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CN100355786C - Polyethylene glycol-modified thymus peptide 1 derivatives - Google Patents

Polyethylene glycol-modified thymus peptide 1 derivatives Download PDF

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CN100355786C
CN100355786C CNB200410037523XA CN200410037523A CN100355786C CN 100355786 C CN100355786 C CN 100355786C CN B200410037523X A CNB200410037523X A CN B200410037523XA CN 200410037523 A CN200410037523 A CN 200410037523A CN 100355786 C CN100355786 C CN 100355786C
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derivative
zadaxin
glu
ser
polyoxyethylene glycol
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CN1690079A (en
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钟慧娟
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Jiangsu Best Pharmaceutical Co ltd
Lianyungang Hongchuang Pharmaceutical Co ltd
Jiangsu Hansoh Pharmaceutical Group Co Ltd
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Jiangsu Hansoh Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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Abstract

The present invention relates to a long-acting thymic peptide-1 derivative, a preparation method for salts thereof suitable for medical application and an application thereof. The long-acting thymic peptide derivative is prepared from a thymic peptide derivative and polyethyleneglycol by covalent linkage, the structure of which is defined in the specification. The precursor thymic peptide-1 derivative used for polyethyleneglycol modification can be prepared by a chemical synthesis method and be prepared by a recombinant DNA method.

Description

Polyethyleneglycol modified thymus peptide 1 derivative
Technical field
The present invention relates to the preparation and the application of long-acting thymulin 1 derivative.Be specifically related to polyethyleneglycol modified thymus peptide 1 derivative.
Technical background
Thymus gland is intravital important immune organ, grows and keep in the immune normal function at lymphsystem to play an important role.Especially anti-infective, antitumor, autoimmune disorder and organ transplantation etc. has special meaning on.Along with organismic age is aging, thymus gland gradually atrophy is degenerated, and tumour infects, and sickness rate such as immunological disease increase gradually.In recent years the foreign scholar purifies Zadaxin and is several individual composition, and the biologic activity of finding thymus peptide 1 wherein is than the active high 10-1000 of Zadaxin mixture times.Thymus peptide 1 belongs to Zadaxin the 5th component, has immunoregulation effect, can advance sensitized cell generation lymphokine external, has immunoregulation effect, generates lymphokine at the external sensitized cell that can promote, as α-IFN, and γ-INF, IL-2; Strengthen the expression of cytokine high-affinity IL-2; Terminal deoxynucleotidyl transferase (TdT) activity of regulation and control bone marrow precursor and splenocyte; Strengthen the Thy1 and the Lyt1 of bone marrow precursor, 2,3, expression; Quicken the generation of NK cell, promote the NK cell activity; Can promote the reaction of mixed lymphocytes by the activity that strengthens helper cell; Apoptosis in the antagonism thymocyte ripening process.Use the result in the body and show that also it is significant in T cell development and function weight; Can promote the expression of lymphocytic emiocytosis IL-2 and IL-2; Strengthen host's anti-infection ability; Promote virus sweep; Raise immunity; Vital role such as anti-oxidant, anticancer growth are arranged; Often be applied to treat tumour clinically, antiviral and immune deficiency patient's assisting therapy, and can be used as the vaccine toughener and use.(Chien, R-N et al, Hepatology, 1998 such as Chien; 27:1383-138) treat chronic hepatitis B patient with thymus peptide 1, its HBV DNA serum clearance rate and HBeAg negative conversion rate are higher than the control group of not treating with thymus peptide 1, and pathological examination finds that also treatment group inflammation and fiber generate all remarkable end of situation in control group.Also have one's own knack at Zadaxin al aspect the treatment hepatic fibrosis.Find to have only IFN that certain curative effect is arranged at present, but it exists shortcomings such as low reactivity and dose limitation, (Sherman, K.E. etc., Hepatology, 1998 such as Sherman to hepatic fibrosis; 27:1128-35) IFN and thymus peptide 1 are share treatment chronic hepatitis C, random packet, double blind control, observe the every detection index of combination group and all be better than singly with IFN group and untreated control group, wherein HCV RNA clearance rate for this reason 37.1%, organizes really 16.2% apparently higher than single with group IFN.Thymus peptide 1 also has therapeutic action to liver cancer, (Stefanini G.F. etc., Hepatol-Gastroenterology, 1998 such as Stefanini; 45:209-15) treated not 12 routine liver cancer patients down, found its survival time significant prolongation with it.
In recent years have the product of thymus peptide 1 both at home and abroad, trade(brand)name " Zadaxin ", artificial chemosynthesis does not contain other serum products such as albumin etc.Clinical application treatment hepatitis B, hepatitis C, immunodeficiency virus infection and some tumours, as melanoma, lung cancer, leukemia, phosphor shaped epithelial cell cancer, colorectal carcinomas etc., effect are comparatively remarkable.But the pharmacokinetic property of thymus peptide 1 itself is relatively poor, is excluded than weak point residence time in vivo, and its transformation period is 2 hours.The experimentation on animals proof can not be brought into play its due drug effect fully.This just needs frequent injection thymus peptide 1, and the inconvenience and the treatment cost that therefore cause treating raise, because the common patient that costs an arm and a leg is difficult to bear, therapeutic quality descends simultaneously.Studies show that body internal thymus peptide 1 is to be derived by prothoracic gland peptide 1 in the body, prothoracic gland peptide 1 has identical life activity with thymus peptide 1, and its difference just is varying in size of their molecular weight.Prothoracic gland peptide 1 its activity in vivo may be lasting.Because the bigger eliminating time of its molecular weight is longer.Therefore, one of solution to the problems described above is researched and developed long lasting thymus peptide 1 derivative exactly, prolongs the thymus peptide 1 residence time and stability in vivo with the method for chemically modified, will reach to reduce the treatment cost, improves the purpose of therapeutic quality.
