) Pen Gly Pro wherein (ox) denotes intramolecularly cyclised, pen denotes D-penicillamine and Acm denotes acetamidomethyl; and

Leu Leu Phe Gly Pro Xxx He Leu Leu Tyr Ser Pro Hey Phe

Leu Leu Phe Gly Pro Hey He Leu Asn Arg Leu Leu Phe Gly Pro Cys (Me,O) He Leu Leu Tyr Ser Pro Cys(Me,O₂) Phe Val He Met(O) Cys Leu Thr Val He Met(O₂) Met(O₂) Leu Thr

Leu Leu Tyr Ser Pro Aaa Phe (Leu Leu Phe Gly Pro Aaa Ile)₂ Met Leu Phe Ser Pro Hey Trp

wherein Xxx denotes a residue of formula (III) in which m is 1, n is 0 and R₇ is SH and Aaa denotes a residue of formula (II) in which one of R₅ and Rg is H and the other is methyl, n is 0 and R₇ is SH.

It is to be understood that the amino acid residues located at the N and C terminals respectively of the above peptides do not contain modifications thereto.

When A and/or B represent an amino acid residue or a sequence of amino acid residues, the amino acid residue or sequence of amino acid residues can include naturally occurring amino acid residues, such as those described hereinabove or analogues thereof or can include non-naturally occurring amino acid residues, such as synthetic amino acid residues and analogues thereof, or amino acid residues or sequences of amino acid residues including both naturally occurring amino acid residues and/or analogues thereof and non- naturally occurring amino acid residues and/or analogues thereof. The skilled addressee will also appreciate that included within the scope of the invention are peptides in which intramolecular bridges between, contiguously aligned or spaced apart, on one hand an R_g grouping or cysteine residue, and on the other hand a R₈ amino acid residue. Such peptides represent an oxidised form of peptides of the invention.

The skilled addressee will also appreciate that the dimers of the invention may be in parallel or anti-parallel form and may include heterodimers, and can be linked through covalent bonds between on one R_g or cysteine amino acid residue, and on the other hand a R₈ amino acid residue. Also included within the ambit of the invention are pharmaceutically acceptable salts of peptides of the invention or physiologically functional derivatives thereof together with a pharmaceutically acceptable carrier therefor.

The skilled addressee will further appreciate that the peptides of the invention will include within their ambit variants of the formula (I) which contain one or more modifications of the peptide backbone and which retain the immunomodulatory properties according to the invention. Such modificiations have been reviewed for example by A.F. Spatola "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins"; B. Weinstein, Ed; Marcel Dekker, New York, 1983, Vol 1, Chapter 5; Robert A Wiley et al, "Peptidomimetics derived from natural products" Medicinal Research Reviews, Vol 13, No. 3, 327-384 (1993) John Wiley & Sons Inc; and Youe Kong Shue et al, "Double Bond Isoteres of the Peptide Bond" Bioorganic and Medicinal Chemistry, Vol 1, No. 3, 161-171 (1993) Pergamon Press Ltd.

The monomers of formula (I) can be made synthetically, for example by chemical means, or through the use of recombinant DNA technology. Alternatively, they can be isolated from polypeptides or proteins and the like. For example the monomers can be made by a chemical process in which individual amino acid residues or fragments of the monomers are joined to form peptide bonds and wherein protecting groups are employed at the beginning and/or end of the process. 29

The amino acids R_g are known or can be prepared by methods known per se.

The peptides of the invention can be administered with or without transport agents. Preferably, peptides of the invention are administered orally, intra-tracheally, nasally, or systemically free from added transport agents. More preferably, the peptides of the invention are administered intra-tracheally, nasally, or orally. Most preferably, the peptides of the invention are administered orally. "Transport agents" includes added means for delivery such as vesicular delivery systems, micro particles, liposomes, and like systems which are designed to carry drugs ( eg peptides) to the epithelial cell lining or endothelial cell lining. "Transport agents" also includes chemicals or additional peptide sequences which may form an association with, or are fused to, or are complexed with the peptides and which help to maintain physiological integrity of peptide sequences of the invention, for example, presenting the peptides in a prepro- or pro- form or fusing the peptides to carrier proteins, eg glucosyl transferase, or complexed to chemical agents, such as cyclodextrins and the like. Preferably, peptides of the invention are administered to the recipient as free peptides along with the usual adjuvants, excipients and diluents commonly found in pharmaceutical formulations. Thus, peptides of the invention can be delivered by oral or systemic administration in simple oral or systemic formulations comprising adjuvants, diluents and excipients commonly employed in oral and systemic dosage forms. Preferably, the peptides are administered in an oral dosage form free from added transport agents.

Mucosal associated lymphoid tissue (MALT) is also found in the epithelial cell linings of the gastrointestinal tract, ie, oesophagous, stomach, duodenum, ileum, and colon; bronchiole linings in the lung; and in the linings of the nasal passages. Without the intention of being bound by theory, it is thought that the peptides of the invention interact with MALT and thereby set in train a sequence of immunomodulating events which results in a therapeutic effect against certain diseases. The immunomodulatory response can be immunoinhibitory or immunostimulatory in effect. The immunomodulatory response has been shown to be indicative for therapy against cancer. The peptides of the invention having an immunomodulatory effect are indicated as being advantageous in the treatment of cancers of mesenchymal origin such as sarcoma, eg, fϊbrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma or mesotheliosarcoma; leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma or Hodgkins disease; sarcomas like leiomysarcoma or rhabdomysarcoma, tumours of epithelial origin (Carcinomas) such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma choriocarcinoma, semonoma or embryonal carcinoma; and tumours of the central nervous system like glioma, meningoma, medulloblastoma, schwannoma or ependy oma. Peptides of the invention are indicated on the basis of their activity for the treatment of malignancies such as melanoma, mammary carcinoma, gastrointestinal carcinoma such as colonic carcinomas, glioma, bladder carcinoma and squamous cell carcinoma of the neck and head region. Furthermore, peptides according to the invention are indicated for therapy in the treatment of acute and/or chronic infections associated with autoimmune disease and autoimmune disease per se such as non-obese diabetes, systemic lupus erythematosus, sclerodermia, Sjogren's syndrome, dermatomyositis or multiple sclerosis, rheumatoid arthritis, artheriosclerosis, and psoriasis, asthma, rhinitis, fibrosis, chronic bronchitis, hepatitis, post- infectious anergy, acquired immune deficiency diseases such as AIDS, HIV and post traumatic immunological anergy.

The peptides according to the present invention may, if appropriate, be used together with a traditional therapy regime, such as with methotrexate (MTX). Moreover, the peptides according to the present invention, being immunomodulatory in action, may be advantageously employed as adjuvants in various forms of vaccine preparations and in formulations designed to inhibit rejection of organs in transplants.

In another aspect of the invention, there is provided a method of inducing a modulated immune response in a mammal which comprises administering to the epithelial cell lining of the mammal a dose of a peptide according to the invention, enough to induce said modulated immune response and thereby a therapeutic effect.

In a further aspect of the invention there is provided a method of inducing a modulated immune response in a mammal which comprises 1 ) identifying a mammal in need of modulation of its immune response and 2) administering to at least one epithelial cell lining of the mammal a dose of a peptide according to the invention, enough to induce said immunomodulatory response and thereby a therapeutic effect. Preferably, the epithelial cell lining to which the peptide is administered is the epithelial cell lining of the gastroinestinal tract. Most preferably, the peptide is administered to the MALT.

In a preferment there is provided a method of inducing a modulated immune response in a mammal which comprises administering to MALT of the mammal a dose of a peptide according to the invention, said peptide being free from added transport agents and being sufficient to induce said modulated immune response and thereby a therapeutic effect.

In a further aspect of the invention there is provided use of a peptide according to the invention, in the preparation of a medicament suitable for the treatment of disease. Particular forms of cancer which may be treated with peptides of the invention are listed hereinabove.

