ES2575563T3

Treatment of t-cell mediated diseases

Abstract

A method of synthesizing a diketopiperazine, which comprises contacting a solution of a protein with an enzyme that cleaves the two N-terminal or C-terminal amino acids of the protein under effective conditions to produce the diketopiperazine, where the protein is albumin, immunoglobulin. or erythropoietin.

ES2575563T3, drawing sheet 1 — Sheet 1 of 9

Term

Term ended

Projected expiry passed 14 May 2024, 2.4 years ago.

3 claims: 1 independent, 2 dependent

1
ES 2 575 563 T3 REIVINDICACIONES 1. Un método para sintetizar una dicetopiperazina, que comprende poner en contacto una solución de una proteína con una enzima que escinde los dos aminoácidos N-terminales o C-terminales de la proteína en condiciones 5 eficaces para producir la dicetopiperazina, donde la proteína es albúmina, inmunoglobulina o eritropoyetina.
2
El método de la reivindicación 1, donde la enzima es una dipeptidil peptidasa o una carboxipeptidasa.
3
El método de la reivindicación 1 o 2, donde la dicetopiperazina se purifica a partir de la solución.

Independent claims3

337 paragraphs in 17 sections, as filed

ES 2 575 563 T3

DESCRIPTION

Treatment of T-lymphocyte-mediated diseases

Field of the invention

The present invention relates to a method for synthesizing a diketopiperazine. Furthermore, the treatment of T lymphocyte mediated diseases and the inhibition of T lymphocyte activation using certain diketopiperazines is described, but not as part of the invention. Pharmaceutical compositions comprising certain diketopiperazines are also described, but not as part of the invention. Also described, but not as part of the invention, are methods of making improved protein and peptide pharmaceutical compositions to increase or decrease the content of diketopiperazines in the compositions and the resulting improved pharmaceutical compositions.

Background

T-lymphocyte-mediated diseases represent a large number of immune system disorders. In particular, T lymphocytes are believed to be cells that initiate and perpetuate autoimmune diseases. Autoimmune diseases are a group of eighty chronic, serious diseases that, in the United States alone, affect millions of people. Autoimmune diseases are characterized by the reactivity of the immune system towards endogenous (auto) antigens. These immune responses to autoantigens are maintained by persistent or recurrent activation of autoreactive T lymphocytes and, directly or indirectly, autoreactive T lymphocytes are responsible for the characteristic tissue damage and destruction seen in autoimmune diseases. Although many treatments have been proposed for autoimmune and other T-cell mediated diseases, there is still a need for additional treatments.

Summary of the invention

The present invention is defined in the appended claims. One method of treating T-cell mediated diseases comprises administering, to an animal in need thereof, an effective amount of a diketopiperazine having the following formula:

OR

Or in which:

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

(a) a side chain of an amino acid, in which the amino acid is glycine, alanine, valine, norvaline, aaminoisobutyric 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, p-aminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine; however, provided that when R1 be the side chain of asparagine or glutamine, then R2 cannot be the side chain of lysine or ornithine, and when R1 be the side chain of lysine or ornithine, then R2 cannot be the asparagine or glutamine side chain;

(b) R1 is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline and / or R2 is -CH2-CH2-CH2-0 -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline; or (c) a derivative of a side chain of an amino acid, wherein the amino acid is one of those listed in (a), and the derivatized side chain has:

(i) a -NH2 group replaced by a -NHR group3 or -N (R3) 2, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

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

ES 2 575 563 T3 (iii) a -COOH group replaced by a -COOR group3, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(iv) a -COOH group replaced by a -CON (R4)2, in which each R4 they may independently be H, or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(v) a -SH group replaced by -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2-CH (NH2) -COOH;

(vi) a -CH group2- replaced by a group -CH (NH2) - or a group -CH (OH) -;

(vii) a -CH3 group replaced by a -CH group2-NH2 or a -CH group2-OH; and / or (viii) an H that is attached to a carbon atom replaced by a halogen; or a physiologically acceptable salt thereof.

A method for inhibiting T-lymphocyte activation is further described. The method comprises administering, to an animal in need thereof, an effective amount of a diketopiperazine of formula I or a physiologically acceptable salt thereof.

Further described is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a diketopiperazine having the following formula:

(H) in which:

R5 and R6, which can be the same or different, is each:

(a) a side chain of an amino acid, in which the amino acid is glycine, alanine, valine, norvaline, aaminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine , threonine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, paminophenylalanine, tyrosine, tryptophan, thyroxine or ornithine; however, provided that when R5 be the side chain of asparagine or glutamine, then R6 cannot be the side chain of lysine or ornithine, and when R5 be the side chain of lysine or ornithine, then R6 cannot be the asparagine or glutamine side chain;

(b) R5 is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline and / or R6 is -CH2-CH2-CH2-or -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline; or (c) a derivative of a side chain of an amino acid, wherein the amino acid is one of those listed in (a), and the derivatized side chain has:

(i) a -NH group2 replaced by a -NHR group3 or -N (R3)2, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(ii) a -OH group replaced by a -O-PO3H group2 u -OR3, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(iii) a -CH group2- replaced by a group -CH (NH2) - or a group -CH (OH) -;

(iv) a -CH3 group replaced by a -CH group2-NH2 or a -CH group2-OH; and / or (v) an H that is attached to a carbon atom replaced by a halogen; or a physiologically acceptable salt thereof.

ES 2 575 563 T3

Another method of treating a disease mediated by T lymphocytes is further described. The method comprises administering, to an animal in need thereof, an effective amount of a pharmaceutical composition comprising a protein or a peptide normally found in the animal, the protein or peptide having been treated so that the composition also comprises at least one diketopiperazine derived from the protein or peptide.

A method of inhibiting the activation of T lymphocytes is further described. The method comprises administering, to an animal in need thereof, an effective amount of a pharmaceutical composition comprising a protein or a peptide normally found in the animal, having the protein or peptide has been treated such that the composition also comprises at least one diketopiperazine derived from the protein or peptide.

The invention provides methods for the synthesis of diketopiperazines. One method, not within the scope of the invention, comprises heating a solution of a protein or a peptide under conditions effective to generate the formation of a diketopiperazine. In one embodiment, the method comprises contacting a solution of a protein or peptide with an enzyme that cleaves the two N-terminal amino acids or the two C-terminal amino acids of the protein or peptide under conditions effective to produce a diketopiperazine. , where the protein is albumin, immunoglobulin, or erythropoietin.

An improved pharmaceutical composition of a protein or a peptide is further described. The improvement is that the composition comprises a lower content of diketopiperazines.

A method of manufacturing an improved pharmaceutical composition of a protein or a peptide is also described. The method comprises removing from the composition at least part of the diketopiperazines present in the composition.

A method of manufacturing an improved pharmaceutical composition of a protein or a peptide is also described. The method comprises treating a solution of the protein or peptide to increase the content of diketopiperazines in the composition.

An improved pharmaceutical composition of a protein or a peptide is further described. The improvement is that the composition comprises a higher content of diketopiperazines.

Brief description of the figures

Figure 1. Plot of counts against ERK1 / 2 concentration for TriPS cells (CD4 + T lymphocyte line isolated from influenza immunized donors that is specific for hemagglutinin) isolated on day 20 after challenge with anti-CD3 OKT3 antibodies and incubated with 25 ng of phorbol myristic acid (PMA), HC-RBL (fraction of heated human colostrum with molecular weight less than 3 kD and containing MR-DKP) at a 1:10 dilution and 0.5 mM DA-DKP for 15 minutes at 37 ° C.

Figure 2. Bar graph showing inhibition of tumor necrosis factor α (TNFa) and IL16 secretion by TriPS cells 12 days after challenge with anti-CD3 OKT3 antibodies. It indicates the inhibition of both TNFα and IL-16 secretion by the DA-DKP band from human colostrum (HC) 2626 (containing MR-DKP). The maximum release observed using HC 2626 at 1: 100 and 1: 1,000 dilutions is due to the lytic effect of the high concentrations of human colostrum. No lysis is observed using 0.5 mM DA-DKP, and secretion of TNFa and IL-16 is reduced.

