CN103157112B - Application of sialic acid derivatives in preparation of polyethylene glycol (PEG) lipid derivative modifying agent - Google Patents

Abstract

The invention belongs to the field of pharmaceutical preparation, in particular to the phenomenon that the speed of blood cleaning is increased be means of eliminating or weakening a polyethylene glycol (PEG) agent using sialic acid derivatives. When the PEG agent such as PEG lipidosome, PEG emulsion and PEG nanoparticles are repeatedly injected, the cleaning speed of a secondary injection agent in the blood can be greatly increased, namely, the ABC phenomenon appears. The ABC phenomenon can be eliminated and even weakened after the sialic acid derivatives are decorated on the surface of the PEG agent.

Description

Sialic acid derivative modifies the application in preparation at preparation PEG lipid derivate

Technical field

The invention belongs to field of pharmaceutical preparations, be specifically related to the application of sialic acid derivative in preparation PEG lipid derivate modification preparation, the acceleration blood that PEG chemical preparation could be eliminated or weaken to said preparation removes phenomenon.

Background technology

Polyethylene Glycol (polyethylene glycol, PEG) polyoxyethylene alcohol or polyoxyethylene glycol is called, be that oxirane and monoethylene glycol (or two ethylene glycol) are polymerized under base catalyst catalysis, molecular mass is different because the degree of polymerization is different, usually, between 200 ~ 35 000, its chemical general formula is HOCH ₂(CH ₂oCH ₂) n CH ₂oH.

Polyethylene Glycol water solublity is good and can be compatible preferably with a lot of organic constituents, also has low toxicity, to body nonirritant, feature that immunogenicity is low, therefore has a wide range of applications at art of pharmacy.As can be increased the dissolubility of medicine as solvent, as lubricant and the adhesive of tablet, as the framework material of sustained-release preparation, as the substrate of ointment, or as the stabilizing agent of protein medicaments.PEG is that the only a few ratified through FDA (Food and Drug Adminstration) (FDA) can as one of synthetic polymer of injection drug in body, 1991 the 1st kind with polyethyleneglycol modified protein drug PEG ADA Adenosine deaminase obtain FDA approval listing, be used for the treatment of immunization programs for children Defect, multiple PEG modified protein medicine has been had to go on the market at present, as Oncaspar, (the Hu Yongxiang such as PEG-IFN a-2b and PEGization granulocyte colony-stimulating factor, Niu Jinliang, Zhang Wenbo, Deng. the progress [J] of polyethyleneglycol modified drug technique. Chinese biochemical drug magazine, 2004, 25 (6): 369.373).

Polyethylene Glycol or derivatives thereof is used for the modification of drug molecule, immunogenicity and the antigenicity of protein and peptide drugs can be reduced; Increase the stability of medicine, reduce enzyme degradation; The plasma half-life of prolong drug, reduces blood concentration fluctuation; Improve drug disposition distribution, intensifier target tropism etc.If Polyethylene Glycol or derivatives thereof is modified and also can obtain similar effect in carrier surface.Such as, liposome has good biocompatibility, the distribution in vivo of institute's entrapped drug can be changed and improve the therapeutic index of medicine, but conventional liposome enters after in body more easily by mononuclear phagocyte system (mononuclear phagocyte system, MPS) absorb, cause its rapidly from blood remove and MPS be correlated with organ (as liver and spleen) accumulation.If polyethyleneglycol lipid derivates is modified in surface of liposome (being called PEGization liposome), thus form " conformation cloud " (conformational clouds) and hydration shell, for liposome provides larger sterically hindered and cover its surface hydrophobic binding site, reduce MPS to the identification of liposome and picked-up, extend its circulation time in vivo, thus can better by EPR(enhanced permeability and retention effect) effect of effect or targeting group realizes the Targeting distribution of liposome medicament, also make the physics of medicine simultaneously, chemistry and biology stability improves.At the bottom of nineteen ninety-five, the Evacet Doxil of PEGization ^?the treatment of the Kaposi's sarcoma caused for breast carcinoma, ovarian cancer, myeloma, acquired immune deficiency syndrome (AIDS) in U.S.'s listing is ratified through FDA.

In view of the unique advantage of PEGization carrier/drug, and the considerable Social benefit and economic benefit that commercialized product brings, countries in the world government all drops into a large amount of manpowers, financial resources and resource, carry out research, the particularly PEGization research in conjunction with the 21 century main direction such as active targeting part, gene transfection, cell-penetrating peptides, RNA interference treatment of the aspects such as such as PEGization liposome, PEGization dendroid (dendrimer) drug-supplying system, PEGization nanoparticle, PEGization micelle, PEG chemical medicine thing, PEGization enzyme.