The peptides according to the invention may be used in combination with surgery; pre- or, more preferably, post-operationally. In a preferment, there is provided use of a peptide according to the invention free from added transport agents in the preparation of a medicament suitable for the treatment of disease, in particular cancer and rheumatoid arthritis.

5

In a further embodiment of the invention there is provided a method of making a peptide of the invention by a chemical process in which individual amino acid residues or fragments of peptides of the invention are joined to form peptide bonds and wherein protecting groups are employed at the beginning and/ or end of the process.

10

In another embodiment of the invention there is provided as a further alternative aspect of the invention a physiologically active peptide according to the invention, preferably free from added transport agents, for use in therapy, for example, in cancer or rheumatoid arthritis therapy. In a preferment, there is provided a peptide of the invention for use in 15 therapy, for example in cancer therapy or rheumatoid arthritis therapy.

The amount of a peptide according to the invention which is required in cancer or rheumatoid arthritis therapy will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being 0 treated, the route of administration, and nature of the formulation, the mammal^" s body weight, surface area, age, and general condition and the particular peptide to be administered. A suitable effective dose of peptides of the invention generally lies in the range of from about 0.0001 μmol/kg to about lOOOμmol/kg bodyweight, preferably from about 0.003 to about 300 μmol/kg body weight, e.g. in the range of from about 0.001 to

25 100 μmol/kg bodyweight, for example, 0.03 to 3.0 μmol/kg bodyweight. The total dose may be given as a single dose or multiple doses, e.g two to six times per day. For example, for a 75 kg mammal (e.g. a human) the dose range would be about 2.25 μmol/kg/day to 225 μmol/kg/day and a typical dose could be about 100 μmol of peptide. If discrete multiple doses are indicated treatment might typically be 25 μmol of a peptide of the

30 invention given up to 4 times per day. In an alternative administrative regimen, peptides of the invention may be given on alternate days or even once or twice a week. The skilled addressee will appreciate that an appropriate administrative regimen would be at the discretion of the physician or veterinary practitioner.

Whilst it is possible for the active peptide to be administered alone, it may be preferable to present the active peptide in a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise a peptide of the invention or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and substantially non-deleterious to the recipient thereof. The skilled addressee will appreciate that free acid addition salts (e.g. hydro-halo salts) of peptides referred to herein as well as base salts are encompassed within the ambit of the invention. Most preferably the salts will be pharmaceutically acceptable.

Suitable acid addition salts include those formed from hydrochloric, hydrobromic, nitric, perchloric, sulphuric, citric, tartaric, phosphoric, lactic, benzoic, glutamic, oxalic, aspartic, pyruvic, acetic, succinic, fumaric, maleic, oxaloacetic, isethionic, stearic, phthalic, methanesulphonic, p-toluene sulphonic, benzenesulphonic, lactobionic, glucuronic and trifluoracetic acids. Suitable base salts include inorganic base salts such as alkali metal (e.g. sodium and potassium) salts and alkaline earth metal (e.g. calcium) salts; organic base salts e.g. phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine and diethanolamine salts; and amino acid salts e.g. lysine and arginine. Most preferably, the salts will be pharmaceutically acceptable.

The present invention, therefore, further provides a pharmaceutical formulation comprising a peptide of the invention together with a pharmaceutically acceptable carrier therefor.

Naturally, the skilled addressee will appreciate that any pharmaceutical formulation comprising a peptide of the invention can include more than one peptide of the invention. 8/12219

34

Thus, a pharmaceutical formulation may comprise at least two peptides of the invention or a cocktail of peptides of the invention.

There is also provided a method for the preparation of a pharmaceutical formulation comprising bringing into association one or more peptides of the invention, or a physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.

The peptides of the invention and physiologically functional derivatives thereof may be administered by any route appropriate to the condition to be treated, suitable routes including oral, intra-tracheal, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, intraperitoneal, and epidural). It will be appreciated that the route may vary with, for example, the condition of the recipient. Preferred formulations are those suitable for oral, nasal or intra-tracheal administration. Most preferred formulations are those suitable for oral administration.

Formulations for topical administration in the mouth include lozenges comprising the peptide(s) in a flavoured basis, usually sucrose and acacia and tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth washes comprising the peptide(s) in a suitable liquid carrier.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, lozenges comprising the peptide(s) in a flavoured base, usually sucrose and acacia and tragacanth; pastilles comprising the active ingredient(s) in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth washes comprising the active ingredient(s) in a suitable liquid carrier. Each formulation generally contains a predetermined amount of the active peptide(s); as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like. 35

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active peptide(s) in a free-flowing form such as a powder or granules, optionally mixed with a binder, (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered peptide(s) moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.

A syrup may be made by adding the active peptide(s) to a concentrated, aqueous solution of a sugar, for example, sucrose, to which may also be added any necessary ingredients. Such accessory ingredient(s) may include flavourings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol, for example, glycerol or sorbitol.

In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.

Emulgents and emulsion stabilisers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl mono- stearate and sodium lauryl sulphate.

The choice of suitable oils or fats for the formulation is based on achieving the desired therapeutic properties, since the solubility of the active compound in most oils likely to be „8/,1_.2-.2,1_.«9 PC

36

used in pharmaceutical emulsion formulations is low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branch-chained esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft parafϊn and/or liquid parafin or other mineral oils can be used.

Formulations for rectal administration may be presented in any suitable form e.g. as a suppository with a suitable base comprising peptide(s) of the invention in admixture with a neutral fatty base, for example cocoa butter, or, for example in admixture with a salicylate, or in the form of solutions and suspensions. In an alternative, formulations in the form of gelatin rectal capsules comprising active peptide(s) of the invention in admixture with vegetable oil(s) or paraffin oil can be used.

Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns. Where the particle size relates to an active substance in particle form per se, the particle size may be in the range of from 2 to 500 microns. Coarse powder formulations can be administered by rapid inhalation through the nasal passage from a container of the powder held up close to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Thus, peptides of the invention may be formulated in pressurised metered dose inhalers or dry powder inhalers for oral or nasal inhalation or in liquid formulations for nebulisation. The active peptide(s) is micronised or otherwise processed to a particle size suitable for inhalation therapy (mass median diameter < 10 μm). In the case of pressurised metered dose inhalers the micronised peptide(s) can be suspended in a liquefied propellant or a mixture of liquefied propellants. Such propellants can also, but not necessarily act as solvents. In either case, the micronised peptide(s) can be filled into a container equipped, for example with a metering valve.

Suitable propellants include those commonly employed in the art, such as, hydrofluoroalkanes (HFAs). The HFA propellants can be present in any mixture which is appropriate for delivering peptide(s) of the invention to MALT. Examples of suitable HFAs for use in the invention include tetrafluoroethane (eg propellant 134a (Hoechst)) and heptafluoropropane (eg propellant 227 (Hoechst)). Naturally, the skilled addressee will appreciate that appropriate concentrations of surfactants can also be present in such formulations, for example, sorbitan trioleate, lecithin, oleic acid and the like, the use of surfactants being to increase the physical stability of the peptide(s) preparation. The formulation can also contain solvents, such as ethanol, to improve the solubility of the peptide(s) in the chosen propellant.

Active peptides of the invention may be delivered through inhaling devices suitable for dry powder inhalation, such as portable inhaler devices and the like. In such dry powder formulations, the active peptide(s) of the invention can be used either alone or in combination with a carrier, such as lactose, mannitol, or glucose. The selection of carrier is not critical, provided that the physiological action of the peptide(s) of the invention is substantially unimpaired. Other additives may also be included in powder formulations as required e.g. to maintain stability etc. Again, such additives should be such so as not to substantially interfere with the physiological and hence therapeutic effect of the peptide(s) of the invention. The inhaling device can be of any type known in the art, such as a single dose inhaler having a predetermined dose or a multi-dose inhaler wherein the dose is measured by a metering unit within the inhaler or is delivered from an assembly of predetermined doses. Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable acid addition salt of a peptide(s) of the invention that is isotonic with the blood of the recipient.

