CN102558389B - 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 derivative (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 acquired immune deficiency syndrome (AIDS) thereof that contain the carboxyl reduction derivative of this lower molecular weight fucosylated glycosaminoglycan.
Background technology
Human immunodeficiency virus (human immunodeficiency virus, HIV) infect due to acquired immune deficiency syndrome (AIDS) (aquired immunodeficiency syndrome AIDS) is the major disease of serious harm human life health.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 (as hiv reverse transcriptase/proteinase inhibitor) of AIDS, but the clinical treatment of AIDS still exist be difficult to cure, problems such as treatment no response, resistance and serious toxic side effect.Therefore, the clinical novel therapeutic medicine that presses for particularly needs to form complementary novel targets new mechanism original new drug with existing clinical application.
In the inverase research and development, mainly concentrate on reverse transcriptase inhibitors 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 inhibitor, entry inhibitors and ripe inhibitor etc. grinding novel targets new mechanism inverase.
In the new mechanism inverase, need not to stride the advantage that film enters the drug transport aspect of cell owing to exist, HIV entry inhibitors especially receive publicity (PNAS, 2003,100:10598-10602; 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, hereinafter to be referred as FGAG), referring to that a class is extracted from echinoderms body wall or internal organ obtains, has the chondroitin sulfate of being similar to backbone structure, but have glycosaminoglycan derivative (J.Biol.Chem., 1988,263 (34): 18176-181783 and J.Biol.Chem. that the side chain sulfated fucose replaces, 1991,266 (21): 13530-13536).
The FGAG of natural origin has significant HIV (human immunodeficiency virus)-resistant activity (lst International AIDS SocietyConference on HIV Pathogenesis and Treatment, 2001, Jul 8-11, Abstract No.247), yet FGAG also has non-selective pharmacologically active widely, 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 potent HIV (human immunodeficiency virus)-resistant activity, evades it to the influence of 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 (human immunodeficiency virus)-resistant activity, but do not pointed out it whether to influence blood coagulation system the FGAG depolymerization in stichopus japonicus source by directly.Chinese patent application CN201110114860.4 provides a kind of novel low molecular weight amount glycosylation chondroitin sulfate LGC, and this compound and the composition 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 at the open report of anti-HIV application facet.
Summary of the invention
The object of the invention provides a kind of carboxyl reduction derivative (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 derivative of this lower molecular weight fucosylated glycosaminoglycan, this derivative and medicinal compositions thereof can be in conjunction with the conserved regions CD4i of the surface glycoprotein gp120 of HIV-1, the efficient HIV-1 virus invasion procedure that stops, and do not influence coagulation process, can be used for prevention or treatment acquired immune deficiency syndrome (AIDS).
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of mixture with homology glycosaminoglycan derivative of formula (I) structure is 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 -
Carboxyl reduction derivative and the pharmacy acceptable salt thereof of aforesaid lower molecular weight fucosylated glycosaminoglycan, wherein:
The monose of the carboxyl reduction derivative of described 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-glucosyl group+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-glucosyl group: the molar ratio range of (D-glucosyl group+D-glucal acidic group) is 20%～100%;
The weight average molecular weight range of described lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative is 3000～20000Da; Polydispersity index is between 1.0 to 1.8.
The weight average molecular weight range of described lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative 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 derivative 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 derivative.
The carboxyl reduction derivative 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 derivative that extracts the fucosylated glycosaminoglycan that obtains or the carboxyl reduction derivative 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 derivative 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 raw material, by 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 by depolymerization reaction; Or
(2) depolymerization product with the fucosylated glycosaminoglycan in echinoderms body wall and/or internal organ source is raw material, obtains the carboxyl reduction product of the fucosylated glycosaminoglycan of desired molecule weight range by the reaction of uronic acid carboxyl reduction.
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 to carry out in acid water or water-bearing media, under the carbodiimide effect, react with reductive agent, generate the carboxyl reduction derivative 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 described 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 temperature range of reaction is 10 ℃～80 ℃.
The present invention provides a kind of pharmaceutical composition of anti-HIV-1 simultaneously, contains carboxyl reduction derivative 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 acquired immune deficiency syndrome (AIDS) thereof.And be the application of pharmaceutical composition in the medicine of preparation prevention or treatment acquired immune deficiency syndrome (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, alkaline-earth metal etc., and similarly, described 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 derivative of the fucosylated glycosaminoglycan depolymerization product extracted of Echinodermata Holothuroidea animal body wall and/or internal organ and/or is depolymerization product again behind the described FGAG carboxyl reduction.
