Covalent modification of proteins by polyethylene glycols


 

(57) Abstract:

The invention relates to biotechnology and medicine. Compounds having the General formula R1- X - R2where X - ones polymeric spacer, and R1and R2- receptor antagonist interleukin-1 or TNF, produced by the interaction of R1and R2containing or modified so that they contain reactive cysteine, ones with a spacer to be able to form thioester linkages. Mutiny receive recombinant means. The compounds can reduce the rate of excretion of therapeutic agent. 12 C. and 24 C.p. f-crystals, 24 ill., table 4.

The invention relates to polypeptides that are covalently linked to long-chain polymers, such as methoxypolyethyleneglycol. This invention also describes methods and reagents for the interaction of activated polymer molecules with various biologically important peptides.

Discovered that many proteins that have been identified and isolated from humans and animals, have promising medicinal and therapeutic opportunities. Much progress has been made in the methods Ident pure form and in relatively large quantities. As methods of using such potentially valuable substances, there are a lot of obstacles to obtaining these compounds in a form suitable for use in clinical models.

For example, it was found that many of these proteins have only a short period of half-life from blood serum. Mostly proteins derived from serum through the kidneys. The systematic introduction of relatively large amounts of protein, especially alien to man, may cause immunogenic reactions, which can lead to problems such as rapid excretion of protein from the body through the formation of immune complexes. In the case of other proteins problems of solubility and aggregation also interfere with the optimal dose of protein.

One of the most promising ways of solving these problems is the covalent addition of one or more chains of inert polymer to the desired polypeptide. The most commonly used polymer is polyethylene glycol (PEG) or monomethoxypolyethylene (MPEG) /1/. PEG is ideal for these purposes because of its proven nontoxicity. Other researchers use for such purposes is the modification of proteins with polyethylene glycol ("tahilramani") resulted in a protein of the desired characteristics. For example, it was shown that pegylation of IL-2 reduces the excretion of IL-2 without affecting significantly the activity of the cytokine. Reduced excretion leads to increased efficiency compared with non-pegylated substance /3/.

Increasing the half-life of the peroxide dismutase (PD) of the serum was a decisive obstacle to the use of PD for the treatment of various symptoms. A large number of studies have shown that tahilramani PD will reduce the speed of removing /4/.

Aggregation of immunoglobulin G (IgG) is the factor that leads to serious side effects in patients who were administered intravenous IgG. It was shown that tahilramani IgG reduces aggregation of proteins, removing this problem /5/.

It was also shown through tahilramani affect the immunogenicity of the protein. Obuchowski with staff studied the immunogenicity and the period of removal from the blood pegylated catalase bovine liver /6/.

Attach the PEG groups to these various proteins slows down the excretion by increasing the size of the molecule is pegylated protein. Up to a certain size, the rate of glomerular filtration of proteins back about Avila, not how many PEG groups joined to the protein, and the total molecular weight of the modified protein. This is confirmed by studies of inference process, which varied as the size of the lateral PEG-chains, and the number of PEG chains associated with IL-2. /3/.

Numerous studies of the removal processes, immunogenicity, aggregation and physical properties of pegylated proteins indicate that the PEG forms a flexible hydrophilic shell around the protein. Chain PEG become highly hydrated and give pegylated proteins with a higher apparent molecular weight than expected, and shield the charges on the protein.

Due to numerous encouraging results that have been obtained in this area, created a directory of methods of attaching PEG-units to the polypeptides. A key element of these methods is to "activate" the terminal OH-groups of the glycol. This activation is necessary in order to form the connection between the PEG group and the polypeptide. In the vast majority of such techniques activate PEG-component in order to make possible reaction with free primary amino groups of polypeptides. Most of these Svoboda-chains. For example, it was found that to suppress the immunogenicity, it is desirable to use from 15 to 50 moles of polymer per mole of polypeptide /7/.

Due to the fact that each polypeptide is usually attached many PEG-chains, and because each protein usually present a large number of lysine residues, proteins easily PageLayout with homogeneous reaction products /8/. This lack of specificity of the reaction leads to numerous complications. Among them is that tahilramani often leads to a significant loss of activity of the protein. Presumably, joining relevant to the lysine residue could change the active center of the protein, making it inactive.

