CN1791420A

CN1791420A - Treatment of T-cell mediated diseases

Info

Publication number: CN1791420A
Application number: CN 200480013249
Authority: CN
Inventors: 戴维·巴-奥尔; 拉斐尔·巴-奥尔; 理查德·希蒙科维茨
Original assignee: DMI Biosciences Inc
Current assignee: DMI Biosciences Inc
Priority date: 2003-05-15
Filing date: 2004-05-14
Publication date: 2006-06-21
Also published as: ZA200509184B; CN101279094A

Abstract

The invention provides a method of treating T-cell mediated diseases and a method of inhibiting the activation of T-cells using certain diketopiperazines. The invention also provides methods of synthesizing diketopiperazines and pharmaceutical compositions comprising certain diketopiperazines. The invention further provides methods of making improved pharmaceutical compositions of proteins and peptides by either increasing or decreasing the content of diketopiperazines in the compositions and the resultant improved pharmaceutical compositions.

Description

The treatment of the disease that T-is cell-mediated

Technical field

The present invention relates to treat with some diketopiperazines the activation of the cell-mediated disease of T-and inhibition T-cell. The present invention also relates to comprise the pharmaceutical composition of some diketopiperazines and relate to the method for synthesizing diketopiperazines. The invention still further relates to the method for the improvement pharmaceutical composition for preparing albumen and peptide with the content of diketopiperazines in increase or the minimizing composition, and relate to the improvement pharmaceutical composition of gained.

Background of invention

The cell-mediated disease of T-has represented a large amount of disease of immune system. Particularly, the T-cell is considered to cause and keep the cell of autoimmune disease. Autoimmune disease one group of 80 kinds of serious chronic disease for only just tormenting the millions of people in the U.S.. Autoimmune disease is characterised in that the reactivity of immune system and endogenous (self) antigen. These to the immune response of autoantigen by the continuing or send out again activation and keep of the T-cell of oneself-reaction, and directly or indirectly, the T-cell of this oneself-reaction is responsible to the feature organization damage and fracture seen in the autoimmune disease. Although proposed a variety of therapies of the cell-mediated disease of autoimmune disease and other T-, still had the needs for other therapies.

Summary of the invention

The invention provides the method for the cell-mediated disease for the treatment of T-. The method comprises and gives acceptable salt on the diketopiperazine with following formula of effective dose or its physiology to its animal of needs:

Wherein:

R ¹And R², can be identical or different, each is:

(a) amino acid whose side chain, wherein this amino acid is glycine, alanine, valine, norvaline, α-aminoacid, 2,4-DAB, 2,3-DAB, leucine, isoleucine, nor-leucine, serine, homoserine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulling, phenylalanine, p-aminobenzene alanine, tyrosine, tryptophan, thyroxine, cysteine, homocysteine, methionine, penicillamine or ornithine; Yet condition is to work as R¹During for the side chain of asparagine or glutamine, R so²Can not be the side chain of lysine or ornithine, and work as R¹During for the side chain of lysine or ornithine, R²Can not be the side chain of asparagine or glutamine;

(b)R ¹For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline with adjacent loops nitrogen, and/or R²For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline with adjacent loops nitrogen; Or

(c) derivative of amino acid whose side chain, wherein this amino acid be described in (a) those one of, and the side chain of this derivatization has following group:

(i)-NH ₂Group quilt-NHR³Or-N (R³) ₂Group replaces, wherein each R³Can be substituted or unsubstituted alkyl, cycloalkyl, Heterocyclylalkyl, aryl, alkaryl, aralkyl or heteroaryl independently;

(ii)-OH group quilt-O-PO₃H ₂Or-OR³Group replaces, wherein each R³Can be substituted or unsubstituted alkyl, cycloalkyl, Heterocyclylalkyl, aryl, alkaryl, aralkyl or heteroaryl independently;

(iii)-COOH group quilt-COOR³Group replaces, wherein each R³Can be substituted or unsubstituted alkyl, cycloalkyl, Heterocyclylalkyl, aryl, alkaryl, aralkyl or heteroaryl independently;

(iv)-COOH group quilt-CON (R⁴) ₂Group replaces, wherein each R⁴Can be the substituted or unsubstituted alkyl of H, cycloalkyl, Heterocyclylalkyl, aryl, alkaryl, aralkyl or heteroaryl independently;

(v)-SH group quilt-S-S-CH₂-CH(NH ₂)-COOH or-S-S-CH₂-CH ₂-CH(NH ₂)-COOH replaces;

(vi)-CH ₂-group quilt-CH (NH₂)-or-CH (OH)-group replaces;

(vii)-CH ₃Group quilt-CH₂-NH ₂Or-CH₂-OH group replaces; And/or

(viii) H that is connected on the carbon atom is replaced by halogen.

The present invention also provides the method that suppresses the T-cell-stimulating. The method comprises and gives acceptable salt on the formula I diketopiperazine of effective dose or its physiology to its animal of needs.

The present invention also provide comprise pharmaceutically acceptable carrier and have the diketopiperazine of following formula or its physiology on the pharmaceutical composition of acceptable salt:

Wherein:

R ⁵And R⁶, can be identical or different, each is:

(a) amino acid whose side chain, wherein this amino acid is glycine, alanine, valine, norvaline, α-aminoacid, 2,4-DAB, 2,3-DAB, leucine, isoleucine, nor-leucine, serine, homoserine, threonine, lysine, hydroxylysine, histidine, arginine, homoarginine, citrulling, phenylalanine, p-aminobenzene alanine, tyrosine, tryptophan, thyroxine or ornithine; Yet condition is to work as R⁵During for the side chain of asparagine or glutamine, R so⁶Can not be the side chain of lysine or ornithine, and work as R⁵During for the side chain of lysine or ornithine, R⁶Can not be the side chain of asparagine or glutamine;

(b)R ⁵For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline with adjacent loops nitrogen, and/or R⁶For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline with adjacent loops nitrogen; Or

(iii)-CH ₂-group quilt-CH (NH₂)-or-CH (OH)-group replaces;

(iv)-CH ₃Group quilt-CH₂-NH ₂Or-CH₂-OH group replaces; And/or

(v) H that is connected on the carbon atom is replaced by halogen.

The invention provides the another kind of method of the cell-mediated disease for the treatment of T-. The method comprises and is included in the animal the normal albumen of finding or the pharmaceutical composition of peptide to what its animal of needs gave effective dose, described albumen or peptide are processed, make described composition also comprise at least a diketopiperazine that is derived from this albumen or peptide.

The present invention also provides a kind of method of the T-of inhibition cell-stimulating. The method comprises and is included in the animal the normal albumen of finding or the pharmaceutical composition of peptide to what its animal of needs gave effective dose, described albumen or peptide are processed, make described composition also comprise at least a diketopiperazine that is derived from this albumen or peptide.

In addition, the invention provides the method for synthetic diketopiperazines. In one embodiment, the method is included in heating albumen or peptide solution under the condition that effectively causes diketopiperazine formation. In second embodiment, the method is included under the condition of effective generation diketopiperazines, and two N-enzyme terminal or two C-end amino acids of this albumen of albumen or peptide solution and cut-out or peptide is contacted.

The present invention also provides the improvement pharmaceutical composition of albumen or peptide. Described improvement is that said composition comprises the diketopiperazines that reduces content.

In addition, the invention provides the method for the improvement pharmaceutical composition of preparation albumen or peptide. The method comprises removes at least some diketopiperazineses that exist in said composition from said composition.

The present invention also provides the method for the improvement pharmaceutical composition of preparation albumen or peptide. The method comprises the content of the solution of this albumen or peptide being processed to improve diketopiperazines in the said composition.

The present invention also provides the improvement pharmaceutical composition of albumen or peptide. Described improvement is the diketopiperazines that said composition comprises increases content.

Description of drawings

Fig. 1: Trips (separate from influenza immunity donor to the specific CD4+T-cell line of hemagglutinin) cell count is to ERK1/2 concentration graphy figure, this TriPS cell be separated in the 20th day after stimulating with anti--CD3OKT3 antibody obtain and with 25ng Buddhist ripple myristic acid (phorbal myristic acid) (PMA), HC-RBL (heated people's colostrum part, molecular weight is less than 3kD and contain MR-DKP) and the DA-DKP of 0.5mM with dilution in 1: 10 cultivated 15 minutes in 37 ℃.

Fig. 2: what column diagram showed is to stimulate rear 12 days to the inhibition of Trips emiocytosis tumor necrosis factor α (TNF α) and IL-16 with anti--CD3OKT3 antibody. Shown the inhibition of people's colostrum (HC) 2626 (containing MR-DKP) DA-DKP band to TNF α and IL-16L secretion. It is splitting action owing to people's colostrum of high concentration that the maximum that use is observed with the HC2626 of 1: 100 and 1: 1000 dilution discharges. Do not observe cracking when using the DA-DKP of 0.5mM, and TNF α and IL-16L secretion have been reduced.

