CN1626549A

CN1626549A - Carbowax alcoholized ramification of TimopEntin, combination of medication and application

Info

Publication number: CN1626549A
Application number: CN 200310117356
Authority: CN
Inventors: 潘和平; 王良友; 邵宁生; 柳川; 高亚萍
Original assignee: Institute of Basic Medical Sciences of AMMS
Current assignee: Institute of Basic Medical Sciences of AMMS
Priority date: 2003-12-11
Filing date: 2003-12-11
Publication date: 2005-06-15

Abstract

本发明公开了胸腺五肽的聚乙二醇化衍生物，含它们的药物组合物及用途。本发明的胸腺五肽的聚乙二醇化衍生物为在胸腺五肽的N－端氨基、C－端羧基或侧链的氨基或羧基偶联有引入氨基或羧基的聚乙二醇或偶联有聚乙二醇化修饰的氨基酸，或在胸腺五肽的N端或C端引入半胱氨酸，偶联有马来酰胺基聚乙二醇、乙烯基聚乙二醇或碘代乙酰基聚乙二醇。本发明的胸腺五肽的聚乙二醇化衍生物可用于制备类风湿性关节炎、变应性皮炎、肿瘤、某些病毒感染疾病等与免疫缺陷、免疫功能低下有关疾病的治疗或预防药物。The invention discloses pegylated derivatives of thymopentin, their pharmaceutical composition and application. The PEGylated derivatives of thymopentin of the present invention are polyethylene glycol or conjugated with amino or carboxyl groups introduced into the N-terminal amino group, C-terminal carboxyl group or side chain amino or carboxyl group of thymopentin. Amino acids modified by PEGylation, or cysteine introduced at the N-terminal or C-terminal of thymopentin, coupled with maleamide-based polyethylene glycol, vinyl polyethylene glycol or iodoacetylpolyethylene glycol ethylene glycol. The PEGylated derivatives of thymopentin of the present invention can be used to prepare rheumatoid arthritis, allergic dermatitis, tumors, certain viral infection diseases and other diseases related to immunodeficiency and immune function.

Description

The polyethylene glycol derivative of thymopeptide-5 contains their pharmaceutical composition and purposes

Technical field

The present invention relates to a kind of compound, relate in particular to the polyethylene glycol derivative of thymopeptide-5, also relate to the pharmaceutical composition and the purposes that contain them.

Background technology

Thymopoietin (Thymopoietin) is by a kind of polypeptide hormone that contains 49 amino-acid residues of thymic epithelial cells's excretory, and (Thymopentin TP5) is the 32-36 amino acid fragment of thymopoietin to thymopeptide-5.TP5 is the reactive site of thymopoietin, its function is identical with thymopoietin, it is a kind of at the lymphocytic immunostimulant of T, can promote the ripe and differentiation of T cell, and impel the multiple lymphokine of sophisticated T emiocytosis (as interleukin-2 and gamma-interferon etc.), can also promote the generation of interleukin-2 acceptor.In the impaired animal model of immunologic balance, TP5 has the equilibrated of making it characteristics.No matter be that immune response is too high or too low, TP5 all can realize balance adjustment, makes it very promising in the relevant human disease treatment of immunologic balance imbalance.The TP5 molecular weight is 679.9, and its aminoacid sequence is as follows:

Arg-Lys-Asp-Val-Tyr

Thymopeptide-5 can be induced prothymocyte and the peripheral T cell of activation, has significant clinical related immune and regulates active.Clinical study shows that thymopeptide-5 is all effective in treatment rheumatoid arthritis, allergic dermatitis, some virus infection and meat shape knurl.It can reduce the patient's of serious recurrent herpes simplex virus infection recurrence rate and recurrence time.Its antagonism tumor chemoradiotherapy and prevention major operation postoperative infection have tangible clinical effectiveness.Can prevent long-term diabetic subject's complication and promote its rehabilitation.To the patient of early stage acquired immunodeficiency syndrome, the benefit of thymopeptide-5 maximum is the immunological competence that can increase patient.Its security is fine, and a large amount of research has confirmed not have obvious toxic and side effects.

TP5 is a peptide medicament, is easily degraded by hydrochloric acid in gastric juice, must parenteral admin.TP5 is very fast in human plasma to reduce to amino acid by proteolytic enzyme and aminopeptidase, and the transformation period is about 30 seconds.Dosage is big, the cycle is long.Therefore, TP5 is modified, prolonging it has application promise in clinical practice in vivo action time.

