CN102190721A

CN102190721A - Long-acting growth hormone

Info

Publication number: CN102190721A
Application number: CN2010101173300A
Authority: CN
Inventors: 徐明波; 王俊玲; 杨仲璠; 吴彦卓
Original assignee: BEIJING SL LISHENG PHARMACEUTICAL Co Ltd; BEIJING SHUANGLU PHARMACEUTICAL Co Ltd
Current assignee: BEIJING SL LISHENG PHARMACEUTICAL Co Ltd; BEIJING SHUANGLU PHARMACEUTICAL Co Ltd
Priority date: 2010-03-03
Filing date: 2010-03-03
Publication date: 2011-09-21

Abstract

The invention discloses a polyethylene glycol-modified bivalent growth hormone, aiming at providing a protein medicament with low immunogenicity, high activity, long half life period, low medicine feeding frequency and stability. The polyethylene glycol-modified bivalent growth hormone is mainly used for treating growth retardation diseases. Dual ends of polyethylene glycol are activated; and two growth hormone molecules are connected to form a bivalent flexible molecule by connecting the dual ends of the polyethylene glycol with a free amine group of the protein.

Description

A kind of long lasting growth hormone

Technical field

The present invention relates to a kind of polyethyleneglycol modified pharmaceutical grade protein.Be specially the both-end activated polyglycol and modify tethelin.

Background technology

Human growth hormone (hGH) be find twentieth century twenties by people's prepituitary gland oxyphie excretory important hormone, strand, the non-saccharification hydrophilic protein formed by 191 amino-acid residues, between 53-165 and 182-189 amino acid, respectively there is a disulfide linkage to connect, relative molecular mass is about 21700 dalton, pI4.9-5.2.Make its partial hydrolysis with Chymotrypsin or trypsin treatment tethelin, activity is not lost, and the activity of visible growth hormone does not need whole molecule.Experiment confirm, 1～134 amino acid section peptide chain of human growth hormone N-end is for active essential, and one section peptide chain that C-holds may shield, and makes that tethelin is unlikely in circulation of blood to be destroyed by enzyme.Human growth hormone molecule quite stable, its activity can keep the several years under freezing condition, places the 48h no change in room temperature.Between the mammiferous tethelin of different genera tangible species specificity is arranged, have only the tethelin of primates that the people is had activity.

The mechanism of action of hGH is commonly considered as being undertaken by cAMP, with the running that can change amino acid and meta-bolites after target cell combines, induce the synthetic of some specific protein and nucleic acid.

1, the growth promoting function of human growth hormone

The most tangible effect of human growth hormone is the growth that promotes bone, cartilage and tissue.Synthesizing of its energy stimulating protein and collagen, promote tissue to amino acid whose picked-up and utilization in the recycle system.The people childhood and when adult the level of hGH there is no great difference (the conventional reference value that detects in serum or the blood plasma be: adult man＜2 μ g/L; Adult female＜10 μ g/L; Children＜20 μ g/L), but when the young stage that the life long hair is educated, body tissue is more responsive to hGH, and hGH depends on the situation of epiphyseal cartilage in growth to the growth promoting function of bone.People GH when childhood and finishing period grows to long bone of limbs promoter action is arranged, so the birth back mainly promotes the growth of trunk and backbone subsequently to growth in stature quickening before this in sexual maturing period; This moment, the growth of body length was comparatively rapid, and the growth of four limbs and backbone finishes substantially when extremely growing up.Though it is necessary that hGH belongs to the prolongation of keeping bone, be not directly effect.It is by SM in the blood or claims sulphation factor (Sulfationfactor) to realize.This is a kind of peptide hormone, and molecular weight 4000Da can promote SO ₄ ^2-, H-thymidine, H-uridine and C-leucine enter cartilage, increases collagenous tissue and protein synthesis, promotes the division of chondrocyte, liver, muscle and fibroblast.The content of SM-C and transporter thereof is regulated and control by hGH.

2, hGH promotes protein synthesis

HGH can promote that amino acid enters cell, quickens proteinic synthesizing, and reduces the discharge of urea, creatine and nitrogen, impels the running balance that keeps nitrogen in the body.In proteinic building-up process, it is synthetic that hGH can strengthen tRNA, promotes amino acid whose picked-up, and tetracycline can not be blocked this effect, and prompting hGH is not promotion cell certain new protein of interior formation but strengthens amino acid whose running.Regular Insulin can promote that amino acid enters cell, but this effect will be arranged when hGH exists.