At least one system it was shown that tahilramani can lead to the occurrence of steric hindrance at the active centers. In other words, a relatively small substrates can approach the protein, while the activity of proteins that react with large substrates, can dramatically affect the chaotic tahilramani /7/. Selective tahilramani active sites of these proteins could lead to modified substances, when acquiring tahilramani Zelichenko use, the mixture of diverse proteins, which occurs when the conduct of non-specific tahilramani, leads to difficulties in obtaining product with reproducible and measurable properties. This makes it extremely difficult treatment assessment and obtaining information about the dosage and effectiveness.

It was found that in some cases the introduction of multidimensional complexes that contain more than one biologically active polypeptide or medication, can lead to positive synergistic effects. For example, a complex containing two identical related polypeptide, may have substantially greater affinity for the ligand or active center, which he relates, compared with one-dimensional polypeptide. For this reason, it is desirable to have multidimensional protein complexes to increase the affinity of the protein for this ligand in addition to increasing the molecular weight of the complex.

Proteins often exhibit their biological effects through interaction with other proteins. In cases where quite simple complex of two proteins, in order to achieve a biological effect, it was possible to simulate the physiological effects of endogenous the KSA, containing more than two protein to reproduce the function of endogenous proteins using recombinante received exogenous equivalents is more difficult due to the fact that complexes of higher order are often unstable. In such cases, it may be advantageous to use custom made varieties containing two components of the complex to simulate biologically active complex.

After the described invention, at least three research groups reported receiving crosslinked proteins in which the extracellular portion of one of the TNF receptors attached to the heavy chain of immunoglobulin IgG human or mouse, which are then sewn together by disulfide bonds /9/. In each case, these proteins are expressed in an expressive systems of animal cells and found that they are significantly more effective in the inhibition of TNF than just one-dimensional soluble receptor. Similar methods were used to obtain similar crosslinked proteins of the CD4 protein /10/, CR1 protein /11/ and CR2 protein /12/.

It is shown that these cross-linked proteins, constructed from two polypeptide chains and part of the antibody immunoglobulin IgG, are promising is the speed of removing the complex from the body, in addition to the obvious increased affinity of proteins to their ligands. However, proteins are made this way so far been obtained only by the expression of expressive systems of the animal cell expression of the fused gene. It was necessary that the portion of the protein, which is IgG, was properly Packed after expression. In addition, the fixed heavy chain portion of the IgG antibody, which serves as the link - spacer or linker between the polypeptide units, it is not possible to change the length, size and geometry of the spacer. When this apparent synergistic effect achieved by two-dimensional protein by changing the spatial orientation of the polypeptides of the benefits of a synergistic effect can probably be optimized. And, finally, cross-linked proteins can be antigenic and/or have low solubility. Heavy chain antibodies is not biologically inert.

Described other two-dimensional or "bivalent" complexes. One such two-dimensional group of compounds called "Virology". These compounds include very short polypeptide units, which are connected by short polyglycidyl a spacer or linker. One of the polypeptide denizleasing of 65 amino acids - and the other is an inhibitor of recognition anion-binding associte (ESA) /13/.

C-reactive protein (CRP) is a protein ostrofsky serum, built from five 23 to a sub-units. PSA can cause precipitation reaction and agglutination, and can also interact with Clg for activation of the classical pathway of complement.

Using bis(sulfosuccinimidyl)substrate or 3.3-dithio(sulfosuccinimidyl) and as cross-linking agents, the obtained cross-linked oligomers PSA /14/.

We have also studied the formation of two bivalent ligands for conjugation opioid receptors. Ones pharmacophore - naltexone or Oxymorphone were connected by a short ethylenoxide or the glycine spacers /15/. Tetrapeptide enkephalins, United short methylene bridges were designed for conjugation of opioid receptors and has been shown to have more selectivity and affinity for Delta receptors than the original Delta ligand /16/.

