Treatment of T-cell mediated diseases

[0001] The invention relates to a medicament (pharmaceutical composition) containing purified diketopiperazines. The treatment of T cell mediated diseases, inhibition of T cell activation using certain diketopiperazines, methods for synthesizing diketopiperazines and sp persons for the production of improved pharmaceutical compositions of proteins and peptides to increase or decrease the diketopiperazine content of the compositions, and thus obtained, are described in the following. improved pharmaceutical compositions.

BACKGROUND [0002] T cell mediated diseases represent a large number of disorders of the immune system. It is especially believed that T cells are cells that start and consolidate the course of autoimmune diseases. Autoimmune diseases are a group of eighty serious chronic diseases that affect millions of people in the United States alone. Autoimmune diseases are characterized by the responsiveness of the immune system to endogenous (auto) antigens. These immune responses to autoantigens are maintained by the sustained or recurrent activation of autoreactive T cells and, directly or indirectly, autoreactive T cells are responsible for the characteristic tissue damage and destruction observed in autoimmune diseases. Despite this,

SUMMARY OF THE INVENTION [0003] The invention is defined in the claims. Described is a non-constitutive part of the invention for the treatment of diseases mediated by T cells.

The method comprises administering to an animal in need thereof an effective amount of diketopiperazine having the following formula:

<img file="PL2537524T3_D0001.tif" />

wherein:

each from R¹ and R², which may be the same or different, is:

- (a) the side chain of the amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, β-aminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine or ornithine; however, provided that when R¹ is the side chain of asparagine or glutamine, then R² can not be a side chain of lysine or ornithine, and when R¹is the side chain of lysine or ornithine, then R² it may not be a side chain of asparagine or glutamine;

(b) R.¹ is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline and / or R² is -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline; or (c) a side chain derivative of an amino acid, wherein the amino acid is one of those specified in (a) and the derived side chain has:

(i) the -NH2 group replaced by the -NHR group³ or -N (R.³) 2, each R being³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(ii) a -OH replaced by a -O-PO3H2 or -OR group³, each R³ it may be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iii) a -COOH group replaced with a -COOR group³, each R³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iv) the -COOH group replaced with the -CON group (R.⁴) 2, each R being⁴ can independently be H or substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(v) -SH replaced with -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2CH (NH2) -COOH;

(vi) a -CH2- group replaced with a -CH (NH2) - or -CH (OH) - group;

(vii) -CH3 replaced by a -CH2-NH2 or -CH2-OH group; and / or (viii) H which is attached to a halogen replaced carbon; or a pharmaceutically acceptable salt thereof. Further described is a method for inhibiting T-cell activation that is not part of the invention. The method comprises administering to an animal in need thereof an effective amount of diketopiperazine of formula I or a physiologically acceptable salt thereof. Further described is not part of the invention

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and diketopiperazine having the following formula:

<img file="PL2537524T3_D0002.tif" />

wherein:

R⁵ and R⁶that can be the same or different, each is:

(a) the side chain of the amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, βaminophenylalanine, tyrosine, tryptophan, thyroxin or ornithine; however, provided that when R⁵ is the side chain of asparagine or glutamine, then R⁶can not be a side chain of lysine or ornithine, and when R⁵ is the side chain of lysine or ornithine, then R⁶ it may not be a side chain of asparagine or glutamine;

(b) R.⁵ is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline and / or R⁶ is -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline; or (c) a side chain derivative of an amino acid, wherein the amino acid is one of those specified in (a) and the derived side chain has:

(i) the -NH2 group replaced by the -NHR group³ or -N (R.³) 2, each R being³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(i) a -OH replaced by a -O-PO3H2 or -OR group³, each R³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iii) -CH2- replaced by a -CH (NH2) - or -CH (OH) - group;

(iv) a -CH3 group replaced with a -CH2-NH2 or -CH2-OH group; and / or (v) H which is attached to a halogen replaced carbon; or a physiologically acceptable salt thereof.

[0004] Hereinafter, a method of treating T cell mediated diseases, not forming part of the invention, is described. The method comprises administering to an animal in need thereof an effective amount of a pharmaceutical composition comprising a protein or peptide normally found in the animal, protein or peptide treated so that the composition also contains at least one diketopiperazine obtained from a protein or peptide.

[0005] Further described is a non-method of inhibiting T-cell activation. The method comprises administering to an animal in need thereof an effective amount of a pharmaceutical composition comprising a protein or peptide normally found in an animal, protein or peptide treated such that the composition also comprises at least one diketopiperazine obtained from a protein or peptide.

[0006] Also described are methods of synthesizing diketopiperazines that are not part of the invention. In one method, the method comprises heating the protein or peptide solution under effective conditions to induce the formation of diketopiperazine. In a second method, the method comprises contacting a protein or peptide solution with an enzyme that cleaves two N-terminal or two C-terminal amino acids of a protein or peptide under effective conditions to produce diketopiperazine.

[0007] The invention provides an improved drug that contains purified diketopiperazines.

[0008] Further described is a method for the preparation of an improved pharmaceutical composition of a protein or peptide which is not part of the invention. The method comprises removing at least a portion of the diketopiperazines present in the composition from the composition.

[0009] Further described is a method for preparing an improved pharmaceutical composition of a protein or peptide which is not part of the invention. The method includes treating a protein or peptide solution so as to increase the diketopiperazine content of the composition.

[0010] Also described is an improved pharmaceutical composition of a protein or peptide that is not part of a pharmaceutical composition. The improvement is that the composition contains an increased content of diketopiperazines.

BRIEF DESCRIPTION OF THE FIGURES [0011]

Fig. 1 Counting-ERK1 / 2 concentration charts for TriPS cells (CD + 4 T-cell line isolated from a influenza-immunized donor that is specific for hemagglutinin) isolated on day 20 after stimulation with OKT3 anti-CD3 antibody and incubated with 25ng of acid myristic phorbol (PMA), HC-RBL (fraction of heated human colostrum with a molecular weight less than 3 kD and containing

MR-DKP) at a dilution of 1:10 and 0.5 mM DA-DKP for 15 minutes at 37 ° C.

Fig. 2. Bar graph showing inhibition of tumor necrosis factor α (TNFa) and IL-16 from TriPS cells 12 days after stimulation with OKT3 anti-CD3 antibody. It indicates the inhibition of both secretion of TNFα and IL-16 in human colostrum (HC) 2626 (containing MR-DKP), the DA-DKP band. The maximum release observed with HC 2626 at dilutions 1: 100 and 1: 1000 results from the lytic effect

- high concentrations of human colostrum. With the use of 0.5 mM DA-DKP, no lysis was observed, and the secretion of TNFα and IL-16 is reduced.

Fig. 3. Bar graph showing inhibition of TNFα secretion from TriPS cells 10 days after stimulation with OKT3 anti-CD3 antibody. Indicates that HC RBL and DA-DKP should be further investigated for a titratable response as observed with HC 2626. May indicate strong activity.

Fig. 4. Bar graph showing inhibition of TNFα secretion from TriPS cells at different times after stimulation with OKT3 anti-CD3 antibody. Indicates that the DA-DKP and HC RBL effect is inhibitory early in the stimulation cycle, while later in the cycle (day 14) the effect is stimulatory. HC 2626 causes inhibition at any time, presumably due to other components.

Fig. 5. Bar graph showing the inhibition of TNF? Secretion from H4 # 9.25 cells (CD4 + T cell line isolated from brain tissue during autopsy, from a patient with multiple sclerosis, which is specific for the basic myelin protein) on day 7-10 after stimulation with anti antibody -CD3 OKT3. Indicates that secretion of TNFα from the T cell line is also inhibited by HC 2626, HC RBL and DA-DKP.

DETAILED DESCRIPTION OF THE PREVIOUS EMBODIMENTS [0012] The invention provides a medicament containing purified diketopiperazines.

[0013] Further described is a non-part of the invention for treating diseases mediated by T cell cells. "Cure" is used herein to reduce (fully or partially) the symptoms, duration or severity of the disease, including treating the disease or preventing the disease.