By means of precedent, modify thymus peptide 1 derivative with same technology and will improve its medicine, thereby improve drug effect for character with polyethyleneglycol modified albumen success.WO 03/037272 discloses relevant method and application of modifying thymus peptide 1 itself with superpolymer.Specifically, be that about 20,000 polyoxyethylene glycol is modified one of contained five amino of thymus peptide 1 itself or modified wherein a plurality of amino simultaneously with molecular weight.(a N end is amino exactly because thymus peptide 1 has five amino; four lysine side-chain amino); adding its chemically modified sets up on these five amino bases; so having only by the chemosynthesis of complexity and protection mechanism, the selection specificity of this modifying method could realize; thereby; to its cost height that carries out scale operation, it is very little to reach business-like possibility.This need seek modification thymus peptide 1 derivative and the preparation method who is easier to prepare industrial.
Relatively find in view of above-mentioned observation with through research, by means of the single-minded chemical modification method of reaction, with the effect that the C of polyethyleneglycol modified thymus peptide 1 brings in prothoracic gland peptide 1 and thymus peptide 1 in the analogue body, understand the bioactive possibility that minimum degree ground reduces or influence thymus peptide 1 itself like this.For this reason, has only the precursor of modifying with the derivative rather than the thymus peptide 1 conduct itself of thymus peptide 1.In other words, add the tie point of modification reaction or the Methionin in its sequence, thereby replace the specificity of facilitating modification reaction with arginine at the C of thymus peptide 1 end.These preparations of modifying used precursor can be finished with any technology well known in the art, preferably with chemical process or use recombinant DNA method.
The object of the invention is to provide a class to hold and a class new compound of generation with polyethyleneglycol modified thymus peptide 1 derivative C, and their preparation method has the characteristics of the single-minded concise production process of reaction.They can be used as injection formulations or other preparation, are used for the treatment of diseases and the assisting therapy of immunologic hypofunction.
Summary of the invention
In order to overcome the deficiencies in the prior art part, the present invention relates to polyethyleneglycol modified thymus peptide 1 derivative of a kind of usefulness and pharmacologically acceptable salt thereof, this polyoxyethylene glycol is modified at the C of thymus peptide 1 derivative end.
Described polyethyleneglycol modified thymus peptide 1 derivative and pharmaceutical salts thereof is characterized in that the C end of thymus peptide 1 derivative can be connected an end or two end of polyoxyethylene glycol.The C of thymus peptide 1 derivative end can be connected an end of polyoxyethylene glycol in addition.The molecular weight ranges of polyoxyethylene glycol is 5,000-80, and 000, be preferably 8,000-60,000, more preferably 10,000-50,000, most preferred is 20,000-40,000.
Polyethyleneglycol modified thymus peptide 1 derivative of the present invention and pharmaceutical salts thereof is characterized in that having the following formula structure:
A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-B-Asp-Leu-C-Glu-D-E-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
Wherein A is H or Ac;
B, C, D, E are Lys or Arg,
X is selected from (Gly) n, (Gly-Ser) n, and (Gly-Gly-Ser) n, (Ser-Gly-Gly) n,
n＝0-10
Y is Cys or high Cys or Lys or Arg or His, and Y is polyethyleneglycol modified residue;
Z is OH or NH2.
Shown in structure be the described sequence of SEQ ID NO:1-12.
Shown polyethyleneglycol modified thymus peptide 1 derivative and pharmaceutical salts thereof, wherein
A is Ac;
B, C, D, E are Lys;
n＝0；
Y is the Cys that PEG40K modifies.
Perhaps,
A is Ac;
B, C, D, E are Arg;
n＝0；
Y is the Lys that PEG40K modifies.
The invention still further relates to the polyethyleneglycol modified thymus peptide 1 derivative and the preparation method of pharmaceutical salts thereof, comprising following steps:
Supply the precursor thymus peptide 1 derivative of modification with chemical synthesis process or gene engineering research preparation; Precursor thymus peptide 1 derivative C end is derived with polyoxyethylene glycol.
This method may further comprise the steps:
(1) preparation or acquisition (a) contain the polyoxyethylene glycol of reversal of the Michael addition acceptor and contain the thymus peptide 1 derivative that the body halfcystine is given in reversal of the Michael addition; Or (b) contain reversal of the Michael addition and give the polyoxyethylene glycol of body and the thymus peptide 1 derivative of reversal of the Michael addition acceptor;
(2) carry out reversal of the Michael addition, with its acceptor or react for the polyoxyethylene glycol and the thymus peptide 1 derivative of syntaxy, form polyethyleneglycol modified thymus peptide 1 derivative.
Reaction type is explained
In the method, precursor thymus peptide 1 derivative C holds and polyoxyethylene glycol is derived is to modify point by halfcystine, homocysteine, Methionin, arginine or the Histidine of precursor thymus peptide 1 derivative C end as the polyoxyethylene glycol chemistry, derives with polyoxyethylene glycol.
Form and covalently boundly should be selected from following method by reacting single-minded modification reaction:
＜1〉has polyoxyethylene glycol or the thymus peptide 1 derivative reaction that forms asymmetric cystine linkage ability;
＜2〉polyethyleneglycol derivative with activated carboxylic and the Zadaxin that contains C end Methionin or Histidine
The reaction of 1 derivative covalent attachment;
＜3〉polyethyleneglycol derivative with aldehyde radical passes through reductive amination process with the thymus peptide 1 derivative that contains C end Methionin;
＜4〉thymus peptide 1 derivative that contains C end Methionin of the polyethyleneglycol derivative with isocyanide or isothiocyano group by with amino addition reaction, form urea or thiocarbamide and connect;
＜5〉have the carbonyl activated polyglycol and contain C end Methionin or histidine reaction and form the ammonia ester and be connected.
＜6〉polyethyleneglycol derivative with 2-ketone group-phenylacetic aldehyde reacts with the thymus peptide 1 derivative that contains the C terminal arginine;
＜7〉have the thymus peptide 1 derivative that contains sulfydryl with contain easy leaving group because of polyoxyethylene glycol generation nucleophilic substitution reaction.
The row reversal of the Michael addition is to contain the polyoxyethylene glycol of activatory cystine linkage and the thymus peptide 1 derivative reaction that contains halfcystine.