3, hGH is to the metabolic influence of fat and sugar

HGH can impel the steatolysis of animal, suppresses glucose oxidase, and the consumption, the hGH that reduce glucose are complicated to carbohydrate metabolism.The hGH of physiological level can stimulate the insulin B cell, causes insulin secretion, strengthens the utilization of glucose, and excessive hGH then suppresses the utilization of glucose.

Human growth hormone extracts and tries out in children's nanism and starts from 1958.After update product purity and carry out clinical trial, progressively formally got permission clinical application from 1975 in various countries in the clinical effective back of affirmation.Because the human growth hormone that extracts comes in dead person's pituitary gland, clinical consumption is difficult to guarantee, so people begin to attempt to produce human growth hormone with chemosynthesis and gene recombination method.Wherein preceding method efficient is very low, no practical value.Back method has but realized industrialization in 1981 under the effort of U.S. Genentech company etc.

Recombinant somatropin's commodity that Genentech company produces Somatrem by name, in clinical trial, be proved to be and the human growth hormone physiologically active that extracts identical.U.S. Lilly company and Denmark Novo Nordisk company etc. have also succeeded in developing the recombinant human somatropin in succession.The suitability for industrialized production of human growth hormone has now all adopted gene recombination technology, its general method is the dna fragmentation of chemosynthesis human growth hormone, again with molecule clone technology amplification, clone, obtain the complete genome of human growth hormone, change intestinal bacteria over to by plasmid, promptly obtain the pure product of target protein through cultivation, fermentation and aftertreatment.

But solubleness is low, poor stability, transformation period short, have the performance of its route of administration of drawbacks limit such as immunogenicity and drug effect.The PEG modification technique reaches its maturity at present, its solvability of albumen after the modification and stability increase, the ability of anti-enzymic hydrolysis strengthens, reduced immunogenicity even elimination, the transformation period prolongs, and increases the therapeutic index of medicine, enlarge clinical use, bioavailability improves, and toxic side effect reduces, and thermostability and mechanical stability increase.Based on this, utilize me the recombinant human somatropin to be modified the PEG modification technique platform of company's comparative maturity.

Summary of the invention

The object of the present invention is to provide a kind of polyethyleneglycol modified two valency recombinant human somatropins, this modified outcome has not only prolonged the transformation period of reorganization life hormone, improved stability, reduced immunogenicity, and having reduced the administration number of times and the dosage of this medicine, single administration be injected biological activity and the long lasting pharmacological action that multiple dosing has high-strength promotion animal body growth every day than needing.

The present invention is by activating the PEG both-end, form a kind of new modifier ALD-PEG-ALD, method reference J.MiltonHarris, Evelyn C.Struck.Synthesis and Characterrization of Poly (ethylene Glycol) Derivatives, adopt this novel modifier that the recombinant human somatropin is modified, obtain a kind of couple of valency protein modification medicine GH-PEG-GH, its activity is 1.7 times of GH.

Embodiment

Embodiment 1 both-end activated polyethylene glycol obtains ALD-PEG-ALD

With molecular weight is that 20000 dalton's pharmaceutical grade polyoxyethylene glycol are substrate, add oxalyl chloride, DMSO is-60 ℃ to-70 ℃ reactions, reaction finishes back triethylamine cancellation, dichloromethane extraction, saturated sodium-chlor is washed, and is concentrated into driedly, adds diethyl ether and separates out the dry both-end activated polyglycol ALD-PEG-ALD that generates of solid suction filtration.

Embodiment 2 recombinant human somatropins' purifying

Preparation 20mM pH8.0Tris-HCl, buffer A, by thalline: extraction buffer (g/ml) is ultrasonication in 1: 20; ultrasonic by the omnidistance time 40min of work 1s 1s at intermittence, 1200-1600W, 30 ℃ of overheating protections; it is centrifugal that 4900rpm*35min is pressed in broken back, collecting precipitation, washing inclusion body.

