CN102247401A - Low molecular weight glycosylated chondroitin sulfate and its purpose in preparation of anti-HIV-1 medicament - Google Patents
Low molecular weight glycosylated chondroitin sulfate and its purpose in preparation of anti-HIV-1 medicament Download PDFInfo
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Abstract
The invention discloses a low molecular weight glycosylated chondroitin sulfate, whose weight average molecular weight is 3000-15000Da. The monosaccharide composition comprises acetyl galactosamine (D-GalNAc), glucuronic acid (D-GlcUA), fucose (L-Fuc), or its sulfuric ester (expressed in -OS03<->), wherein the mole ratio of D-GalNAc to D-GlcUA to L-Fuc to -OS03<-> is 1: (1+/-0.3): (1+/-0.3): (3.5+/-0.5). The low molecular weight glycosylated chondroitin sulfate has a strong anti-HIV-1 virus activity, is a gp120 entry inhibitor, and can be used for preventing and/or treating AIDS. The invention also provides a method for preparing the low molecular weight glycosylated chondroitin sulfate and its composition preparation. Glycosylated chondroitin sulfate is depolymerized by the peroxide method to obtain a low molecular weight product, and then low molecular and/or high-molecular impurities of the product are removed by gel separation or ultrafiltration method. The low molecular weight glycosylated chondroitin sulfate and its medicinal composition can be prepared in the form of an injection, a lyophilized powder or a suppository.
Description
Technical field
The present invention relates to a kind of low-molecular-weight glycosyl chondroitin sulfate (Low molecular weight Glycosylated Chondroitin sulfate, LGC) and preparation method thereof, contain Pharmaceutical composition and the purposes in preventing and/or treating the AIDS-treating medicine preparation thereof of LGC.
Background technology
HIV (human immunodeficiency virus) (human immunodeficiency virus, HIV-1) infect due to acquired immune deficiency syndrome (AIDS) (aquired immunodeficiency syndrome AIDS) is the major disease of serious harm human life health.UNAIDS's issue on November 23rd, 2010 report claims to estimate that the whole world has 3,330 ten thousand people's aids infection poison, has had 2,500 ten thousand people's death since confirming acquired immune deficiency syndrome (AIDS) first in June, 1981.In China, by the end of the year 2009, estimate existing 740,000 adults and childhood infection HIV (human immunodeficiency virus) (Human Immunodeficiency Virus), 4.8 ten thousand is newly-increased infection the in 2009.Among these patients, estimate at 10.5 ten thousand for the acquired immune deficiency syndrome (AIDS) case, 2.6 ten thousand died from 2009 the acquired immune deficiency syndrome (AIDS) related diseases because of.The clinical treatment of acquired immune deficiency syndrome (AIDS) is except to complication treatment, anti-infective therapy, the immune modulating treatment, and its topmost effective Therapeutic Method is the highly efficient anti-virus treatment.Data shows, from associating antiretroviral therapy (HAART in 1996, at least three kinds of ucleosides/non-nucleoside antiretroviral drugs and protease inhibitor coupling) come out since 2006, aids patient was increased to 13.3 from 0.26 from the mean survival time from making a definite diagnosis.Although the treatment of acquired immune deficiency syndrome (AIDS) makes great progress, defective such as be difficult to cure, treatment no response, drug resistance occur, toxic and side effects is remarkable but still exist.At present, owing to reasons such as treatment no response or drug resistance generations, the HAART treatment is invalid to about 30% HIV sufferers.
For the patient that HAART is failed to respond to any medical treatment can access effective Drug therapy, the clinical novel anti HIV-1 medicine that presses for, its described novel anti HIV-1 should be different with the pharmacotoxicological effect target spot of antiretroviral drugs and protease inhibitor.
Glycosylation chondroitin sulfate (Glycosylated chondroitin sulfate, GCs) be a kind of similar thing of glycosaminoglycans that has the fucose side substitution, the similar chondroitin sulfate of its polysaccharide main chain, the disaccharide construction unit that is made of hexuronic acid, aminohexose is connected to form in turn, and all can there be the Sulfation group in main chain and side chain sugar hydroxyl.Natural GCs is mainly derived from echinoderm body wall or internal organs, it has [→ 4) the backbone structure unit of D-GlcUA (β 1 → 3) D-GalNAc (1 →], its side chain sulfated fucose then is connected in D-GlcUA (J Biol Chem with (α 1 → 3) glycosidic bond, 1988,263 (34): 18176-83 and J Biol Chem, 1991,266 (21): 13530-6).Natural GCs has remarkable anticoagulant active, is characterized in having potent inhibition endogenous factors X enzyme (internal factor tenase, f.Xase) activity, and antithrombase (f.IIa) activity that exists significant HCII to rely on.CN101735336A discloses the method for preparing oligomeric fucosylated glycosaminoglycan as the Chinese patent publication number, it is by preparing with the catalytic peroxide depolymerization of period 4 transition metal ions depolymerization fucosylated glycosaminoglycan in aqueous media, this preparation method reaction condition gentleness, repeatability and good stability, cracking selectivity height, the product quality homogeneous is controlled.The oligomeric fucosylated glycosaminoglycan that obtains is not less than 80% with GalNAc as the polysaccharide molecular number of reducing end under neutral, and weight average molecular weight is about 6,000Da～20, and 000Da, PDI are 1.0～2.0.
This shows that research low-molecular-weight glycosyl chondroitin sulfate (LGC) and preparation method thereof, the Pharmaceutical composition that contains LGC and the purposes in preventing and/or treating the AIDS-treating medicine preparation thereof are significant.
Summary of the invention
The object of the invention at first provides a kind of low-molecular-weight glycosyl chondroitin sulfate (LGC) and the purposes of pharmaceutically acceptable salt in anti-1 type HIV (human immunodeficiency virus) (HIV-1) medication preparation thereof, wherein said LGC is the mixture with homology glycosaminoglycans derivant of formula (I) structure
In the formula (I):
D-GlcUA-β 1-is β-D-glucuronic acid-1-base;
D-GalNAc-β 1-is β-D-N-acetylamino galactosamine-1-base;
L-Fuc-α 1-is α-L-fucose-1-base;
R
1For-H or D-GalNAc-β 1-;
R
3, R
5, R
5, R
6, R
7Be independently of each other-H or-SO
3 -
R
2Can be-OH ,-4-O-D-GlcUA, also can be formula (II) or (III) shown in group:
Wherein, R ' is a L-Fuc-α 1-substituted radical, and has the sulfate group of cotype (1) structure on the L-Fuc-α 1-;
R " be-4-[L-Fuc (α 1-3)] D-GlcUA-β 1-group, there is the sulfate group of cotype (1) structure on its L-Fuc-α 1-,
R
3, R
4With above definition.