[0014] T-cell mediated diseases include transplant rejection, graft versus host disease, delayed type hypersensitivity reactions (such as delayed type allergic reactions), T cell mediated diseases and autoimmune diseases. Pulmonary diseases mediated by T cells include sarcoidosis, hypersensitivity pneumonitis, acute interstitial pneumonitis, pulmonary alveolitis, pulmonary fibrosis, idiopathic pulmonary fibrosis and other diseases characterized by inflammatory lung injury. Autoimmune diseases include multiple sclerosis, neuritis, polymyositis, psoriasis, acquired vitiligo, Sjogren's syndrome, rheumatoid arthritis, type 1 diabetes, autoimmune pancreatitis, inflammatory bowel diseases (e.g.

[0015] A T cell mediated disease is treated by administering to an animal in need thereof an effective amount of diketopiperazine having the following formula:

<img file="PL2537524T3_D0003.tif" />

wherein:

R¹ and R²that can be the same or different, each is:

(a) the side chain of the amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, aspartic acid , asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, β-aminophenylalanine, tyrosine, tryptophan, thyroxin, cysteine, homocysteine, methionine, penicillamine or ornithine; however, provided that when R¹ is the side chain of asparagine or glutamine, then R² can not be a side chain of lysine or ornithine, and when R¹is the side chain of lysine or ornithine, then R² it may not be a side chain of asparagine or glutamine;

(b) R.¹ is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline and / or R² is -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline; or (c) a side chain derivative of an amino acid, wherein the amino acid is one of those specified in (a) and the derived side chain has:

(i) the -NH2 group replaced by the -NHR group³ or -N (R.³) 2, each R being³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(ii) a -OH replaced by a -O-PO3H2 or -OR group³, each R³can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iii) a -COOH group replaced with a -COOR group³, each R³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iv) the -COOH group replaced with the -CON group (R.⁴) 2, each R being⁴ can independently be H or substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(V) -SH replaced with -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2CH (NH2) -COOH;

(vi) a -CH2- group replaced with a -CH (NH2) - or -CH (OH) - group;

(vii) -CH3 replaced by a -CH2-NH2 or -CH2-OH group; and / or (viii) H which is attached to a halogen replaced carbon; or a pharmaceutically acceptable salt thereof.

[0016] By "replaced" is meant that with reference to a side chain amino acid pattern, a particular group is replaced by another specific group. For example, the isoleucine side chain pattern is -CH (CH3) -CH2-CH3. -CH3 is replaced by the group -CH2-OH, then the formula of the derived side chain-isoleucine derivative would be -CH (CH3) -CH2-CH2-OH As another example, the alanine side chain formula is -CH3. If one of the hydrogen atoms is replaced by a chlorine atom, then the derivative side chain of alanine would be -CH2-Cl. Note that the side chain of the glycine is -H, and if H is replaced by a chlorine atom (or other halogen), the side chain obtained would be -Cl, along with a chlorine atom attached to the carbon in the ring (e.g., R.¹ = -Cl) [0017] Diketopiperazines where R is preferred¹, R² or both are the side chain of aspartic acid or glutamic acid or a derivative of such a side chain, wherein the -COOH group is replaced by a group

-COOR³ or the -CON group (R.⁴) 2, with R being³ and R⁴ are defined above. Of this group of compounds, diketopiperazines having side chains with aspartic and alanine (Asp-Ala DPK or DA-DKP) are the most preferred, side chains with glutamic acid and alanine (Glu-Ala DKP or EA-DKP), tyrosine lateral chains. and aspartic acid (Tyr-Asp DKP or YD-DKP), side chains with tyrosine and glutamic acid (Tyr-Glu DKP or YE-DKP) and derivatives of aspartic acid or glutamic acid side chains from among the four diketopiperazines, with the group -COOH is replaced by the -COOR group³ or the -CON group (R.⁴) 2, with R being³ and R⁴defined above.

[0018] Also preferred are diketopiperazines, where both Rs¹ and R² are hydrophobic side chains (e.g., phenylalanine side chain) or hydrophobic side chain derivatives. By "hydrophobic side chain derivatives" it is meant that the side-chain derived is hydrophobic, especially diketopiperazines where R is particularly preferred.¹ and / or R², which may be the same or different, each is a side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine and / or R¹ and / or R² is -CH2-CH2-CH2- and together with the adjacent atom (adjacent atoms) form proline. Of this group of compounds, diketopiperazines with side chains of glycine and leucine (Gly-Leu DKP or GLDKP), proline and phenylalanine (Pro-Phe DKP or PF-DKP) and alanine and proline (Ala-Pro DKP or AP-DKP) are most preferred. ).

[0019] Additional preferred diketopiperazines are those where R¹, R² or both are a methionine side chain, an arginine side chain or a derivative of these side chains. The most preferred of these is diketopiperazine, where R¹ is the side chain of methionine and R² is the arginine side chain (Met-Arg DKP or MR-DKP).

[0020] By "side chain" of an amino acid is understood the portion of the amino acid attached to

And nh₂-ch-cooh common skeleton of all the amino acids listed above. For example, the glycine side chain is -H, the alanine side chain is -CH3, and the serine side chain is

-CH2OH.

[0021] By "hydrophobic" is meant a side chain or a side chain derivative that is not charged at physiological pH and is repelled by the aqueous solution.

By &amp; quot; alkyl &amp; quot; is meant a saturated straight-chain or branched hydrocarbon containing 1-10 carbon atoms, preferably 1-6 carbon atoms. Lower alkyl means a saturated straight-chain or branched hydrocarbon containing 1-6 carbon atoms. [0023] By "cycloalkyl" is meant a saturated cyclic hydrocarbon containing at least one ring, each ring containing at least three carbon atoms. Preferably, the cycloalkyl contains one ring of 4-8 carbon atoms.

[0024] By "heterocycloalkyl" is meant a cycloalkyl having one or more ring atoms of at least one ring replaced by O, S or N.

[0025] By "aryl" is meant an aromatic group having at least one aromatic ring (e.g., phenyl).

[0026] By "alkylaryl" is meant a lower alkyl having H replaced with an aryl (e.g., -CH2-C6H5 or -CH3CH (C6H5) CH3).

[0027] By "aralkyl" is meant an aryl having H replaced by a lower alkyl (e.g., -C6H4CH3).

[0028] By "heteroaryl" is meant an aryl having one or more ring carbon atoms on at least one of the rings replaced by O, S or N.

[0029] By "substituted" it is meant that the molecule is substituted with one or more substituents selected from the following group: -OH, NH2, -SH, -COOH and / or halogen atoms.

[0030] By "halogen" is meant chlorine, fluorine, bromide or iodine. Preference is given to chlorine or bromine.

[0031] Diketopiperazines of the formula I are effective in the treatment of diseases mediated by T cells, because they inhibit the activation of T cells. For this reason, diketopiperazines of the formula

-9I can be used to treat inflammation or inflammatory diseases that are caused by, exacerbated by or include activated T cells. "Inhibition" is used herein to mean reducing (completely or partially) or preventing.

[0032] Methods of preparing diketopiperazines are well known in the art and these methods can be used to synthesize the diketopiperazines described herein. See, e.g., U.S. Patents 4,694,081, 5,817,751, 5,990,112, 5,932,579 and 6,555,543, U.S. Patent Application Publication 2004/0024180, PCT Applications WO 96/00391 and WO 97/48685 and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374 (1998).

[0033] For example, diketopiperazines can be prepared first by synthesizing dipeptides. The dipeptides may be synthesized using methods well known in the art, using L-amino acids, D-amino acids or a combination of D- and L-amino acids. Preferred are synthetic methods for solid phase peptides. Of course, dipeptides are also available on the market from a variety of sources, including DMI Synthesis Ltd., Cardiff, United Kingdom (in-house synthesis), Sigma-Aldrich, St. Louis, Missouri (mainly own synthesis), Phoenix Pharmaceuticals, Inc., Belmont, California (proprietary synthesis), Fisher Scientific (proprietary synthesis) and Advanced ChemTech, Louisville, Kentucky.

[0034] Once the dipeptide has been synthesized or purchased, it is cyclized to form diketopiperazine. You can do this in a number of ways.

[0035] For example, U.S. Patent Application Publication 2004/0024180 discloses a method for cyclizing dipeptides. In brief, the dipeptide is heated in an organic solvent with simultaneous removal of water by distillation. Preferably, the organic solvent is an azeotrope of a low boiling point of water, such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol, 2-butanol, t-butanol, butyloester acetic acid, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethyl acetal, dimethylacetal, acetic acid ethyl ester, heptane, methyl isobutyl ketone, 3-pentanol, toluene and xylene. The temperature depends on the rate of the reaction with which the cyclization takes place and on the type of the azeotroping agent used. The reaction is preferably carried out at 50-200 ° C, more preferably 80-150 ° C. PH range, wherein cyclisation can be easily determined by one skilled in the art. Preferably it will be 2-9, preferably 3-7.

