Aldehyde derivatives of sialic acid, methods of their obtainment, conjugates of aldehyde derivatives of sialic acid, and pharmaceutical composition based on them

FIELD: chemistry.

SUBSTANCE: invention concerns aldehyde derivatives and conjugates of di-, oligo- or polysaccharide, of the general formula (I), methods of obtaining them, and pharmaceutical composition based on them and capable of staying in blood flow for prolonged time. , where R is -CH(CHO)CH2OH, -CH2CHO, -CH(CH2NHR1)CH2OH, -CH(CH2NHNHR1)CH2OH, -CH(CH=NNHR1)CH2OH, -CH2CH2NHR1, -CH2CH=N-NHR1, -CH2CH2NHNHR1; R1 is polypeptide or albumen; GlyO is a sialic acid bond; R3 is H; R4 is OH; n is 2 or more.

EFFECT: obtaining pharmaceutical composition based on aldehyde derivatives of sialic acid capable of staying in blood flow for prolonged time.

20 cl, 7 tbl, 22 dwg, 10 ex

 

The present invention relates to derivatives of compounds such as polysaccharides having at least a terminal sialic links and preferably composed of units of sialic acid containing aldehyde group to react with the substrates on pampering terminal and methods for their preparation. Derivatives suitable for transformation into other reactive derivatives for conjugation with such substrates containing an amino group as peptides, proteins, drugs, systems for delivery of drugs (for example, liposomes, viruses, cells such as animal cells, microorganisms, synthetic polymers, etc.

Policially acid (PSA) is a natural unbranched polymers of sialic acid produced by certain strains of bacteria and in certain cells in mammals (Roth et al., 1993). You can get them with different degree of polymerization, n = about 80 or more residues of sialic acid to n=2, incomplete acid hydrolysis or splitting neuraminidase, or fractionation of natural produced by bacteria of the species of the polymer. The composition of different polisialovoi acids also changed so that there is a homopolymer form, i.e., alpha-2,8-linked Polivanova acid comprising capsular polysaccharide shtam the K1 and E. coli and In groups of meningococci, which is also found in embryonic form of the cell adhesion molecules neuron (N-CAM). There are also heteropolymer forms, such as alternating alpha-alpha 2,8-2,9 Polivanova acid strain of E. coli K92 and polysaccharides From the group N. meningitidis. Sialic acid can also be found in alternating copolymers with monomers, different from sialic acid, such as group W135 or group Y of N. meningitidis. Policially acids have important biological effects, including avoidance of pathogenic bacteria from the immune system and complement system and regulation giocatori adhesion of immature neurons during embryonic development (where the polymer has anti-adhesive action) [Muhlenhoff et. al., 1998; Rutishauser, 1989; Troy, 1990, 1992; Cho and Troy, 1994], although the receptors polisialovoi acids in mammals is not known. Alpha-2,8-linked Polivanova acid strain E. coli K1 is also known as "colomina acid and its (various lengths) is used as an example of the present invention.

Among bacterial polysaccharides alpha-2,8 linked form polisialovoi acid is the only non-immunogenic (not calling no response of T-cells or the formation of antibodies in mammals, even when conjugated to immunogenic carriers of proteins), which may affect its status as the polymerase is and mammals (as well as bacterial). A shorter form of the polymer (up to n=4) found in the cell surface gangliosides, which are widely distributed in the body and are effective for reporting and maintaining immunological tolerance towards polisialovoi acid. In recent years biological properties polisialovoi acids, in particular biological properties of alpha-2,8 linked homopolymer polisialovoi acid, were used to modify the pharmacokinetic properties of the protein or low molecular weight molecules of medicinal substances [Gregoriadis, 2001; Jain et al., 2003; US-A-5846951, WO-A-0187922]. Deriving polisialovoi acid leads to a strong improvement from the point of view of the half-life period of a certain number of therapeutic proteins, including catalase and asparaginase [Fernandes and Gregoriadis, 1996 and 1997], and also makes possible the use of such proteins in spite of pre-existing antibodies that have arisen as spam (and in some cases inevitable) consequence of previous exposure to therapeutic protein [Fernandes and Gregoriadis, 2001]. In many respects, the modified properties polysialylated proteins comparable to proteins, derivatives of polyethylene glycol (PEG). For example, in this and in another case increases the half, and proteins and peptides are more stable is the compared to proteolytic cleavage, but the preservation of biological activity seems great in PSA compared with PEG [Hreczuk-Hirst et al., 2002]. Also, questions arise about the use of PEG to therapeutic agents, which should be introduced constantly, because PEG is only subjected to very slow biological destruction [Beranova et al., 2000] and form high molecular weight tend to accumulate in tissues [Bendele et al., 1998; Convers et al., 1997]. It was found that polietilenglikolya proteins lead to the formation of anti-PEG antibodies, which can also affect the delay time of the conjugate in the bloodstream [Cheng et al., 1990]. Although PEG historically used as parenteral entered polymer conjugated with drugs, requires a better understanding of its immunotoxicology, pharmacology, and metabolism [Hunter and Moghimi, 2002; Brocchini, 2003]. There are also doubts about the use of PEG to therapeutic agents, which may require high dosages, because the accumulation of PEG can lead to toxicity. Therefore, alpha-2,8-linked Polivanova acid (PSA) offers an attractive alternative to PEG, representing immunologically invisible biologically erodible polymer, which is a natural part of the human body, and which is destroyed tissue neuroaminidase to sialic acid, toksichnaja of saccharide.

The group of applicants described in previous scientific publications and issued patents benefit from the use of natural polisialovoi acids for improving the pharmacokinetic properties of the protein therapeutic agents [Gregoriadis, 2001; Fernandes and Gregoriadis, 1996, 1997, 2001; Gregoriadis et al., 1993, 1998, 2000; Hreczuk-Hirst et al., 2002; Mital, 2004; Jain et al., 2003, 2004; US-A-05846951; WO-A-0187922]. Now, the applicants describe new PSA derivatives that make possible new compositions and methods for producing PSA-derived proteins (and other forms of therapeutic agent). These new substances and methods particularly applicable to obtain the PSA-derived therapeutic agents intended for use in humans and animals, in which primary importance is a chemical and molecular drug of interest due to safety requirements, medical ethics and regulatory bodies (the Management under the control over products and medicines (FDA), EMEA).

Previously described methods for attachment of polysaccharides to therapeutic agents such as proteins [Jennings and Lugowski, 1981; US-A-5846951; WO-A-0187922]. Some of these methods depend on the chemical derivative "not refreshing" end of the polymer to obtain the aldehyde residue, reactive against the protein (figure 1). This is because regenerating the end of the PSA on the natives of polysaccharides has a low reactivity to proteins under mild conditions, necessary to maintain protein conformation and chemical integrity of the PSA protein during conjugation. Neostanovimie terminal link of sialic acid, because it contains vicinal diols, can be easily (and selectively) oxidized periodata obtaining Manualidades shape, which is substantially more reactive to proteins and which includes suitable reactive element for attachment of proteins by reductive amination and other chemical reactions. Applicants have previously described in US-A-5846951 and WO-A-0187922. The reaction is illustrated in figure 1, where

(a) shows the oxidation of kolominova acid (alpha-2,8 linked polisialovoi acid E. coli) periodates sodium obtaining reactive against the protein aldehyde on non end and

(b) shows a selective reduction of a Schiff's base by lamborghini.com sodium obtaining a stable irreversible covalent bond with the amino group of a protein.

From the various methods which have been described for attaching polisialovoi acids to therapeutic agents [US-A-5846951; WO-A-0187922], none of them is specifically designed for conjugation by regenerating the end because of its low reactivity with respect to terapeuticas the m proteins. Although the reaction is theoretically suitable, the achievement of acceptable outputs conjugate by reaction of proteins with Polyethylen reducing end of the PSA requires a reaction time, which does not seem favourable for the stability of the protein. Secondly, the necessary concentration of the reagent (excess polymer), which may be inaccessible or uneconomical. However, despite the inefficiency of this reaction, the applicants have noticed that it can lead to side products during conjugation reactions designed to obtain conjugates with protein through the introduced aldehyde (opposite) non the end of the polymer. The potential of such by-products is evident in the published studies on catalase, insulin and asparaginase [Fernandes and Gregoriadis, 1996, 1997, 2001; Jain et al. 2003], in which policital natural (not chemically modified) form of the polymer gives rise to a protein conjugates with low efficiency (less than 5% of the protein becomes derivatives, see further below in the examples and comparison table 1) during reductive amination.

Reactivity of the reducing end of kolominova acid, although low in relation to protein targets, sufficient to cause concern in the manufacture of chemically defined conjugates of such a t is PA, to be preferred from the point of view of administrative bodies in therapeutic use in humans and animals. Unlike the natural polymer kolominova acid, which in a weak degree is monofunctional, the form of PSA, oxidized periodate (with the aldehyde at one end and policital on the other), inevitably leads to the production of complex products, which seriously complicate the task of obtaining certain molecular and pharmaceutically acceptable conjugates (figure 2). Figa is a schematic diagram showing the formation of by-products during polysilicone (the original way). Fig.2b is a more detailed schematic diagram showing the formation of by-products during polysilicone (the original way), specifically

i) asymmetric dimer;

ii) a linear polymer;

iii) a branched polymer and

iv) a variety of more complex structures.