With molar ratio computing, the contained D-GlcUA of described LGC, D-GalNAc, three kinds of monosaccharide residues of L-Fuc and contained-OSO
3 -The proportion of base is 1: (1 ± 0.3): (1 ± 0.3): (3.5 ± 0.5);
N is that average is about 3～17 integer;
The weight average molecular weight range of described LGC is 3000～15000Da.
The molecular weight of the present invention's LGC can adopt efficient gel chromatography (HPLPC) to detect.From the anti-HIV-1 activity intensity and avoid the hematology to influence consideration, in weight average molecular weight, the molecular weight ranges of the low-molecular-weight glycosyl chondroitin sulfate that the present invention selects is about 3,000～15,000Da (being the average about 3～17 of the n of homologue shown in the formula (I)), preferred molecular weight range is about 5,000～8,000Da (average about 5～9 of the n of homologue shown in the formula (I)).
The polydispersity index of the present invention's LGC (PDI, the ratio of weight average/number-average molecular weight is Mw/Mn) generally between 1.0 to 1.8; The PDI of preferred LGC is between 1.1 to 1.5.
The present invention's LGC can be the salt of its pharmaceutically acceptable alkali metal, alkaline-earth metal etc., and similarly, described LGC also can be the ester that makes the formation of itself and alkaline organic group.The pharmaceutically acceptable salt of the preferred LGC of the present invention is sodium salt, potassium salt or the calcium salt of LGC.
LGC of the present invention is that the depolymerization product of the GCs that extracts of Echinodermata Holothuroidea animal body wall and the end of described GCs depolymerization product are reduced the amidized product of cheese.
The present invention discovers that described LGC has potent anti-HIV-1 activity, and it suppresses HIV-1 experiment strain, the lymphocytic IC of clinical separation strain infected person
50Value is about 10～100nmol/L extremely, and under this drug effect concentration, there is not significant anticoagulating active in described LGC.
The present invention discovers that further described LGC is the HIV-1 entry inhibitors, and this pharmaceutical requirements with clinical required novel targets anti-HIV-1 is consistent; The present invention detects proof by bio-molecular interaction, and the pharmacotoxicological effect target spot of described LGC is the outer membrane glycoprotein gp120 of HIV-1; Adopt solubility CD4 (sCD4) and CD4i monoclonal antibody 14b to interact and detect proof, the distinguishing feature of described LGC is that it is in conjunction with glycoprotein gp120 conserved region CD4i.CD4 is the adherent CD4 of mediation HIV-1
+Surface of cell membrane receptor, CD4i are gp120-CD4 zygotic induction naked positions, and 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 cell membrane, gp41 resets folding cause outer virionic membrane and CD4
+Cell membrane merges, and realizes finally that thus HIV-1 invades step.
Obviously blocking-up CD4i HIV-1 capable of blocking invades CD4
+Cell.Because the high conservative of CD4i, help avoiding drug resistance to produce at the HIV-1 entry inhibitors of CD4i.At present, be the HIV-1 vaccine of target and the emphasis direction that the HIV-1 entry inhibitors has become the treating AIDS drug development with CD4i, so the present invention's LGC have important use as the HIV-1 entry inhibitors of overriding resistance and is worth.
Work as R
2Can be-OH or-during the 4-O-D-GlcUA group, only there is uv absorption in LGC of the present invention in the near infrared region, is unfavorable for setting up the analyzing detecting method based on the UV detection technique.Convenient for description, this description is called LGC-1 with this type of LGC homologue.Obviously, LGC-1 exist reproducibility glycosyl end-D-GlcUA or-D-GalNAc.
Work as R
2Be formula (II) or (III) shown in during group, there is the uv absorption at about 280nm place in LGC of the present invention, this description is called LGC-2 with this type of LGC homologue.Obviously, LGC-2 is that corresponding LGC-1 reproducibility glycosyl end is reduced amidized product.When containing a certain amount of LGC-2 among the LGC of the present invention's formula (I) structure, owing to there is the uv absorption at 280nm place, set up the spectrophotography detection method easily, these methods for set up reliably, quality control, pharmacokinetic analysis technology etc. are significant accurately.In general, when LGC-2 accounts for 30% when above of whole LGC, the detection method that is fit to quality control, pharmacokinetic analysis needs can be set up easily.
R of the present invention
2For-OH or-LGC-1 of 4-O-D-GlcUA is actually the depolymerization product of the GCs that Echinodermata Holothuroidea animal body wall extracts; Described R
2For formula (II) or (III) LGC-2 of group be actually the product of described GCs depolymerization product through terminal reductive amination gained.
Result of study of the present invention shows, GCs depolymerization product (LGC-1) is handled back (LGC-2 or contain LGC-2) through part or all of terminal reductive amination, its anti-HIV-1 active near and slightly be better than the GCs depolymerization product of correspondence.For this reason, described GCs depolymerization product and part or all of terminal reductive amination product thereof all can be used for preparing the anti-HIV-1 medicine, and are included within the scope of the invention thus.
The further purpose of the present invention provides a kind of low-molecular-weight glycosyl chondroitin sulfate (LGC) and pharmaceutically acceptable salt thereof, and described LGC is the homology glycosaminoglycans derivant mixture of formula defined above (I) structure, wherein, in molar percentage, R
2For formula (II) or (III) LGC-2 of group account for 30%～100% of whole formulas (I) chemical compound.
The further purpose of the present invention also provides a kind of low-molecular-weight glycosyl chondroitin sulfate (LGC) and pharmaceutically acceptable salt thereof, and described LGC is the mixture of the homology glycosaminoglycans derivant of formula defined above (I) structure, wherein, and R
2Be formula (II) or (III) LGC-2 of group, i.e. R
2Be not-OH or-4-O-D-GlcUA.
Further purpose of the present invention provides the preparation method of described low-molecular-weight glycosyl chondroitin sulfate (LGC).The preparation method of described LGC is to extract from Holothuroidea animal body wall to obtain GCs, then adopt the peroxide depolymerization depolymerization of metal ion catalysis to obtain GCs depolymerization product (LGC-1), gained LGC-1 can select to carry out the reaction of further reductive amination, to obtain the aminated LGC product of all or part of terminal cheese (LGC-2 or contain LGC-2).
Described preparation method can include but not limited to following steps:
Step 1: from Echinodermata Holothuroidea animal body wall, extract and obtain acid mucopolysaccharide, i.e. glycosylation chondroitin sulfate (GCs).