Figure 3. Bar graph showing inhibition of TNFa secretion by TriPS cells 10 days after challenge with anti-CD3 OKT3 antibody. Indicates that HC RBL and DA-DKP need to be further examined for titratable response as seen with HC 2626. May indicated potent activity.

Figure 4. Bar graph showing inhibition of TNFa secretion by TriPS cells at different times after stimulation with anti-CD3 OKT3 antibodies. It indicates that early in the stimulation cycle, the effect of DA-DKP and HC RBL is inhibitory, while later (day 14), the effect is stimulating. HC 2626 inhibits at all times, presumably due to other constituents.

Figure 5. Bar graph showing inhibition of TNFa secretion by H4 # 9.25 cells (CD4 + T lymphocyte line isolated from autopsy brain tissue of a multiple sclerosis patient that is specific for myelin basic protein) in on days 7-10 after challenge with the anti-CD3 OKT3 antibody. It indicates that the secretion of TNFα by this line of T lymphocytes is also inhibited by HC 2626, HC RBL and DADKP.

Detailed Description of Currently Preferred Embodiments

ES 2 575 563 T3

A method of treating T-lymphocyte-mediated diseases is described. Treat is used herein in the sense of reducing (in whole or in part) the symptoms, duration or severity of a disease, including curing the disease or disease prevention.

T-cell mediated diseases include graft rejection, graft-versus-host disease, unwanted delayed-type hypersensitivity reactions (such as delayed-type allergic reactions), T-cell mediated lung diseases, and autoimmune diseases. T-cell mediated lung diseases include sarcoidosis, hypersensitivity pneumonitis, acute interstitial pneumonitis, alveolitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, and other diseases characterized by inflammatory lung damage. Autoimmune diseases include multiple sclerosis, neuritis, polymyositis, psoriasis, vitiligo, Sjógren's syndrome, rheumatoid arthritis, type 1 diabetes, autoimmune pancreatitis, inflammatory bowel diseases (eg, Crohn's disease and ulcerative colitis), celiac disease, glomerulonephritis , scleroderma, sarcoidosis, autoimmune thyroid diseases (for example, Hashimoto's thyroiditis and Graves' disease), myasthenia gravis, Addison's disease, Autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, and systemic lupus erythematosus.

T-lymphocyte-mediated diseases are treated by administering, to a needy animal, an effective amount of a diketopiperazine having the following formula:

R '(I) in which:

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

(a) a side chain of an amino acid, in which the amino acid is glycine, alanine, valine, norvaline, aaminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine , threonine, aspartic acid, asparagine, glutamic acid, glutamine, Usine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, p-aminophenylalanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine; however, provided that when R1 be the side chain of asparagine or glutamine, then R2 cannot be the side chain of Usine or ornithine, and when R1 be the side chain of Usine or ornithine, then R2 cannot be the asparagine or glutamine side chain;

(iii) a -COOH group replaced by a -COOR group3, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylated, arylalkyl or heteroaryl;

(iv) a -COOH group replaced by a -CON (R4) 2, in which each R4 they may independently be H, or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylated, arylalkyl or heteroaryl;

(v) a -SH group replaced by -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2-CH (NH2) -COOH;

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

ES 2 575 563 T3 (vii) a -CH3 group replaced by a -CH2-NH2 group or a -CH2-OH group; and / or (viii) an H that is attached to a carbon atom replaced by a halogen; or a physiologically acceptable salt thereof.

By "replaced" is meant that, with reference to the formula of an amino acid side chain, the specified group is replaced by the other specified group. For example, the formula for the isoleucine side chain is -CH (CH3) -CH2-CH3. If the terminal -CH3 group is replaced by a -CH2-OH group, then the resulting derivatized isoleucine side chain formula 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 resulting derivatized alanine side chain would be -CH2-CI. It should be noted that the glycine side chain is -H and if this H is replaced by a chlorine atom (or other halogen), the resulting side chain-will be Cl, with the chlorine atom attached to the ring carbon (e.g. , R1 = Cl)

Dicetopiperazines in which R1, R2 or both is the side chain of aspartic acid or glutamic acid or a derivative of said side chain in which the group -COOH is replaced by a group -COOR3 or a group -CON (R4) 2, where R3 and R4 are defined above. Of this group of compounds, the most preferred are the diketopiperazines comprising the aspartic acid and alanine side chains (Asp-Ala DKP or DADKP), the glutamic acid and alanine side chains (Glu-Ala DKP or EA-DKP), the tyrosine and aspartic acid side chains (Tyr-Asp DKP or YD-DKP), the tyrosine and glutamic acid side chains (Tyr-Glu DKP or YEDKP) and aspartic acid derivatives or glutamic acid side chains of these four diketopiperazines in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, where R3 and R4 have been defined above.

Dicetopiperazines in which R1 and R2 they are both hydrophobic side chains (eg, phenylalanine side chain) or hydrophobic side chain derivatives. By hydrophobic side chain derivative is meant that the derivatized side chain is hydrophobic. In particular, diketopiperazines are preferred in which each R1 and / or R2, which may be the same or different, is the 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 nitrogen atom (s), form proline. Of this group of compounds, the most preferred are the diketopiperazines comprising the side chains of glycine and leucine (Gly-Leu DKP or GL-DKP), proline and phenylalanine (Pro-Phe DKP or PF-DKP), and alanine and proline (Ala-Pro DKP or AP-DKP).

Other preferred diketopiperazines are those in which R1, R2 or both is the methionine side chain, the arginine side chain, or a derivative of these side chains. The most preferred of this group is a diketopiperazine, in which R1 is the methionine side chain and R2 is the arginine side chain (Met-Arg DKP or MR-DKP).

Side chain of an amino acid means the part of the amino acid linked to the common main chain I

NHj-CH-COOH of all the amino acids mentioned above. For example, the glycine side chain is -H, the alanine side chain is -CH3, and the serine side chain is -CH2OH.

By hydrophobic is meant a side chain or side chain derivative that is uncharged at physiological pH and is repelled by an aqueous solution.

By alkyl 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.

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 a ring of 4-8 carbon atoms.

By heterocycloalkyl is meant a cycloalkyl having one or more of the ring carbon atoms of at least one of the rings replaced by an O, S or N.

By aryl is meant an aromatic group having at least one aromatic ring (eg phenyl).

By alkylaryl is meant a lower alkyl having an H replaced by an aryl (for example, -CH2-C6H5 or -CH3CH (C6H5) CH3).

By arylalkyl is meant an aryl having an H replaced by a lower alkyl (eg, -C6H4-CH3).

ES 2 575 563 T3

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

By substituted it is meant that the residue is substituted with one or more substituents selected from the following group: OH, NH2, -SH, -COOH and / or a halogen atom.

By halogen is meant chlorine, fluorine, bromine or iodine. Chlorine or bromine is preferred.

The diketopiperazines of formula I are effective in the treatment of diseases mediated by T lymphocytes, as they inhibit the activation of T lymphocytes. Consequently, the diketopiperazines of formula I can also be used to treat inflammation and inflammatory diseases that are caused by, aggravated by or involving activated T lymphocytes. Inhibit is used herein in the sense of reducing (in whole or in part) or preventing.

Methods of making diketopiperazines are well known in the art, and these methods can be employed to synthesize the diketopiperazines described herein. See, for example, US Patent Nos. 4,694,081, 5,817,751, 5,990,112, 5,932,579, and 6,555,543, US Patent Application Publication Number 2004/0024180, PCT Applications WO 96 / 00391 and WO 97/48685, and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374 (1998).

For example, diketopiperazines can be prepared by first synthesizing dipeptides. Dipeptides can be synthesized by methods well known in the art using L-amino acids, D-amino acids, or a combination of D- and L-amino acids. Solid phase peptide synthesis methods are preferred. Of course, dipeptides are also commercially available, from numerous sources, including DMI Synthesis Ltd., Cardiff, UK (synthesis on demand), Sigma-Aldrich, St. Louis, MO (primarily, Customer Demand Synthesis), Phoenix Pharmaceuticals, Inc., Belmont, CA (Customer Demand Synthesis), Fisher Scientific (Customer Demand Synthesis), and Advanced ChemTech, Louisville, KY.