Fig. 3: column diagram shows to stimulate rear 10 days with anti-CD3OKT3 antibody, to the inhibition of Trips emiocytosis TNF α. Shown needs further investigate HC RBL and DA-DKP as with the being seen drop reaction of HC2626 (titratable response as seen with HC2626). May show potential activity (May indicated a potent activity).

Fig. 4: column diagram shows with anti--CD3 OKT3 antibody stimulates rear different time to the inhibition of Trips emiocytosis TNF α. Shown early stage at the thorn flyback cycle, DA-DKP and HC RBL are inhibiting, and are activations in the late period (the 14th day) in this cycle. HC2626 is inhibiting in institute if having time, and the chances are for this owing to other composition.

Fig. 5: column diagram shows the 7th～10 day H4#9.25 cell (separate from multiple sclerosis patient's corpse brain tissue to the specific CD4+T-cell line of MBP ELISA) TNF secretion α after stimulating with anti--CD3OKT3 antibody is suppressed situation. Shown in the secretion of TNF α from this T-cell line and also suppressed by HC 2626, HC RBL and DA-DKP.

Present detailed description of preferred embodiments

The invention provides the method for the cell-mediated disease for the treatment of T-. " treatment " used herein refers to reduce symptom, duration or the seriousness of (completely or partially) disease, comprises curing this disease, perhaps prevents this disease.

The cell-mediated disease of T-comprises graft rejection, graft versus host disease(GVH disease), unwanted delayed allergy (such as the delayed allergy reaction), cell-mediated PUD D and the autoimmune disease of T-. The cell-mediated PUD D of T-comprises sarcoidosis, hypersensitivity pneumonia, acute interstitial pneumonia, pulmonary alveolitis, pulmonary fibrosis, idiopathic pulmonary fibrosis and the other diseases take the inflammatory injury of lungs as feature. Autoimmune disease comprises multiple sclerosis, neuritis, polymyositis, psoriasis, Leucoplakia, house Glenn syndrome, rheumatoid arthritis, type 1 diabetes, autoimmunity pancreatitis, inflammatory bowel disease (for example cloning disease and ulcerative colitis), CD, glomerulonephritis, chorionitis, sarcoidosis, AITD (for example Hashimoto thyroiditis and Graves disease), myasthenia gravis, Addison disease, the autoimmunity ommochrome retinitis (uveoretinitis), pemphigus vulgaris, PBC, pernicious anaemia and systemic lupus erythematosus.

Acceptable salt is treated the cell-mediated disease of described T-on the diketopiperazines with following formula by its animal of needs being given effective dose or its physiology:

Wherein:

R ¹And R², can be identical or different, each is:

(vi)-CH ₂-group quilt-CH (NH₂)-or-CH (OH)-group replaces;

(vii)-CH ₃Group quilt-CH₂-NH ₂Or-CH₂-OH group replaces; And/or

(viii) H that is connected on the carbon atom is replaced by halogen.

" by replacing " refers to, according to the formula of amino acid whose side chain, described special groups is replaced by described other special groups. For example, the formula of the side chain of isoleucine is-CH (CH₃)-CH ₂-CH ₃ If should end-CH₃Base quilt-CH₂-OH base replaces, and the formula of the leucine side chain of deriving different of gained is exactly-CH (CH so₃)-CH ₂-CH ₂-OH. As another example, the side chain formula of alanine is-CH₃ If one of described hydrogen atom is replaced by the chlorine atom, the side chain of the alanine of deriving of gained is exactly-CH so₂-Cl. The side chain of noting glycine is-H, and if this H replaced by chlorine (or other halogens) atom, the side chain of gained is exactly-Cl, (R is for example linked and encircled on the carbon to this chlorine atom¹＝-Cl)。

Preferably following diketopiperazines, wherein R¹、R ²Or these two is the side chain of aspartic acid or glutamic acid, or like this side chain-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative that group replaces, wherein R³And R⁴Defined in the above. In this group compound, following diketopiperazines most preferably, it comprises the derivative of the side chain of the aspartic acid of the side chain (Tyr-Glu DKP or YE-DKP) of side chain (Tyr-Asp DKP or YD-DKP), tyrosine and glutamic acid of side chain (Glu-Ala DKP or EA-DKP), tyrosine and aspartic acid of side chain (Asp-Ala DKP or DA-DKP), glutamic acid and alanine of aspartic acid and alanine and these four kinds of diketopiperazineses or glutamic acid, in these derivatives-and COOH group quilt-COOR³Group or-CON (R⁴) ₂Group replaces, wherein R³And R⁴Defined in the above.

Also have, preferably following diketopiperazines, wherein R¹And R²The two all is hydrophobic side chains (for example side chain of phenylalanine) or hydrophobic side chains derivative. " hydrophobic side chains derivative " refers to that the side chain that is derivatized is hydrophobicity. Particularly, preferably following diketopiperazines, wherein R¹And/or R²Can be identical or different, R¹And R²Respectively be side chain and/or the R of glycine, alanine, valine, norvaline, α-aminoacid, leucine, isoleucine, nor-leucine or phenylalanine¹And/or R²For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms. In this group compound, most preferably comprise the diketopiperazines of the side chain 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).

In addition preferred diketopiperazines be following those, R wherein¹、R ²Or these two is the derivative of the side chain of methionine, arginic side chain or these side chains. R most preferably in this group¹Side chain and R for methionine²Diketopiperazines (Met-Arg DKP or MR-DKP) for arginic side chain.

Above referring to be connected to, amino acid whose " side chain " list the amino acid moiety on all amino acid whose common skeletons. For example, the side chain of glycine is-H that the side chain of alanine is-CH₃, and the side chain of serine is-CH₂OH。

" hydrophobicity " refers to that side chain or side chain derivative are not charged and repelled by aqueous solution under physiological pH.

" alkyl " refers to contain 1～10, the saturated straight chain of preferred 1～6 carbon atom or branched-chain hydrocarbons. " low alkyl group " refers to contain saturated straight chain or the branched-chain hydrocarbons of 1～6 carbon atom.

" cycloalkyl " refers to contain at least one ring filling cyclic hydrocarbon, and each ring contains at least three carbon atoms. Preferably, this cycloalkyl contains the ring of 4～8 carbon atoms.

" Heterocyclylalkyl " refers to that one or more ring carbon atoms of at least one ring are by the cycloalkyl of O, S or N replacement.

" aryl " refer to have at least one aromatic rings aromatic group of (for example phenyl).

" alkaryl " refer to the low alkyl group that H replaced by aryl (for example-CH₂-C ₆H ₅Or-CH₃CH(C ₆H ₅)CH ₃)。

" aralkyl " refer to the aryl that H replaced by low alkyl group (for example-C₆H ₄-CH ₃)。

" heteroaryl " refers to that one or more ring carbon atoms of at least one ring are by the aryl of O, S or N replacement.

" be substituted " and refer to that one or more substituting group that described part is selected from following radicals replaces :-OH, NH₂,-SH ,-COOH and/or halogen atom.

" halogen " refers to chlorine, fluorine, bromine or iodine. Chlorine or bromine preferably.

The diketopiperazines of formula I is being effectively aspect the cell-mediated disease for the treatment of T-, because their suppress the activation of T-cell. Therefore, also can be used for treating T-cell by activation that cause, that increase the weight of or involve inflammation or the inflammatory disease of T-cell for the diketopiperazines of formula I. " inhibition " used herein refers to reduce (intactly or partly) or prevents.

The method for preparing diketopiperazines is well known in the art, and these methods can be used for synthesizing diketopiperazines of the present invention. See for example U.S. Patent number 4,694,081,5,817,751,5,990,112,5,932,579 and 6,555,543, U.S. Patent Application Publication No. 2004/0024180, PCT application WO 96/00391 and WO97/48685, and the Bioorg.Med.Chem.Letters such as Smith, 8,2369-2374 (1998), the complete disclosure of these documents is hereby incorporated by.

For example, diketopiperazines can prepare by at first synthetic dipeptides. This dipeptides can synthesize with L-amino acid, D-amino acid or D-and the amino acid whose combination of L-by method well known in the art. Solid-phase peptide synthesis preferably. Certainly, dipeptides also can obtain from a large amount of source commerce, comprises DMI Synthesis Ltd., Cardiff, UK (entrusting synthetic), Sigma-Aldrich, St.Louis, MO (mainly entrusting synthetic), Phoenix Pharmaceuticals, Inc., Belmont, CA (entrusting synthetic), Fisher Scientific (entrusting synthetic) and Advanced ChemTech, Louisville, KY.

After described dipeptides is synthesized or has bought, formed diketopiperazine by cyclisation. This can finish by many kinds of technology.