Extracting GCs from Holothuroidea animal body wall can carry out with reference to the known method in this area, generally comprise but be not limited to following steps: defat, alkali and/or enzyme are handled and are obtained extracting solution, lower alcohol and/or ketone precipitation obtain the raw sugar extract, can select to carry out Deproteinization after gained raw sugar extract redissolves and handle (isoelectric point method, the protein denaturant sedimentation method etc.), (oxidizing process etc.) are handled in decolouring, remove small molecular weight impurity (ultrafiltration dialysis, gel filtration etc.), purification (gel chromatography and/or the chromatography of ions) etc., the gained purified product can also select to adopt the metal cation salt of acquisition particular types such as ion exchange (as sodium salt, potassium salt, calcium salt etc.), obtain the GCs end-product by drying under reduced pressure and/or freeze-drying at last.
Step 2: the GCs of peroxidating depolymerization step (1) gained is to obtain its depolymerization product LGC-1.
Because the particularity of GCs chemical constitution, glycosaminoglycans depolymerization method commonly used such as enzyme process depolymerization, nitrous acid depolymerization, soda acid depolymerization all is difficult to be applied to the GCs depolymerization.At present, the depolymerization method of GCs mainly is the peroxide depolymerization.For avoiding separating the influence of collecting process to the GCs feature structure, the peroxide depolymerization that the preferred LGC-1 preparation method of the present invention is a metal ion catalysis, this method is (numerical value of the n in the formula (I) reduces) except the degree of polymerization that reduces construction unit, substantially the monosaccharide that does not influence depolymerization product is formed, the constitutional repeating unit characteristic, do not influence feature chemical functional groups such as side chain fucose substituent group and sulfate group, promptly do not influence formula (I) general structure.
Specifically, the preferred GCs depolymerization of the present invention is that step 1 gained GCs is made into mass fraction is 0.05～10% aqueous solution, in gained GCs solution, be added to the metal ion catalyst (metal cation salt: GCs) of mass fraction 0.01～0.1%, add about 0.5～5% peroxide of mass fraction (peroxide: GCs), be reflected under about 20～60 ℃ and carry out.When treating depolymerization product molecular weight (in Mw and/or Mn), add after the catalyst chelating agen cessation reaction or directly add lower alcohol/ketone and make product precipitation to the desired molecule weight range.After collecting precipitation and the redissolution, gel chromatography, ion exchange chromatography or ultrafiltration dialysis method purification, products therefrom can be selected to obtain corresponding alkali metal ion salt by ion exchange.Obtain GCs depolymerization product LGC-1 thus.
In general, among the LGC-1 with the inventive method preparation, detect spectrogram according to product NMR and calculate, in molar percentage, R
2For the chemical compound of-OH can account for more than 85% of LGC-1 total amount.
In the described depolymerization method of step 2, the preferred peroxide of the present invention is a hydrogen peroxide; Preferred metal ion catalyst is bivalent cupric ion (salt).
In the preparation method of LGC-1 of the present invention, the depolymerization velocity-stabilization of reactant GCs, the molecular weight of product narrowly distributing, products therefrom is easy to separation and purification, can the highly purified LGC product of mass production.
Step 3: step 2 gained GCs depolymerization product LGC-1 can select to carry out terminal reductive amination and handle, to obtain to contain R
2Be the formula (II) or (III) LGC-2 of group.
Among the present invention, the terminal reductive amination step of described LGC-1 is: the GCs depolymerization product is dissolved in the buffer of pH7～9, adds stoichiometric tyramine and sodium cyanoborohydride successively, room temperature or reacting by heating.After reaction finishes, the separation and purification product, and be translated into required alkali metal ion salt.Figure below is that example illustrates its reductive amination process with end-D-GalNAc:
The separation and purification of described step (3) product is meant in the purified product, with molar ratio computing, and R
2For formula (II) or (III) formula of group (I) chemical compound account for more than 30% of whole formulas (I) chemical compound usually.
Because the present invention's LGC has potent anti-HIV-1 activity, the further purpose of the present invention provides the pharmaceutical composition that contains LGC, the LGC of the present invention or its pharmaceutically acceptable salt that contain effective dose in the described pharmaceutical composition, and pharmaceutically acceptable excipient.
Among the present invention, the pharmaceutical composition that contains LGC 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 LGC 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 dosage form 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 LGC anti-HIV-1 activity and drug administration by injection thereof according to the present invention, the LGC content in its single-dose preparations can be about 5～150mg.
In the local administration preparation, the Pharmaceutical composition dosage form can be selected suppository, gel, ointment, liniment etc.Active component LGC content in these pharmaceutical formulations can capable technical staff active drug concentration decision during according to local application.
Because the machine-processed target spot of the present invention's LGC anti-HIV-1 is different from existing clinical application, therefore can unite use with existing clinical application, comprise and the blanking time of the administration in turn 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.
The present invention's low-molecular-weight glycosyl chondroitin sulfate (LGC) 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.
Good effect summary of the present invention: the present invention at first finds, the active LGC of described potent anti-HIV-1, and it suppresses HIV-1 experiment strain, clinical separation strain and the lymphocytic IC of persister infected person
50Value is about 10～100nmol/L extremely, and under this drug effect concentration, there is not anticoagulating active substantially in its LGC.Discover that further described LGC is the HIV-1 entry inhibitors, this pharmaceutical requirements with clinical required novel targets anti-HIV-1 is consistent.In addition, the present invention also finds, as the HIV-1 entry inhibitors, the characteristics of described LGC are in conjunction with the outer membrane glycoprotein gp120 conserved region CD4i of HIV-1, because the high conservative of CD4i, the emphasis direction that has become the treating AIDS drug development at HIV-1 vaccine and the HIV-1 entry inhibitors of CD4i, therefore, the present invention's LGC has important use for the HIV-1 entry inhibitors of development overriding resistance and is worth.
Description of drawings
Fig. 1 is the GCs in Thelenota ananas (Jaeger) source and the HPGPC collection of illustrative plates of low molecular weight product LGC thereof.
Fig. 2 is LGC-1A
1H NMR detects collection of illustrative plates.
Fig. 3 is LGC-1A
13C NMR detects collection of illustrative plates.
Fig. 4 is LGC-1A DEPT135 ° and detects collection of illustrative plates.
Fig. 5 is LGC-1A
1H-
1H COSY detects collection of illustrative plates.
Fig. 6 is LGC-1A
1H-
13C HSQC detects collection of illustrative plates.
Fig. 7 is the inhibitory action datagram that LGC forms the inductive syncytium of HIV-1, the inhibitory action that demonstration LGC forms the inductive syncytium of HIV-1.