Once the dipeptide has been synthesized or acquired, it is cyclized to form a diketopiperazine. This can be done through a variety of techniques.

For example, US patent application publication number 2004/0024180 describes a method of cyclization of dipeptides. Briefly, the dipeptide is heated in an organic solvent while removing the water by distillation. Preferably, the organic solvent is a low-boiling azeotrope with water, such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol, 2-butanol, t-butanol, acetic acid butyl ester, 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 reaction rate at which the cyclization is carried out and the type of azeotropic separating agent used. The reaction is preferably carried out at 50-200 ° C, more preferably at 80-150 ° C. The pH range in which the cyclization takes place can be easily determined by the person skilled in the art. It will advantageously be from 2 to 9, preferably from 3 to 7.

When one or both amino acids of the dipeptide have, or are derivatized to have, a carboxyl group in its side chain (eg, aspartic acid or glutamic acid), the dipeptide is preferably cyclized as described in US Patent No. 6,555,543. Briefly, the dipeptide, with the side chain carboxyl still protected, is heated under neutral conditions. Generally, the dipeptide will be heated to about 80 ° C to about 180 ° C, preferably about 120 ° C. The solvent will be a neutral solvent. For example, the solvent can comprise an alcohol (such as butanol, methanol, ethanol, and higher alcohols, but not phenol) and an azeotropic cosolvent (such as toluene, benzene, or xylene). Preferably, the alcohol is butan-2-ol, and the azeotropic cosolvent is toluene. Heating is continued until the reaction is complete, and these times can be determined empirically. Generally, the dipeptide will be cyclized by heating under reflux for about 8 to 24 hours, preferably about 18 hours. Finally, the protecting group is removed from the diketopiperazine. Thus, the use of strong acids (mineral acids such as sulfuric acid or hydrochloric acid), strong bases (alkaline bases, such as potassium hydroxide or sodium hydroxide) and strong reducing agents (for example, hydride) should be avoided. lithium aluminum) in order to maintain the chirality of the final compound.

The dipeptides created in solid phase resins can be cyclized and released from the resin in one step. See, for example, US Patent No. 5,817,751. For example, the resin having an N-alkylated dipeptide attached is suspended in toluene or toluene / ethanol in the presence of acetic acid (eg 1%) or triethylamine (eg 4%). Generally, basic cycling conditions are preferred because of their faster cycling times.

To prepare the diketopiperazine of formulas I and II, in which the amino acid side chains are derivatized, amino acid derivatives can be used in the synthesis of the dipeptides, the dipeptides can be derivatized and / or

ES 2 575 563 T3 diketopiperazines can be derivatized as is known in the art. See, for example, the references cited above.

Other methods of dipeptide cyclization and diketopiperazine preparation are known in the art, and can be used in the preparation of diketopiperazines useful in the practice described herein. See, for example, the references mentioned above. Furthermore, many diketopiperazines can be prepared as described below from proteins and peptides. Dicetopiperazines are also commercially available from, for example, DMI Synthesis Ltd., Cardiff, UK (synthesis on demand).

The diketopiperazines of formulas I and II include all possible stereoisomers that can be obtained by varying the configuration of individual chiral centers, axes, or surfaces. In other words, the diketopiperazines of formulas I and II include all possible diastereomers, as well as all optical isomers (enantiomers).

The physiologically acceptable salts of the diketopiperazines can also be used. Physiologically acceptable salts 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, stearic, lactic, malic, tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic and the like) or bases (such as hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or organic cations derived from N, N-dibenzylethylenediamine, D-glucosamine or ethylenediamine). The salts are prepared in a conventional manner, for example, by neutralizing the free base form of the compound with an acid.

As noted above, a diketopiperazine, or a physiologically acceptable salt thereof, can be used to treat a disease mediated by T lymphocytes or to inhibit the activation of T lymphocytes. For this, a diketopiperazine, or a physiologically acceptable salt thereof, to an animal that needs it. 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 making such determinations is well within the art. Those skilled in the art understand that the dose amount will vary with the particular compound employed, the disease or condition to be treated, the severity of the disease or condition, the route (s) of administration, the rate of compound excretion, duration of treatment, identity of any other drugs being administered to the animal, age, size and species of the animal, and similar factors known in the medical and veterinary arts. In general, a suitable daily dose of a diketopiperazine-type compound will be that amount of the compound that is the lowest effective dose to produce a therapeutic effect. However, the daily dosage will be determined by an attending physician or veterinarian within the scope of medical judgment. If desired, the effective daily dose can be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. Administration of the compound should be continued until an acceptable response is obtained.

The compounds (i.e., the diketopiperazines and physiologically acceptable salts thereof) can be administered to an animal patient for therapy by any suitable route of administration, including oral, nasal, rectal, vaginal, parenteral (e.g., via intravenous, intraspinal, intraperitoneal, subcutaneous or intramuscular), intracisternal, transdermal, intracranial, intracerebral and topical (including buccal and sublingual). The preferred routes of administration are oral and intravenous.

Although a compound can be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). Pharmaceutical compositions comprise a compound or compounds as an active ingredient in admixture with one or more pharmaceutically acceptable carriers and, optionally, with one or more other compounds, drugs or other materials. Each vehicle must be acceptable in the sense of being compatible with the rest of the ingredients of the formulation and not harmful to the animal. Pharmaceutically acceptable carriers are well known in the art. Regardless of the selected route of administration, the compounds are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. See, for example, Remington's Pharmaceutical Sciences.

Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion. , or as an elixir or syrup, or as lozenges (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds as an active ingredient. A compound (s) can also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, lozenges, powders, granules and the like), the active ingredient (i.e. one or more diketopiperazines and / or physiologically acceptable salts thereof) is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or

ES 2 575 563 T3 dicalcium phosphate and / or any of the following: (1) fillers or diluents such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (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) dissolution retarding 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 sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type can be employed 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.

A tablet can be made by compression or molding, optionally with one or more auxiliary ingredients. Compression tablets can be prepared using a binding agent (eg, gelatin or hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (eg, cross-linked sodium starch glycolate or sodium carboxymethylcellulose), surfactant, or dispersant. Molded tablets can be made by molding in a suitable machine a mixture of powdered compound moistened with an inert liquid diluent.

Tablets, and other solid dosage forms of pharmaceutical compositions, such as lozenges, capsules, pills, and granules, optionally, can be marked or prepared with coatings and shells such as enteric coatings and other coatings well known in the formulation art. pharmaceutical. They can also be formulated so as to provide a slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in variable proportions to provide the desired release profile, other polymeric matrices, liposomes and / or microspheres. They can be sterilized by, for example, filtration through a bacteria retention filter. These compositions can also optionally contain opacifying agents and can be of such a composition that they release the active principle alone, or preferentially, in a certain part of the gastrointestinal tract, optionally, in a delayed manner. Examples of inclusion compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form.

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, liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, peanut, corn, germ, olive oils, of castor and sesame), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.

In addition to inert diluents, oral compositions can also include adjuvants such as wetting, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

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

Formulations of pharmaceutical compositions for rectal or vaginal administration can be presented as a suppository, which can be prepared by mixing one or more compounds with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature but liquid at body temperature and therefore that melts in the rectum or vaginal cavity, releasing the active compound. Formulations that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing vehicles such as those known in the art to be appropriate.

Dosage forms for topical or transdermal administration of the compounds include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops, and inhalants. The active ingredient can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any buffer, or propellant that may be needed.

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

ES 2 575 563 T3

Powders and sprays can contain, in addition to the active principle, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can further contain the usual propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Transdermal patches have the additional advantage of providing a controlled release of compounds into the body. Such dosage forms can be prepared by dissolving, dispersing, or otherwise incorporating one or more compounds in a suitable medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound through the skin. The speed of such flow can be controlled either by providing a rate controlling membrane or by dispersing the compound in a polymeric matrix or in a gel.