[0036] When one or both of the dipeptide amino acids have or are derivatised to have a carboxyl group on their side chain (e.g., aspartic acid or glutamic acid), the dipeptide is preferably cyclized as described in US Patent No. 6,555,543. Briefly, the dipeptide together with the further side group protected carboxyl is heated under neutral conditions. Typically, the dipeptide will be heated from about 80 ° C to about 180 ° C, preferably about 120 ° C. The solvent will be an inert solvent. For example, the solvent may contain an alcohol (such as butanol, methanol, ethanol and higher alcohols, but not phenol) and an azeotropic co-solvent (such as toluene, benzene or xylene). Preferably, the alcohol is butane-2-ol, and the azeotropic co-solvent is toluene. Heating is continued until the reaction is completed and such time can be determined empirically. Typically, the dipeptide will cyclize by refluxing for about 8-24 hours,

Preferably about 18 hours. Finally, the protected group is removed from diketopiperazine. By doing this avoid the use of strong acids (mineral acids such as sulfuric or hydrochloric acid), strong bases (alkaline bases such as potassium hydroxide or sodium hydroxide) and a strong reducing agents (eg. Lithium aluminum hydride) to maintain the chirality of the final compound.

[0037] The dipeptides produced on the solid phase resins can be cyclized and released from the resin in one step. See, e.g., US Patent No. 5,817,751. For example, a resin having an attached N-alkylated dipeptide is suspended in toluene or toluene / ethanol in the presence of acetic acid (e.g., 1%) or triethylamine (e.g., 4%). Typically, basic cyclization conditions are preferred due to their faster cyclisation time.

[0038] To prepare diketopiperazine of formulas I and II, wherein the amino acid side chains are derivatives, amino acid derivatives can be used in the synthesis of dipeptides, dipeptides can be derivatives and / or diketopiperazine can be derivatives according to the knowledge in the art. See, e.g. references cited above.

[0039] Other methods for cyclizing dipeptides and producing diketopiperazines are known in the art and can be used to prepare the diketopiperazines used in the practice of the invention. See, e.g., the references cited above. In addition, many of the diketopiperazines suitable for use in the invention can be prepared as described below from proteins and peptides. Further, diketopiperazines for use in the practice of the invention can be obtained commercially from, e.g., DMI Synthesis Ltd., Cardiff, United Kingdom (in-house synthesis).

[0040] Diketopiperazines of formulas I and II include all possible stereoisomers that can be obtained by changing the configuration of individual chiral centers, axes or surfaces. In other words, diketopiperazines of formulas I and II include all possible diastereoisomers as well as all optical isomers (enantiomers).

[0041] Physiologically acceptable salts of diketopiperazine include conventional non-toxic salts, such as salts derived from inorganic acids (such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric and the like), organic acids (such as acetic, propionic, succinic, glycolic acids, stearic, lactic, malic, tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic and the like) or bases (such as hydroxide, carbonate or bicarbonate or a pharmaceutically acceptable metal cation or organic cations obtained from N, N-diebenzylethylenediamine , D-glucosamine or ethylenediamine). Salts are prepared in a conventional manner, e.g., neutralizing the form of the free base of the compound with the acid.

[0042] As noted above, diketopiperazine or a physiologically acceptable salt thereof can be used to treat a T-mediated disease or inhibit T cell activation. To do so, diketopiperazine or its physiologically acceptable salt is administered to an animal in need of treatment. Preferably, the animal is a mammal, such as a rabbit, goat, dog, cat, horse or human. Effective dosage forms, modes of administration and dosage amounts for compounds can be determined empirically, and the performance of such

-11 specific areas are within the scope of knowledge of the field. It is understood by those skilled in the art that the dosage will vary with the particular compound employed, the disease or disease being treated, the severity of the disease or disease, the route (s) of administration, the rate of compound release, duration of treatment, identification of any other drugs administered to the animal, age , size and species of animal and such similar factors known in the field of medicine and veterinary. In general, a suitable daily dose of the compound will be that amount of the compound that is the lowest effective dose to produce a therapeutic effect. However, the daily dose will be determined by the attending physician or veterinarian in terms of sound medical judgment. If necessary, the effective daily dose can be given as two, three, four, five,

[0043] Administration of the compound should continue until an acceptable response is achieved.

[0044] The compounds (i.e., diketopiperazines and their physiologically acceptable salts) can be administered to an animal patient for therapy by any appropriate route, including orally, intranasally, rectally, vaginally, parenterally (e.g., intravenously, intraosseously, intraperitoneally, subcutaneously, or intramuscularly), into the subarachnoid space, percutaneously, intracranial, intracerebral and locally (including buccal and sublingual). Preferred routes of administration are oral and intravenous.

[0045] Although it is possible that the described compound be administered alone, it is preferred to administer the compound as a pharmaceutical formulation (composition). The medicaments of the invention contain diketopiperazine as an active ingredient in admixture with one or more pharmaceutically acceptable carriers and, optionally, with one or more compounds, drugs or other materials. Each conveyor must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and must not be detrimental to the animal Pharmaceutically acceptable conveyors are well known in the art Regardless of the route of administration chosen, the compounds are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art See, e.g., Remington's Pharmaceutical Sciences.

[0046] Formulations suitable for oral administration can be in the form of capsules, sachets, pills, tablets, powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid or as liquid emulsions of oil in water or water in oil or as an elixir or syrup or as pellets (using an inert base such as gelatin or glycerol or sucrose and acacia) and the like, each containing a predetermined amount of the compound or compounds described herein as the active ingredient. The compound or compounds may also be administered in a bolus, electuary or paste.

[0047] In solid dosage forms of the invention for oral administration (capsules, tablets, pills, drags, powders, granules and the like), the active ingredient (i.e., one or more diketopiperazines and / or their physiologically acceptable salts) is mixed with one or more a pharmaceutically acceptable carrier, such as

Sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or diluents such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid, (2) binders such as, for example, carboxymethylcellulose, alginates , gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) sustained release agents such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate and mixtures thereof; and (1) colorants. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain buffering agents. Solid compositions of a similar type may be used as fillers in soft and hard filled gelatin capsules, using excipients such as lactose or milk sugars as well as high molecular weight polyethylene glycols. and the like.

[0048] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a binder (e.g. gelatine or hydroxypropylmethylcellulose, lubricant, inert diluent, preservative, decomposer (e.g., sodium starch glycollate or cross-linked sodium carboxymethylcellulose), surface active agent or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0049] Tablets and other solid dosage forms of the pharmaceutical compositions of the invention, such as dragees, capsules, pills and granules, may optionally be coated or prepared with coatings or shells, such as enteric release coatings and other coatings well known in the art of pharmaceutical formulations. They can also be formulated so as to provide a slow or controlled release of the active ingredient contained therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be sterilized, for example, by filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and the composition may only release the active ingredient or preferentially in a particular part of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form.

[0050] Liquid dosage forms for oral administration of the compounds include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other

- solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, groundnut oil) , maize, sprouts, olive oil, castor and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof.

[0051] In addition to inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preserving agents.

[0052] Suspensions, in addition to the active ingredient, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth and mixtures thereof.

[0053] The dosage formulations of the invention for rectal or vaginal administration may be a suppository, which may be prepared by mixing one or more compounds with one or more suitable non-irritating excipients or carriers containing, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate and which are solid at room temperature, but liquid at body temperature and thus melt in the rectal or vaginal cavity and release the active compound. Formulations of the invention that are suitable for vaginal administration also include pessary, tampon, creme, gels, pastes, foams or spray formulations containing such carriers as those known in the art that are suitable.

[0054] Dosage forms for topical or transdermal administration of compounds include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any buffers or propellants that may be required.

[0055] Ointments, pastes, creams and gels may contain, in addition to the active ingredient, auxiliary substances, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures thereof.

Powders and sprays may contain, in addition to the active ingredient, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorocarbons and unstable unstable hydrocarbons, such as butane and propane.

[0058] Transdermal patches have the additional advantage of providing controlled release of compounds to the body. Such dosage forms can be prepared by dissolving, dispersing or incorporating otherwise one or more compounds

In a suitable substrate, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the release of the compound on the skin. The rate of such flow can be controlled either by providing a membrane controlling the rate or dispersion of the compound in the polymer matrix or gel.

