CN102558389A - Low molecular weight carboxyl-reduced derivatives of fucosylated glycosaminoglycans and preparation method and applications of low molecular weight carboxyl-reduced derivatives - Google Patents
Low molecular weight carboxyl-reduced derivatives of fucosylated glycosaminoglycans and preparation method and applications of low molecular weight carboxyl-reduced derivatives Download PDFInfo
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Abstract
The invention discloses low molecular weight carboxyl-reduced derivatives of fucosylated glycosaminoglycans (LCRG). The extent of carboxyl reduction is not less than 20%. Weight-average molecular weight of the LCRG is about 3000-20000Da, and monosaccharides comprise acetyl galactosamine (GalNAc), glucose (Glc) or glucuronic acid (GlcUA) and fucose (Fuc) or sulfates of fucose (shown as -OSO3-). The mole ratio of GalNAc, Glc (containing GlcUA), Fuc and -OSO3- is about 1: (1+-0.3): (1+-0.3): (3.0+-1.0). The LCRG is a potent human immunodeficiency virus (HIV) Type 1 entry inhibitor which acts on conserved regions and has the advantages of high activity against HIV Type 1, high therapeutic index and no-drug-resistance, and the LCRG can be used for preventing or curing HIV. The invention further provides a preparation method of the LCRG. The carboxyl-reduced derivatives of fucosylated glycosaminoglycans and medicinal compositions of the carboxyl-reduced derivatives can be prepared into injection agents, lyophilized powder or suppository and the like.
Description
Technical field
The invention belongs to medical technical field; Specifically; Carboxyl reduction verivate (the Low molecular weight carboxyl-reduced derivatives offucosylated glycosaminoglycans that relates to a kind of lower molecular weight fucosylated glycosaminoglycan; LCRG) and preparation method thereof, and the pharmaceutical composition and the application in the medicine of preparation prevention or treatment AIDS thereof that contain the carboxyl reduction verivate of this lower molecular weight fucosylated glycosaminoglycan.
Background technology
Human immunodeficiency virus (human immunodeficiency virus, HIV) infect due to AIDS (aquired immunodeficiency syndrome AIDS) is the healthy major disease of serious harm human life.Two during the last ten years; HIV virus and infect relevant fundamental research and all obtained huge advance made about applied basic research and the clinical practice of the pharmacological agent (like hiv reverse transcriptase/proteinase inhibitor) of AIDS, but the clinical treatment of AIDS still exist be difficult to cure, treatment no response, resistance and serious problems such as toxic side effect.Therefore, the clinical novel therapeutic medicine that presses for particularly need form complementary novel targets new mechanism original new drug with existing clinical application.
In the inverase research and development, mainly concentrate on RTI and proteinase inhibitor class medicine in early days, the emphasis of new drug development is progressively transferred to novel targets new mechanism medicine initiative direction in the recent period.The major cause that novel targets new mechanism medicine draws attention is: because target spot is different from clinical medicine, can be used for having the medicine resistance now or treating unresponsive patient; Can avoid producing crossing drug resistant with existing medicine; Because target spot and mechanism are different from existing medicine, new drug and clinical application coupling might produce synergy.At present, mainly comprise chemokine receptor anagonists, intergrase chain transfer suppressor factor, entry inhibitors and ripe suppressor factor etc. grinding novel targets new mechanism inverase.
In the new mechanism inverase, need not to stride the advantage that film gets into the transport of drug aspect of cell, HIV entry inhibitors especially receive publicity (PNAS, 2003,100:10598-10602 owing to exist; Med.Res.Rev., 2009,29:369-393).In theory, suppress receptor in target cell CD4 that target spot that HIV invades can comprise HIV, co-receptor (CCR5, CXCR4), HIV membranin gp120, gp41 etc.
Fucosylated glycosaminoglycan (Fucosylated Glycosaminoglycan is hereinafter to be referred as FGAG) is meant that one type extract to obtain from echinoderms body wall or internal organ; Has the CHS of being similar to backbone structure; But have the substituted TGSS C3 verivate of side chain sulfated fucose (J.Biol.Chem., 1988,263 (34): 18176-181783 and J.Biol.Chem.; 1991,266 (21): 13530-13536).
The FGAG of natural origin has significant HIV-resistant activity (lst International AIDS SocietyConference on HIV Pathogenesis and Treatment; 2001, Jul 8-11, Abstract No.247); Yet FGAG also has non-selective widely pharmacologically active; As influence blood coagulation system and activate biologically active pdgf (Blood, 2006,107 (10): 3876-3882; Blood, 2009,114 (14): 3092-3100 and Thromb.Haemost., 1988,59 (3): 435-439), this has limited the application and development of FGAG as inverase.For this reason, the investigator attempts FGAG is carried out the structural modification transformation both at home and abroad, on the basis that keeps strong effect HIV-resistant activity, evades its influence to the blood coagulation system.International conference document (Int Conf AIDS.1991 Jun16-21; 7 (1): 178, abstract no.M.A.1345) reported a kind of lower molecular weight FGAG depolymerization product, it has the activity of stronger inhibition HTLV-IIIB virus strain, but still has anticoagulant active to a certain degree.European patent EP 0410002A1, has obtained low-molecular-weight product, and discloses its HIV-resistant activity, but do not pointed out it whether to influence blood coagulation system the FGAG depolymerization in stichopus japonicus source through directly.One Chinese patent application CN201110114860.4 provides a kind of novel low molecular weight amount glycosylation CHS LGC, and this compound and the compsn that contains LGC can be used as the medicine of anti-HIV-1.Yet not seeing has pair FGAG carboxyl to carry out that structural modification obtains its low-molecular-weight derivant and in the public reported of anti-HIV application facet.