Pharmaceutical formulations include those suitable for administration by inhalation or insufflation or for nasal or intraocular administration. For administration to the upper (nasal) or lower airways by inhalation, the compounds are conveniently administered from an insufflator, nebulizer or pressurized container or other convenient means of administration of an aerosol spray. Pressurized containers may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition 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, for example, capsules or cartridges, or, for example, in gelatin or blister packs of which the powder can be administered with the aid of an inhaler, insufflator or an inhaler of metered doses.

For intranasal administration, the compounds can be administered via nasal drops or a liquid spray, such as via a plastic bottle atomizer or metered dose inhaler. The most common atomizers are the Mistometer (Wintrop) and Medihaler (Riker).

Drops such as eye drops or nasal drops can be formulated with an aqueous or non-aqueous base which also comprises one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently administered from pressurized containers. The drops can be administered by means of a simple bottle with a dropper cap for the eyes or by means of a plastic bottle adapted to the administration of liquid content drop by drop by means of a specially shaped closure.

Pharmaceutically acceptable pharmaceutical compositions suitable for parenteral administration comprise 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 reconstituted into sterile injectable solutions or dispersions just prior to of their use, which may contain antioxidants, buffers, solutes that make the formulation isotonic with the blood of the desired recipient, or thickening or suspending agents.

Examples of suitable aqueous and non-aqueous carriers that can be employed in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

These compositions may also contain adjuvants such as wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. Furthermore, prolonged absorption of the injectable dosage form can be achieved by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material that has low water solubility. The absorption rate of the drug then depends on its dissolution rate which, in turn, may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot 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 employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). The

ES 2 575 563 T3 depot injectable formulations are also prepared by trapping the drug in liposomes or microemulsions that are compatible with body tissue. Injectable materials can be sterilized, for example, by filtration through a bacteria retention filter.

The formulations can be presented in sealed unit dose or multiple dose containers, for example ampoules and vials, and can be stored in a lyophilized state that only requires the addition of the sterile liquid carrier, for example water for injection, immediately prior to delivery. use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above.

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

Accordingly, diketopiperazines can be prepared by heating solutions of albumin, immunoglobulin, erythropoietin, and other proteins and peptides. For example, a solution of albumin, immunoglobulin, erythropoietin, or other protein or peptide is prepared in phosphate buffer at neutral pH. Preferably, the solution is a concentrated solution (eg, about 100-500 mM) to achieve protonation of the N-terminal and / or C-terminal amino acids. The solution is heated at 60 ° C for from about 2 hours to several days, preferably about 4 days, to cause formation of the diketopiperazines. Preferably, denaturation of the protein should be avoided. This can be achieved by using shorter times and / or by adding caprylic acid or N-acetyltryptophan at about 0.02M for each.

The diketopiperazines synthesized according to the present invention can be prepared by contacting a solution of albumin, immunoglobulin, or erythropoietin, or with an enzyme that can cleave the two N-terminal amino acids of the protein (for example, dipeptidyl peptidases) or a enzyme that can cleave the two C-terminal amino acids of the protein (eg, carboxypeptidases). Suitable dipeptidyl peptidases and carboxypeptidases are commercially available, for example, from Sigma. The reaction should be carried out at a pH of 6-8, preferably in a buffer, such as phosphate buffer, at a temperature high enough to accelerate the reaction, but not so high as to cause denaturation of the protein (for example , 37 ° C).

The amino acid sequences of numerous proteins and peptides are known, and a protein or peptide with the desired N-terminal and / or C-terminal sequence can be selected to give the desired diketopiperazine (s) using any of the methods. . Furthermore, peptides with a desired sequence can be synthesized by well known and used methods.

Dicetopiperazines can be purified from solutions containing them, including commercially available pharmaceutical compositions comprising albumin, immunoglobulin, and erythropoietin, by well-known methods such as size exclusion chromatography (eg Centricon filtration), chromatography affinity (for example, using a column of beads that has attached to it an antibody or antibodies directed to the desired diketopiperazine (s), or an antibody or antibodies directed to the truncated protein or peptide), anion exchange or cation exchange. Purified diketopiperazines can be used and incorporated into pharmaceutical compositions as described above.

Instead of purifying the diketopiperazines, for the treatment of a T-lymphocyte-mediated disease, pharmaceutical compositions comprising albumin, immunoglobulin, erythropoietin and / or other proteins and / or peptides normally found in the recipient animal can be administered, and can be used to inhibit the activation of T lymphocytes. Although compositions comprising these proteins and / or peptides that are currently commercially available can be used if they contain diketopiperazines, it is highly preferable to treat albumin, immunoglobulin, erythropoietin, and / or other proteins and / or peptides as described above. to increase the content of the desired diketopiperazine / s before the administration of the compositions thus improved. The animal is preferably human, and the proteins and / or peptides are preferably human proteins and / or peptides. Oral administration of the composition (s) is preferred.

Effective dosage amounts of protein and / or peptide compositions can be determined empirically, and making such determinations is in the art. In particular, to determine an effective dosage amount of a protein and / or peptide composition, the amount of one or more diketopiperazines present in the composition can be measured, and an amount of the composition sufficient to administer can be administered to the animal. an effective amount of the diketopiperazine / s. Those skilled in the art understand that the dosage amount will vary with the particular composition employed, the disease or condition to be treated, the severity of the disease or condition, the route (s) of administration, the

ES 2 575 563 T3 rate of excretion, duration of treatment, identity of any other drugs being administered to the animal, age, size and species of the animal, and similar factors known in the medical and veterinary arts. In general, a suitable daily dose of a protein and / or peptide composition will be the amount that is the lowest effective dose to produce a therapeutic effect. However, the daily dosage will be determined by an attending physician or veterinarian within the scope of medical judgment. If desired, the effective daily dose can be administered as two, three, four, five, six or more subdoses, administered separately at appropriate intervals throughout the day. Administration should continue until an acceptable response is achieved.

As noted above, diketopiperazines have been found to be found in intravenous pharmaceutical compositions of albumin, immunoglobulin, and erythropoietin available on the market, where the manufacture of these compositions consists of one or more heating steps (for example, for sterilization ). Dicetopiperazines are probably also present in other protein and peptide pharmaceutical compositions in which the manufacture of the compositions involves heating steps. As described herein, many diketopiperazines have the ability to inhibit the activation of T lymphocytes. Therefore, in many situations, it may be undesirable to administer compositions of albumin, immunoglobulin, erythropoietin, or other diketopiperazine-containing proteins or peptides to patients. For example, albumin is often given to patients with trauma, immunoglobulin is often given to patients with infections or immune deficiencies, and erythropoietin is given to patients with anemic cancer or chronic diseases whose immune systems are often compromised. Accordingly, a method of extracting at least part of, preferably essentially all, the diketopiperazines from said compositions is provided. Dicetopiperazines can be extracted as described above (eg by size exclusion chromatography (eg Centricon filtration), affinity chromatography (eg using a column of beads having an antibody or antibodies attached thereto targeting the desired diketopiperazine (s), or an antibody or antibodies targeting albumin, immunoglobulin, erythropoietin, or other protein or peptide), anion exchange or cation exchange) to produce improved compositions of albumin, immunoglobulin, erythropoietin, and other proteins and peptides.

Examples

Example 1: Absorption of Asp Ala DKP (DA-DKP) and Glu Ala DKP (EA-DKP) from rat intestine

Rat intestine was marginally isolated from the pyloric sphincter to the rectum and perfused via the mesenteric artery with an erythrocyte-based perfusion fluid containing bovine serum albumin. The effluent perfusion fluid from the intestine was collected by portal vein cannulation and recirculated (after reoxygenation). After a period of equilibrium, a solution (approximately 1 ml) containing approximately 1 mg of Asp-Ala diketopiperazine (DA-DKP) or 1.4 mg of Glu-Ala diketopiperazine (EADKP) was administered by injection into the lumen. of the duodenum.

After dosing, serial samples of the perfusion fluid were collected at time intervals of up to 2 hours after dosing. Said samples were centrifuged and plasmas were assayed for both cyclic dipeptides by liquid chromatography mass spectrometry (LC-MS).