Thymus peptide 1 derivative that described confession is polyethyleneglycol modified and pharmaceutical salts thereof can be used for treating with the immune system thing replys relevant disease, the immune system thing is replied relevant disease comprise influenza, chronic hepatitis, immunologic hypofunction, tumour virus infects.
Reaction type is explained
Figure C20041003752300111
Figure C20041003752300121
In other words, content of the present invention provides polyethyleneglycol modified thymus peptide 1 derivative of a kind of usefulness and pharmacologically acceptable salt thereof, and this polyoxyethylene glycol is modified at the C of thymus peptide 1 derivative end, preferably has the following formula structure:
5 10
A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser
15 20
-Glu-Ile-Thr-Thr-B-Asp-Leu-C-Glu-D-E-Glu
25 30
-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
Wherein A is H or Ac;
B, C, D, E are Lys or Arg,
X is selected from (Gly) n, (Gly-Ser) n, and (Gly-Gly-Ser) n, (Ser-Gly-Gly) n,
n＝0-10
Y is Cys or high Cys or Lys or Arg or His, and Y is polyethyleneglycol modified residue;
Z is OH or NH2,
If Y is Cys or high Cys then polyoxyethylene glycol is with thioether bond or cystine linkage and covalent attachment; If Y is Lys, then polyoxyethylene glycol is with amido linkage or secondary amine and covalent attachment; If Y is His, then polyoxyethylene glycol is to form the acyl imidazoles and covalent attachment with the imidazoles of Histidine nitrogen surrounded by mountains; If Y is Arg, then polyoxyethylene glycol becomes heterocycle to connect by type.
Polyethyleneglycol modified thymus peptide 1 derivative and pharmacologically acceptable salt thereof are preferably the described sequence of SEQ ID NO:1-12, and best optionally amino acid or group have to give a definition:
A is Ac, B, and C, D, E are Lys, n=0, Y are the Cys that PEG40K modifies;
Or
A is Ac, B, and C, D, E are Arg, n=0, Y are the Lys that PEG40K modifies.
The molecular weight ranges of used polyoxyethylene glycol is 5 among the present invention, 000-80,000.Be 8 wherein with molecular weight ranges, 000-60,000 is good, with 10,000-50,000 is better, with 20,000-40,000 is best.
The C of thymus peptide 1 derivative end can be connected an end or two end of polyoxyethylene glycol among the present invention, is good with the modifying method that is connected an end.
Polyethyleneglycol modified thymus peptide 1 derivative provided by the invention is an amphoteric substance, and one of ordinary skill in the art utilize known technology can use this area acidity or basic cpd commonly used to react salify with it.
Usually the acid of the formation acid salt that adopts is: hydrochloric acid, and Hydrogen bromide, hydroiodic acid HI, carbonic acid, phosphoric acid, tosic acid, methylsulfonic acid, benzoic acid, to bromophenyl carbonic acid, carbonic acid, succsinic acid, lemon acid, phenylformic acid, acetate, gifblaar poison etc.The example of this class salt comprises vitriol, pyrosulphate, hydrosulfate, sulphite, hydrosulphite, phosphoric acid salt, hydrophosphate, dihydrogen phosphate, metaphosphate, pyrophosphate salt, hydrochloride, bromide, iodide, acetate, trifluoroacetate, propionic salt, caprate, octylate, acrylate, formate, isobutyrate, caproate, enanthate, propiolate, benzoic acid salt, malonate, succinate, suberate, sebacate, fumarate, maleate, butine-1,4-diacid salt, acetylene-1,6-diacid salt, benzoate, chloro benzoate, tolyl acid salt, dinitro-benzoate, hydroxy benzoate, methoxybenzoic acid salt, benzene caproate, phenpropionate, benzenebutanoic acid salt, Citrate trianion, lactic acid salt, r-hydroxybutyric acid salt, glycol hydrochlorate, tartrate, mesylate, propanesulfonic acid salt, naphthalene-1-sulfonate, naphthalene-2-sulfonic acid salt, mandelate etc., preferred acid salt is polyethyleneglycol modified thymus peptide 1 derivative and hydrochloride, Hydrogen bromide, acetate, the salt that trifluoroacetic acid, especially acetate form.
Alkaline matter also can form salt with polyethyleneglycol modified thymus peptide 1 reaction, and these alkaline matters comprise ammonium, the oxyhydroxide of basic metal or alkaline-earth metal, and carbonate, supercarbonate etc.Sodium hydroxide is typically arranged, potassium hydroxide, ammonium hydroxide, yellow soda ash, salt of wormwood etc.
The preparation method of thymus peptide 1 derivative of the present invention can use any method well known in the art to be prepared, and this preparation method is preferably solid phase and liquid phase chemical synthesis method.Solid phase method comprises the amino acid that uses Fmoc or Boc protection, carries out the synthetic of aminoacid sequence with polypeptide synthesis or manual synthesis method, again through excision, through high performance liquid phase (HPLC) separation and purification, freeze-drying.The gained polypeptide is to carry out polyethyleneglycol modified intermediate precursor.Thymus peptide 1 derivative of the present invention preferably has the aminoacid sequence of SEQ ID NO:1-24.
The also available gene engineering research preparation of thymus peptide 1 derivative of the present invention, its step comprises;
A. press the aminoacid sequence synthetic gene fragment of thymus peptide 1 derivative;
B. gene fragment is through connecting, and plasmid construction is cultivated by solid, transforms, and the clone obtains bacterial strain;
C. bacterial strain is through fermentation, broken wall, and extracting obtains look and contains body;
D. look is contained the body cracking, separate crude product, give high performance liquid phase instrument (HPLC) separation and purification, after the freeze-drying gained is to carry out polyethyleneglycol modified intermediate precursor.
The polyethyleneglycol modified thymus peptide 1 derivative preparation method of usefulness of the present invention, comprising:
1, preparation or acquisition (a) contain the polyoxyethylene glycol of reversal of the Michael addition acceptor and contain the thymus peptide 1 derivative that the body halfcystine is given in reversal of the Michael addition; Or (b) contain reversal of the Michael addition and give the polyoxyethylene glycol of body and the thymus peptide 1 derivative of reversal of the Michael addition acceptor;
2, carry out reversal of the Michael addition, with its acceptor or the polyoxyethylene glycol and the Zadaxin of giving syntaxy
1 derivative reacts, and forms polyethyleneglycol modified thymus peptide 1 derivative, as the polyoxyethylene glycol that contains maleimide derivative forms product with containing the thymus peptide 1 derivative addition reaction of half deamination acid.