Summary of the invention
The object of the invention provides a kind of carboxyl reduction verivate (Lowmolecular weight carboxyl-reduced derivatives of fucosylated glycosaminoglycans of lower molecular weight fucosylated glycosaminoglycan; LCRG) and preparation method thereof; And the pharmaceutical composition that contains the carboxyl reduction verivate of this lower molecular weight fucosylated glycosaminoglycan; This verivate and medicinal compsns thereof can combine the conserved regions CD4i of the surface glycoprotein gp120 of HIV-1; Efficient prevention HIV-1 virus invasion procedure, and do not influence coagulation process, can be used for prevention or treatment AIDS.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of mixture with homology TGSS C3 verivate of formula (I) structure is the carboxyl reduction derivative L CRG and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan,
In the formula (I):
N is that average is about 3～22 integer;
R is independently of each other-H or-SO
3 -
The carboxyl reduction verivate and the pharmacy acceptable salt thereof of aforesaid lower molecular weight fucosylated glycosaminoglycan, wherein:
The monose of the carboxyl reduction verivate of said lower molecular weight fucosylated glycosaminoglycan is formed and is comprised D-glucose and/or D-glucuronic acid, D-2-deoxidation-2-acetylamino galactosamine sulfuric ester, L-Fucose sulfuric ester; With molar ratio computing, monose is formed and with the proportional range of contained sulfate group is: (D-glucone+D-glucal acidic group): (D-2-deoxidation-2-acetylamino galactosamine base): (L-fucosido): (sulfate group)=1: (1 ± 0.3): (1 ± 0.3): (3.0 ± 1.0); And the D-glucone: the molar ratio range of (D-glucone+D-glucal acidic group) is 20%～100%;
The weight average molecular weight range of said lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate is 3000～20000Da; Polydispersity index is between 1.0 to 1.8.
The weight average molecular weight range of said lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate is 6000～15000Da; The polydispersity index value is between 1.1 to 1.5.
Above-mentioned pharmacy acceptable salt is basic metal, alkaline earth salt and the organic ammonium salt of the carboxyl reduction verivate of lower molecular weight fucosylated glycosaminoglycan.
Above-mentioned pharmacy acceptable salt is sodium salt, sylvite or the calcium salt of lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate.
The carboxyl reduction verivate of above-mentioned lower molecular weight fucosylated glycosaminoglycan is the depolymerization product of Echinodermata Holothuroidea animal body wall and/or the internal organ carboxyl reduction verivate that extracts the fucosylated glycosaminoglycan that obtains or the carboxyl reduction verivate of fucosylated glycosaminoglycan depolymerization product.
Above-mentioned Echinodermata Holothuroidea animal includes but not limited to Thelenota ananas (Jaeger), huge Thelenota ananas (Jaeger), stichopus japonicus, lattice Pi Shi sea cucumber, black newborn ginseng, Bohadschia argus Jaeger, Stichopus chloronotus (Brandt)., red abdomen sea cucumber, Chinese sea cucumber, Haiti melon and rough sea cucumber.
The carboxyl reduction verivate of above-mentioned lower molecular weight fucosylated glycosaminoglycan and the preparation method of pharmacy acceptable salt thereof comprise:
(1) fucosylated glycosaminoglycan with echinoderms body wall and/or internal organ source is a raw material; Through the carboxyl reduction product of uronic acid carboxyl reduction reaction acquisition fucosylated glycosaminoglycan, gained carboxyl reduction product obtains the end product of desired molecule weight range through depolymerization reaction; Or
(2) depolymerization product with the fucosylated glycosaminoglycan in echinoderms body wall and/or internal organ source is a raw material, obtains the carboxyl reduction product of the fucosylated glycosaminoglycan of desired molecule weight range through the reaction of uronic acid carboxyl reduction.
The above-mentioned lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product and the preparation method of pharmacy acceptable salt thereof; The described uronic acid carboxyl reduction reaction in its method (1) and (2) is in acid water or water-bearing media, to carry out; Under the carbodiimide effect, react with reductive agent; Generate the carboxyl reduction verivate of fucosylated glycosaminoglycan; Wherein carbodiimide includes but not limited to 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), N, N '-dicyclohexyl carbodiimide (DCC) and aromatic series carbodiimide and their salt; Reductive agent is any reductive agent with reduction carboxylic acid halides ability, and these reductive agents include but not limited to hydroborate, sulfo-hydroborate and metal hydride.
Wherein in reaction system; The massfraction of said fucosylated glycosaminoglycan is 0.05%～15%; The concentration (in mass) of carbodiimide in reaction system is and the mass ratio of fucosylated glycosaminoglycan 1: 0.05 to 1: 20, and the concentration of reductive agent (in mass) is and the mass ratio of fucosylated glycosaminoglycan 1: 0.05 to 1: 20.The TR of reaction is 10 ℃～80 ℃.
The present invention provides a kind of pharmaceutical composition of anti-HIV-1 simultaneously, contains carboxyl reduction verivate or its pharmacy acceptable salt of the above-mentioned lower molecular weight fucosylated glycosaminoglycan of the present invention of effective anti-HIV-1 dosage, and pharmaceutical excipient.
The present invention also provides lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product and the application of pharmacy acceptable salt in the medicine of preparation prevention or treatment AIDS thereof.And be the application of pharmaceutical composition in the medicine of preparation prevention or treatment AIDS that effective constituent and pharmaceutical excipient are formed with lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product and pharmacy acceptable salt thereof.
The molecular weight of the carboxyl reduction derivative L CRG of lower molecular weight fucosylated glycosaminoglycan can adopt efficient gel chromatography (HPGPC) to detect.In weight-average molecular weight, the molecular weight ranges of the LCRG that the present invention selects is about 3,000～20; 000Da (being the average about 3～22 of the n of homologue shown in the formula (I)); Preferred molecular weight range is about 6,000～15,000Da (average about 3～16 of the n of homologue shown in the formula (I)).
The polydispersity index of LCRG (PDI, the ratio of weight average/number-average molecular weight is Mw/Mn) generally between 1.0 to 1.8; The PDI of preferred LCRG is between 1.1 to 1.5.
LCRG can be the salt of its pharmaceutically acceptable basic metal, earth alkali metal etc., and similarly, said LCRG also can be the ester that makes the formation of itself and alkaline organic group.The pharmacy acceptable salt of the preferred LCRG of the present invention is sodium salt, sylvite or the calcium salt of LCRG.