At first sight it would seem simple to clean the expected reaction product from various by-products, described in figure 2, however, there are no direct ways of cleaning because of the physico-chemical properties of the expected forms (size, charge and so on) are largely the same, in fact almost identical compared to the planned forms of the product. This would eliminate the need for cleaning the expected types from the reaction mixture by such means as ion-exchange chromatography and helpanimals chromatography (who share on the basis of charge and size, respectively) and would exclude many other cleaning methods. Currently, applicants have solved the stated problem by the development of a new method of conjugation of polysaccharides containing groups of sialic acid on pampering terminal end, with proteins, in accordance with the low reactivity of the reducing end can be used as a beneficial effect, which eliminates the difficulty of obtaining the product described in Fig 2(b) using an established method (figure 1) reductive amination of proteins with oxidized periodata natural kolominova acid.

Jennings and Lugovski in U.S. patent 4356170 describe the preparation of derivatives of bacterial polysaccharides with proteins due to activated the reducing terminal of the link, including preliminary recovery and then a stage of oxidation. They suggest this approach if the terminal regenerating element is a N-acetyl mannosamine, glucose, glucosamine, rhamnose and ribose.

In European patent application EP-A-0454898 the amino group of the protein is associated with ALD is gidney group, obtained by reduction and partial oxidation of the reducing terminal sugar residue of glycosaminoglycan. The glucosaminoglycans, thus treated, include hyaluronic acid, chondroitin sulfate, heparin, heparansulfate and dermatooncology. None of these compounds does not contain the level of sialic acid on pampering terminal end.

The invention provides a new method for producing aldehyde derivative of sialic acid in which the original substance containing a link sialic acid pampering at its terminal end, is subjected to successive stages:

a) restore restored to the open ring of the vitality of the terminal link of sialic acid with the formation of the vicinal diol group; and

b) selective oxidation of the vicinal diol group, obtained in stage a) to obtain the aldehyde group.

The original substance is preferably a di-, oligo-, or polysaccharide, although the invention may have application for other starting substances.

The original substance used in the method according to the invention should preferably contain link of sialic acid on pampering terminal end attached to the adjacent link by means of the eighth carbon atom. In stud and b) 6,7-diol group is oxidized with the formation of aldehyde 7 carbon atom.

In an alternative embodiment, in which the level of sialic acid on pampering terminal end is connected with the adjacent link through 9 carbon atoms, in stage b) receive 7,8-diol group and oxidize with obtaining aldehyde at 8 carbon atom.

In the method according to the invention, in which the starting material is a di-, oligo - or polysaccharide, preferably starting material had on the non-terminal end sharenow group containing vicinal diol group, and in which the starting material is subjected to a preliminary stage before stage a), selective oxidation of the vicinal diol group to the aldehyde, in accordance with the than in stage a) aldehyde also restore to the formation of the hydroxy-group, which is not part of the vicinal diol group. The invention is particularly appropriate in the case, if the terminal link of the reducing end of the original substance is a link sialic acid. In an alternative embodiment, the source material may be vicinal diol group, which remains in this form for non terminal sahariana link source materials for stage a). It will not be modified by the stage of recovery, but it will be oxidized at the stage of oxidation with about what adowanie aldehyde group. The product is bifunctional and can show useful therapeutic activity due to its ability to bind substrates by the reaction of the two aldehyde groups with acceptable functional groups of the substrate.

According to the second aspect of the invention provide a new way in which sialic acid source materials having terminal sialic acid at the non terminal end, is subjected to the following stages:

c) stage non selective oxidation of the terminal link of sialic acid 7,8-vicinal diol group for the formation of 7-aldehyde; and

d) a stage of recovery 7-aldehyde group to the corresponding alcohol.

This aspect of the invention provides derivatives of sialic acid, which are passivated non terminal end, enabling activation of the reducing terminal end for subsequent reactions. Activation may represent a recovery/oxidation, for example, the first aspect of the invention, with optional subsequent stages of transformation of the aldehyde group to another group, such as amination with the formation of the amine. Can be a stage to activate the reducing terminal end.

Preferably this second aspect invented the I is part of the way, in which the original substance has a regenerating terminal link and subsequently requires to be conjugated with another molecule through the specified link. In this method restores the terminal link usually activate, for example, by reaction, which would otherwise have activated the part of the non terminal units of sialic acid, if it does not lead to passivation. This reaction, for example, is a selective oxidation of the vicinal diol fragment and it is carried out after stage d).

In the invention, the preferred polysaccharide starting material may contain in the molecule links other than sialic acid. For example, levels of sialic acid can be interleaved with other sharedname links. It is preferable however that the polysaccharide contained substantially only the links of sialic acid. Preferably they are connected to 2→8 and/or 2→9.

Preferably the polysaccharide starting material has at least 2, more preferably at least 5, more preferably at least 10, for example at least 50 sharidny links. For example, the polysaccharide may include at least 5 units of sialic acid.

Policylevel acid can be obtained from any source, preferably a natural source of the ICA, such as bacterial source, such as E. coli K1 or K92, meningococci group, or even cow's milk, or N-CAM polymer of sialic acid can be heteropolymers, such as group 135 or group V N. meningitides. Polivanova acid may be in the form of a salt or free acid. It can be in the hydrolyzed form, such that the molecular weight decreased after discharge from a bacterial source. Polivanova acid may be a substance with a wide range of molecular masses, such that the polydispersity was more than 1.3, for example, was 2 or more. Preferably the polydispersity of molecular weight is less than 1.2, for example, is to 1.01.

Part polisialovoi acids, with a wide range of molecular weight, can be fractionated low polydispersity, i.e. into fractions of different average molecular weight. Fractionation is a preferable anion-exchange chromatography with elution suitable primary buffer. Applicants found suitable anion exchange environment i) preparative environment, such as a strong ion-exchange substance on the basis of activated agarose containing side groups of the Quaternary ammonium ion (i.e. a strong base). Lucindy buffer is not reactive, and preferably a Ki is volatile, so that the desired product can be distinguished from the base of each fraction by evaporation. Suitable examples are amines, such as triethanolamine. The selection may represent, for example, freeze drying. The fractionation method suitable for polisialovoi acid as the starting material as well as its derivatives. Technological method can thus be used before or after the required stages of the method according to the invention.

Applicants believe that the first ion-exchange chromatography is used to obtain fractions of ionic polysaccharides with molecular weight of more than about 5 kDa, in particular polisialovoi acid with such a molecular weight (MW). According to an additional aspect of the present invention provide a method for fractionation of a part of an ionisable polysaccharide with molecular weight (MW) higher than 5 kDa when using ion-exchange chromatography with lucioni buffer base or acid, which is preferably volatile. Preferably the polysaccharide contains groups of carboxylic acids and ion exchange is an anionic exchange. Preferably lucindy buffer contains amine, preferably triethanolamine. More preferably the polysaccharide isolated from the fractions by means of freeze drying. This method can be applied for fracc is onirovaniya kolominova acid (CA), containing other reactive fragments (imide of maleic acid or iodoacetate etc) or other nature (for example, textresult) or synthetic (for example, polyglutamine acid, polylysine in the latter case, when using cation-exchange chromatography) charged polymers. Applicants believe that the first ion-exchange chromatography (IEC) is used to separate ionic polysaccharides in combination with technological methods of deposition and/or by means of ultrafiltration. Methods IEC should also remove the by-products of receipt as endotoxins, which remain in commercially available PSA and SA.

In the pre-stage oxidation stage and (C) selective oxidation should preferably under such conditions that largely did not occur splitting in the middle part of the main hydrocarbon chain long chain (polymer) of the original substance, so that there was no decrease in molecular weight. You can use the enzymes responsible for carrying out this stage. It is most convenient to oxidation was represented by chemical oxidation. The reaction can be carried out with immobilizerturning reagents such as perruthenate-based polymer. The most direct method is carried out with the dissolved reagents. The oxidizer is a city of enat or preferably, periodat. The oxidation can be carried out by periodate at a concentration in the range from 1 mm to 1 M, at pH in the range from 3 to 10, a temperature in the range from 0 to 60°in the range from 1 minute to 48 hours.

In the way of stage a) is a stage in which the level of sialic acid on pampering the end of the restore. Usually link to pampering the end of the original substance is in the form of catalogo rings and recovery in stage a) reveals the ring and restores the ketone to the alcohol. Thus, the hydroxyl group at the 6-carbon atom is part of a vicinal diol fragment.

Under suitable conditions, the recovery (stages a) and (d)) we can use hydrogen with catalysts or preferably hydrides, such as boron. They can be immobilizerturning, as, for example, Amberlite (trade mark) - borohydride on the substrate. As the reducing agent, preferably using hydrides of alkali metals such as sodium borohydride at a concentration in the range from 1 μm to 0.1 M, at pH ranging from 6.5 to 10, at a temperature in the range from 0 to 60°in the range from 1 minute to 48 hours. Reaction conditions are chosen so that the lateral carboxyl groups of the source materials were not recovered. When the pre-stage oxidized whom I held, the obtained aldehyde group is reduced to an alcohol group, not part of the vicinal diol group. Other suitable reducing agents are cyanoborohydride in acidic conditions, such as cyanoborohydride on a polymer substrate or cyanoborohydride alkali metal, L-ascorbic acid, meta sodium bisulfite, L-selectride, triacetoxyborohydride etc.