Useful formulations also comprise concentrated solutions or solids containing peptide(s) of the invention which upon dilution with an appropriate solvent give a solution for parenteral administration as above.

The invention will now be illustrated by the following non-limiting examples.

It is to be understood that where no group is shown at the N- and C- terminals of peptides of the invention herein described that the N-terminal is in the amino form (NH2) and that the C-terminal is in the carboxyl form (-COOH).

In the synthetic examples below:

Peptides of the present invention were prepared using standard solid phase sequentional coupling techniques on a Millipore 9050 automatic peptide synthesizer (for further information about this technique see for example Jones, J. The Chemical Synthesis of

Peptides, pp 132-156, first edition, Oxford University Press, 1991 and R. Epton (ed)

Innovation and Perspectives in Solid Phase Synthesis, SPCC (UK) Ltd 1990).

Penicillamine was purchased from Bachem. Oxidation of thiols into disulfides was accomplished using general oxidation techniques (e.g. Andreu, D et al. Methods in

Molecular Biology, Vol 35: Peptide Synthesis Protocols edited by M.W. Pennington and

B.M. Dunn Humana Press Inc., Totowa, NJ.).

The C-terminal aminoacid was purchased coupled to a resin which consisted of a crosslinked polystyrene backbone grafted with polyethyleneglycol chains, functionalized with either the linker p-hydroxymethyl phenoxyacetic acid (TentaGel S PHB-Aaa-Fmoc, Sheppard, R.C., Williams, B.J. Acid-labile Resin Linkage Agents for Use in Solid Phase Peptide Synthesis. Int. J. Peptide Protein Res. 1982, 20, 451-454) from RAPP Polymere or a more sterically hindered trityl handle (TentaGel S Trt-Aaa-Fmoc) from RAPP Polymere. N^α-Fmoc-protected L-amino acid pentafiuorophenyl esters used were purchased from Bachem and Biosearch. DMF and 20% piperidine DMF in peptide reagent quality was purchased from Biosearch. The coupling reagent 1 -hydroxybenzotriazole (HOBT) came from Fluka. Synthesis were performed on a Millipore 9050 Plus PepSynthesizer.

In the Examples below ox denotes oxidised. The amino acid residues are in L- form unless otherwise specified.

Example 1

Synthesis of H-Leu-Leu-Phe-Gly-Pro-Xxx-Ile-OH wherein Xxx=

The N-Fmoc protected derivative of Xxx were prepared according to Marshall G. et al. J. Med. Chem. _ϋ_ 137 (1995)

The C-terminal aminoacid Tentagel S PHB-Ile-Fmoc (1.0 g, 0.21 mmol/g. 0.21 mmol) on resin, drypacked in the synthesizer column, was allowed to swell in DMF for 30 minutes. The synthesizer worked with consecutive deblocking, washing and coupling cycles consisting of 8 min recycling with 20% piperidine/DMF for each Fmoc-deblocking followed, after wash, by activation of the N^α-Fmoc-protected L-amino acid pentafiuorophenyl ester (0.8 mmol) with HOBT (0.9 mmol) and by activation of the N^α- Fmoc-protected special amino acid (0.4 mmol) with HOBT (0.5 mmol) and diisopropylcarbodiimide (DIPCDI, 0.5 mmol) The activated amino acids were added to the column and recycled 30 min each (12h for the special aminoacid). The synthesizer ended the synthesis with a deblocking of the N-terminal Fmoc-group and a final wash with DMF. The resulting peptide on the resin was transferred to a sintered glas funnel where it was washed twice with MeOH (2 x 10 ml) and three times with CH₂C1₂ (3 x 10 ml). The resin was allowed to dry under vacuum over night after which the peptide was side chained deprotected and cleaved from the resin using ethanedithiol/TFA 5/95 (20 ml) at room temperature for 3 h. The resin was filtered off and washed with 3 x 30 ml of acetic acid. The combined acidic fraction was evaporated after which the residue was triturated 3 times with ether.

The crude peptide was dissolved in H₂O/CH₃CN 1/1 and lyophilized. The resulting material was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C18 25 cm x 20 mm id reversed phase column (0.1% TFA CH₃CN - 0.1% TFA/H₂0 90 - 10, 10 ml/min, 220 nm). The combined HPLC fraction was lyophilized leaving 25 mg of the title compound.

Synthesis of Examples 2 - 8 below was accomplished following a similar protocol as per Example 1 to give the following products:

Example 2 Leu Leu Phe Gly Pro Xxx He Xxx= 1 S, 2S

Example 3 Leu Leu Phe Gly Pro Xxx He Xxx= 1 S, 2R Example 4 Leu Leu Phe Gly Pro Xxx He Xxx= 1 R, 2S

Example 5 Leu Leu Tyr Ser Pro Xxx Phe Xxx= 1 R, 2R

Example 6 Leu Leu Tyr Ser Pro Xxx Phe Xxx= 1 S, 2S

Example 7 Leu Leu Tyr Ser Pro Xxx Phe Xxx= 1 S, 2R

Example 8; Leu Leu Tyr Ser Pro Xxx Phe Xxx= 1 R, 2S

Example 9: Leu Leu Phe Gly Pro Xxx He (ox, dimer) Xxx= IR, 2R

Leu Leu Phe Gly Pro Xxx He (50 mg, 63.4 μmol) was prepared following the protocol as per example 1. The peptide was dissolved in 5% aqueous acetic acid (25 ml) and the pH of the solution was adjusted to 6 with ammonium carbonate. Dimethylsulfoxide (5 ml) was added and the mixture was stirred at room temperature for 16 h. Upon completion, the 41

reaction mixture was concentrated in vacuo until ca 5 ml remained. The crude product was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C18 25 cm x 20 mm id reversed phase column (0.1 % TFA/CH₃CN - 0.1 % TFA/H₂0 90 - 10, 10 ml/min, 220 nm). The combined HPLC fraction was lyophilised to give the title peptide as 5 a white powder.

Synthesis of Examples 10 - 16 below was accomplished following a similar protocol as per Example 9 to give the following products:

o Example 10 Leu Leu Phe Gly Pro Xxx He (ox, dimer) Xxx= 1 S, 2S

Example 1 1 Leu Leu Phe Gly Pro Xxx He (ox, dimer) Xxx= 1 S, 2R

Example 12 Leu Leu Phe Gly Pro Xxx He (ox, dimer) Xxx= 1 R, 2S

Example 13 Leu Leu Tyr Ser Pro Xxx Phe(ox, dimer) Xxx= IR, 2R

Example 14 Leu Leu Tyr Ser Pro Xxx Phe(ox, dimer) Xxx= 1 S, 2S s Example 1 Leu Leu Tyr Ser Pro Xxx Phe(ox, dimer) Xxx= 1 S, 2R

Example 16 Leu Leu Tyr Ser Pro Xxx Phe(ox, dimer) Xxx= 1 R, 2S

Synthesis of Examples 17- 25 below was accomplished following a similar protocol as per Example 1 with homocysteine (Hey, purchased from Bachem) as special aminoacid. 0 Fmoc-Hcy-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/HOBT mediated coupling with a coupling time of lh. The products were as follows:

Example 17: Leu Leu Phe Gly Pro Hey He

Example 18 Leu Leu Tyr Ser Pro Hey Phe 5 Example 1 Leu Leu Phe Asp Pro Hey He

Example 20 Gly Pro Cys Hey Pro Gly

Example 21 Ala Pro Hey Hey Val Pro

Example 22 Pro Asp Hey Cys He Pro

Leu Leu Phe Gly Pro Hey He Leu Asn Arg o Example 24 Val Val Phe Ala Pro Hey Tyr Example 25: Phe Hey He Hey Lys

Example 26: Met Leu Phe Ser Pro Hey Trp (ox. dimer) The monomeric peptide was synthesised following a similar protocol as per Example 17. Oxidation into dimer was accomplished following a similar protocol as per Example 9.