For example, U.S. Patent Application Publication No. 2004/0024180 has illustrated a kind of method of cyclisation dipeptides. Briefly, described dipeptides is heated in organic solvent simultaneously by dephlegmate part. Preferably, this organic solvent is the low azeotropic mixture that boils of water, such as acetonitrile, allyl alcohol, benzene, phenmethylol, just-butanols, 2-butanols, uncle-butanols, butyl acetate, carbon tetrachloride, chlorobenzene chloroform, cyclohexylamine, 1,2-dichloroethanes, diethyl acetal, dimethyl-acetal, ethyl acetate, heptane, methyl iso-butyl ketone (MIBK), 3-amylalcohol, toluene and dimethylbenzene. Described temperature depends on the type of reaction speed that cyclisation occurs and used entrainer. Reaction is preferably carried out more preferably 80～150 ℃ at 50～200 ℃. The pH scope at place occurs and can easily be determined by those skilled in the art in cyclisation. Advantageously, pH is 2-9, preferred 3-7.

When one or two amino acid of described dipeptides has or derives by when having carboxyl (for example aspartic acid or glutamic acid) at its side chain, described dipeptides is preferably according to U.S. Patent number 6,555, and 543 illustrated carry out cyclisation. Briefly, side chain carboxyl group still protected this dipeptides is heated under neutrallty condition. Typically, with this dipeptides in about 80 ℃～about 180 ℃ of heating, preferably approximately 120 ℃. Described solvent is neutral flux. For example, described solvent can comprise alcohol (such as butanols, methyl alcohol, ethanol and higher alcohol, but not being phenol) and azeotropic cosolvent (such as toluene, benzene or dimethylbenzene). Preferably, described alcohol is fourth-2-alcohol, and described azeotropic cosolvent is toluene. Heating is continued until and reacts completely, and these times can be determined by rule of thumb. Typically, described dipeptides came cyclisation in about 8～24 hours preferred 18 hours by refluxing. At last, described blocking group is removed from this diketopiperazine. When doing like this, should avoid using strong acid (inorganic acid, such as sulfuric acid or hydrochloric acid), highly basic (alkali-metal alkali (alkaline bases), such as potassium hydroxide or NaOH) and strong reductant (for example lithium aluminium hydride reduction), to keep the chirality of final compound.

The dipeptides for preparing on the solid-phase resin can come cyclisation and discharges from resin by a step. See for example U.S. Patent number 5,817,751. For example, will be with the Resin Suspension of N-alkylation dipeptides in the toluene or toluene/ethanol that have acetic acid (for example 1%) or triethylamine (for example 4%) to exist. Typically, preferred alkaline cyclisation conditions is because they have the faster cyclisation time.

For the derived diketopiperazine of the amino acid whose side chain of preparation formula I and II, in dipeptides synthetic, can use amino acid derivativges, dipeptides can be derivatized and/or diketopiperazines can be derivatized, as known in the art. See for example above-mentioned those lists of references of quoting.

The additive method of cyclisation dipeptides and preparation diketopiperazines is known in the art, and can use in the preparation to diketopiperazines useful in the present invention practice. See for example above-mentioned those lists of references of quoting. In addition, being suitable for a lot of diketopiperazineses of using in the present invention can be according to following explanation by albumen and peptide preparation. Also have, the diketopiperazines that uses in the present invention's practice can be from for example DMI Synthesis Ltd., Cardiff, the commercial acquisition of UK (entrusting synthetic).

The diketopiperazines of formula I and II except comprise can obtain by the configuration that changes individual chiral centre, axis or surface, also comprise all possible stereoisomer. Use another kind of saying, the diketopiperazines of formula I and II comprises all possible diastereomer and all optical isomers (enantiomer).

Acceptable salt also can use in the present invention's practice on the physiology of diketopiperazines of the present invention. Acceptable salt comprises conventional non-toxic salt on the physiology, such as the salt that is derived from inorganic acid (such as hydrochloric acid, hydrobromic, sulfuric acid, phosphoric acid, nitric acid etc.), be derived from the salt of organic acid (such as acetic acid, propionic acid, butanedioic acid, glycolic (glycolic), stearic acid, lactic acid, malic acid, tartaric acid, citric acid, glutamic acid, aspartic acid, benzoic acid, salicylic acid, oxalic acid, ascorbic acid etc.), or be derived from alkali (such as pharmaceutically acceptable metal cation or be derived from N, organic cations hydroxide, carbonate or the bicarbonate of N-dibenzyl-ethylenediamin, GLUCOSAMINE or ethylenediamine). These salt are with the method preparation of routine, for example by the free alkali form with the described compound of acid neutralization.

As above explanation, on diketopiperazine of the present invention or its physiology acceptable salt can be used for treating the cell-mediated disease of T-or be used for suppressing the activation of T-cell. In order to do like this, the animal that needs are treated gives acceptable salt on diketopiperazine or its physiology. Preferably, described animal is mammal, such as rabbit, goat, dog, cat, horse or people. The effective dosage forms of the compounds of this invention, administering mode and dosage can determine by rule of thumb, and to make such decision be within the technology of this area. Those skilled in the art is to be understood that the medicine such as the evaluation of the other drug that dosage will give along with duration of the drainage rate of the seriousness of the compound of concrete use, the disease that will treat or illness, disease or illness, the approach that gives, compound, treatment, to described animal, age, size and the kind of described animal and the known factor of veterinary applications and changes. Usually, the suitable daily dose of the compounds of this invention effectively produces the lowest dose level of result for the treatment of for this compound. Yet this daily dose will be determined in suitable medical judgment scope by attending doctor or animal doctor. If expectation, effectively daily dose can with two, three, four, five, six or more fractionated dose whole day give respectively with suitable compartment of terrain. The giving to last till of this compound reached till acceptable the replying.

The animal patient that the compounds of this invention (being acceptable salt on diketopiperazines and its physiology) can be treated with any suitable method of administration, that method of administration comprises is oral, intranasal, rectum, vagina, non-in intestines (for example vein, backbone in, abdominal cavity, subcutaneous or muscle), brain, in skin, encephalic, brain and (comprising buccally and the hypogloeeis) administration of part. Preferred method of administration is oral and intravenously administrable.

Be possible although give separately the compounds of this invention, still preferably give this compound with the form of pharmaceutical preparation (composition). Pharmaceutical composition of the present invention be included in one or more pharmaceutically acceptable carriers and randomly in the mixture of one or more other compounds, medicine or other materials as a kind of compound of the present invention of active component or multiple compounds. Every kind of carrier must be " acceptable " mean with preparation in other compositions compatible and harmless to described animal. Pharmaceutically acceptable carrier is known in the art. No matter select which kind of method of administration, the compounds of this invention is all by well known to a person skilled in the art that conventional method makes pharmaceutically acceptable formulation. See for example Remington ' s Pharmaceutical Sciences.

Being suitable for the oral preparation of the present invention that gives can be for the form of capsule, cachet, pill, tablet, powder, granule or as the form of solution or the suspending agent in water-based or non-aqueous liquid or oil-in-water or water-in-oil emulsion, or as the form of elixir or syrup, or lozenge (uses inert base, such as gelatin and glycerine, or sucrose and Arabic gum) etc. form, each contains one or more compounds of the present invention of scheduled volume as active component. One or more compounds of the present invention also can be used as large ball, electuary or cream and give.

In the solid dosage forms of the present invention of oral administration (capsule, tablet, pill, lozenge, powder, granule etc.), active component (being acceptable salt on one or more diketopiperazineses of the present invention and/or its physiology) mixes mutually with one or more pharmaceutically acceptable carriers, such as natrium citricum or Dicalcium Phosphate, and/or following arbitrary: (1) filler or replenishers, such as starch, lactose, sucrose, glucose, sweet mellow wine and/or silicic acid; (2) adhesive, such as, for example carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or Arabic gum; (3) wetting agent is such as glycerine; (4) disintegrant is such as agar, calcium carbonate, potato or tapioca, alginic acid, some silicate and sodium carbonate; (5) solution retarding agent is such as paraffin; (6) sorbefacient is such as quaternary ammonium compounds; (7) wetting agent, such as, for example cetanol and glycerin monostearate; (8) absorbent is such as kaolin and bentolite clay; (9) lubricant is such as talcum powder, calcium stearate, dolomol, solid polyethylene glycol, NaLS and its mixture; And (10) colouring agent. In the situation of capsule, tablet and pill, this pharmaceutical composition also can comprise buffer. The solid composite of similar type can be as the filler that is filled into the soft hard gelatin capsule that uses lactose or the auxiliary materials such as lactose class (milk sugars) and high molecular weight polyethylene glycol.

Tablet can randomly suppress with one or more compounding ingredients or molding prepares. The tablet of compacting can use adhesive (for example gelatin or HPMC), lubricant, inert diluent, anticorrisive agent, disintegrant (for example Sodium Carboxymethyl Starch or Ac-Di-Sol), surfactant or dispersant to prepare. Molded tablet can prepare by will carry out molding with the powder compounds mixture that inert liquid diluent is got wet in the machine that is fit to.