Between any pre-stage oxidation and stage of recovery (a) and after stage b), and between the stage of oxidation) and the stage of recovery (d), and between stage d) and any subsequent stage of oxidation of the corresponding intermediate must be separated from the respective oxidizing and reducing agents, respectively, before the subsequent stage. If stage is carried out in the liquid phase, the separation can be conducted by conventional means, such as spending an excess of oxidizing agent when using glycol, dialysis polysaccharide and ultrafiltration for concentration of aqueous solution. The mixture of products from the stage of recovery can be again separated by dialysis and ultrafiltration. You can offer reactions carried out using immobilized oxidizing or reducing reagents, resulting in direct isolation of the product.

Phase selective oxidation stage b) is conveniently carried out the ü under conditions similar to the preliminary stage of oxidation, as described above. Similarly, the oxidizing agent should be spent before the product selection when using ethylene glycol. The product is then allocate such suitable methods, such as dialysis and ultrafiltration.

The method according to the first aspect of the invention and the preferred alternative implementation of the second aspect, which includes the subsequent stage of oxidation after stage d) to enable the vitality terminal Zaharenko link leads to the production of activated derivative containing a reactive aldehyde fragment derived from the vitality of the terminal end. The preferred method involving oxidation, then recovery, then stage oxidation leads to the production of activated product having one reactive aldehyde fragment. If you do not conduct the preliminary stage of oxidation and the original substance contains nevosstanovlenie terminal element, which contains vicelow diol group (e.g., sialic acid), the product will contain the aldehyde group on each of the terminal ends, which can be used.

Aldehyde groups suitable for conjugation to substrates containing an amino group, or hydrazine powered connections. Ways, the cat is where the activated product of the oxidation steps in consequence of the conjugates with the substrate, form an additional aspect of the invention. Preferably the reaction of conjugation, as described in previous publications of the applicants, as described above, which includes conjugation with amine to form a Schiff's base, preferably followed by recovery with the formation of a secondary amine fragment. The method is particularly valuable for proteins that can be modified in which the amino group is an Epsilon-amino group of the lysine groups or N-terminal amino group. The method is particularly valuable for proteins that can be modified, or peptide therapeutically active agents, such as cytokines, growth hormones, enzymes, hormones, antibodies or fragments. Alternatively, the method can be used to obtain derivatives of the systems of drug delivery, such as liposomes, for example, by reaction of the aldehyde with the amino group of components forming the liposome. Other systems of drug delivery is described in an earlier application of the applicant, US-A-5846951. Other substances that can be modified include viruses, bacteria, cells, including cells of the animal, and synthetic polymers.

Alternatively, the substrate may contain hydrazine powered group, in this case the product is a hydrazone. It can be restored if desired for extra niceley stability to the alkyl hydrazide.

In another preferred embodiment, at the stage of oxidation b) or a subsequent stage of oxidation (d) should be the reaction of one or each of the aldehyde groups with a connection via a linker comprising the amino or hydrazide group and another functional group suitable for selective receipt of the derived proteins or other therapeutically active compounds, or delivery systems of drugs. Such a linker may, for example, include a compound containing a Deputy functional groups for specific reaction with sulphhydryl groups and dibasic organic group linking the amine or hydrazide group and a functional group. In the reaction residue of the aldehyde with the amino or hydrazide group is formed reactive conjugate suitable for binding with the substrate containing Tilney (sulfhydryls) group. Such conjugates have a special value for selective or site-specific obtain derivatives of peptides and proteins.

Deriving proteins and delivery systems of drugs can lead to increased half-life, improved stability, reduced immunogenicity and/or control the solubility and hence bioavailability and pharmacokinetic properties, or can improve the solubility of activehost or viscosity solutions, containing a derivative of the active substance.

According to the invention also provide a new connection, which is an aldehyde derivative of di-, oligo - or polysaccharide, including fragments of sialic acid in which the terminal link of the reducing end is an OR group in which R is chosen from

-CH2CH2Other1CH2CH=N-other1and CH2CH2NHNHR1in which R1represents H, C1-24alkyl, aryl With2-6alkanoyl or polypeptide, or protein, which is connected through the N-terminal end or the amino group of the side chain of the lysine residue, drug delivery, or an organic group containing a functional substitute, adapted to react with sulfhydryl group and, preferably, the terminal fragment on non the end was passivated.

A new connection may include the middle part of the chain of sharidny links between the two terminal links. The links in the middle part of the chain composed of links of sialic acid or, alternatively, may include other sacharine links in addition to the terminal units, which are derived from units of sialic acid. The connection can be usually the Holocene, as described above in relation to the first aspect of the invention.

A new connection can be polysialylated substrate comprising at least one group polisialovoi acid (polysaccharide), conjugate with each substrate molecule conjugation, including the connection of the secondary amine, hydrazone or acylhydrazides through revitalizing the terminal end polisialovoi acid and substantially not containing a crosslinking non end polisialovoi acid with another molecule or substrate. The substrate may consist, for example, biologically active compound, such as pharmaceutically active compound, in particular a peptide or protein therapeutic agent or drug delivery. Such active substances are widely described below.

A new connection can have a General formula I

in which R is selected from

-CH2CH2Other1CH2CH=N-other1and CH2CH2NHNHR1in which R1represents H, C1-24alkyl, aryl With2-6alkanoyl or polypeptide, or protein, which is connected through the N-terminal end or γ-amino group of a lysine residue, drug delivery, or an organic group containing options the regional Deputy, adapted for reaction with sulfhydryl group;

R3and R4selected from the

i) R3represents N and R4represents IT;

ii) where R is CH(CH2OH)CH2HE or-CH2SNO, R3and R4together are =O;

iii) where R is CH(CH2OH)CH2Other1or-CH2CH2Other1, R3represents H and R4is an-other1;

iv) where R is a-CH(CH2OH)CH2NHNHR1or-CH2CH2NHNHR1, R3represents H and R4represents-NHNHR1; or

v) -CHCH=N-other1, R3and R4together are =N-other1; AC represents an acetyl,

n represents 0 or more; and

GlyO is glucosyloxy group.

If R is a group

the compound of General formula I is a polysaccharide that is derived polisialovoi acids with aldehyde group on pampering terminal link.

If R is a group

CH2CH=N-other1or CH2CH2NHNHR1the compound is a conjugate formed by reaction of the aldehyde derivative polisialovoi acid HYDR what Zid R 1NHNH2. Hydrazide preferably represents an acyl hydrazide (R1contains a terminal carbonyl group).

If R is a group

the compound is a conjugate formed by reaction of the aldehyde derivative polisialovoi acid with a primary amino group containing compound R1NH2.

R1may be a residue of the peptide or protein therapeutic agent, such as antibody or fragment, enzyme or other biologically active compounds, as described above. The group R1may include linker fragment between the active compound and polisialovoi acid.

Alternatively, R1may be a residue of the linker reagent, for example, to obtain the derived polisialovoi acid suitable for conjugation with groups that differ from the amino or hydrazides, on the active compounds. The examples represent a linker reagents of the formula

what is a N-connection imide maleic acid, in which R2represents a dibasic organic group, for example, Allen oligo(alkoxy)alkane, or, preferably, landiolol group, for example With2-12-landiolol GRU is PU.

The present invention is most applicable if a new connection is monofunctional and passivated at the terminal link of non end. In these compounds R3represents N and R4is a HE. R can be any of the values specified above. Glucosamine group, preferably, include links sialic acid and, more preferably, consist only of such links associated 2-8 or 2-9, for example alternating 2-8/2-9 with each other.

The invention further provides compositions, including new connections and thinner, as well as pharmaceutical compositions comprising the novel compounds in which R1has biological activity, and a pharmaceutically acceptable excipient. The pharmaceutical compositions can be administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally, intradermally, topically or vnutritrahealno.

In the second aspect of the invention provides a new compound that is a product of the method according to the second method aspect, which has the General formula II

in which

AC represents acetyl;

m is 0 or more;

Gly1About represents glycosyl; and

R5is an organic GRU is PU, preferably the recovered form of the terminal reducing Zaharenko link, its oxidized derivative, which is an aldehyde or reaction product of such aldehyde, which represents, for example, an amine or hydrazide.

Preferably R5selected from the same groups as R above. Alternatively, R5is a group III, connected via one carbon, 8 or 9, with

(whereby the other of the carbon, 8 or 9, substituted by hydroxyl:

which is a product of the recovery opening cycle reducing terminal sialic acid.

Preferably the group Gly1O include links sialic acid, most preferably consist of chains of sialic acid. The value of m is preferably 2 or more, more preferably is 5-1000, such as 10-500, preferably from 10 to 50.

The new method is particularly valuable for the creation of a monofunctional polisialovoi acid (PSA). This is based on the understanding of the tautomeric equilibrium, the reducing end rings PSA, for example, kolominova acid (SA), which is described in figure 3. Revitalizing the end of the sialic acid residue PSA spontaneously forms a ketone with the opening to which ICA through tautomerizations (figure 3). When the dynamic equilibrium between the ring and linear structures of sialic acid residue of the reducing end at any given moment ketone fragment was present only in part of PSA molecules. However, as noted above, here it is emphasized that the reactivity of polumetla reducing end is insufficient for practical use joining PSA to proteins on the basis of which the previously described methods do not use that part of the polymer to attach proteins or other drugs. Thus, as illustrated in figure 3, the solution of the terminal sialic acid residue on pampering the end polisialovoi acid is in tautomeric equilibrium. The form of an open ring, although present in small surplus in equilibrium is weakly reactive towards amino groups of proteins and leads to the formation of covalent adducts with proteins in the presence of cyanoborohydride sodium.