[0059] Medicaments include those suitable for administration by inhalation or insufflation or for intranasal or intraocular administration. For administration to the upper (nasal) or lower airways by inhalation, the compounds described are suitably delivered by means of a presiveser, nebulizer or pressurized package or other suitable means for delivering the aerosol spray. Pressurized packagings may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for delivering a metered amount.

[0060] Alternatively, for administration by inhalation or insufflation, the drug may take the form of a dry powder, for example, a powder mixture of one or more compounds and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin packs or blisters, from which the powder may be administered by means of an inhaler, insufflator or metered dose inhaler.

[0061] For nasal administration, the compounds may be administered by means of nasal drops or a liquid spray, such as by means of an atomiser with a plastic bottle or a metered dose inhaler. Typical atomisers are Mistometer (Wintrop) and Medihaler (Riker).

[0063] Drops, such as eye drops or nasal drops, may be formulated with an aqueous or nonaqueous base, also containing one or more disintegrating agents, solubilizing agents, or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered using a simple bottle with a dropper cap or with a plastic bottle adapted to deliver liquid contents in drops using a specially shaped closure.

[0064] Medicaments of the invention suitable for parenteral administration contain one or more compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders that can be dissolved in sterile injectable or dispersion solutions right prior to use, which may contain antioxidants, buffers, solutes that render the formulation isotonic to the blood of the intended recipient, or suspending or thickening agents.

[0065] Examples of suitable aqueous and non-aqueous conveyors that can be used in the medicaments of the invention include water, ethanol, polyhydric alcohols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and their suitable mixtures, vegetable oils, such as olive oil olive and organic esters for injection, such as

-Ethyloleinate. Proper fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.

[0066] These compositions may also contain adjuvants such as wetting agents, emulsifying agents and disintegrants. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that can delay absorption, such as aluminum monostearate and gelatin.

[0067] In some cases, to prolong the effect of the drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material having poor water solubility. Accordingly, the rate of drug absorption depends on its dissolution rate, which may ultimately depend on the size of the crystals and the crystalline form. Alternatively, delayed absorption of the parenterally administered drug is achieved by dissolving or suspending the drug in an oil carrier.

[0069] Depot injectable forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations are also prepared by enclosing the drug in liposomes or microemulsions that are compatible with body tissue. Injectable materials can be sterilized, for example, by filtration through a bacteria-retaining filter.

[0070] The formulations may be presented in a dose in airtight containers with a unit dose or multiple doses, for example, ampoules and vials, and may be stored under lyophilized conditions requiring only the addition of a sterile liquid transporter, for example injection water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0071] Diketopiperazines have been found to be present in commercially available intravenous pharmaceutical compositions containing albumin, immunoglobulin and erythropoietin. The diketopiperazines present in these pharmaceutical preparations are formed by the heating steps often used in the production of these pharmaceutical compositions. Heating results in the cleavage and cyclization of the two N-terminal and / or two C-terminal amino acids of the proteins to form diketopiperazines.

[0072] Consequently, diketopiperazines can be prepared by heating solutions of albumin, immunoglobulins, erythropoietin and other proteins and peptides. For example, a solution of albumin, immunoglobulin, erythropoietin or another protein or peptide is prepared in a phosphate buffer at neutral pH. Preferably, the solution is concentrated

A solution (e.g., about 100-500 mM) to achieve protonation of the N-terminal and / or C-terminal amino acid. The solution is heated at 60 ° C for from about 2 hours to several days, preferably about 4 days, to form diketopiperazin formation. Preferably, protein denaturation should be avoided. This can be achieved by using shorter times and / or adding caprylic acid or N-acetyltryptophan at about 0.02 M for each.

[0073] Diketopiperazines can also be prepared by contacting a solution of albumin, immunoglobulin, erythropoietin or another protein or peptide with an enzyme that cleaves two N-terminal amino acids of a protein or peptide (e.g., dipeptidyl peptidase) or an enzyme that can cleave two C-terminal amino acids from a protein or a peptide (e.g., carboxypeptidase). Suitable dipeptidyl peptidases and carboxypeptidases are commercially available e.g. from Sigma. The reaction should be carried out at pH 6-8, preferably in a buffer such as a phosphate buffer, at a temperature high enough to accelerate the reaction, but not so high as to denature the protein (e.g., 37 ° C).

[0074] The amino acid sequences of many proteins and peptides are known, and a protein or peptide with the desired N-terminal and / or C-terminal sequence can be chosen to obtain the desired diketopiperazine (desired diketopiperazines) using any of the methods. In addition, peptides with the desired sequence can be synthesized using well-known and used methods.

[0075] According to the invention, diketopiperazines are purified from commercially available solutions containing albumin, immunoglobulin and erythropoietin by well-known methods, such as size exclusion chromatography (e.g. Centricon filtration), affinity chromatography (e.g. using a column of beads having attached to them) an antibody or antibodies directed against the desired diketopiperazine (diketopiperazines) or an antibody or antibodies against a truncated protein or peptide), anion exchange or cation exchange. The purified diketopiperazines are used and included in the drugs as described above.

[0076] Instead of purifying diketopiperazines, pharmaceutical compositions containing albumin, immunoglobulin, erythropoietin and / or other proteins and / or peptides usually found in an animal recipient can be administered for the treatment of T cell mediated disease and can be used to inhibit T cell activation. containing these proteins and / or peptides that are currently available on the market can be used if they contain diketopiperazines, it is highly preferred to subject albumin, immunoglobulin, erythropoietin and / or peptides to treatment as described above to increase the content of the desired diketopiperazine (diketopiperazines) before administration of such improved of the composition. The animal is preferably a human, and the proteins and / or peptides are preferably human proteins and / or peptides. Oral administration of the composition is preferred.

[0077] Effective dosage amounts of protein and / or peptide compositions can be determined empirically, and the determination of such terms is well within the knowledge of the field. In particular, to determine the effective dosage amount of the protein and / or peptide composition, one can measure the amount of one or more diketopiperazines present in the composition, and the amount of composition

-The final dose to deliver the effective amount of diketopiperazine (diketopiperazines) can be given to the animal. It is understood by those skilled in the art that the dosage will vary with the particular composition being used, the disease or disease being treated, the severity of the disease or disease, the route (s) of administration, the rate of secretion, duration of treatment, identification of any other drugs administered to the animal, age, size and species of animal and such similar factors known in the field of medicine and veterinary. In general, a suitable daily dose of the protein and / or peptide will be that amount of the compound that is the lowest effective dose to produce a therapeutic effect. However, the daily dose will be determined by the attending physician or veterinarian in terms of sound medical judgment. If necessary, the effective daily dose may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day. Administration should continue until an acceptable response is achieved.

[0078] As noted above, diketopiperazines have been found in commercially available intravenous pharmaceutical compositions with albumin, immunoglobulin and erythropoietin, where the production of these compositions includes one or more heating steps (e.g., sterilization). Diketopiperazines are also likely to be present in other pharmaceutical compositions of proteins and peptides, where the preparation of the composition includes heating steps. As described, many diketopiperazines have the ability to inhibit T cell activation. Therefore, in many situations, it may not be desirable to administer albumin, immunoglobulin, erythropoietin or other diketopiperazine containing protein or peptide compositions to patients. For example, albumin is often given to patients suffering from an injury, immunoglobulin is often administered to patients suffering from infection or immunodeficiency, and erythropoietin is administered to patients with anemia, cancer or chronically ill, whose immune systems are often impaired. Consequently, a method is described for removing at least a portion, preferably substantially all of the diketopiperazines from such compositions. Diketopiperazines can be removed as described above (e.g., by size exclusion chromatography (e.g. Centricon filtration), affinity chromatography (e.g., using a column with beads having an antibody attached thereto or antibodies directed against the desired diketopiperazine (diketopiperazines) or antibody or antibodies directed against against albumin, immunoglobulin, erythropoietin or another protein or peptide), exchange of anions or exchange of cations,

EXAMPLES

EXAMPLE 1: Absorption of Asp Ala DKP (DA-DKP) and Glu Ala DKP (EA-DKP) from rat's intestine.

