JPH07505179A - Oligosaccharides with growth factor binding affinity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The following two summer sugars The present invention relates to the fields of biochemistry and medicine. More specifically, the present invention applies to biological systems. Any biologically active protein or polypeptide that exists, especially the eloquent cell complex. specific growth factors or cytokines such as long-term growth factor (FGF). The present invention relates to a class of novel oligosaccharide products with binding affinity and methods for their preparation. Also , the present invention relates to the use of such oligosaccharide products, especially in medicine.

weight In complex multicellular organisms, such as humans and other animals, cell development, migration, growth, and proliferation are Various aspects of reproduction, sometimes involving cell interactions, are commonly referred to as growth factors. generally specialized soluble proteins secreted by cells of related tissues or Under the control of various extracellular mesoietors or cytokines that are polypeptides There are many cases. In other words, it is often adjusted accordingly.

These growth factors, many of which have already been isolated using DNA recombination techniques and then (synthesized) are thought to act through various mechanisms, but in general Typically, the effect is due to the initial interaction with specialized receptors or binding sites on the surface of target cells. result from the interaction and the target cell is thereby activated to initiate a chain of intracellular biochemical events. Seems to give rise to chains or sequences.

These protein growth factors consist of (particularly high binding to heparin) (characterized by compatibility) is referred to in the general term eloquent cell growth factor (FGF). called, among them, have partial amino acid sequence identity but different isoelectric points The two main forms of FGF are acidic and basic cell growth factor (aFGF). Valve cell growth factor (brGF) has been recognized. FGF is widely used in mammals. Located in mammalian tissues, it is an important signal generator involved in the regulation of cell growth and differentiation. It is thought to function and consist of both normal and diseased physiological conditions as children. Acts as a potent mitogen to stimulate proliferation on a range of cell types, D. Gospodaro on some of the traits and properties of FGF. Ce1l Biology Reviews f199 by wicz 1) Found in "A" Tsuzumi, 305-314.

In particular, basic eloquent cell growth factor (bFGF) is involved in embryonic development, wound healing and tumor development. appears to have an important role in processes such as growth and involves cell proliferation various diseases or degenerative conditions (e.g., diabetic u!I membrane disease, post-cataract surgery) crystalline groove opacification, post-angioplasty gyral stenosis, llff1 tumor angiogenesis and various forms of In particular, it was thought to be directly involved in inflammation (including chronic inflammation). It's a special cell Surface tyrosine kinase receptor IKa 1O-5 (10pM), e.g. is an expression product of fig, and by binding to a receptor that generates an intracellular signal. gives signals to cells. However, bFGF and similar growth factors That is, the mode of action of cytokines is complex, and they can be found on the surface of mammalian cells or Heparan sulfate proteoglycan (HSPG) in the extracellular matrix [K-5-5 It is thought that interaction with the heparan sulfate component of 0nil is also involved. In recent research, For example, in cells with insufficient heparan sulfate (HS) synthesis, fig receptors Does not react with bFGF, but adds heparin or heparan sulfate (HS) It was found that reactivity could be restored by At least in many cases, such It is clear that long-term factors need to be activated before they can exert their biological effects. It seems to be easy. Polysaccharides such as HS and heparin are interacting partners b. induces structural changes in growth factors such as FGF, which bind to signal transduction receptors It has also been suggested that this is a prerequisite for

Therefore, in accordance with this, at least a dual receptor mechanism for the action of bFGF is proposed. A model with a mechanism was proposed. However, conventionally, the bFG of the HS assumption The nature of the F binding site is not completely clear. HS is probably N-1O- The most complex consisting of linear polysaccharide chains with a regular domain arrangement rich in sulfate groups is a major mammalian glycosaminoglycan (GAG), in which basic disaccharide The repeating unit is a low sulfate compound dominated by N-acetylated disaccharide (G), cA-GlcNAc). N-sulfated glucosamine (i.e., GlcA/IdoA) separated by the -GlcNSOm), consisting of glucuronic acid or iduronic acid bound to GlcNSOm. bF Since GF is a heparin-binding growth factor, sulfur containing a species of "heparin-like" region consisting of The oxidized domain is expected to provide the most likely location of the bFGF binding site.

On the other hand, the size of these domains, their sulfation patterns and their size As the ronic acid content varies considerably, a strong interaction with bFGF may result in antithrombin A special 5 in heparin known to have high affinity for III Tightly defined binding domains confer specialized binding domains in a manner similar to saccharide sequences The possibility arises that a sulfated monosaccharide isomer sequence is required. Endothelial cell induced HS It has already been proven by affinity chromatography that it binds strongly to bFGF. from Engelbreth Ho1m Swarm (EHS) tumor. Weaker interactions with HS have also been reported, but the structure for such interactions is unknown. The full architectural requirements are not yet known.

Compared to bFGF, acidic cell growth factor (aFGF) is generally less potent. However, despite this, a wide range of cells (particularly mesoderm-derived It is known as an active mitogen and differentiation factor for cell types). acid line eloquence Cell growth factors are present in various tissues and have approximately the same affinity, such as bFGF. evarin, cell surface heparan sulfate or Strongly binds to extracellular matrix Δ, balan sulfate proteoglycan. Mechanism of interaction It will be clear that the molecule is the same as bFGF. Numerous other growth factors viz. Cytokines also contain heparan sulfate or similar sulfated glycosaminoglycans in the extracellular matrix. lycans or cell surface proteoglycans, here again this is a physiological This may be a necessary prerequisite for biological activity under normal conditions.

These growth factors, cytokines such as FGF, maintain normal physiological conditions. react in a manner that maintains or restores or promotes a disease state considered to have an important widespread role in controlling or regulating cellular processes. therefore, it is possible to control or modify their activity for the purpose of therapeutic treatment. The idea that it can be done is appealing. Thus, for example, cell surface signal transduction binding The development and use of drugs that suppress growth factor activity by blocking receptors is already being considered. If this is the case, for example in wound treatment, the activity of growth factors Use and administration of exogenous growth factors as therapeutic agents when increased giving is considered. Other possibilities to block activity or reduce activity In terms of gender, it is possible to use drugs that act as antagonists or agonists; is a combination of such growth factors and proteoglycans or glycosaminoglycans (and for example, heparan sulfate). This preliminary combination This appears to be necessary before binding to cell surface signal transducing receptors can occur.

Also for this purpose, the use and administration of heparin, which acts as a competitive inhibitor The possibility of this is also conceivable, at least in principle. However, heparin or heparan sulfate itself) are particularly unsuitable for use as drugs in this situation. Ru. This is especially true due to its complexity and the large number of disaccharide sequences in its molecular composition. Due to the heterogeneity of This is because it lacks specificity.What is necessary for use as a drug is convenient and without the risk of promoting unexpected or unwelcome side effects. At low levels, a very high degree of binding to specific glycosaminoglycans of the growth factor High purity or near-purity of relatively small molecular compounds of known composition with specificity of It is a nearly homogeneous drug. In other words, high specific binding affinities or or having the smallest molecule size consistent with a high ratio of biological activity to size. would be most desirable. Such agents therefore of value that blocks or prevents subsequent binding to the body and reduces growth factor activity. or, if so, cell surface signal transduction receptors. to stimulate growth factor activity by promoting subsequent growth factor binding to the body It might work. In addition, if you want to administer exogenous growth factors for therapeutic purposes, For example, perhaps in wound treatment or various other tissue repair applications, this may be the case during administration. Growth factors, such as It may be advantageous to synthesize them. This way, it can be administered together with growth factors. can bind to the glycosaminoglycan binding site of growth factors with a high degree of specificity. Good morning.

Similar to the parent molecule, heparan sulfate fragments of at least one species are also sensitive to FGG growth factors. It can be expected that heparan sulfate has a specific binding affinity consisting of partially depolymerized by enzymes such as hebarinase, heparitinase) It is known that oligosaccharide drugs are produced using relatively short oligosaccharides. Oligosaccharide drugs are generally available in a wide range of different compositions and sizes. consists of a complex mixture of molecular species. Therefore, these drugs are heparin sulfate It is less suitable for use on its own or as a drug than heparin. like this Various fractionations and partial purifications of oligosaccharide drugs or mixtures have been carried out in experiments. , a special structural trait that confers high specificity binding affinity for FGF growth factors. The problem is a lack of more detailed knowledge about the Oligosaccharide products with optimal efficiency and potential as drugs or therapeutic agents This has delayed the development of drugs better suited for certain medical uses.

The present invention studies human skin cotton eloquent cells heparan sulfate, FGF and similar A clear protein with specific binding affinity for heparin or heparan sulfate-binding growth factors. This was done during an experiment in which the precise oligosaccharide structure was isolated and characterized. the result , the present invention provides FG in medical use, particularly in connection with the treatment of the various conditions mentioned above. For use as an F growth factor modulator, it is more effective than any previously known oligosaccharide drug. Suitable oligosaccharide products can be prepared.

Abbreviation Throughout the specification, including the claims, the following abbreviations are used:

GAG - glycosaminoglycan; HS - heparan sulfate; HS PG - heparan sulfate proteoglycan; bFGF - basic line eloquence Cell growth factor; aFGF - acidic eloquent cell growth factor; dp - degree of polymerization (for example For example, in the case of dimorphs, dp=2, etc.); GlcA-D-glucuronic acid; IdoA-L-iduronic acid; IdoA (2S)-L-iduronic acid 2-sulfate; GlcNAc-N-a Cetyl D-glucosamine; GlcNSOs N-sulfated D-glucosamine; GlcNSOz (6S) -N-sulfated D-glucosamine 6-sulfate; G lcA (2S) D-glucuronic acid 2-sulfate; nHexA - unsaturated uron acid residue nG1cA - derived from the saturated residue GicA in the original polymer chain For example, heparitinase cleavage (described below) Based on the known specificity of ;aMan++-terminal 2.5-anhydromannose reduction with N a B H4 2,5-Anhydro-D-mannitol formed by the symbol used here (n) indicates whether the monosaccharide residue is unsaturated or not, and the symbol (±63) is 0 Indicates whether the residue can or cannot be sulfated at the 6 position.

Omega plate for light As mentioned above, the present invention generally relates to highly specific proteins or polypeptides. peptide binding affinity, especially HS-binding typified by growth factors such as FGF. Proteins or polypeptides with high specificity for synthetic proteins or polypeptides Novel oligosaccharide products with peptide binding affinity are provided.

More particularly, - according to an aspect, the present invention provides a method for treating fibrillar cell growth factor (FGF). - Oligosaccharide products with special binding affinities, especially essentially approximately homogeneous with respect to GF binding affinity and preferably arranged in a continuous sequence. at least 4, preferably 6 disaccharide units containing sulfated disaccharide units containing N-sulfated glucosamine residues (±6S) and 2-〇- An oligosaccharide chain consisting of sulfated iduronic acid residues. Provides ligosaccharide products.