LCRG is the carboxyl reduction verivate of the fucosylated glycosaminoglycan depolymerization product extracted of Echinodermata Holothuroidea animal body wall and/or internal organ and/or is the depolymerization product again behind the said FGAG carboxyl reduction.
The present invention discovers that it is active that LCRG has strong effect anti-HIV-1, and it suppresses HIV-1 experiment strain, the lymphocytic EC of clinical separation strain infected person
50Value can be to about 2.5～100nM, and therapeutic index is greater than 1.0 * 10
4And under this drug effect concentration, there is not significant anticoagulating active in LCRG, does not influence blood coagulation system.
The external timesharing administration of the present invention discovers that LCRG acts on virus absorption and fusing stage, is the HIV-1 entry inhibitors, can effectively stop the invasion procedure of HIV-1 to host cell.Since act on the extracellular, therefore, as the advantage that has lower toxic side effect on the anti-HIV-1 clinical drug.The present invention detects further proof through interaction of biomacromolecules, and the pharmacotoxicological effect target spot of LCRG is the outer membrane glycoprotein gp120 of HIV-1; Adopt solubility CD4 (sCD4) and CD4i monoclonal antibody 17b and 48d etc. to interact and detect proof, the distinguishing feature of LCRG is that it combines gp gp120 conserved regions CD4i.
CD4 is the adherent CD4+ surface of cell membrane of a mediation HIV-1 acceptor; CD4i is gp120-CD4 zygotic induction naked position; The gp120 that exposes this position can further combine CD4+ surface of cell membrane co-receptor CCR5 or CXCR4; And causing HIV-1 gp gp41 to get into cytolemma, gp41 resets folding outer virionic membrane and the CD4+ cytolemma of causing and merges, and realizes finally that thus HIV-1 invades step.Obviously, blocking-up CD4i HIV-1 capable of blocking invades host CD4+ cell.Because the high conservative property of CD4i, help avoiding resistance to produce to the HIV-1 entry inhibitors of CD4i.Therefore, LCRG of the present invention has important use value as the HIV-1 entry inhibitors of overriding resistance.
The method that the present invention prepares LCRG is that the glucuronic acid carboxyl to FGAG carries out the method that reduction reaction prepares reductive derivative; FGAG reacts with reductive agent under the carbodiimide effect in water or water-bearing media; Products therefrom is used Fenton or Fenton-like reaction depolymerization, obtains title product through purifying.Specifically may further comprise the steps:
1) in water or water-bearing media, FGAG reacts with reductive agent under the carbodiimide effect;
2) carboxyl reduction product and peroxide reactions in the presence of catalyzer, the control reaction conditions prepares low molecular weight product;
3) stopped reaction, the low molecular weight product of collection and purifying desired molecule weight range.
Above-mentioned steps 1) and 2) order can change, promptly be to prepare the lower molecular weight sample by first depolymerization prototype FGAG, then depolymerization product is carried out carboxyl reduction reaction, obtain end product through the step 3) separation and purification.
In the step 1), the FGAG in said sea cucumber source is meant the acidic mucopolysaccharide that contains the Fucose component that from the said animal of Echinodermata Holothuroidea, extracts preparation.Its constitutional features is: in its monose is formed; Glucuronic acid (GlcUA) and 2-deoxidation-2-amino-N-ethanoyl-semi-lactosi or its sulfuric ester (GalNAc) exist mol ratio [1: (1 ± 0.3)] such as to be bordering on; And GlcUA and GalNAc interconnect the formation polysaccharide main chain with β 1 → 3 with β 1 → 4 glycosidic link respectively, and Fucose (Fuc) or its sulfuric ester are connected on the main chain GlcUA with side chain α 1 → 3 glycosidic link.
The difference of sea cucumber kind and tissue-derived difference or process for extracting can cause the difference of the aspects such as monose proportion of composing, side chain existence form and polysaccharide sulfated degree of FGAG; But these differences all do not relate to the constitutional features that there is carboxyl in FGAG, therefore do not influence the enforcement and the application of esterification process according to the invention.The source echinoderms of FGAG according to the invention can be selected from but be not limited to Thelenota ananas (Jaeger) (Thelenota ananasJaeger); Huge Thelenota ananas (Jaeger) (Thelenota anax H.L.Clark); Stichopus japonicus (ApostichopusjaponicusSelenaka); Lattice Pi Shi sea cucumber (Pearsonothuria graeffei Semper); Hojothuria leucospilota (Holothurialeucospilota Brandt); Black breast ginseng (Holothuria nobilis Selenka); Bohadschia argus Jaeger (Bohadschiaargus Jaeger); Stichopus chloronotus (Brandt). (Stichopus chloronotus Brandt); Red abdomen sea cucumber (Holothuria edulisLesson); China sea cucumber (Holothuria sinica Liao); Haiti melon (Acaudina molpadioidesSemper) and rough sea cucumber (Holothuria scabra Jaeger) etc.Obviously; It will be appreciated by those skilled in the art that; For other kind sea cucumber that originates in all over the world; The fucosylated glycosaminoglycan that meets the said structure characteristic in its source all can adopt carboxyl reduction method according to the invention to obtain required carboxyl reduction FGAG verivate, and therefore, the inventive method does not receive the restriction of specific sea cucumber kind.
In the step 1), carboxyl reduction is in acid water or water-bearing media, to carry out, and under the carbodiimide effect, reacts with reductive agent, to generate the carboxyl reduction verivate of FGAG.
The massfraction of said FGAG in reaction system is about 0.05%～15%.
Said carbodiimide can be in the acid-reaction system with FGAG on the carboxyl of glucuronic acid residue generate carboxylic acid halides, be reduced into alcoholic extract hydroxyl group to be easy to further to be reduced agent, and then form the carboxyl reduction product of said FGAG.These carbodiimide include but not limited to 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), N, and N '-dicyclohexyl carbodiimide (DCC) and aromatic series carbodiimide and their hydrochloride or other salt are preferably EDC.
The concentration (in mass) of said carbodiimide in reaction system, with the mass ratio of FGAG be 1: 0.05～1: 20.