The present invention discovers that LCRG has potent anti-HIV-1 activity, 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 the host cell of HIV-1.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 by 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 is in conjunction with glycoprotein gp120 conserved regions CD4i.
CD4 is the CD4+ surface of cell membrane acceptor that mediation HIV-1 adheres to, CD4i is gp120-CD4 zygotic induction naked position, the gp120 that exposes this position can be further combined with CD4+ surface of cell membrane co-receptor CCR5 or CXCR4, and cause HIV-1 glycoprotein gp41 to enter 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 of CD4i, be conducive to avoid resistance to produce at 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 target 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, namely 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 described sea cucumber source refers to extract the acidic mucopolysaccharide that contains the Fucose component of preparation from the described animal of Echinodermata Holothuroidea.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 are interconnected to constitute polysaccharide main chain with β 1 → 3 and 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 extracting method 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 enforcement and the application of esterification process of the present invention.The source echinoderms of FGAG of the present 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 feature in its source all can adopt carboxyl reduction method of the present invention to obtain required carboxyl reduction FGAG derivative, therefore, the inventive method is not subjected to the restriction of specific sea cucumber kind.
In the step 1), carboxyl reduction is to carry out in acid water or water-bearing media, reacts with reductive agent under the carbodiimide effect, to generate the carboxyl reduction derivative of FGAG.
The massfraction of described FGAG in reaction system is about 0.05%～15%.
Described 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 described 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 described carbodiimide in reaction system, with the mass ratio of FGAG be 1: 0.05～1: 20.
Described 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 sodium borohydride.
The concentration (in mass) of described 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 described 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; Temperature range 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 to carry out in aqueous media, adopts metal ion as the depolymerization reaction of 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 derivative 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 by method purifying known in the art (CN 101735336A), for example removes impurity such as small molecule salt by dialysis method or ultrafiltration process, or be further purified by gel chromatography or DEAE ion exchange chromatography etc.
In the described dialysis impurity elimination treating processes, can select 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 determine according to the particular procedure condition, is no less than 6 hours usually.
Gained LCRG product can also be by cationic exchange to be prepared into single salt form, as sodium salt, sylvite or calcium salt etc.
The salification process of LCRG product can adopt and earlier sample is exchanged into Hydrogen, and neutralizing with corresponding alkali then obtains the salt of LCRG correspondence; Also preferably the dynamic ion exchange salt forming method directly is exchanged into salt at post, wherein can select to adopt storng-acid cation exchange resin.Sample and wash-out all can carry out according to a conventional method on resin column pre-treatment, the sample.In the inventive method on the preferred sample sample 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 (human immunodeficiency virus)-resistant activity structure activity study, illustrates the molecular weight size to the influence of HIV (human immunodeficiency virus)-resistant activity, with the FGAG carboxyl reduction derivative of preferred high-efficiency low-toxicity.
Gained LCRG product of the present invention can detect by the NMR method.NMR detects the existence that can also be advantageously used in confirming the glucosyl group (Glc) in the product.Also can adopt the ratio of electrical conductivity method titration sulfate group and carboxyl, calculate the carboxyl reduction degree accordingly.
Because the present invention's LCRG has potent anti-HIV-1 activity, 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 described 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 preparation.In these preparations, the preferred pharmaceutical composition formulation is the per os of aqueous solution for injection, freeze-dried powder injection and respirability canal drug administration or nasal mist 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 compositions formulation can be selected suppository, gelifying agent, ointment, liniment etc.Activeconstituents LCRG content in these medicinal preparationss can capable technician active drug concentration during according to local application determine.
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 turn of existing anti-HIV-1 medicine, also can with the administration simultaneously of these medicines, perhaps form drug combination preparation jointly with existing anti-HIV-1 medicine.
The present invention's lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative has potent anti-HIV-1 activity, can be used as the anti-HIV-1 medicine and is used for the treatment of and prevents 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;
The restraining effect data plot that the synplasm that Fig. 5 induces for the HIV-1 of LCRG-1 forms, the restraining effect that the synplasm that the demonstration medicine is induced HIV-1 forms;
When Fig. 6 is LCRG-1 timesharing administration to HIV-1
IIIBPlasmodial inhibiting rate;
Fig. 7 is the HIV-1 of LCRG-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 analytical reagent; Chondroitin-4-suleate, glucose (Glc), glucuronic acid (GlcUA), acetamido semi-lactosi (GalNAc) and Fucose monose reference substances such as (Fuc) are sigma company product; DCl and D
2O is Norell company product.
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 sodium borohydride, 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, 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) test.
Use specific conductivity volumetry detection-OSO further
3 -/-COO
-Mol ratio, calculate accordingly 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 substantially.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, illustrates 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 analytical reagent.