Furthermore, each of the sulfated disaccharide units is IdoA (2S)-C1,4-Gl c　N　S　Oa, and furthermore, the oligosaccharide chain is 10 in total. Preferably, it consists of a sequence of less than two disaccharide units. Preferred specific example So, are these oligosaccharide chains a sequence of six disaccharide units in total? four of which are included in the above continuous sequence of sulfated disaccharide units. However, in the most preferred embodiment, a total of seven disaccharide units may be used. There are a number of included in the

Also, the predominant majority of oligosaccharide chains are all of the same length and are O-sulfated at C6. The glucosamine residue content (if any) is less than 20% and more Preferably it is less than 5%. The oligosaccharides according to the invention are generally has almost complete resistance to depolymerization by heparitinase; It is not resistant to depolymerization by enzymes. And this is human line eloquent cell heparan sulfate ・Can be obtained from the proteoglycan (HSPG) heparan sulfate (HS), For this purpose, partial depolymerization of heparitinase and enzymes to the maximum extent is carried out, and then Size differentiation is performed using e.g. gel filtration size exclusion chromatography and then As an immobilized ligand for selected fractions collected from the size differentiation stage, FGF binding fragments were separated by affinity chromatography using FGF growth factor. and then into a salt concentration range that falls under a salt concentration gradient, advantageously sequentially stepwise components. The fragments were separated with respect to FGF binding affinity by selective elution over Recover the fragments that are bound together and optionally further use the methods described above. Add at least one step of size differentiation and selection of recovered product.

Alternatively, a binding parent specific for eloquent cell growth factor (FGF) according to the invention Compatible oligosaccharide products may be defined as having the following characteristics: i.e. , (aHi consists of the following molecular species.

X is nHexA GlcNSOa (±6S), Y is IdoA (2S) -GlcNSO- (±63), and Z is IdoA-GlcR (±63) or IdoA (2S)-GlcR (±63), where R is N5Os or NAc, where n is in the range of 4-7.

(b) the content of monosaccharide residues with 6-0-sulfate groups, if any, is less than 20%; It is full.

(c) Digesting heparan sulfate with heparitinase to achieve its partial depolymerization to the maximum extent. and then the resulting oligosaccharide mixture is subjected to e.g. gel filtration size exclusion chromatography. The size was resolved using chromatography and ranged from 12 to 18 monosaccharide residues.

Preferably, the oligosaccharide product further has the following characteristics:

Y is mostly IdoA (2S)-GlcNSOs, and n is 5 or 6. ri, a total of 7 2@class 12/sotokai certain powers), a l/'+4, 4 or less selected from.

[rtl GlcA-GlcNSO, -(IdoA[2Sl-GlcNSO,] , -IdoA-GlcR.

Here, R is NSO or NAc.

Oligosaccharides according to the invention are in particular oligosaccharides having the following disaccharide sequence: It includes the main component of the product or drug (hereinafter referred to as Oligo-II).

nGlcA-β], 4-GlcNSOa-C1,4-[1doA 12S)- a1.4-GlcNSO3) s-al, 4-Id.

A-al, 4-G1. cR or nG], cA-01,4-[GlcNSO,-al, 4-IdoA (2S) ] a-a l, 4-GlcR where Ri, tNAc or NSO.

And, the small change has at least the same specific binding affinity for bFG and is relatively high.

Although the oligo Pis according to the present invention also have a weak binding affinity for bFGF, The main oligosaccharide drugs (hereinafter referred to as oligo-M and oligo-L) that are It also encompasses related highly sulfated oligosaccharides such as the constituents. These are few At least Oligo-M contains an oligosaccharide chain having the following sequence:

nGlcA-GlcNSO-[±6Sl-[IdoA f2s)-GlcN SOs] 4-IdoA-GlcR (±6S) where R is generally NAc Yes, but NSO! It may be.

The main component of Oligo-L is thought to consist of the following sequence.

nGlcA-GlcNSO, -IdoA-GlcNAc (6S) -GlcA -GlcNSOs (6S) - [1doA (2S1-GlcNSO,], -1doA-C1cR (±6S) and n GlcA-GlcNSO, -IdoA f2S) -GlcNSO, -1d oA-GlcNAc (6S)-GlcA-G1. cNSQ, (6S)-Id oAf2S)-GlcNSOs-1doA-GlcR(±EIS) where R is Generally, it is NAc, but it may also be NSO.

Oligosaccharide products according to the invention may be isolated from natural sources or synthesized. .

With respect to isolation from natural sources, the present invention further relates to the isolation of heparan sulfate, In a relatively homogeneous state purified from eel glycosaminoglycan, the glycosaminoglycan Binds to noglycan or corresponding proteoglycan in multicellular biological systems have specific binding affinity for the selected bioactive protein or polypeptide. This is a method for separating oligosaccharides that are Collect a fraction that is thought to contain oligosaccharides consisting of less than 10 disaccharide units. (d.) the affinity chromatography substrate from step (a) or prepares the substrate from step (C) containing a particular number of disaccharide units ranging from 4 to 9. A selected fraction or a set of selected fractions is contacted with the immobilized kumbaku substance or poly of glycosaminoglycans that also have at least some binding affinity for the peptide extracting and retaining sized oligosaccharide fragments on said substrate or substrate; (e) Affinity chromatography while gradually increasing salt concentration or salt gradient eluting the substrate or substrate; (f) Fraction or distillate containing oligosaccharide fragments in the selected maximum dripping salt concentration range and, optionally, (g) from step (f). of a selected fraction or set of fractions in place of the reaction mixture obtained from step (b). Selecting step (c) using the selected fraction or set of fractions collected in step (f) and optionally repeat steps (d), (e) and (f). The stage of further purification through repetition. glycosaminoglycans by a chemical method that causes deamination in situ. Partial depolymerization may be carried out.

However, at least in the case of heparin sulfate, the preferred selective cleavage reagent is polysaccharide The lyase enzyme is baritinase. This is Seikagaku Co. in Tokyo. Rporation is commercially available under the name r) Ieparinase IJ. Or, Sigma Chemical Co.

is commercially available under the name rHeparinase IIIJ, and according to the EC classification 4.2.2.8. This enzyme works on the non-reducing side of GicA-containing disaccharides, even if For example, GlcNAc-C1,4-G present in the low sulfation region], The cosidic bond will be selectively separated, but generally L-iduronic acid or 2-sulfated monoiduronic acid (i.e., IdoA or IdoA (2'S)) It does not separate the bonds of sulfated disaccharides containing . This means that 2-sulfation Heparin sulfate to separate glycosidic bonds between disaccharides containing L-iduronic acid (EC4, 2, 2, 7). If you want a closer look, check out RJ Linhardt et al f19901B iochemistry '29.2611-2671), A few heparitinase enzymes are known, and these have approximately the same binding specificity. However, the depolymerization efficiency varies depending on, for example, the size of the substrate molecule. But long Generally speaking, throughout this specification, including the claims, unless otherwise stated, , the term "heparitinase" is used as rHeparitinase IJ Seikagaku (: Enzymes distributed by the organization or the same glycerin Used to indicate any equivalent enzyme with cosidic binding specificity.

polysaccharide or oligosaccharide glycosidic linkages, e.g. 1.4 linkages, heparitina In connection with enzymatic cleavage, such as heparinase and heparin sulfate, What you want to get is that in one fragment, the non-reducing end immediately adjacent to the broken bond However, monosaccharide residues generally have a double bond formed between C4 and C5. This means that it becomes unsaturated. However, this unsaturation It is unlikely to affect factor binding affinity in any way and probably affects the stability of the molecule. It might make an impact.

Oligosaccharides or oligosaccharide products according to the invention generally have a defined composition. It has a complex structure and can easily be further purified if necessary. Also those Given its size and unique growth factor binding affinity, it has great potential in the pharmaceutical field. used as growth factor inhibitors or activators and mobilizers, e.g. very well suited to. Therefore, the oligosaccharides or oligosaccharides according to the invention Ligosaccharide products may be used as therapeutic agents to control or modulate the activity of FGF, especially bFGF. It is expected to be particularly valuable as a medical therapy. This applies, for example, to diabetic retina. symptoms, post-angioplasty ilestenosis, lens capsule opacification, proliferative vitreoretinopathy, arthritis and other chronic inflammatory conditions, cancer cell growth, tumor angiogenesis, moderate muscular dystrophy, Alzheimer's disease Immerosis and various viral infections (for example) lerpes SimpL In the clinical treatment of symptoms such as ex type 1), FGF-dependent cell growth This can become a problem when there is a need to control or suppress proliferation.

This also applies, for example, to wound treatment, fracture treatment, nerve regeneration, duodenal ulcers or or venous ulcers, various ocular and retinal diseases, atherosclerosis, degenerative muscles. To promote treatment or tissue repair in the clinical treatment of diseases, conditions such as ischemia or endogenous FG to protect tissue from severe damage during radiotherapy. Problems also arise when it is necessary to stimulate FGF or administer exogenous FGF for activation. There is a possibility that it will happen. For these purposes, the present oligosaccharide product (or its pharmaceutical (acceptable salts) can be made as pharmaceuticals if necessary; such Uses also fall within the scope of the invention.

Further description to enable those skilled in the art to more easily understand the nature of the invention and enable it to be practiced more easily. In order to achieve this, the following describes some of the background experimental work carried out by the inventors of the present invention. and its various practical details. In connection with this explanation, Reference must be made to the attached drawings.

The hard surface of the drawing Figure 1: This figure shows native, partially depolymerized bFGF on an experimental bFGF-affinity column. In these experiments, the H3J chain (control-panel A ) or HS treated with heparin sulfate (panel B) or treated with hebaritinase. A sample of 3H-labeled HS (panel C) was labeled with a bFGF affinity parent as described below. The Japanese color needle was raised. The binding substance is sodium chloride as shown in panel AC (dashed line). It was eluted with a stepwise gradient of . A compound that retains a high affinity for bFGF. Heparitinase oligosaccharide (panel C, 1-1.5M, soluble in NaCl) (see fractions 22-35) and then dialyzed with 5pectra por 71000-Mr cut-off, Spectrum UK), Lyophilized. Next, their size is 4n+l in 0.5MNH,HCO, by gel filtration on a Blo-Ge1 P6 column (IX120 cm) at a flow rate of /h. It was confirmed.

Figure 2: This figure shows the relationship between the affinity of HS oligosaccharides for bFGF in the experiment. In these experiments, the 3H-labeled H3 chain The strands were treated with baritinase and subjected to Blo-Ge1 P6 chromatography. Therefore, it was divided into sizes. Vice hebaritiner of size dp14 and dpi 4 The oligosaccharide fractions were pooled and then subjected to bFGF affinity affinity chromatography. I split it up. Elute with 0°75M, 1.0M and 21.25M NaCl Three main fractions were obtained. These are, respectively, Shown by M (medium) and oligo H (high) affinity chromatography. These b Affinity for FGF was determined as is (solid line) and after heparinase treatment (dashed line). ) and reapply to the affinity column (with a stepwise concentration gradient of sodium chloride). Figure 3 tested by (panel A, broken, IJl): This figure shows the experimental b Blo-Ge1 P6 group of HS oligosaccharides with different affinities for FGF romatography results are shown, and in these experiments the relative Relatively low affinity (Oligo-S), medium affinity (Oligo-M) and high affinity The HS oligosaccharide (dpi2-14) with (oligo-H) is explained in relation to Figure 2. Prepared as described. These size distributions are shown in the as-is state (solid line) and in the hepatic state. Blo-Ge1 P6 chromatography after linease treatment (dashed line) Therefore, it was confirmed.