Said reductive agent can be for having any reductive agent of reduction carboxylic acid halides ability, and these reductive agents include but not limited to hydroborate, sulfo-hydroborate, metal hydride etc., are preferably Peng Qinghuana.
The concentration (in mass) of said reductive agent in reaction system, with the mass ratio of FGAG be 1: 0.05～1: 20.
In the reduction reaction process of FGAG, the reductive agent reactant can be before reaction disposable all joining in the reaction system, also can adopt to continue or the intermittence mode progressively joins the reductive agent reactant in the reaction system.The present invention preferably continues the mode of reductive agent reactant according to controlled rate to join in the reaction system.
The common process parameter of said reduction reaction process is: the pH scope of the system of carbodiimide and FGAG effect is 1～7, and is preferred 2～6, optimum 4.75; TR is 10 ℃～80 ℃; Reaction times is 20 minutes～8 hours; Reaction can be carried out under normal pressure or pressurized conditions; Reaction can be selected to carry out under nitrogen, the protection of inert gas, also can be under condition of normal pressure communicates with atmospheric environment to carry out.
Step 2) in, depolymerization is in aqueous media, to carry out, and adopts the depolymerization reaction of metals ion as the catalyst superoxide, to generate lower molecular weight carboxyl reduction product.
The depolymerization method of FGAG carboxyl reduction product: it is 0.05%～10% the aqueous solution that FGAG carboxyl reduction product is made into massfraction; In obtained aqueous solution, add superoxide FGAG to massfraction 0.05%～5%; Add as the transition metal ion salt FGAG of catalyzer or prototype carboxyl reduction verivate to massfraction 0.01%～1%; Under 20～60 ℃, react to FGAG or prototype reductive derivative by depolymerization to the desired molecule weight range; The separation and purification depolymerization product, and be converted into the form of required pharmacy acceptable salt.
Reaction product LCRG can be through method purifying known in the art (CN 101735336A), for example removes impurity such as small molecule salt through dialysis method or ultrafiltration process, or be further purified through gel chromatography or DEAE ion exchange chromatography etc.
In the said dialysis impurity elimination treating processes, can select the dialysis membrane or the ultra-filtration membrane bag of suitable molecular weight cut-off according to the requirement of target LCRG molecular weight size, preferred molecular weight cut-off is 3000Da.Dialysis time needs to confirm according to the particular procedure condition, is no less than 6 hours usually.
Gained LCRG product can also be through cationic exchange to be prepared into single salt form, like sodium salt, sylvite or calcium salt etc.
The salification process of LCRG product can adopt and be exchanged into Hydrogen to sample earlier, and neutralizing with corresponding alkali then obtains the corresponding salt of LCRG; Also preferably the dynamic ion exchange salt forming method directly is exchanged into salt on post, wherein can select to adopt strongly acidic cationic exchange resin.Appearance all can be undertaken by ordinary method with wash-out on resin column pre-treatment, the sample.In the inventive method on the preferred sample appearance massfraction be about 2%～5%.
The inventive method can obtain the FGAG carboxyl reduction oligomerization product of a series of different molecular weight sizes, can be used as the HIV-resistant activity structure activity study, illustrates the active influence of molecular weight size antagonism HIV, with the FGAG carboxyl reduction verivate of preferred high-efficiency low-toxicity.
Gained LCRG product of the present invention can detect through the NMR method.NMR detects the existence that can also be advantageously used in confirming the glucone (Glc) in the product.Also can adopt the ratio of electrical conductivity method titration sulfate group and carboxyl, calculate the carboxyl reduction degree in view of the above.
Because it is active that the present invention's LCRG has strong effect anti-HIV-1; The further purpose of the present invention provides the pharmaceutical composition that contains LCRG; The LCRG of the present invention or its pharmacy acceptable salt that contain effective dose in the said pharmaceutical composition, and pharmaceutically acceptable vehicle.
Among the present invention, the pharmaceutical composition that contains LCRG can be the preparation that is administered systemically, and also can be local administration preparation.
Be administered systemically in the preparation, the pharmaceutical composition of LCRG can be to be fit to intravenously administrable, subcutaneous and/or intramuscular injection drug-delivery preparation, also can be respiratory tract administration prepn.In these preparations, the preferred pharmaceutical composition formulation is per os or nasal mist of aqueous solution for injection, freeze-dried powder injection and respirability canal drug administration etc.In the pharmaceutical composition that is administered systemically, the bioavailability of LCRG anti-HIV-1 activity and drug administration by injection thereof according to the present invention, the LCRG content in the single-dose preparations can be about 5～150mg
In the local administration preparation, the medicinal compsns formulation can be selected suppository, gelifying agent, ointment, liniment etc.Activeconstituents LCRG content in these medicinal prepnss can capable technician active drug concentration decision during according to local application.
Because the machine-processed target spot of the present invention's LCRG anti-HIV-1 is different from existing clinical application; Therefore can unite use with existing clinical application; Comprise and the pitch time of the administration in order of existing anti-HIV-1 medicine; Also can with the administration simultaneously of these medicines, perhaps become drug combination preparation with existing anti-HIV-1 medicine mutual group.
It is active that the present invention's lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate has strong effect anti-HIV-1, can be used as the anti-HIV-1 medicine and be used for treating and preventing AIDS.
Description of drawings
Fig. 1 is the heavy water hydrochloride IR collection of illustrative plates of Thelenota ananas (Jaeger) FGAG and carboxyl reduction product C RG-1 thereof;
Fig. 2 is the HPGPC collection of illustrative plates of Thelenota ananas (Jaeger) FGAG and lower molecular weight carboxyl reduction product LCRG-1 thereof;
Fig. 3 is Thelenota ananas (Jaeger) FGAG and LCRG-1's thereof
1H NMR spectrogram;
Fig. 4 is Thelenota ananas (Jaeger) FGAG and LCRG-1's
13C NMR spectrogram;
Fig. 5 is the restraining effect data plot that LCRG-1 forms HIV-1 inductive synplasm, shows the restraining effect that medicine forms HIV-1 inductive synplasm;
When Fig. 6 is LCRG-1 timesharing administration to HIV-1
IIIBPlasmodial inhibiting rate;
Fig. 7 is that LCRG-1 is to HIV-1
IIIBInvade active influence;
Embodiment
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with following embodiment of the present invention, but do not limit the scope of the invention with this.