Wherein this method preferably is prepared by following steps:
(1) have polyoxyethylene glycol or the thymus peptide 1 derivative that forms asymmetric cystine linkage ability, as the polyoxyethylene glycol that contains the activatory cystine linkage forms product with the thymus peptide 1 derivative reaction that contains halfcystine;
(2) polyethyleneglycol derivative with activated carboxylic reacts the formation amido linkage and covalent attachment formation product, inner contained four the Methionins application arginine replacement of thymus peptide 1 this moment with the thymus peptide 1 derivative that contains C end Methionin or Histidine;
(3) polyethyleneglycol derivative with aldehyde radical holds the Zadaxin al derivative of Methionin to form product by reductive amination process with containing C, and four inner contained Methionins of thymus peptide 1 this moment are replaced because of arginine.
(4) polyethyleneglycol derivative with isocyanide or isothiocyano group contains the Zadaxin of C end Methionin
1 derivative is by forming product with amino addition, and four Methionins of thymus peptide 1 inside should be smart ammonia and replaced this moment;
(5) have the polyethyleneglycol derivative of 2-ketone group-phenylacetic aldehyde and the product of the thymus peptide 1 derivative reaction formation that contains the C terminal arginine;
(6) have the thymus peptide 1 derivative that contains sulfydryl and easy leaving group because of (as I, Br, the product of polyoxyethylene glycol generation nucleophilic substitution reaction formation Cl).
Compound of the present invention is with polyethyleneglycol modified thymus peptide 1 derivative, also is included as to carry out polyethyleneglycol modified and new intermediate that produce, is to be applicable to the various diseases that treatment is replied the immune system thing and to refer to immune system thing seized with terror.Enhancing immunity compound of the present invention can be used for forfeiture immunity and immunosuppressant patient's recombinant immune function, and can be used for the treatment of immune deficiency disorder, as influenza, chronic hepatitis, immunologic hypofunction, diseases such as tumour virus infection, particularly for the treatment tumour, the patient of virus infection can make assisting therapy.
The invention provides the new polyethyleneglycol modified thymus peptide 1 derivative of a class, utilize the C of polyethyleneglycol modified thymus peptide 1 derivative to bring in simulation prothoracic gland peptide 1.Experiment showed, that through drug effect medicine generation polyethyleneglycol modified thymus peptide 1 derivative has the drug action of thymus peptide 1 fully.If thymus peptide 1 has only nearly 2 hours with the transformation period in the subcutaneous syringe body, but the transformation period prolongs so far 2-3 days in vivo with the polyethyleneglycol modified thymus peptide 1 derivative for preparing among the present invention.Its consumption of making the immunostimulant assisting therapy can be measured with conventional dose titration method, and its consumption can be defined as in the scope in 1-100mg/ kg body weight/week, wherein with 5-50mg/ kg body weight/Zhou Weijia, with 20mg/ kg body weight/week be optimum.Can be with polyethyleneglycol modified thymus peptide 1 derivative and use with suitable medicinal fluid such as water system injection.Its medicine has obtained very big improvement for character, thereby has really produced long-acting thymulin 1 derivative.
Relatively find with the thymus peptide 1 derivative that the N end is polyethyleneglycol modified, polyethyleneglycol modified thymus peptide 1 derivative and the preparation method of C end provided by the present invention is easy to preparation, overcome the polyethyleneglycol modified thymus peptide 1 derivative of N end is difficult for extensive industry production, is difficult to obtain in industrial production problem, and therapeutic activity is stronger.
Description of drawings
Fig. 1 is for being subjected to test product standard ELISA calibration curve.
Fig. 2 is macaque sc PEG-TA1 (64 a μ g.kg-1) back serum antigen concentration-time curve.
Embodiment
Implementing below is to further elaboration of the present invention, rather than the restriction to inventing.
Embodiment one:
The preparation of Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-A sn-Glu-Cys-PEG40K
Step 1: the preparation of solid state chemistry synthesis method is for polyethyleneglycol modified precursor---C end halfcystine thymus peptide 1 derivative:
(1) amino acid monomer that is adopted
Fmoc-Ala-OH Fmoc-Lys(Boc)-OH
Fmoc-Asn(Trt)-OH Fmoc-Ser(tBu)-OH
Fmoc-Asp(OtBu)-OH Fmoc-Thr(tBu)-OH
Fmoc-Cys(Trt)-OH Fmoc-Val-OH
Fmoc-Glu(OtBu)-OH Fmoc-Ile-OH
Fmoc-Leu-OH
Abbreviation expression in the following formula:
Fmoc:9-fluorenylmethyloxycarbonyl this (9-fluorenylmethoxycarbonyl)
Boc: tertbutyloxycarbonyl (tert-butyloxycarbonyl)
Trt: trityl (trityl)
OtBu: the tertiary butyl
TBu: the tertiary butyl (tert-butyl)
(2) synthetic equipment and the reagent that is adopted
Instrument: the synthetic employing manual method of peptide sequence is carried out, and reacting appliance is autocracy.
Reagent: N, dinethylformamide (DMF), methylene dichloride (DCM), hexahydropyridine, Virahol, methyl alcohol, di-isopropyl, carbodiimide (N, N-Diisopropylcarbodimide), I-hydroxybenzotriazole (HOBt)
(3) operation
Be equipped with in the synthetic flask of peptide that fritted glass filter and neck be equipped with mechanical stirrer in the bottom and add king's resin (Fmoc-Cys (Trt)-OH-Wang resin) 0.6mMol/g resin that 10g is connected to Fmoc-Cys (Trt)-OH, 6mmol).Use 150ml N, dinethylformamide (DMF) washs this resin.The DMF solution (300ml) of the hexahydropyridine with 20% divides secondary, and each came process resin in 10 minutes at every turn, to remove amido protecting group Fmoc.Divide washing resin three times with DMF 450ml again, drain.To be dissolved in 15mmol Fmoc-Asn (the Trt)-OH (8.95g) among the 150ml DMF; 15mmol I-hydroxybenzotriazole water and thing (2.3g) and 15mmol N, N-sec.-propyl carbodiimide (2.34ml) formed Fmoc-Asn (Trt)-Cys (Trt) king resin in two hours with the resin reaction of having removed the amido protecting gene.It is negative that ninhydrin reaction should show.Then, proceed solid phase synthesis according to the following steps.Wherein, a seed amino acid is linked on the peptide chain that increases on the resin (explanation is arranged in addition) according to this.Except, the each washing with 20 volume of solvent or reagent.Unless otherwise indicated, all amino acid whose derivatives are the L configuration.