Because most of PEG chemical preparation needs multiple dosing in clinical practice, (the Dams ETM such as Dams, Laverman P, Oyen WJG, et al. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes [J]. J Pharmacol exp Ther, 2000,292:1071 – 1079) with ⁹⁹ _mtc labelling liposome, pharmacokinetic parameters after research rat duplicate injection PEGization liposome and tissue distribution situation of change, it is unexpected that the liposome blood plasma level finding that second time is injected show very big reduction (concentration when 20 minutes is about inject first 10%), and liver intake rises sharply to 46.2 ± 9.8% from 8.1 ± 0.8%, this is and accelerates blood removing (Accelerated Blood Clearance, ABC) phenomenon.Current research thinks that PEGization liposome causes the possible mechanism of ABC phenomenon as follows: the PEGization liposome injected first, as TI-2 antigenic activation splenic marginal zones specific b cells, this B cell proliferation secretes " anti-PEG IgM " after being divided into plasma cell, the PEG of the surface of liposome that this specific antibody was injected afterwards with several days is combined, activating complement system subsequently, by Complement C_3 fragment, the picked-up of MPS to it is accelerated to the opsonic action of PEGization liposome, PEGization liposome is made from blood, to remove (She Zhennan fast, Zhai Wenjun, Deng Yihui, " accelerate blood to remove " the immunologic mechanism analysis [J] in phenomenon. Shenyang Pharmaceutical University's journal, 2011. 28 (9): 760-768.).

More research shows, ABC phenomenon is not that PEGization liposome is proprietary, (the LU such as Lu, W., WAN, J., SHE, Z., et al., Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle [J]. Journal of Controlled Release, 2007, 118 (1): 38-53.) research also creates ABC phenomenon when finding the duplicate injection in Mice Body of nanoparticle (CBSA-NP) that PEG and the PLA of cation Bovine Serum Albumin Modified is cross-linked to form, (the KOIDE such as KOIDE, H., ASAI, T., HATANAKA, K., et al., Particle size-dependent triggering of accelerated blood clearance phenomenon [J]. International Journal of Pharmaceutics, 2008,362 (1-2): 197-200.) find that the micelle of PEGization also can induce the generation of ABC phenomenon, the research of our seminar also proves that the micelle of PEGization and solid lipid nanoparticle can produce strong ABC phenomenon (national natural science fund subsidy project 81072602) for the duplicate injection of rat and beasle dog.As can be seen here, ABC phenomenon is that PEGization nano-carrier is common.Although mainly concentrate on the pharmaceutical carrier of the PEGization such as PEG liposome, PEG nanoparticle at present to the research of ABC phenomenon, but (the Cheng such as Cheng, T. L., P. Y. Wu, et al. (1999). " Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM. " Bioconjug Chem 10 (3): 520-528.) find that PEGization albumen can produce anti-PEG antibody, thus cause protein drug retention time in vivo to shorten.This phenomenon and ABC phenomenon may have close to even identical mechanism of production, so ABC phenomenon may be the common problem that PEGization carrier or even PEG chemical medicine thing exist.

The change of the PEG chemical preparation pharmaco-kinetic properties that ABC phenomenon causes is a problem that can not be ignored in its clinical practice, can have a strong impact on Drug therapy safety, effectiveness and patient compliance.(the Semple SC such as Semple, Harasym TO, Clow KA, et al. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic acid [J]. J Pharmacol Exp Ther, 2005, 312:1020 – 1026) research prove, duplicate injection encapsulating oligonucleotide (ODN), the PEGization liposome of pDNA or RNA ribozyme can induce strong immunne response, the shortening of preparation blood circulation time and mouse death rate is caused significantly to increase.We find in research work, beasle dog when duplicate injection PEGization Emulsion, nanoparticle and liposome, the generation of ABC phenomenon often along with serious allergic phenomena, as vomiting, diarrhoea, facial congestion and edema and sleepy etc.

In order to find the solution of ABC phenomenon, researcheres have done large quantity research to the generation mechanism of ABC phenomenon and influence factor.Think at present the phospholipid dosage of ejection preparation, injection interval, carrier surface PEG modify density, PEG chain length, the particle diameter of PEGization carrier, PEGization carrier charged situation etc. all can have an impact (low to the generation of ABC phenomenon and intensity, the wangkai universe, Huang Weiwei, et al., ABC phenomenal research progress [J] of polyethyleneglycol modified liposome. Acta Pharmaceutica Sinica, 2010. 45 (6): 677-683.).

the dosage of phospholipidthere is obvious inverse correlation (Ishida T in the degree that the first dosage of the discovery such as Ishida PEGization liposome and ABC phenomenon occur, Masuda K, Ichikawa T, et al. Accelerated clearance of a second injection of PEGylated liposomes in mice [J]. Int J Pharm, 2003,255:167 – 174).Rat injects PEGization liposome first, phospholipid dosage is respectively 0(HEPES salt buffer), 0.001,0.01,0.1,1,5 μm of ol/kg, inject radiolabeled PEGization liposome (phospholipid dosage is 5 μm of ol/kg) after 5 days, result shows that the blood of biphasic injection PEGization liposome removes degree and liver aggregate amount obviously increases with the reduction of first dosage.When first dosage phospholipid is higher than 1 μm of ol/kg, the PEGization liposome blood removing speed of biphasic injection and liver aggregate amount no longer continue to increase.And there is certain contradiction in the result of the research of Laverman etc. and above-mentioned Ishida: inject phospholipid dosage first and be respectively 0.05, 0.5, the PEGization liposome of 5.0 μm of ol/kg, biphasic injection PEGization liposome (phospholipid dosage 5.0 μm of ol/kg) Shi Junneng produces ABC phenomenon (Laverman P, Carstens MG, Boerman OC, et al. Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection [J]. J Pharmacol Exp Ther, 2001, 298:607 – 612).When fixing injected dose is first 5.0 μm of ol phospholipid/kg, when the PEGization liposome dosage of biphasic injection is 15 or 50 μm of ol phospholipid/kg, the ABC phenomenon of generation obviously weakens than 5.0 μm of ol phospholipid/kg groups.Nearest document (Repeated Injection of High Doses of Hemoglobin-Encapsulated Liposomes (Hemoglobin Vesicles) Induces Accelerated Blood Clearance in a Hemorrhagic Shock Rat Model DRUG M ETABOLISM AND DISPOSITION 2011,39(3): 484 – 489) report, even if high dose also can produce ABC phenomenon.