The carboxyl reduction of [embodiment 1] fucosylated glycosaminoglycan
1.1 material
FGAG: the FGAG in Thelenota ananas (Jaeger), stichopus japonicus, hojothuria leucospilota source, press literature method (J.Biol.Chem., 1991,266 (21): 13530-13536) extract preparation.Purity is all greater than 98% (HPGPC, area normalization method).
1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), 1-phenyl-3-methyl-5-pyrazolones ketone (PMP), HCl, NaBH
4, agents useful for same such as NaCl: be commercially available AR; Chondroitin-4-suleate, glucose (Glc), glucuronic acid (GlcUA), acetamido semi-lactosi (GalNAc) and Fucose monose reference substances such as (Fuc) are the sigma Company products; DCl and D
2O is the Norell Company products.
1.2 method
Respectively take by weighing FGAG 300mg and be dissolved in 48ml water respectively, 0.1M HCl transfers pH to 4.75.Add 905mg solid EDC in about 5min time-histories, and transfer pH to make it keep 4.75 with 0.1M HCl.Slowly add 3.6g solid NaBH in the stirring
4, make solution under 50 ℃ of water-baths, keep 2h.Add small amount of acetic acid and destroy excessive Peng Qinghuana; Small molecular weight impurity is removed in deionized water dialysis (molecular weight cut-off 3500Da); And freeze-drying obtains the carboxyl reduction product; Numbering is respectively CRG-1, CRG-2 and CRG-3, and its quality is 235mg (yield 78.3%), 256mg (yield 85.3%) and 249mg (yield 83%) respectively.
Whether hexuronic acid changes into the judgement employing liquid cell IR method of glucose in the product.Detecting instrument is FT-IR EQUINOX-55ATR (liquid cell) (Bruker, a Germany); Each sample of FGAG and CRG and each 15mg of reference sample chondroitin-4-suleate (CS-4S) are dissolved in 0.25ml 1.0mol/L DCl/D
2Among the O, and the reference method (Carbohydr.Res., 1978,63:13-27) make an experiment.
Use specific conductivity volumetry detection-OSO further
3 -/-COO
-Mol ratio, calculate in view of the above carboxyl reduction degree (Zhang Weijie, saccharide complex Biochemical Research technology (second edition), Zhejiang: press of Zhejiang University, 1999,409-410).
PMP derivatize HPLC method is analyzed monose composition (chemical journal, 2007,65 (23): 2761-2764) before adopting post.Chromatographiccondition is Agilent 1200 high performance liquid chromatographs, Waters Symmetry C18 (150mm * 4.6mm * 5 μ m) chromatographic column, 30 ℃ of column temperatures; Moving phase 0.1M acetate (pH 5.5) damping fluid-acetonitriles (78: 22), flow velocity 1ml/min; Detect wavelength 250nm; Sampling volume 40 μ l.
1.3 result
Fig. 1 has provided the IR spectrogram of stichopus japonicus FGAG and carboxyl reduction product thereof, does not see the carbonyl signal on the glucose uronic acid among the figure, and hence one can see that, and carboxyl reduction is complete basically.The IR spectrogram of the carboxyl reduction product of other two kinds of sea cucumber FGAG is similar, does not also see the carboxyl signal.
The electrical conductivity method data presentation has only the flex point of a sulfate group, explains that the carboxyl of glucuronic acid is reduced fully among the FGAG.The monose compositional analysis data presentation of sample is not seen glucuronic acid before and after the carboxyl reduction behind the carboxyl reduction, has only glucose, acetamido semi-lactosi and Fucose, and three's mol ratio is: 1: 1: 1.05.
[embodiment 2] superoxide depolymerization prepares low molecular weight product
2.1 material
CRG-1: the FGAG carboxyl reduction product in Thelenota ananas (Jaeger) source, by the preparation of [embodiment 1] method.Purity 98% (HPGPC, area normalization method), weight-average molecular weight (Mw), 68,840.
H
2O
2, CH
3COONa3H
2O, NaCl, NaOH, Cu (CH
3COO)
2H
2Agents useful for same such as O: be commercially available AR.
2.2 method
Prepare oligomeric carboxyl reduction product: each 5.0g of Thelenota ananas (Jaeger) CRG-1 is dissolved in the 180ml aqueous solution in the round-bottomed flask, and 35 ℃ of water bath heat preservations and lasting even the stirring add the cupric chloride (Cu of 50mM concentration then respectively respectively
2+) solution, in 2 hours, drip 15% H then with 10ml/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8.The continuously stirring reaction added 500mg EDTA disodium salt and mixing respectively after 4 hours under the above-mentioned condition in reaction solution, and frozen water cools off, and adds 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal must the deposition, 100ml 60% washing with alcohol twice, centrifugal must the deposition.Resolution of precipitate becomes sodium salt through 001 * 7 type resin cation exchange in 150ml water, dialysed 6 hours with the dialysis membrane of molecular weight cut-off 3500Da then, holds back product and concentrates, and lyophilize obtains the 3.82gLCRG-1 sample.
Physics and chemistry detects: HPGPC detection molecules amount and distribution; Conductometric titration detection-OSO
3 -/-COO
-Mol ratio.
NMR detects:
Instrument, AVANCE AV 400 superconduction nuclear magnetic resonance spectrometers (600MHz of Switzerland Bruker company);
Solvent, D
2O 99.9 Atom%D (Norell company);
Interior mark, trimethylsilyl-propionic acid (TSP-D
4); Temperature, 27 ℃.
2.3 result
Detected result sees the following form 1.HPGPC spectrogram 2, the NMR spectrogram is seen accompanying drawing 3 and Fig. 4.
Can be known that by table 1 and Fig. 2 data FGAG carboxyl reduction product can obtain its oligomerization product LCRG-1 through the hydrogen oxidation catalyst depolymerization, and yield is higher, MWD is narrower, and electrical conductivity method does not detect the existence of carboxyl.Pre-column derivatization HPLC method monose compositional analysis result shows (table 1), and carboxyl reduction method provided by the invention can realize complete carboxyl reduction.