1) with the 20% hexahydropyridine solution that is dissolved in DMF said resin is washed.
2) with stirring 30 minutes in the 20% hexahydropyridine solution that is dissolved in DMF;
3) with DMF washing three times;
4) respectively be the Fmoc-Glu of 15mmol (OtBu)-OH among the DMF with being dissolved in, I-hydroxybenzotriazole water and thing and N, N-di-isopropyl carbodiimide stirring 120 minutes;
5) use the washed with isopropyl alcohol secondary;
6) with DMF washing three times;
7) color reaction of test triketohydrindene hydrate, positive in this way, repeating step 4-6; As feminine gender, then carry out next synthesis cycle.
Repeat synthesis cycle with following Fmoc-amino acid and in each circulation step 7 with corresponding amino acid according to this;
Fmoc-Ala-OH， Fmoc-Glu(OtBu)-OH， Fmoc-Glu(OtBu)-Ort，
Fmoc-Val-OH， Fmoc-Val-OH， Fmoc-Glu(OtBu)，
Fmoc-Lys(Boc)-OH， Fmoc-Lys(Boc)-OH， Fmoc-Glu(OtBu)-OH，
Fmoc-Lys(Boc)-OH， Fmoc-Leu-OH， Fmoc-Asp(OtBu)-OH，
Fmoc-Lys(Boc)-OH， Fmoc-Thr(tBu)-OH， Fmoc-Thr(tBu)-OH，
Fmoc-Ile-OH， Fmoc-Glu(OtBu)-OH， Fmoc-Ser(tBu)-OH，
Fmoc-Ser(tBu)-OH， Fmoc-Thr(tBu)-OH， Fmoc-Asp(OtBu)-OH，
Fmoc-Val-OH， Fmoc-Ala-OH， Fmoc-Ala-OH，
Fmoc-Asp(O+Bu)-OH， Fmoc-Ser(+Bu)-OH，
Thereby at last with finishing whole synthesis cycle after the acetate acetylize.Obtain the thymus peptide 1 king resin that shielded C end halfcystine has been revised like this.
The heavy 40g of Ac-Ser (tBu)-Asp (OtBu)-Ala-Ala-Val-Asp (OtBu)-Thr (tBu)-Ser (tBu)-Ser (tBu)-Glu (OtBu)-Ile-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asn (Trt)-Cys (Trt)-Wang resin.
Inferior peptide resin (5.0g) that obtains with contain 2% thioanisole, 2% methyl alcohol, 4% tri isopropyl silane, 2% dimercaptoethane 50ml trifluoroacetic acid mixes stirring 2 hours.After removing trifluoroacetic acid, go out the product crude product with ether sedimentation.Crude product is dissolved in the Ammoniom-Acetate damping fluid that 100ml concentration is 0.2M.After the filtration, the stand-by preparation of supernatant liquor HPLC separates purification.
Divide flow velocity with 10ml/, detect, use Vydac at the 234nm place, C18 lean on (2.2 * 25cm, 10u).With linear gradient 0.5% acetonitrile/1 minute wash-out (buffer A: 0.01% trifluoroacetic acid aqueous solution; Buffer B: 0.01% trifluoroacetic acid acetonitrile solution).The composition that will contain product merges, and it is 99% product 0.4g that freeze-drying gets purity.With liquid matter (LC-MS) coupling molecular weight is measured and to be shown having correct molecular weight (M+2H) 2+=1606, (M+H) +=3210.(molecular weight of calculating is=3210, referring to Fig. 2).After this peptide acid hydrolysis, hydrolysising condition is: use 6N HCl 110 ℃ of hydrolysis 24 hours.Carry out amino acid analysis and show that this peptide has the amino acid ratio of expectation.
Step 2: polypeptide precursor and polyoxyethylene glycol reaction, the thymus peptide 1 derivative of synthetic modification:
The methoxy poly (ethylene glycol) (molecular weight is 40,000 Dalton) of holding halfcystine thymus peptide 1 and 500mg to revise with maleimide 50mgC is dissolved in oneself in 5ml 0.1M phosphate buffered saline buffer.React after two hours, desalt, and then isolate product with anti-phase preparation high-efficient liquid phase technique with Superdex.Condition is; Vydac C18 post (2.2 * 25cm, 10 μ); Linear gradient 0.5% acetonitrile/minute, acetonitrile and water all contain 0.01% trifluoroacetic acid; Detect: 234mm; Flow velocity: 10 ml/min.Get 490mg after the product freeze-drying.