Inclusion body is dissolved in 8M urea, among the 20mM Tris-HCl, PH9.0 at room temperature stirred sex change 1 hour, centrifugal removal insolubles, supernatant dilutes with 10% glycerine of 4 times of volumes, 20mMTris-HCl PH8.0, and 4 ℃ are spent the night, use the Q column purification, GH monomer and dimer can be used 250mM NaCl wash-out, further use the S-200 can be with dimer and monomer separation, and monomer can be used for modification reaction.

Embodiment 3 modification reactions

The pure product concentration adjustment of GH is to 5.0mg/ml, and add 20mM hydrogen boron sodium cyanide, fully dissolving, GH: ALD-PEG-ALD=1 in molar ratio: add ALD-PEG-ALD at 1,1: 5,1: 10, and 4 ℃ of reaction 4hr are to determine the optimum proportion of two kinds of reagent.Use non-reduced electrophoresis detection, found that to be mainly two modified outcomes, scanning analysis modification rate reaches 30%.Utilize the S-300 sieve chromatography that modifier GH-PEG-GH is separated with the GH of other many decorating molecules and unmodified, target substance can be used non-reduced electrophoresis detection, separates the pure product freeze-drying in back and preserves.

The check of embodiment 4 systemic anaphylaxis

60 of cavys, male and female half and half, body weight 250-300g is divided into three groups, GH and GH-PEG-GH is pressed three times of intraperitoneal administrations of human dosage, the next day once, be administered three times, make its sensitization, GH and GH-PEG-GH are pressed six times of human dosage with sensitization after the 14th day, excited respectively at the intravenous injection of cavy shank shin in 21 days, and observed also record result in the 30min.

Table 1 cavy systemic allergy test progression judging criterion

Reaction order	Reaction symptom
		0	No significant reaction
1	Have only and slightly grab nose, tremble and erect hair
		2	Have several times and cough, grab nose, tremble and erect hair
3	Repeatedly cough continuously, with expiratory dyspnea or spasm, tremble, tic etc.
		4	Spasm, tic, gatism, shock death

Annotate: anaphylaxis reaches more than 2 grades, and anaphylaxis is positive

Cavy anaphylaxis presentation of results, GH group have repeatedly cough continuously of cavy, with expiratory dyspnea or spasm, tremble, explanation such as tic has serious systemic anaphylaxis, anaphylaxis reaches 3 grades, GH has high immunogenicity; The GH-PEG-GH anaphylaxis is a zero level, and immunogenicity is negative, illustrates to modify the strong immunogenicity that can reduce or eliminate this macromole foreign protein.

The long-acting of embodiment 5 recombinant human somatropin's concatermers is observed

To remove pituitary gland rat (no endogenous growth hormone) is animal model, observes bioactive functions and the long-acting pharmacological action of polyethyleneglycol modified recombinant human growth hormone at the promotion growth of animal of body.

Method: to extract big white mouse pituitary is animal model, evenly is divided into 8 groups at random by body weight, 10 every group.Contrast 1 group of (the disposable rhGH of giving high dose group) disposable recombinant human somatropin of giving (rhGH) 9.4IUkg ^-1Contrast 2 groups (model group) and only give solvent (physiological saline that contains 0.1% bovine serum albumin); Recombinant human somatropin's concatermer is by estimating to tire 3.0IUmg ^-1Calculate, dosage is high dose group 9.4IUkg ^-1, middle dosage group 4.7IUkg ^-1, low dose group 2.3IUkg ^-1, three dosage groups are disposable administration; Each dosage group of positive control rhGH is divided 6 times, and be administered once every day, continuous 6d, and administration summation dosage is respectively high dose group 9.4IUkg ^-1, middle dosage group 4.7IUkg ^-1, low dose group 2.3IUkg ^-1