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 continue evenly to stir 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, the NaOH solution control pH value scope with 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 precipitation, 100ml 60% washing with alcohol twice, centrifugal must the precipitation.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.
By table 1 and Fig. 2 data as can be known, FGAG carboxyl reduction product can obtain its oligomerization product LCRG-1 through the hydrogen oxidation catalyst depolymerization, and yield is higher, and molecular weight distribution 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 and
13The spectral data of C NMR as seen, be that glucosyl group H5 and 60.4ppm place are glucosyl group C5 at the 3.35ppm place, having confirmed that the carboxyl among the FGAG has been reduced into alcoholic extract hydroxyl group, namely is to have realized that the glucal acidic group (GlcUA) among the FGAG is transformed into glucosyl group (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 (human immunodeficiency virus)-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 microplate reader (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 according to a conventional method
IIIB, titration also calculates viral TCID
50After the packing of virus storage liquid, put-70 ℃ of preservations.Cell and virus is frozen and recovery according to a conventional method all.
3.2 method
(1) cytotoxicity of the C8166 of LCRG-1 detects: 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 microplate reader is measured the OD value, calculates CC
50(50%Cytotoxicconcentration namely causes the drug level of half necrocytosis).
(2) the HIV-1 of LCRG-1 induces active detection of inhibition of C8166 pathology: 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, inverted microscope is counted synplasm under next five visual field of hundred multiplying powers.Calculate LCRG-1 inhibition HIV-1 and induce the EC of C8166 pathology
50(50%Effective Concentration namely suppresses the LCRG-1 concentration that half synplasm forms).In conjunction with (1) gained CC above
50Value is calculated selectivity index (CC
50/ EC
50, Therapeutic Index, TI).
(3) the HIV-2 of LCRG-1 induces active detection of inhibition of C8166 pathology: above (2) described method detects the inhibition activity that the HIV-2 of LCRG-1 induces the C8166 pathology together.
(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, counting synplasm number under inverted microscope.
(5) SPR method detection LCRG-1 and gp120 interact: LCRG-1 and gp120 interaction detect (BIAcore 3000 biosensor system) by 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, with the damping fluid HBS-EP dilution of the sample of different concns.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 by BIAcore 3000 software analysis systems.
(6) the coagulation function of LCRG-1 influences: activated partial thromboplastin time (APTT) method detects the coagulation function of LCRG-1 to be influenced.Practical situation according to sample are mixed with series concentration with deionized water dissolving, test at 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 (human immunodeficiency virus)-resistant activity: accompanying drawing 5,6,7 and table 2 in the result show that LCRG-1 is the new texture activeconstituents of potent inhibition HIV-1, the EC of its anti-HIV-1
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 (human immunodeficiency virus)-resistant activity detected result
(2) action target spot of LCRG-1 anti-HIV-1 activity: the SPR detected result shows, LCRG-1 can high-affinity in conjunction with gp120, CD4 is not then had significant combination; Solubility CD4 (sCD4) is in conjunction with behind the gp120, and LCRG-1 further strengthens in conjunction with the avidity of CD4i, shows that LCRG-1 can induce the gp120 conserved regions CD4i of exposure in conjunction with CD4.Because CD4i has high conservative, can effectively avoid the resistance due to the viral DNA sudden change to produce in conjunction with this regional gp120 inhibitor.
(3) influence of the coagulation function of LCRG-1: table 3 result shows that 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 illustrating does not influence coagulation function.
Table 3 LCRG-1 anticoagulant active detected result
The anti-HIV-1 activity of [embodiment 4] serial molecular weight size LCRG
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 by the elder generation's depolymerization of control reaction times, the carboxyl reduction method that provides according to [embodiment 1] is carried out reduction reaction respectively then, by 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 ion-exchange salify, (LCRG-2～LCRG-6), yield is all greater than 65% finally to obtain the sodium salt of the LCRG of 5 different molecular weight sizes; 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 (human immunodeficiency virus)-resistant activity detection method: according to [embodiment 3] described method above, detect the cytotoxicity of the C8166 of LCRG-2～LCRG-6 and the inhibition activity of HIV-1 being induced the C8166 pathology respectively.
4.3 result
Molecular weight and the distribution thereof of stichopus japonicus series LCRG see Table 4, and the activity of its anti-HIV-1 sees 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 low molecular weight product of a series of different molecular weight sizes according to method provided by the invention, and its molecular weight distribution 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 of the present 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 relation of the molecular weight of these samples and anti-HIV-1 as can be known, 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 |
| Make 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 intermediate content.Qualified back filtering with microporous membrane with 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, gets finished product.