the concentration of PEG-DSPEthe PEGization liposome producing ABC phenomenon can be induced at present to be all long circulating material with PEG-DSPE, therefore to have researcher to investigate the impact of ratio on ABC phenomenon of PEG-DSPE in prescription specially.Inject first PEG-DSPE in PEGization liposome account for prescription lipid molar ratios be respectively 0,5,10,15%(phospholipid dosage is 0.001 μm of ol/kg), (phospholipid dosage is 5.0 μm of ol/kg within 5 days, to inject PEGization liposome afterwards, the molar ratio of PEG-DSPE in prescription is 5%), found that the ratio of the degree that ABC phenomenon occurs and PEG-DSPE exists obvious inverse correlation: the liposome (i.e. conventional liposome) containing 0% PEG-DSPE does not produce ABC phenomenon; Liposome containing 5% PEG-DSPE can be induced and be produced obvious ABC phenomenon, and significantly increases with liver aggregate amount.The content (10 of further increase PEG-DSPE, 15%), compared with 5% group, the clearance rate of biphasic injection liposome and liver assemble and present reduction trend, but still be significantly higher than matched group (P<0.01) (Ishida T, Harada M, Wang XY, et al. Accelerated blood clearance of PEGylated liposomes following preceding liposome injection:Effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes [J]. J Control Release, 2005, 105:305 – 317).Ishida etc. also find that the ratio increasing PEG-DSPE can weaken the induction of PEGization liposome to ABC phenomenon.Compared with the liposome of low with PEG-DSPE content in initial dose (being less than 5 mol%), if PEG-DSPE content high (being greater than 10 mol%) in injecting lipid body first, the liver accumulation of the PEGization liposome then injected thereafter reduces (Ishida T, Ichikawa T, Ichihara M, et al. Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice [J]. J Control Release, 2004, 95:403 – 412).

the molecular weight of PEGthere are some researches show, inject PEG2000-DSPE and the PEG5000-DSPE modified liposome (0.001 μm of ol phospholipid/kg) that mol ratio is 5% first, the labelling liposome (5 μm of ol phospholipid/kg) injected PEG2000-DSPE afterwards and modify for 5 days, all produce obvious ABC phenomenon (not investigating biphasic injection PEG5000-DSPE modified liposome) (Ishida T, Maeda R, Ichihara M, et al. Accelerated clearance of PEGylated liposomes in rats after repeated injections [J]. J Control Release, 2003, 88:35 – 42), therefore think that the molecular weight of PEG does not affect induction ABC phenomenon.The conclusion of Ishida etc. is on the contrary: inject the degree of PEG5000-DSPE modified liposome induction ABC phenomenon first well below injecting PEG2000-DSPE modified liposome first, the liver aggregate amount of PEG5000-DSPE and PEG2000-DSPE modified liposome group is respectively 35.3 ± 3.8% and 78.7 ± 8.8%, shows that the increase of PEG molecular weight in injecting lipid body first can weaken ABC phenomenon.

particle diameterthe research of Dams etc. shows: ABC phenomenon is with to inject the particle diameter of PEGization liposome, surface nature and radioactive label first all irrelevant.Unlabelled little (85 nm) or large (400 nm) PEGization liposome all can cause the blood removing speed of biphasic injection PEGization liposome significantly to increase, and produce obvious ABC phenomenon.

inject time interval and injecting continuouslydams etc. find to induce the double injection minimum interval of ABC phenomenon to be 5 days; When Ishida etc. prove that double injection is spaced apart 7 days, ABC phenomenon is the strongest.Separately studies have reported that, after the 35 days second time injection PEGization liposomees in rat interval, third time injection PEGization liposome is carried out at interval for 4 days or 7 days, though its blood removing speed slightly increases, but there was no significant difference, and liver spleen aggregate amount does not change, illustrate that repeatedly duplicate injection can not continue to cause ABC phenomenon.And inject PEGization liposome every day until the 4th time does not all produce ABC phenomenon.

different animals modelthe reported animal for studying ABC phenomenon comprises rat, mice and Rhesus Macacus.The research of Dams etc. shows, rat and Rhesus Macacus all can produce ABC phenomenon, and mice then can not; And the result of Ishida etc. shows to produce obvious ABC phenomenon in Mice Body.

Pertinent literature and our experiment show, can be alleviated even avoid the PEG chemical medicine thing of biphasic injection that ABC phenomenon occurs by the interval or adjustment injected dose extending double injection administration.In addition, the generation of ABC phenomenon can also be avoided when some cytotoxic drug is loaded in PEGization liposome.But overcome ABC phenomenon by these means and will greatly limit the range of application of PEGization modification technique.We also find can eliminate or alleviate ABC phenomenon (Xu H by can rupture PEG lipid derivate modified liposome or vesicle; Wang KQ; Deng YH; et al. Effects of cleavable PEG-cholesterol derivatives on the accelerated blood clearance of PEGylated liposomes. Biomaterials, 2010; , but the PEG lipid derivate that can rupture is little for extending the blood circulation time effect of liposome 31 (17): 4757-63.).Can say, not search out now the promising approach solving PEGization carrier/drug ABC phenomenon.