Studies show that some protein medicaments can keep biological activity preferably after polyoxyethylene glycol (PEG) is modified, and can the biological intravital transformation period of significant prolongation.Polyethyleneglycol modified adenosine deaminase went on the market through the FDA approval in 1991, and calendar year 2001, polyethyleneglycol modified Interferon, rabbit went on the market through the FDA approval.Peptide medicament and protein medicaments have many similarities on structure and properties, the Pegylation of peptide medicament is modified the Pegylation modification that research is later than protein medicaments, obtained effect preferably at present, therefore the peptide medicament of Pegylation modification has good application prospects, does not still have the report of the thymopeptide-5 of Pegylation modification at present.

Summary of the invention

One of purpose of the present invention provides the polyethylene glycol derivative of thymopeptide-5, the amino acid that polyoxyethylene glycol that amino that the structure of this derivative is amino for the N-at thymopeptide-5 holds, C-holds carboxyl or side chain or carboxyl coupling have introducing amino or carboxyl or coupling have Pegylation to modify; Or at the N of thymopeptide-5 end or C end introducing halfcystine, coupling has maleoyl amido polyoxyethylene glycol, vinyl polyoxyethylene glycol or iodo ethanoyl polyoxyethylene glycol.

The compound of polyethylene glycol derivative of the present invention for having formula (I) structure comprises that the amino in site is through the compound of PEG covalent modification arbitrarily among the TP5, and wherein said amino comprises the amino and lysine side-chain amino of N-end:

[PEG-X-(CH2)mCO-NH]z-TP5??(I)

Wherein PEG represents: RO (CH2CH2O) n-CH2CH2, R=H or CH3, n=5-1000; X=O, NH or NHCO; M=0-6; Z=1-2.

Polyethylene glycol derivative of the present invention, also the compound for having formula (II) structure comprises that the carboxyl in site is through the compound of PEG covalent modification arbitrarily among the TP5, wherein said carboxyl comprises the side chain carboxyl group of C-end carboxyl and aspartic acid:

TP5-[CO-Y-PEG]z?????????(II)

Wherein the PEG definition is the same; Y=O or NH; Z=1-2.

Polyethylene glycol derivative of the present invention also is the compound with formula (III) structure, and promptly the N-of TP5 end is introduced halfcystine after the compound that PEG-MAL, PEG-VS or PEG-IODO modify:

PEG-M-Cys-TP5???????????(III)

Wherein

Or

Or

The PEG definition is the same; Cys is a halfcystine, links to each other with M group covalency by the side chain sulphur atom.

Polyethylene glycol derivative of the present invention also is the compound with formula (IV) structure, and promptly the C-of TP5 end is introduced halfcystine after the compound that PEG-MAL, PEG-VS or PEG-IODO modify

TP5-Cys-M-PEG??????????????(IV)

PEG wherein, Cys, the M definition is the same.

Another object of the present invention provides the preparation method of polyethylene glycol derivative.

The used PEG-OH structure of the present invention is: RO (CH ₂CH ₂O) n-CH ₂CH ₂-OH, R=H or CH ₃, n=5-1000.Molecular-weight average can be used as commercialization reagent by the PEG-OH of hundreds of to several ten thousand and buys PEG-NH ₂Can buy or be translated into PEG-NH earlier by following reaction ₂, obtain PEG-NHCOCH with the succinyl oxide reaction again ₂CH ₂COOH can be used as carboxyl group with this compound then, can it be coupled to the N-end of polypeptide on solid phase, after trifluoroacetic acid cracking and RPLC purifying get product.

Introduce the amino acid of amino, carboxyl or the modification of preparation PEGization earlier (as Fmoc-Lys (NH-COCH at the end of PEG ₂-PEG)-and OH, Fmoc-Asp (CO-NH-PEG)-OH), be coupled in the peptide sequence with liquid phase or solid phase method again and go, can realize the modification that the N-end is amino, C-holds carboxyl, Lys side chain amino, Asp or Glu side chain carboxyl group to polypeptide.With PEG-NH ₂Get maleoyl amido polyoxyethylene glycol (PEG-MAL), vinyl polyoxyethylene glycol (PEG-VS) or iodo ethanoyl polyoxyethylene glycol (PEG-IODO) with maleic anhydride, vinyl chlorination sulfoxide, iodo acetic anhydride respectively.