In a preferred embodiment of the invention, for the best achievements of protein conjugation described products with PSA, applicants have created a chemically modified form of polisialovoi acid, which is monofunctional. The new form includes chemical modification of both ends of natural molecules polisialovoi acid. In ex is contrast to the original form of the reaction (figure 1), in which the polymer becomes conjugated mainly from 2 to 8 position, with the most remote "regenerating end", a new form of polymer attachment occurs exclusively in the opposite orientation.

New preferred monofunctional form polisialovoi acid or other polysaccharide aldehyde derivative is more suitable for the synthesis and manufacture of pharmaceutically acceptable product because it eliminates considerable complexity, which otherwise occurs when using plastic molds with non-modified reducing end (figure 2). Obtaining new forms of the polymer (figure 4) includes selective oxidation, preferably by periodate, as in the foregoing descriptions of the applicants for the introduction of the aldehyde function on a non end. In contrast to the earlier technology shown in figure 1, this aldehyde fragment then destroy recovery, for example a borohydride. At the other end of the polymer phase reduction with borohydride also simultaneously locks the ring structure of the reducing end through the restoration of polumetla. Simultaneous recovery of the ketone to hydroxyl fragment introduces a new diol functional group, which is now subject of selection the mu oxidation in the second stage of oxidation. When a natural polymer was successfully oxidized periodata restored by borohydride and oxidized a second time by periodate, a new form of the polymer, which really is a monofunctional, having at least one reactive group (aldehyde) only a pampering end (figure 3).

The reaction activity of the protein (when the recovery aminating) various intermediates described in method ' double oxidation" 4 described in table 2. In particular, the data show that intermediate "CAOR" (colomina acid - Polivanova acid - oxidized/reduced), obtained by reduction with borohydride oxidized periodata polymer is inert to protein targets, ensuring that both the aldehyde and policitally fragment was destroyed when restoring a borohydride. In the second cycle of oxidation periodata "inert with respect to the protein of intermediate CAOR get a new derived polisialovoi acid (CAORO), which again is reactive to proteins (table 2) and, moreover, it really is monofunctional in nature, having only one aldehyde group on "pampering the end of the polymer being the reaction-inactive to proteins on the other end. Monofunctional PSA may lead to the exclusively uniquely oriented joining proteins at unrecoverable end and can lead to unintentionally crosslinked proteins (figure 5). This new reaction scheme (figure 4), known as the method of "double oxidation allows you to elegantly avoid the need for cleaning the expected product from different inadvertently obtained products (see figure 2), which can be completely avoided presents a new reaction scheme.

The subsequent is a brief description of the drawings.

Figa is a reaction scheme showing the activation of prior art non terminal link of sialic acids;

fig.1b is a reaction scheme showing the reductive amination of prior art aldehyde fragment of the product of reaction scheme 1A when using fragment protein-amine;

figa is a schematic diagram showing the possible adverse reactions occurring in the reaction fig.1b involving regenerating end;

fig.2b schematically represents a possible by-products of side reactions figa;

figure 3 is a reaction scheme showing the tautomerism between Cetelem and forms a closed ring of the reducing terminal of the level of sialic acid PSA;

figa is a reaction scheme showing the preferred oxidation reaction recovery oxidation PSA

fig.4b gives an idea about the suitable conditions for the stages of the circuit of Fig.4 and explains the abbreviations used for the original substances, intermediates and final products;

figure 5 is a schematic diagram similar to fig.2b, but showing the reaction products of Fig.4;

Fig.6 shows the result of analysis by the method of gel chromatography (GPC) of the products of example 1;

Fig.7 shows the results of denaturing electrophoresis in polyacrylamide gel (SDS-PAGE) of example 2;

Fig shows the pharmacokinetic half-life of the conjugates testedin vivoin mice in example 3;

Fig.9 shows the results of the IEC for kolominova acid (SA) to 22.7 kDa in example 2 comparison;

figure 10 shows the initial results of polyacrylamide gel electrophoresis (PAGE) for SA 22,7 kDa in example 2 comparison;

11 shows the original results of polyacrylamide gel electrophoresis (PAGE) for several substances SA loaded and separated fractions, as in example 2.2 comparison;

Fig shows the GPC chromatogram for 3 of fractions of CA, isolated as in example 2.2 comparison;

Fig shows the original results PAGE for two of the samples used in Fig, and other samples of CA and CAO, as described in example 2.2 comparison;

Fig shows the results of ultrafiltration SA 22,7 kDa, as the description is about the example 2.4 comparison;

Fig shows SDS PAGE, for example, 5;

Fig shows the results of SDS PAGE for fractionated conjugates of growth hormone GH-CA obtained in example 5; and

Fig shows the results of example 7.

The invention is additionally illustrated by the accompanying examples.

Examples

Materials

Ammonium carbonate, ethylene glycol, polyethylene glycol (8 kDa), cyanoborohydride sodium (>98% purity), meta-periodate sodium and molecular weight markers were obtained from Sigma Chemical Laboratory, UK. Used colomina acid, linear α-(2→8)-linked policially acid E. coli (22,7 kDa on average, high polydispersity of 1.34, 39 kDa, polydispersity of 1.4; 11 kDa and a polydispersity 1,27) were from Camida, Ireland, radioactive iodide (Na125I) was purchased from Amersham, UK. Other substances included 2,4 dinitrophenyl hydrazine (Aldrich Chemical Company, UK), tubular dialysis unit (within the cut-off of 3.5 kDa and 10 kDa; Medicell International Limited, UK), Sepharose HiTrap SP, PD-10 columns (Pharmacia, UK), Tris-glycine polyacrylamide gels (4-20% and 16%), Tris-glycine sodium dodecyl sulphate flowing buffer and loading buffer (Novex, UK). Deionized water was obtained from a water treatment plant Elgastat Option 4 (Elga Limited, UK). All used reagents were of analytical purity. For spectrophotometric measurements in the analysis of proteins or SA used spectropho is Omer for reading plates (Dynex Technologies, UK). Outbred CD1 mice (aged 8-9 weeks; body weight 29-35 g) were purchased from Charles River (UK) and acclimatized for at least one week before using.

Ways

Determination of proteins and kolominova acid

Quantitative assessment polisialovoi acids (as sialic acid) with resorcinol reagent conducted resorcinol way [Svennerholm, 1957], as described in other publications [Gregoriadis et al., 1993; Fernandes and Gregoriadis, 1996, 1997]. Fab (protein) was measured on the basis of the BCA colorimetric method.

Example comparison 1

Covalent PSA-protein conjugates were obtained by reductive amination with cyanoborohydride sodium using natural forms polisialovoi acid (colomina acid, SA) from E. coli, through its weakly reactive reducing end. SA = colomina acid; CAO = oxidized colomina acid as in Fernandes and Gregoriadis, 1996; Jain et al., 2003. Cyanoborohydride sodium (NaCNBH3used at a concentration of 4 mg ml-1.

The results are presented in table 1. The molar ratio in column 1 was the ratio of the initial CA(OH) to protein (n=3, ± standard deviation).

Table 1
ReceivingThe degree of modification with CA molar ratio (SA:b the Lok)
Catalase + CAO + NaCNBH3(10:1)0,77±0,16
Catalase + CAO + NaCNBH3(50:1)2,59±0,08
Catalase + SA + NaCNBH3(50:1)0,55±0,05
Catalase + SA (50:1)0,65±0,04
Insulin + CAO + NaCNBH3(25:1)1,60±0,14
Insulin + CAO + NaCNBH3(50:1)1,65±0,14
Insulin + CAO + NaCNBH3(100:1)1,74±0,12
Insulin + SA + NaCNBH3(25:1)0,20±0,02
Insulin + SA + NaCNBH3(50:1)0,21±0,04
Insulin + SA + NaCNBH3(100:1)0,24±0,06

Example 1 Obtaining monofunctional polisialovoi acid:

1A Activation of kolominova acid

Freshly prepared 0.1 M solution of metaperiodate sodium (NaIO4) was mixed with CA (100 mg CA/ml NaIO4) at 20°and the reaction mixture was stirred with a magnetic stirrer for 15 minutes in the dark. Then the reaction mixture was added twice the amount of ethylene glycol to spend excess NaIO4and the mixture was left to mix at 20°even within 30 minutes. Oxidized clomidbuy acid was subjected to extensive dialysis (cut-off of 3.5 KDa tubular and the preparations for dialysis) (24 hours) against 0,01% buffer ammonium carbonate (pH 7.4) at 4° C. Ultrafiltration (over limit cut-off of 3.5 kDa) was used for the concentration of the solution of CAO from tubular dialysis unit. After concentration to the desired volume of the filtrate liofilizirovanny and kept at -40°C until further use.