The tablet of pharmaceutical composition of the present invention and other solid dosage forms such as lozenge, capsule, pill and granule, can randomly be come impression or preparation with dressing and shell, such as other dressings of knowing in enteric coating and the pharmaceutical formulations field. They also can discharge with the slow or control that active component is provided by preparation, use therein for example release profile type, other polymer substrates, liposome and/or the microballoon of HPMC so that expectation to be provided of different proportion. They can for example filter to sterilize by sterilizing filter. These compositions also can randomly contain opacifier and also can be only or preferentially in GI certain part randomly with the composition of the mode release of active ingredients that postpones. The example of the embedding composition that can use comprises polymeric material and wax. This active component also can be present in the micro-capsule form.

The oral administration liquid dosage form of the compounds of this invention comprises pharmaceutically acceptable emulsion, microemulsion, solution, suspending agent, syrup and elixir. Except active component, this liquid dosage form can contain common inert diluent in this area, such as, for example water or other solvents, solubilizer and emulsifying agent, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, phenmethylol, Ergol, propane diols, 1,3-butanediol, oil (especially continuous seed, peanut, corn, seed, olive, castor-oil plant and sesame oil), glycerine, tetrahydrofuran base methyl alcohol, polyethylene glycol and fatty acid esters of sorbitan and its mixture.

Except inert diluent, described Orally administered composition can comprise that also assistant is such as wetting agent, emulsifying agent and outstanding floating agent, sweetener, flavor enhancement, colouring agent, aromatizing agent and anticorrisive agent.

Suspending agent, except described active component, for example can contain outstanding floating agent, such as isooctadecanol ethoxylate, polyoxyethylene sorbitol and Isosorbide Dinitrate, microcrystalline cellulose, inclined to one side aluminium hydroxide (aluminum metahydroxide), bentonite, agar and tragacanth and its mixture.

The preparation that pharmaceutical composition of the present invention is used for rectum or vagina administration can be suppository, this suppository can be by mixing one or more the compounds of this invention to prepare with non-irritating excipient or the carrier that one or more are fit to, this excipient or carrier comprise for example cocoa butter, polyethylene glycol, suppository wax or salicylate, and be solid and be liquid during at body temperature when room temperature, therefore in rectum or vaginal canal, melt and release of active compounds. The preparation that the present invention is fit to vagina administration also comprises and contains pessary, liniment, cream, gel, paste, foaming agent or the spray for suitable carrier known in this field.

The compounds of this invention is used for local or comprises powder, spray, ointment, paste, cream, lotion, gel, solution, patch, drops and inhalant through the formulation that skin gives. Active component can mix with pharmaceutically acceptable carrier under aseptic condition, and with buffer or the propellants of any needs.

Ointment, paste, cream and gel can also comprise excipient except active component, such as animal or plant fat, oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silicic acid, talcum powder and zinc oxide or its mixture.

Powder and spray can also contain excipient except active component, such as the mixture of lactose, talcum powder, silicic acid, aluminium hydroxide, calcium silicates and polyamine powder or these materials. Spray can also contain propellant commonly used such as CFC and volatile unsubstituted hydrocarbon such as butane and propane.

Have to provide through the skin patch and make the compounds of this invention controllable delivery to the additional advantages of human body. Such formulation can by with the dissolving of one or more the compounds of this invention, disperse or be incorporated in suitable Media Ratio such as the elastomeric matrices material with other forms to prepare. Also can improve the transdermal flow of described compound by enough sorbefacients. But the speed of this flow can be controlled by providing rate-controlling membrane maybe this compound to be distributed in polymer substrate or the gel.

Pharmaceutical preparation comprise be suitable for sucking or be blown into give or intranasal or intraocular give those. For being administered to (nose) or lower respiratory tract by suction, the compounds of this invention is sent spray from insufflator, sprayer or pressurized package or other easily and is sent installing easily. Pressurized package can comprise suitable propellant such as dicholorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other gas that is fit to. In the situation of pressurized aerosol, dosage unit can be measured by the mode that the amount of valve to send metering is provided.

Perhaps, for sucking or being blown into administration, described composition can adopt the form of dry powder, for example one or more compounds of the present invention and the mixture of powders of the powder matrix that is fit to such as lactose or starch. This powder composition for example can be present in the form of UD capsule or cartridge case or for example in gelatin or the blister-pack, this powder can administration from above-mentioned under the help of inhalator, insufflator or metered dose inhaler.

For intranasal administration, the compounds of this invention can give by the method for collunarium or liquid spraying, such as relying on plastic bottle sprayer or metered dose inhaler. Typical sprayer is Mistometer (Wintrop) and Medihaler (Riker).

Drops such as eye drops or nasal drop, can prepare with the water-based that also comprises one or more dispersants, solubilizer or outstanding floating agent or non-aqueous matrix. Liquid spray is sent from pressurized package easily. Drops can rely on simply adds a cover eye drop bottle or plastic bottle comes administration, and this plastic bottle is the dropping that is adapted to liquid contents in the mode of special shape closure.

Being suitable for the non-pharmaceutical composition of the present invention that gives through intestines comprises one or more the compounds of this invention and unites one or more pharmaceutically acceptable sterile isotonic water-based or non-aqueous solution, dispersion, suspension or emulsion, or exactly can be distributed to again before use powder in sterile injectable solution or the dispersion, solute or outstanding floating agent or thickener that they can contain antioxidant, buffer, preparation and experimenter's blood etc. are oozed.

The water-based that is fit to that can use in pharmaceutical composition of the present invention or the example of non-aqueous carrier comprise water, ethanol, polyalcohol (such as glycerine, propane diols, polyethylene glycol etc.) and its suitable mixture, vegetable oil such as olive oil and injectable organic ester such as ethyl oleate. Suitable flowability can be for example by with coating material such as lecithin if dispersion is by keeping its required granular size and by keeping with surfactant.

These compositions also can contain assistant such as wetting agent, emulsifying agent and dispersant. Also expectation comprises that isotonic agent is such as sucrose, sodium chloride etc. in composition. In addition, the delay absorbent can make this injectable drug preparation produce the prolongation absorption such as the adding of aluminum monostearate and gelatin.

In some cases, for the effect of prolong drug, expectation delays the absorption of medicine from subcutaneous or intramuscular dose. This can be by realizing with the liquid suspension with weak water miscible crystalline form or amorphous material. The speed of drug absorption just depends on dissolution rate so, and in turn dissolution rate can depend on grain size and crystal formation. Perhaps, the non-delay that gives medicine through intestines absorb be by with this medicine dissolving in or be suspended in the oily carrier and realize.

Injectable bank (depot) preparation prepares by the micro-capsule matrix that forms medicine at biodegradable polymer in such as polylactic acid-polyglycolic acid. Depend on the character of ratio and the concrete polymer that uses of medicine and polymer, can control the speed that medicine discharges. The example of other biological degradable polymer comprises poly-(ortho esters) and poly-(acid anhydride). Injectable depot formulations is also by preparing the medicine embedding to the liposome compatible with body tissue or micro emulsion. Described injectable material can be sterilized with the method for for example filtering by sterilizing filter.

The container that preparation may reside in the sealing of UD or multiple dose is for example in ampoule and the phial, and can with only need just to add before use sterile liquid carrier for example the condition of the freeze-drying of water for injection store. Interim injection solution and suspending agent can be prepared by aseptic powdery, granule and the tablet of above-mentioned explanation type.

Have been found that the diketopiperazines that is suitable for the present invention's application appears at some commercially available veins that contain albumin, immunoglobulin (Ig) and hematopoietin and gives in the pharmaceutical composition. The diketopiperazines that occurs in these pharmaceutical preparations is to form by preparing heating steps commonly used in these pharmaceutical compositions. Heating causes two N-ends and/or two C-end amino acids of albumen to rupture and cyclisation formation diketopiperazines.

Accordingly, the diketopiperazines of usefulness can prepare by the solution of heating white albumen, immunoglobulin (Ig), hematopoietin and other albumen and peptide in the present invention. The neutral pH phosphate buffered liquor that has for example prepared albumin, immunoglobulin (Ig), hematopoietin or other protein or peptide. Preferably, this solution is that (for example about 100～500mM) to realize the protonated of N-end and/or C-end amino acid for concentrated solution. This solution was heated about 2 hours～several days at 60 ℃, preferably approximately 4 days, to cause the formation of diketopiperazines. Preferably should avoid albuminous degeneration. This can be by with the time that shortens and/or by adding sad or each about 0.02M of AT realizes.

The diketopiperazines that the present invention uses also can be by making albumin, immunoglobulin (Ig), hematopoietin or other albumen or peptide solution with can maybe can contact to prepare from the enzyme (carboxypeptidase) of this albumen or two C-end amino acids of peptide cut-out from the enzyme (for example dipeptidyl peptidase) that this albumen or peptide cut off two-terminal amino acids. The dipeptidyl peptidase and the carboxypeptidase that are fit to can be buied, for example available from sigma. Reaction should be in pH6～8, preferably carry out in such as phosphate buffer at buffer solution, and temperature is high enough to accelerates this reaction but not high enough to making albuminous degeneration (for example 37 ℃).