Example 27: Pro Gly Gly Pro (intramolecularly oxidized)

Pro Gly Hey Hey Gly was synthesised following a similar protocol as per

Example 17. Intramolecular oxidation of the monomer was accomplished following the protocol as per example 9, but this time in higher dilution. The peptide (0.05mmol) was dissolved in 5% aqueous acetic acid (100 ml) and the pH of the solution was adjusted to 6 with ammonium carbonate. Dimethylsulfoxide (5 ml) was added and the mixture was stirred at room temperature for 16 h. Upon completion, the reaction mixture was concentrated in vacuo until ca 5 ml remained. The crude product was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C18 25 cm x 20 mm id reversed phase column (0.1% TFA/CH₃CN - 0.1% TFA/H₂0 90 - 10, 10 ml/min. 220 nm). The combined HPLC fraction was lyophilised to give the title peptide as a white powder.

Examples 28 and 29 were synthesised following a similar protocol as per Example 27 to give the following products.

Example 28: Pro Gly Hey Cys Gly Pro (intramolecularly oxidized)

Example 29: Phe Hey Le uu Gly Pro Hey Pro (intramolecularly oxidized)

Example 30: Leu Leu Phe Gly Pro Hey He

I

Leu Leu Phe Gly Pro Cys He 8/12219

43

The two monomers were synthesized following a similar protocol as per Example 1. To prepare the hetero dimer the general procedure of Ruiz-Gayo was used (Ruiz-Gayo et al, 1988, Tetrahedron Letters, 29, 3845-3848). The unprotected thiol function on Leu Leu Phe Gly Pro Cys He was activated with ditihiopyridine resulting in the S-pyridyl derivative Leu Leu Phe Gly Pro Cys(SPyr) He. This derivative was reacted with the second peptide chain resulting in the disulfide which, after purification on HPLC, resulted in the final product.

Synthesis of Examples 31 -34 below was accomplished following a similar protocol as per Example 1 with S-methyl-cysteine (purchased from Bachem) as special aminoacid. Fmoc- Cys(Me)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/ HOBT mediated coupling with a coupling time of lh. The products obtained were as follows:

Example 3 i Leu Leu Phe Gly Pro Cys(Me) He Example 32 Leu Leu Tyr Ser Pro Cys(Me) Phe

Example 33 Pro Gly Cys(Me) Cys(Me) Gly Pro

Example 34 Leu Ala Phe Glu Pro Cys(Me) Met

Synthesis of Examples 35-38 below was accomplished following a similar protocol as per Example 31 with S-methyl-cysteine (purchased from Bachem) as special aminoacid. Fmoc-Cys(Me)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/ HOBT mediated coupling with a coupling time of lh. Oxidation into sulfoxide was accomplished with sodium periodate in water/MeOH (Drabowicz, I and Mikolajczyk, M. Org. Prep. Proc Int. vol 14 1982 45-89). The products obtained were as follows: Example 35: Leu Leu Phe Gly Pro Cys(Me,O) He

Example 36 Gly Pro Cys(Me,O) Cys(Me,O) Pro Gly

Example 37 Leu Leu Tyr Ser Pro Cys(Me,0) Phe

Example 38 Leu Leu Phe Arg Pro Cys(Me,0) He Synthesis of Examples 39-42 below was accomplished following a similar protocol as per Example 31 with S-methyl-cysteine (purchased from Bachem) as special aminoacid. Fmoc-Cys(Me)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/ HOBT mediated coupling with a coupling time of 1 h. Oxidation into sulfones was accomplished with hydrogen peroxide in water (Pomerantz, A and Conner, R: JACS vol 61 (1939) 3388). The products obtained were as follows:

Example 39: Leu Leu Phe Gly Pro Cys(Me,0₂) He

Example 40 Leu Leu Tyr Ser Pro Cys(Me,O₂) Phe Example 41 Leu Leu Phe Trp Pro Cys(Me,O₂) He

Example 42 Ala Pro Cys(Me,O₂) Cys(Me,O₂) Val Pro

Synthesis of Examples 43- 46 below was accomplished following a similar protocol as per Example 1 with S-tBu cysteine (purchased from Bachem) as special aminoacid. Fmoc-Cys(tBu)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/ HOBT mediated coupling with a coupling time of lh. The products obtained were as follows:

Example 43 Leu Leu Phe Gly Pro Cys(tBu) He Example 44 Leu Leu Tyr Ser Pro Cys(tBu) Phe

Example 45 Leu Leu Phe Gly Pec Cys(tBu) He

Example 46 He Cys Tyr Cys(tBu) Glu

Synthesis of Examples 47- 50 below was accomplished following a similar protocol as per Example 1 with Methionine sulfoxide (purchased from Bachem) as special aminoacid. Fmoc-Met(O)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/HOBT mediated coupling with a coupling time of lh. The products obtained were as follows:

Example 47: Leu Leu Phe Gly Pro Met(O) He Example 48: Leu Leu Tyr Ser Pro Met(O) Phe Example 49: Leu Leu Phe Ala Pro Met(O) He Example 50: Val He Met(O) Cys Leu Thr

Synthesis of Examples 51 - 54 below was accomplished following a similar protocol as per Example 1 with Methionine sulfone (purchased from Bachem) as special aminoacid. Fmoc-Met(O₂)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/HOBT mediated coupling with a coupling time of lh. The products obtained were as follows:

Example 5 ^'. Leu Leu Phe Gly Pro Met(O₂) He

Example 52 Leu Leu Tyr Ser Pro Met(O₂) Phe

Example 53 Leu Leu Phe Ala Pro Met(O₂) He

Example 54 Val He Met(O₂) Met(O₂) Leu Thr

Synthesis of Examples 55-66 below was accomplished following a similar protocol as per Example 1 with different special aminoacids. Fmoc-Aaa(Trt)-OH was coupled to the aminoacid(s) on the resin through a DIPCDI/HOBT mediated coupling with a coupling time of lh. Synthesis of the aminoacids were accomplished using literature methods (Morell et al. JOC vol 42 (1977) 355, Lago et al. JOC vol 57 (1992) 3493). The oxidation into dimers of Examples 58, 62 and 66 were accomplished following a similar protocol as per Example 9. The products obtained were as follows:

Example 55 Leu Leu Phe Gly Pro Aaa He Example 56 Leu Leu Tyr Ser Pro Aaa Phe

Example 57 Pro Gly Aaa Aaa Gly Pro

Example 58 Leu Leu Phe Gly Pro Aaa He (ox, dimer)

Example 59 Leu Leu Phe Gly Pro Aaa He

Example 6Q Leu Leu Tyr Ser Pro Aaa Phe

Example 61 Pro Gly Aaa Aaa Gly Pro Example 62 Leu Leu Phe Gly Pro Aaa He (ox, dimer)

Example 63: Leu Leu Phe Gly Pro Aaa He

Example 64: Leu Leu Tyr Ser Pro Aaa Phe Example 65: Pro Gly Aaa Aaa Gly Pro

Example 66: Leu Leu Phe Gly Pro Aaa He (ox, dimer)

Synthesis of Examples 67 and 68 below were accomplished following a similar protocol as per Examples 1 and 9. Fmoc-(Se)-pMeBzl-Selenocysteine were synthesized through lit. methods (Marzoni et al. Synthetic Com. ___5(16) 2475 (1995), Ahmad R. et al. JOC 42 1228 (1977)). (Se)-p-MeBzl protected selenocystein was coupled to the aminoacid(s) on the resin through a DIPCDI/HOBT mediated coupling with a coupling time of 4h. The products obtained were as follows:

Example 67: Leu Leu Tyr Ser Pro Aaa Phe (ox, dimer) Aaa= Seleno-cysteine Example 68: Leu Leu Phe Gly Pro Aaa He (ox, dimer) Aaa= Seleno-cysteine