The results showed that, after only 2 hours of infusion, the amounts of DA-DKP and EA-DKP that had been absorbed from the intestinal lumen into the circulatory system corresponded to 95% and 100% (actually, 112%), respectively. , of the administered dose.

Therefore, both cyclic peptides are rapidly and efficiently absorbed from the intestinal lumen into the blood, with no evidence of metabolism during transport through the intestinal wall. Therefore, these potential therapeutics can be administered orally.

The rapid absorption of DA-DKP and EA-DKP unchanged from the gastrointestinal tract into the blood, combined with the lack of first-pass hepatic clearance of both compounds in the isolated perfused rat liver (data not shown) demonstrates that clearance presystemic is low. Therefore, oral dosing will be an ideal route of administration.

On the other hand, studies with perfused rat kidney isolates showed that, unlike many linear chain peptides, which are extensively metabolized by renal peptidases, the renal clearance of the two cyclic dipeptides is relatively slow.

Taken together, these data suggest that a low daily dose diketopiperazine dosing regimen is likely adequate for therapeutic purposes.

Preliminary pharmacokinetic data in rats after oral administration coincided with the above for both cyclic dipeptides, with Tmax values of 30-60 minutes and Cmax values of 4-6 pg / ml (DA-DKP) and 0.6-1 , 1 pg / ml

ES 2 575 563 T3 (EA-DKP) after oral dosing at 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 in which the concentration reaches a maximum, and Cmax is the maximum concentration reached; both were calculated from a curve fitting equation for the data obtained).

Preliminary data suggest that DA-DKP and other diketopiperazines cross the blood-brain barrier. Therefore, DA-DKP and other diketopiperazines of the invention should be useful for treating disorders of the nervous system, such as multiple sclerosis.

Example 2: Inhibition of human T-cell cytokine production in vitro by human colostrum fractions containing Met-Arg DKP (MR-DKP) and by Asp-Ala DKP (DA-DKP)

A. Materials

The present example demonstrates that DA-DKP, human colostrum (HC 2626) containing MR-DKP and a low molecular weight fraction of human colostrum (HC RBL; a fraction of human colostrum containing components of molecular weights less than 3,000 prepared by Centricon filtration of defatted colostrum) which also contains MR-DKP, inhibited cytokine production from 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 the physiological response to inflammation. They are also sometimes found in human intravenous immunoglobulin (IVIg), human albumin, and other biological preparations.

B. Inhibition of T-cell cytokine production

Two different human CD4 positive T cell clones were tested. One of the cell lines (TRiPS) was isolated from an influenza immunized donor, and is specific for the hemagglutinin peptide 307-319. The other cell line (H4 # 9.25) was isolated from autopsy brain tissue of a multiple sclerosis donor, and is specific for myelin basic protein (amino acids 87-99). Both T cell clones produce interleukin 8 (IL-8), IL16, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) after in vitro stimulation with either (1) specific antigen plus presenting cells HLA DR2 positive; (2) anti-CD3 plus anti-CD28 antibodies.

T lymphocyte cell lines were stimulated for passage using approximately 4 x 105 cells on day 18-20 after a previous stimulation. Cells were washed once in cold Iscove's Modified Dulbecco's Minimum Essential Medium (IMDM, Sigma) plus 10% Fetal Bovine Serum (FBS; American Type Culture Collection (ATCC)) and resuspended in 1.0 ml. of cold IMDM medium containing a 1: 500 dilution of OKT3 anti-CD3 monoclonal antibody (prepared from mouse ascites fluid). Cells were incubated with the antibody for 30 minutes on ice, then washed with cold medium without FBS and combined with approximately 2 x 106 4000R irradiated normal human donor peripheral blood leukocytes (PBL), as feeder cells, in medium plus 50 U / ml human IL-2 (Xenometrix). The cultures were expanded by adding fresh IMDM medium with FBS plus IL-2 on day 3. The day of culture was measured from the day of OKT3 challenge. Cells can be used for experiments from day 7 (at maximum proliferation), usually on day 14 (most sensitive to restimulation) and until day 21 (resting cells approaching senescence).

Activation experiments were performed by removing an aliquot of cells and washing twice with heated IMDM medium (37 ° C). For each specific assay, 2 x 10 were previously incubated5 Viable cells in a total volume of 0.9 ml of heated IMDM medium containing the specified amount of treatment additive (eg, HC 2626, DA-DKP, PMA, etc.) for 15 minutes at 37 ° C. Then a 2 x 10 aliquot was added.5 Dynabeads CD3 / CD28 (Dynal), as an activation stimulus, in 0.1 ml of warmed IMDM, and the cultures were incubated overnight (18 hours) at 37 ° C. Cell culture supernatants were collected after agglomeration of cells by centrifugation. The cytokine content was assayed by specific ELISA (eg TNFa, IFNy, IL-8, IL-16; Endogen).

As shown in Figures 1-5, human colostrum (HC 2626) inhibited cytokine production in vitro by both T-lymphocyte cell lines in a dose-dependent manner. As also shown in Figures 1-5, HC RBL and DA-DKP inhibited cytokine production in vitro by both T lymphocyte lines in a dose dependent manner early in the stimulation cycle. However, the effects of HC RBL and DADKP at a later time in the cycle (day 14 or later) were stimulating (see Figure 4). Both HC 2626 and HC RBL contain MR-DKP (as determined by mass spectrometry), but HC 2626 contains other components (including caseins, which are relatively dephosphorylated proteins that can therefore be anti-inflammatory, as described in co-pending application 10 / 723,247, filed November 25, 2003), in addition to MR-DKP, which may be responsible for its inhibitory effects at a later time in the cell cycle. Therefore, HC RBL and HC 2626 (containing both MR-DKP), MR-DKP, and DA-DKP should be useful in down-modulating the inflammatory response of cytokines in autoimmune and / or T-lymphocyte-mediated diseases. , such as multiple sclerosis, since all of them inhibit cytokine production by T lymphocytes early in the stimulation cycle.

ES 2 575 563 T3

These results also suggest that HC RBL, HC 2626, MR-DKP, and DA-DKP will selectively affect antigen-specific T cells without affecting resting T cells.

C. Mechanism of action

The mechanism of action of DA-DKP and HC 2626 (containing MR-DKP) was investigated. For this, 1 x 106 TriPS cells from day 18 for 30 minutes at 37 ° C, either without any addition (Without anything), with addition of CD3 / CD28 Dynabeads (CD3 / CD28 beads), with CD3 / CD28 beads and 0.5 mM DA-DKP, or with addition of CD3 / CD28 beads and 1: 500 dilution of HC 2626. After incubation, cells were lysed in Cell-Lytic mammalian cell extraction reagent (Sigma).

The cell extracts were then incubated separately with Hypromatrix matrices in duplicate for 2 hours at room temperature, followed by two washes following the manufacturer's protocol (Hypromatrix). The Hypromatrix matrix is a nylon membrane transferred with antibodies against the transcription factors listed in Table 1 (manufactured on customer request by Hypromatrix). A cocktail of antibodies specific for phosphorylated tyrosine, phosphorylated serine, and phosphorylated threonine (Zymed) was added and incubated for 1 hour. Then, a biotin-labeled anti-immunoglobulin antibody was added. After the anti-immunoglobulin biotin was washed away, streptavidin-peroxidase was added, and the matrices were washed one last time before the addition of a peroxidase-reactive luminescent substrate.

The results were visualized by exposure to the film and scored as 0 (negative) or + to ++++ (positive) as presented in Table 2. As shown in Table 2, some activation of transcription factors of cytokines (ERK1 / 2) and release of preformed cytokines were inhibited by HC 2626 (containing MR-DKP) and DA-DKP.