A large amount of albumen and the amino acid sequence of peptide are known, and can select to have with either method N-end and/or the albumen of C-end sequence or the diketopiperazine (class) that peptide is used for producing expectation of expectation. The method that also can enoughly know of peptide with expectation sequence is synthesized and is used.

Described diketopiperazines can comprise purifying from the pharmaceutical composition that contains albumin, immunoglobulin (Ig) and hematopoietin from the solution that contains them, by the method known such as size exclusion chromatography (for example Centricon filters), affinity chromatography (for example use magnetic bead (beads) with for a kind of antibody of the diketopiperazines of expecting or Multiple Antibodies or for the pillar of one or more antibody of the albumen of brachymemma or peptide), anion exchange or cation exchange. Sublimed diketopiperazines can be used or be incorporated in the pharmaceutical compositions of above-mentioned explanation.

Replacement is to the purifying of diketopiperazines, and the pharmaceutical compositions that comprises albumin, immunoglobulin (Ig) and hematopoietin and other albumen of usually finding and/or peptide in animal subject can be used in the activation for the treatment of the cell-mediated disease of T-and can be used for suppressing the T-cell. If contain diketopiperazines and just can be used although comprise the current commercially available composition of these albumen and/or peptide, it is highly preferred that before giving these improved composition the content of according to above-mentioned explanation albumin, immunoglobulin (Ig) and hematopoietin and/or other albumen and/or peptide being processed to carry high expected diketopiperazines. Animal is the people preferably, and described albumen and/or the preferred people's albumen of peptide and/or peptide. The oral administration of composition is preferred.

The effective dose of described albumen and/or peptide combinations can determine by rule of thumb, and does within the technology that such decision is this area. Especially, in order to determine the effective dose of albumen and/or peptide combinations, the amount of one or more diketopiperazineses that exist in the described composition can be measured, and the composition of the amount of this effective dose diketopiperazines can be given enough to produce to animal. Those skilled in the art is to be understood that the medicine such as the evaluation of the other drug that dosage will give along with duration of the drainage rate of the seriousness of the compound of concrete use, the disease that will treat or illness, disease or illness, the approach that gives, compound, treatment, to described animal, age, size and the kind of described animal and the known factor of veterinary applications and changes. Usually, the suitable daily dose of albumen and/or peptide combinations is the lowest dose level that effectively produces result for the treatment of. Yet this daily dose will be determined in suitable medical judgment scope by attending doctor or animal doctor. If expectation, effectively daily dose can with two, three, four, five, six or more fractionated dose whole day give respectively with suitable interval. Administration should last till and reached till acceptable the replying.

Illustrate that as above the known commercially available vein that comprises albumin, immunoglobulin (Ig) and hematopoietin gives to have found in the pharmaceutical composition diketopiperazines, relate to one or more heating stepses (for example sterilization) in the preparation of these compositions. Diketopiperazines also may appear in the preparation of composition and relate in other albumen and peptide medicine composite of heating steps. Such as this paper explanation, a lot of diketopiperazineses have the ability that suppresses the T-cell activation. Therefore, under a lot of conditions, do not wish patient is given albumin, immunoglobulin (Ig), hematopoietin or contains other albumen of diketopiperazines or the composition of peptide. For example, albumin often gives those patients who suffers from wound, and immunoglobulin (Ig) often suffers from the patient of infection or immune deficiency, and hematopoietin gives anaemia cancer or chronic disease patient that immune system is often compromised. Accordingly, the invention provides and from these compositions, remove methods that at least some preferably all remove diketopiperazines basically. This diketopiperazines can be removed size exclusion chromatography for example (for example Centricon filters), affinity chromatography (for example use bead with for a kind of antibody of the diketopiperazines of expectation or Multiple Antibodies or for the pillar of one or more antibody of the albumen of brachymemma or peptide), anion exchange or cation exchange to produce the improved composition of albumin, immunoglobulin (Ig), hematopoietin and other albumen or peptide according to top explanation.

Embodiment

Embodiment 1:Asp Ala DKP (DA-DKP) and Glu Ala DKP (EA-DKP) are from the absorption of rat intestine

With the pyloric sphincter of rat intestine to the rectum section free and by mesenteric artery in order to the perfusate perfusion that contain bovine serum albumin(BSA) of red blood cell for the basis. Collect perfusate and the recirculation (again after the oxygenation) of from intestines, flowing out with portal catheterization. After the balance period, the about 1ml injection of solution that will contain the Glu-Ala diketopiperazine (EA-DKP) of the Asp-Ala diketopiperazine (DA-DKP) of about 1mg or 1.4mg is administered in this duodenal tube chamber.

After administration, according to certain hour every collecting a series of perfusate sample until after the administration 2 hours. Centrifugal and blood plasma is used for the analyses of these two kinds ring dipeptides with the method for liquid chromatography and mass spectrometry (LC-MS) with these samples.

The result shows, behind perfusion only 2 hours, DA-DKP and EA-DKP reached respectively 95% and 100% (in fact being 112%) that gives dosage from the amount that enteric cavity is absorbed into circulation.

Therefore, two kinds of ring dipeptides all are absorbed rapidly and have effectively entered into blood from enteric cavity, in the proof that does not have metabolism from the process of intestines wall transhipment. Therefore can carry out these potential treatments by oral.

Unconverted DA-DKP and EA-DKP enter into the quick absorption of blood and lack at two kinds of compounds of rat liver of the perfusion that separates from intestines and stomach and firstly cross liver and remove (not listing data), remove lower before the display system. Therefore oral administration will be desirable method of administration.

In addition, to studies show that of the Perfused Rat kidney that separates, do not resemble much by the linear peptides of the extensive metabolism of kidney peptase, the kidney clearance rates of two kinds of ring dipeptides are relatively low.

The low daily dose administration scope of all these Notes of Key Data diketopiperazineses may be enough for therapeutic purposes.

Preliminary pharmacokinetic data available after the Oral Administration in Rats administration and the above-mentioned data consistent of two kinds of dipeptides, after oral dose 1.1～3.7mg/kg body weight (DA-DKP) and 1.5～4.8mg/kg body weight (EA-DKP), T_maxValue is 30～60 minutes and C_maxValue is 4～6 μ g/ml (DA-DKP) and 0.6～1.1 μ g/ml (EA-DKP), (T_maxThe time that reaches Cmax, C_maxIt is the Cmax that reaches; The two all is to be calculated by the corresponding equation of the curve of the data obtained to get. )

Preliminary data prompting DA-DKP and other diketopiperazineses pass through blood-brain barrier. Therefore, DA-DKP and other diketopiperazineses of the present invention should be useful such as multiple sclerosis to the treatment the nervous system disease.

Embodiment 2: contain the people's colostrum part of Met-Arg DKP (MR-DKP) and Asp-AlaDKP (DA-DKP) in the generation of the external people of inhibition T-lymphocyte factor

The A material

This embodiment illustration DA-DKP, contain people's colostrum (HC 2626) of MR-DKP and also contain low molecular weight part (the HC RBL of people's colostrum of MR-DKP; Contain people's colostrum part that the molecular weight that filters preparation by skimmed colostrum Centricon is lower than 3000 component) suppress the generation of people T-lymphocyte factor. DA-DKP and MR-DKP are from DMI Synthesis, Ltd., and Cardiff, UK. obtains. The little natural generation compound of these two kinds of diketopiperazineses for producing in the physiologic response process to inflammation. They also are found in immunoglobulin (Ig) (IVIg), human albumin and the other biological goods that people's vein gives sometimes.

B. suppressing the T-cell factor produces

Two kinds of different CD-4 positive human T-lymphocyte clones are tested. A kind of (TRiPS) of these cell lines be from influenza immunity donor, separate and to hemagglutinin peptide 307～319th, specific. Another cell line (H4#9.25) be from the autopsy brain tissue of the donor of multiple sclerosis, separate and be specific to myelin basic protein (amino acid 87～99). Add with (1) specific antigen the aobvious positive cell of HLA-DR2-or (2) anti--CD3 adds anti--CD28 antibody after stimulated in vitro, two kinds of T-lymphocyte clones all produce interleukin 8 (IL-8), IL-16, interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α).

Behind priming the 18th～20 day with about 4 * 10⁵The stimulation that individual cell carries out described T-lymphocyte strain is gone down to posterity. Add 10% hyclone (FBS with cold Iscove Modfied Dulbecco Minimal Essential Medium (IMDM, Sigma); American Type Culture Collecti (ATCC)) with cell washing once, and be resuspended in contain dilution in 1: 500 anti--the cold IMDM culture medium of 1.0ml of CD3 monoclonal antibody OKT3 (by the preparation of mouse ascites liquid) in. Cell is being cultivated washing with antibody on ice and is being added in 350u/ml human IL-2's (Xenometrix) the culture medium, as feeder cells, with about 2 * 10⁶The PBL of normal person's donor of individual 4000R-irradiation merges. Contain the fresh IMDM culture medium expansion cultivation that FBS adds IL-2 by adding at the 3rd day. The fate of cultivating calculated from the same day that stimulates with OKT3. Can be used in experiment since the 7th day (maximum propagation) cell, typically the 14th day (the most responsive to stimulating again) and until the 21st day (resting cell closes on aging).