Sialic acid (Sialic acid described in the present invention; SA); formal name used at school is called " N-acetylneuraminic acid "; also known as saccharic acid; a class nine carbon monosaccharide; also be the general name of N-or the O-derivant of all neuraminic acids or ketone group-deoxidation nonanone saccharic acid (KDN), sialic general molecular formula is simultaneously:

The derivant relevant with sialic acid exceedes over one hundred kind, wherein topmost two kinds of sialic acides are 5-acetylaminohydroxyphenylarsonic acid 3,5-dideoxy-D-glycerol-D-gala ketononose (NANA, Neu5Ac, N-n acetylneuraminic acid n) and 3-deoxidation-D-glycerol-D-gala ketononose (KDN), N-n acetylneuraminic acid n is wherein more common in vertebrate.Remaining sialic acid derivative is all derivative on the basis of these two kinds of compounds forming.Polysialic acid is the linear homopolymer that sialic acid monomer connects with α-2,8 and/or α-2,9 ketoside key (Ketosidic linkage).

Sialic acid is extensively present in the tissue of various biology, is the main component forming cell surface compound saccharic, is mostly connected to the end of glycoprotein, glycolipid and oligosaccharide with α-glycosidic bond by the hydroxyl of 2-position anomeric carbon.Ganglioside is that the mankind understand comparatively deep sialic acid derivative, it contains sialic glycosyl sphingolipid as a class, content in brain is very abundant, it not only can promote neural cellular differentiation, neurite outgrowth and Synaptic formation, and take part in neural plasticity wither joint and brain injury after functional rehabilitation.Monostalotetrahexosylgangliside (Monosialylganglioside GM ₁, be called for short GM ₁) be study ganglioside the most deep so far, its structural formula is as follows:

At present, GM ₁be widely used in the treatment (as the monosialoganglioside sodium injection that Shandong Qilu Pharmaceutical Co., Ltd. of China produces, commodity are called " Shen Jie ") of parkinson disease, apoplexy, neonatal hypoxic ischemic encephalopathy, cerebral trauma, spinal cord injury and peripheral neuropathy.Allen, T.M. scientist (Allen is waited, T.M. and Chonn, A. Large unilamellar liposomes with low uptake into the reticuloendothelial system (1987) FEBS Lett. 223,42-46) the earliest by GM ₁modify for surface of liposome, increase liposome (EPC: CH, LUV, 0.17 μm)) circulation time 3 ~ 10 times, reduce RES and absorb (blood/RES(Blood/RES) ratio by EPC: CH(2:1) 0.13 bring up to EPC: CH:GM ₁(2:1:0.14) 1.7; DSPC:CH(2:1) 0.007 brings up to DSPC:CH:GM ₁(2:1:0.14) 3.2).

Except GM ₁outward, common at present ganglioside also has GM ₂, GM ₃, GD ₁, GD ₂, GD ₃deng, concrete classification and structure see the following form, and wherein GM represents containing 1 sialic acids groups, and GD represents containing 2 sialic acids groups, and GT represents containing 3 sialic acids groups, and GQ represents containing 4 sialic acids groups.Cer represents neural acid amide residue, and the sugar of composition oligonucleotide chain is mainly D-Fructus Vitis viniferae (Glc), D-galactose (Gal), N-acetylgalactosamine (GalNAc), N-acetyl-neuraminate (NeuNAc) and NeuGc ALPHA2-3Gal (NeuGc).

Sialic derivant also comprises sialic cholesterol glycoside (SA-CHOL), Japanese Unexamined Patent Publication No. 62-265229 (1987); And flat 1-93529 (1989) describes the Synthesis and characterization method of this compound.

The bonding structure being present in the N-acetyl-neuraminate in natural ganglioside is α type, but the bonding structure having synthesized N-acetyl-neuraminate by chemical synthesis process is in recent years episialo (episialo) complex carbohydrate of β type.Japan Patent (KOKOKU) 1-49354 and United States Patent (USP) 4968786 report episialo ganglioside epiGM ₃synthesis and characterization method; Japan Patent (KOKAT) 61-282391 and United States Patent (USP) 4751290 report episialo ganglioside epiGM ₄synthesis and characterization method; Japan Patent (KOKAT) 63-452931 and United States Patent (USP) 4730058 report episialo ganglioside epiGM ₅synthesis and characterization method.

Due to sialic acid bear electricity and be prevalent in surface of cell membrane, be the main source of cell membrane negative charge, therefore researcher thinks that sialic acid is relevant with the adhesion of cell.Between cell and cell, between cell and environment, sialic acid residues plays a part to stand in the breach.Verified: cell in growth, differentiation, old and feeble, the process such as to cancerate, often with the change of surface recombination sugar.Sialic acid is also relevant with the life-span of cell, and the sialic acid content of immaturity and just ripe erythrocyte surface, higher than the erythrocyte of aging, is studied and also found to shorten to several hours through the red blood cell life span of sialidase process from original 120 days.In addition, cell surface sialic acid content is relevant with malignant degree, the tumor cell that namely surface of cell membrane sialic acid content is high, and metastatic is also high.Sialic biological function can be divided into 3 classes substantially: the information transmission between the receptor acting that sialic acid itself can be identified, cell contacts played masking action with by stoping or weakening cell or molecule to its specific recognition position.Based on this understanding, Chinese patent 200780033741.8 describes and uses Polysialic acid modified protein medicine to increase the scheme of protein drug curative effect; Chinese patent CN91101721.6 provides the purposes using episialo ganglioside (epiGM) to prepare cancer diagnosis medicine.