PEG-MAL obtains by following reaction:

TP5 can be synthetic with solid phase or liquid phase polypeptide synthesis, is easy to introduce a Cys at N-end or C-end in building-up process.The peptide chain that will contain Cys is soluble in water, transfers pH7-8 with sodium bicarbonate, adds 3 times of normal PEG-MAL or PEG-VS or PEG-IODO, and stirring at room is reacted, and gets the derivative of the TP5 of PEG modification with the RPLC purifying.The reaction of PEG-MAL modification Cys-TP5 is as follows:

The Wang resin that the present invention relates to, Rink resin, DCC, HOBT, HBTU, NMM, TMBS, TFA and common Fmoc-amino acid obtain by buying.

The implication of above-mentioned each english abbreviation is as follows: PEG-polyoxyethylene glycol, mPEG-mono methoxy polyethylene glycol, Arg-arginine, the Asp-aspartic acid, the Cys-halfcystine, Lys-Methionin, Tyr-tyrosine, the Val-Xie Ansuan, the Boc-tertbutyloxycarbonyl, Fmoc-fluorenylmethyloxycarbonyl, DCC-dicyclohexylcarbodiimide, the HOBT-1-hydroxybenzotriazole, HBTU-2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethyl-urea phosphofluoric acid, the NMM-N-methylmorpholine, TFA-trifluoroacetic acid, TMBS-bromotrimethylsilane, the Ts-Cl-Tosyl chloride, the RP-HPLC-RPLC.

In the present invention, all amino acid are L-type amino acid.

According to the present invention, following compounds is preferred in the polyethylene glycol derivative of thymopeptide-5:

mPEG ₂₀₀₀-NHCOCH ₂CH ₂CO-TP5

mPEG ₅₀₀₀-NHCOCH ₂CH ₂CO-TP5

Cys(mPEG ₂₀₀₀-MAL)-TP5

Cys(mPEG ₅₀₀₀-MAL)-TP5

TP5-Cys(mPEG ₂₀₀₀-MAL)-NH ₂

TP5-Cys(mPEG ₅₀₀₀-MAL)-NH ₂

Another object of the present invention provides the pharmaceutical composition of the polyethylene glycol derivative that contains TP5, and said composition comprises polyethylene glycol derivative and the medical dressing of TP5.

Another object of the present invention is the purposes that discloses the polyethylene glycol derivative of TP5 and comprised their pharmaceutical composition.The present inventor observes the biological activity that part of compounds has the short mouse lymphocyte propagation of good in-vitro and inducing mouse lymphocytic emiocytosis IFN-γ and IL-2 by activity rating.The pharmaceutical composition that contains them can be used for the treatment or the prevention of relative diseases such as immune deficiency, immunologic hypofunction.The polyethylene glycol derivative of TP5 combines with one or more vehicle and makes the formulation that is applicable to the people, as making injection with N.F,USP MANNITOL as vehicle, through relative diseases such as subcutaneous administration treatment immune deficiency, immunologic hypofunctions.With immune deficiency or immunologic hypofunction diseases associated rheumatoid arthritis, allergic dermatitis, tumour, hepatitis B, hepatitis C, acquired immune deficiency syndrome (AIDS) etc. are arranged.

The polyethylene glycol derivative of TP5 of the present invention has the chemical structure novelty, and biological activity is stable, and characteristics such as biological intravital long half time have good application prospects.

Embodiment

Following embodiment represents illustrative embodiment of the present invention, but the present invention is not subjected to the restriction of these embodiment.The used solid-phase synthesized carrier Wang of embodiment resin is the ACT product; DCC, HOBT, HBTU, Fmoc-protection amino acid are Shanghai gill biochemical products; TFA is the ACROS product, and TMBS is the Fluka product, and molecular-weight average is that 2000 and 5000 mPEG-OH is the Sigma product.