Embodiment two:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Arg-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Lys-PEG40K
Step 1: the synthetic preparation of solid state chemistry is held Methionin 14,17,19,20 for polyethyleneglycol modified precursor C, the thymus peptide 1 derivative that arginine replaces:
The solid state chemistry synthesis method sees that embodiment 1 solid state chemistry prepares C end halfcystine thymus peptide 1 derivative.Synthetic with 10g be connected to Fmoc-Lys (Boc)-OH king's resin (the 0.6mmol/g resin, 6mmol).Synthesize according to following Fmoc amino acid and in each circulation step and repeat synthesis cycle with corresponding amino acid:
Fmoc-Asn(Tre)-OH， Fmoc-Ala-OH， Fmoc-Glu(OtBu)-OH，
Fmoc-Glu(OtBu)-Ort，
Fmoc-Val-OH， Fmoc-Val-OH， Fmoc-Glu(OtBu)，
Fmoc-Arg(Pbf)-OH， Fmoc-Arg(Pbf)-OH， Fmoc-Glu(OtBu)-OH，
Fmoc-Arg(Pbf)-OH， Fmoc-Leu-OH， Fmoc-Asp(OtBu)-OH，
Fmoc-Arg(Pbf)-OH， Fmoc-Thr(tBu)-OH， Fmoc-Thr(tBu)-OH，
Fmoc-Ile-OH， Fmoc-Glu(OtBu)-OH，Fmoc-Ser(tBu)-OH，
Fmoc-Ser(tBu)-OH， Fmoc-Thr(tBu)-OH， Fmoc-Asp(OtBu)-OH，
Fmoc-Val-OH， Fmoc-Ala-OH， Fmoc-Ala-OH，
Fmoc-Asp(O+Bu)-OH， Fmoc-Ser(+Bu)-OH，
After using the acetate acetylize at last, thereby finish whole synthesis cycle, obtain shielded polypeptide resin like this, heavy 40g.The method that the crude product polypeptide cuts from resin is similar to the method among the embodiment one.The separation purification preparation method of the crude product polypeptide of gained is also similar with the method for being given among the embodiment one.This polypeptide is hydrolyzed, and hydrolysising condition is: use 6NHCL 110 ℃ of hydrolysis 24 hours.Carry out amino acid analysis and show, this peptide has the amino acid ratio of expectation.
Polypeptide precursor and polyoxyethylene glycol form polyethyleneglycol modified thymus peptide 1 derivative, and 50mgC is held Methionin, and the thymus peptide 1 derivative that 14,17,19,20 arginine join generation is dissolved in 5ml 0.In the 1M sodium hydrogen carbonate solution.To this solution add 550mg with contain carboxyl through N-maloyl imines activated polyglycol (molecular weight 40,000Dalton).
Step 2: polypeptide precursor and polyoxyethylene glycol reaction, the thymus peptide 1 derivative of synthetic modification:
Reaction PH should be controlled between the 8-9.React and desalt with Superdex after 4 hours, and then isolate product with anti-phase preparation high-efficient liquid phase technique.Condition is: Vydac C18 post (2.2 * 25cm, 10 μ); Linear gradient 0.5% second eyeball/minute, second eyeball and water all contain 0.01% trifluoroacetic acid; Detect; 234nm; Flow velocity 10 ml/min.Draw 490mg after the product freeze-drying.
Embodiment three effect experiments
With the positive contrast of Zadaxin, whether test sample has the power of regulating effect and effect to the influence of T lymphocyte generation cytokine to determine sample to the lymphocytic immunocompetence of T.
[experimental principle]
The T lymphocyte is being subjected to mitogen concanavalin A (ConA in the lymphocyte of vitro culture, Concanavalin A) after the stimulation, can cause the T lymphocyte activation, produce that cytokine is synthetic, the variation of the cell behavior of cytokine receptor expression, cytodifferentiation and cell proliferation.The proliferative response of production of cytokines and cell is the reflection of the functional status and the cell activation of immunocyte.Detection means has characteristics such as reliability, good repeatability and simplicity, so produce and the mensuration of cell proliferative response is come judgement sample lymphocytic function effect effect is widely used to T by cytokine always.
[experiment material]
1. sample: Zadaxin, PEG sample dilute with RPMI-1640, are diluted to the experiment desired concn respectively.
2. animal: the ICR mouse is adopted in experiment, and is male, 6-8 age in week, purchases the Shanghai Experimental Animal Center in the Chinese Academy of Sciences, animal conformity certification book number: SCXK (Shanghai) 2002-0010.Animal is raised in this institute and cleans a grade Animal House after purchasing and, generally raise 3-4 days after, be used for testing.
2. reagent: ConA (Concanavalin A) is available from Sigma company.
The RPMI RPMI-1640 is the preparation of GIBCO product, contains the foetal calf serum of 10% deactivation, HEPES damping fluid 10mM, and penicillin 100IU/mL, Streptomycin sulphate 100 μ g/mL, glutamine 2mM, alpha-mercapto ethanol 50 μ M, PH 7.2.
ELISA such as cytokine IFN-, IL-2 test kit (Becton Dickinson co. production).
[experimental technique]
The preparation of splenocyte suspension: aseptic taking-up spleen, with frosted glass plate mouse 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 * 106 cells/ml.
One, IL-2 and IFN-γ Determination on content (ELISA method)
Obtain culture supernatant:
Get mouse boosting cell, transfer to 5 * 106/ml, add 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 (20ug/ml) 0.5ml/ hole.370C cultivated 24 hours in the CO2 incubator, and is centrifugal, receives culture supernatant.
Two, ELISA detection method:
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 40C.
Sealing: remove antibody-solutions, wash 3 times, add confining liquid (PBS/10%FBS), the 100ul/ hole 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 H2SO4), the 25ul/ hole.
OD value: put in the microplate reader,, measure the OD value in 450nm, correction 570nm place.
[experimental result]
Sample detects the influence that ConA inducer T lymphocyte cytokine produces:
IFN-r(pg/ml) IL-2(pg/ml)
Sample Concentration (ug/ml) Mean SD Mean SD
Contrast 4122 77 2920 24
Zadaxin 0.001 0.01 0.1 1 10 100 4177 4118 4106 4836 4832 4095 177 134 191 208 36 427 3231 3582 3434 3692 3614 3840 30 25 184 19 304 476
The PEG sample 0.001 0.01 0.1 1 10 100 4809 4603 4141 4949 3874 5467 151 234 200 367 3 73 3505 3438 3339 3214 3348 3582 296 117 134 55 293 25
[experiment conclusion]
Produce in the cytokine experiment at ConA activation-inducing T cell, the Zadaxin sample has certain promoter action when the IFN-of T cell is created in 1ug/ml and 10ug/m, and the C-thymosin2 sample is compared the worker FN-that promotes the T cell preferably and produced at a plurality of concentration units and with the Zadaxin sample.
Produce in the cytokine experiment at ConA activation-inducing T cell, 2 liang of samples of Zadaxin and C-thymosin do not have evident difference to the promoter action of the IL-2 generation of T cell.