1b Restoring kolominova acid

Oxidized clomidbuy acid (CAO, an increase of 22.7 kDa) was restored in the presence of sodium borohydride. Freshly prepared 0.15 mm solution of sodium borohydride (NaBH4; in 0.1 M NaOH, diluted to a pH of 8 to 8.5 by dilution diluted solution of H2SO4) was mixed with CAO (100 mg CA/ml) at 20°and the reaction mixture was stirred for 2 hours in the dark. Due to the completion of the reaction the pH was lowered to 7. Oxidized/restored clomidbuy acid (CAOR) dialyzed (cut-off tubular dialysis unit molecular weight of 3.5 KDa) against a 0.01% solution of a buffer of ammonium carbonate with pH (7) if 4°C. For a concentration CAOR solution of the tubular dialysis unit used ultracentrifugation. The filtrate liofilizirovanny and kept at 4°C until further use. The definition of any of the content of the aldehyde was determined as described in "determining the oxidation SA.

1C Re-oxidation SA

After confirmation of the absence of the aldehyde content in the oxidized/Voss is set out kolominova acid (CAOR) it was again oxidized in the same way, as described in the activation of kolominova acid, except that CAOR incubated with a solution of periodate for a longer time (up to 1 hour). Oxidation CAORO product was measured on the dried powder obtained in this stage.

1d determination of the oxidation state of CA and derivatives

A qualitative assessment of the degree of oxidation of kolominova acid was performed with 2,4-dinitrophenylhydrazine (2,4-DNPH), which when interacting with carbonyl compounds led to poorly soluble 2,4-dinitrophenylhydrazones. Unoxidized (SA), and oxide (CAO), restored (CAOR) and re-oxidized (CAORO) (5 mg each) was added to 2,4-DNPH reagent (1.0 ml), the solutions were shaken and then left at 37°until a crystalline precipitate [Shriner et al., 1980]. Degree (quantitative) oxidation of SA was measured using the method [Park and Johnson, 1949], based on the recovery ferricyanide ions in alkaline solution to ferric ferrocyanide (Persian blue), which is then measured at 630 nm. In this case, as the standard used glucose.

1E Helpanimals chromatography

Samples of kolominova acid (SA, CAO, CAOR and CAORO) was dissolved in NaNO3(0.2 M), CH3CN (10%; 5 mg/ml) and was chromatographically two GMPWXLcolumns with the definition on the basis of the index of refraction (SE the (GPC) system: VE1121 GPC pump solvent, VE3580RI detector and compared using Trisec software 3 (Viscoteck Europe Ltd). Samples (5 mg/ml) was filtered through 0.45 µm nylon membrane and missed at 0.7 cm/min 0.2 M NaNO3and CH3CN (10%) as the mobile phase.

Results

Colomina acid (SA), Polivanova acid, is a linear alpha-2,8-linked homopolymer residue N-acetylneuraminic acid (Neu5Ac) (figa). However, periodit is a strong oxidizing agent and despite the fact that he is selective [Fleury and Lang, 1932] with respect to hydrocarbons containing a hydroxyl group attached to adjacent carbon atoms, it can cause time-dependent cleavage of internal Neu5Ac residues. In this regard, in the present work, the time of oxidation of kolominova acid limited 15-60 minutes when using 100 mm periodate at room temperature [Lifely et al., 1981]. Moreover, since periodic decomposes when exposed to light with the formation of more reactive compounds [Dyer, 1956], the reaction mixture was kept in the dark. The integrity of the internal alpha-2,8-linked Neu5Ac residues after treatment with periodate and borohydride were analyzed using gel chromatography and the chromatogram obtained for the oxide (CAO), oxidized restored (CAOR), double-oxidized (CAORO) is exist compared with those for native SA. It was found (6)that oxidized (15 minutes) (CAO) (6b), restored (CAOR) (6s), double-oxide (1 hour) (CAORO) (6d) and native (6A) CA actually possess identical elution profiles without evidence that successive stages of oxidation and reduction has resulted in significant fragmentation of the polymer chain. Small peaks correspond buffer salts. Quantitative measurements of the oxidation state of the SA carried out the restoration of ferricyanide ion in alkaline solution to ferrocyanide (Persian blue) [Park and Johnson, 1949] using glucose as standard [results shown in table 2]. Table 2 shows that oxidized colomina acid has more than stoichiometric (>100%) the amount of the reducing agent, i.e. 112% mol apparent aldehyde content, including the combined restoring force of the reducing end of polumetla and put aldehyde (the other end). The CAOR was not observed reaction activity, it shows that neutralization as aldehyde and polumetla CAO was successfully implemented by the restoration of the borohydride. After the second cycle of oxidation periodata the aldehyde content in the polymer was stable at 95% CAORO (when the error of the experiment 10%), indicating the successful introduction of a new aldehyde fragment to pampering the end.

Financial p the tats quantitative studies of the intermediates kelemenova acid in the dual way of oxidation when using ferricyanide (table 2) coincided with the results of the qualitative experiments, conducted with 2,4-dinitrophenylhydrazine, which gave light yellow precipitate after 10 minutes of reaction at room temperature with native CA and intense orange colour with the forms of the polymer containing aldehyde.

Table 2
Types CAThe oxidation state
Colomina acid (SA)16,1±0,63
Oxidized colomina acid (CAO)112,03±equal to 4.97
Restored colomina acid (CAOR)0; netdetective
Oxidized restored oxidized colomina acid (CAORO)95,47±7,11

Table 2: Degree of oxidation of various intermediates of kolominova acid in the reaction scheme for the dual oxidation using glucose as a standard (100%, 1 mol of aldehyde per mole of glucose n=3±standard deviation).

Example 2 - Getting conjugates Fab-colomina acid

Fab was dissolved in 0.15 M phosphate-saline buffer solution (PBS) (pH 7.4) and covalently linked with various kolominova acids (CA, CAO, CAOR and CAORO) via reductive amination in the presence of cyanoborohydride sodium (NaCNBH3). Clomidbuy acid from each stage of the synthesis (and the original substance and the products of each of examples 1A to C) together with Fab molar relations SA:Fab 100:1 was introduced into the reaction in 0.15 M PBS (pH 7.4; 2 ml)containing cyanoborohydride sodium (4 mg/ml) in sealed vessels under stirring with a magnetic stirrer at 35±2°in the furnace. The mixture was then subjected to precipitation with ammonium sulfate ((NH4)2SO4) by slowly adding salt with continuous stirring to obtain a 70% wt./about. the saturation. The samples mixed for 1 hour at 4°C, centrifuged for 15 minutes (5000·g) and the pellets, containing polysialylated Fab, suspended in a saturated solution (NH4)2SO4and again centrifuged for 15 minutes (5000·g). Selected precipitation was re-dissolved in 1 ml of 0.15 M Na-phosphate buffer with the addition of 0.9% NaCl (pH 7.4; PBS) and actively deliberately (24 hours) at 4°again with the same PBS. The dialysates were then examined on the content of sialic acid and Fab and the output of the conjugate were expressed as molar ratio CA:Fab. The control consisted of conjugation source of protein method in the presence of oxygenated SA or in the absence of SA under the described conditions. Stirring was maintained at a minimum in order to avoid concomitant denaturation of the protein. Polysialylated Fab additionally characterized gel chromatography, ion exchange chromatography and by denaturing polyacrylamide gel electrophoresis (SDS-PAGE).

2b, the Ion exchange chromatography the

Samples (0.5 ml) at initial time (control) and after 48 hours of reaction mixtures was subjected to ion-exchange chromatography (IEC) on a cation exchange column (SP Sepharose (1 ml; flow rate 1 ml/min; binding/wash buffer 50 mm sodium phosphate, pH 4.0; lucindy buffer, 50 mm sodium phosphate, pH 4.0, containing 1 M horida sodium). The column was washed, was suirable and erwerbende fractions were analyzed for calcium content and protein (Fab). For desalting of samples prior to use in the column used a PD-10 column.

2C SDS-polyacrylamide gel electrophoresis

The polyacrylamide gel electrophoresis (SDS-PAGE) (MiniGel, vertical gel setting, the model VGT 1, the supply model Consort E132; VWR, UK) was used to determine changes in the molecular size Fab when polysialylated. SDS-PAGE of Fab and its conjugates (SA, CAO, CAOR and CAORO) of the samples in the initial moment of time (control) and after 48 hours of reaction mixtures, as well as a control method (unoxidized CA) was performed using 4-20% polyacrylamide gel. The samples were calibrated in a wide range of molecular weight markers.

In previous experiments [Jain et. Al., 2003; Gregoriadis, 2001] with other proteins, it was found that the optimal outputs molecular conjugation CA:Fab (derived from immunoglobulin (IgG) sheep) require a temperature of 35±2 C in 0.15 M PBS buffer at pH 6-9 within 48 hours. Minovia samples (Schiff's base), formed under these conditions between the aldehyde polymer and protein was successfully restored NaCNBH3with stable secondary amine [Fernandes and Gregoriadis, 1996; 1997]. Effects on protein oxidized periodata natural SA leads to the formation of metastable adduct joining Schiff bases of SA-protein (as described for polysilicone catalase) [Fernandes and Gregoriadis, 1996]. Thus in the reaction of the oxidized form of SA with Fab applicants first obtain metastable adduct joining Schiff bases by incubation of the oxidized polymer with Fab for 48 hours at 37°With that then reinforce selective restore (by reductive amination) with NaCNBH3(which restores kinoway the structure of the Schiff's base, but not the aldehyde fragment of the polymer). In order to characterize the reactivity against different protein intermediates SA fashion double oxidation", Fab was subjected to reductive aminating in the presence of natural CA (SA), oxidized CA (CAO), oxidized restored SA (CAOR) and "double-oxidized" SA (CAORO). For these studies are used to 22.7 kDa PSA at a molar ratio of CA: Fab (100:1). After 48 hours incubation in the presence of NaCNBH3conjugates Fab is delali from the reaction mixtures by precipitation with ammonium sulfate (as described in the Examples) and the results were expressed in terms of molar ratios of CA:Fab in the resulting conjugates (table 3).