FGAG and LCRG-1 in the annex map 3 and 4
1H NMR with
13The spectral data of C NMR is visible; At the 3.35ppm place is that glucone H5 and 60.4ppm place are glucone C5; Having confirmed that the carboxyl among the FGAG has been reduced into alcoholic extract hydroxyl group, promptly is to have realized that the glucal acidic group (GlcUA) among the FGAG is transformed into glucone (Glc).
The monose mol ratio of table 1 FGAG, CRG-1 and LCRG-1 and physics and chemistry detected result
Mw: weight-average molecular weight; Mn: number-average molecular weight; PDI: molecular weight polydispersity index (PDI=Mw/Mn)
The HIV-resistant activity of [embodiment 3] lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product LCRG-1
3.1 material
(1) test sample: the LCRG-1 (Mw 12930Da) of [embodiment 2] preparation is dissolved in the water for injection, and being mixed with concentration is the 25mg/ml storage solutions, preserves down for 4 ℃.
(2) reagent: MTT (3-(4,5)-dimethylthiahiazo-2-y1)-2,5-diphenyltetrazoliumbromide), SDS (Sodium Dodecyl Sulfate), DMSO reagent such as (Dimethyl sulfoxide) be commercially available, analytical pure.Blood coagulation Quality Control blood plasma, activated partial thromboplastin time (APTT) are measured test kit, and thrombin time (TT) is measured test kit, and prothrombin time test kit (PT-dry powder) is German TECO GmbH company and produces; Other all reagent are commercially available analytical pure.
Instrument: MC-4000 coagulo meter (German Megtron company); ELx800ELISAReader ELIASA (U.S. Bao Te company)
(3) cell and virus: C8166, HIV-1
IIIB/2802/2840/9495And HIV-2
CBL-20/RODDeng by Britain Medical Research council, AIDS Reagent Project is so kind as to give.Prepare HIV-1 by ordinary method
IIIB, titration also calculates viral TCID
50After the packing of virus storage liquid, put-70 ℃ of preservations.The all frozen and recoveries of cell and virus by ordinary method.
3.2 method
(1) LCRG-1 detects the cytotoxicity of C8166: C8166 cell suspension (4 * 10
5/ ml) 100 μ l mix 37 ℃ of following 5%CO with the LCRG-1 of 100 μ l series concentration
2Cultivated three days, mtt assay detects cytotoxicity.The 570/630nm ELIASA is measured the OD value, calculates CC
50(50%Cytotoxicconcentration promptly causes the drug level of half necrocytosis).
(2) LCRG-1 induces active detection of inhibition of C8166 pathology to HIV-1: with C8166 cell suspension (8 * 10
5/ ml) 50 μ l mix with the LCRG-1 of 100 μ l different concns, add 50 μ l HIV-1 dilution supernatant, and M.O.I. is 0.0091, and the blank hole is set simultaneously, 37 ℃ of following 5%CO
2Cultivated three days, next five visual field of hundred multiplying powers of inverted microscope are the counting synplasm down.Calculate LCRG-1 inhibition HIV-1 and induce the EC of C8166 pathology
50(50%Effective Concentration promptly suppresses the LCRG-1 concentration that half synplasm forms).In conjunction with preceding text (1) gained CC
50Value is calculated SI (CC
50/ EC
50, Therapeutic Index, TI).
(3) LCRG-1 induces active detection of inhibition of C8166 pathology to HIV-2: the said method of again (2) detects LCRG-1 and induces the inhibition of C8166 pathology active to HIV-2.
(4) the active detection of inhibition of C8166 pathology induced in LCRG-1 timesharing administration to HIV-1: on 96 porocyte culture plates, every hole adds 4 * 10
4Individual C8166 cell and 20 μ l HIV-1
IIIBSupernatant diluent (M.O.I.=8) places 37 ℃, 5%CO
2Cultivate in the incubator.In infection preceding 2 hours, infect the LCRG-1 that the back added testing sample 10 μ g/ml in 0,1,2,4,8,16,24 hours respectively, each time is established three repeating holes, and in infection back 72 hours, in inverted microscope counting synplasm number down.
(5) SPR method detection LCRG-1 and gp120 interact: LCRG-1 and gp120 interaction detect (BIAcore 3000 biosensor system) through the method for surface plasma body resonant vibration technology SPR; With behind the CM-5 chip activation gp120 being fixed on the CM-5 chip surface, the sample of different concns is diluted with damping fluid HBS-EP.The sample that dilution is good is placed on the specimen holder, and setting the application of sample flow velocity is 10 μ l/min, selects inject mode sample introduction.Finish the back and carry out kinetic constant through BIAcore 3000 software analysis systems.
(6) LCRG-1 influences coagulation function: activated partial thromboplastin time (APTT) method detects LCRG-1 to be influenced coagulation function.Practical situation according to sample are mixed with series concentration with deionized water dissolving, test on the MC-4000 coagulo meter according to the method that provides in APTT, PT, three test kit specification sheetss of TT blood coagulation respectively, to investigate anticoagulant active.
3.3 result
(1) LCRG-1 HIV-resistant activity: the result shows in accompanying drawing 5,6,7 and the table 2, and LCRG-1 is the strong new texture activeconstituents that suppresses HIV-1, the EC of its anti-HIV-1 of imitating
50Be about 13nM (0.17 μ g/ml); Do not see significant cytotoxicity according to preparation maximum concentration medicine (25mg/ml), its therapeutic index can reach more than 150,000; To the inhibition activity of HIV-2 then a little less than.Timesharing administration result shows that LCRG-1 acts on virus absorption and fusing stage, shows that it is entry inhibitors (accompanying drawing 6).
Table 2 LCRG-1 HIV-resistant activity detected result
(2) the active action target spot of LCRG-1 anti-HIV-1: the SPR detected result shows that LCRG-1 can combine gp120 by high-affinity, does not combine CD4 is then significant; After solubility CD4 (sCD4) combined gp120, LCRG-1 combined the avidity of CD4i further to strengthen, and shows that LCRG-1 can combine CD4 to induce the gp120 conserved regions CD4i of exposure.Because CD4i has high conservative property, can effectively avoid the resistance due to the viral DNA sudden change to produce in conjunction with this regional gp120 suppressor factor.