Embodiment 1 mPEG ₅₀₀₀-NHCOCH ₂CH ₂CO-TP5's is synthetic

Weighing m PEG ₂₀₀₀-OH 40.0g (20mmol) places the 250ml reaction flask, adds 100mlCH ₂Cl ₂, add 15mlEt again after the solid dissolving ₃N (100mmmol) and 19.0g Ts-Cl (100mmol), the stirring at room reaction.After the TLC monitoring reaction was complete, rotary evaporation removed and desolvates, and added the 100ml anhydrous diethyl ether and was settled out solid, got 28.5gmPEG ₂₀₀₀-OTs, yield 71%

With 18.0gmPEG ₂₀₀₀-OTs (9mmol) is dissolved in 50ml DMF, adds 5.0g (27mmol) potassium phthalimide, and 120 ℃ were reacted 4 hours.The pressure reducing and steaming solvent is dissolved in the 50ml dehydrated alcohol with resistates, adds the 13.5ml hydrazine hydrate, back flow reaction 4 hours.Rotary evaporation removes and desolvates, and resistates is dissolved in CH ₂Cl ₂, be settled out solid with anhydrous diethyl ether, again with dehydrated alcohol-ether recrystallization, get 14.2gmPEG ₂₀₀₀-NH ₂, yield 78%.

With 5.0gmPEG ₂₀₀₀-NH ₂Be dissolved among the 10mlDMF, add the 0.38g succinyl oxide, 40 ℃ were reacted 0.5 hour, and the pressure reducing and steaming solvent adds anhydrous diethyl ether and is settled out solid, gets 4.8gmPEG ₂₀₀₀-NHCOCH ₂CH ₂COOH, yield 96%.

100mg Wang resin (0.05mmol) is a solid phase carrier, Fmoc-Arg (Mtr), Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Val-OH, Fmoc-Tyr (tBu) is a raw material, DCC-HOBT makes condensing agent, according to the aminoacid sequence of TP5,, add mPEG by the synthetic H-Arg (Mtr) of the Fmoc solid-phase peptide synthesis of standard-Lys (Boc)-Asp (OtBu)-Val-Tyr (tBu)-Wang resin ₂₀₀₀-NHCOCH ₂CH ₂COOH (0.1mmol), HBTU (0.1mmol), NMM (0.15mmol) reacted after 48 hours, and ninhydrin method detects negative, and stopped reaction is dry with the washing of gained peptide resin.With being lytic reagent, 0 ℃ was reacted 90 minutes with 5ml meta-cresol-TMBS-TFA, and the filtering resin is removed TFA with the filtrate rotary evaporation, and add anhydrous diethyl ether and be settled out solid, filter collection solid, soluble in water, frost drying gets white sticky solid 40mg.The RP-HPLC purifying gets target product 12.4mg.

MPEG ₂₀₀₀-NHCOCH ₂CH ₂CO-TP5 analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 2731, and it is about 44 that adjacent two peak molecular weight differ, and has the typical structure feature of polyoxyethylene glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.04 (1); Val, 0.88 (1); Lys, 1.01 (1); Tyr, 0.95 (1); Arg, 1.00 (1).

Implement 2 mPEG ₅₀₀₀-NHCOCH ₂CH ₂CO-TP5's is synthetic

Weighing m PEG ₂₀₀₀-OH 50.0g (10mmol) places the 250ml reaction flask, adds 50mlCH ₂Cl ₂, add 7.5ml Et again after the solid dissolving ₃N (50mmmol) and 9.5g Ts-Cl (50mmol), the stirring at room reaction.After the TLC monitoring reaction was complete, rotary evaporation removed and desolvates, and added the 100ml anhydrous diethyl ether and was settled out solid, got 33.5gmPEG ₅₀₀₀-OTs, yield 67%

With 30.0g mPEG ₅₀₀₀-OTs (6mmol) is dissolved in 30ml DME, adds 3.33g (18mmol) potassium phthalimide, and 120 ℃ were reacted 4 hours.The pressure reducing and steaming solvent is dissolved in the 50ml dehydrated alcohol with resistates, adds the 4.0ml hydrazine hydrate, back flow reaction 4 hours.Rotary evaporation removes and desolvates, and resistates is dissolved in CH ₂Cl ₂, be settled out solid with anhydrous diethyl ether, again with dehydrated alcohol-ether recrystallization, get 22.5gmPEG ₂₀₀₀-NH ₂, yield 75%.

With 10.0gmPEG ₂₀₀₀-NH ₂Be dissolved among the 20ml DMF, add the 0.3g succinyl oxide, 40 ℃ were reacted 0.5 hour, and the pressure reducing and steaming solvent adds anhydrous diethyl ether and is settled out solid, gets 9.8gmPEG ₅₀₀₀-NHCOCH ₂CH ₂COOH, yield 96%.