Figure 4: This figure shows the bFGF-conjugated HS oligo that was deaminoated in the experiment. The results of Blo-Ge1 P6 chromatography of saccharides are shown. These experiments , low affinity for bFGF prepared as described in connection with FIG. (Oligo-shi), medium affinity (Oligo-M), and high affinity (Oligo-H). Oligosaccharides were treated with nitrous acid and analyzed using Blo-Ge1 P6 chromatography. I divided it. These disaccharides (dp2) are partially bisulfated species (main peak) and and unsulfated species.

Figure 5: This figure shows bFGF-conjugated oligosaccharides after hebaridinase IV depolymerization. (Oligo-H and Oligo-M) Blo-Ge1 P6 chromatography - show the results.

Figure 6: This figure shows dp=2 after preparation by Blo-Ge1 P6 gel filtration. bFG of heparitinase-resistant oligosaccharides for various sizes of ~dp=14 The results of F affinity chromatography are shown.

Figure 7: This figure shows growth factor activation for bFGG and aFGF, respectively. Graph showing the effect and size of related H3-binding oligosaccharides and binding affinities It is.

FIG. 8; This figure shows the bFGF parent as mentioned in the examples described below. Heparitinase quenching of 3H-labeled line eloquent cells H3 prior to affinity chromatography. Representative results of Blo-Ge1 P6 gel filtration of compounds are shown.

Heel rope wish theory skin ヱjiro example Initial background experimental work that led to the present invention involved human skin cotton eloquent cells and human tissue. An investigation was conducted using heparan sulfate derived from alternative bFGF as a raw material.

Human recombinant bFGF is produced by Ke, Y, et al. (19901Bi.

chem Biophys, Res, Comm, 171.963-971 It was prepared in a manner similar to that described in . In other words, recombinant bF GF can be detected by heparin sepharose chromatography and bacterial cell chromatography. Purified by reverse phase or cation exchange HPLC from lysate, human bFGF Amino acids 1-155 (Abraham, J, Aet al, t198 6] PKK encoding EMBOJ (see “2.2523-2528) 233-2-bFGF construct (A+nann, E et al, f198 5) Gene, 40.183-190 t3 reference) and 5DS - Generated a single compound of MK 17 kDa on PAGE. amino acid The sequence matches that of human bFGF, and the recombinant protein is derived from the complete organism. It had activity.

``H-glucosamine biosynthetic radiolabeled HSPG and HS chain, Turnbull & Gallagher's paper (Turnbull, J. E, et al, f1991) Biochem, JA 273 , 553-559). was prepared from a fusion culture of. The content of this paper is incorporated as reference material.

Among the experimental techniques used, depolymerization of H3 chains with heparitinase; or low p)-1 nitrite and oligosaccharides at P6 or P2 of Blo-Ge1 Similar gel chromatography was performed by Turnbull J, E, et al. , (19911 Biochem, J, 277, 297-303) It was carried out as described. Also, Inoue & Nagasawa (Inoue, Y, eLal (1,976) Carbohydrate Res, 46.87-95), the chemical N-d esul, phaLion/re-N-acetylation was performed.

This work also involved affinity chromatography and 2M! strong anion exchange of bFGF-Affi-Ge1 was used for affinity chromatography. 10 affinity substrates were used. To prepare this affinity substrate, Bio-Rad 1ml packed Affi-Gel 10”’activation affinity from the laboratory The gel was washed with 5 volumes of double distilled water using centrifugal force at 800 g for 1 min. Washed twice. Heparin (500u g) was mixed with bFGF (500 g in 3I Added to nl O, 6M NaCl, 25miJ Na, HPO4, pH 6, 6) and mixed with 1 ml of packed Affi-Gel 10, washed overnight at 4°C. Add 2ml of 4M Tris-HCL pH 8.0 to remove any unreacted material on the gel. I blocked the group. Add 5 mg of heparin to bind bFGF and store Stabilized 20% (w / v) N a N z of 465 μm as an agent . The gel was diluted with 10 bottles of 2M NaCl in 10mM Tris-H (:1, pH 6.5). Washed with Rium. bFGF was not detected in the wash solution by reverse-phase HPLC. First, this indicates a high degree of coupling efficiency.

Affinity chromatography was performed as follows.

Pump approximately 1 ml of bFGF-Affi-Gel 10 affinity substrate into the glass column. [Bed size 6mm x 35mm], flow rate of 0.25ml/min, 10m Samples were loaded onto the column in M Tris-HCI, pH 6,5. unbound The material was eluted by collecting five 1 ml fractions. The bonding material is 0.5m Stepwise concentration gradient of sodium chloride (color in 0,25M step) at a flow rate of 1/min. Elution was performed using 0-2M NaCl in buffer. Five 1 ml fractions each Collected at concentrations. The column was filled with 10 Mg/ml heparin (Sigmal, 0.0 A running bag containing 1% (w/v) sodium azide and 0°2M NaCl It was stored in a refrigerator at 4°C.

In order to analyze the disaccharide composition of HS oligosaccharides, HS oligosaccharides were treated with heparitinase. A mixture of enzyme, heparitinase II and heparin (Seikaga, Tokyo, Japan) ku　K.

(obtained from GYO Co.) was completely enzymatically depolymerized. Blo-Ge1 disaccharide Collected by P2"" chromatography and loaded onto a Propac PAI analytical column (4X 250 mm; separated by HPLC on Dionex, UKi. 1ml Equilibration was carried out in the mobile phase (double-distilled water adjusted to pH 3,5 with HCI) at /min. After injecting the sample, the disaccharide was added to a linear concentration gradient of sodium chloride in the same mobile phase. Eluted with O-LM (O-LM over 45 minutes).

The eluent was evaluated for UV absorbance (A2. for unlabeled disaccharide order) and About radioactivity (Radiomatic Flo-one/Beta A-20 0detector) was monitored inline. Disaccharides released by nitrite treatment As mentioned earlier (Pijler, G. et al. (19871B) iochem, J., 248.67-69 and Bienkowski, M. , J, et al, Biochem, J, Q6几 356-365), separated by HPLC.

In early experiments, HSPG metabolically labeled with 'H-glucosamine was It was purified from the culture medium of the dermatophyte. The H3 chain is mediated by pronase treatment of HSPG. and loaded onto an affinity column prepared with human recombinant bFGF as described above. Filled. Combined substances range from 0.25M to 2.0M in 1’U floor. Elution was performed stepwise with NaCl concentration. The majority of HS binds strongly to bFGF, and the main The peak elution was 1.25M NaCl (see Figure 1A). Similar elution distribution Obtained for HSPG without any treatment (results not shown). this thing showed that heparan sulfate (HS) linkages are the major determinants of proteoglycan binding to bFGF. This shows that it is a constant factor. Hyaluronic acid is a bFGF column and fibroblast inducer. Does not bind to chondroitin and dermatan sulfate is in the range of O-0.5M NaCl It was eluted with However, commercially available heparin is mainly 1.25M, 1.5M Eluted with NaCl (data not shown). These results indicate that bFGF N-sulfate This shows a unique interaction with chemical polysaccharides. The importance of the N-sulfate group in nitrous acid is Deamino acid cleavage or N-desulfation/re-N-ac of HS Confirmed by the finding that etylation terminated high affinity interactions (Results not shown).

The problem of identifying the bFGF-binding domain within H3 is Treated with heparitinase, an enzyme that selectively cleaves polysaccharides. . As already mentioned, hevarinases act on the N-sulfated region and the 2-0- Sulfated iduron salt, i.e. GlcNSOi (±6S)-al, 4-I It specifically cleaves disaccharides including doA (2S). The main product is 9-10 kDa It is a oligosaccharide. On the contrary, heparitinase is a Gl cA-containing disaccharide, in principle, cleaves GlcNAc-al, 4-GlcA, but , type JGJ, cNSOs (±6S1-aL, 4-IdoA (±25) ],.

N-sulfuric acid and N-sulfuric acid will not be attacked.

Hebarinase cleavage of S] produces a product with significantly reduced affinity for bFGF. and elution occurred in the range of 0.25-0.75M NaCl (Figure 1B, 1.1). i&,). The effect of heparitinase digestion is even more pronounced; most of this material is Either it failed to bind to the system or eluted with 0.25-0.75M NaCl ( (See Figure 1C). However, a small fraction of oligosaccharides within the heparitinase digest shows affinity for bFGF, which is associated with untreated H3 (1,O-1,5M Na (eluting in the C1 range). Gel filtration size for Blo-Ge1 P6 Exclusion chromatography shows that these high affinity products are present in six and seven Preferentially two oligosaccharides, dpi2 and dp14, with a size equal to the disaccharide unit. It was shown that it consists of class fractions. These fractions are derived from human skin line eloquent cells H3. It is the largest fraction present in significant amounts within the Chinase digest. This data is H, The extended N-sulfation sequence in S has the highest affinity for bFGF. contains a binding site and that IdoA (f2s) makes an important contribution to the interaction. It shows.

Binding specificity of oligosaccharides We will examine the specificity of oligosaccharide interactions in more detail and investigate the associated structural features. To study the symptoms, some acute heparitinase oligosaccharides were added to Blo-G. Directly from the heparitinase digest of e1P6 gel filtration size exclusion chromatography It was prepared in close contact. Selection of size dpL2, dp141 (12.5% of total product) The components were pooled and resolved again by bFGF affinity chromatography. . Elutes with 075M, 1.0M, 1.25M NaCl, oligo-low), Three major oligosaccharides with affinity for oligo-M (medium) and oligo-H (high) fractions were identified. Reapplication of these fractions to the column allows for bFGF to be Different affinities were confirmed (see Figure 2). Oligosaccharides of size dp14 is mainly present in the oligo-H fraction, and the oligo-M and oligo-L fractions are predominantly dpi2 (see Figure 3).

Hevalinase treatment significantly reduces the binding of these oligosaccharides to bFGF. (See Figure 2). The degree of depolymerization (Figure 3) is closely related to the loss of affinity. was. The presence of the major product (Fig. 3) of size dp4 (IP6) is due to the cleavage of internal bonds. Indicates a decision.

Disaccharide composition of oligosaccharides The disaccharide composition of 1-1, M, and L oligosaccharides (see Table 1) was It was evaluated by saccharide lyase depolymerization and strong anion exchange HPLC. calculate The molar ratios are shown in Table 2. The most striking feature of this analysis is the Type Pro HeyJi ( 2S)-C1,4-GlcNSO, disulfated disaccharides, especially oligo-H, oligo - Disulfated disaccharide in M (approximately 74% and 60% of 2Fl units, respectively) The content was high. The hebarinase sensitivity (Figure 2.3) of these fractions is: Most of the disulfated HexA residues are initially I prior to cleavage of their glycosidic bonds. doA (2S) and was shown to be unsaturated. IdoA in natural H3 Since the content of (2S)-disaccharides is about 10-12%, these results The residues were found to be about 7 times more abundant in Oligo-H and about 6 times more abundant in Oligo-M. ing. The total concentration of nHexA (2Sl-al, 4-GICNSO3 is H,M , is strongly associated with the various bFGF affinities of oligosaccharides of L (Table 1 and Fig. 2 reference). Conversely, the 6-0-sulfated derivative nHexA-α1.4-GlcNA c (6S), nHexA-crl, and 4-GlcNSO-(6S). The joint strength showed a significant inverse relationship. These account for 26% of oligo disaccharides. However, it is a minor component in Oligo-H (Table 1.2). N-acetylation 2 The amount of sugar 1nHexA-C1,4GlcNAc is the same for each oligosaccharide drug. This corresponds to approximately 1/fragment (Table 1.2).