Animal experiment study shows, the reduction of ganglioside lipid level reduces relevant with early malnutrition and learning capacity, and supplements the learning behavior that sialic acid can improve animal.Enough sialic acid supplies may be even more important for the normal development of low birth weight infant brain function.After baby due, the sialic acid in breast milk is for ensureing that their normal development is most important.Investigation display, in childbirth stepmother parent, As time goes on Sialic Acid Level becomes downward trend.Therefore, at pregnancy period and the enough sialic acides of pregnant rear lasting absorption, can help to maintain the Sialic Acid Level in body.So a purposes of current sialic acid derivative is namely as supplementary, as described in Chinese patent 200510069325.6.

Summary of the invention

The present inventor finds unexpectedly in the research to PEG chemical preparation ABC phenomenon, and modified by sialic acid derivative when PEG chemical preparation surface, can weaken the ABC phenomenon even eliminating PEG chemical preparation, the sialic acid derivative described in invention has the structure of formula (1):

Wherein R ₁-OCH ₂cHOH, the carbon be wherein connected with hydroxyl is connected with No. 7 carbon, or-OCHCH ₂oH, the carbon be wherein connected with ehter bond is connected with No. 7 carbon, when n be 1 or general formula (1) in except R ₅when structure is in addition in end group, R ₁oHCH ₂oHCH-;

R ₂n-acetyl group, N-glycolyl, amino or hydroxyl; R ₃h, acetyl group, methyl; R ₄h, hydroxyl or fluorine, R ₄can be α configuration with the connecting key of No. 3 carbon, or beta comfiguration; R ₅the ceramide with oligonucleotide chain residue, glycolipid, lipid residue, protein or peptide, medicine or drug delivery system; Glycosidic bond in formula (1) on No. 2 carbon or ketoside key can be α configuration, or beta comfiguration; N is an integer between 1 ~ 100.

As preferably, R in general formula (1) compound ₁-OCH ₂cHOH, the carbon be wherein connected with hydroxyl is connected with No. 7 carbon, or-OCHCH ₂oH, the carbon be wherein connected with ehter bond is connected with No. 7 carbon; Or OHCH ₂oHCH-; R ₂n-acetyl group, N-glycolyl or hydroxyl; R ₃h, acetyl group; R ₄be H or hydroxyl, work as R ₄when being hydroxyl, its connecting key can be α configuration, or beta comfiguration; Wherein R ₅ceramide, glycolipid, the lipid residue with oligonucleotide chain residue; Glycosidic bond wherein on No. 2 carbon or ketoside key can be α configuration, or beta comfiguration; N is an integer between 1 ~ 100.

As most preferably, formula (1) compound is GM ₁, GM ₂, GM ₃, GM ₄, GM ₅, epiGM ₁, epiGM ₂, epiGM ₃, epiGM ₄, epiGM ₅or SA-CHOL.

It is PEGization Emulsion that PEG lipid derivate of the present invention modifies preparation, PEGization microemulsion, PEGization liposome, PEGization solid lipid nanoparticle, PEGization nanostructured carrier, PEGization micelle or PEGization vesicle, prepare these preparations material used and comprise various phospholipid, various surfactant, polymer substance.

the beneficial outcomes that the present invention brings

Obviously can weaken after using sialic acid derivative and PEG lipid derivate jointly to modify preparation or even eliminate ABC phenomenon completely.

Sialic acid derivative, particularly ganglioside wherein, belong to the endogenous material of organism, so can be absorbed and degrade after it enters body as pharmaceutical carrier or drug molecule trim, can not increase the security risks of modified preparation.

This patent sialic acid derivative used is mostly endogenous material, enters after in body and can be utilized by body, namely can be used as the supplementary of human body with PEG chemical preparation.

Accompanying drawing explanation

Fig. 1 is not containing GM in embodiment 1 ₁pEGization Emulsion inject (representing with control) first and the Drug-time curve (n=3) of biphasic injection after being separated by 3 days, 5 days and 7 days.

Fig. 2 is containing GM in embodiment 1 ₁pEGization Emulsion inject (representing with control) first and the Drug-time curve (n=3) of biphasic injection after being separated by 3 days, 5 days and 7 days.

Fig. 3 is not containing GM in embodiment 2 ₁the Drug-time curve (n=3) of biphasic injection of the different first dosage of PEGization Emulsion, wherein the expression of control group with phospholipid dosage be 2 μm of ol/kg carry out single injection time Drug-time curve.