100mg Wang resin (0.05mmol) is a solid phase carrier, Fmoc-Arg (Mtr), Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Val-OH, Fmoc-Tyr (tBu) is a raw material, DCC-HOBT makes condensing agent, according to the aminoacid sequence of TP5,, add mPEG by the synthetic H-Arg (Mtr) of the solid-phase peptide synthesis of standard-Lys (Boc)-Asp (OtBu)-Val-Tyr (tBu)-Wang resin ₅₀₀₀-NHCOCH ₂CH ₂COOH (0.1mmol), HBTU (0.1mmol), NMM (0.15mmol) reacted after 48 hours, and ninhydrin method detects negative, and stopped reaction is dry with the washing of gained peptide resin.With being lytic reagent, 0 ℃ was reacted 90 minutes with 5ml meta-cresol-TMBS-TFA, and the filtering resin is removed TFA with the filtrate rotary evaporation, and add anhydrous diethyl ether and be settled out solid, filter collection solid, soluble in water, frost drying gets white sticky solid 65mg.The RP-HPLC purifying gets target product 23.7mg.

MPEG ₅₀₀₀-NHCOCH ₂CH ₂CO-TP5 analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 5595, and it is about 44 that adjacent two peak molecular weight differ, and has the typical structure feature of polyoxyethylene glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.07 (1); Val, 0.96 (1); Lys, 1.06 (1); Tyr, 0.85 (1); Arg, 0.84 (1).

Implement 3 Cys (mPEG ₂₀₀₀-MAL)-TP5 synthetic

With 5.0gmPEG ₂₀₀₀-NH ₂Be dissolved in the 10ml dioxane, add maleic anhydride 2.0g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent adds the 50ml anhydrous diethyl ether, and cooling is settled out solid, and filter collection solid gets 4.9g after the drying.The gained solid is dissolved in the 15ml diacetyl oxide, adds the 5.0g sodium acetate, 100 ℃ of stirring reaction 45min.The pressure reducing and steaming solvent dissolves resistates with methylene dichloride, the elimination insolubles adds proper amount of active carbon in filtrate, place 30min, the filtering gac is concentrated into filtrate dried, add anhydrous diethyl ether, be settled out solid, get light yellow solid 2.5gmPEG after filter collection, the drying ₂₀₀₀-MAL, yield 50%.

With 100mg Wang resin (0.05mmol) is solid phase carrier, Fmoc-Cys (Trt), Fmoc-Arg (Mtr)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Val-OH, Fmoc-Tyr (tBu)-OH is a raw material, DCC-HOBT makes condensing agent, according to the aminoacid sequence of TP5, by the synthetic H-Cys (Trt) of the Fmoc solid-phase peptide synthesis of standard-Arg (Mtr)-Lys (Boc)-Asp (OtBu)-Val-Tyr (tBu)-Wang resin.Make lysate with EDT-meta-cresol-TMBS-TFA, 0 ℃ was reacted 90 minutes, and the filtering resin is removed TFA with the filtrate rotary evaporation, and add anhydrous diethyl ether and be settled out white solid, filter collection solid, soluble in water, frost drying gets white dry powder 38mg.

Will be soluble in water through the Cys-TP5 behind the RP-HPLC purifying, transfer pH to 7-8 with sodium bicarbonate, add 3 normal mPEG ₂₀₀₀-MAL, room temperature reaction is with RP-HPLC monitoring reaction process and separated product.

Cys (mPEG ₂₀₀₀-MAL)-TP5 analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 2832, it is about 44 that adjacent two peak molecular weight differ, and has the typical structure feature of polyoxyethylene glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.06 (1); Val, 0.91 (1); Lys, 1.03 (1); Tyr, 0.89 (1); Arg, 1.01 (1).