TABLE 1: HYPROMATRIX MATRIX (CUSTOMER REQUEST): PROTEINS FOR PHOSPHORYLATION

<td>NUMBER</td><td>ACRONYM</td><td>COMPOUND</td>

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

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

<td> 3</td><td>CBP</td><td>csk binding protein (PAG); transiently (and low-level) Tyr-dephosphorylated integral membrane protein to release csk</td>

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

<td> 5</td><td>csk</td><td>COOH end src kinase; phosphorylated in Ser364, also phosphorylated on Tyr (activity?) - phosphorylates and inactivates lck</td>

<td> 6</td><td>ERK1</td><td>kinase related to extracellular signals</td>

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

<td> 8</td><td>NFATC</td><td>activated T-cell nuclear factor; intact anergy</td>

<td> 9</td><td>c-jun</td><td>AP-1 constituent activated by TcR activation; phosphorylated by JNK-MAPK on Ser63</td>

<td> 10</td><td>IkB-o</td><td>NFkB inhibitor</td>

<td> 11</td><td>pIKB-α</td><td>Ser phosphorylated and inactivated NFkB inhibitor</td>

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

<td> 13</td><td>pI3 / p85 kinase</td><td>activated by glucocorticoids and p2-adrenergic R</td>

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

<td> 16</td><td>Rap1</td><td>Negative TcR regulatory GTPase</td>

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

<td> 18</td><td>fyn</td><td>cell membrane bound immediate TcR signal kinase</td>

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

<td> 20</td><td>Kinase ZAP70</td><td>CD3Z flag; phosphorylated in? by lck / fyn, ZAP70 phosphorylates LAT (linker for T lymphocyte activation) in Tyr and Tyr in sLP-76</td>

ES 2 575 563 T3

TABLE 2: RESULTS

<td>COMPOUND</td><td>NOTHING</td><td>CD3 / CD28</td><td>DKP</td><td>HC2626</td>

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

<td>pI3 / p85 kinase</td><td> +</td><td> ++</td><td> +</td><td> ++</td>

<td>ZAP70 kinase</td><td> --</td><td> --</td><td> --</td><td> --</td>

Example 3: Inhibition of the production of human cytokine T cells in vitro by Gly-Leu DKP (GL-DKP) and DKP Ala-Pro (AP-DKP)

GL-DKP and AP-DKP (obtained from DMI Synthesis, Ltd, Cardiff, UK) were assayed as described in Example 2 using TriPS and H4 # 9.25 cell lines. GL-DKP and AP-DKP were found to inhibit in vitro cytokine production by these two T-lymphocyte cell lines in a dose-dependent manner. The mechanism of action is currently under investigation as described in Example 2, and both the activation of the cytokine transcription factor and the release of previously formed cytokine appear to be affected.

Example 4: Inhibition of human T-cell cytokine production in vitro by Asp Ala DKP (DA-DKP) and Tyr Glu DKP (YE-DKP)

Normal human lymphocytes were isolated from the peripheral blood of a normal human donor with Histopaque (Sigma). Then they were suspended, 3-4 x 105 of lymphocytes in 1 ml of IMDM medium without serum. Cells were stimulated by adding 25 µl of a 1: 2000 dilution of anti-CD3 antibody (Pharmingen, San Diego, CA) and incubating for 18 hours at 37 ° C.

Subsequently, one of the three DKP preparations and dexamethasone (final concentration of 10-5 M) to triplicate cultures. The three DKP preparations were:

1. DA-DKP (obtained from DMI Synthesis, Ltd, Cardiff, UK; final concentration of 25 pg / ml in cultures).

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

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

ES 2 575 563 T3

After the addition of the DKP or dexamethasone preparations, the cultures were incubated for 18 hours at 37 ° C. The amounts of IL-2, IFNy and TNFa released in each culture were then measured by ELISA (Pierce Biotechnology, Rockford, IL 61105).

The results are presented in the following Table 3. As can be seen, the greatest reduction in the release of the three cytokines was obtained with DKP-y-glob. Flow cytometry relative to the number of CD69 + T lymphocytes (CD69 is a marker found in activated T lymphocytes) also showed that DKP-y-glob reduced the number of CD69 + T lymphocytes by approximately 90%, compared with a reduction in the approximately 50% by dexamethasone, despite internalization of the T-cell receptor complex.

TABLE 3

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

<td>Nothing</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>DKP-y-glob</td><td>0.24 ± 0.25 (91% reduction)</td><td>2.1 ± 0.1 (99% reduction)</td><td>1.6 ± 0.6 98% reduction</td>

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

The following clauses are also disclosed, but not as part of the present invention:

1. A method of treating a T-lymphocyte-mediated disease, which comprises administering, to an animal in need thereof, an effective amount of a diketopiperazine having the following formula:

in which:

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

(b) R1 is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline, R2 is -CH2-CH2-CH2-0 -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline; or both R1 as R2 they are each independently -CH2-CH2-CH2-0 -CH2-CH (OH) -CH2-, and together with the adjacent ring nitrogens form proline and hydroxyproline; or (c) a derivative of a side chain of an amino acid, wherein the amino acid is one of those listed in (a), and the derivatized side chain has:

(i) a -NH2 group replaced by a -NHR group3 or -N (R3) 2, in which each R3 it can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylated, arylalkyl or heteroaryl;

(ii) an -OH group replaced by a -O-PO3H2 or -OR group3, in which each R3 it can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylated, arylalkyl or heteroaryl;

(iii) a -COOH group replaced by a -COOR group3, in which each R3 it can be, independently, a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylated, arylalkyl or heteroaryl;

ES 2 575 563 T3 (iv) a -COOH group replaced by a -CON (R4) 2, in which each R4 they may independently be H, or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(v) a -SH group replaced by -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2-CH (NH2) -COOH;

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

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

two. The method in Clause 1, in which R1, R2 or both is the aspartic acid side chain, the glutamic acid side chain, or a derivative of an aspartic acid or glutamic acid side chain, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4)2.

3. The method in Clause 2, where R1 is the side chain of aspartic acid or a derivative of the side chain

4 2 of aspartic acid, in which the -COOH group is replaced by a -COOR group or -CON (R) 2 group, and R is the alanine side chain.

4. e | Clause 2 method, in the whatthat r1 is the acid side chain asparctic or a3derived from chain sidetwo of aspartic acid, in which the -COOH group is replaced by a -COOR group or a -CON (R) 2 group, and R is the tyrosine side chain.

5. The method in Clause 2, where R1 is the side chain of glutamic acid or a derivative of the side chain of glutamic acid, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, and R2 is the alanine side chain.

6. The method in Clause 2, where R1 is the side chain of glutamic acid or a derivative of the side chain of glutamic acid, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, and R2 is the tyrosine side chain.

7. The method in Clause 2, where R1 is the side chain of aspartic acid or glutamic acid, and R2 is the alanine side chain.

8. The method in Clause 2, where R1 is the side chain of aspartic acid or glutamic acid, and R2 is the tyrosine side chain.

9. The method in Clause 1, in which R1 and R2 they are both a hydrophobic side chain or a hydrophobic side chain derivative.

10. The method of Clause 9, in which:

(a) R1 and R2, which may be the same or different, is each the side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine;

(b) R1 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, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine, and R2 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline.

eleven. The method in Clause 10, in which R1 is the glycine side chain, and R2 is the side chain of leucine.

12. The method in Clause 10, in which R1 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline, and R2 is the phenylalanine side chain.

13. The method in Clause 10, in which R1 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline, and R2 is the alanine side chain.

14. The method in Clause 1, in which R1, R2 or both is the methionine side chain, the arginine side chain, or a derivative of these side chains.

fifteen. The method in Clause 14, in which R1 is the methionine side chain and R2 is the arginine side chain.

ES 2 575 563 T3

16. The method of any one of Clauses 1-15, wherein the animal is a human.

17. The method of any one of Clauses 1-15, wherein the T-cell mediated disease is graft rejection, graft-versus-host disease, an unwanted delayed-type hypersensitivity reaction, T-cell-mediated lung disease or an autoimmune disease.

18. The method of any one of Clauses 1-15, wherein the T-cell mediated disease is multiple sclerosis, neuritis, polymyositis, psoriasis, vitiligo, Sjogren's syndrome, rheumatoid arthritis, type 1 diabetes, autoimmune pancreatitis, diseases inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, glomerulonephritis, scleroderma, sarcoidosis, autoimmune thyroid diseases, Hashimoto's thyroiditis, Graves disease, myasthenia gravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, or systemic lupus erythematosus.