Deamino acid cleavage at low pH nitrite almost exclusively involves oligo-H, oligo-M 2@ class products were made with fractions (see Figures 4A and 4B; 99% and 9%, respectively). 5%). In contrast, both disaccharides (76%) and tetrasaccharides [24%] - is the major product from the L fraction (Figure 4C). Therefore, in H and M fractions Virtually all internal gysosaminigose bonds within oligosaccharides are GlcNSO. , with a residue, the N-acetylation unit is clearly at the reducing end of the fragment. (see below). This means that the results show a large proportion for oligosaccharides (60- 70%), which shows the presence of internal N-acetylglucosamine. Ru.

The disaccharides released from the H, M, and L fractions by nitrous acid also undergo strong anion exchange. The identity of the component uronic acid residues was determined by chromatography. Oligo-H is standard IdoA (2S)-aMa, 71% labeled product eluting at n++ position Souvenir production. The remaining labeled products are nGlcA-aManR and IdoA- It eluted as a non-sulfated peak corresponding to GlcNAc. Oligo-M and Oligo-M The goals were 61% and 37% for IdoA and (2S)-aManR, respectively. was produced. Therefore, the disaccharide IdoA (2S)-aMan in each fraction The content of ++ was determined by lyase depolymerization of nHexA (2S1-C1,4 - Same size as that of GlcNSO3 2. Place the HS oligosaccharide dice in a bowl that has become resistant to FGF. , similar to bFGF has low (low oligo-L), medium (9 oligo-M), and high (oligo-H) affinity for HS oligosaccharides (dp12-14) were prepared as described in connection with Figure 2. . As mentioned above, the disaccharide composition was determined by strong anion exchange HPLC. Go-L Oligo-M Oligo-HnHexA-GIcNAc i3.6 16. 0 11.3nl+exA-GlcNAc f6sl 12.4 2.6 0. 9nHexA-GlcNSO, 27,311,37,5nHexA-GlcNS Oa(6S) 14.0 4.5 1.4nHexA f2s)-GlcNS O-31,059,874,2n HexA (2Sl-GlcNSO, (6 S) 0.6 2.2 1.02 Saccharide yield % 98.9 96.4 96.3 Table λ 2nd floor of HS Oligosushi who became bFGF and became a student. ″′′ The ratio table shows the average relative molar ratio of component disaccharides in HS oligosaccharides (disaccharide composition data in Table 1). (based on data). The predominant average size of these oligosaccharides is shown.

Disaccharide Oligo-L Oligo-M Oligo-I] nHexA-GlcNAc O ,8? , 1.00 0.820HexA-GlcNAc f6s) [1,7 50,160,07nHexA-GlcNSO,1,650,700,55nH exA-GlcNSOx f6s) 0.85 0.28 0.10nHexA [2S)-GlcNSOa i, aa 3.72 5.39n HexA (2S) - GlcNSOa f6s) 0.04 0.14 0.07 (Mo 1 mol oligosaccharide) 6 6 7 A novel enzyme, heparitinase IV (Se These oligosaccharides were synthesized using - characterized Kens. This enzyme is a hevarinase [i.e., GlcNSO3[ ±6Sl-a (similar binding specificity for 1-41-IdoA f2sl small substrates containing sensitive bonds (e.g., tetrasaccharides and hexasaccharides) It is quite efficient in cutting.

Treatment of oligo-I with the enzyme heparitinase IV results in a high degree of depolymerization. , disaccharide, and tetrasaccharide products (as shown in Figure 5, 69% of the H level, 31%). These results support the proposed priority sequence for Oligo-H. The ratio of the number of disaccharides to tetrasaccharides shows good agreement with the expected ratio (5:1) based on 4.5:1 is shown.

Treatment of oligo-M with heparitinase IV also results in a high degree of depolymerization, Mainly disaccharides and tetrasaccharides, but some hexasaccharides (respectively, ”) The results show that the expected ratio (4: 1:0) and 4:1.4:0 for the 2.4.6 sugar order. The molar ratio of 3 is shown.

The above-mentioned experimental work was performed on the sulfated oligosaccharide fraction (oligo-H ) is particularly strong against bFGF (consisting of a sequence of seven disaccharides that bind) showed that. The preferential structural unit in this oligosaccharide is IdoA (2Sl -al, 4-GlcNSOs (74% of disaccharides; Table 1), and 2-〇-sulfate Both the group and the N-sulfate group appear to be essential for binding activity. deamidation Analysis of the disaccharide composition following cleavage revealed that the identity of the uronic acid component of this disaccharide was nH. exA-C1,4-GlcNso, and nHexA-al, 4-GlcNAc. It was confirmed that

Because oligo-H is the product of heparitinase digest, it is the major or most The sequence of oligosaccharide component(s) is estimated to be as follows: Ta.

1'1GlcA-β1.4-GlcNSOi-al, 4-[IdoA (2S l-a 1.4-GlcNSOsJs-al, 4-Ido`-al, -GlcR or, nGlcA-al, 4-[GICNSO3-al, 4(doA[2S)]a-a 1,4-GlcR where R is generally NAc, but may also be N5Oa.

These structures were confirmed sequentially, but without significantly affecting specificity binding affinity. Table 1.51 of disaccharides containing GlcNAc (6S) The presence of a 6-〇-sulfate group in either of the Changes can occur. Polysaccharide lyase depolymerization of these sequences is shown in Table 1. resulting in a disaccharide composition for oligo-H that closely matches that of The fact that the G1cNAc residue is placed at the reducing end of the sequence (position 4) is sensitive to deamino acid cleavage (Fig. 4A). ), which indicates the existence of a single continuous sequence of N-sulfated disaccharides. Indicates presence. If GlcNAc is placed somewhere else in the sequence, e.g. position 2 However, if placed, deamino acid cleavage would result in a disaccharide order plus a tetrasaccharide product. (e.g., Fig. 4C, when observed as an oligomer) ) For oligo-M, the main or predominant oligosaccharide linkage has the following sequence: It was confirmed that there was one.

nGlcA-GlcNSOs l±6S1-[IdoA (2S)-Glc NSO, ], -IdoA-GlcR (±6S) where R is generally NAc However, it may also be NSO@.

This is an extended continuous sequence of IdoA (2Sl-C1,4-GlcNSOs units). It is believed that this is the first time that the cans have been identified within the HS. 6-〇-Surprising effect of sulfate group It is clear that the GlcNSOs residues are often sulfated with C6. , that is, [IdoA (2Sl-C1,4-GlcNSO-(6Sl)]n This oligosaccharide was distinguished from the typical N-sulfation sequence in heparin. Separate. The oligo-H sequence confirmed here is not necessarily the best for bFGF. It does not necessarily represent the minimal sequence for proper binding and may represent the total activity of bFGF. (measured by its ability to bind to flg receptors) is about the same size as oligo-H. (i.e., dp14-dp16) That seems noteworthy. The related cytokine acid FGF also falls within this size range. is activated by heparin oligosaccharides, and the types of oligosaccharides shown here are It was also found that they combine with the class.

The structural requirements for optimal high-affinity interaction with bFGF are Comparing the composition with oligosaccharides showing intermediate and low affinity for bFGF It can be indicated by These oligosaccharides are also resistant to heparitinase digests. As certain oligosaccharide products, the same basic 2 of IdoA-GlcNSOs Contains saccharide repeats. Oligo-H and Oligo-M have similar degrees of sulfation (respectively , 1.6.1.5 sulfate/disaccharide), but Oligo-M has oligo-H compared to 74% of IdoA (2Sl-al, 4-GlcNSO-). Contains about 60% of type 21 in form.

Approximately 10% of the amino sugars within Oligo-M are 6-0-sulfated (Table 1), with total sulfate ion, there is a large offset to the lower concentration of IdoA (2S). two The only other detectable difference in the fractions is size, with oligo-M replacing oligo-H. It contains predominantly 6 disaccharides compared to 7 (Figure 3). Fragment size and The combined effect of the abundance of oligo-doA (2S) is greater than that of oligo-H with bFGF. This is considered to be an important property that facilitates strong interactions. IdoA (2S) The importance of Here, 3% of the disaccharides in the star contain this component and are consecutive in the sequence. Whether or not it is possible to obtain two or fewer basic IdoA-GlcNso, disaccharide reactions. It appears to be a recovery unit. However, oligo-L is GlcNSOs (6S) and GlcNAc (6S) (Table 1), even higher It is frequently sulfated (1.3 sulfates/2@). In addition, oligomers have some peculiarities. The main oligosaccharide component that has FGF binding activity and has the above-mentioned sequence is May have some utility.

The data thus obtained provide some relevant insight into the structural heterogeneity of HS.

Differences in 〇-sulfation of large N-sulfated oligosaccharides contribute to the complex mechanism of H3 biosynthesis. As a result, 2- and 6-sulfotransferases can be regulated independently. Gl c A special sequence consisting of N S Os and IdoA (2S) is bFG Although it seems to have strong binding to F, it has a different sulfation pattern. sequences, especially those with a mixture of 2-16-sulfate isomers, are may interact with binding proteins and other members of the FGF family HS sequences that are somewhat different from those preferentially recognized by bFGF and It is thought that there is a possibility of binding to thus retaining the same basic traits However, it is clear that there is room for certain structural changes. In particular, b For FGF (or aF G, F), a higher proportion of hexasulfated glucose Cosamine residues appear to be quite detrimental to binding affinity and also Oligo-M and sequences similar to oligo-M also have a useful degree of specific binding parentage. gives oligosaccharides with compatibility, but oligo-H is not very expensive.

Identification of specific binding sequences in glycosamineoglycans (GAGs) is important for understanding biological fractions. Antithrombin-II in heparin The sequence of the I-binding region is a major advance in this field (Lindahl, 198).

Interactions are singular and determined primarily by relatively non-singular electrostatic forces Rather, it requires distinct sugar sequences and sulfation patterns. anti Thrombin-III acts on heparin in a manner similar to the HS/heparin activity of bFGF. Therefore, it is activated. Thus, the unique interaction with GAGs is a latent protein. The oligosaccharides of the present invention can also be converted into active forms. F, for example, has evidence that it is effective in activating bFGF. Ta. However, the activation ability is limited to oligosaccharides consisting of less than 6 disaccharide units. does not seem to exist, at least not to a significant extent. Tampa against HS When a protein ligand or similar GAG is known, it is disclosed herein in accordance with the present invention. method (specific polysaccharide cleavage and size partitioning by affinity chromatography) ) can also isolate protein-binding domains in these other cases and It should be considered useful for marking.

biological activity In relation to the biological activity of heparitinase-resistant oligosaccharides, FGF-stimulated mitosis Using a mitogenic HS-dependent biological assay, we determined the origin of aFGF and bFGF. Further research was conducted on the relationship between gosaccharide affinity and activity. This test method is 3T3 lines grown in the presence of sodium chlorate (which inhibits polysaccharide sulfation) Although fibroblasts do not respond to aFGF or bFGF, they do not respond to HS or heparin in the culture medium. The responsiveness ("H - based on the fact that thymidine uptake) is restored. It is something that By doing this, exogenous HS oligosaccharide that activates FGF You can test your abilities. The number of oligosaccharides with structures ranging from and affinity for FGF, especially size dp6.8.10.12.14.1 6 and larger size (from porcine mucosal cells H3) resistance to heparitin Zeoligosaccharides were studied using this test method. bFGF, aFGF Preliminary results for both are shown in FIG. This is an oligosaccharide larger than dpi2 This indicates that those smaller than ctpio are inactive, whereas those smaller than ctpio are active. are doing. This indicates that the specific Oligosaccharides with larger structures (and structures similar to those described here) only those that can fully biologically activate FGF, and the smaller structures, even though they have binding affinities of This suggests that it does not activate growth factors.

preparation Indeed, the oligosaccharides of the present invention can be used in gel filtration as described herein in connection with research experimental work. Using techniques of perchromatography and FGF affinity chromatography, It would be convenient if it could be prepared from purified heparin sulfate (both natural and recombinant). , generally heparin sulfate does not need to be radioactively labeled for pure preparative purposes. There's no need. Oligosaccharides derived by heparitinase cleavage are due to unsaturated terminal uronic acid residues that exhibit strong absorption at 232 nm). Can be easily monitored.