[0079] The rat intestine from the pylorus of the pylorus to the rectum was marginally isolated and perfused through a mesenteric artery with a perfusate containing bovine serum albumin. Perfusate flowing from the intestine was collected by cannulation of the portal vein and submitted

- circulatory (after re-oxygenation). After the equilibration period, a solution (approximately 1 ml) containing approximately 1 mg of Asp-Ala diketopiperazine (DA-DKP) or 1.4 mg of Glu-Ala diketopiperazine (EA-DKP) was administered by means of an injection into the duodenum.

[0080] After dosing, further doses of perfusate were collected at intervals up to 2 hours after dosing. These samples were centrifuged and the plasma tested for both cyclic dipeptides by tandem mass spectrometry chromatography (LCMS).

[0081] The results showed that after only 2 hours of perfusion, the amounts of DA-DKP and EA-DKP that were absorbed from the intestinal lumen were 95% and 100% (actually 112%) of the dose administered, respectively.

[0082] Therefore, both cyclic peptides are rapidly absorbed from the intestine and efficiently from the intestinal lumen into the blood, with no trace of metabolism during transport through the intestinal wall. Therefore, strong therapeutics can be administered orally.

[0083] Rapid absorption of unchanged DA-DKP and EA-DKP from the gastrointestinal tract into the blood in combination with a lack of hepatic first pass clearance for both compounds in isolated rat liver subjected to perfusion (data not shown) shows that pre-systemic clearance is low. As a consequence, oral dosing will be an ideal route of administration.

[0084] Furthermore, studies with isolated rat kidneys subjected to perfusion have shown that, unlike many straight chain peptides that undergo extensive metabolism by renal peptidases, the renal clearance of both cyclic dipeptides is relatively low.

[0085] In sum, these data suggest that a dosing schedule for low daily doses of diketopiperazines is likely to be suitable for therapeutic applications.

[0086] Preliminary pharmacokinetic data in rats after oral administration corresponded to the above for both cyclic dipeptides, with Tmax values ​​of 30-60 minutes and Cmax values ​​of 4-6 Lig / ml (DA-DKP) and 0.6-1,1 Lig / ml (EA-DKP) after oral dosage of 1.1-3.7 mg / kg body weight (DA-DKP) and 1.5-4.8 mg / kg body weight (EA-DKP) (Tmax is the time when the concentration reaches the maximum and Cmax is the maximum concentration achieved, both are calculated from the curve fit equation for the obtained data).

[0087] Preliminary data suggest that DA-DKP and other diketopiperazines pass the blood-brain barrier. Accordingly, DA-DKP and other diketopiperazines of the invention should be useful in treating disorders of the nervous system, such as multiple sclerosis.

EXAMPLE 2: Inhibition of human cytokine cytokine production in vitro with a human colostrum fraction containing Met-Arg DKP (MR-DKP) and with Asp-Ala DKP (DADKP)

A. Materials

[0088] This example demonstrates that DA-DKP, human colostrum (HC 2626) containing MR-DKP and a low molecular weight human mammalian colostrum fraction (HC RBL; a human colostrum fraction containing components with molecular weights less than 3000, prepared from Centricon degreased colostrum filtration) also containing MR-DKP inhibited cytokine production of human T lymphocytes. DA-DKP and MR-DKP were obtained from DMI Synthesis, Ltd., Cardiff, UK. These two diketopiperazines are small naturally occurring compounds generated during a physiological response to ignition. They are also sometimes found in human intravenous immunoglobulin (IVIg), human albumin and other biological preparations.

B. Inhibition of T cell cytokine production [0089] Two different clones of human CD4-positive T lymphocytes were tested. One of the cell lines (TRiPS) was isolated from a donor immunized against influenza and is specific for the 307-319 hemagglutinin peptide. Another cell line (H4 # 9.25) was isolated from brain tissue at autopsy, from a donor with multiple sclerosis, and is specific for the basic myelin protein (amino acids 87-99). Both T cell clones produce interleukin 8 (IL-8), IL-16, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) after in vitro stimulation or with (1) specific antigen plus HLA-presenting cells DR2-positive, or (2) anti-CD3-plus anti-CD28 antibodies.

[0090] T cell lines were stimulated to the passage using approximately 4x10⁵ cells on days 18-20 after previous stimulation. The cells were washed once in cold Iscove's Modified Dulbecco Minimal Essential Medium (IMDM, Sigma) plus 10% fetal bovine serum (FBS; American Type Culture Collection (ATCC)) and resuspended in 1.0 ml cold IMDM medium containing 1: 500 dilution of antibody monoclonal anti-CD3 OKT3 (prepared from ascites fluid in mice). The cells were incubated with the antibody for 30 minutes on ice, washed with cold medium without FBS and combined with approximately 2x10⁶ irradiated 4000R peripheral blood leukocytes (PBL) from the normal human donor, as the cells of the nutrient layer, in the medium plus 50 U / ml human IL-2 (Xenomatrix). Cultures were expanded by the addition of fresh IMAM medium with FBS plus IL-2 on day 3. The breeding day is counted from the day of stimulation with OKT3. Cells can be used for experiments starting from day 7 (at maximum proliferation), typically on day 14 (most sensitive to restimulation) and up to day 21 (cells at rest reaching the aging stage).

[0091] Activation experiments were carried out by collecting a sample of cells and washing with twice warmed (37 ° C) IMDM media. For each specific test, 2x10⁵ viable cells were pre-incubated in a total volume of 0.9 ml warm IMDM medium containing a predetermined quantity of therapeutic supplement (e.g., HC 2626, DA-DKP, PMA, etc.) for 15 minutes at 37 ° C. Dynabeads (Dynal) was then added as a stimulus to 0.1 ml of heated IMDM, and the cultures were incubated overnight (18 hours) at 37 ° C. The supernatants from the cell culture were then collected after the cells were precipitated by means of centrifugation. The cytokine content was examined using a specific ELISA (e.g., T1F, IFNγ, IL-8, IL-16, Endogen).

[0092] As shown in Figs. 1-5, human colostrum (HC 2626) inhibited the production of in vitro cytokines by both T cell lines in a dose-dependent manner. As also shown in Figs. 1-5, HC RBL and DA-DKP inhibited cytokine production in vitro by both T cell lines in a dose-dependent manner at an early stage of the stimulatory cycle. However, the effects of HC RBL and DA-DKP later on the cycle (day 14 or later) were stimulatory (see Fig. 4). Both HC 2626 and HC RBL contain MR-DKP (as determined by mass spectrometry), however, HC 2626 contains other ingredients (including caseins, which are relatively dephosphorylated proteins, which can therefore be anti-inflammatory as described in the same waiting for the patent to be granted application 10 / 723,247, filed on November 25, 2003), apart from MR-DKP which may be responsible for its inhibitory effects at a later stage of the cell cycle. Consequently, HC RBL and HC2626 (both containing MR-DKP), MD-DKP and DA-DKP should be useful in modulating downstream inflammatory cytokine response in T-cell mediated diseases and / or autoimmune diseases such as multiple sclerosis, because they all inhibit the production of cytokines by T cells at an early stage of the stimulatory cycle. These results also suggest that HC RBL, HC 2626, MR-DKP and DA-DKP will selectively affect T cells specific for antigens without affecting resting T cells. MD-DKP and DA-DKP should be useful in modulating down-inflammatory cytokine responses in T-cell mediated diseases and / or autoimmune diseases such as multiple sclerosis as they all inhibit cytokine production by T cells at an early stage of the stimulatory cycle. These results also suggest that HC RBL, HC 2626, MR-DKP and DA-DKP will selectively affect T cells specific for antigens without affecting resting T cells. MD-DKP and DA-DKP should be useful in modulating down-inflammatory cytokine responses in T-cell mediated diseases and / or autoimmune diseases such as multiple sclerosis as they all inhibit cytokine production by T cells at an early stage of the stimulatory cycle. These results also suggest that HC RBL, HC 2626, MR-DKP and DA-DKP will selectively affect T cells specific for antigens without affecting resting T cells.

C. Mechanism of action [0093] The mechanism of action of DA-DKP and HC 2626 (containing MR-DKP) was investigated. For this purpose, 1x10 was incubated⁶ TRiPS cells from day 18. for 30 minutes at 37 ° C, or without anything ("Nil"), or with added Dynabeads CD3 / CD28 (CD3 / CD28 beads), or with CD3 / CD28 and 0.5 mM DA beads -DKP, or with CD3 / CD28 beads and HC2626 added at a dilution of 1: 500. After incubation, the cells were lysed in Cell-Lytic Mammalian Cell Extraction Reagent (Sigma).