Table 3< / br>
The synthesis of compounds Fab (protein) kolominova acid
The analyzed types CAReached the molar ratio during conjugation (SA:Fab)
Colomina acid (SA)0,21:1 (weakly reactive)
Oxidized colomina acid (CAO)2,81:1 (highly reactive)
Oxidized restored colomina acid (CAOR)Not definable (reactivity liquidated)
Oxidized restored oxidized colomina acid (CAORO)2,50:1 (newfound high reactivity)

From table 3 it is obvious that if used natural oxygenated CA (in the presence of cyanoborohydride), observed a significant conjugation, but at a low level (the result of which was the molar ratio of CA:Fab 0,21:1) due to the reaction with polyacetylenes group SA on her pampering the end.

The formation of conjugates CA-Fab was further proved by joint precipitation of the two fragments adding (NH4)2SO4(SA itself is not precipitated in the presence of this salt). Proof conjugation also received jonoob the military chromatography (IEC, not shown) and polyacrylamide gel electrophoresis (SDS-PAGE; Fig.7).

For ion-exchange chromatography polysialylated Fab obtained by precipitation (NH4)2SO4re-dissolved in a buffer of sodium phosphate (50 mm, pH 4.0) and subjected to ion-exchange chromatography (IEC) using SP Sepharose HiTrap column (cation exchange). In contrast to the results showing the complete dissolution of CA (wash water) and Fab (buervenich fractions), and SA and Fab in the reaction samples 48 hours together were suirable in fractions of wash water, showing the presence of CA-Fab conjugate.

Fig.7 describes the analysis of the antibodies described above Fab conjugates. These data confirm that the distribution of the molecular masses of the two conjugates are extremely similar (as expected, since the by-products obtained from asymmetric bifunctional SA, constitute only a small percentage of the total part of the molecule). From Fig.7 it is obvious that, independently, have a Fab conjugates of asymmetric bifunctional SA (i.e. oxidized periodata natural SA) or monofunctional PSA was formed conjugates with a wide range of molecular weight greater than the molecular weight is not translated in derivatives Fab control. This is consistent with the known natural polymer polydispersity, OPI is Anna in previous publications of the applicant. 7 also confirms that reductive amination of monofunctional SA leads to the production Fab conjugate with output comparable to the output of the previously described method based on oxidized periodata natural SA (described in figure 1). From Fig.7 it is obvious that only trace amounts untranslated in the derived Fab remain in each sample conjugate. Trace amounts remaining Fab was removed from these conjugates ion exchange chromatography prior studiesin vivo(example 3 below).

Example 3 - the study ofin vivo

In samples of Fab fragment of sheep immunoglobulin (IgG) or conjugates with CAO or CAORO was injected radioactive tag125I in the following way:

10% by volume of each of these samples was selected (˜100 μl) and placed in fresh IODO-gen tubes. 20 μl of the sample PBS containing 200 µci125I (NaI)was added to the protein or conjugate and the tubes were closed and left incubated at room temperature for 10 minutes. The contents of the test tubes and then transferred to 500 μl of centrifugal filters (cut-off molecular weight of 3.5 kDa) and the samples were centrifuged at 6500 rpm in microcentrifuge. Eluent was discarded and the volume of retentate (above the membrane) brought up to 500 μl. This method was repeated 5 more times, measured radioactivity above (protein) and below (free is hydrated iodine) membranes for sample 5 ál when using counter Pakard Cobra Gamma. If the number of quanta free125I was less than 5% of the number of quanta in the conjugated fraction, additional purification is not carried out. If free125I was >5%, the cleaning cycle was repeated and the samples were analyzed again.

CD1 mice (body weight 29-35 g) were administered a dose of 40 μg (100 μl volume of PBS) of protein each mouse (˜1.6 mg/kg) intravenously (via tail vein) in a single injection and selected 50 μl blood samples (using treated heparin graduated capillary) at time intervals of other tail vein and placed in 500 μl of PBS. Last bleeding was a bleeding to obtain a sufficient number of quanta. The samples are then centrifuged at 3000 rpm for 10 minutes and the resulting supernatant was removed and placed in tubes for counter gamma-quanta. The samples were calculated with the models presented injectable protein in Pakard Cobra II auto gamma counter-ray. The registered numbers of photons expressed as a percentage of the original injectable dose.

Samples labeled with radioactive iodine Fab, CAO and CAORO Fab conjugates were administered as intravenous injection to mice for control of half-life from blood flow. On Fig shown pharmacokinetics native Fab compared to conjugates Fab-kolominova acid obtained by the original method (using CAO) and a new method of double oxidation (using CAORO). The presented results show that CAO-Fab and CAORO-Fab lead to a pronounced and significantly longer retention time in the bloodstream compared to that observed in the case of unmodified Fab, leading to an increase in 6,28 time and 5,28 times (respectively) AUC values compared with native Fab.

Example 4 - Synthesis of conjugate imide maleic acid

CAORO synthesized in example 1C above, was introduced into the reaction with 5 molar equivalents of hydrazide N-[β-multimediaphoto acid] in 0.1 M sodium acetate for 2 hours at 37°C. the resulting hydrazone was besieged in ethanol, re-suspended in sodium acetate and again besieged in ethanol, re-dissolved in water and subjected to freeze dry. The product is suitable for site-specific conjugation with thiol groups of fragments of cysteine in proteins and peptides. Aldehyde derivative monofunctional polisialovoi acid can also be introduced into the reaction with the linker compound containing hydrazide fragment and N-maleimides fragment with the formation of a stable hydrazone containing active maleimide functionality, suitable for reaction with Tilney group.

An example of comparison of 2 - Fractionation of kolominova acid using ion exchange chromatography (SA, an increase of 22.7 kDa, polydispersity 1,34)

Example relative to the Oia 2.1 large-Scale fractionation

XK50 column (Amersham Biosciences, UK) were filled with 900 ml of Q Sepharose FF (Amersham Biosciences) and balanced 3 volumes of column wash buffer (20 mm triethanolamine; pH 7.4) at a flow rate of 50 ml/min CA (25 grams in 200 ml wash buffer) was loaded into the column at 50 ml / min via syringe entrance. Following this, the column was washed with 1.5 volumes (1350 ml) wash buffer.

Related SA was suirable 1.5 column volumes of different lucianic buffers (triethanolamine buffer 20 mm pH 7.4 with NaCl in the range from 0 mm to 475 mm in increments of 25 mm) and, finally, 1000 mm NaCl in the same buffer to remove residual and other residues (if any are present).

The samples were concentrated to 20 ml by ultrafiltration at high pressure through kDa membrane (Vivascience, UK). In these samples the buffer was replaced with deionized water using repeated ultrafiltration at 4°C. the Samples were analyzed on the average molecular weight and other parameters gel chromatography (GPC) as described in example 1E) and native electrophoresis acrylamide gel (stained with alcian blue).

Example 2.2 comparison

Fractionation on a smaller scale

The following samples were fractionally using identical wash water and gradients on a smaller scale (matrix up to 75 ml; 0.2 to 3 grams of kolominova acid):

alminova acid (SA, an increase of 22.7 kDa, polydispersity of 1.34; SA, 39 kDa, polydispersity of 1.4), colomina acid-aldehyde (CAO, an increase of 22.7 kDa, polydispersity 1,34), monofunctional colomina acid (CAORO, an increase of 22.7 kDa; polydispersity 1,34); Amin kolominova acid (SA-NH2, an increase of 22.7 kDa, polydispersity 1,34), maleimide kolominova acid (ITSELF, as for example 4 and a molecular weight of CA was monitored during the whole process).

Narrow fractions of SA obtained using the procedure described above, was oxidized with 10 mm of periodate and were analyzed by gel chromatography (GPC) and native PAGE to change the size of the polymer.

Results

Table 4< / br>
Ion-exchange chromatography SA 22,7:< / br>
zoom (matrix 75 ml, 3 g, SA)
Lucianne buffers (20 mm triethanolamine buffer + mm NaCl, pH 7.4)Molecular weight (M.W.)The polydispersity% share
325 mm125861,09177,4%
350 mm208841,0373,2%
375 mm255421,014 registered5,0%
400 mm284081,0244,4%
425 mm **7,4%
450 mm437601,0322,3%
475 mm429211,0960,2%

* not held

Clomidbuy acid and its derivatives (22,7 kDa) was successfully fractionally in a variety of narrow fractions with a polydispersity less than 1.1, with an average molecular mass of 46 kDa with different % stake. Figures 9 and 10; table 4 shows the results of separation 22,7 kDa substances in scale 75 ml. Fig.9 represents the result of the GPC and figure 10 is a native PAGE.

This method also allows a scale from 1 ml to 900 ml of the matrix is virtually identical to the profile fractionation for each of the scales (not all results shown).