Practicing the present invention, and according to the present invention, an agent having a relatively high affinity for bFGF Specific examples of preferred methods of preparing oligosaccharide products are described in more detail below. Ru. Here we begin with a preliminary purification of the heparan sulfate source material.

Compliance example Preliminary purification of Dandan A commonly applicable procedure for extraction and purification of PG and GAC is connective tissue Two detailed papers covering the method both for cultured cells (Hein et al. egard lk Sommarin, 1987. Methods in Enzymology, 144.319-372; Yanag≠Tang■Amaga= @et al., 1987. Methods in Enzymology, 138 ．． 279-2891, but the preferred approach for the purposes of this example is The following describes the purification of HS from skin line eloquent cells grown in culture medium. .

A fused culture of eloquent cells was grown in Eagle's minimum essential medium at 37°C [CO□/ai r, 1:19). This medium contains 15% (v/v) donor-calf Citrus serum, 2mM-glutamine, 1mM-sodium pyruvate, non-essential amino acids , penicillin (100uits/ml, streptomycin f100ug/ml) ml) was added.

Cells can be harvested at the fusion site after biosynthetic radioactivity leveling if necessary [ Na”5O4 (e.g. at 10-50 uci/ml) or [3H1 gluco Samin (for example, 10-20 LL (:i/ml)) or both by culturing for 72 hours]. HS can be extracted from both the culture medium and the cell layer.

After removing the medium, the cell layer was soaked in warm (37°C) phosphate-buffered saline (PB). Washed twice with S). Centrifuge this mixed solution (200xg, 10 minutes) , cells and other debris are pelleted, and the resulting supernatant is used as a medium extract. becomes. HS was incubated with 0.05% (w/v) trypsin for 30 min in PBS at 37°C. It can be efficiently extracted from the cell layer by processing for minutes. thus Centrifuge the resulting supernatant cells as above, and carefully remove the supernatant. Pe After washing the cells twice with PBS, the combined supernatant was used as a tryptic extract of the cell layer. Ru.

Initial purification of the soluble extract of the crude material by anion exchange chromatography . Transfer the sample in PBS to a DEAE-3ephacel column (1cm x 5cm) Wash with 20mM phosphoric acid (0.3M NaCl in 1 solution, pH 6,8). to elute proteins and hyaluronic acid as contaminants. combined A concentration gradient of PG and GAG in 20 mM phosphate buffer, 0.3-1. Elute with 0M NaCl. A fraction corresponding to HS (typically about 0.53MN aC1) were collected, pooled and eluted for 5 ephads with distilled water as eluent. Desalt on ex G-25 column (2.5 cm x 40 cm) and lyophilize.

Traces of contaminated GAGs (for example, chondroitin sulfate and dermatan sulfate) are Treated with 1 unit of clondroitinase ABC for 3-4 at 7°C. It can be removed by

The protein core of the HSPG is then removed using chondroitinase ABC nibs. Add lonase (5 mg/ml final concentration) and calcium acetate for 24 hours at 37°C. Just let it digest for a while. The H3 chain was removed by IMN after eluting contaminants with 0.3NaCl. Repair by stepwise elution from DEAE-3ephacel with aCl. H.S. The fraction containing S is then heated at 100°C for 10 min and then subjected to dialysis (S pectrapor 7 high purity dialysis membrane) or the above 5e Desalt on a pbadex G-25 column and lyophilize.

1. Depolymerization of HS for selective production of ioligosaccharides A. Purified as above. In biosynthesis, H3 chains labeled with 3H or SO4 or both are Paritinase, for example, sold by Seikagaku Kogya Co. in Tokyo. Heparitinase I (E, C, 4, 2, 2, 81 treated with relatively low sulfuric acid) The cleavage is carried out within the region of Leaving the more highly sulfated domains intact. More specifically, freeze-drying A sample of HS (7 x 10'dpm) was diluted with 0.2mM calcium acetate. in 20(')LLl of 100mM sodium acetate at pH 7.0 containing and treated with baritinase T (5 milliunits) for 16 hours at 37°C. A further aliquot of 5 milliunits of heparitinase was then added and incubated at 37°C for 1 hour. Incubate for hours. Digestion is usually complete in 3-4 hours, but usually lasts 16 hours. complete cleavage of all hebaritinase-sensitive junctions must be performed. . The progress of this reaction is shown in the case of unlabeled HS at the non-reducing end of the digestion products. 4.5 - Increase in absorbance at 282 nm due to formation of unsaturated hexrone residue Can be monitored by measuring. Extinguish by heating at 100℃ for 2 minutes. The process ends.

Another chemical method for selective preparation of sulfated domains from HS is to convert polysaccharides into After special de-N-acetylation, the resulting N-unsubstituted glucosamine It involves making special cuts with the residue. The methodological details have already been explained in detail. (311akleand Conrad (19841, Biochem , J, 217. 187-197; Guo and Conrad (198 91CAna Lytical Biochemistry, 176, 96-104). easy In other words, de-N-acetylation is 1% (W/V) hydrazine sulfate. Sanated at 96°C for approximately 4 hours in 70% (w/v) aqueous hydrazine containing acid. Perform hydrazinolysis by heating the pull. dried in an air stream Afterwards, the mixture is neutralized by adding 500mM sulfuric acid. Then the sump The N-unsubstituted glucosamine residue produced by deamino acid cleavage at pH 4.0 Special cleavage with distillate. This method produces intact hexuronate residues at the non-reducing end. and a 2,5-anhydromannose residue at the reducing end. It produces oligosaccharides different from those prepared by palitinase treatment. 2. 5-Anhydromannose residues are usually reduced with NaBH4 radiolabel 2, converted into an Erannhydro-D-mannitol derivative. At this stage, if necessary, oligosaccharides are added using Nal 1″lL as a reducing agent. Can be introduced to

Oligosaccharides by gel filtration, Oligo@ products of heparitinase or chemical treatment methods are purified by gel filtration chromatography. Partially dissolved based on the surms by graphography, resulting in individual bead formation. It consists of an open compound of oligosaccharides consisting of a fixed number of disaccharide units, and the size ranges upward from disaccharides, each containing one complete disaccharide unit. differs from the following by an increment of . the purpose of the analysis (e.g., sample approximately 10 For filling up to mg), use Blo-Ge1 PG or 13io-Gel A column (1 x 120 cm or IX 240 cm) are suitable, but for separation of larger volumes For this purpose, it is recommended to scale up the column by appropriately increasing the diameter of the column. sump The gel was loaded on top of the gel, and the NILIICO was loaded at a flow rate of 4 ml/h to 500 ml/J. Elute by Collect 1 ml of fractions and add liquid if HS is labeled. A small sample is taken from each fraction for stilation counting. a Alternatively, measure the absorbance of individual fractions at 232 nm or monitor them continuously with a UV monitor. Unlabeled HS ori Detects gosaccharides. Fixed size (determined by pre-calibration with standard values) The fractions corresponding to the oligosaccharide peaks (reduced) are pooled and lyophilized. Figure 8 is Blo-Ge of the hebaritinase digest of the 3H-labeled linear eloquent cells H3 described above. 1 Representative results for gel filtration of PG are shown.

Determined amount of disaccharide of primary gosaccharide by bFGFI chromatography Individual peaks containing oligosaccharide mixtures consisting of similar units were compared to bFGF. further split based on affinity. Immobilized on Affi-Gel 10 The preparation of analytical scale affinity substrates containing bFGF was previously described. Preparation For use in Kale, a similar procedure is carried out, but the amount of bFGF bound and The amount of gel depends on the required sample capacity. Below, this special implementation Example of line eloquent cell H3 or bFG for isolation of 3H labeled HS oligosaccharides A case where F-Af f i -Ge 110 is used will be explained.

Approximately 1 ml of bFGF-Affi-Gel 10 affinity substrate was added to the glass column. 6mm in diameter x 35mm). 10mM) 0° in squirrel HCI Load the sample onto the column at a flow rate of 25 ml/min. to elute unbound material. Collect five 1 ml fractions. The binding substance is concentrated at a flow rate of 0.5 ml/min. Elute with a gradient of sodium chloride (0-2M NaCl in column buffer) do. This can be achieved by using a discrete stepwise concentration gradient (e.g. 250mM NaCl). step or other suitable increments). This is convenient. Five 1 ml fractions are collected at each concentration. or, To elute the bound fragments and collect 1 ml fractions, a linear continuous concentration gradient ( For example, in the case of a total volume of 50 rnl) may be used. liquid cinderosis meter Sample a small amount from each fraction for counting (or UV absorbance detection).

Figure 6 shows different sizes (dp) prepared by Blo-Ge1 P6 gel filtration. bFGF affinity chroma of heparitinase-resistant oligosaccharide peak of 2-dp14) Representative results of tography are shown. Ori with the same affinity for bFGF Pool selected fractions containing gosaccharides] 5 pectrapor 710100 O-cut-off membrane (evaporated using Pierce Ltd. Perform dialysis against distilled water or 500mM NH at a flow rate of 10ml/hour. Gel filtration was performed again on the Blo-Ge1 P2 column eluted with ,HCO, Desalt by drying and lyophilize. The fraction of particular interest is di) = 1 2, dp=14.

Additional cosmetics: Additional purification of oligosaccharides with selected specific affinities for bFGF Further analysis of gel filtration chromatography and bFGF affinity chromatography This can be achieved by following steps. This has been done, for example, in preliminary experiments. It was. For this preliminary experiment, Figure 2 shows that following the second application to the affinity column. (low (750mM fraction), medium (1000mM fraction) by bFGF affinity step bFGF parent of oligosaccharides selected for ) and high (≧1250mM fraction) It shows a compatibility distribution. Additionally, if desired, two additional techniques can also be used: That is, strong anion exchange (SAX) H which separates mainly according to anion exchangeability PLC and concentration gradient polyacrylamide that preferentially separates according to molecular size. By applying gel electrophoresis (PAGE), oligosaccharides can be reduced to an apparently solid state. Moreover, it can be further purified.