Fig. 8 is the toxic action datagram of LGC to C8166, shows the toxic action of LGC to C8166.
Fig. 9 is that surface plasma resonance method (SPR) detects LGC-gp120 interaction datagram, and display surface plasma resonance method (SPR) detects LGC-gp120 and interacts.
The specific embodiment
Following examples are the detailed descriptions to content of the present invention, and described embodiment does not constitute limitation of the scope of the invention.
The preparation of [embodiment 1] low-molecular-weight glycosyl chondroitin sulfate (LGC)
1.1 material:
Thelenota ananas (Jaeger) (Thelenota ananas Jaeger), commercially available product is removed the dry body wall of internal organs;
H
2O
2, CH
3COONa3H
2O, NaCl, NaOH, Cu (CH
3COO)
2H
2Agents useful for same such as O: be commercially available analytical reagent.
1.2 method:
(1) glycosylation chondroitin sulfate (GCs) preparation: get the dry body wall of echinoderm Thelenota ananas (Jaeger), press literature method (J Biol Chem, 1991,266 (21): 13530-6) prepare GCs, yield 0.75%, purity 98% (HPGPC, area normalization method), weight average molecular weight (Mw), 65,820.
(2) low-molecular-weight glycosyl chondroitin sulfate (LGC) preparation: get step (1) gained GCs 5.0g and add in the round-bottomed flask, add the 180ml distilled water and make it dissolving, 35 ℃ of water bath heat preservations add 1% copper chloride solution (5ml) at the uniform velocity stirring, and drip 15% H in 60min
2O
210ml, the NaOH solution control pH value scope with 1N in the course of reaction is 7.2～7.8.The continuous stirring reaction added 400mg disodium EDTA cessation reaction after 1 hour in reactant liquor, subsequently, the frozen water cooling, adding concentration is 95% (v/v) ethanol 540ml, centrifugal must the precipitation.
Gained is precipitated with after twice of 100ml 80% washing with alcohol, add 150ml water and make it dissolving, become sodium salt through 001 * 7 type resin cation exchange, dialysed 6 hours with the dialyzer of molecular cut off 3500Da then, holding back product concentrates, lyophilization obtains depolymerization sample LGC-1A 4.02g, and yield is 80.4%.
(3) GCs in Thelenota ananas (Jaeger) source and depolymerization product LGC-1A physical and chemical parameter thereof, monosaccharide composition and structural analysis detect with wave spectrum: efficient gel chromatography (HPGPC) detection molecules amount and distribution; Conductance method detection-OSO
3 -/-COO
-Mol ratio; Optical rotation is measured according to two appendix VI E of Chinese Pharmacopoeia (2005 editions) method; Intrinsic viscosity is used dark type viscometer and is measured.
The Elson-Morgon method detects acetylamino galactosamine (D-GalNAc) content, and carbazole method detection glucuronic acid (D-GlcUA) content (Zhang Weijie, saccharide complex Biochemical Research technology (second edition), Zhejiang: publishing house of Zhejiang University, 1999,19-20);
1H NMR methyl peak integral area calculates the D-GalNAc/L-Fuc mol ratio.The AVANCE AV of Switzerland Bruker company 400 superconduction nuclear magnetic resonance spectrometers (400MHz) detect NMR spectrogram (testing conditions, solvent D
2O, 99.9Atom%D (Norell company); Interior mark, trimethylsilyl-propionic acid (TSP-d4); 45 ℃ of temperature).
The HPGPC of GCs and depolymerization product LGC-1A thereof detects spectrogram and sees accompanying drawing 1, and physical and chemical parameter, monosaccharide are formed testing result and seen Table 1;
1H/
13The detection spectrogram of C NMR and relevant spectrum thereof is seen accompanying drawing 2～6,
1H/
13The signal ownership of CNMR spectral data sees Table 2.
Table 1 testing result shows that compare with GCs, LGC-1A molecular weight and intrinsic viscosity significantly reduce, and monosaccharide was formed maintenance stable (about 1: 1: 1: 3.5).
The GCs in table 1. Thelenota ananas (Jaeger) source and the physical and chemical parameter of LGC-1A, monosaccharide are formed testing result
Shown in the table 2
1H/
13The signal ownership of C NMR spectral data shows the feature chemical constitution basically identical of GCs, LGC-1A.Below be that example is sketched its signal ownership and structure elucidation with LGC-1A.
1Among the H NMR, 5.2～5.7ppm presents three groups of stronger signal peaks, and this is the α-fucose terminal hydrogen signal of dissimilar Sulfations, and wherein about 5.6,5.3ppm signal is corresponding Fuc2S4S and Fuc4S terminal hydrogen respectively.The 5.35ppm peak that has obvious acromion is the stack of Fuc3S and Fuc4S terminal hydrogen signal; About 5.18ppm place signal is the α-terminal hydrogen signal of main chain free-end.Main chain β-terminal hydrogen signal appears at about 4.4～4.6ppm place.The Fuc methyl proton signal appears at about 1.0～1.3ppm place; The GalNAc acetyl protons appears at about 1.9～2.0ppm.The sugar ring hydrogen that sulfate group replaces the position appears at about 4.6～5.0ppm, and about 3.6～4.6ppm signal then replaces the stack of the sugar ring hydrogen of position for the non-sulfuric acid ester group.
By
1H-
1H COSY spectrum as seen, the H2 of GlcUA and H3 signal peak position all＞3.6ppm, (3.35～3.55ppm) to the about 0.3～0.4ppm of low field displacement, shows that there is side substitution in this position, and is that the sugar ring replaces but not Sulfation replaces with respect to the respective signal of chondroitin sulfate compounds GlcUA.
1H-
1The HTOCSY spectrum is clear to show that the coupling between the further proton signal of GlcUA is relevant; The also clear coupling that has shown between Fuc terminal hydrogen and the H3 of spectrogram.
1H-
1H NOESY spectrum has then shown the coupling between the H4 of the H3 of H3, GlcUA terminal hydrogen and GalNAc of each Fuc terminal hydrogen and GlcUA and GalNAc terminal hydrogen and GlcUA respectively, show that the annexation between the monosaccharide: GlcUA and GalNAc are interconnected to form main chain with β (1 → 3) and β (1 → 4) glycosidic bond respectively, Fuc then is connected in GlcUA with α (1 → 3) glycosidic bond.