Fractionation of larger polymer (SA, 39 kDa, polydispersity of 1.4) resulted in obtaining samples up to 90 kDa. This method can be successfully used for fractionation of even large quantities of polymer. 11 shows the results of native PAGE 3 submitted samples SA and fractions separated by ion exchange chromatography, analyzed in table 4. The results PAGE shows that the ion-exchange fractions have a narrow polydispersity. This is consistent with the GPC data shown in Fig that show the results for 3 of the factions, otdeleniia 22,7 kDa CA. The withholding amounts shown in table 5.

Table 5
SampleMolecular weightThe polydispersity
118727150161,25
227677250951,10
340950402791,02

22,7 kDa substances were separated on a large scale. When using the GPC analyzed fractions from ion exchange. Allocated the following fractions are shown in table 6.

All narrow fractions were successfully oxidized 10 mm periodate and samples taken at various stages of the method of obtaining and analyzed GPC and native PAGE showed no changes in molecular weight and polydispersity. Data for some of the samples shown in Fig.

2.3 Deposition of kolominova acid

Differential precipitation with ethanol was used to precipitate kolominova acids with different chain length.

Results

Differential precipitation with ethanol showed that smaller SA demanded more ethanol (EtOH). Broadly polydisperse polymers 22,7 kDa besieged 70% EtOH, giving an output >80% of the obtained polymer. The concentration of EtOH 80% was required on what I deposition > 80% with a lower molecular weight of 6.5 kDa (polydispersity <1,1). This method also removes any salt, contaminating the product.

2.4 Fractionation of kolominova acid filtering

Samples 22,7 kDa was purified by ultrafiltration on membranes with different limits cut-off (5, 10, 30, 50, and 100 kDa). In all cases, retentate were analyzed by GPC and native PAGE.

Results

Samples 22,7 kDa was purified by ultrafiltration on membranes with different limits clipping that showed a decrease in polydispersity polymer and a shift towards higher molecular weight with increasing limits cut-off membrane (Fig).

You can also use a combination of methods and chromatography ion pairs for the fractionation of polymers.

Example 5 - Synthesis of conjugates of growth hormone (GH)-kolominova acid (wide and narrow dispersion)

To obtain conjugates GH used oxidized clomidbuy acid (CAO, an increase of 22.7 kDa) and oxidized clomidbuy acid with a narrow dispersion (NCAO; an increase of 22.7 kDa, polydispersity = 1,09; of 40.9 kDa, polydispersity = 1,02)obtained in example 2.2 comparison.

Obtaining conjugates of growth hormone - kolominova acid

The growth hormone was dissolved in 0.15 M PBS (pH 7.4) and covalently linked with various kolominova acids (CAO and NCAO). Different SA (22,7 kDa, CAO; 22,7 CD is & of 40.9 kDa, NCA) separately added to GH (2 mg) in molar ratios of CA: GH (12,5:1), cyanoborohydride sodium was added to a final concentration of 4 mg/ml. of the Reaction mixture was tightly closed and stirred using a magnetic stirrer for 24 hours at 35±2°C. the Mixture is then precipitated with ammonium sulfate ((NH4)2SO4) by slowly adding salt with continuous stirring to achieve a 70% wt./about. saturation, stirred for 1 hour at 4°C, then centrifuged (5000·g) for 15 minutes, and the pellets re-suspended in a saturated solution (NH4)2SO4and again centrifuged for 15 minutes (5000·g). Selected precipitation was re-dissolved in 1 ml PBS pH 7.4 and subjected to active dialysis (24 hours) at 4°against the same buffer. Controls included the conjugation of a native protein in the presence of oxygenated SA or in the absence of SA. The shaking was minimized to avoid concomitant denaturation of the protein. Polysialylated GH was characterized by SDS-PAGE. Polysialylated GH was subjected to ion-exchange chromatography as described in example 2 comparison, and fractions of the product were subjected to SDS-PAGE.

Results

The results (Fig) show that in the control cell (GH) migration of the sample was the same as for fresh GH. In the bands conjugate is there are changes, that usually indicates an increase in mass, inherent politicaleconomy GH. Band width was significantly narrower in the case of conjugates usadepartment polymers compared to conjugates shirokogabaritnykh polymers. Moreover, GH conjugates (shirokolistvennymi polymer) were divided into different samples by anion-exchange chromatography (Fig).

Example 6 - Synthesis of conjugates of insulin-colomina acid

Activated policylevel acid (oxidized colomina acid (CAO)and monofunctional policylevel acid (oxidized restored oxidized colomina acid (CAORO))obtained in example 1 was used to obtain rh-insulin conjugates.

Obtaining conjugates of insulin-colomina acid

Insulin was dissolved in a minimum volume of 15 mm HCl, followed by dilution with 0.15 M PBS (pH 7.4) and covalently linked with various kolominova acids (SA, CAO and monofunctional CAORO). Clomidbuy acid (22,7 kDa) with insulin (2 mg) in molar ratios of CA:insulin (25:1) were introduced into a reaction within 48 hours in 0.15 M PBS (pH 7.4; 2 ml)containing cyanoborohydride sodium (4 mg/ml) in sealed vessels under stirring with a magnetic stirrer at 35±2°in the incubator. The mixture was then subjected to precipitation with ammonium sulfate ((NH4)2SO4by slow EXT the effect of salt with continuous stirring to achieve 70% wt./about. the saturation. The samples were stirred for 1 hour at 4°C, then centrifuged (5000·g) for 15 minutes and the pellets suspended in a saturated solution (NH4)2SO4and again centrifuged for 15 minutes (5000·g). The obtained precipitation was again dissolved in 1 ml of 0.15 M sodium phosphate buffer with the addition of 0.9% NaCl (pH 7.4; PBS) and subjected to extensive dialysis (24 hours) at 4°against the same PBS. The dialysates were then analyzed for content of sialic acid and protein and output conjugation expressed in terms of molar ratio CA:insulin (as in example 1). Controls included the conjugation of a native protein in the presence of oxygenated SA or in the absence of SA under the described conditions. The shaking was minimized to avoid concomitant denaturation of the protein. Policyrelevant insulin is additionally characterized by ion-exchange chromatography and SDS-PAGE. The results were expressed in terms of molar ratio CA:insulin in the resulting conjugates (table 7).

Table 7< / br>
The synthesis of compounds insulin (a protein) kolominova acid
The analyzed types CAReached the molar ratio during conjugation (SA:insulin)
Colomina acid (SA)of 0.7:1 (weakly reactive)
Oxidized colomina acid (CAO)1,60:1 (highly reactive)
Oxidized restored oxidized colomina acid (CAORO) (monofunctional)of 1.35:1 (newfound high reactivity)

From table 7 it is evident that if used natural oxygenated CA (in the presence of cyanoborohydride), observed a significant conjugation, but at a low level (the result of which was the molar ratio of CA:insulin 0,07:1) due to the reaction with polyacetylenes group SA on her pampering the end.

The formation of conjugates CA-insulin was further confirmed by joint precipitation of the two fragments adding (NH4)2SO4(SA itself is not precipitated in the presence of this salt). Proof conjugation also received ion exchange chromatography (IEC) and polyacrylamide gel electrophoresis (SDS-PAGE).

Example 7 - researchin vivo

Insulin and polysialylated insulin, obtained in example 6, were investigated for their ability to lower blood glucose in normal T/O outbred mice (body weight 22-24 grams). Animals were divided into groups of five, were injected subcutaneously (s.c.) insulin (0.3 units each mouse in 0.9% sodium chloride solution or the same equivalent protein in the form of policial the new insulin) and measured the level of glucose in blood samples at time intervals during use of the kit for determination of glucose (Accu-Chek Advantage, Roche, UK).

Results

The pharmacological activity polysialylated insulin components was compared with the activity of unmodified insulin in normal mice, which were injected the drug subcutaneously and the blood was collected at time intervals. Glucose levels in the blood of mice for 3 insulins shown in Fig. Points on the graph show the mean for 5 samples and error bars represent s.e.m. value. The results on Fig clearly show that polysialylated insulin (obtained by the original method (using CAO) and a new way of double oxidation (using monofunctional CAORO)) cause a more prolonged decrease of glucose levels in the blood. Thus, whereas glucose levels, reaching the lowest values over 0.75 hour, return to normal levels within two hours after injection of unmodified insulin, glucose levels in mice which were injected polysialylated protein, while also reached the lowest values over 0.75 hour, and returned to normal level after 6 hours. These results show that CAO-insulin and CAORO-insulin result in a noticeable and significantly longer retention time in the bloodstream compared to the unmodified insulin, leading to increase in the area under the curve, CPA is to the native insulin.

Literature

1. Bendele, A., Seely, J., Richey, C, Sennello, G., Shopp, G., Renal tubular vacuolation in animals treated with polyethylene-glycol conjugated proteins, Toxicological sciences, 42 (1998) 152-157.

2. Beranova, M., Wasserbauer, R., Vancurova, D., Stifter, M., Ocenaskova, J., Mora, M., Biomaterials, 11 (2000) 521-524.

3. Brocchini, S., Polymers in medicine: a game of chess. Drug Discovery Today, 8, (2003)111-112.

4. Cheng T, Wu, M., Wu,P., Chern, J, Roffer, SR., Accelerated clearance of polyethylene glycol modified proteins by anti-polyethylene glycol IgM. Bioconjugate chemistry, 10 (1999) 520-528.