(3) LCRG-1 is to the influence of coagulation function: table 3 result shows, under effective anti-HIV-1 virus dosage (0.1～8.0 μ g/ml), it is active not find that significant APTT, PT and TT prolong for LCRG-1, and explaining does not influence coagulation function.
Table 3 LCRG-1 anticoagulant active detected result
The anti-HIV-1 of [embodiment 4] serial molecular weight size LCRG is active
4.1 material
Series lower molecular weight carboxyl esterification product sample: the depolymerization method that stichopus japonicus FGAG provides according to [embodiment 2] obtains the depolymerization product of serial different molecular weight size through the depolymerization of controlling reaction time elder generation; The carboxyl reduction method that provides according to [embodiment 1] is then carried out reduction reaction respectively; Through control and EDC reaction pH value; Conditions such as the add-on of NaBH4 and reaction times can prepare the sample of different carboxyl reduction degree.Behind separation and purification IX salify, (LCRG-2～LCRG-6), yield is all greater than 65% finally to obtain the sodium salt of LCRG of 5 different molecular weights size; Electrical conductivity method and pre-column derivatization methods analyst monose proportion of composing calculate carboxyl reduction degree (referring to [embodiment 1]) with this.
Other materials and reagent see above [embodiment 1～3].
4.2 method
HIV-resistant activity detection method:, detect LCRG-2～LCRG-6 respectively and induce the inhibition of C8166 pathology active to the cytotoxicity of C8166 and to HIV-1 according to the said method of preceding text [embodiment 3].
4.3 result
Table 4 is seen in molecular weight and the distribution thereof of stichopus japonicus series LCRG, and the activity of its anti-HIV-1 is seen table 5.
The molecular weight of the lower molecular weight carboxyl reduction product of table 4 stichopus japonicus FGAG and distribution thereof and monose are formed
The result of table 4 shows, can obtain the big or small low molecular weight product of a series of different molecular weights according to method provided by the invention, and its MWD is narrower.Electrical conductivity method detection computations result shows that the carboxyl reduction degree is greater than 20%.
The active result of the anti-HIV-1 of the lower molecular weight carboxyl reduction product of table 5 stichopus japonicus FGAG
Table 5 experimental result shows that under experiment condition according to the invention, 5 samples are very low to the toxicity of C8166 cell, and the activity of external anti-HIV-1 is very strong, and therapeutic index is all greater than 1 * 10
4Relatively the molecular weight of these samples and the relation of anti-HIV-1 can know that molecular weight reduces its anti-HIV-1 activity and weakens to some extent, but still stronger.Hence one can see that, and preferred LCRG molecular weight is not less than 4000Da.
The freeze-dried products of [embodiment 5] lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product
5.1 material
Lattice Pi Shi sea cucumber (Pearsonothuria graeffei Semper) LCRG-7, Mw 8,050.Depolymerization method preparation by FGAG carboxyl reduction product in [embodiment 2].
5.2 prescription
| The supplementary material title | Consumption |
| LCRG-7 | 50g |
| Water for injection | 500ml |
| Process altogether | 1000 |
5.3 preparation technology
The LCRG-7 that takes by weighing recipe quantity adds to the full amount of water for injection, and stirs to make dissolving fully the sterilization of intermittent type pressure sintering.The medicinal carbon of adding 0.6% stirs 20min; Use B and 3.0 μ m millipore filtration decarbonization filterings to remove thermal source.Survey midbody content.Qualified back is with the filtering with microporous membrane of 0.22 μ m; Can in the control cillin bottle, every bottle of 0.5ml, pouring process monitoring loading amount, half tamponade is put in the freeze drying box, carries out freeze-drying by the freeze-drying curve of setting, tamponade, outlet rolls lid, visual inspection is qualified, finished product.
Freeze-drying process: with the sample inlet, fall the dividing plate temperature, keep 3h to-40 ℃; Cold-trap is reduced to-50 ℃, begins to be evacuated to 300 μ bar.Begin distillation: 1h at the uniform velocity is warming up to-30 ℃, keeps 2h; 2h at the uniform velocity is warming up to-20 ℃, keeps 8h, and vacuum keeps 200～300 μ bar; Carry out drying: 2h again and be warming up to-5 ℃, keep 2h, vacuum keeps 150～200 μ bar; 0.5h be warming up to 10 ℃, keep 2h, vacuum keeps 80～100 μ bar; 0.5h be warming up to 40 ℃, keep 4h, vacuum is evacuated to minimum.
The vaginal suppository of [embodiment 6] lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product
6.1 material
The lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product ([embodiment 2] gained LCRG-1) in Thelenota ananas (Jaeger) source, its weight-average molecular weight 12930Da.Reagent such as ethylparoben and glycogelatin is commercially available, medicinal rank.
6.2 prescription
| The supplementary material title | Consumption |
| LCRG-1 | 100g |
| Water for injection | 400ml |
| Ethylparoben | 2.0g |
| Glycogelatin adds to | 4000g |
| Process altogether | 1000 pieces |
6.3 preparation method
Get lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product and add the dissolving of injection water, add the ethylparoben stirring and dissolving, add an amount of glycerine again and stir, slowly add in the gelatin glycerine matrix, fully stir, be incubated 55 ℃, irritate mould, every piece heavy 4g.
The gelifying agent of [embodiment 7] lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product
7.1 material
The lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product ([embodiment 4] gained LCRG-3) in stichopus japonicus source, its weight-average molecular weight 11005Da.Reagent such as cross linked sodium polyacrylate, PEG 400, Morpan BB and glycerine are commercially available, medicinal rank.