13In the C-NMR spectrum, the C1 peak of GlcUA and GalNAc appears at about 102～106ppm, shows the beta configuration of its end of the bridge hydrogen; (the C1 signal of about 101～99ppm) Fuc conforms to its end of the bridge hydrogen α-configuration at relative High-Field place.Carbonyl carbon (C7) in carbonyl carbon in the GlcUA carboxyl (C6) and the GalNAc acetylamino appear at much at one position (～177ppm); Fuc methyl carbon signal appears at about 18～19ppm; Methyl carbon signal in the GalNAc acetylamino then appears near about 25ppm.The C2 signal of GalNAc4S6S appears at about 54ppm, shows the feature that its deoxidation and acetylamino replace; More weak GalNAc4S C2 signal comes across 55ppm.
DEPT90 ° of spectrogram shown～177ppm carbonyl carbon quaternary carbon and about 18 and the primary carbon character of the methyl carbon at 25ppm place.DEPT135 ° of visible GalNAc4S6S of spectrogram secondary carbon (C6, CH
2) signal appears at 70.1ppm, do not exist the secondary carbon signal of GalNAc4S of 6-position sulfate group then to appear at 64.0ppm.Calculate according to two kinds of C6 signal intensitys, GalNAc4S only accounts for the about 5% of GalNAc total amount, and this result approaches the ratio calculated according to the C2 signal intensity.
The C1 of visible Fuc and the coupling between the GlcUAH3 in the HMBC spectrum, the coupling between GlcUAC1 and the GalNAcH-3; Coupling between GalNAc C1 and the GlcUAH4; But the coupling from this spectrogram between identification Fuc, GlcUA, GalNAc end of the bridge hydrogen and GlcUAC3, GalNAc C3 and the GlcUAC3 confirms further that thus THG forms the annexation between the monosaccharide.
Comprehensive THG-1 hydrogen spectrum, carbon spectrum and relevant composing as seen thereof, this product is formed in the monosaccharide for three kinds, and GlcUA and GalNAc interconnect by β (1 → 3) and β (1 → 4) glycosidic bond and form the polysaccharide main chain, form main chain disaccharide construction unit thus.Obviously, the similar chondroitin sulfate of THG backbone structure, its aminohexose is mainly GalNAc4S6S, and only about 5% aminohexose does not have 6-position sulfate group and replaces (GalNAc4S).H2, H3 chemical shift and combination according to GlcUA
1H-
1H NOESY,
1H-
13C HM C can judge that Fuc is connected in GlcUA with α (1 → 3) glycosidic bond.Nearly all GlcUA all exists the side chain fucosido to replace, and GlcUA, Fuc monosaccharide ratio were near 1: 1 among the THG, so all there is a side chain fucose substituent group in each disaccharide construction unit of THG main chain.
The GCs in table 2. Thelenota ananas (Jaeger) source and LGC-1A's
1H/
13CNMR detects data (δ [ppm])
The preparation of [embodiment 2] serial molecular weight LGC-1
2.1 material:
The GCs in Thelenota ananas (Jaeger) source is with embodiment 1 described method preparation.
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:
(1) serial molecular weight LGC-1 sample preparation: four parts of each 5g of GCs in Thelenota ananas (Jaeger) source adopt the described method depolymerization of embodiment 1 step (2), but the time point of cessation reaction are respectively 90,120,360,420min.The LGC-1 product for preparing thus is numbered respectively: LGC-1B, LGC-1C, LGC-1D and LGC-1E.The yield of described depolymerization reaction is all greater than 70%.
(2) the LGC-1 product detects: HPGPC detection molecules amount and distribution; Conductance method detection-OSO
3 -/-COO
-Mol ratio; Optical rotation is measured according to two appendix VI E of Chinese Pharmacopoeia (2005 editions) method; Intrinsic viscosity is used dark type viscometer and is measured.
2.3 result:
2.2 the testing result of described step (2) products therefrom LGC-1B～E sees the following form 3.Testing result shows that the GCs in Thelenota ananas (Jaeger) source is higher through the yield that the depolymerization of hydrogen peroxide depolymerization obtains product LGC-1, and molecular weight distribution is narrower, and the electrical conductivity method testing result shows that sulfate group does not have significant change, and intrinsic viscosity reduces with molecular weight.
The GCs in table 3. Thelenota ananas (Jaeger) source and depolymerization product LGC's thereof
Physicochemical datas such as molecular weight and distribution thereof, optical rotation and intrinsic viscosity detect
The terminal reductive amination of [embodiment 3] LGC-1A
3.1 material:
LGC-1A: embodiment 1 preparation depolymerization GCs product.
Tyramine hydrochloride, sodium cyanoborohydride: be commercially available analytical reagent.
3.2 method:
(1) the terminal reductive amination of LGC-1A: GCs depolymerization product LGC-1A 1000mg is dissolved in 35ml0.2mM phosphate buffer (PBS, pH 8.0) in, add excessive 800mg tyramine and 300mg sodium cyanoborohydride in the stirring respectively, the about 100hr of reaction in 35 ℃ of waters bath with thermostatic control.After reaction finishes, add 95% ethanol 105ml, centrifugal must the precipitation is after the gained precipitation is washed twice with 95% ethanol 30ml, with 35ml 0.1%NaCl redissolution gained precipitation, the centrifugal insoluble matter that goes, Seph adex on the supernatant
TMG-100 post, 0.1%NaCl eluting, UV detector detect collects the eluting stream part that has λ 280nm light absorption, and part lyophilizing of gained stream obtains LGC-2A 652mg.
(2) product physics and chemistry and wave spectrum detect: HPGPC detection molecules amount and distribution; Conductance method detection-OSO
3 -/-COO
-Mol ratio; The Elson-Morgon method detects acetylamino galactosamine (D-GalNAc) content, and the carbazole method detects glucuronic acid (D-GlcUA) content,
1HNMR methyl peak integral area calculates D-GalNAc/L-Fuc mol ratio (with embodiment 1).AVANCE AV 400 superconduction nuclear magnetic resonance spectrometers detect NMR spectrogram (solvent D
2O, interior mark TSP-d4,45 ℃ of temperature).
3.3 result
In the LGC-1A inventory, LGC-2A efficiency of pcr product about 65%;
Product component testing result shows, D-GalNAc: D-GlcUA: L-Fuc :-OSO
3 -Be about 1.00: 1.13: 1.00: 3.80, Mw is about 14380, and PDI is about 1.42, and the Theoretical Calculation of this and the LGC-1A construction unit degree of polymerization about 15 is basically identical as a result;
1H?MR(D
2O，δ[ppm])：7.25(2’，6’H)；6.91(3’，5’H)；5.65，5.36，5.28(L-Fucα1H)；3.38(8’H)；2.82(7’H)；2.02(D-GalNAc，CH
3)；1.30～1.32(L-Fuc，CH
3)。L-Fuc methyl hydrogen and phenyl ring hydrogen integration show that than about 10.5 the products therefrom reducing end under neutral all is reduced the cheese ammonification.