Freeze-drying process: with the sample inlet, fall the dividing plate temperature to-40 ℃, keep 3h; Cold-trap is down 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 |
| Make 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 evenly, 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, poly(oxyethylene glycol) 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 |
| Poly(oxyethylene glycol) 400 (PEG-400) | 80.0g |
| Morpan BB | 10.0ml |
| Glycerine | 100.0g |
| Distilled 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, after SDB-L-400 adding 700ml water grinds well in mortar, with matrix and PEG-400, glycerine, LCRG-3 mixing, add water to 1000g, can namely.
Claims (13)
1. mixture with homology glycosaminoglycan derivative of formula (I) structure, described mixture is 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 -
R ' is-CH independently of each other
2OH or-COOH.
2. carboxyl reduction derivative and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan as claimed in claim 1, wherein:
The monose of the carboxyl reduction derivative of described 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-glucosyl group+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-glucosyl group: the molar ratio range of (D-glucosyl group+D-glucal acidic group) is 20%～100%;
The weight average molecular weight range of described lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative is 3000～20000Da; Polydispersity index is between 1.0 to 1.8.
3. lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative as claimed in claim 1 or 2 and pharmacy acceptable salt thereof, it is characterized in that the weight average molecular weight range of described lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative is 6000～15000Da; The polydispersity index value is between 1.1 to 1.5.
4. amine glycan carboxyl reduction derivative and the pharmacy acceptable salt thereof of the fucosylated sugar of lower molecular weight as claimed in claim 1 or 2, it is characterized in that described pharmacy acceptable salt is an alkali metal salt, alkaline earth salt and the organic ammonium salt of the carboxyl reduction derivative of lower molecular weight fucosylated glycosaminoglycan.
5. carboxyl reduction derivative and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan as claimed in claim 4, it is characterized in that described pharmacy acceptable salt is sodium salt or sylvite or the calcium salt of lower molecular weight fucosylated glycosaminoglycan carboxyl reduction derivative.
6. carboxyl reduction derivative and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan as claimed in claim 1 or 2, it is characterized in that, the carboxyl reduction derivative of described lower molecular weight fucosylated glycosaminoglycan is the depolymerization product of extracting the carboxyl reduction derivative of the fucosylated glycosaminoglycan that obtains with Echinodermata Holothuroidea animal body wall and/or internal organ, or with the carboxyl reduction derivative of the fucosylated glycosaminoglycan depolymerization product in echinoderms body wall and/or internal organ source.
7. carboxyl reduction derivative and the pharmacy acceptable salt thereof of lower molecular weight fucosylated glycosaminoglycan as claimed in claim 6, it is characterized in that described Echinodermata Holothuroidea animal is 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. as the carboxyl reduction derivative 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 raw material, by 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 by depolymerization reaction; Or
(2) depolymerization product with the fucosylated glycosaminoglycan in echinoderms body wall and/or internal organ source is raw material, obtains the carboxyl reduction product of the fucosylated glycosaminoglycan of desired molecule weight range by 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 to carry out at acid aqueous phase, under the carbodiimide effect, react with reductive agent, generate the carboxyl reduction derivative of fucosylated glycosaminoglycan, wherein carbodiimide is 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), N, N '-dicyclohexyl carbodiimide (DCC), aromatic series carbodiimide; Reductive agent is reductive agent sulfo-hydroborate or the metal hydride with reduction carboxylic acid halides ability.
10. preparation method as claimed in claim 8, wherein in reaction system, the massfraction of described fucosylated glycosaminoglycan is 0.05%～15%, the consumption of carbodiimide in reaction system is the mass ratio 1:0.05 to 1:20 with fucosylated glycosaminoglycan, the consumption of reductive agent is and the mass ratio 1:0.05 to 1:20 of fucosylated glycosaminoglycan that the temperature range of reaction is 10 ° of C～80 ° C.
11. the pharmaceutical composition of an anti-HIV-1 contains the claim 1 of effective anti-HIV-1 dosage or carboxyl reduction derivative and its pharmacy acceptable salt of 2 or 4 described lower molecular weight fucosylated glycosaminoglycans, and pharmaceutical excipient.
12. claim 1 or 2 or 4 described lower molecular weight fucosylated glycosaminoglycan carboxyl reduction products and pharmacy acceptable salt thereof the application in the medicine of preparation prevention or treatment acquired immune deficiency syndrome (AIDS).
13. the application of the described pharmaceutical composition of claim 11 in the medicine of preparation prevention or treatment acquired immune deficiency syndrome (AIDS).
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