[0094] Subsequently, the cell extracts were incubated separately with the double Hypromatrix matrices for 2 hours at room temperature, and then washed twice with the manufacturer's protocol (Hypromatrix). The Hypromatrix matrix is ​​a nylon membrane with the applied antibodies against the transcription factors listed in Table 1 (own, manufactured by Hypromatrix). A cocktail of antibodies specific for phosphorylated tyrosine, phosphorylated serine and phosphorylated threonine (Zymed) was added and incubated for 1 hour. An anti-immunoglobulin antibody, biotin labeled, was then added. After washing the anti-immunoglobulin-biotin, streptavidinucleotoxidase was added and the matrices were washed for the last time before adding the luminescent peroxidase reactive substrate.

[0095] The results were visualized by means of membrane exposure and were scored as 0 (negative) or + to ++++ (positive) as shown in Table 2. As shown in Table 2, some cases of activation of transcription cytokine factors (ERK1 / 2) ) and release of the pre-formed cytokine were inhibited by HC 2626 (containing MR-DKP) and DA-DKP.

TABLE 1: HYPROMATRIX MATERIAL (OWN): PHOSPHORILE PROTEINS

<td>NUMBER</td><td>ACRONYM</td><td>RELATIONSHIP</td>

<td>1</td><td>Act 1/2</td><td>protein kinase B, anti-apoptotic kinase</td>

<td>2</td><td>c-Cbl</td><td>TcR inhibitory pathway; phosphorylation Tyr²⁹² activates binding and inactivation of Syk and ZAP-70</td>

<td>3</td><td>CBP</td><td>csk binding protein (PAG); integral membrane protein temporarily (and at low dephosphorylates Tyr to release csk</td>

<td>4</td><td>CREB</td><td>the cAMP response element binding protein; phosphorylation (unk) to activate / down-regulate the IL-2 promoter</td>

<td>5</td><td>csk</td><td>COOH-terminal; src kinase; phosphorylated Se³⁶⁴, also phosphorylated Tyr (activity?) - phosphorylates and inactivates lck</td>

<td>6</td><td>ERK1</td><td>extracellular kinase associated with the signal</td>

<td>7</td><td>c-fos</td><td>an AP-1 component activated by TcR stimulation; phosphorylation at both N- and C-unk residues</td>

<td>8</td><td>NFATC</td><td>nuclear factor activated T cells; intact in anergy</td>

<td>9</td><td>c-jun</td><td>an AP-1 component activated by TcR activation; phosphorylated by JNK-MAPK at Ser⁶³</td>

<td>10</td><td>Ik B-α</td><td>NFk B inhibitor</td>

<td>11</td><td>pIk B-α</td><td>Cheese-phosphorylated and inactivated NFk B inhibitor</td>

<td>12</td><td>p38 MAPK</td><td>mitogen-activated protein kinase</td>

<td>13</td><td>PI3 kinase / p85</td><td>activated by glucocorticoids and e2-adrenergic-R</td>

<td>14</td><td>PTEN</td><td>cytoplasmic 3'-inositol phosphatase; the tumor suppressor gene antagonizes PI 3 'kinase by converting PI-PO back to inactive forms</td>

<td>15</td><td>c-Raf-1</td><td></td>

<td>16</td><td>Rap1</td><td>negative GTC-aza regulatory TcR</td>

<td>17</td><td>Ras</td><td>kinase; inactivated during anergy</td>

<td>18</td><td>fyn</td><td>direct TcR signal kinase bound to the cell membrane</td>

<td>19</td><td>lck</td><td>direct TcR signal kinase bound to the cell membrane, the active form is phosphorylated Tyr³⁹⁵; inactivated by phosphorylation of csk at C-terminal Tyr</td>

<td>20</td><td>ZAP70 kinase</td><td>signaling device from CD3Z; phosphorylated on? by lck / fyn, ZAP70 phosphorylates LAT (linker for T-cell activation) with Tyr and Tyr on SLP-76</td>

TABLE 2: RESULTS

<td>RELATIONSHIP</td><td>NILE</td><td>CD3 / CD28</td><td>DKP</td><td>HC2626</td>

<td>Act 1/2</td><td>+</td><td>++</td><td>+++</td><td>++</td>

<td>c-Cbl</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>CBP</td><td>+</td><td>++</td><td>++</td><td>++</td>

<td>CREB</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>csk</td><td>+</td><td>++</td><td>+</td><td>+</td>

<td>ERK1</td><td>+</td><td>+</td><td>+</td><td>+</td>

<td>c-fos</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>NFATC</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>c-jun</td><td>++</td><td>+</td><td>+</td><td>+</td>

<td>Ik B-α</td><td>++</td><td>++</td><td>+</td><td>+</td>

<td>pIk B-α</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>p38 MAPK</td><td>++</td><td>+++</td><td>+++</td><td>+++</td>

<td>p3 / p85 kinase</td><td>+</td><td>++</td><td>+</td><td>++</td>

<td>PTEN</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>c-Raf-1</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>Rapla</td><td>+</td><td>++</td><td>++</td><td>+</td>

<td>Ras</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td>fyn</td><td>+</td><td>+</td><td>+</td><td>+</td>

<td>lck</td><td>-</td><td>-</td><td>-</td><td>-</td>

<td></td><td></td><td></td><td></td><td></td>

ZAP70 kinase

EXAMPLE 3: Inhibition of cytokine production of human Tin lymphocytes in vitro by GlyLeu DKP (GL-DKP) and Ala-Pro DKP (AP-DKP) [0096] GL-DKP and AP-DKP (obtained from DMI Synthesis, Ltd., Cardiff, UK ) was tested as described in Example 2 using the TRiPS and H4 cell lines # 9.25. It was found that GL-DKP and AD-DKP inhibited the production of in vitro cytokines by both T cell lines in a dose-dependent manner. The mechanism of action is currently being investigated as described in Example 2 and appears to affect both the activation of the cytokine transcription factor and the release of the preformed cytokine.

EXAMPLE 4: Inhibition of human T cell cytokine production in vitro by Asp Ala DKP (DA-DKP) and Tyr Glu DKP (YE-DKP) [0097] Normal human lymphocytes were isolated from the peripheral blood of a regular human donor with Histopaque (Sigma). Then 3-4 x 10⁵ lymphocytes were suspended in 1 ml of IMDM medium without serum. Cells were stimulated by adding 25 μl of anti-CD3 antibody at a 1: 2000 dilution (Pharmingen, San Diego, California) and incubating for 18 hours at room temperature.

37 ° C.

Next, up to three identical cultures, one of three DKP preparations and dexamethasone (final concentration 10^-5 M). The three DKP preparations were:

1. DA-DKP (obtained from DMI Synthesis, Ltd., Cardiff, Great Britain, final concentration 25 μg / ml in cultures).

2. DKP-ZLB, a preparation of 25% albumin (obtained from ZLB Bioplasma, AG 3000 Bern 22, Switzerland) was heated for 4 days at 60 ° C, after which it was found that it contained 0.5 mM DA-DKP, as determined by using mass spectrometry (final concentration of 14 μg / ml DA-DKP in cultures).

-243. DKP-Y-glob - γ-globulin preparation (obtained from Sigma, number G-4386) containing 12 mg / ml γ-globulin in phosphate buffered saline, pH 7.4, was filtered using a Centricon 3000 filter and filtrate was used (containing components having a MW of less than 3,000). The filtrate contained mass 292, which is the mass of Ty-Glu DKP (YE-DKP), as determined by means of anion exchange HPLC coupled to mass spectrometry with a negative potential electrospray. The filtrate was used in cultures with a final dilution of 1: 4.

[0099] After the addition of DKP or dexamethasone preparations, the cultures were incubated for 18 hours at 37 ° C. Thereafter, the amounts of IL-2, IFNγ and TNFa secreted into each culture were measured by ELISA (Pierce Biotechnology, Rockford, Illinois 61105).