The terminal reductive amination of [embodiment 4] LGC-1B～1E
4.1 material
LGC-1B, LGC-1C, LGC-1D and the LGC-1E of LGC-1: embodiment 2 preparations.
Tyramine hydrochloride, sodium cyanoborohydride: be commercially available analytical reagent.
4.2 method
Get each 500mg of LGC-1A, 1C, 1D and 1E and be dissolved in respectively in the 20ml 0.2mM phosphate buffer (PBS, pH 8.0), add excessive 400mg tyramine and 150mg sodium cyanoborohydride in the stirring respectively, the about 60h of reaction in 35 ℃ of waters bath with thermostatic control.After reaction finishes, the centrifuging and taking supernatant, add 95% ethanol 80ml respectively, centrifugal must the precipitation, after the gained precipitation is washed twice with 95% ethanol 15ml, with 20ml distilled water redissolution gained precipitation, the centrifugal insoluble matter that goes, the supernatant lyophilizing, obtain respectively LGC-2B, 2C, 2D and 2E each 468,452,410 and 422mg.
4.3 result
In LGC-1B～1E, LGC-2B～2E efficiency of pcr product about 84%～93%;
Detect according to LGC-1B～1D number-average molecular weight and AVANCE AV 400 superconduction nuclear magnetic resonance spectrometers
1The L-Fuc methyl hydrogen that H NMR spectrogram shows is calculated with phenyl ring hydrogen integration ratio, and the ratio that LGC-2B, 2C, 2D and 2E reducing end under neutral are reduced the cheese ammonification is respectively about 52%, 43%, 37% and 32%.
The anti-HIV-1 activity of [embodiment 5] low-molecular-weight glycosyl chondroitin sulfate LGC-1A
5.1 material and reagent
(1) LGC-1A (Mw 13820Da) of test sample embodiment 1 preparation is dissolved in the water for injection, and being mixed with concentration is the 25mg/ml storage solutions, preserves down for 4 ℃.
(2) (3-(4,5)-dimethylthiahiazo-2-y1)-2,5-diphenyltetrazolium bromide, SDS (Sodium Dodecyl Sulfate), DMSO reagent such as (Dimethyl sulfoxide) are commercially available to reagent MTT, analytical pure.The standard rabbit plasma, rabbit platelet poor plasma, the special bio tech ltd of Guangzhou stamen; APTT detection kit (ellagic acid), Shanghai Sun Bio-Tech Co., Ltd.'s product, lot number 090327.
(3) cell and viral C8166, HIV-1
IIIB/2802/2840/9495And HIV-1-2
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.The all frozen according to a conventional method and recovery of cell and virus.
5.2 method
(1) LGC-1A detects the cytotoxicity of C8166: C8166 cell suspension (4 * 10
5/ ml) 100 μ l mix 37 ℃ of following 5%CO with the LGC-1A of 100 μ l series concentration
2Cultivated three days, mtt assay detects cytotoxicity.570/630nm ELx800ELISA Reader microplate reader is measured the OD value, calculates CC
50(50%Cytotoxic concentration promptly causes the drug level of half cell death).
(2) LGC-1A induces active detection of inhibition of C8166 pathological changes to HIV-1: with C8166 cell suspension (8 * 10
5/ ml) 50 μ l mix with the LGC-1A of 100 μ l variable concentrations, 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, five visuals field of next Radix Achyranthis Bidentatae rate of inverted microscope are the counting syncytium down.Calculate LGC inhibition HIV-1 and induce the EC of C8166 pathological changes
50(50%Effective Concentration promptly suppresses the LGC-1A concentration that half syncytium forms).In conjunction with (1) gained CC above
50Value is calculated selectivity index (CC
50/ EC
50Value, Therapeutic Index, TI).
(3) LGC-1A induces active detection of inhibition of C8166 pathological changes to HIV-2: above (2) described method detects LGC-1A induces the C8166 pathological changes to HIV-2 inhibition activity together.
(4) LGC-1A influences coagulation function: activated partial thromboplastin time (APTT) method detects LGC-1A to be influenced coagulation function.It is an amount of to get LGC-1A, and normal saline is configured to the testing sample solution that concentration is 25 μ g/ml.Get the testing sample solution 20 μ l that prepare, add 180 μ l normal man blood plasma, immediately behind the mixing, according to the appended description method of APTT detection kit (ellagic acid), LGC under the effective anti-HIV-1 drug level of the last detection of BICO-dual pathways coagulo meter (Minivolt company, Italy) is to the influence of coagulation function.
(5) SPR method detection LGC-1A and gp120 interact: LGC and gp120 interaction detect (BIAcore 3000biosensor 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 buffer HBS-EP dilution of the sample of variable concentrations.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.
5.3 result
(1) LGC-1A antiviral activity: the result shows in accompanying drawing 7,8 and the table 4, and LGC-1A is the new construction active component of potent inhibition HIV-1, the IC of its anti-HIV-1
50Be low to moderate about 9.4nM (0.13 μ g/ml); Do not see significant cytotoxicity (referring to accompanying drawing 8 of the present invention) according to preparation maximum concentration medicine (25mg/ml), its therapeutic index can reach more than 150,000; To the inhibition activity of HIV-1-2 then a little less than.
Table 4LFC-1A HIV (human immunodeficiency virus)-resistant activity testing result
(2) LGC-1A is to the influence of coagulation function: table 5 result shows, under effective anti-HIV-1 virus dosage (0.1～8 μ g/ml), it is active not find that significant APTT prolongs for LGC-1A.
Table 5LFC-1A anticoagulant active testing result
| LFC-1A(μg/ml) | 0 | 0.1 | 1 | 4 | 8 |
| APTT(s) | 23.2 | 23.2 | 23.6 | 24.9 | 25.1 |
(3) the active action target spot of LGC-1A anti-HIV-1: the SPR testing result shows, LGC-1A 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 LGC-1A further strengthens in conjunction with the affinity of CD4i, shows that LGC-1A can induce the pg120 conserved region CD4i of exposure in conjunction with CD4.Because CD4i has high conservative, can effectively avoid the drug resistance due to the viral DNA sudden change to produce (seeing accompanying drawing 9) in conjunction with this regional gp120 inhibitor.