Example 69: Synthesis of Leu Leu Phe Gly Pro Pen He

The C-terminal aminoacid Tentagel S PHB-Ile-Fmoc (0.95 g, 0.22 mmol/g, 0.2rnmol) on resin, drypacked in the Synthesizer column, was allowed to swell in DMF for 30 minutes. The Synthesizer worked with consecutive deblocking, washing and coupling cycles consisting of 8 min recycling with 20% piperidine/DMF for each Fmoc-deblocking followed, after wash, by activation of the N^α-Fmoc-protected L-amino acid pentafiuorophenyl ester (0.8 mmol) with HOBT (0.9 mmol) and by activation of N^α-Fmoc- Pen(Trt)-OH (0.8 mmol) with HOBT (0.9 mmol) and diisopropylcarbodiimide (DIPCDI, 0.9 mmol) The activated amino acids were added to the column and recycled 30 min each (4h for the penicillamine). The synthesizer ended the synthesis with a deblocking of the N- terminal Fmoc-group and a final wash with DMF. The resulting peptide on the resin was transferred to a sintered glass funnel where it was washed twice with MeOH (2 x 10 ml) and three times with CH₂C1₂ (3 x 10 ml). The resin was allowed to dry under vacuum over night after which the peptide was side chained deprotected and cleaved from the resin using ethanedithiol/TFA 5/95 (20 ml) at room temperature for 3 h. The resin was filtered off and washed with 3 x 10 ml of acetic acid. The combined acidic fraction was evaporated after which the residue was triturated 3 times with ether.

The crude peptide was dissolved in H₂O/CH₃CN 1/1 and lyophilized. The resulting material was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C18 25 cm x 20 mm id reversed phase column (0.1% TFA/CH₃CN - 0.1% TFA/H₂0 90 - 10, 10 ml/min, 220 nm). The combined HPLC fraction was lyophilized leaving 78 mg of the title compound.

Synthesis of Examples 70 - 92 below was accomplished following a similar protocol as per Example 69 to give the following products:

Example 70 Met Leu Phe Ser Pro Pen Trp

Example 71 Val Val Phe Ala Pro Pen Tyr Example 72 Leu Ala Phe Glu Pro Pen Met

Example 73 Lys Pen Arg Pen Lys

Example 74 Pro Asp Pen Pen He Pro

Example 75 Pro Gly Cys Pen Gly Pro

Example 76 Leu Leu Phe Gly Pro pen He (pen = D-penicillamine) Example 77 Gly Pro Pen Pen Pro Gly Example 78 Leu Pen Ala Pen Val

Example 79 He Pen Tyr Pen Glu

Example 80 Leu Leu Tyr Ser Pro Pen Phe

Example 81 Phe Pen He Cys Lys Example 82 Phe Cys He Pen Lys

Example 83 Leu Leu Phe Gly Pro Pen He Leu Asn NH2

Example 84 Ala Pro Pen Pen Val Pro

Example 85 Phe Pen He Pen Lys

Example 86 Leu Leu Phe Asp Pro Pen He Example 87 Leu Leu Phe Ala Pro Pen He

Example 88 Leu Leu Phe Arg Pro Pen He

Example 89 Leu Leu Phe Gly He Pen He Example 90 Leu Leu Phe Gly Pec Pen He

Example 91 Leu Leu Phe Gly Pro Pen He Leu Asn Arg Example 92 Leu Leu Phe Trp Pro Pen He

Example 93 Leu Leu Phe Gly Pro Pen He (ox, dimer)

Leu Leu Phe Gly Pro Pen He

Leu Leu Phe Gly Pro Pen He (80mg, 0.1 mmol) was prepared following the protocol as per Example 69. The peptide was dissolved in 5% aqueous acetic acid (25 ml) and the pH of the solution was adjusted to 6 with ammonium carbonate. Dimethylsulfoxide (5 ml) was added and the mixture was stirred at room temperature for 16 h. Upon completion, the reaction mixture was concentrated in vacuo until ca 5 ml remained. The crude product was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5 Cl 8 25 cm x 20 mm id reversed phase column (0.1% TFA/CH₃CN - 0.1% TFA/H₂0 90 - 10, 10 ml/min, 220 nm). The combined HPLC fraction was lyophilised to give the title peptide as a white powder. Synthesis of Examples 94-95 below was accomplished following a similar protocol as per Example 93 to give the following products:

Example 94: Met Leu Phe Ser Pro Pen Trp (ox, dimer) Example 95: Leu Leu Phe Gly Pro pen He (ox, dimer, D-penicillamine)

Example 96: Gly Pro Pen Pen Pro Gly (intramolecularly oxidized)

Gly Pro Pen Pen Pro Gly w uas synthesised following a similar protocol as per Example 69. Intramolecular oxidation of the monomer was accomplished following the protocol as per Example 93, but this time in higher dilution. The peptide (0.05mmol) was dissolved in 5% aqueous acetic acid (100 ml) and the pH of the solution was adjusted to 6 with ammonium carbonate. Dimethylsulfoxide (5 ml) was added and the mixture was stirred at room temperature for 16 h. Upon completion, the reaction mixture was concentrated in vacuo until ca 5 ml remained. The crude product was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C18 25 cm x 20 mm id reversed phase column (0.1% TFA/CH₃CN - 0.1% TFA/H₂O 90 - 10, 10 ml/min, 220 nm). The combined HPLC fraction was lyophilised to give the title peptide as a white powder.

Synthesis of Examples 97-105 below was accomplished following a similar protocol as per Example 96 to give the following intermolecularly oxidised products:

Example 97: Pro Gly Gly Pro (ox)

Example 98: Pro Gly Pen Pen Gly Pro (ox)

Example 99: Thr Pro Pe un Pen Phe Ala (ox) u Example 100: Val He Pen Pen Leu Thr (ox)

Example 101 : Pro Gly P uen Cys Gly Pro (ox)

Example 102: Phe Cys L ueu Gly Pro Pen Pro (ox)

Example 103: Gly Pro Pen Cys Pro Gly (ox)

Example 104: Leu Pen Ala Pen Val (ox)

Example 105: He Pen Thr Pen Glu (ox)

Example 106: Pro Gly Pen Pen Gly Pro (ox, Head to Head dimer)

I I Pro Gly Pen Pen Gly Pro

To prepare the parallel homodimer a single peptide chain with an Acm (acetamidomethyl) protecting group on one of the penicillamines and with the other penicillamine unprotected (H-Phe-Pen-Leu-Gly-Pro-Pen(Acm)-Pro-OH) was synthesized using the same protocol as per Example 69. The monomer was dimerized through oxidation of the free penicillamines using the same protocol as per Example 93. The second disulfidebond was accomplished using the protocol of Ruiz-Gayo (Ruiz-Gayo et al, 1988, Tetrahedron Letters, 29, 3845- 3848) in which a onepot deprotection and oxidation of the Acm protected penicillamine with iodine in 80% aqueous acetic acid resulted in a crude product which was purified on HPLC.

Example 107: Phe Pen Leu Gly Pro Pen Pro (ox, Head to Head dimer)

I I

Phe Pen Leu Gly Pro Pen Pro was synthesised following a similar protocol as per Example 106.

Example 108: Leu Leu Tyr Ser Pro Pen Phe (ox)

I Leu Leu Tyr Ser Pro Cys Phe

The two monomers were synthesized following a similar protocol as per Example 69. To prepare the hetero dimer the general procedure of Ruiz-Gayo was used (Ruiz-Gayo et al, 1988, Tetrahedron Letters, 29, 3845-3848). The unprotected thiol function Leu Leu Tyr Ser Pro Cys Phe was activated with ditihiopyridine resulting in the S-pyridyl derivative Leu Leu Tyr Ser Pro Cys(S-Pyr) Phe This derivative was reacted with the second peptide chain resulting in the disulfide which, after purification on HPLC, resulted in the final product.