[0100] The results are shown in Table 3 below. As can be seen, the greatest reduction in the secretion of all three cytokines was obtained from DKP-Y-glob. Flow cytometry with the analysis of CD69 + T cell count (CD69 is a marker on activated T cells) also showed that DKP-Y-glob reduced the amount of CD69 + T cells by about 90% compared to a decrease of about 50% by dexamethasone despite being internalized the T cell receptor complex

TABLE 3

<td>Stimulation</td><td>Treatment</td><td>U / ml IL-2</td><td>pg / ml IFNy</td><td>pg / ml TNFa</td>

<td>Nile</td><td>-</td><td>0.24 ± 0.1</td><td>2.3 ± 0.9</td><td>2.8 ± 0.5</td>

<td>CD3</td><td>-</td><td>2.6 ± 0.5</td><td>289 ± 35</td><td>98 ± 3.2</td>

<td>CD3</td><td>DA-DKP</td><td>1.4 ± 0.3</td><td>306 ± 17</td><td>74 ± 4.7</td>

<td>CD3</td><td>DKP-ZLB</td><td>1.4 ± 0.4</td><td>311 ± 18</td><td>130 ± 2.9</td>

<td>CD3</td><td>DKP-Y-glob</td><td>0.24 ± 0.25 (91% reduction)</td><td>2.1 ± 0.1 (decrease by 99%)</td><td>1.6 ± 0.6 (reduction by 98%)</td>

<td>CD3</td><td>dexamethasone</td><td>0.9 ± 0.1 (reduction by 65%)</td><td>76 ± 7.32 (decrease by 74%)</td><td>4.1 ± 0.3 (decrease by 96%)</td>

[0101] Further provided below are not part of the invention:

A method of treating a T cell mediated disease comprising administering to an animal in need thereof an effective amount of diketopiperazine having the following formula:

<img file="PL2537524T3_D0004.tif" />

wherein:

R¹ and R²that can be the same or different, each is:

(a) the side chain of the amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, βaminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine or ornithine; however, provided that when R¹ is the side chain of asparagine or glutamine, then R² can not be a side chain of lysine or ornithine, and when R¹ is the side chain of lysine or ornithine, then R² it may not be a side chain of asparagine or glutamine;

(b) R.¹ is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline, R² is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline or both R¹ and R² are each independently -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adjacent ring nitrogen form proline or hydroxyproline; or (c) a side chain derivative of an amino acid, wherein the amino acid is one of those specified in (a) and the derived side chain has:

(i) the -NH2 group replaced by the -NHR group³ or -N (R.³) 2, each R being³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(ii) a -OH replaced by a -O-PO3H2 or -OR group³, each R³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iii) a -COOH group replaced with a -COOR group³, each R³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(Iv) the -COOH group replaced by the -CON group (R.⁴) 2, each R being⁴ can independently be H or substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(v) -SH replaced with -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2CH (NH2) -COOH;

(vi) a -CH2- group replaced with a -CH (NH2) - or -CH (OH) - group;

(vii) -CH3 replaced by a -CH2-NH2 or -CH2-OH group; and / or (viii) H which is attached to a halogen replaced carbon; or or a pharmaceutically acceptable salt thereof.

2. The method according to Point 1, whereby R¹, R² or both are an aspartic acid side chain, a glutamic acid side chain or an aspartic acid or glutamic acid side chain derivative, where the -COOH group is replaced by a -COOR group³ or the -CON group (R.⁴) 2.

3. The method according to Item 2, wherein R¹ is an aspartic acid side chain or an aspartic acid side chain derivative, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the alanine side chain.

4. The method according to Item 2, wherein R¹ is an aspartic acid side chain or an aspartic acid side chain derivative, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the tyrosine side chain.

5. The method according to Item 2, wherein R¹ is a side chain of glutamic acid or a side chain derivative of glutamic acid, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the alanine side chain.

6. The method according to Item 2, wherein R¹ is a side chain of glutamic acid or a side chain derivative of glutamic acid, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the tyrosine side chain.

7. The method according to Item 2, wherein R¹ is the side chain of aspartic acid or glutamic acid and R² is the alanine side chain.

8. The method according to Item 2, wherein R¹ is the side chain of aspartic acid or glutamic acid and R² is the tyrosine side chain.

9. The method according to Item 1, wherein R¹ and R² they are both a hydrophobic side chain or derivative of a hydrophobic side chain.

10. The method according to Item 9, where:

-27 (a) R¹ and R²which may be the same or different, each being a side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine;

(b) R.¹ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R² is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline; or (c) R¹ is the side chain of glycine, alanine, valine, norvaline, aaminobutyric acid, leucine, isoleucine, norleucine or phenylalanine and R² is -CH2-CH2CH2- and together with the adjacent nitrogen atom forms proline.

11. The method according to Item 10, wherein R¹ is the side chain of glycine and R² it is a leucine side chain.

12. The method according to Item 10, wherein R¹ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R² is the phenylalanine side chain.

13. The method according to Item 10, wherein R¹ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R² is the alanine side chain.

14. The method according to Item 1, wherein R¹, R² or both are a methionine side chain, an arginine side chain or a derivative of these side chains.

15. The method according to Item 14, wherein R¹ is the side chain of methionine and R² is the arginine side chain.

16. The method according to any of items 1-15, wherein the animal is a human.

17. The method of any of Claims 1-15, wherein the T-mediated disease is transplant rejection, graft versus host disease, delayed type hypersensitivity reactions, T cell mediated disease or autoimmune disease.

18. The method according to any of items 1-15, wherein the disease mediated by T cells is multiple sclerosis, neuritis, polymyositis, psoriasis, acquired vitiligo, Sjogren's syndrome, rheumatoid arthritis, type 1 diabetes, autoimmune pancreatitis, inflammatory diseases. intestines, Crohn's disease, ulcerative enteritis, celiac disease, glomerulonephritis, scleroderma, sakoidosis, autoimmune thyroid disease, Hashimoto type thyroiditis, Graves' disease, myasthenia gravis, Addison's disease, autoimmune retinal uveitis or systemic lupus erythematosus.

19. The method according to Items 1-15, wherein the T cell mediated disease is pulmonary fibrosis or idiopathic pulmonary fibrosis.

20. A method of inhibiting T-cell activation comprising administering to an animal in need thereof an effective amount of diketopiperazine having the following formula:

<img file="PL2537524T3_D0005.tif" />

wherein:

R¹ and R²that can be the same or different, each is:

(a) the side chain of the amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, βaminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine or ornithine; however, provided that when R¹ is the side chain of asparagine or glutamine, then R² can not be a side chain of lysine or ornithine, and when R¹ is the side chain of lysine or ornithine, then R² it may not be a side chain of asparagine or glutamine;

(b) R.¹ is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline, R² is -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline or both R¹ and R² are each independently -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adjacent ring nitrogen form proline or hydroxyproline; or (c) a side chain derivative of an amino acid, wherein the amino acid is one of those specified in (a) and the derived side chain has:

(i) the -NH2 group replaced by the -NHR group³ or -N (R.³) 2, each R being³can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(ii) a -OH replaced by a -O-PO3H2 or -OR group³, each R³can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iii) a -COOH group replaced with a -COOR group³, each R³ can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(Iv) the -COOH group replaced by the -CON group (R.⁴) 2, each R being⁴ can independently be H or substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(v) -SH replaced with -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2CH (NH2) -COOH;

(vi) a -CH2- group replaced with a -CH (NH2) - or -CH (OH) - group;

(vii) -CH3 replaced by a -CH2-NH2 or -CH2-OH group; and / or (viii) H which is attached to a halogen replaced carbon; or a pharmaceutically acceptable salt thereof.

21. The method according to Item 20, wherein R¹, R² or both are an aspartic acid side chain, a glutamic acid side chain or an aspartic acid or glutamic acid side chain derivative, where the -COOH group is replaced by a -COOR group³ or the -CON group (R.⁴) 2.

22. The method according to Item 21, wherein R¹ is an aspartic acid side chain or an aspartic acid side chain derivative, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the alanine side chain.

23. The method according to Item 21, wherein R¹ is an aspartic acid side chain or an aspartic acid side chain derivative, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the tyrosine side chain.

24. The method according to Item 21, wherein R¹ is a side chain of glutamic acid or a side chain derivative of glutamic acid, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the alanine side chain.

25. The method according to Item 21, wherein R¹ is a side chain of glutamic acid or a side chain derivative of glutamic acid, with the -COOH group being replaced by the -COOR group³ or a CON (R.⁴) 2, and R² is the tyrosine side chain.

26. The method according to Item 21, wherein R¹ is the side chain of aspartic acid or glutamic acid and R² is the alanine side chain.