Implement 4 TP5-Cys (mPEG ₂₀₀₀-MAL)-NH ₂Synthetic

With 100mg Rink resin (0.05mmol) is solid phase carrier, Fmoc-Cys (Trt), Fmoc-Arg (Mtr)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Val-OH, Fmoc-Tyr (tBu)-OH is a raw material, DCC-HOBT makes condensing agent, according to the aminoacid sequence of TP5, by the synthetic H-Arg (Mtr) of the Fmoc solid-phase peptide synthesis of standard-Lys (Boc)-Asp (OtBu)-Val-Tyr (tBu)-Cys (Trt)-Rink resin.Make lysate with EDT-meta-cresol-TMBS-TFA, 0 ℃ was reacted 90 minutes, and the filtering resin is removed TFA with the filtrate rotary evaporation, and add anhydrous diethyl ether and be settled out white solid, filter collection solid, soluble in water, frost drying gets white dry powder 31mg.

Will be through the TP5-Cys-NH behind the RP-HPLC purifying ₂Soluble in water, transfer pH to 7-8 with sodium bicarbonate, add 3 normal mPEG ₂₀₀₀-MAL, room temperature reaction is with RP-HPLC monitoring reaction process and separated product.

TP5-Cys (mPEG ₂₀₀₀-MAL)-NH ₂Analyze through MALDI-TOF-MS, a series of peaks are arranged near 2831, it is about 44 that adjacent two peak molecular weight differ, and has the typical structure feature of polyoxyethylene glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.09 (1); Val, 0.99 (1); Lys, 1.08 (1); Tyr, 0.84 (1); Arg, 0.83 (1).

The activity rating of the polyethylene glycol derivative of embodiment 5 TP5

1, ³The H-TdR method of mixing detects the proliferative response to mouse spleen lymphocyte

The preparation of splenocyte suspension: aseptic taking-up mouse spleen, with frosted glass plate spleen is ground, make splenocyte suspension.After the splitting erythrocyte, wash three times counting (viable cell is more than 95%).With containing 10%FBS (foetal calf serum) RPMI1640 nutrient solution splenocyte concentration is adjusted to 5 * 10 ⁶Cell/ml.

The cell suspension that in 96 orifice plates, adds 100 μ l, the sample of 50 μ l, 50 μ lConA, contrast adds the nutrient solution that 50 μ l contain 10% serum, and cumulative volume is 200 μ l.Cell is in containing 5%CO ₂37 ℃ of incubators in cultivate, cultivate to finish preceding 12 hours, every hole adds 25 μ l ³H-thymidylic acid (2 * 10 ⁴Bq).Continue to be cultured to experiment and finish, then that culture plate is frozen in-20 ℃ of refrigerators; Cell with freeze thawing during mensuration is collected on the glass fibre membrane with the cell harvesting instrument, add on Beta numeration instrument (MicroBetaTrilux, PerkinElmer Life Sciences), read behind the scintillation solution mix cell DNA [ ³H]-the thymidine amount, represent the situation of cell proliferation with the cpm value.

Table 1 ³The H-TdR method of mixing detects the proliferative response result to mouse spleen lymphocyte

The sample name	Concentration (μ g/ml)	T lymphocyte Con A stimulation of CP M mean value	??SD	The comprehensive activity assessment enhancing/inhibition of T cell proliferation per-cent
The sample name	Concentration (μ g/ml)	T lymphocyte Con A stimulation of CP M mean value	??SD		Contrast	??-	?31578	??2484	???-
					Contrast	??-	?31578	??2484	???-
					??W01	??1	?35379	??3248	???12％
	??10	?32998	??2067	???4％	??W01	??1	?35379	??3248	???12％
	??10	?32998	??2067	???4％		??100	?30806	??264	???-2％
						??100	?30806	??264	???-2％
					??W02	??1	?34216	??157	???8％
	??10	?35029	??507	???11％	??W02	??1	?34216	??157	???8％
	??10	?35029	??507	???11％		??100	?35402	??5027	???12％
						??100	?35402	??5027	???12％
					??W03	??1	?35444	??1324	???12％

????10	????34007	??978	????8％
????10	????34007	??978	????8％		????100	????32351	??2369	????2％
					????100	????32351	??2369	????2％
				??W04	????1	????37083	??2457	????17％
????10	????35123	??161	????11％	??W04	????1	????37083	??2457	????17％
????10	????35123	??161	????11％		????100	????35577	??3046	????13％
					????100	????35577	??3046	????13％
				??W05	????1	????33612	??2421	????6％
????10	????32725	??870	????4％	??W05	????1	????33612	??2421	????6％
????10	????32725	??870	????4％		????100	????34457	??2481	????9％
					????100	????34457	??2481	????9％
				??W06	????1	????37989	??1717	????20％
????10	????37984	??1159	????20％	??W06	????1	????37989	??1717	????20％
????10	????37984	??1159	????20％		????100	????37806	??605	????20％
					????100	????37806	??605	????20％
				??W07	????1	????34176	??1893	????8％
????10	????35283	??2644	????12％	??W07	????1	????34176	??1893	????8％
????10	????35283	??2644	????12％		????100	????33816	??2892	????7％