7.2 prescription
| The supplementary material title | Consumption |
| LCRG-3 | 10.0g |
| Cross linked sodium polyacrylate (SDB-L-400) | 10.0g |
| PEG 400 (PEG-400) | 80.0g |
| Morpan BB | 10.0ml |
| Glycerine | 100.0g |
| Zero(ppm) water adds to | 1000g |
7.3 preparation method
Get the dissolving of LCRG-3 adding distil water; Take by weighing PEG-400, glycerine and put in the beaker low-grade fever to dissolving fully; Add lower molecular weight glycosyl chondroitin sulfate cellulose solution mixing, SDB-L-400 adds after 700ml water grinds well in mortar, with matrix and PEG-400, glycerine, LCRG-3 mixing; Add water to 1000g, can promptly gets.
Claims (13)
1. the mixture with homology TGSS C3 verivate of formula (I) structure is the carboxyl reduction derivative L CRG and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan,
In the formula (I):
N is that average is 3～22 integer;
R is independently of each other-H or-SO
3 -
2. the carboxyl reduction verivate and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan as claimed in claim 1, wherein:
The monose of the carboxyl reduction verivate of said lower molecular weight fucosylated glycosaminoglycan is formed and is comprised D-glucose and/or D-glucuronic acid, D-2-deoxidation-2-acetylamino galactosamine sulfuric ester, L-Fucose sulfuric ester; With molar ratio computing, monose is formed and with the proportional range of contained sulfate group is: (D-glucone+D-glucal acidic group): (D-2-deoxidation-2-acetylamino galactosamine base): (L-fucosido): (sulfate group)=1: (1 ± 0.3): (1 ± 0.3): (3.0 ± 1.0); And the D-glucone: the molar ratio range of (D-glucone+D-glucal acidic group) is 20%～100%;
The weight average molecular weight range of said lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate is 3000～20000Da; Polydispersity index is between 1.0 to 1.8.
3. according to claim 1 or claim 2 lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate and pharmacy acceptable salt thereof; It is characterized in that the weight average molecular weight range of said lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate is 6000～15000Da; The polydispersity index value is between 1.1 to 1.5.
4. like the amine glycan carboxyl reduction verivate and the pharmacy acceptable salt thereof of claim 1 or the fucosylated sugar of 2 or 3 described lower molecular weights; It is characterized in that said pharmacy acceptable salt is basic metal, alkaline earth salt and the organic ammonium salt of the carboxyl reduction verivate of lower molecular weight fucosylated glycosaminoglycan.
5. like the carboxyl reduction verivate and the pharmacy acceptable salt thereof of claim 1 or 2 or 3 described lower molecular weight fucosylated glycosaminoglycans; It is characterized in that said pharmacy acceptable salt is sodium salt, sylvite or the calcium salt of lower molecular weight fucosylated glycosaminoglycan carboxyl reduction verivate.
6. like the carboxyl reduction verivate and the pharmacy acceptable salt thereof of each described lower molecular weight fucosylated glycosaminoglycan of claim 1 to 5; It is characterized in that the carboxyl reduction verivate of said lower molecular weight fucosylated glycosaminoglycan is the depolymerization product of Echinodermata Holothuroidea animal body wall and/or the internal organ carboxyl reduction verivate that extracts the fucosylated glycosaminoglycan that obtains or the carboxyl reduction verivate of fucosylated glycosaminoglycan depolymerization product.
7. the carboxyl reduction verivate and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan as claimed in claim 6; It is characterized in that said Echinodermata Holothuroidea animal includes but not limited to Thelenota ananas (Jaeger), huge Thelenota ananas (Jaeger), stichopus japonicus, lattice Pi Shi sea cucumber, black newborn ginseng, Bohadschia argus Jaeger, Stichopus chloronotus (Brandt)., red abdomen sea cucumber, Chinese sea cucumber, Haiti melon and rough sea cucumber.
8. like the carboxyl reduction verivate of each described lower molecular weight fucosylated glycosaminoglycan of claim 1 to 7 and the preparation method of pharmacy acceptable salt thereof, comprising:
(1) fucosylated glycosaminoglycan with echinoderms body wall and/or internal organ source is a raw material; Through the carboxyl reduction product of uronic acid carboxyl reduction reaction acquisition fucosylated glycosaminoglycan, gained carboxyl reduction product obtains the end product of desired molecule weight range through depolymerization reaction; Or
(2) depolymerization product with the fucosylated glycosaminoglycan in echinoderms body wall and/or internal organ source is a raw material, obtains the carboxyl reduction product of the fucosylated glycosaminoglycan of desired molecule weight range through the reaction of uronic acid carboxyl reduction.
9. the preparation method of lower molecular weight fucosylated glycosaminoglycan carboxyl reduction product as claimed in claim 8 and pharmacy acceptable salt thereof; It is characterized in that; The described uronic acid carboxyl reduction reaction in its method (1) and (2) is in acid water or water-bearing media, to carry out; Under the carbodiimide effect, react with reductive agent; Generate the carboxyl reduction verivate of fucosylated glycosaminoglycan, wherein carbodiimide includes but not limited to 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), N, N '-dicyclohexyl carbodiimide (DCC) and aromatic series carbodiimide and their salt; Reductive agent is any reductive agent with reduction carboxylic acid halides ability, and these reductive agents include but not limited to hydroborate, sulfo-hydroborate and metal hydride.
10. preparation method as claimed in claim 8; Wherein in reaction system; The massfraction of said fucosylated glycosaminoglycan is 0.05%～15%; The concentration (in mass) of carbodiimide in reaction system is and the mass ratio of fucosylated glycosaminoglycan 1: 0.05 to 1: 20, and the concentration of reductive agent (in mass) is and the mass ratio of fucosylated glycosaminoglycan 1: 0.05 to 1: 20.The TR of reaction is 10 ℃～80 ℃.
11. the pharmaceutical composition of an anti-HIV-1 contains carboxyl reduction verivate or its pharmacy acceptable salt of each described lower molecular weight fucosylated glycosaminoglycan of claim 1～5 of effective anti-HIV-1 dosage and pharmaceutical excipient.
12. the described lower molecular weight fucosylated glycosaminoglycan of claim 1-5 carboxyl reduction product and pharmacy acceptable salt thereof the application in the medicine of preparation prevention or treatment AIDS.
13. the application of the described pharmaceutical composition of claim 11 in the medicine of preparation prevention or treatment AIDS.
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