Fig. 4 is containing GM in embodiment 2 ₁the Drug-time curve (n=3) of biphasic injection of the different first dosage of PEGization Emulsion, wherein the expression of control group with phospholipid dosage be 2 μm of ol/kg carry out single injection time Drug-time curve.

detailed description of the invention:

In embodiment, the abbreviation of each composition used is as follows

TN vitamin E Nicotinate

MCT median chain triglyceride oil

S ₇₅soybean phospholipid S75

MPEG-DSPE methoxy poly (ethylene glycol) PHOSPHATIDYL ETHANOLAMINE

CF calcein

CHOL cholesterol

GMS 18 (alkane) sour glycerine ester

PEG Polyethylene Glycol

Below in conjunction with embodiment, the present invention is described in further detail; but these embodiments can not be understood to limit scope of the present invention; protection scope of the present invention limited by claims, and any change on the claims in the present invention basis is all protection scope of the present invention.

When studying ABC phenomenon, the selection of model drug is most important, because the immunoreation of ABC phenomenon and organism is in close relations, so we should select body particularly immune system fanout free region or the less medicine of injury.Also to consider whether to be convenient to be loaded in selected PEGization carrier also can reflect carrier situation of change in vivo really simultaneously.Consider, we select vitamin E Nicotinate (Tocopheryl Nicotinate is called for short TN), and (Calein is called for short CF to calcein )with coenzyme Q10 (Coenzyme Q10 is called for short Q10).

embodiment 1

GM ₁aBC is affected

Emulsion is prepared according to table 1 prescription composition

Table 1 TN Emulsion prescription

Composition/prescription	1	2
			TN（mg）	12	12
MCT（mg）	30	30
			S 75（mg）	7.0	7.0
mPEG-DSPE (mg)	2.8	2.8
			10%GM 1Injection	1.8 mL	-
5% glucose injection	-	1.8 mL

MPEG-DSPE modifies the preparation technology of Emulsion: by recipe quantity aqueous phase (5 % glucose injection) 55 DEG C of pre-stand-by heats.Recipe quantity oil phase (TN, MCT, S75, mPEG-DSPE) is stirred to whole dissolving at 55 DEG C.Under stirring, aqueous phase is added oil phase, high speed dispersion, obtain colostrum.Probe Ultrasonic Searching (200 w × 2 min; 400 w × 6 min) after process, cross 0.22 μm of microporous filter membrane degerming and get final product.

GM ₁with the preparation technology of the co-modified Emulsion of mPEG-DSPE: with the GM of 10% ₁injection is as aqueous phase, and all the other preparation technologies are identical with the technique that mPEG-DSPE modifies Emulsion.

Dosage regimen: after male Wistar rat labelling is weighed, in tail vein injection Emulsion, inject first after 3 days, 5 days or 7 days and carry out second time injection, phospholipid dosage is 2 μm of ol/kg, and before injection 30min in socket of the eye venous blood sampling 400 μ L, centrifugalize upper serum after standing 2h, 5min, 15min, 30min, 1h, 2h, 4h socket of the eye venous blood sampling 400 μ L after injection, be placed in the 1.5 mL centrifuge tubes scribbling heparin, centrifugalize upper plasma ,-20 DEG C of preservations are to be measured.

Standard curve: get inner mark solution, blank Dog Plasma is appropriate, the TN solution of a series of concentration of accurate preparation, after extraction and isolation process, gets supernatant and carries out HPLC analysis.With drug level c(μ g/mL) is abscissa, the peak area ratio of medicine and internal standard substance ( a _s / A _i ) be vertical coordinate, carry out linear regression by weighted least-squares method, and calculate regression beeline equation and be a _s / A _i =0.0629*C+0.000356 (1.0 ~ 100.0 μ g/mL), R=0.9976.

Sample treatment: get plasma sample 100 μ L in 2 mL centrifuge tubes, adds interior mark liquid 50 μ L, methanol 150 μ L, normal hexane 600 μ L.Vortex 5 min mixes, and in 10, centrifugal 10 min of 000 rpm, pipette normal hexane layer 500 μ L, nitrogen volatilizes.Add mobile phase 100 μ L, vortex 1 min mixes, and in 10, centrifugal 10 min of 000 rpm, get supernatant, carry out HPLC analysis.

The results are shown in Figure 1 and Fig. 2, Fig. 1 represent and only inject (representing with control) first with the Emulsion that mPEG-DSPE modifies and the Drug-time curve of biphasic injection after being separated by 3 days, 5 days and 7 days; Fig. 2 represents containing using GM ₁the Emulsion jointly modified with mPEG-DSPE is injected first (representing with control) and the Drug-time curve of biphasic injection after being separated by 3 days, 5 days and 7 days.As seen from the figure, the duplicate injection of the Emulsion of mPEG-DSPE modification can produce strong ABC phenomenon; GM is added in PEGization Emulsion ₁after, if second time injection is carried out at interval for 3 days, do not observe ABC phenomenon, if interval 5 days or 7 days, ABC phenomenon weakens, and the ABC phenomenon intensity there was no significant difference produced.

embodiment 2

The prescription of Emulsion used and preparation technology are with " embodiment 1 ".

Dosage regimen: after male Wistar rat labelling is weighed, in tail vein injection Emulsion, phospholipid dosage is 0.1 μm of ol/kg, 0.5 μm of ol/kg, 1 μm of ol/kg or 2 μm ol/kg, inject first after 7 days and carry out second time injection, phospholipid dosage is 2 μm of ol/kg, before injection, 30min is in socket of the eye venous blood sampling 400 μ L, centrifugalize upper serum after standing 2h, 5min, 15min, 30min, 1h, 2h, 4h socket of the eye venous blood sampling 400 μ L after injection, be placed in the 1.5 mL centrifuge tubes scribbling heparin, centrifugalize upper plasma ,-20 DEG C of preservations are to be measured.Analytical method is with " embodiment 1 ".