2, inducer T lymphocyte carefully produces the mensuration (ELISA method) of IL-2 and IFN-γ

Get mouse boosting cell, transfer to 5 * 10 ⁶/ ml adds in 24 orifice plates 1ml/ hole; Each concentration of each sample is added on 24 orifice plates respectively, the 0.5ml/ hole; Add stimulator ConA (20 μ g/ml) 0.5ml/ hole.370 ℃, CO ₂Cultivated 24 hours in the incubator, centrifugal, receive culture supernatant.

Antibody sandwich: with antibody dilution, 96 hole enzyme plates add the antibody (Capture Antibody) after the dilution, 50 μ l/ holes with the diluted liquid of bag.Shrouding spends the night in 4 ℃.

Sealing: remove antibody-solutions, wash 3 times, add confining liquid (PBS/10%FBS), 100 μ l/ holes with washing lotion (PBS/Tween solution).Room temperature 1 hour.

Standard substance and sample: deblocking liquid, wash 3 times with washing lotion (PBS/Tween solution), add standard substance and sample, 50 μ l/ holes.Room temperature 2 hours.

Detect antibody and enzyme: remove standard substance and sample, wash 3 times, add detection antibody (Detection Antibody) and enzyme (HRP) after diluting, 50 μ l/ holes, room temperature 1 hour with washing lotion (PBS/Tween solution).

The substrate colour developing: remove to detect antibody and enzyme, wash 3 times with washing lotion (PBS/Tween solution), adding is dissolved in citric acid, the substrate of hydrogen peroxide (TMB), 50 μ l/ holes.Room temperature, lucifuge, 30 minutes.After the colour developing, add stop buffer (2N H ₂SO ₄), 25 μ l/ holes.

OD value: put in the microplate reader,, measure the OD value in 450nm, correction 570nm place.

Table 2 sample is to the result that influences of ConA inducer T lymphocyte cytokine generation

Sample	Concentration (μ g/ml)	??IFN-γ(pg/ml)	??IL-2(pg/ml)
		??IFN-γ(pg/ml)	??IL-2(pg/ml)	??Mean????SD	??Mean????SD
		Contrast	??-	??Mean????SD	??Mean????SD	??4122????77	??2920????24
??W01	??1 ??10 ??100	Contrast	??-	??3968????34 ??5660????651 ??4565????123	??3425????12 ??3487????259 ??3639????168	??4122????77	??2920????24
??W01	??1 ??10 ??100	??W02	??1 ??10 ??100	??3968????34 ??5660????651 ??4565????123	??3425????12 ??3487????259 ??3639????168	??4742????221 ??4952????33 ??3787????142	??3504????49 ??3421????166 ??3525????105
??W03	??1 ??10 ??100	??W02	??1 ??10 ??100	??4764????316 ??4346????257 ??4671????203	??3309????116 ??3257????535 ??3090????84	??4742????221 ??4952????33 ??3787????142	??3504????49 ??3421????166 ??3525????105
??W03	??1 ??10 ??100	??W04	??1 ??10 ??100	??4764????316 ??4346????257 ??4671????203	??3309????116 ??3257????535 ??3090????84	??4329????107 ??4760????762 ??3990????88	??3231????18 ??3356????37 ??3141????97
??W05	??1 ??10 ??100	??W04	??1 ??10 ??100	??4590????100 ??3660????255 ??4658????73	??3039????48 ??3133????60 ??3399????12	??4329????107 ??4760????762 ??3990????88	??3231????18 ??3356????37 ??3141????97
??W05	??1 ??10 ??100	??W06	??1 ??10 ??100	??4590????100 ??3660????255 ??4658????73	??3039????48 ??3133????60 ??3399????12	??4006????42 ??4203????203 ??4586????1448	??2958????78 ??2916????66 ??2778????12
??W07	??1 ??10 ??100	??W06	??1 ??10 ??100	??3938????194 ??4317????841 ??4040????400	??2790????30 ??2841????6 ??2891????6	??4006????42 ??4203????203 ??4586????1448	??2958????78 ??2916????66 ??2778????12