Reclaim an aliquot cell and carry out activation experiment with temperature (37 ℃) IMDM culture medium washed twice. For each specific test, with 2 * 10⁵Individual living cells in the warm IMDM culture medium of the cumulative volume 0.9ml that contains specified quantitative treatment additive (for example, HC 2626, DA-DKP, PMA etc.) in 37 ℃ of precultures 15 minutes. Add afterwards and have 2 * 10⁵The warm IMDM 0.1ml of individual CD3/CD28 Dynabeads (Dynal) conduct activation stimulus and described culture are in 37 ℃ of incubated overnight (18 hours). By centrifugal make cell precipitation after the supernatant of collecting cell culture. With specific ELISA (for example, TNF α, IFN γ, IL-8, IL-16; Endogen) analysis of cells factor content.

Show that such as Fig. 1～5 people's colostrum (HC 2626) suppresses the generation of the cell in vitro factor of these two kinds of T-lymphocyte strains in dose-dependent mode. Show such as Fig. 1～5 that also HC RBL and DA-DKP are with the generation of dose-dependent mode in the cell in vitro factor of these two kinds of T-cell lines of early stage inhibition of thorn flyback cycle. Yet HC RBL and DA-DKP are (the seeing Fig. 4) of spread effect in this late period in cycle (the 14th day or slower). HC 2626 and HC RBL contain MR-DKP (as determining with mass spectrography), but HC 2626 is except the MR-DKP that possibility cell cycle inhibitory action in late period is responsible for, contain other components and (be included as the casein of corresponding dephosphorylation albumen thereby Anhydroalkannin, jointly examining explanation in the application 10/723,247 such as what submit on November 25th, 2003). Accordingly, HC RBL and HC 2626 (both containing MR-DKP), MR-DKP and DA-DKP should be to being useful in T-downward modulation cell-mediated and/or that autoimmune disease is replied such as the inflammatory cytokine in the multiple sclerosis, because they produce in the cell factor that the thorn flyback cycle all suppresses the T-cell in early days. These results also point out HC RBL, HC 2626, MR-DKP and DA-DKP optionally to affect antigen-specificity T-cell and do not affect tranquillization T-cell.

C. the mechanism of action

The mechanism of action to DA-DKP and HC 2626 (containing MR-DKP) is studied. In order to do like this, with 1 * 10⁶The TRiPS cell was cultivated 30 minutes in 37 ℃ in individual the 18th day, simultaneously what does not add (" completely without "), add CD3/CD28 Dynabeads (CD3/CD28 magnetic bead), add CD3/CD28 magnetic bead and 0.5mM DA-DKP, perhaps add the HC 2626 of CD3/CD28 magnetic bead and dilution in 1: 500. After the cultivation, cell is extracted cracking in the reagent (Sigma) at the mammalian cell of lysis.

Then this cell extract is cultivated with the antibody chip (Hypromatrix Array) that repeats discretely in room temperature, afterwards according to the scheme washed twice of manufacturing firm. This antibody chip is the nylon membrane (Hypromatrix is customized) of crossing with the antibody trace of the transcription factor of listing in the table 1. Adding was cultivated 1 hour phosphorylated tyrosine, phosphorylation serine and the specific mixtures of antibodies of phosphorylation threonine (Zymed). Then add with biotin labeled and resist-immune globulin antibody. After flush away is somebody's turn to do anti--immunoglobulin (Ig)-biotin, add streptavidin-peroxidase, and before adding peroxidase-reaction luminous substrate, chip is carried out last washing.

By the exposure film can make visual result and as shown in table 2 with the result according to 0 (feminine gender) or +～++ ++ (positive) scoring. As shown in Table 2, the release of the activation of some cell transcription factors (ERK1/2) and preformation (pe-formed) cell factor is suppressed by HC 2626 (containing MR-DKP) and DA-DKP.

Table 1:Hypromatrix array (customized): the albumen that is used for phosphorylation

Number	Acronym	Compound
Number	Acronym	Compound	1	Akt 1/2	Protein kinase B, anti--the Apoptosis kinases
2	c-Cb1	TcR suppresses path; Tyr²⁹²Phosphorylation triggers combination and the inactivation of Syk and ZAP-70	1	Akt 1/2	Protein kinase B, anti--the Apoptosis kinases
2	c-Cb1		3	CBP	Csk-is in conjunction with albumen (PAG); Inherent memebrane protein momently (and low-level) tyrosine take off-phosphorylation to be to discharge csk
4	CREB	The cAMP response element binding protein; Be phosphorylated (unk) to activate/downward modulation IL-2 promoter	3	CBP
4	CREB		5	csk	COOH-end src kinases; The Ser of phosphorylation³⁶⁴With the Tyr of phosphorylation (active (activity whether? )) make lck phosphorylation and inactivation
6	ERK1	The extracellular signal associated kinase	5	csk
6	ERK1	The extracellular signal associated kinase	7	c-fos	Stimulated the AP-1 composition that activates by TcR; N-and C-unk residue all are phosphorylated
8	NFATC	The nuclear factor of the T-cell of activation; Complete incompetent state	7	c-fos
8	NFATC		9	c-jun	By the AP-1 composition of TcR activation activation; Ser⁶³By the JNK-MAPK phosphorylation
10	IκB-α	The inhibitor of NF κ B	9	c-jun
10	IκB-α	The inhibitor of NF κ B	11	pκB-α	The NF kB inhibitor of the Ser-of phosphorylation and inactivation
12	P38MAPK	Mitogen-activated protein kinase	11	pκB-α
12	P38MAPK	Mitogen-activated protein kinase	13	PI3 kinases/p85	Activated by glucocorticoid and beta 2-adrenergic receptor
14	pten	Kytoplasm 3 '-inositol phosphatase; Tumor suppressor gene is by transforming back PI-PO the antagonism PT 3 ' kinases of inactive form	13	PI3 kinases/p85	Activated by glucocorticoid and beta 2-adrenergic receptor

15	c-Raf-1
15	c-Raf-1		16	Rap 1	Negative TcR regulation and control GTP enzyme
17	Ras	Kinases; At incompetent state inactivation	16	Rap 1	Negative TcR regulation and control GTP enzyme
17	Ras	Kinases; At incompetent state inactivation	18	fyn	Cell membrane is in conjunction with instant TcR signal kinases (immediate TcR Signal kinase)
19	lck	The cell membrane combination is TcR signal kinases immediately, and activity form is the Tyr of phosphorylation³⁹⁵ At C-end Tyr by the deactivation of csk phosphorylation	18	fyn
19	lck		20	The ZAP70 kinases	Signaling molecule from CD3 ζ; By lck/fyn at unknown position (at?) phosphorylation, ZAP70 makes LAT (T-cell activation linker) in tyrosine site phosphorylation and makes the tyrosine phosphorylation of SLP-76.

Table 2: result

Compound	ML	CD3/CD28	DKP	HC2626
Compound	ML	CD3/CD28	DKP	HC2626	Akt
1/2	+	++	+++	++	Akt
1/2	+	++	+++	++	c-Cbl	--	--	--	--
CBP	+	++	++	++	c-Cbl	--	--	--	--
CBP	+	++	++	++	CREB	--	--	--	--
csk	+	++	+	+	CREB	--	--	--	--
csk	+	++	+	+	ERK1	+	+	+	+
c-fos	--	--	--	--	ERK1	+	+	+	+
c-fos	--	--	--	--	NFATC	--	--	--	--
c-jun	++	+	+	+	NFATC	--	--	--	--
c-jun	++	+	+	+	IκB-α	++	++	+	+
pIκB-α	--	--	--	--	IκB-α	++	++	+	+
pIκB-α	--	--	--	--	p38MAPK	++	+++	+++	+++
PI3 kinases/p85	+	++	+	++	p38MAPK	++	+++	+++	+++
PI3 kinases/p85	+	++	+	++	pten	--	--	--	--
c-Raf-1	--	--	--	--	pten	--	--	--	--
c-Raf-1	--	--	--	--	Rap1	+	++	++	+
Ras	--	--	--	--	Rap1	+	++	++	+
Ras	--	--	--	--	fyn	+	+	+	+
lck	--	--	--	--	fyn	+	+	+	+
lck	--	--	--	--	The ZAP70 kinases	--	--	--	--

Embodiment 3:Gly-Leu DKP (GL-DKP) and Ala-Pro DKP (AP-DKP) are in the generation of external inhibition people T-lymphocyte factor

According to use TRiPS illustrated among the embodiment 2 and H4#9.25 cell line GL-DKP and AP-DKP (from DMI Synthesis, Ltd., Cardff, UK obtains) are tested. Find that GL-DKP and AP-DKP produce with the cell in vitro factor that dosage dependence mode suppresses these two kinds of T-lymphocyte strains. This mechanism of action as described in example 2 above current place under study for action, and the release of the cell factor of the activation of cell factor transcription factor and preformation all is affected.