[embodiment 6] serial molecular weight LGC-1 and LGC-2 anti-HIV-1 are active to be detected
6.1 material:
LGC-1A, 1B, 1C, 1D, 1E and LGC-2A, 2B, 2C, 2D, 2E series molecular weight LGC sample, embodiment 1～4 products therefrom; Other reagent is with 5.1 (3) of embodiment 5.
6.2 method:
Detect the LGC series of samples cytopathic inhibition activity of C8166 and cytotoxicity thereof are induced in HIV-1IIIB experiment strain, its detection method is with 5.2 of embodiment 5.
6.3 result:
Experimental result sees Table 6.Experimental result shows that under this experiment condition, LGC-1 and LGC-2 series of samples are less to the toxicity of C8166 cell, its CC
50All greater than about 25mg/ml; The activity of external anti-HIV-1 is stronger, its IC
50Value at about 8.3nM (LGC-2A) between the 30.8nM (LGC-1E); Therapeutic index is higher, all greater than 15000.
In addition, this experimental result shows that also (1) relatively LGC-1 anti-HIV-1 serial and the LGC-2 series of samples is active as seen, and its anti-HIV-1 activity improves with mean molecule quantity and strengthens; (2) the anti-HIV-1 specific activity between LGC-1 series and the LGC-2 series of samples as seen, the active corresponding LGC-1 sample of LGC-2 sample is active slightly strong.
Because the LGC sample weakens with the molecular weight reduction the influence of blood coagulation system, so description of the present invention points out that its preferred LGC molecular weight (Mw) is about 5000～8000.
The external anti-HIV-1 activity of the serial molecular weight LGC of table 6.
The LGC preparation in [embodiment 7] different genera source and the active detection of anti-HIV-1 thereof
7.1 material
Radix Morinae Bulleyanae (Stichopus japonicus), the dry body wall of hojothuria leucospilota (Holothuria leucospilota), commercially available product.
7.2 the LGC preparation in Radix Morinae Bulleyanae, hojothuria leucospilota source
With the LGC in embodiment 1 method (1) and (2) preparation Radix Morinae Bulleyanae and hojothuria leucospilota source, and abbreviate LGC-respectively as
StAnd LGC-
Hl
Detect 7.3 physics and chemistry and anti-HIV-1 are active
The physics and chemistry testing result shows, LGC-
StAnd LGC-
HlMolecular weight be respectively 8650Da and 8595Da;
1H,
13C NMR and relevant analysis of spectrum thereof show, LGC-
StAnd LGC-
HlHave the basic structural feature of the glycosylation chondroitin sulfate compounds of similar embodiment 1 described LGC-1A, but position and the quantity of respectively forming the sulfate group on the monosaccharide residue there are differences LGC-
StBe mainly D-GalNAc4S6S with the D-GalNAc in the LGC-1A main chain, and LGC-
HlThere is a certain amount of D-GalNAc4S in the structure; With LGC-
StAnd LGC-
HlDifference, side chain fucose substituent group type is except L-Fuc2S4S and L-Fuc4S, and LGC-1A also contains the L-Fuc3S side chain, and lacks the L-Fuc3S4S side chain.
The active testing result of anti-HIV-1 shows LGC-
StThe LGC-1C activity intensity suitable with its molecular weight is approximate, and slightly is better than the also proximate LGC-of molecular weight
Hl
The different chemical structures feature of table 7LGC is to the active influence of anti-HIV-1
+: the side-chain radical that has described type;-: do not exist or the less side-chain radical that has described type
The freeze-dried products of [embodiment 8] low-molecular-weight glycosyl chondroitin sulfate
8.1 material
The low-molecular-weight glycosyl chondroitin sulfate (LGC-hl) in embodiment 7 gained hojothuria leucospilotas source, its weight average molecular weight 9500Da.
8.2 prescription
| The supplementary material title | Consumption |
| LGC- hl | 50g |
| Water for injection | 500ml |
| Make altogether | 1000 |
8.3 preparation technology
The hojothuria leucospilota low-molecular-weight glycosyl chondroitin sulfate that takes by weighing recipe quantity adds to the full amount of water for injection, and stirs to make dissolving fully.The medicinal carbon of adding 0.6% stirs 20min; Use buchner funnel and 3.0 μ m microporous filter membrane decarbonization filterings.Survey intermediate content.Qualified back filtering with microporous membrane with 0.22 μ m; Fill 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 lyophilizing 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 9] low molecule glycosylation chondroitin sulfate
9.1 material
The low-molecular-weight glycosyl chondroitin sulfate (embodiment 3 gained LGC-2A) in Thelenota ananas (Jaeger) source, its weight average molecular weight 14380Da.Reagent such as ethyl hydroxybenzoate and glycerin gelatine is commercially available, medicinal rank.
9.2 prescription
| The supplementary material title | Consumption |
| LGC-2A | 100g |
| Water for injection | 400ml |
| Ethyl hydroxybenzoate | 2.0g |
| Glycerin gelatine adds to | 4000g |
| Make altogether | 1000 pieces |
9.3 preparation method
Get low-molecular-weight glycosyl chondroitin sulfate and add the dissolving of injection water, add the ethyl hydroxybenzoate stirring and dissolving, add an amount of glycerol again and stir evenly, slowly add in the gelatin glycerol substrate, fully stir, be incubated 55 ℃, irritate mould, every piece heavy 4g.
The gel of [embodiment 10] low molecule glycosylation chondroitin sulfate
10.1 material
The low-molecular-weight glycosyl chondroitin sulfate (embodiment 4 gained LGC-2C) in Radix Morinae Bulleyanae source, its weight average molecular weight 9250Da.Reagent such as cross linked sodium polyacrylate, PEG400, benzalkonium bromide and glycerol are commercially available, medicinal rank.
10.2 prescription
| The supplementary material title | Consumption |
| ?LGC-2C | 10.0g |
| Cross linked sodium polyacrylate (SDB-L-400) | 10.0g |
| PEG400 (PEG-400) | 80.0g |
| Benzalkonium bromide | 10.0ml |
| Glycerol | 100.0g |
| Distilled water adds to | 1000g |
10.3 preparation method
Get the dissolving of low-molecular-weight glycosyl chondroitin sulfate adding distil water, take by weighing PEG-400, glycerol and put in the beaker slight fever to dissolving fully, add low-molecular-weight glycosyl chondroitin sulfate cellulose solution mixing, after SDB-L-400 adding 700ml water grinds well in mortar, with substrate and PEG-400, glycerol, low-molecular-weight glycosyl chondroitin sulfate mixing, add water to 1000g, fill promptly.