27. The method according to Item 21, wherein R¹ is the side chain of aspartic acid or glutamic acid and R² is the tyrosine side chain.

28. The method according to Item 20, wherein R¹ and R² they are both a hydrophobic side chain or derivative of a hydrophobic side chain.

29. The method according to Clause 28, wherein:

-30 (a) R¹ and R²which may be the same or different, each being a side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine;

(b) R.¹ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R² is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline; or (c) R1 is the side chain of glycine, alanine, valine, norvaline, aaminobutyric acid, leucine, isoleucine, norleucine or phenylalanine and R² is -CH2CH2-CH2-i and together with the adjacent nitrogen atom forms proline.

30. The method according to Item 29, wherein R¹ is the side chain of glycine, and R² it is a leucine side chain.

31. The method according to Item 29, wherein R¹ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R² is the phenylalanine side chain.

32. The method according to Item 29, wherein R¹ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R² is the alanine side chain.

33. The method according to Item 20, wherein R¹, R² or both are a methionine side chain, an arginine side chain or a derivative of these side chains.

34. The method according to Item 33, wherein R¹ is the methionine side chain, and R² is the arginine side chain.

35. The method according to any of Points 20-34, wherein the animal is a human.

36. The method of any of Claims 20-34, wherein the diketopiperazine is used to treat inflammation or an inflammatory disease that is caused or exacerbated at least in part by the activation of T-cells.

37. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and diketopiperazine having the following formula:

<img file="PL2537524T3_D0006.tif" />

wherein:

R⁵ and R⁶that can be the same or different, each is:

(a) the side chain of the amino acid, wherein the amino acid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine, threonine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline,

-Phenylalanine, β-aminophenylalanine, tyrosine, tryptophan, thyroxine or ornithine; however, provided that when R⁵ is the side chain of asparagine or glutamine, then R⁶ can not be a side chain of lysine or ornithine, and when R⁵ is the side chain of lysine or ornithine, then R⁶ it may not be a side chain of asparagine or glutamine;

(b) R.⁵ is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline, R⁶ is -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adhering nitrogen of the ring forms proline or hydroxyproline or both R⁵ and R⁶ are each independently -CH2-CH2-CH2- or -CH2CH (OH) -CH2- and together with the adjacent ring nitrogen form proline or hydroxyproline; or (c) a side chain derivative of an amino acid, wherein the amino acid is one of those specified in (a) and the derived side chain has:

(i) the -NH2 group replaced by the -NHR group³ or -N (R.³) 2, each R being³can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(ii) a -OH replaced by a -O-PO3H2 or -OR group³, each R³can independently be substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, aralkyl or heteroaryl;

(iii) -CH2- replaced by a -CH (NH2) - or -CH (OH) - group;

(iv) a -CH3 group replaced with a -CH2-NH2 or -CH2-OH group; and / or (v) H which is attached to a halogen replaced carbon; or a pharmaceutically acceptable salt thereof.

38. Composition according to Clause 37, wherein R⁵ and R⁶ they are both a hydrophobic side chain or derivative of a hydrophobic side chain.

39. Composition according to Clause 38, where:

(a) R⁵ and R⁶which may be the same or different, each being a side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine;

(b) R.⁵ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R⁶is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline; or (c) R⁵ is the side chain of glycine, alanine, valine, norvaline, aaminobutyric acid, leucine, isoleucine, norleucine or phenylalanine, and R⁶ is -CH2CH2-CH2-i and together with the adjacent nitrogen atom forms proline.

-3240. Composition according to Item 39, wherein R⁵ is the side chain of glycine, and R⁶it is a leucine side chain.

41. Composition according to Item 39, wherein R⁵ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R⁶ is the phenylalanine side chain.

42. Composition according to Item 39, wherein R⁵ is -CH2-CH2-CH2- and together with the adjacent nitrogen atom forms proline and R⁶ is the alanine side chain.

43. Composition according to Clause 37, wherein R⁵, R⁶ or both are a methionine side chain, an arginine side chain or a derivative of these side chains.

44. Composition according to Clause 43, wherein R⁵ is the methionine side chain, and R⁶is the arginine side chain.

45. A method for treating a T-mediated disease comprising administering to an animal in need thereof an effective amount of a pharmaceutical composition comprising a protein or peptide normally found in the animal, protein or peptide treated so that the composition also comprises at least one diketopiperazine obtained from a protein or peptide.

46. ​​The method according to Item 45, wherein the protein is albumin.

47. The method according to Item 45, wherein the protein is immunoglobulin

48. The method according to Item 45, wherein the protein is erythropoietin.

49. The method of any of Claims 45-48, wherein the pharmaceutically acceptable composition is administered orally.

50. The method according to any of Points 45-48, wherein the animal is a human and the protein or peptide is a human protein or peptide.

51. A method of inhibiting T-cell activation comprising administering to an animal in need thereof an effective amount of a pharmaceutical composition comprising a protein or peptide normally found in the animal, protein or peptide treated so that the composition also comprises at least one diketopiperazine obtained from a protein or peptide.

52. The method according to Item 51, wherein the protein is albumin.

53. The method according to Item 51, wherein the protein is immunoglobulin.

54. The method according to Item 51, wherein the protein is erythropoietin.

55. The method of any of Clauses 51-54, wherein the pharmaceutical composition is administered orally.

56. The method of any of Clauses 51-54, wherein the animal is a human and the protein or peptide is a human protein or peptide.

57. A method of synthesizing diketopiperazine comprising heating a protein or peptide solution under effective conditions to induce the formation of diketopiperazine.

-3358. The method according to Item 57, wherein the protein is albumin.

59. The method according to Item 57, wherein the protein is immunoglobulin.

60. The method according to Item 57, wherein the protein is erythropoietin.

61. The method according to Item 57, wherein the diketopiperazine is purified from solution.

62. The method according to Items 57-61, wherein the solution is heated for four days at 60 ° C.

63. A method of synthesizing diketopiperazine comprising contacting a protein or peptide solution with an enzyme that cleaves two N-terminal or two C-terminal amino acids of a protein or peptide under effective conditions to produce diketopiperazine.

64. The method according to Item 63, wherein the protein is albumin.

65. The method according to Item 63, wherein the protein is immunoglobulin

66. The method according to Item 63, wherein the protein is erythropoietin.

67. The method according to Item 63, wherein the enzyme is a dipeptidyl peptidase.

68. The method according to Item 63, wherein the enzyme is a carboxypeptidase.

69. The method according to any of Items 63-68, wherein the diketopiperazine is purified from solution.

70. An improved pharmaceutical protein or peptide composition, the improvement includes a reduced diketopiperazine content in the composition.

71. The method according to Item 70, wherein the protein is albumin.

72. The method according to Item 70, wherein the protein is immunoglobulin.

73. The method according to Item 70, wherein the protein is erythropoietin.

74. A method of producing an improved pharmaceutical protein or peptide composition, the method comprising removing at least a portion of the diketopiperazines present in the composition from the composition.

75. The method according to Item 74, wherein the protein is albumin.

76. The method according to Item 74, wherein the protein is immunoglobulin.

77. The method according to Item 74, wherein the protein is erythropoietin.

78. A method of producing an improved pharmaceutical protein or peptide composition, the method comprising treating the protein or peptide solution so as to increase the diketopiperazin content.

79. The method of Paragraph 78, wherein the solution is heated under conditions effective to effect the formation of diketopiperazines.

80. The method according to Item 79, wherein the solution is heated for four days at 60 ° C.

-3481. The method of Paragraph 78, wherein the solution is contacted with an enzyme that cleaves two N-terminal or two C-terminal amino acids of a protein or peptide under effective conditions to produce diketopiperazines.

82. The method according to Item 81, wherein the enzyme is a dipeptidyl peptidase.

83. The method according to Item 81, wherein the enzyme is a carboxypeptidase.

84. The method according to Item 78, wherein the protein is albumin.

85. The method according to Item 78, wherein the protein is immunoglobulin

86. The method according to Item 78, wherein the protein is erythropoietin.

87. An improved pharmaceutical protein or peptide composition, the improvement includes an increased diketopiperazine content in the composition.

88. The composition according to Item 87, wherein the protein is albumin.

89. The composition according to Item 87, wherein the protein is immunoglobulin.

90. The composition according to Item 87, wherein the protein is erythropoietin.

91. A composition according to any of Points 87-90, which is suitable for oral administration.

Dorota Rzążewska Patent attorney