Thus, the purity of the bound oligosaccharides can be determined by SAX HPLC chromatography. In order to increase the For analytical columns (4 x 250 mm) or semi-preparative columns (9 x 250 mm) and separate it. Mobile phase (p with HCI) (adjusted to 3,5) at 1 ml/min. After equilibration in double-distilled water), inject the Zamble and transfer the oligosaccharides to the same mobile layer. Elute with a linear gradient of sodium chloride (0-2M over 8 minutes) at . melt Syneresis is useful for detecting unlabeled oligosaccharides using ultraviolet absorbance (A,...). or monitor inline for radioactivity (e.g. (:an Radiomatic Flo-one by berra Packard Ltd. Using an inline monitor like the Beta A-200detector ) to monitor.

The concentration gradient PAGE method has been described in previous publications [e.g., J.E. & Gallagh er, J, T, (19881Biochem, J, 251.597-6 08; Turnbull, J.E. & Gallagher, J.T. (19X0) Bi ochem, J, 265.715-724; Turnbull et a l (19931, “Approaches to the@5truct ural analysis of GAGS' in Extracellu lar Matrix Macromolecules: `　Practic al Approach, 0xford University Press ;　Turnbull　[1993), “Oligosa momo mapping and 5 sequence analysis of GAG s”, in Methods in Mo1ecular@Biology: Membrane Methods, Humans Press, Cha pter 24). This methodology is a seemingly simple thing. Luspecie is a very powerful technique for dissolving large oligosaccharide chain compounds. This can be eluted directly from the gel using suitable equipment or Electrotransfer from the gel to a nylon membrane as described in the above references. Large amounts of oligosaccharides can be recovered from the membrane by elution with salt or Various combinations of these techniques can be applied to the preparation scale for separation. according to the present invention (defined oligosaccharide products (specific sequences and bF Final purification of homogeneous preparations with defined affinities for growth factors such as GF can be carried out to a very high degree.

Basic features of oligosaccharide sequences have been identified and unique FGF binding affinities ( In the case of Rigo-H, it is characterized by producing As far as this knowledge is concerned, these oligosaccharides and similar It is also possible to synthesize objects, and to meet the requirements of using ordinary synthesis methods, "tailoring" is possible. - Please understand that it is possible to say "maid". For example, these compounds These units can be related to construct three types of main disaccharide units. −-]　Low] “-one piece Precursors of nits are designated as Ig, Ig, 6, and these are -methylchloroacetyl-ni (OMCA) protected terminal group, e.g. MCAO and OAC groups are selected Alternatively, it can be converted into an -OH group by, for example, pyridine or hydrazine, respectively. , the required number of B units are first coupled together, and then the required terminal units are connected to each other. It is possible to build chains by selectively combining them, and the structures created in this way can Perform deacylation, 〇-sulfation, hydrogenolysis, and N-sulfation as necessary for the product. I can. Thereby, a final product can be obtained. About the synthesis method A general explanation of See rHeparin J, pages 51 et seq.

Rito Nurihara In general, mammals from therapeutic uses and therapeutic needs of the oligosaccharide products of the present invention. For administration to patients, an effective growth factor-binding amount of active oligosaccharides (pharmaceutically potent salt form) may be administered in any suitable manner, e.g., orally, parenterally (subcutaneously). (including intramuscular, intravenous), topical, or slow-release administration for implantation. It will be configured as a pharmaceutical formulation ready for administration by the device.

Such preparations may be given in unit dosage form and may be provided by those well known in the pharmaceutical field. may consist of a pharmaceutical composition prepared by any of the methods , in which case the active oligosaccharide component(s) is a comparable pharmaceutically acceptable component. in intimate association with at least one other component that serves as a carrier, diluent or additive. Matched or mixed. Alternatively, such formulations may be comparable or comparable. Contains a protective envelope of relatively inert pharmaceutically acceptable substances; whether or not combined or mixed with any other ingredients within the rope. The active oligosaccharide component(s) may also be included.

Here, for pharmaceutical use, the oligosaccharides of the present invention can be obtained by heparitinase cleavage. It should be noted that it is preferable to make the non-reducing end unsaturated, as shown in FIG.

This means that this form is more resistant to biometamorphosis, which can reduce effectiveness. This is because there is a wealth of evidence that can be held. However, this is not true for all applications. It's not necessarily necessary.

Formulations of the invention suitable for oral administration are, for example, capsules, cachets, tablets or It can be presented in discrete units such as lozenges. Each unit has a predetermined amount of active ingredient. Accommodate minutes. The preferred type is that capsules provide the most effective means of oral administration. It is a formulation. For parenteral administration, the formulation should be packaged in sealed ampoules ready for use. The active oligosaccharide may be a sterile liquid drug containing a predetermined amount of the active oligosaccharide.

The amount of oligosaccharide products of the present invention and the dosage regimen necessary for effective therapeutic use are limited. There are many different types of medical or veterinary medicine that ultimately treat mammals for each specific use. At the discretion of the medical practitioner. Considered by such practitioners, e.g. The factors to be treated are specific to the specific disease being treated (and growth factor stimulation or whether growth factor suppression is necessary) as well as the route and type of administration of the pharmaceutical agent. It also includes the type, i.e., weight, surface area, age and general condition of the mammal. Ru. However, for example, an effective bFGF-suppressing dose suitable for antitumor therapy is Probably in the range of about 1.0 to 75 mg per kilogram of body weight, which is preferred. or about 5-40 mg, and the most suitable dose is 10-30 mg/kg. in range. For example, for daily treatment, the total daily dose may be a single dose, multiple doses, in multiple doses, e.g. - 2 to 6 times per day, or for any selected period of time. Can be given by intravenous infusion. For example, for a 75 kg mammal, The dosage range is likely to be about 75 to loomg per day, with a typical The dosage will normally be about 100 mg per day. Individual multiple doses indicated In this case, treatment typically involves administering 50 mg of the oligosaccharide product as a tablet as described above. , given 4 times per day in capsule, liquid (e.g. syrup) or injection form. It might turn out to be a good thing.

As pointed out earlier, the necessary treatment is to administer growth factors such as bFGF and For example, when it is necessary to promote tissue repair, such as in wound treatment applications. In some cases, the active oligosaccharide component may be administered together with growth factors.

For wound treatment applications and where it is desired to increase or stimulate growth factor activity. Other medical uses, such as fracture treatment, nerve regeneration, duodenal ulcer, venous ulcer, Various ocular and retinal diseases, amploic TJJ artery sclerosis, degenerative muscle diseases, ischemia , therapeutic applications, or to protect tissue from severe damage during radiation therapy. Medical uses of the oligosaccharide components or products of the invention, apart from their uses in connection with their uses. are probably most often focused on inhibiting growth factor activity, and these Drugs or compositions containing gosaccharides may cause harmful growth or cell proliferation, as discussed above. symptoms that result from or are exacerbated by the activity of growth factors that promote reproduction; For example, diabetic membrane disease, capsular opacity, proliferative vitreoretinopathy, tumor angiogenesis, Cancer cell growth, cancer cell metastasis, rheumatoid arteritis, mild muscular dystrophy Alzheimer's disease, various viral infections (e.g. Herpes Simple, type) or re-stenosis following angioplasty or other chronic inflammation. It is expected to be particularly useful in treating conditions such as stenosis.

As already understood, the present invention provides a number of different aspects, including: Novel and inventive materials containing novel ingredients, expressly or implicitly disclosed herein; It includes all features and aspects singly or in combination with each other.

Furthermore, the scope of the invention may be determined by the illustrated embodiments or by way of explanation herein. It is not intended to be limited by the terms and expressions used.

Distillate amount Distillate amount +0 50 60 70 80 90 Distillate amount Distillate amount Distillate amount (iml) = 10 15 20 25: o 35 fraction molecule fi (Lml) 0100200300405aO (What, blood) ! Rolo: Ri0300MAj Rolo (n(, mll Ofu Submission of translation of written amendment (Article 184-8 of the Patent Act) 1. Indication of patent application PCT/GB931005973, patent applicant Address: London, United Kingdom, NL 14, PA Cambridge Terrace 6-10 Cambridge House Name: Cancer Research Campaign Technology 4, Agent: 107 Address: 2-2-21 Akasaka, Minato-ku, Tokyo (1) 1 translation of the written amendment Shodan-kun Ikuzuka-Ryo 12 Oligos with unique binding affinity for eloquent cell growth factor (FGF) It is a sugar product obtained by the depolymerization of heparan sulfate by heparitinase. are nearly homogeneous with respect to FGF binding affinity, and are essentially continuous series. at least six disaccharide units including sulfated disaccharide units of Contains non-heparin oligosaccharides, these sulfated 211 Wl units). each of I\1-sulfated glucosamine residue (±63) and 2-0-sulfated glucosamine residue (±63) Glucosamines in oligosaccharide chains that are 0-sulfated at C6 and consist of acid residues. Oligos characterized in that the content of carbon residues (if any) is less than 20%. Sugar products.

2. In the oligosaccharide product according to claim 1, the sulfated 2@ class Each of the nits is IdoA (2Sl-αI, 4-GICNSO3). Characteristic oligosaccharide products.

3. In the oligosaccharide product according to claim 1 or 2, the oligosaccharide product according to claim 1 or 2, characterized in that the gosaccharide chain consists of a sequence of less than 10 disaccharide units Oligosaccharide products.

4. Oligosaccharide production according to any one of claims 1 to 3 It shows almost complete resistance to depolymerization by heparitinase, but Oligosaccharide products 5 characterized in that they are not susceptible to depolymerization by heparinase; Any one of claims 1 to 4 may include an oligosaccharide product according to one of claims 1 to 4. in which at least a preferential majority of said oligosaccharide chains are all of the same length. An oligosaccharide product characterized by:

6. Oligosaccharide product according to any one of Claims 1 to 5 In this case, almost all of the oligosaccharide chains have a sequence of six 2@ class units. An oligosaccharide product characterized in that it consists of

7. Oligosaccharide production according to any one of claims 1 to 6 in which the oij oligosaccharide chain comprises at least four of the sulfated disaccharide units. An oligosaccharide product characterized in that it contains a continuous sequence of .

8. Oligosaccharide product according to any one of claims 1 to 4 In this case, almost all oligosaccharide chains are sequences of seven disaccharide units. and at least five of these disaccharide units are the aforementioned sulfated disaccharide units. An oligosaccharide product characterized in that it is comprised in a continuous sequence of units.

94 Oligosaccharide production according to any one of claims 1 to 8 〇-sulfated at C6 in the oligosaccharide chain (if any) in The content of glucosamine residues added is less than 20% of the total number of glucosamine residues. An oligosaccharide product characterized by:

10. In the oligosaccharide product according to claim 9, (if any) The content of glucosamine residue in the oligosaccharide chain 〇-sulfated at C6 is An oligosaccharide product characterized in that it contains less than 5% of the total number of samin residues.

11. Oligosaccharide raw material according to any one of claims 1 to 10 The product was subjected to enzymatic partial depolymerization to the maximum extent with heparitinase and then size separation using, for example, gel filtration size exclusion chromatography and then sizing. For the selected fraction(s) recovered from the fractionation step, the immobilized ligand FGF binding was cleaved by affinity chromatography using FGF growth factor. The pieces are separated and then subjected to a salt concentration gradient, advantageously successive stepwise concentration gradients. Selective elution was performed over a range of salt concentrations and the fragments were analyzed for FGF binding affinity. to collect the strongest bonded fragments and optionally generate further collections. by performing at least one additional step of object sizing and selection. By further purifying the recovered product, human eloquent cell heparan sulfate protein was purified. Theoglycan (H3PG) can be obtained from heparan sulfate (HS). Characterized oligosaccharide products.