Embodiment 4:Asp Ala DKP (DA-DKP) and Tyr Glu DKP (YE-DKP) are in the generation of external inhibition people T-lymphocyte factor

From the peripheral blood of normal person's donor, separate normal person's lymphocyte with Histopaque (Sigma). Then, with 3-4 * 10⁵Individual this lymphocyte is suspended among the IMDM of 1ml serum-free. Come irritation cell by the anti-CD 3 antibodies (Pharmingen, San Diego, CA) that dilutes at 1: 2000 that adds 25 μ l, and cultivated 18 hours in 37 ℃.

Then (final concentration is 10 with a kind of and dexamethasone in three kinds of DKP preparations^-5M) join in three parts of cultures. Described three kinds of DKP preparations are:

1.DA-DKP (from DMI Synthesis, Ltd., Cardiff, UK obtains; Final concentration in culture is 25 μ g/ml).

2.DKP-ZLB, a kind of 25% albumin preparation is (from ZLB Bioplasma, AG 3000 Berne 22 Switzerland obtain), it has heated 4 days in 60 ℃, finds that through mass spectrometric determination it contains 0.5mM DA-DKP (final concentration of DA-DKP is 14 μ g/ml in the culture) afterwards.

3.DKP-gamma globulin---the gamma globulin preparation that contains the 12mg/ml gamma globulin in the phosphate buffered saline (PBS) of pH 7.4 (obtains from Sigma, numbering G-4386) filters with Centricon 3000 filters, and use this filtrate (containing molecular weight less than 3000 component). Measure such as anion exchange HPLC and negative electrojet mass spectrometry, this filtrate is contained one 292 molecular weight, and it is the molecular weight of Tyr-Glu DKP (YE-DKP). This filtrate final dilution with 1: 4 in culture uses.

Add after DKP preparation or the dexamethasone, this culture was cultivated 18 hours in 37 ℃. Then use ELISA (IL 61105 for Pierce Biotechnology, Rockford) to measure to be discharged into IL-2, IFN γ in every kind of culture and the amount of TNF α.

The result shows in the following Table 3. As can be seen, the maximum of using the DKP-gamma globulin to obtain all these three kinds of release of cytokines reduces. Observe CD69+T-cell (CD69 is the mark of finding at the T-cell of activation) although the flow cytometry of quantity also shows T-cell receptors compound internalization, the DKP-gamma globulin approximately reduced by 90% CD69+T-cell quantity, and the ground dexamethasone of comparing has reduced about 50%.

Table 3

Stimulate	Process	U/ml IL-2	pg/ml IFNγ	pg/ml TNFα
Stimulate	Process	U/ml IL-2	pg/ml IFNγ	pg/ml TNFα	Nil		0.24±9.1	2.3±0.9	2.8±9.5
CD3		2.6±0.5	289±35	98±3.2	Nil		0.24±9.1	2.3±0.9	2.8±9.5
CD3		2.6±0.5	289±35	98±3.2	CD3	DA-DKP	1.4±0.3	306±17	74±4.7
CD3	DKP-ZLB	1.4±0.4	311±18	130±2.9	CD3	DA-DKP	1.4±0.3	306±17	74±4.7
CD3	DKP-ZLB	1.4±0.4	311±18	130±2.9	CD3	The DKP-gamma globulin	0.24 ± 0.25 (91% reduction)	2.1 ± 0.1 (99% reduction)	1.6 ± 0.6 (98% reduction)
CD3	Dexamethasone	0.9 ± 0.1 (65% reduction)	76 ± 7.32 (74% reduction)	4.1 ± 0.3 (96% reduction)	CD3	The DKP-gamma globulin	0.24 ± 0.25 (91% reduction)	2.1 ± 0.1 (99% reduction)	1.6 ± 0.6 (98% reduction)

Claims

1. method for the treatment of the cell-mediated disease of T-, it comprises and gives acceptable salt on the diketopiperazine with following formula of effective dose or its physiology to its animal of needs:

Wherein:

R ¹And R², can be identical or different, each is:

(b)R ¹For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline, R with adjacent loops nitrogen²For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline, perhaps R with adjacent loops nitrogen¹And R²Be independently of one another-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline with adjacent loops nitrogen; Or

(c) derivative of amino acid whose side chain, wherein this amino acid be described in (a) those one of, and the side chain of this derivatization has:

(iv)-COOH group quilt-CON (R⁴) ₂₂Group replaces, wherein each R⁴Can be the substituted or unsubstituted alkyl of H, cycloalkyl, Heterocyclylalkyl, aryl, alkaryl, aralkyl or heteroaryl independently;

(v)-SH group quilt-S-S-CH₂-CH(NH ₂)-COOH or

-S-S-CH ₂-CH ₂-CH(NH ₂)-COOH replaces;

(vi)-CH ₂-group quilt-CH (NH₂)-or-CH (OH)-group replaces;

(vii)-CH ₃Group quilt-CH₂-NH ₂Or-CH₂-OH group replaces; And/or

(viii) H that is connected on the carbon atom is replaced by halogen.

2. the process of claim 1 wherein R¹、R ²Or these two is the side chain of aspartic acid, the side chain of glutamic acid, the perhaps derivative of the side chain of aspartic acid or glutamic acid, in this derivative-COOH group quilt-COOR³Group or-CON (R⁴) ₂Group replaces.

3. the method for claim 2, wherein R¹For the side chain of aspartic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the aspartic acid that group replaces, and R²Side chain for alanine.

4. the method for claim 2, wherein R¹For the side chain of aspartic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the aspartic acid that group replaces, and R²Side chain for tyrosine.

5. the method for claim 2, wherein R¹For the side chain of glutamic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the glutamic acid that group replaces, and R²Side chain for alanine.

6. the method for claim 2, wherein R¹For the side chain of glutamic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the glutamic acid that group replaces, and R²Side chain for tyrosine.

7. the method for claim 2, wherein R¹Be side chain aspartic acid or glutamic acid, and R²Side chain for alanine.

8. the method for claim 2, wherein R¹Be side chain aspartic acid or glutamic acid, and R²Side chain for tyrosine.

9. the process of claim 1 wherein R¹And R²It all is the derivative of hydrophobic side chains or hydrophobic side chains.

10. the method for claim 9, wherein:

(a)R ¹And R², can be identical or different, each is the side chain of glycine, alanine, valine, norvaline, α-aminoacid, leucine, isoleucine, nor-leucine or phenylalanine;

(b)R ¹For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms, and R²For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms; Or

(c)R ¹Be the side chain of glycine, alanine, valine, norvaline, α-aminoacid, leucine, isoleucine, nor-leucine or phenylalanine, and R²For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms.

11. the method for claim 10, wherein R¹Be the side chain of glycine, and R²Be leucic side chain.

12. the method for claim 10, wherein R¹For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms, and R²Side chain for phenylalanine.

13. the method for claim 10, wherein R¹For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms, and R²Side chain for alanine.

14. the process of claim 1 wherein R¹、R ²Or these two is the derivative of the side chain of methionine, arginic side chain or these side chains.

15. the method for claim 14, wherein R¹Be the side chain of methionine, and R²Be arginic side chain.

16. each method in the claim 1～15, wherein said animal is behaved.

17. each method in the claim 1～15, the cell-mediated disease of wherein said T-are graft rejection, graft versus host disease(GVH disease), unwanted delayed allergy, cell-mediated PUD D or the autoimmune disease of T-.

18. each method in the claim 1～15, the cell-mediated disease of wherein said T-are multiple sclerosis, neuritis, polymyositis, psoriasis, Leucoplakia, qualified human relations syndrome, rheumatoid arthritis, type 1 diabetes, autoimmunity pancreatitis, inflammatory bowel disease, clone's disease, ulcerative colitis, CD, glomerulonephritis, chorionitis, sarcoidosis, AITD, Hashimoto thyroiditis, Graves disease, myasthenia gravis, Addison disease, the autoimmunity ommochrome retinitis, pemphigus vulgaris, PBC, pernicious anaemia or systemic lupus erythematosus.

19. each method in the claim 1～15, the cell-mediated disease of wherein said T-is pulmonary fibrosis or idiopathic pulmonary fibrosis.

20. a method that suppresses the T-cell-stimulating, it comprises and gives acceptable salt on the diketopiperazine with following formula of effective dose or its physiology to its animal of needs:

Wherein:

R ¹And R², can be identical or different, each is:

(iv)-COOH group quilt-CON (R₄) ₂Group replaces, wherein each R⁴Can be the substituted or unsubstituted alkyl of H, cycloalkyl, Heterocyclylalkyl, aryl, alkaryl, aralkyl or heteroaryl independently;

(v)-SH group quilt-S-S-CH₂-CH(NH ₂)-COOH or

-S-S-CH ₂-CH ₂-CH(NH ₂)-COOH replaces;

(vi)-CH ₂-group quilt-CH (NH₂)-or-CH (OH)-group replaces;

(vii)-CH ₃Group quilt-CH₂-NH ₂Or-CH₂-OH group replaces; And/or

(viii) H that is connected on the carbon atom is replaced by halogen.