19. The method of any one of Clauses 1-15, wherein the T-cell mediated disease is pulmonary fibrosis or idiopathic pulmonary fibrosis.

twenty. A method of inhibiting the activation of T lymphocytes comprising administering, to an animal in need thereof, an effective amount of a diketopiperazine having the following formula:

(I) in which:

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

(¡I) an -OH group replaced by a -O-PO3H2 or -OR group3, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(Ii) a -COOH group replaced by a -COOR group3, in which each R3 it may independently be a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(¡V) a -COOH group replaced by a -CON (R4) 2, in which each R4 they may independently be H, or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, arylalkyl or heteroaryl;

(v) a -SH group replaced by -SS-CH2-CH (NH2) -COOH or -SS-CH2-CH2-CH (NH2) -COOH;

ES 2 575 563 T3 (vi) a -CH2- group replaced by a -CH (NH2) - group or a -CH (OH) - group;

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

twenty-one. The method in Clause 20, in which R1, R2 or both is the aspartic acid side chain, the glutamic acid side chain, or a derivative of an aspartic acid or glutamic acid side chain, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4)2.

22. The method in Clause 21, in which R1 is the aspartic acid side chain or a derivative of the aspartic acid side chain, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, and R2 is the alanine side chain.

2. 3. The method in Clause 21, in which R1 is the aspartic acid side chain or a derivative of the aspartic acid side chain, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, and R2 is the tyrosine side chain.

24. The method in Clause 21, in which R1 is the side chain of glutamic acid or a derivative of the side chain of glutamic acid, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, and R2 is the alanine side chain.

25. The method in Clause 21, in which R1 is the side chain of glutamic acid or a derivative of the side chain of glutamic acid, in which the -COOH group is replaced by a -COOR group3 or a group -CON (R4) 2, and R2 is the tyrosine side chain.

26. The method in Clause 21, in which R1 is the side chain of aspartic acid or glutamic acid, and R2 is the alanine side chain.

27. The method in Clause 21, in which R1 is the side chain of aspartic acid or glutamic acid, and R2 is the tyrosine side chain.

28. The method in Clause 20, in which R1 and R2 they are both a hydrophobic side chain or a hydrophobic side chain derivative.

29. The method of Clause 28, in which:

30. The method in Clause 29, in which R1 is the glycine side chain, and R2 is the side chain of leucine.

31. The method in Clause 29, in which R1 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline, and R2 is the phenylalanine side chain.

32. The method in Clause 29, in which R1 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline, and R2 is the alanine side chain.

33. The method in Clause 20, in which R1, R2 or both is the methionine side chain, the arginine side chain, or a derivative of these side chains.

3. 4. The method in Clause 33, in which R1 is the methionine side chain and R2 is the arginine side chain.

35. The method of any one of Clauses 20-34, wherein the animal is a human.

36. The method of any one of Clauses 20-34 in which diketopiperazine is used to treat inflammation or an inflammatory disease that is caused or aggravated, at least in part, by the activation of T lymphocytes.

ES 2 575 563 T3

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

(II) in which:

R5 and R6, which can be the same or different, is each:

(a) a side chain of an amino acid, in which the amino acid is glycine, alanine, valine, norvaline, aaminoisobutyric acid, 2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine, homoserine , threonine, usine, hydroxylysine, histidine, arginine, homoarginine, citrulline, phenylalanine, paminophenylalanine, tyrosine, tryptophan, thyroxine or ornithine; however, provided that when R5 be the side chain of asparagine or glutamine, then R6 cannot be the side chain of Usine or ornithine, and when R5 be the side chain of Usine or ornithine, then R6 cannot be the asparagine or glutamine side chain;

(b) R5 is -CH2-CH2-CH2- or -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline, R6 is -CH2-CH2-CH2-0 -CH2-CH (OH) -CH2- and, together with the nitrogen of the adjacent ring, forms proline or hydroxyproline; or both R5 as R6 they are each independently -CH2-CH2-CH2-0 -CH2-CH (OH) -CH2-, and together with the adjacent ring nitrogens form proline and hydroxyproline; or (c) a derivative of a side chain of an amino acid, wherein the amino acid is one of those listed in (a), and the derivatized side chain has:

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

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

38. The method in Clause 37, in which R5 and R6 they are both a hydrophobic side chain or a hydrophobic side chain derivative.

39. The composition of Clause 38, in which:

(a) R5 and R6, which may be the same or different, is each the side chain of glycine, alanine, valine, norvaline, α-aminobutyric acid, leucine, isoleucine, norleucine or phenylalanine;

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

40. The composition of Clause 39, in which R5 is the glycine side chain and R6 is the side chain of leucine.

ES 2 575 563 T3

41. The composition of Clause 39, in which R5 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline, and R6 is the phenylalanine side chain.

42. The composition of Clause 39, in which R5 is -CH2-CH2-CH2- and, together with the adjacent nitrogen atom, forms proline, and R6 is the alanine side chain.

43. The composition of Clause 37, in which R5, R6 or both is the methionine side chain, the arginine side chain, or a derivative of these side chains.

44. The composition of Clause 43, in which R5 is the methionine side chain and R6 is the arginine side chain.

Four. Five. A method of treating a T-lymphocyte-mediated disease, which 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, the protein or peptide such that the composition also comprises at least one diketopiperazine derived from the protein or peptide.

46. The method of Clause 45, in which the protein is albumin.

47. The method of Clause 45, in which the protein is immunoglobulin.

48. The method in Clause 45, where the protein is erythropoietin.

49. The method of any one of Clauses 45-48, wherein the pharmaceutical composition is administered orally.

fifty. The method of any one of Clauses 45-48, wherein the animal is a human and the protein or peptide is a human protein or a human peptide.

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

52. The method of Clause 51, in which the protein is albumin.

53. The method in Clause 51, in which the protein is immunoglobulin.

54. The method in Clause 51, where the protein is erythropoietin.

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

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

57. A method of synthesis of a diketopiperazine which comprises heating a solution of a protein or a peptide under conditions effective to generate formation of the diketopiperazine.

58. The method of Clause 57, in which the protein is albumin.

59. The method in Clause 57, in which the protein is an immunoglobulin.

60. The method in Clause 57, where the protein is erythropoietin.

61. The method of Clause 57, wherein the diketopiperazine is purified from solution.

62. The method of any one of Clauses 57-61, in which the solution is heated for four days at 60 ° C.

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

64. The method of Clause 63, in which the protein is albumin.

ES 2 575 563 T3

65. The method of Clause 63, in which the protein is an immunoglobulin.

66. The method in Clause 63, where the protein is erythropoietin.

67. The method of Clause 63, wherein the enzyme is a dipeptidyl peptidase.

68. The method of Clause 63, wherein the enzyme is a carboxypeptidase.

69. The method of any one of Clauses 63-68 wherein the diketopiperazine is purified from solution.

70. An improved pharmaceutical composition of a protein or peptide, which comprises a lower content of diketopiperazines in the composition.

71. The composition of Clause 70, in which the protein is albumin.

72. The composition of Clause 70, in which the protein is an immunoglobulin.

73. The composition of Clause 70, in which the protein is erythropoietin.

74. A method of manufacturing an improved pharmaceutical composition of a protein or a peptide, which method comprises removing from the composition at least part of the diketopiperazines present in the composition.

75. The composition of Clause 74, in which the protein is albumin.

76. The composition of Clause 74, in which the protein is an immunoglobulin.

77. The composition of Clause 74, in which the protein is erythropoietin.

78. A method of manufacturing an improved pharmaceutical composition of a protein or peptide, which method comprises treating a solution of the protein or peptide to increase the content of diketopiperazines.

79. The method of Clause 78, in which the solution is heated under conditions effective to generate the formation of diketopiperazines.