12. An ointment with a unique binding affinity for fibrosal vesicle growth factor (FGF) is a oligosaccharide product; (a) Predominantly consists of the following molecular species, where X is nHexA-GlcNSOs (± 63), Y is IdoA(2S)-GlcNSOs (±6S), and Z is IdoA-GlcR (±65) or IdoA (2S)-GlcR (±6S ), where R is N50M or NAc and n ranges from 4 to 7. .

(b) the content of monosaccharide residues with 6-0-sulfate groups, if any, is less than 20%; full, (C) Heparan sulfate is digested with heparitinase to achieve its partial depolymerization to the maximum extent. and then the resulting oligosaccharide mixture is subjected to e.g. gel filtration size exclusion chromatography. Size-resolved using totography and selected within the range of 12-18 monosaccharide residues Collect the fraction containing oligosaccharide chains with a specific size and select this The obtained fraction was subjected to affinity chromatography using immobilized FGF ligand. Stronger <FGF binding by elution under a salt gradient over a range of salt concentrations Select fractions containing bound substances that separate only at the highest salt concentration are recycled. Oligos characterized in that they can be obtained by a process consisting of Sugar products.

13. In the oligosaccharide product according to claim 12, Y is predominantly Id oAf2s)-GlcNSO.

14. In the oligosaccharide product according to claim 12 or 13, An oligosaccharide product characterized in that n is 5 or 6.

15. The oligosaccharide product according to claim 14, wherein all of the molecular species are An oligosaccharide product characterized in that it consists of seven 2@ class units.

16. In the oligosaccharide product according to claim 12 or 13, n is 4, and the total number of disaccharide units of the molecular species is 6. Ligosaccharide products.

17. Oligosaccharide according to any one of claims 12 to 16 In the product, the content of residues with 6-0-sulfate groups, if any, is An oligosaccharide product characterized in that it contains less than 5% of the total number of oligosaccharide residues.

18, has a unique binding affinity for filamentous cell growth factor (FGF), All are composed of oligosaccharide chains, and these oligosaccharide chains are 14 monomers in length. sugar residues, each containing GlcNSOs [±6S+Ido bound to the residues. A (an integral continuous sequence of 5 or 6 disaccharide units consisting of 2Sl residues) containing less than 20% glucosamine residue (terminal or internal) Oligosaccharide products characterized by being oxidized.

19. The oligosaccharide product according to claim 18, wherein the oligosaccharide series ln l GlcA-GlcNSOa-[IdoA(2S)-GlcNSOsls-I doA-GlcRl and (selected from nl GlcA-[GlcNSO, -IdoA(2Sl]a-GlcR) characterized in that R is NSO or NAc. Oligosaccharide products.

20. Oligos with unique binding affinity for filamentous cell growth factor (FGF) oligosaccharide products, which are almost all monosaccharide residues 12 in length. It consists of a saccharide chain, containing an integral continuous sequence of four disaccharide units, each containing two The saccharide is composed of 15oA (2S) bound to GlcNSO, (±6S) residue. less than 20% glucosamine residue (terminal or internal) is 6-0-sulfated. An oligosaccharide product characterized by:

2. In the oligosaccharide product according to claim 20, preferential oligosaccharides The sugar linkage sequence (nl GlcA-GlcNSO-(±6Sl-[1doA (2S)-Gl cNSO,)4-IdoA-GlcR (±6Sl, where R is NSO3 or or NAc.

22. Relatively high specific binding to basic eloquent cell growth factor (bFGF) consisting of an oligosaccharide chain having a disaccharide sequence substantially as follows: An oligosaccharide product characterized by:

nGlcA-β], 4-GlcNSO-al, 4-[IdoAf2S)-al, 4-GlcNSOsls-al, 4-Id.

A-al, 4-GlcR or nGlcA-[GlcNSOs-IdoA (2Sl)] a-GlcR here In this case, R is NSO or NAc, or its small change is small (both bFG have the same relatively high specific binding affinity for F. An oligosaccharide product characterized by:

23. From glycosaminoglycans such as heparan sulfate, 23. A method for separating oligosaccharide products according to any one of clauses 22 to 22, comprising: This oligosaccharide property is purified and consists of small oligosaccharides in a relatively homogeneous state, These oligosaccharides bind to the glycosaminoglycans or Linear cell growth factors ((a) that bind to the corresponding proteoglycans in biological systems ) Containing a sample of the linear cell growth factor as an immobilized affinity ligand creating an affinity chromatography substrate or substrate for (b) Treating the glycosaminoglycan with a selective cleavage reagent to achieve relatively low sulfuric acid (C) selectively severing the polysaccharide chain in the polysaccharide region; and (C) for example, size separation of the product of step (b) by filtration size exclusion chromatography; Selectively, it is thought to contain oligosaccharides consisting of less than 10 disaccharide units. (d) collecting the fraction from step (a); and (d) affinity chromatography from step (a). The substrate or substrate is modified to contain a specific number of disaccharide units ranging from 4 to 9. In contact with the selected fraction or selected fraction set from floor (C), the fixed wire eloquent fine Glycosaminoglycans that have at least some binding affinity for cell growth factors extracting and retaining the sized oligosaccharide fragments on said substrate or substrate; , (e) Affinity chromatography while gradually increasing salt concentration or salt concentration gradient eluting the substrate or substrate; (f) Fractions containing oligosaccharide fragments that elute within the selected maximum eluting salt concentration range. and, optionally, step (g) ( f) of the reaction mixture obtained from step (b). Alternatively, the selected fraction or set of fractions recovered in step (f) may be used to carry out step (c). ) and optionally repeat steps (d), (e) and Steps that are further refined by repeating (f) A method characterized by comprising:

2. In the method according to claim 23, the glycosaminoglycan is Heparan sulfate derived from heparan sulfate proteoglycan of animal cells, The eloquent cell growth factor of mammalian cells when activated under physiological conditions A substance that stimulates mammalian cells through binding interactions with signal-transmitting receptors on their surface. A method characterized by being an itkine.

2. The method according to claim 24, wherein the selective cleavage reagent is heparitina Heparan sulfate is quenched until cleavage of the heparitinase-selective bond is completed. Process 2 characterized in that partial depolymerization is achieved to the greatest extent by , the method according to claim 24, wherein the selective cleavage reagent is nitrous acid. , which after prior treatment of heparan sulfate with an N-type acetylating agent at a pH of about 4. A method characterized by reacting with a polypi compound and cleaving it at a free amine group. Law.

2. The method according to any one of claims 23 to 26 In this case, the line eloquent cell growth factor is the basic line eloquent cell growth factor (bFGF). A method characterized by:

2. The method according to any one of claims 23 to 27 In this case, the fraction collected from the size separation step was divided into an ori- um consisting of seven disaccharide units. A method characterized in that the product is believed to contain gosaccharides.

2. The method according to any one of claims 23 to 27 In this case, the fraction collected from the size-splitting step is divided into olefins consisting of six disaccharide units. A method characterized in that the product is believed to contain oligosaccharides.

30. Oligosaccharide according to any one of claims 1 to 22 products, such as wound treatment, fracture treatment, nerve regeneration, duodenal ulcers, static For pulsatile ulcers, various ocular and retinal diseases, antiromantic arteriosclerosis, and ischemic symptoms. treatment is required to protect tissue from severe damage during radiation therapy or radiation therapy. active FGF for promoting therapy or tissue repair in the treatment of mammals with An oligosaccharide product characterized in that it is used therapeutically as an active stimulant.

31, comprising the oligosaccharide product of claim 30, which is used to carry out the above treatment. It is characterized in that it is combined with an exogenous FGF growth factor that is administered together with the treatment. Medical composition.

32, the oligosaccharide according to any one of claims 1 to 22 For example, diabetic retinopathy, capsular opacity, proliferative vitreoretinopathy. , tumor angiogenesis, cancer cell growth, cancer cell metastasis, rheumatoid arteritis, mild Muscular dystrophy, Alzheimer's disease, various viral infections [e.g. 11erpes Simplex type li or post-angioplasty stenosis cell growth or is used therapeutically as an active FGF activity inhibitor to control or reduce cell proliferation. An oligosaccharide product characterized by:

33. Oligosaccharides according to any one of Claims 1 and 22 The product or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or includes a therapeutically effective non-toxic agent comprising a vehicle and a FGF activity modulator. A drug or pharmaceutical composition for medical use.

34, the oligosaccharide according to any one of claims 1 to 22 The product can be used to treat diabetic retinopathy, lens capsule opacity, proliferative vitreous W4 membrane disease, and tumor angiogenesis. , cancer cell growth, cancer cell metastasis, rheumatoid arteritis, mild muscular dystrophy Alzheimer's disease, various viral infections [e.g. Herpes disease] Simplex type l) or for use in the treatment of stenosis after angioplasty. or for wound treatment, fracture treatment, nerve regeneration, duodenal ulcers, various ocular and ocular Treatment of conditions such as membranous disease, antiloamic atherosclerosis, degenerative muscle disease, and ischemia. damaged to protect tissue from severe damage during radiotherapy or to protect tissue from severe damage during radiation therapy. Oligosaccharide products for use in promoting repair of damaged tissues.

35, has a unique binding affinity for filamentous cell growth factor (FGF) and is essentially It consists of oligosaccharide chains, and these oligosaccharide chains are related to FGF binding affinity. It is almost homogeneous and contains multiple sulfated disaccharide units between the terminal residues. Contains a sequence of less than 10 disaccharide units, each sulfated disaccharide unit being N -Sulfated glucosamine residue (±63) and 2-0-sulfated iduronic acid residue etc. and less than 5% of said glucosamine residue has 6-0-sulfate groups. Characteristic oligosaccharide products.

36. Pharmaceutical composition used to control the activity of eloquent cell growth factor in mammalian mammals or drugs that promote tissue repair or cell therapy in the treatment of diseases. It is for suppressing growth or angiogenesis, and is defined in claim 35. characterized in that it contains a therapeutically effective amount of an essentially pure oligosaccharide product of A pharmaceutical composition or drug.

37. Wounds, duodenal ulcers, venous ulcers, various ocular, retinal diseases, or ischemia. or to promote healing or tissue repair in cases of fracture healing. or to protect tissues from severe damage during radiation procedures. a method of treating a mammal in need of such treatment; Essentially pure as described in paragraph 35 or any one of paragraphs 1 to 22 a method comprising administering an effective amount of an oligosaccharide product.

38. The method of claim 37, wherein the oligosaccharide product is bFGF. A method characterized in that it is administered together with a drug.