21. the method for claim 20, wherein R¹、R ²Or these two is the derivative of the side chain of the side chain of side chain, glutamic acid of aspartic acid or aspartic acid or glutamic acid, in this derivative-and COOH group quilt-COOR³Group or-CON (R⁴) ₂Group replaces.

22. the method for claim 21, wherein R¹For the side chain of aspartic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the aspartic acid that group replaces, and R²Side chain for alanine.

23. the method for claim 21, wherein R¹For the side chain of aspartic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the aspartic acid that group replaces, and R²Side chain for tyrosine.

24. the method for claim 21, wherein R¹For the side chain of glutamic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the glutamic acid that group replaces, and R²Side chain for alanine.

25. the method for claim 21, wherein R¹For the side chain of glutamic acid or-COOH group quilt-COOR³Group or-CON (R⁴) ₂The derivative of the side chain of the glutamic acid that group replaces, and R²Side chain for tyrosine.

26. the method for claim 21, wherein R¹Be side chain aspartic acid or glutamic acid, and R²Side chain for alanine.

27. the method for claim 21, wherein R¹Be side chain aspartic acid or glutamic acid, and R²Side chain for tyrosine.

28. the method for claim 21, wherein R¹And R²It all is the derivative of hydrophobic side chains or hydrophobic side chains.

29. the method for claim 28, wherein:

30. the method for claim 29, wherein R¹Be the side chain of glycine, and R²Be leucic side chain.

31. the method for claim 29, wherein R¹For-CH₂-CH ₂-CH ₂-and form proline and R with contiguous nitrogen-atoms²Side chain for phenylalanine.

32. the method for claim 29, wherein R¹For-CH₂-CH ₂-CH ₂-and form proline and R with contiguous nitrogen-atoms²Side chain for alanine.

33. the method for claim 20, wherein R¹、R ²Or these two is the derivative of the side chain of methionine, arginic side chain or these side chains.

34. the method for claim 33, wherein R¹Be the side chain of methionine, and R²Be arginic side chain.

35. each method in the claim 20～34, wherein said animal is behaved.

36. each method in the claim 20～34, wherein said diketopiperazines are used for the treatment of at least partially inflammation or inflammatory disease that the activation owing to the T-cell causes or increases the weight of.

37. a pharmaceutical composition, its comprise pharmaceutically acceptable carrier and have the diketopiperazine of following formula or its physiology on acceptable salt:

Wherein:

R ⁵And R⁶, can be identical or different, each is:

(b)R ⁵For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline, R with adjacent loops nitrogen⁶For-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline, perhaps R with adjacent loops nitrogen⁵And R⁶Be independently of one another-CH₂-CH ₂-CH ₂-or-CH₂-CH(OH)-CH ₂-and form proline or hydroxyproline with adjacent loops nitrogen; Or

(c) amino acid whose side chain derivative, wherein this amino acid be described in (a) those one of, and the side chain of this derivatization has:

(iii)-CH ₂-group quilt-CH (NH₂)-or-CH (OH)-group replaces;

(iv)-CH ₃Group quilt-CH₂-NH ₂Or-CH₂-OH group replaces; And/or

(v) H that is connected on the carbon atom is replaced by halogen.

38. the composition of claim 37, wherein R⁵And R⁶It all is the derivative of hydrophobic side chains or hydrophobic side chains.

39. the composition of claim 38, wherein:

(a)R ⁵And R⁶, can be identical or different, each is the side chain of glycine, alanine, valine, norvaline, α-aminoacid, leucine, different shell propylhomoserin, nor-leucine or phenylalanine;

(b)R ⁵For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms, and R⁶For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms; Or

(c)R ⁵Be the side chain of glycine, alanine, valine, norvaline, α-aminoacid, leucine, isoleucine, nor-leucine or phenylalanine, and R⁶For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms.

40. the composition of claim 39, wherein R⁵Be the side chain of glycine, and R⁶Be leucic side chain.

41. the composition of claim 39, wherein R⁵For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms, and R⁶Side chain for phenylalanine.

42. the composition of claim 39, wherein R⁵For-CH₂-CH ₂-CH ₂-and form proline with contiguous nitrogen-atoms, and R⁶Side chain for alanine.

43. the composition of claim 37, wherein R⁵、R ⁶Or these two is the derivative of the side chain of methionine, arginic side chain or these side chains.

44. the composition of claim 43, wherein R⁵Be the side chain of methionine, and R⁶Be arginic side chain.

45. method for the treatment of the cell-mediated disease of T-, it comprises and is included in the animal the normal albumen of finding or the pharmaceutical composition of peptide to what its animal of needs gave effective dose, described albumen or peptide are processed, make described composition also comprise at least a diketopiperazine that is derived from this albumen or peptide.

46. the method for claim 45, wherein said albumen are albumin.

47. the method for claim 45, wherein said albumen are immunoglobulin (Ig).

48. the method for claim 45, wherein said albumen are hematopoietin.

49. each method in the claim 45～48, wherein said pharmaceutical composition are oral administration.

50. each method in the claim 45～48, wherein said animal is behaved, and described albumen or peptide behaviour albumen or peptide.

51. method that suppresses the T-cell-stimulating, it comprises and is included in the animal the normal albumen of finding or the pharmaceutical composition of peptide to what its animal of needs gave effective dose, described albumen or peptide are processed, make described composition also comprise at least a diketopiperazine that is derived from this albumen or peptide.

52. the method for claim 51, wherein said albumen are albumin.

53. the method for claim 51, wherein said albumen are immunoglobulin (Ig).

54. the method for claim 51, wherein said albumen are hematopoietin.

55. each method in the claim 51～54, wherein said pharmaceutical composition are oral administration.

56. each method in the claim 51～54, wherein said animal is behaved, and described albumen or peptide behaviour albumen or peptide.

57. the method for a synthetic diketopiperazine, it is included in heating albumen or peptide solution under the condition that effectively causes diketopiperazine formation.

58. the method for claim 57, wherein said albumen are albumin.

59. the method for claim 57, wherein said albumen are immunoglobulin (Ig).

60. the method for claim 57, wherein said albumen are hematopoietin.

61. the method for claim 57, wherein said diketopiperazine purifying from solution.

62. each method in the claim 57～61, wherein said solution was in 60 ℃ of heating 4 days.

63. the method for a synthetic diketopiperazine, it is included in two N-that make albumen or this albumen of peptide solution and cut-out or peptide under the condition that effectively produces diketopiperazine enzyme terminal or two C-end amino acids and contacts.

64. the method for claim 63, wherein said albumen are albumin.

65. the method for claim 63, wherein said albumen are immunoglobulin (Ig).

66. the method for claim 63, wherein said albumen are hematopoietin.

67. the method for claim 63, wherein said enzyme are dipeptidyl peptidase.

68. the method for claim 63, wherein said enzyme are carboxypeptidase.

69. each method in the claim 63～68, wherein said diketopiperazine purifying from solution.

70. the improvement pharmaceutical composition of an albumen or peptide, described improvement comprise the content that reduces diketopiperazines in the composition.

71. the method for claim 70, wherein said albumen are albumin.

72. the method for claim 70, wherein said albumen are immunoglobulin (Ig).

73. the method for claim 70, wherein said albumen are hematopoietin.

74. comprising, a method for preparing the improvement pharmaceutical composition of albumen or peptide, the method from composition, remove at least some diketopiperazineses that in composition, exist.

75. the method for claim 74, wherein said albumen are albumin.

76. the method for claim 74, wherein said albumen are immunoglobulin (Ig).

77. the method for claim 74, wherein said albumen are hematopoietin.

78. a method for preparing the improvement pharmaceutical composition of albumen or peptide, the method comprises the content of the solution of this albumen or peptide being processed to improve diketopiperazines.

79. the method for claim 78, wherein said solution heats under the condition that causes diketopiperazines formation.

80. the method for claim 79, wherein said solution was in 60 ℃ of heating 4 days.

81. the method for claim 78, wherein described solution contacts with two N-that cut off this albumen or peptide enzyme terminal or two C-end amino acids under the condition that effectively produces diketopiperazines.

82. the method for claim 81, wherein said enzyme are dipeptidyl peptidase.

83. the method for claim 81, wherein said enzyme are carboxypeptidase.

84. the method for claim 78, wherein said albumen are albumin.

85. the method for claim 78, wherein said albumen are immunoglobulin (Ig).

86. the method for claim 78, wherein said albumen are hematopoietin.

87. the improvement pharmaceutical composition of an albumen or peptide, described improvement comprise the content that improves diketopiperazines in the composition.

88. the method for claim 87, wherein said albumen are albumin.

89. the method for claim 87, wherein said albumen are immunoglobulin (Ig).

90. the method for claim 87, wherein said albumen are hematopoietin.

91. each composition in the claim 87～90, it is suitable for oral administration.