80. The Clause 79 method, in which the solution is heated for four days at 60 ° C.

81. The method of Clause 78, in which the solution is contacted with an enzyme that cleaves the two N-terminal amino acids or the two C-terminal amino acids of the protein or peptide under conditions effective to produce the diketopiperazines.

82. The method of Clause 81, wherein the enzyme is a dipeptidyl peptidase.

83. The method of Clause 81, wherein the enzyme is a carboxypeptidase.

84. The method of Clause 78, in which the protein is albumin.

85. The method of Clause 78, in which the protein is an immunoglobulin.

86. The method in Clause 78, where the protein is erythropoietin.

87. An improved pharmaceutical composition of a protein or peptide, an improvement comprising a higher content of diketopiperazines in the composition.

88. The composition of Clause 87, in which the protein is albumin.

89. The composition of Clause 87, in which the protein is an immunoglobulin.

90. The composition of Clause 87, in which the protein is erythropoietin.

91. The composition of any one of Clauses 87-90 that is suitable for oral administration.

115 members in 18 offices

This recordMember, by publication datePriority claim8 more offices are in the list below

Priority claims20

Priority claims
Document	Office	Kind	Date
471017P	United States of America	–	2003-05-15
47101703	United States of America	P	2003-05-15
47101703	United States of America	P	2003-05-15
489270P	United States of America	–	2003-07-21
48927003	United States of America	P	2003-07-21
48927003	United States of America	P	2003-07-21
514930P	United States of America	–	2003-10-27
51493003	United States of America	P	2003-10-27
51493003	United States of America	P	2003-10-27
517338P	United States of America	–	2003-11-04
51733803	United States of America	P	2003-11-04
51733803	United States of America	P	2003-11-04
471017P	–	–	–
489270P	–	–	–
514930P	–	–	–
517338P	–	–	–
US20030471017P	–	–	–
US20030489270P	–	–	–
US20030514930P	–	–	–
US20030517338P	–	–	–

Members115

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AU2004241101A1	Australia	A1	2004-12-02
CA2523467A1	Canada	A1	2004-12-02
CA2525587A1	Canada	A1	2004-12-02
CA3050734A1	Canada	A1	2004-12-02
WO2004103304A2	World Intellectual Property Organization (WIPO)	A2	2004-12-02
WO2004104773A2	World Intellectual Property Organization (WIPO)	A2	2004-12-02
US2005010955A1	United States of America	A1	2005-01-13
US2005119177A1	United States of America	A1	2005-06-02
WO2004103304A3	World Intellectual Property Organization (WIPO)	A3	2005-07-07
EP1622633A2	European Patent Office (EPO)	A2	2006-02-08
EP1623301A2	European Patent Office (EPO)	A2	2006-02-08
KR20060022659A	Republic of Korea	A	2006-03-10
IL171961A0	Israel	A0	2006-04-10
IL171961D0	Israel	D0	2006-04-10
BRPI0410506A	Brazil	A	2006-06-20
CN1791420A	China	A	2006-06-21
JP2007500747A	Japan	A	2007-01-18
ZA200509184B	South Africa	B	2007-04-25
WO2004104773A3	World Intellectual Property Organization (WIPO)	A3	2007-05-31
JP2007516646A	Japan	A	2007-06-21
CN101279094A	China	A	2008-10-08
NZ542886A	New Zealand	A	2009-06-26
EP1622633A4	European Patent Office (EPO)	A4	2010-02-17
AU2004241101B2	Australia	B2	2010-05-13
US7732403B2	United States of America	B2	2010-06-08
US2010143338A1	United States of America	A1	2010-06-10
US2010144611A1	United States of America	A1	2010-06-10
US2010190696A1	United States of America	A1	2010-07-29
AU2010203293A1	Australia	A1	2010-08-12
NZ576931A	New Zealand	A	2010-12-24
CN101947306A	China	A	2011-01-19
JP2011102309A	Japan	A	2011-05-26
HK1148690A1	Hong Kong, China	A1	2011-09-16
CN102210852A	China	A	2011-10-12
JP4800953B2	Japan	B2	2011-10-26
US2012022004A1	United States of America	A1	2012-01-26
US8183209B2	United States of America	B2	2012-05-22
KR20120091266A	Republic of Korea	A	2012-08-17
KR20120101164A	Republic of Korea	A	2012-09-12
CN102727861A	China	A	2012-10-17
EP2517718A2	European Patent Office (EPO)	A2	2012-10-31
EP2517719A1	European Patent Office (EPO)	A1	2012-10-31
EP2517721A2	European Patent Office (EPO)	A2	2012-10-31
EP2517722A2	European Patent Office (EPO)	A2	2012-10-31
US8324167B2	United States of America	B2	2012-12-04
EP2537524A2	European Patent Office (EPO)	A2	2012-12-26
US2013065839A1	United States of America	A1	2013-03-14
US2013071436A1	United States of America	A1	2013-03-21
US2013071448A1	United States of America	A1	2013-03-21
US2013072684A1	United States of America	A1	2013-03-21
EP2537524A3	European Patent Office (EPO)	A3	2013-03-27
US2013079284A1	United States of America	A1	2013-03-28
EP2517718A3	European Patent Office (EPO)	A3	2013-04-03
EP2517721A3	European Patent Office (EPO)	A3	2013-04-10
EP2517722A3	European Patent Office (EPO)	A3	2013-04-24
HK1173394A1	Hong Kong, China	A1	2013-05-16
HK1173669A1	Hong Kong, China	A1	2013-05-24
CN103142599A	China	A	2013-06-12
CN103191409A	China	A	2013-07-10
CN103191410A	China	A	2013-07-10
US8513196B2	United States of America	B2	2013-08-20
HK1178427A1	Hong Kong, China	A1	2013-09-13
HK1178810A1	Hong Kong, China	A1	2013-09-19
US8551953B2	United States of America	B2	2013-10-08
AU2010203293B2	Australia	B2	2014-01-09
AU2013270553A1	Australia	A1	2014-01-16
IL171961A	Israel	A	2014-02-27
IL230815A0	Israel	A0	2014-03-31
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IL231142A0	Israel	A0	2014-03-31
IL231142D0	Israel	D0	2014-03-31
CN101947306B	China	B	2014-06-18
JP2014167014A	Japan	A	2014-09-11
CN104095851A	China	A	2014-10-15
EP2799114A2	European Patent Office (EPO)	A2	2014-11-05
EP2799114A3	European Patent Office (EPO)	A3	2015-02-18
US8962568B2	United States of America	B2	2015-02-24
US8969308B2	United States of America	B2	2015-03-03
CN103191410B	China	B	2015-03-04
CN102727861B	China	B	2015-05-13
KR20150080004A	Republic of Korea	A	2015-07-08
CA2525587C	Canada	C	2015-08-11
HK1203056A1	Hong Kong, China	A1	2015-10-16
HK1203164A1	Hong Kong, China	A1	2015-10-23
US9176643B2	United States of America	B2	2015-11-03
US2016015705A1	United States of America	A1	2016-01-21
EP1622633B1	European Patent Office (EPO)	B1	2016-02-24
EP2517718B1	European Patent Office (EPO)	B1	2016-03-02
EP2517722B1	European Patent Office (EPO)	B1	2016-03-02
EP2517719B1	European Patent Office (EPO)	B1	2016-03-30
ES2572454T3	Spain	T3	2016-05-31
ES2572975T3	Spain	T3	2016-06-03
ES2575563T3This record	Spain	T3	2016-06-29
EP2537524B1	European Patent Office (EPO)	B1	2016-07-06
ES2579635T3	Spain	T3	2016-08-12
PT2537524T	Portugal	T	2016-09-05
AU2013270553B2	Australia	B2	2016-09-15
CN102210852B	China	B	2016-10-19
DK2537524T3	Denmark	T3	2016-10-24
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Showing 100 of 115

Treatment of t-cell mediated diseases

Abstract

Term

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DESCRIPTION

OR

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TABLE 1: HYPROMATRIX MATRIX (CUSTOMER REQUEST): PROTEINS FOR PHOSPHORYLATION

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TABLE 2: RESULTS

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TABLE 3

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Priority claims20

Members115

Numbers

Titles2

Classification