5. Cho, J.W. and Troy, F. A., Polysialic acid engineering: Synthesis of polysialylated neoglycosphingolipid by using the polytransferase from neuroinvasiveE. coliK1, Proceedings of National Academic Sciences, USA, 91 (1994) 11427-11431.

6. Convers, C. D., Lejeune, L., Shum, K., Gilbert, C., Shorr, R.G.L, Physiological effect of polyethylene glycol conjugation on stroma-free bovine hemoglobin in the conscious dog after partial exchange transfusion, Artificial organ, 21(1997) 369-378.

7. Dyer, J.R., Use of periodate oxidation in biochemical analysis, Methods of Biochemical Analysis, 3 (1956) 111-152.

8. Fernandes, A.I.; Gregoriadis, G., Polysialylated asparaginase: preparation, activity and pharmacokinetics, Biochimica et Biophysica Acta, 1341 (1997)26-34.

9. Fernandes, A.I.; Gregoriadis, G., Synthesis, characterization and properties of polysialylated catalase, Biochimica et Biophysica Acta, 1293 (1996) 92-96.

10. Fernandes, A.I.; Gregoriadis, G., The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implications in its pharmacokinetics, International Journal of Pharmaceutics, 217 (2001) 215-224.

11. Fleury, P., Lange, J., Sur I oxydation des acides alcools et des sucres par I acid periodique, Comptes Rendus Academic Sciences, 195 (1932) 1395-1397.

12. Gregoriadis, G., Drug and vaccine delivery systems, in: PharmaTech, World Markets Research Centre Limited, London (2001) 172-176.

13. Gregoriadis, G., Fernandes, A., McCormack, B., Mital, M., Zhang, X, Polysialic acids: Potential for long circulating drug, protein, liposome and other microparticle constructs, in Gregoriadis, G and McCrmack, B (Eds), Targeting of Drugs, Stealth Therapeutic Systems, Plenum Press, New York (1998) 193-205.

14. Gregoriadis, G., Fernandes, A., Mital, M., McCormack, B., Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics, Cellular and Molecular Life Sciences, 57 (2000) 1964-1969.

15. Gregoriadis, G., McCormack, B., Wang, Z., Lifely, R., Polysialic acids: potential in drug delivery, FEBS Letters, 315 (1993) 271-276.

16. Hreczuk-Hirst, D., Jain, S., Genkin, D., Laing, P., Gregoriadis, G., Preparation and properties of polysialylated interferon-α-2b, AAPS Annual Meeting, 2002, Toronto, Canada, M1056.

17. Hunter, A. C, Moghimi, S. M., Therapeutic synthetic polymers: a game of Russian Roulette. Drug Discovery Today, 7 (2002) 998-1001.

18. Jain, S., Hirst, D. H., McCormack, B., Mital, M., Epenetos, A., Laing, P., Gregoriadis, G., Polysialylated insulin: synthesis, characterization and biological activity in vivo, Biochemica et. Biophysica Acta, 1622 (2003) 42-49.

19. Jain, S., Hirst, D. H., Laing, P., Gregoriadis, G., Polysialylation: The natural way to improve the stability and pharmacokinetics of protein and peptide drugs, Drug Delivery Systems and Sciences, 4(2) (2004) 3-9.

20. Jennings, H. J., Lugowski, C, Immunogenicity of groups A, B, and C meningococal polysaccharide tetanus toxoid conjugates, Journal of Immunology, 127(1981)1011-1018.

21. Lifely, R., Gilhert, A.S., Moreno, C.C., Sialic acid polysaccharide antigen ofNeisseria meningitidisandEscherichia coli:esterification between adjacent residues, Carbohydrate Research, 94 (1981) 193-203.

22. Mital, M., Polysialic acids: a role for optimization of peptide and protein therapeutics, Ph.D. Thesis, University of London, 2004.

23. Muflenhoff, M., Ectehardt, M., Gerardy-Schohn, R., Polysialic acid: three-dimensional structure, biosynthesis and function, Current opinions in Structural Biology, 8(1998)558-564.

24. Park, J.T., Johnson, M.J., A submicrodetermination of glucose, Journal of Biological Chemistry, 181 (1949) 149-151.

25. Roth, J., Rutishauser, U., Troy, F.A. (Eds.), Polysialic acid: from microbes to man, Birkhauser Verlag, Basel, Advances in Life Sciences, 1993.

26. Rutishauser, U., Polysialic acid as regulator of cell interactins in: R.U. Morgoles and R.K. Margalis (eds.), Neurobiology of Glycoconjugates, pp 367-382, Plenum Press, New York, 1989.

27. Shriner, R. L, Fuson, R.D.C., Curtin, D.Y., Morill, T.C., The Systematic Identification of Organic Compounds, 6thed., Wiley, New York, 1980.

28. Svennerholm L, Quantitative estimation of sialic acid II: A colorimetric resorcinol-hydrochloric acid method, Biochimca et Biophysica Acta, 24 (1957)604-611.

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39. Troy, F.A., Polysialylation: From bacteria to brain, Glycobiology, 2 (1992) 1-23.

1. The method of obtaining the aldehyde derivative of sialic acid in which the original substance containing a link sialic acid pampering at its terminal end and a terminal saccharide on the non-end, which contains a vicinal diol group, is subjected to successive stages

a) pre-selective oxidation of the vicinal diol group to aldehyde;

b) restore restored to the open ring of the reducing terminal of the level of sialic acid, whereby receive a vicinal diol group, and in which the aldehyde formed in stage a) is also reduced to a hydroxyl group that is not part of the vicinal diol group; and

c) selective oxidation of vicinal diol groups, obtained in stage b) to give the aldehyde group.

2. The method according to claim 1, in which the level of sialic acid on restoring the m terminal end attached to the adjacent link by means of its 8 carbon atoms, whereby in stage b) of 6,7-vicinal diol group is oxidized with the formation of aldehyde 7 carbon atom.

3. The method according to claim 1 or 2, in which sahariano link at the end of the non represents the level of sialic acid.

4. The method according to claim 1 or 2, in which the starting material is a di-, oligo - or polysaccharide.

5. The method according to claim 4, in which the polysaccharide is policylevel acid consisting substantially only of units of sialic acid.

6. The method according to claim 5, in which the polysaccharide contains at least 2, preferably at least 5, or more preferably at least 10, most preferably at least 50 units of sialic acid in the molecule.

7. The method according to any one of claims 1, 2, 5, or 6, in which the initial stage of the oxidation is carried out under such conditions that largely did not occur splitting in the middle part of the polysaccharide chain.

8. The method according to claim 7, in which the preliminary oxidation is carried out in aqueous solution in the presence of periodate at a concentration in the range from 1 mm to 1 M, at a pH in the range from 3 to 10, a temperature in the range from 0 to 60°With in a period of time ranging from 1 min to 48 hours

9. The method according to claims 1, 2, 5, 6, or 8, in which stage b) is carried out under such conditions that the lateral carboxyl groups recognize the aqueous substances were not recovered.

10. The method according to claim 9, in which stage b) is carried out in aqueous solution in the presence of borohydride at a concentration in the range from 1 μm to 0.1 M, at a pH in the range from 6.5 to 10, a temperature in the range from 0 to 60°With in a period of time ranging from 1 min to 48 hours

11. The method of obtaining the aldehyde derivative of sialic acid in which the original substance containing a link sialic acid pampering at its terminal end and a terminal saccharide on the non-end, which contains a vicinal diol group, is subjected to successive stages

a) pre-selective oxidation of the vicinal diol group to aldehyde;

b) restore restored to the open ring of the reducing terminal of the level of sialic acid, whereby receive a vicinal diol group, and in which the aldehyde formed in stage a) is also reduced to a hydroxyl group that is not part of the vicinal diol group; and

C) selective oxidation of vicinal diol groups, obtained in stage b) to give the aldehyde groups and then injected into the reaction with the substrate having a primary amino group or a hydrazide group selected from a protein or peptide, followed, if necessary, recovering the resulting product.

Cab according to claim 11, in which the substrate is a therapeutic peptide.

13. The connection, which is an aldehyde derivative of di-, oligo - or polysaccharide having a General formula I

where R represents a-CH(Cho)CH2OH, -CH2SNO, GlyO is a link sialic acid; R3represents H; R4is a HE; p is 2 or more.

14. The connection, which is a conjugate of the aldehyde derivative of di-, oligo - or polysaccharide having a General formula I

where R represents a-CH(CH2Other1)CH2OH, -CH(CH2NHNHR1)CH2OH, -CH(CH=NNHR1)CH2OH, -CH2CH2Other1, -CH2CH=N-other1, -CH2CH2NHNHR1where R1is a polypeptide or protein; GlyO is a link sialic acid; R3represents H; R4is a HE; p is 2 or more.

15. The connection of item 13 or 14, which is a polysaccharide in which substantially all sacharine links are sialic acid associated 2-8, 2-9, or alternating 2-8/2-9 with each other.

16. The connection 15, which contains at least 2, preferably at least 5, or more preferably is at least 10, most preferably at least 50 units of sialic acid in the polysaccharide chain.

17. The compound according to any one of item 13 or 16, in which R represents a

.

18. The compound according to any one of 14 or 16, in which R represents a

.

19. Connection p, in which R1is a peptide or protein therapeutic agent, preferably an antibody or fragment.

20. Pharmaceutical composition having the ability a long time to stay in the bloodstream, including the connection to item 16 or 19 and a pharmaceutically acceptable excipient.



 

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