39. Diabetes in dairy animals by suppressing FGF growth factor activity sexual retinopathy, capsular opacity, proliferative vitreoretinopathy, tumor angiogenesis, cancer cell growth, cancer cell metastasis, rheumatoid arteritis, mild muscular dystrophy, Alzheimer's disease infections, various viral infections (e.g., Herpes Simplex ype l) or post-angioplasty stenosis, comprising: The book described in item 35 or any one of claims 1 to 22 administering an effective amount of a qualitatively pure oligosaccharide product to a mammal in need of treatment; A method consisting of things.

international search report 11.111. Pottery PCT/(to) 93100597 Continuation of front page (72) Inventor Tumble, Jeremy Ivan Manchester, United Kingdom M211 Wysey, Chorlton-Cumoohardy, Killdeer Road 7

Claims (1)

[Claims] 1. Oligosaccharide with unique binding affinity for fibroblast growth factor (FGF) It is a product consisting essentially of oligosaccharide chains, and these oligosaccharide chains are Almost homogeneous in terms of FGF binding affinity and at least six disaccharide units These disaccharide units form a continuous sequence of sulfated disaccharide units. and each of these sulfated disaccharide units is an N-sulfated glucosamine residue. (±6S) and 2-O-sulfated iduronic acid residue. Gosaccharide products. 2. The oligosaccharide product of claim 1, wherein the sulfated disaccharide unit Each of the cuts is IdoA(2S)-α1,4-GlcNSO3. oligosaccharide products. 3. An oligosaccharide product according to claim 1 or 2, wherein the oligosaccharide product is an ointment characterized in that the saccharide linkage consists of a sequence of less than 10 disaccharide units; Ligosaccharide products. 4. An oligosaccharide product according to any one of claims 1 to 3. shows almost complete resistance to depolymerization by heparitinase; 5. Oligosaccharide products characterized in that they are not subject to depolymerization by parinase. request In the oligosaccharide product according to any one of claims 1 to 4, and that at least a preferential majority of said oligosaccharide chains are all of the same length. An oligosaccharide product characterized by: 6. An oligosaccharide product according to any one of claims 1 to 5. , almost all of the oligosaccharide chains are sequences of six disaccharide units. An oligosaccharide product characterized in that it consists of: 7. An oligosaccharide product according to any one of claims 1 to 6. wherein the oligosaccharide linkage comprises at least four of the sulfated disaccharide units. An oligosaccharide product characterized in that it contains a continuous sequence. 8. An oligosaccharide product according to any one of claims 1 to 4. , almost all oligosaccharide chains are formed from a sequence of seven disaccharide units. and at least five of these disaccharide units are the sulfated disaccharide units. An oligosaccharide product characterized by being comprised in a continuous sequence of knits. 9. An oligosaccharide product according to any one of claims 1 to 8. , O-sulfated at C6 within the oligosaccharide chain (if any) Oligosaccharide production characterized in that the content of glucosamine residues is less than 20% thing. 10. In the oligosaccharide product of claim 9, (if any): The content of glucosamine residues in the oligosaccharide chain O-sulfated at C6 is less than 5%. An oligosaccharide product characterized in that it is full. 11. Oligosaccharide raw material according to any one of claims 1 to 10 The product was subjected to enzymatic partial depolymerization to the maximum extent with heparitinase and then size separation using, for example, gel filtration size exclusion chromatography and then sizing. For the selected fraction(s) recovered from the fractionation step, the immobilized ligand FGF binding was cleaved by affinity chromatography using FGF growth factor. The pieces are separated and then subjected to a stepwise concentration gradient, advantageously a stepwise concentration gradient. Selective elution was performed over a range of salt concentrations and the fragments were analyzed for FGF binding affinity. to collect the strongest bonded fragments and optionally generate further collections. by performing at least one additional step of object sizing and selection. By further purifying the recovered product, human fibroblast heparan sulfate was purified. Theocrican (HSPG) can be obtained from heparan sulfate (HS). Characterized oligosaccharide products. 12. Oligos with specific binding affinity for fibroblast growth factor (FGF) is a sugar product; (a) Predominantly consists of the following molecular species, ▲There are mathematical formulas, chemical formulas, tables, etc.▲ here, X is ∩HexA-GlcNSO3 (±6S), Y is IdoA (2S)-G lcNSO3 (±6S) and Z is IdoA-GlcR (±6S) or Id oA(2S)-GlcR(±6S), where R is NSO3 or NAc , n is in the range of 4 to 7, (b) the content of monosaccharide residues with 6-O-sulfate groups, if any, is less than 20%; full, (c) Digesting heparan sulfate with heparitinase to achieve its partial depolymerization to the maximum extent. and then the resulting oligosaccharide mixture is subjected to e.g. gel filtration size exclusion chromatography. Size-resolved using totography and selected within the range of 12-18 monosaccharide residues Collect fractions containing oligosaccharide chains with a certain size and select Affinity chromatography was performed on the fraction using immobilized FGF ligand, and a certain Stronger FGF binding by elution down a salt gradient over a range of salt concentrations Select fractions containing bound substances that separate only at the highest salt concentration are recycled. Oligos characterized in that they can be obtained by a process consisting of Sugar products. 13. In the oligosaccharide product of claim 12, Y is exclusively Id An oligosaccharide product characterized in that it is oA(2S)-GlcNSO3. 14. In the oligosaccharide product according to claim 12 or 13, An oligosaccharide product characterized in that n is 5 or 6. 15. 15. The oligosaccharide product of claim 14, wherein all of the molecular species are An oligosaccharide product characterized in that it consists of seven disaccharide units. 16. In the oligosaccharide product according to claim 12 or 13, n is 4, and the total number of disaccharide units of the molecular species is 6. Ligosaccharide products. 17. Oligosaccharide according to any one of claims 12 to 16 In the product, the content of residues with 6-O-sulfate groups, if any, is 5%. An oligosaccharide product characterized in that it is less than or equal to 18. It has a unique binding affinity for fibroblast growth factor (FGF), and most All are composed of oligosaccharide chains, and these oligosaccharide chains are 14 monomers in length. sugar residues, each bound to a GlcNSO3(±6S) residue. (2S) Contains an integral continuous sequence of 5 or 6 disaccharide units consisting of residues. with less than 20% glucosamine residue (terminal or internal) 6-O-sulfated An oligosaccharide product characterized by: 19. The oligosaccharide product according to claim 18, wherein the oligosaccharide linkage is (∩)GlcA-GlcNSO3-[IdoA(2S)-GlcNSO3]5- IdoA-GlcR, and (∩)GlcA-[GlcNSO3-IdoA(2S)]6-GlcR, selected biologically active protein or polypeptide that is activated under physiological conditions. by binding interaction with signal-transmitting receptors on the surface of mammalian cells when Cytokines or growth factors that stimulate mammalian cells Law. 25. 25. The method of claim 24, wherein the selective cleavage reagent is heparitinar. Heparan sulfate is digested until cleavage of heparitinase-selective bonds is complete. A process characterized in that partial depolymerization is achieved to the greatest extent by. 26. The method of claim 24, wherein the selective cleavage reagent is nitrous acid. , which after prior treatment of heparan sulfate with an N-deacetylating agent at a pH of about 4. A process characterized by reacting with a polysaccharide to cleave it at a free amino group . 27. The method according to any one of claims 23 to 26 The selected bioactive protein or polypeptide is a fibroblast growth factor. (FGF). 28. The method according to any one of claims 23 to 26 selected bioactive proteins or polypeptides to enhance basal fibroblast growth. A method characterized in that it is a factor (bFGF). 29. The method according to any one of claims 23 to 28 The fraction collected from the size-splitting step is converted into an oligomer consisting of seven disaccharide units. A method characterized in that the product is believed to contain sugars. 30. The method according to any one of claims 23 to 28 The fraction collected from the size-splitting step is divided into an ori- um consisting of six disaccharide units. A method characterized in that the product is believed to contain gosaccharides. 31. Oligosaccharide raw material according to any one of claims 1 to 22 For example, wound treatment, fracture treatment, nerve regeneration, duodenal ulcers, veins, etc. In the symptoms of sexual ulcers, various ocular and retinal diseases, atherosclerosis, and ischemia. treatment is required to protect tissue from severe damage during radiation therapy or radiotherapy. activation of active FGF to promote therapy or tissue repair during the treatment of mammals with An oligosaccharide product characterized in that it is used therapeutically as a sexual stimulant. 32. comprising the oligosaccharide product of claim 31, which is used to carry out the treatment described above. It is characterized in that it is combined with an exogenous FGF growth factor that is administered together with the treatment. Medical composition. 33. Oligosaccharide raw material according to any one of claims 1 to 22 For example, diabetic retinopathy, capsular opacification, proliferative vitreoretinopathy, and tumor Tumor angiogenesis, cancer cell growth, cancer cell metastasis, rheumatoid arteritis, mild muscle stiffness stroke disease, Alzheimer's disease, various viral infections (e.g. He rpes Simplex type 1) or restenosis after angioplasty cell growth or cell growth when treating mammals in need of treatment in connection with For therapeutic use as an active FGF activity inhibitor to control or reduce proliferation An oligosaccharide product characterized by: 34. Oligosaccharide according to any one of Claims 1 to 22 The product or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or is a medical drug comprising a vehicle and a therapeutically effective non-toxic amount of an FGF activity modulator. A drug or pharmaceutical composition for use. 35. Oligosaccharide according to any one of claims 1 to 22 The product can be used to treat diabetic retinopathy, capsular opacity, proliferative vitreoretinopathy, tumor angiogenesis, Cancer cell growth, cancer cell metastasis, rheumatoid arteritis, mild muscular dystrophy disease, Alzheimer's disease, and various viral infections (e.g., Herpes S. complex type 1) or for the treatment of restenosis after angioplasty. Or, wound treatment, fracture treatment, nerve regeneration, duodenal ulcer, various eyeball and retinal diseases. , used to treat conditions such as anti-inflammatory arteriosclerosis, degenerative muscle disease, and ischemia. damaged tissue to protect the tissue from severe damage during radiation therapy. Oligosaccharide products for use in promoting tissue repair. 36. It has a specific binding affinity for fibroblast growth factor (FGF) and is essentially It consists of oligosaccharide chains, and these oligosaccharide chains are related to FGF binding affinity. is nearly homogeneous and contains multiple sulfated disaccharide units between the terminal residues. Contains a sequence of less than 10 disaccharide units, each sulfated disaccharide unit being N - Sulfated glucosamine residue (±6S) and 2-O-sulfated iduronic acid residue, etc. An oligosaccharide product characterized by: 37. Pharmaceutical compositions used to control the activity of mammalian fibroblast growth factors or drugs that promote tissue repair or cell therapy in the treatment of diseases. It is for suppressing growth or angiogenesis, and is for the purpose of suppressing growth or angiogenesis, and as set forth in claim 36. characterized in that it contains a therapeutically effective amount of an essentially pure oligosaccharide product of A pharmaceutical composition or drug. 38. wounds, duodenal ulcers, venous ulcers, various ocular, retinal diseases, or ischemia. or to promote healing or tissue repair in cases of fracture healing. or to protect tissues from severe damage during radiation procedures. a method of treating a mammal in need of such treatment; Essentially pure as described in paragraph 36 or any one of paragraphs 1 to 22 a method comprising administering an effective amount of an oligosaccharide product. 39. 39. The method of claim 38, wherein the oligosaccharide product is bFGF. A method characterized in that it is administered together with a drug. 40. diabetes in mammals by suppressing FGF growth factor activity. Retinopathy, lens capsule opacification, proliferative vitreoretinopathy, tumor angiogenesis, cancer cell growth, cancer Cell metastasis, rheumatoid arteritis, mild muscular dystrophy, Alzheimer's disease infections, various viral infections (e.g., Herpes Simplex ty pe1) or a method for treating restenosis after angioplasty, comprising: The essential elements according to any one of claims 1 to 22 administering an effective amount of the pure oligosaccharide product to a mammal in need of treatment. A method consisting of