Claims (15)
1. low-molecular-weight glycosyl chondroitin sulfate and pharmaceutically acceptable salt thereof the purposes in anti-1 type HIV (human immunodeficiency virus) medication preparation, it is characterized in that: described LGC is the mixture with homology glycosaminoglycans derivant of formula (I) structure,
In the formula (I):
D-GlcUA-β 1-is β-D-glucuronic acid-1-base;
D-GalNAc-β 1-is β-D-N-acetylamino galactosamine-1-base;
L-Fuc-α 1-is α-L-fucose-1-base;
R
1For-H or D-GalNAc-β 1-;
R
3, R
4, R
5, R
6, R
7Be independently of each other-H or-SO
3 -
R
2For-OH ,-4-O-D-GlcUA or formula (II) or (III) shown in group:
Wherein, R ' is a L-Fuc-α 1-substituted radical, and has the sulfate group of cotype (1) structure on the L-Fuc-α 1-; R " be-4-[L-Fuc (α 1-3)] D-GlcUA-β 1-group, there are the sulfate group of cotype (1) structure, R on its L-Fuc-α 1-
3, R
4With above-mentioned definition;
With molar ratio computing, the contained D-GlcUA of described LGC, D-GalNAc, three kinds of monosaccharide residues of L-Fuc and contained-OSO
3 -The proportion of base is 1: (1 ± 0.3): (1 ± 0.3): (3.5 ± 0.5);
N is that average is about 3～17 integer;
The weight average molecular weight range of described LGC is 3000～15000Da.
2. LGC and pharmaceutically acceptable salt thereof the purposes in the anti-HIV-1 medication preparation according to claim 1 is characterized in that described LGC is the mixture with homology glycosaminoglycans derivant of formula (I) structure, wherein contains the R of qualification ratio
2Be formula (II) or (III) formula of group (I) chemical compound.
3. as LGC and the purposes of pharmaceutically acceptable salt in the anti-HIV-1 medication preparation thereof as described in the claim 2, it is characterized in that described qualification ratio is meant, in molar percentage, R
2For formula (II) or (III) formula of group (I) chemical compound account for 30%～100% of whole formulas (I) chemical compound.
4. as LGC and the purposes of pharmaceutically acceptable salt in the anti-HIV-1 medication preparation thereof as described in the claim 1 to 3, it is characterized in that described LGC is that the depolymerization product of the glycosylation chondroitin sulfate that extracts of Echinodermata Holothuroidea animal body wall and/or described depolymerization product reducing end are by the amidized product of all or part of reduction cheese.
5. low-molecular-weight glycosyl chondroitin sulfate and pharmaceutically acceptable salt thereof, described LGC are the mixture of homology glycosaminoglycans derivant of formula (I) structure of claim 1 definition, and, in molar percentage, R
2For formula (II) or (III) formula of group (I) chemical compound account for 30%～100% of whole formulas (I) chemical compound.
6. LGC as claimed in claim 5 and pharmaceutically acceptable salt thereof is characterized in that, in the mixture of the homology glycosaminoglycans derivant of described formula (I) structure, and R
2Be formula (II) or (III) group.
7. as described LGC of claim 5 to 6 and pharmaceutically acceptable salt thereof, it is characterized in that described LGC weight average molecular weight (Mw) scope is 5000～8000Da.
8. as claim 5 to 7 described low-molecular-weight glycosyl chondroitin sulfate and pharmaceutically acceptable salt thereof, it is characterized in that described LGC is that the depolymerization product of the glycosylation chondroitin sulfate that extracts of Echinodermata Holothuroidea animal body wall and/or described depolymerization product reducing end are by the amidized product of all or part of reduction cheese.
9. as the preparation method of described LGC of claim 1 to 8 and pharmaceutically acceptable salt thereof, comprise the steps:
(1) from Echinodermata Holothuroidea animal body wall, extracts acquisition acid mucopolysaccharide, i.e. prototype glycosylation chondroitin sulfate GCs;
(2) peroxidating depolymerization step (1) gained GCs, the GCs depolymerization product of collection and purification desired molecule weight range further is converted into required alkali metal ion salt with the gained depolymerization product, obtains the described LGC of claim 1 thus;
(3) the optional reductive amination that carries out terminal saccharide of the described GCs depolymerization product of step (2), its method is, described GCs depolymerization product is reacted with stoichiometric tyramine in the presence of sodium cyanoborohydride, obtains the described reducing end of claim 4～5 by the amidized product of all or part of reduction cheese.
10. as LGC preparation method as described in the claim 9, it is characterized in that: the described peroxidating depolymerization method of described step (2) is: it is 0.05%～10% aqueous solution that the GCs in Holothuroidea animal body wall source is made into mass fraction, in obtained aqueous solution, add peroxide GCs to mass fraction 0.05%～5%, add transition metal ions salt GCs as catalyst to mass fraction 0.01%～1%, under 20～60 ℃, react to GCs by depolymerization to the desired molecule weight range; Add ethanol or acetone and make the depolymerization product precipitation in reactant liquor, after precipitation was dissolved again, ultrafilter membrane was dialysed and/or is crossed the gel filtration chromatography method and remove little minute impurity, and ion exchange further is converted into required alkali metal ion salt with products therefrom.
11. as LGC preparation method as described in the claim 9, it is characterized in that: the method for the terminal reductive amination of the described GCs depolymerization product of described step (3) is: step (2) gained GCs depolymerization product is dissolved in the buffer of pH 7～9, add stoichiometric tyramine and sodium cyanoborohydride successively, room temperature or reacting by heating.After reaction finishes, the separation and purification product, and be translated into required alkali metal ion salt.
12. as LGC preparation method as described in the claim 11, it is characterized in that: the separation and purification of the product of described step (3) is meant in the purified product, with molar ratio computing, and R
2For formula (II) or (III) formula of group (I) chemical compound account for 30%～100% of whole formulas (I) chemical compound.
13. the pharmaceutical composition of an anti-HIV-1, described pharmaceutical composition contain the described LGC of claim 1～7 or its pharmaceutically acceptable salt of effective anti-HIV-1 dosage, and pharmaceutical excipient.
14., it is characterized in that the dosage form of described pharmaceutical composition is aqueous solution for injection, freeze-dried powder injection or topical with suppository, ointment or gel as pharmaceutical composition as described in the claim 13.
15. as pharmaceutical composition as described in the claim 10 to 11 as aids prevention and/or medicine.
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| CN108285498A (en) * | 2017-01-10 | 2018-07-17 | 九芝堂股份有限公司 | A kind of oligosaccharide compound and preparation method thereof and purposes inhibiting intrinsic coagulation factor X multienzyme complexes |
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Application publication date: 20111123 |