Fig. 3 represents the Drug-time curve of the Emulsion biphasic injection when the first dosage of difference only modified with mPEG-DSPE, wherein the expression of control group with phospholipid dosage be 2 μm of ol/kg carry out single injection time Drug-time curve; Fig. 4 represents and uses GM ₁the Drug-time curve of the Emulsion modified with mPEG-DSPE mixing biphasic injection when different first dosage, wherein the expression of control group with phospholipid dosage be 2 μm of ol/kg carry out single injection time Drug-time curve.Result shows, GM ₁the Emulsion of modification is mixed under above-mentioned various initial dose condition, all without obvious ABC phenomenon with mPEG-DSPE.

embodiment 3the ABC phenomenon of different PEG density formulations

The prescription of Emulsion used is as shown in table 2, and preparation technology is with " embodiment 1 ".

The prescription composition of the different PEG density formulations of table 2

Composition/prescription	1	2	3	4	5	6
							TN (mg)	12	12	12	12	12	12
MCT (mg)	30	30	30	30	30	30
							S 75 (mg)	7.0	7.0	7.0	7.0	7.0	7.0
mPEG-DSPE (mg)	-	0.7	1.4	2.8	5.6	8.4
							10 %GM1 injection (mL)	1.8	1.8	1.8	1.8	1.8	1.8

The selection of laboratory animal, dosage regimen and analytic process are with " embodiment 1 ".Result shows under different surface PEG modifies density, GM ₁add and all make ABC phenomenon weaken or even eliminate.

embodiment 4

The prescription of Emulsion used is as shown in table 3, and preparation technology is with " embodiment 1 ".

The Emulsion prescription of the different mPEG molecular weight of table 3

Composition/prescription	1	2	3
				TN (mg)	12	12	12
MCT (mg)	30	30	30
				S 75 (mg)	7.0	7.0	7.0
mPEG 750-DSPE (mg)	1.5	-	-
				mPEG 5000-DSPE (mg)	-	10.7	-
mPEG 20000-DSPE (mg)	-	-	38.8
				10 %GM1 injection (mL)	1.8	1.8	1.8

Remarks: mPEG ₇₅₀-DSPE molecular weight is 750+748.1=1498.1; MPEG ₅₀₀₀-DSPE molecular weight 5748.1; MPEG ₂₀₀₀₀-DSPE molecular weight 20748.1

The selection of laboratory animal, dosage regimen and analytic process are with " embodiment 1 ".When result shows to use the PEG of different chain length to modify Emulsion, GM ₁add and all make ABC phenomenon weaken or even eliminate.

embodiment 5

GM ₃on the impact of PEGization micelle ABC phenomenon

GM ₃structure is as follows

GM ₃according to document (Zhu Zhen unit Zhang Yongmin, ganglioside GM ₃effective synthesis [J], chemical journal, 2004,24:2909-2916) described in method carry out Synthesis and characterization.

The prescription of prepared micelle is as shown in table 4

Table 4 micelle prescription

Preparation technology: by the Q of recipe quantity ₁₀, S ₇₅, DPPC, mPEG ₂₀₀₀-DSPE, GM ₃dissolve by ethanol in proper amount, after being volatilized by ethanol, in 60 DEG C of water bath sonicator situations, add recipe quantity 5% glucose injection.

Particle size determination instrument is used to record gained micelle particle diameter between 10-30 nm.

Select male beagle dogs as laboratory animal, weigh after fasting 10 h.With Q ₁₀dosage 0.8 mg/kg administration.Blood is got respectively at 0 min, 5 min, 10 min, 15 min, 30 min, 60 min, 90 min, 120 min after administration.Separated plasma after centrifugal ,-20 DEG C of preservations are to be measured.Biphasic injection is carried out after 7 days.

Analytical method: get 0.1 mL Dog Plasma in 1.5 mL centrifuge tubes, add interior mark 10 μ L(vitamin K1 15 μ g/mL), after mixing, add methanol 0.2 mL, normal hexane 0.6 mL, centrifugal 10 min of vortex 3 min, 10000 rpm, pipette normal hexane 0.5 mL in 1.5 mL centrifuge tubes; Add normal hexane 0.6 mL in residual residue again, centrifugal 10 min of vortex 3 min, 10000 rpm, pipette normal hexane 0.6 mL, and merge normal hexane layer, centrifugal concentrating waves most normal hexane, and residue 100 μ L mobile phases dissolve, and get 20 μ L sample introductions.

Due to Q ₁₀for organism endogenous material, so we are on pretreatment to the Q in every beasle dog blood ₁₀concentration has carried out measuring and having deducted this background values when plasma sample is tested.

Result shows, the micelle of prescription 1 injects the injected dose of residue 50% in blood after 30 min first, only remains 7 % of injected dose, there occurs obvious ABC phenomenon after biphasic injection in 30 min blood circulations; The micelle of prescription 2 injects the injected dose of residue 65% in blood after 30 min first, remains 51 % of injected dose, can think and not produce ABC phenomenon after biphasic injection in 30 min blood circulations; The micelle of prescription 3 injects the injected dose of residue 58% in blood after 30 min first, only remains 9 % of injected dose, create obvious ABC phenomenon after biphasic injection in 30 min blood circulations; The micelle of prescription 4 injects the injected dose of residue 66% in blood after 30 min first, remains 56 % of injected dose, can think and not produce ABC phenomenon after biphasic injection in 30 min blood circulations.