Put to death all rats after the 6th administration of positive controls after 24 hours, detect body weight, tail length, liver weight and tibial bone epiphyseal plate width.The result: experimental result shows, with the model group ratio, each dosage group body weight of rhGH concatermer group single administration significantly increases (P＜0.01), the long also phenomenal growth (P＜0.01) of tail, the remarkable broadening of tibial bone epiphyseal plate width (P＜0.01), middle and high dosage group liver weight increases (P＜0.01); 1 group of disposable rhGH high dosage that gives of control group promotes after 6 days that the effect of animal body growth is atomic, and the disposable administration of rhGH concatermer promotes after 6 days that animal body growth effect is stronger, proves the long-acting of rhGH concatermer.Each dosage group of rhGH concatermer is compared with the rhGH corresponding dosage of administration every day, and each growth indexes all has enhancing, promptly is higher than common rhGH with drug effect under the dosage; In the rhGH concatermer the disposable administration of dosage group divide the effect of 6 days multiple dosings suitable to the leading indicator weight increase of body growth and epiphyseal plate broadening and high dose group rhGH, illustrate that the rhGH concatermer has the pharmacological action of high-strength, long lasting promotion body growth than common rhGH.The biological value that calculates the rhGH concatermer with reference to Chinese Pharmacopoeia is 5.2IU/mg.

Result of study shows that repeatedly result of use is suitable for effect that the concatermer single uses and non-modification tethelin, points out such concatermer in the preparation of long-acting albumen and polypeptide application prospect to be arranged.The 7th day rat body weight change data see the following form after preceding 1 day of administration and the administration, and remainder data slightly.Δ bw is weight increase value (Δ bw=bw in the table ₇-bw ₁), bw ₁Be administration d1 body weight before the administration, bw ₇Be d7 body weight after the administration.

Each group of table 2.PEG-rhGH to the influence of removing hypophysis rat body weight (bw) (x ± SD, n=10)

Annotate: compare * *: P＜0.01 with contrast 2 model group; Compare with rhGH single-dose group, ^##: P＜0.01; Compare with corresponding dosage positive control rhGH group, ^★P＜0.05

The test of embodiment 6 immunogenicities

With rhGH and PEG-rhGH immunizing rabbit thereof: cut off the part rabbit hair of rabbit two hind paws, with alcohol and iodine disinfection skin; Immunity for the first time: draw 1ml with the 2ml syringe, the subcutaneous 0.5ml that injects of every batter palm; Immunity for the second time: after 10-14 days, inject Freund's complete adjuvant (FCA) emulsive antigen in the lymphoglandula of both sides nest and groin enlargement at interval, each lymphoglandula is annotated 0.1ml, near subcutaneous 1ml altogether all the other injection lymphoglandula.When enlargement or enlargement were not obvious as lymphoglandula, it was subcutaneous directly to inject both sides nest and groin; Heart blood-collecting method bloodletting after 7-10 days at interval, separation of serum was put 37 ℃ of incubators 1 hour with the blood of Erlenmeyer flask, put 4 ℃ of refrigerators interior 3～4 hours.After treating the blood coagulation blood clot retraction, draw serum with capillary burette.In 3000rpm centrifugal 15 minutes, get supernatant and add sanitas (0.01% Thiomersalate or 0.02% sodium azide, ultimate density), preserve standby in the rearmounted 4 ℃ of refrigerators of packing.

Measure the antibody titer of the immune serum that obtained with the two-phase agar diffusion test.Found that the visible obviously immunoprecipitation line of rhGH antibody 1: 64 o'clock, and the PEG-rhGH immunoprecipitation line that may be seen indistinctly at 1: 4 o'clock be can't see the immunoprecipitation line at 1: 8 o'clock, and the immunogenicity of PEG modification obviously reduction rhGH be described.

Claims

1. polyethyleneglycol modified tethelin, it is characterized in that: this pegylated growth hormone is to be the concatermer of two tethelin molecules of connector with the polyoxyethylene glycol.

2. the described polyethyleneglycol modified tethelin of claim 1 is characterized in that tethelin can derive from animal, also can be gene recombinant human source tethelin, preferred gene recombination human source tethelin.

3. the described polyethyleneglycol modified tethelin of claim 1-2 is characterized in that: used polyoxyethylene glycol both-end is all through overactivation, and can both with the tethelin covalent cross-linking.

4. the described polyethyleneglycol modified tethelin of claim 1-3, it is characterized in that: the polyoxyethylene glycol two ends are activated into the aldehydes or ketones structure, are preferably aldehyde structure.

5. the described polyethyleneglycol modified tethelin of claim 1-4 is characterized in that: used molecular weight polyethylene glycol is from 200-200000 dalton, preferred 2000-10000 dalton, more preferably 2000 dalton.