Embodiment four medicines are for experiment
1. experiment purpose
Observation PEG sample (code name: PEG-TA1) in the intravital pharmacokinetic characteristics of macaque.
2. test materials and method
2.1 be subjected to test product
Being subjected to test product PEG-TA1 is the achromaticity and clarification injection liquid, 4 ℃ of preservations.
2.2 animal
Macaque, Military Medical Science Institute's Experimental Animal Center produce (the moving word of medical officer BDW95002 number), totally 3, female 1, male 2, body weight 3.7 ± 0.5kg.Divide cage to feed, freely drink water, give twice of fresh fruit every day with the monkey standard feed.
2.3 route of administration and dosage
Subcutaneous injection.Dosage converts according to clinical human dosage, and macaque dosage is 64 μ g.kg-1.
2.4 get blood time and specimen preparation
Before medicine, behind the medicine 0.5,1,2,4,6,8,12,24,36,48 h get blood from the hind leg vein.Get behind the blood 30 minutes with centrifugal 760g * 15min under the interior room temperature.Avoid haemolysis.Separation of serum ,-20 ℃ of preservations are to be measured.
2.5 detection method
The ELISA method is measured blood-serum P EG-TA1 concentration.Germany Immundiagnostik company produces Thymosin α 1 test kit.
2.6 the non-chamber of the estimation of pharmacokinetic parameter and the statistical inference of bioequivalence-statistics moments method estimation pharmacokinetic parameter.
3. result
3.1 typical curve
Medicine box is down " S " type curve (Fig. 1) for being subjected to test product that good reactivity is arranged in the 1.6-16000ng.mL-1 scope.Each plate difference bidding directrix curve in the experiment.Calculate EPO normalizing concentration in the unknown serum sample with the typical curve on the same plate.
3.2 Plasma Concentration
Table 1 has been listed 3 macaque sc 64 μ g.kg-1 and has been subjected to test product to go background serum antigen concentration with what the ELISA method recorded.Fig. 2 is the comparison of average serum antigen concentration-time curve.From figure and the table can see that the individual difference value is very big between animal, peak time did not wait from 4-12 hour, mean value then after administration 8h reach the peak.
Go background serum antigen concentration-time to change (ng.mL-1) behind table 1, the macaque sc PEG-TA1 (64 μ g.kg-1)
Time/h 1# 2# 3# Mean±SD RSD％
0.5 1 2 4 6 8 12 24 36 48 169 - 722 636 - 936 2163 1976 1091 926 135 237 565 880 2099 2677 2468 1716 663 463 - 1195 1034 1244 995 - 745 965 985 - 152±24 716±678 773±239 920±306 1547±780 1807±1231 1792±919 1553±525 913±223 695±328 15 95 31 33 50 68 51 34 24 47
3.3 PK parameter
As shown in table 2 according to the pharmacokinetic parameter that average Plasma Concentration-the time data is calculated.
Mean P K parameter behind table 2, the macaque sc PEG-TA1 (64 μ g.kg-1)
Parameter Unit mean±SD RSD％
AUC(0-48h) AUC(0-∝) AUC(48h-∝) AUC(48h-∝)％ MRT CL/F VSS t1/2 Kel ng.h.mL-1 ng.h.mL-1 ng.U.h.mL-1 ％ h mL.h-1.kg-1 mL.kg-1 h - 60221±16896 80742±32324 20521±18277 25.4±17.85 9.2±0.7 6.6±2.8 61±27 20±6 0.03385±0.011 28 40 89 70 8 43 44 31 34
4. conclusion
TA1 after PEGization modification peak time generally postpones, and the terminal phase transformation period calculated value of eliminating is long than the unmodified TA1 original shape medicine of bibliographical information significantly also.
Sequence table
SEQ ID NO： 1 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 2 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 3 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 4 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Arg-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 5 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 6 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Arg-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 7 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Lys-Glu-Arg-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 8 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Lys-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO： 9 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Arg-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO：10 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO：11 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO：12 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Arg-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
SEQ ID NO：13 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：14 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：15 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：16 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Arg-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：17 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Lys-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：18 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Lys-Glu-Arg-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：19 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Arg-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：20 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Arg-Glu-Arg-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：21 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Lau-Arg-Glu-Lys-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：22 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Arg-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：23 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Lys-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Z
SEQ ID NO：24 A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Arg-Asp-Leu-Arg-Glu-Arg-Arg-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-His-Z
A：H，Ac，RCO-
X：(Gly)n，(Gly-Ser)n，(Gly-Gly-Ser)n，(Ser-Gly-Gly)n
Y:Lys, Cys, homoCys, deutero-Lys
Z:OH or NH2

Claims (17)

1, a kind of through polyethyleneglycol modified Zadaxin l derivative and pharmacologically acceptable salt thereof, this polyoxyethylene glycol in polyethyleneglycol modified Zadaxin l derivative is modified at the C of Zadaxin l derivative end, and wherein, the molecular weight ranges of polyoxyethylene glycol is from greater than 5,000 to being less than or equal to 80,000.
2, polyethyleneglycol modified Zadaxin l derivative and pharmaceutical salts thereof as claimed in claim 1 is characterized in that the C end of Zadaxin l derivative can be connected an end or two end of polyoxyethylene glycol.
3, polyethyleneglycol modified Zadaxin l derivative and pharmaceutical salts thereof as claimed in claim 2 is characterized in that the C end of Zadaxin l derivative can be connected an end of polyoxyethylene glycol.
4, as claim l described polyethyleneglycol modified Zadaxin l derivative and pharmaceutical salts thereof, the molecular weight ranges that it is characterized in that polyoxyethylene glycol is 8,000-60,000.
5, polyethyleneglycol modified Zadaxin l derivative and pharmaceutical salts thereof as claimed in claim 4, the molecular weight ranges that it is characterized in that polyoxyethylene glycol is 10,000-50,000.
6, polyethyleneglycol modified Zadaxin l derivative and pharmaceutical salts thereof as claimed in claim 5, the molecular weight ranges that it is characterized in that polyoxyethylene glycol is 20,000-40,000.