Example 109: Pro Pen Pro Gly Leu Pen Phe (ox, Head to Tail dimer)

Phe Pen Leu Gly Pro Pen Pro To prepare the antiparallel homodimer the general procedure of Ruiz-Gayo was used (Ruiz-Gayo et al, 1988, Tetrahedron Letters, 29, 3845-3848). Two single peptidechains each with an Acm (acetamidomethyl) protecting group on one of the penicillamines and with the other penicillamine unprotected (H-Phe-Pen-Leu-Gly-Pro-Pen(Acm)-Pro-OH and H-Phe-Pen(Acm)-Leu-Gly-Pro-Pen-Pro-OH) was synthesized using the same protocol as in Example 69. The unprotected penicillamine on one of the monomers was activated with ditihiopyridine resulting in the S-pyridyl derivative H-Phe-Pen(SPyr)-Leu-Gly-Pro- Pen(Acm)-Pro-OH. This derivative was reacted with the second peptide chain resulting in the first disulfide. The second disulfide bond was accomplished using the same protocol as in Example 106 with iodine in 80% aqueous acetic acid which, after purification on HPLC, resulted in the final product.

Example 1 10: Synthesis of Leu Ala Phe Glu Pro Met Met The C-terminal aminoacid Tentagel S PHB-Met-Fmoc (0.9 g, 0.23 mmol/g, 0.2mmol) on resin, drypacked in the Synthesizer column, was allowed to swell in DMF for 30 minutes. The synthesizer worked with consecutive deblocking, washing and coupling cycles consisting of 8 min recycling with 20% piperidine/DMF for each Fmoc-deblocking followed, after wash, by activation of the N^α-Fmoc-protected L-amino acid pentafiuorophenyl ester (0.8 mmol) with HOBT (0.9 mmol). For D-methionine the N^α- Fmoc-met-OH derivative was used which was coupled to aminoacids on the resin with HOBT (0.9 mmol) and DIPCDI (0.9 mmol). The activated amino acids were added to the column and recycled 30 min each. The synthesizer ended the synthesis with a deblocking of the N-terminal Fmoc-group and a final wash with DMF. The resulting peptide on the resin was transferred to a sintered glas funnel where it was washed twice with MeOH (2 x 10 ml) and three times with CH₂C1₂ (3 x 10 ml). The resin was allowed to dry under vacuum over night after which the peptide was side chained deprotected and cleaved from the resin using ethanedithiol/TFA 5/95 (20 ml) at room temperature for 3 h. The resin was filtered off and washed with 3 x 10 ml of acetic acid. The combined acidic fraction was evaporated after which the residue was triturated 3 times with ether.

The crude peptide was dissolved in H₂O/CH₃CN 1/1 and lyophilized. The resulting material was purified on HPLC using a Gilson 305 and 306 HPLC system with a Kromasil 100-5C 18 25 cm x 20 mm id reversed phase column (0.1 % TFA/CH₃CN - 0.1 % TFA/H₂0 90 - 10, 10 ml/min, 220 nm). The combined HPLC fraction was lyophilized leaving 63 mg of the title compound.

Synthesis of Examples 111 - 134 below were accomplished following a similar protocol as per Example 1 10 to give the following products.

Example 111 Met Leu Phe Ser Pro Met Trp

Example 112 Val Val Phe Ala Pro Met Tyr Example 113 Leu Leu Phe Gly Pro Met He Example 1 14: Gly Pro Met Met Pro Gly Example 1 15: Gly Pro Cys Met Pro Gly Example 1 16: Gly Pro Met Cys Pro Gly Example 1 17: Gly Pro Met He Example 1 18: Leu Leu Tyr Ser Pro Met Phe Example 119: Ala Pro Met Met Val Pro Example 120: Leu Leu Phe Asp Pro Met He Example 121 : Leu Leu Phe Trp Pro Met He Example 122: Leu Leu Phe Arg Pro Met He Example 123: He Cys Thr Met Glu

Example 124: Leu Leu Phe Gly Pec Met He Example 125: Leu Leu Phe Ala Pro Met He Example 126: Leu Leu Phe Gly He Met He Example 127: Phe Met Leu Gly Pro Met Pro Example 128: Leu Leu Phe Gly Pro met He met=D-methionine Example 129: Leu Leu Tyr Ser Pro met Phe Example 130: Pro Gly met met Gly Pro Example 1 1 : Leu Ala Phe Glu Pro met Met Example 132: Phe Met He Met Lys Example 133: Phe Met Leu Gly Pro Met Pro Example 134: Lys Met Arg Met Lys

Synthesis of Examples 135 to 137 below was accomplished following a similar procedure as per Examples 17 to 25.

Example 135: Pro Gly Hey Cys Gly Pro Example 1 6: Pro Gly Hey Hey Gly Pro Example 137: Met Leu Phe Ser Pro Hey Trp. Synthesis of Examples 138 to 146 below was accomplished following a similar protocol as per Example 69 to give the following products:

Example 138: Leu Ala Phe Asn Pro Pen Met Example 139: Pro Glv Pen Cvs Glv Pro

Example 140: Pro Gly Pen Pen Gly Pro

Example 141 ; Gly Pro Pen Cys Pro Gly

Example 142: Thr Pro Pen Pen Phe Ala

Example 143: Val He Pen Pen Leu Thr Example 145: Pro Gly Pen (Acm) Pen Gly Pro

Example 146: Phe Pen Leu Gly Pro Pen(Acm) Pro

Example 147:Delaved Type Hvpersensitivitv fDTHI Te.st

This test was used to show immunomodulatory activity.

The ability of the peptides according to the invention to modulate immune responses can be illustrated by their effect in the delayed type hypersensitivity (DTH) test in mice. The DTH test is used to illustrate immunomodulation, the protocol for which is described, for example, by Carlsten H., et al (1986) Int. Arch. Allergy Appl. Immunol 81 :322, herein incorporated by reference. The peptides were tested at one or more of the following dosages: 0.0003 μmol/kg, 0.003 μmol/kg, 0.03 μmol/kg, 0.3 μmol/kg, and 3.0 μmol/kg.

Male and female Balb/c mice were obtained from Bomholtsgaard (Denmark) with a weight of 18-20 grams each. 4-Ethoxymethylene-2-phenyloxazolin-5-one (OXA) (Sigma Chemicals) was used as the antigen in the DTH test.

The mice were sensitized, Day 0, by epicutaneous application of 150 μl of an absolute ethanol-acetone (3 : 1 ) solution containing 3% OXA on the shaved abdomen. Treatment with peptides according to the invention, or vehicle (phosphate buffer, pH 7.4, containing a mixture of Buffer A and Buffer B in the proportion 63%:37% B (Buffer A: Na₂HP0₄, 0.89

g/lOOml, EDTA 0.05 g/ 100ml "Buffer B: NaH₂P0₄, 0.69 g/lOOml, EDTA 0.05 g/lOOml) was initiated by oral feeding immediately after sensitization and continued once daily (a.m) until Day 6. Seven days after sensitization, both ears of all mice were challenged on both sides by topical application of 20 μl 1% OXA dissolved in peanut oil. Ear thickness was measured prior to and at 24hrs or 48 hrs after challenge using an Oditest spring calliper. Challenges and measurements were performed under light pentobarbital anaesthesia.

The intensity of the DTH reactions was measured according to the method described by van Loveren H., et al (1984) J. Immunol. Methods 67: 31 1 and expressed according to the formula: Tg^g-T-o μm units, where tO, t24 and t48 represent the ear thickness at time 0, +24hrs or +48 hrs after challenge respectively, in individual tests (T). The results are expressed as the mean +/- S.E.M.. The level of significance between means of the groups is obtained by Student's two-tailed t-test. The immunomodulating effect of the peptide is reflected in a significant difference in the increase or decrease in ear thickness as compared to the control (phosphate buffer).

The peptides of Examples 3, 4, 17 to 19, 21 to 24, 29, 31 to 35, 37, 40, 42 to 56, 58, 69 to 71, 73 to 82, 84 to 90, 92, 93, 95, 97, 101 to 104 and 132 to 146 were tested using the DTH test. Some of the peptides were found to exhibit very good effect, good effect or to be effective in this particular test and some were found to exhibit little toward no effect compared with the control in this particular test.