Above-mentioned experiment proves GM ₃add the ABC phenomenon that significantly can weaken PEGization micelle.

embodiment 6

Sialic cholesterol glycoside (SA-CHOL) adopts Japanese Unexamined Patent Publication No. 62-265229 (1987); And the method described in flat 1-93529 (1989) carries out Synthesis and characterization.

The prescription of prepared vesicle is as shown in table 5

Table 5 vesicle prescription

The preparation vesicle that uses mPEG-DSPE to modify and the vesicle jointly modified of SA-CHOL and mPEG-DSPE use the CF of the outer aqueous phase of gel chromatography removing respectively.Select mice as laboratory animal, weigh after fasting 10 h.With CF dosage 0.1mg/kg administration.Blood is got respectively at 0 min, 5 min, 10 min, 15 min, 30 min, 60 min, 90 min, 120 min after administration.Separated plasma after centrifugal ,-20 DEG C of preservations are to be measured.

Analytical method is set up and sample treatment:

The CF solution of variable concentrations is configured, the mice plasma containing same volume in every increment product with PBS (pH 7.4, containing 10% Triton X-100).Spectrofluorophotometer (Ex=490 nm, Em=520 nm) measures fluorescence intensity level F.

Linear equation: F=13.129*C+23.564, R ²=0.9955.

The range of linearity: 1.5-75.0 ng/mL.

Blood plasma 100 μ L, with PBS(pH 7.4, containing 10% Triton X-100) be settled to 5mL, mixing, spectrofluorophotometer (Ex=490 nm, Em=520 nm) measures fluorescence intensity F, substitutes into standard curve, calculates the content of CF in blood plasma.

Result of the test shows: when dosing interval was at 7,14 or 21 days, only can occur obvious ABC phenomenon with the vesicle that mPEG-DSPE carries out modifying, and add SA-CHOL in film material after, ABC phenomenon is eliminated substantially.

embodiment 7

EpiGM ₄carry out synthesizing and characterizing according to the method described in Japan Patent (KOKAT) 61-282391 and United States Patent (USP) 4751290.(calculate molecular weight and be about 1143)

Preparation is containing epiGM respectively ₄not containing epiGM ₄pEGization liposome.

Take HSPC 96 mg, CHOL 32 mg, mPEG ₂₀₀₀-DSPE 32 mg, epiGM ₄6mg, dissolves by ethanol in proper amount, adds the 120 mM calcium acetate solutions that 5 mL are preheated to 50 DEG C, after Probe Ultrasonic Searching process, crosses 0.45,0.22 μm of microporous filter membrane, measures particle diameter; Use G-50 gel column that outer for liposome aqueous phase is replaced into 5%(w/v) sucrose solution; Get 5 mL gradient liposomees, add 35 mg CF powder, hatch 1 h for 65 DEG C, obtain the liposome encapsulating calcein.

Use Wistar rat as experimental animal, result shows, when dosing interval is 7 days and phospholipid dosage is 5 μm of ol/kg, does not have epiGM ₄pEGization liposome create obvious ABC phenomenon, the CF existed in blood during 15 min after biphasic injection only has 20% of injected dose; And use epiGM ₄there is not ABC phenomenon in the PEGization liposome modified.

embodiment 8

With GM ₁modify PEGization solid lipid nanoparticle.

Table 6 PEG solid lipid nanoparticle prescription

Composition/prescription	1	2
			TN（mg）	20	20
GMS（mg）	50	50
			mPEG 2000-DSPE (mg)	7	7
10%GM 1Injection (mL)	-	3
			5% glucose injection adds to	5mL	5mL

Prepare solid lipid nanoparticle that mPEG-DSPE modifies according to a conventional method respectively and with mPEG-DSPE and GM ₁the solid lipid nanoparticle that mixing is modified.Dosage regimen and analytical method are with " embodiment 1 ".

Result shows, only creates obvious ABC phenomenon when injection interval is 7 days with the solid lipid nanoparticle that mPEG-DSPE modifies, and mPEG-DSPE and GM ₁the solid lipid nanoparticle that mixing is modified almost does not have the generation of ABC phenomenon,

embodiment 9

The degree of polymerization is adopted to be the Polysialic acid of 40 ~ 60, by α-2 between sialic acid molecule, 8-glycosidic bond connects, and the coupled reaction between Polysialic acid and lipid fragments is carried out according to the method described in Chinese patent 200780033741.8 and Chinese patent 86100178.8, and lipid fragments used is cetyl.

With synthesized Polysialic acid lipid derivate, the PEGization solid lipid nanoparticle of prescription 1 in " embodiment 8 " is modified, use mice for experimental animal and injection interval is 9 days time, carry out the PEGization solid lipid nanoparticle modified compared to only using mPEG-DSPE, its ABC phenomenon is eliminated substantially.

Claims (1)

1. sialic acid derivative GM ₃modify the application in preparation at preparation mPEG-DSPE, it is characterized in that, described preparation is Emulsion, micelle, vesicle, liposome or solid lipid nanoparticle.