The compound structure of W01-W07 representative is respectively:

W01：TP5

W02：mPEG ₂₀₀₀-NHCOCH ₂CH ₂CO-TP5

W03：mPEG ₅₀₀₀-NHCOCH ₂CH ₂CO-TP5

W04：Cys(mPEG ₂₀₀₀-MAL)-TP5

W05；Cys(mPEG ₅₀₀₀-MAL)-TP5

W06：TP5-Cys(mPEG ₂₀₀₀-MAL)-NH ₂

W07：TP5-Cys(mPEG ₅₀₀₀-MAL)-NH ₂

The result shows: W01, W02, W03, W04, W05, W07 to the influence of ConA inductive T lymphproliferation response a little less than, W06 has enhanced activity (table 1) preferably.Produce in the cytokine experiment at ConA activation-inducing T cell, W01, W02, W03, W04, W05, W06 has certain promotion to the generation of T cell IFN-γ; W01, W02, W03, W04, the W05 sample has certain promotion (table 2) to the IL-2 that the T cell produces.

Claims

1. The pegylated derivatives of thymopentin, characterized in that the N-terminal amino, C-terminal carboxyl or side chain amino or carboxyl of thymopentin is coupled with the polyethylene glycol that introduces amino or carboxyl or PEGylated amino acids; or introduce cysteine at the N-terminal or C-terminal of thymopentin, coupled with maleamide-based polyethylene glycol, vinyl polyethylene glycol or iodoacetylpolyethylene glycol ethylene glycol.

2. The PEGylated derivative according to claim 1, characterized in that it is a compound having the structure of formula (I), including a compound in which the amino group at any position in TP5 is covalently modified with PEG, wherein the amino group includes N-terminal amino group and lysine side chain amino group:

[PEG-X-(CH ₂ )mCO-NH]z-TP5 (I)

Where PEG represents: RO(CH ₂ CH ₂ O)n-CH ₂ CH ₂ , R=H or CH ₃ , n=5-1000; X=O, NH or NHCO; m=0-6; z=1- 2.

3. The PEGylated derivative according to claim 1, characterized in that it is a compound having the structure of formula (II), including a compound in which the carboxyl group at any position in TP5 is covalently modified with PEG, wherein the carboxyl group includes C-terminal carboxyl group and side chain carboxyl group of aspartic acid:

TP5-[CO-Y-PEG]z (II)

Wherein PEG is as defined in claim 2; Y=O or NH; z=1-2.

4. The PEGylated derivative according to claim 1, characterized in that it is a compound having the structure of formula (III), that is, the N-terminus of TP5 is introduced into cysteine and then treated with PEG-MAL, PEG-VS or PEG-IODO modified compounds:

PEG-M-Cys-TP5 (III)

in

or or

The definition of PEG is the same as that in claim 2; Cys is cysteine, which is covalently linked to the M group through the side chain sulfur atom.

5. The PEGylated derivative according to claim 1, characterized in that it is a compound with the structure of formula (IV), that is, the C-terminal of TP5 introduces cysteine and then undergoes PEG-MAL, PEG-VS or PEG-IODO Modified Compounds

TP5-Cys-M-PEG (IV)

The definition of PEG is the same as in claim 2; the definition of Cys and M is the same as in claim 4.

6. The PEGylated derivative according to claim 1, characterized in that it has any one of the following structures:

_mPEG2000 - _NHCOCH2CH2CO _- TP5;

_mPEG5000 - _NHCOCH2CH2CO _- TP5;

Cys( _mPEG2000 -MAL)-TP5;

Cys( _mPEG5000 -MAL)-TP5;

TP5-Cys( _mPEG2000 -MAL) _-NH2 ;

TP5-Cys( _mPEG5000 -MAL) _-NH2 .

7. A pharmaceutical composition, characterized in that it contains the PEGylated derivative as claimed in claim 1 and a medicinal dressing.

8. The PEGylated derivatives as claimed in claim 1 or as components of pharmaceutical compositions can be used in the preparation of rheumatoid arthritis, allergic dermatitis, tumors, certain viral infection diseases, etc. and immunodeficiency, immune function Use of medicines for the treatment or prevention of related diseases.