Claims

1. A physiologically active peptide of formula (I):

A-Rι-R2-R3-(R4)χ-B (I)

wherein each A is independently selected from H, a protecting group, or at least one amino acid residue independently selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains;

R. is a residue of an amino acid selected from glycine (Gly), proline (Pro), aspartic acid (Asp), arginine (Arg), alanine (Ala), isoleucine (He), tryptophan (Trp), serine (Ser), cysteine (Cys), glutamic acid (Glu), asparagine (Asn), and R₈,

R₂ is a residue of an amino acid selected from cysteine (Cys), proline (Pro), isoleucine (He), alanine (Ala), tyrosine (Tyr), threonine (Thr), arginine (Arg), pipecolic acid (Pec), and Rg,

R₃ is a residue of an amino acid selected from cysteine (Cys) and R₈, and

at least one of R_, R₂ and R₃ being R₈ and at most one of R,, R₂ and R₃ being cysteine; and

R₈ is an amino acid residue of formula (II) or formula (III)

Formula (II):

Formula (III):

wherein each of R₅ and Rg is independently selected from H, alkyl, aryl and alkoxy,

R₇ is SOH, SO₂H, SO₃H, SR₉, SeR₉ or TeR<, (wherein R, is H, alkyl, aryl, alkoxy, SR₁₀, SOR₁₀ or SO₂R₁₀, and R₁₀ is H, alkyl, aryl or alkoxy),

n is selected from 0, 1, 2, 3 and 4, and

m is selected from 0, 1 , 2, 3 and 4;

provided that, when R₈ is an amino acid residue of formula (II), when n is 0 and R₇ is SH, R₅ and Rg are not both H;

R₄ is a residue of an amino acid selected from glycine (Gly), phenylalanine (Phe), valine (Val), isoleucine (He), lysine (Lys), proline (Pro), tryptophan (Trp), tyrosine (Tyr), glutamic acid (Glu), leucine (Leu) and methionine (Met);

x is 0 or 1 ;

with the provisos that (i) when R₃ is other than penicillamine (Pen) or methionine (Met) (i.e. it is not a residue R₈ of formula II in which n is 0, R₇ is SH and RJ, and R₆ are both methyl or in which n is 1, R₇ is SCH₃ and R₅ and Rg are both H), and R. is serine (Ser) and R₂ is threonine (Thre) or Rι and R₂ are both isoleucine (He), x is 1 ;

(ii) when R₃ is penicillamine (Pen), and R. is serine (Ser) and R₂ is proline (Pro) or threonine (Thr), or R. is asparagine (Asn) and R₂ is arginine (Arg), x is 1 ;

(iii) when the or each R₈ is methionine, x is 1, and

R] is a residue of an amino acid selected from glycine (Gly), proline (Pro), aspartic acid (Asp), arginine (Arg), alanine (Ala), tryptophan (Trp), serine (Ser), methionine (Met), cysteine (Cys), and glutamic acid (Glu),

R₂ is a residue of an amino acid selected from cysteine (Cys), proline (Pro), isoleucine (He), methionine (Met), pipecolic acid (Pec), threonine (Thr), and arginine (Arg), and

R₃ is a residue of an amino acid selected from cysteine (Cys) and methionine (Met), and

R₄ is a residue of an amino acid selected from phenylalanine (Phe), valine (Val), isoleucine (He), proline (Pro), tryptophan (Trp), tyrosine (Tyr), glutamic acid (Glu), methionine (Met), and lysine (Lys);

except that, when R₃ is methionine and R. is alanine (Ala), R₄ is not phenylalanine (Phe), or R₂ is threonine (Thr), R₄ is not lysine (Lys);

each B is independently selected from the group consisting of OH, NH , an oxygen or a nitrogen carrying a protecting group, or at least one amino acid residue selected from the group of amino acid residues having aliphatic side chains, aliphatic hydroxyl side chains, basic side chains, acidic side chains, secondary amino groups, amide side chains, aromatic side chains, and sulphur containing side chains; provided that when x is 0, B is OH, NH₂, or an oxygen or a nitrogen carrying a protecting group;

the entire peptide sequence containing 3 to 30 amino acid residues; or

a homo- or heterodimer thereof.

2. A peptide according to claim 1 wherein R. to R₄ are residues of the amino acids selected from the following:

R, selected from Gly, Pro, Asp, Arg, Ala, He, Ser, Glu, R₈, R₂ selected from Cys, Pro, He, Pec, R₈, R₃ selected from Cys, R₈, R₄ selected from Gly, Phe, Val, He, Pro, Lys, Leu;

at least one of R R₂ and R₃ being R₈ and R_g being Met, Pen, homocysteine (Hey), a residue of the formula (II) wherein R₅ and Rg are both H, n is 0 and R₇ is SCH₃ (Cys(Me)). SOCH₃ (Cys(Me,0)), or SO₂CH₃ (Cys(Me,O₂)), wherein R₅ and Rg are both H, n is 1 and R₇ is SOCH₃ (Met,O) or SO₂CH₃ (Met,0₂), or wherein one of R₅ and Rg is H and the other is methyl, n is 0 and R₇ is SH or a residue of formula (III) wherein m is 1 , n is 0 and R₇ is SH.

3. A peptide according to claim 1 wherein R. to R₄ are residues of amino acids as follows:

R, selected from Gly, Pro, Ser, Asp, Ala, Glu, R₈,

R₂ selected from Cys, Pro, He, R₈,

R₃ selected from Cys, R₈,

R, selected from Gly, Phe, He, Pro, Lys, Met; at least one of R., R₂ and R₃ being R₈ and R₈ being Pen or Met.

4. A peptide according to claim 1 wherein R, to R₄ are residues of amino acids as follows:

R, selected from Gly, Pro, Asp, Ala, Ser, Met, Glu, R₂ selected from Pro, Met, He, R₃ Met, R₄ selected from Phe, Val, He, Pro, Tyr, Met, Lys.

5. A peptide according to claim 1 wherein R, to R₄ are residues of amino acids as follows:

Ri selected from Gly, Asp, Ser, Met, Glu, R₂ selected from Pro, He, R₃ Met, R» selected from Phe, He, Met, Lys.

6. A peptide according to claim 1 wherein R. to R₄ are residues of the amino acids selected from the following:

R, selected from Gly, Pro, Asp, Arg, He, Pen, R₂ selected from Cys, Pro, He, Pen, Pec, R₃ selected from Cys, Pen, R₄ selected from Gly, He, Pro, Lys, Leu;

at least one of R., R₂ and R₃ being Pen.

7. A peptide according to claim 1 wherein Rj to R₄ are residues of amino acids as follows:

R, selected from Gly, Pro, Pen, R₂ selected from Cys, Pro, He, Pen, R₃ selected from Cys, Pen, R₄ selected from Gly, He, Pro, Lys;

at least one of R., R₂ and R₃ being Pen.

8. A peptide according to claim 1 wherein Ri to R₄ are residues of the amino acids selected from the following:

R, selected from Gly, He, Ser, R₂ selected from Pro, R₈ R₃ selected from Cys, R₈, Rst, selected from Phe, He, Trp, Leu;

at least one of R₂ and R₃ being R₈ and R₈ being homocysteine (Hey), a residue of the formula (II) wherein R₅ and Rg are both H, n is 0 and R₇ is SCH₃ (Cys(Me)), SOCH₃ (Cys(Met,0)), or SO₂CH₃ (Cys(Me,0₂)), wherein R₅ and Rg are both H, n is 1 and R₇ is SOCH₃ (Met,O) or SO₂CH₃ (Met,O₂), or wherein one of R₅ and Rg is H and the other is methyl, n is 0 and R₇ is SH or a residue of formula (III) wherein m is 1 , n is 0 and R₇ is SH.