7, as claim 1-3 any described polyethyleneglycol modified a Zadaxin l derivative and a pharmaceutical salts thereof, it is characterized in that having the following formula structure:
A-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-B-Asp-Leu-C-Glu-D-E-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-X-Y-Z
Wherein A is H or Ac;
B, C, D, E are Lys or Arg,
X is selected from (Gly) n, (Gly-Ser) n, and (Gly-Gly-Ser) n, (Ser-Gly-Gly) n,
n＝0-10
Y is Cys or high Cys or Lys or Arg or His, and Y is polyethyleneglycol modified residue;
Z is OH or NH2.
8, polyethyleneglycol modified Zadaxin l derivative and pharmaceutical salts thereof as claimed in claim 7, structure shown in it is characterized in that are the described sequence of SEQ ID NO:1-12.
9, polyethyleneglycol modified Zadaxin l derivative and the pharmaceutical salts thereof shown in claim 7 is characterized in that:
A is Ac;
B, C, D, E are Lys;
n＝0；
Y is the Cys that PEG4OK modifies.
10, polyethyleneglycol modified Zadaxin l derivative and the pharmaceutical salts thereof shown in claim 7 is characterized in that:
A is Ac;
B, C, D, E are Arg;
n＝0；
Y is the Lys that PEG4OK modifies.
11, the polyethyleneglycol modified Zadaxin l derivative of the described confession of claim 1-10 and the preparation method of pharmaceutical salts thereof, comprising following steps:
Supply the precursor Zadaxin l derivative of modification with chemical synthesis process or gene engineering research preparation;
Precursor Zadaxin l derivative C end is derived with polyoxyethylene glycol.
12, preparation method according to claim 11 is characterized in that this method is following concrete steps:
(1) preparation or acquisition (a) contain the polyoxyethylene glycol of reversal of the Michael addition acceptor and contain the Zadaxin l derivative that the body halfcystine is given in reversal of the Michael addition; Or (b) contain reversal of the Michael addition and give the polyoxyethylene glycol of body and the Zadaxin l derivative of reversal of the Michael addition acceptor;
(2) carry out reversal of the Michael addition, with its acceptor or react for the polyoxyethylene glycol and the Zadaxin l derivative of syntaxy, form polyethyleneglycol modified Zadaxin l derivative.
13, according to claim 11 or 12 described preparation methods, it is characterized in that precursor Zadaxin l derivative C holds and polyoxyethylene glycol is derived is to modify point by halfcystine, homocysteine, Methionin, arginine or the Histidine of precursor Zadaxin l derivative C end as the polyoxyethylene glycol chemistry, derives with polyoxyethylene glycol.
14. preparation method according to claim 12 is characterized in that forming and covalently boundly should being selected from following method by reacting single-minded modification reaction:
＜1〉has polyoxyethylene glycol or the Zadaxin l derivatives reaction that forms asymmetric cystine linkage ability;
＜2〉polyethyleneglycol derivative with activated carboxylic reacts with the Zadaxin l derivative covalent attachment that contains C end Methionin or Histidine;
＜3〉polyethyleneglycol derivative with aldehyde radical passes through reductive amination process with the Zadaxin l derivative that contains C end Methionin;
＜4〉the Zadaxin l derivative that contains C end Methionin of the polyethyleneglycol derivative with isocyanide or isothiocyano group by with amino addition reaction, form urea or thiocarbamide and connect;
＜5〉have the carbonyl activated polyglycol and contain C end Methionin or histidine reaction and form the ammonia ester and be connected.
＜6〉polyethyleneglycol derivative with 2-ketone group-phenylacetic aldehyde and the Zadaxin l derivatives reaction that contains the C terminal arginine;
＜7〉have the Zadaxin l derivative that contains sulfydryl with contain easy leaving group because of polyoxyethylene glycol generation nucleophilic substitution reaction.
15, preparation method according to claim 13 is characterized in that the row reversal of the Michael addition is to contain the polyoxyethylene glycol of activatory cystine linkage and the Zadaxin l derivatives reaction that contains halfcystine.
16, the application in the preparation medicine according to polyethyleneglycol modified Zadaxin l derivative of the described confession of claim 1-10 and pharmaceutical salts thereof, wherein this medicine can be used for treating with the immune system thing and replys relevant disease.
17, application according to claim 16 is characterized in that to the disease that the immune system thing is replied be influenza, chronic hepatitis, and immunologic hypofunction, tumour virus infects.
CNB200410037523XA 2004-04-28 2004-04-28 Polyethylene glycol-modified thymus peptide 1 derivatives Expired - Fee Related CN100355786C (en)

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DK1891104T3 (en) * 2005-05-04 2009-11-30 Lonza Ag Solid-phase bound thmosin-alpha1 and its synthesis
CN100577685C (en) * 2005-11-10 2010-01-06 中国人民解放军军事医学科学院毒物药物研究所 Long-acting thymosin alpha1 polyethylene glycol modifier
CN102757500B (en) * 2005-11-10 2014-06-25 中国人民解放军军事医学科学院毒物药物研究所 Pegylation modifier of long-acting thymosin alpha1
CN101062950B (en) * 2006-04-25 2010-04-21 江苏豪森药业股份有限公司 Polyethylene glycol modified thymus peptide 1 derivative
US20100184653A1 (en) * 2008-07-14 2010-07-22 Jiangsu Hansen Pharmaceutical Co., Ltd. Derivates of Polyethylene Glycol Modified Thymosin Alpha 1
WO2010033227A1 (en) * 2008-09-19 2010-03-25 Nektar Therapeutics Polymer conjugates of thymosin alpha 1 peptides
MX2011003117A (en) 2008-09-19 2011-04-21 Nektar Therapeutics Polymer conjugates of therapeutic peptides.
CN102268067A (en) * 2011-06-29 2011-12-07 河北师范大学 Pegylated derivatives of short peptide having leucine-aspartic acid-valine (LDV) sequence
CN108239147B (en) * 2016-12-27 2023-11-10 江苏豪森药业集团有限公司 Process for preparing thymosin alpha1 derivatives

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