9. A peptide according to any of claims 1 to 8 wherein x is 1.

10. A peptide according to any of claims 1 to 8 of formula:

A-R.-Rg-Cys-R^B

A-R,-Cys-R₈-R₄-B

(wherein A, R R₈, R₄, and B are as defined in any of claims 1 to 8).

1 1. A peptide according to any of claims 1 to 8 of formula:

A-R₈-R₂-Rg-R₄-B A-R₈-R₂-Cys-R₄-B

A-Cys-R₂-R₈-R₄-B

(wherein A, R₈, R₂, R,, and B are as defined in any of claims 1 to 8).

12. A peptide according to any of claims 1 to 1 1 wherein the or each peptide sequence is from 4 to 20 amino acid residues in length.

13. A peptide according to any of claims 1 to 1 1 wherein the or each peptide sequence is from 4 to 15 amino acid residues in length.

14. A peptide according to any of claims 1 to 13 in the form of a cyclised monomer in which one amino acid residue Rg forms an intramolecular bridge with another R₈ residue or with a cysteine residue.

15. A peptide according to claim 1 of the formula:

Val Val Phe Ala Pro Met Tyr

Leu Leu Tyr Ser Pro Met Phe

Ala Pro Met Met Val Pro

Leu Leu Phe Asp Pro Met He

Leu Leu Phe Gly Pro met He Leu Leu Tyr Ser Pro met Phe

Pro Gly met met Gly Pro

Leu Ala Phe Glu Pro met Met

Phe Met He Met Lys

wherein met denotes the residue of D-methionine.

16. A peptide according to claim 1 of the formula:

Leu Leu Phe Gly Pro Pen He Phe Pen He Cys Lys Phe Pen He Pen Lys Leu Leu Phe Asp Pro Pen He Leu Leu Phe Arg Pro Pen He Leu Leu Phe Gly Pec Pen He (Leu Leu Phe Gly Pro pen Ile)₂ ; Pro Gly Cys Pen Gly Pro (ox)

Pro Gly P uen Cys Gly Pro (ox)

Phe Cys L ueu Gly Pro Pen Pro (ox)

Pro Gly Pen Cys Gly Pro Pro Gly Pen Pen Gly Pro Gly Pro Pen Cys Pro Gly Val He Pen Pen Leu Thr Pro Gly Pen(Amc) Pen Gly Pro

wherein (ox) denotes intramolecularly cyclised, pen denotes D-penicillamine and Acm denotes acetamidomethyl.

17. A peptide according to claim 2 of the formula:

Leu Leu Phe Gly Pro Xxx He

Leu Leu Tyr Ser Pro Hey Phe

Leu Leu Phe Gly Pro Hey He Leu Asn Arg

Leu Leu Phe Gly Pro Cys (Me,O) He Leu Leu Tyr Ser Pro Cys(Me,O₂) Phe

Val He Met(O) Cys Leu Thr

Val He Met(O₂) Met(O₂) Leu Thr

Leu Leu Tyr Ser Pro Aaa Phe

(Leu Leu Phe Gly Pro Aaa Ile)₂ Met Leu Phe Ser Pro Hey Trp wherein Xxx denotes a residue of formula (III) in which m is 1 , n is 0 and R₇ is SH and Aaa denotes a residue of formula (II) in which one of R₅ and Rg is H and the other is methyl, n is 0 and R₇ is SH.

18. A peptide according to claim 1 specifically identified herein.

19. A peptide according to any of claims 1 to 18 free from an added transport agent.

20. Acid addition salts of peptides according to any of claims 1 to 19.

21. Acid addition salts according to claim 20 selected from the group consisting of salts of hydrochloric, hydrobromic, nitric, perchloric, sulphuric, citric, tartaric, phosphoric, lactic, benzoic, glutamic, oxalic, aspartic, pyruvic, acetic, succinic, fumaric, maleic, oxaloacetic, isethionic, stearic, phthalic, methanesulphonic, p-toluene sulphonic, benzenesulphonic, lactobionic, glucuronic, and trifluoroacetic acids.

22. Base salts of peptides according to any of claims 1 to 19.

23. Base salts according to claim 22 selected from the group consisting of alkali metal and alkaline earth salts, organic base salts, and amino acid salts.

24. An oral dosage form comprising an immunomodulatory peptide according to any of claims 1 to 23.

25. An oral dosage form according to claim 24 wherein the amount of the immuno-modulatory peptide needed to induce an observable level of modulated immune response in a mammal when administered orally is less than the amount of the same immunomodulatory peptide when administered parenterally which is needed to achieve a similar observable level of modulated immune response in the said mammal.

26. An oral dosage form according to claim 24 or 25 not including an added transport agent.

27. An intra-tracheal dosage form comprising an immunomodulatory peptide according to any of claims 1 to 23.

28. An intra-tracheal dosage form according to claim 27 wherein the amount of the immunomodulatory peptide needed to induce an observable level of modulated immune response in a mammal when administered intra-tracheally is less than the amount of the same immunomodulatory peptide when administered parenterally which is needed to achieve a similar observable level of modulated immune response in the said mammal.

29. An intra-tracheal dosage form according to claim 27 or 28 not including an added transport agent.

30. A nasal dosage form comprising an immunomodulatory peptide according to any of claims 1 to 23.

31. A nasal dosage form according to claim 30 wherein the amount of the immuno-modulatory peptide needed to induce an observable level of modulated immune response in a mammal when administered nasally is less than the amount of the same immunomodulatory peptide when administered parenterally which is needed to achieve a similar observable level of modulated immune response in the said mammal.

32. A nasal dosage form according to claim 31 not including an added transport agent.

33. Pharmaceutical formulation comprising at least one peptide according to any of claims 1 to 23 or a salt thereof together with a pharmaceutically acceptable carrier therefor.

34. Pharmaceutical formulation according to claim 33 wherein the pharmaceutical formulation is for oral administration.

35. A peptide according to any of claims 1 to 23 or a salt thereof for use in therapy.

36. A peptide according to any of claims 1 to 23 or a salt thereof for use in cancer therapy.

37. A peptide according to any of claims 1 to 23 or a salt thereof for use in rheumatoid arthritis therapy.

38. A peptide according to any of claims 1 to 23 or a salt thereof in an oral dosage form for use in therapy.

39. A peptide according to any of claims 1 to 23 or a salt thereof in an oral dosage form for use in cancer therapy.

40. A peptide according to any of claims 1 to 23 or a salt thereof in an oral dosage form for use in autoimmune disease therapy.

41. Use of a physiologically active peptide according to any of claims 1 to 23 or a salt thereof in the preparation of a medicament suitable for the treatment of disease.

42. Use of a physiologically active peptide according to any of claims 1 to 23 or a salt thereof in the preparation of a medicament suitable for the treatment of cancer.

43. Use of a physiologically active peptide according to any of claims 1 to 23 or a salt thereof in the preparation of a medicament suitable for the treatment of autoimmune disease.

44. Use of a physiologically active peptide according to any of claims 1 to 23 or a salt thereof free from an added transport agent in the preparation of a medicament suitable for the treatment of rheumatoid arthritis.

45. A method for the preparation of a pharmaceutical formulation comprising bringing into association at least one peptide according to any of claims 1 to 23 or at least one salt thereof, and a pharmaceutically acceptable carrier therefor.

46. A method of inducing an immunomodulatory response in a mammal which comprises identifying a mammal in need of an immunomodulatory response; and administering to the epithelial cell lining of the mammal a dose of a physiologically active peptide according to any of claims 1 to 23 or salt thereof enough to induce said modulated immune response and thereby a therapeutic effect.

47. A method according to claim 46 which comprises administering to the MALT of the mammal a dose of a physiologically active peptide according to any of claims 1 to

23 or salt thereof in an amount sufficient to induce a modulated immune response and thereby a therapeutic effect.

48. A method of making a peptide according to any of claims 1 to 23 by a chemical process in which individual amino acid residues or fragments of peptides of the invention are joined to form peptide bonds and wherein protecting groups are employed at the beginning and/ or end of the process.