By stitching through the remainder of the sugars was obtained in multidimensional forms of the glycoprotein cell surface CD4. As a cross-linking agent used bismaleimide what about the common glycoprotein cell surface, which is a ligand for the T lymphocyte CD2. The LFA Dep.-3 with their associated ligands forms a protein micelles of the eight monomers, which increased their ability to interact with cells on which surface is located CD2 /18/.

Using relatively similar technique, one group studied the inhibitory effect of a synthetic polypeptide, which included repeated pentapeptidnogo unit. The polymer was synthesized by polymerization in the presence of diphenylphosphinite to reach about 10,000 daltons. This polymerized Pentapeptide is one of the most important structures in several biological reactions /19/.

A further obstacle to the development of an effective exogenous proteins, which would increase or competed with endogenous substances, is that exogenous proteins should preferably be introduced systematically, than to be localized in the appropriate place. This can lead to a lower efficiency and increase side effects. Several research groups have reported getting bioactive proteins into the corresponding sites by linking them with other proteins that natural about the future of proteins.

After the appearance of the present invention, it was reported that to create linked superantigens antibodies were used spacers or linkers of polyethylene glycol. Monoclonal antigen reactive against cell carcinoma of the rectum, was attached to staphylococcal enterotoxin bacterial superantigen. These complexes are not likely to use benefits associated with other bivalent complexes (for example, higher molecular weight, synergistic effects of bivalents), and for the conjugation of certain areas superantigens. Described for the formation of these related superantigens how tahilramani gives the complex, which contains a wide mixture of substances. The combination of this antibody and this superantigen was carried out using Succinimidyl 3-(2-pyridyldithio) propionate and hydrophilic spacer PEG-based chain of 24 atoms. In accordance with this method, each antibody were added from 7 to 18 connecting elements, and each of superantigens reacted with one or two lysine /20/. Using this method, it would be impossible to distinguish one type of reaction product in order of optimzie a wide variety of medical indications, are inhibitors of factor necrophila (TNF) and receptor antagonists of interleukin-1 (IL-1ra). It is shown that these substances have a positive effect in the treatment of diseases induced by TNF and IL-1, respectively. Among the indications identified as belonging to caused by either TNF or IL-1, be the syndrome of respiratory disorders in adults, pulmonary fibrosis, rheumatoid arthritis, inflammatory bowel and septic shock.

Pending application, to which reference is here /21/, describes the class of naturally occurring protein inhibitors of TNF and the method of obtaining them in significant quantities with a high degree of purity. In particular, in the above-mentioned application are described in detail two subseries of TNF inhibitors, designated as 30 kDa inhibitor of TNF and 40 kDa inhibitor of TNF. In addition to having a full length of the chain protein of 40 kDa inhibitor of TNF, also received two truncated but biologically active form 40 kDa inhibitor of TNF. These proteins, which have the full chain length of the protein were removed 51 and 53 amino acids with terminal carboxyl groups, denoted respectively as 40 kDa-TNF 51 and 40 kDa inhibitor of TNF 53.

Nah adnych protein inhibitors of IL-1 and the method of obtaining them in significant quantities with a high degree of purity. In particular, in the application described three such inhibitor of interleukin-1, which are receptor antagonists of interleukin-1 (IL-1ra's), namely, IL-1ra , IL-1ra and IL-1rax.

Two additional classes of substances that are potentially useful for treatment of various medical conditions, are inhibitors of interleukin-2 and inhibitors of complement. Potential inhibitors of interleukin-2 include receptors for interleukin-2 extracellular domain of the receptor of interleukin-2 receptor antagonists of interleukin-2, antibodies that recognize interleukin-2, and fragments of any of these substances, which contain binding function of IL-2. Potential inhibitors of the complement system include receptor CR1, the extracellular domain of R1 and CR1 fragment, which contains the function of complement fixation.

In /23/ described receptor of interleukin-2 and methods of its allocation. Also described gene encoding the receptor for interleukin-2 and methods for recombinant obtain /24/.

You can assume that to a certain extent soluble extracellular domain of any receptor of interleukin-2 will act as an inhibitor of the action of interleukin-2 cytokine. Interleukin-2 is one of the most Aharon the growth of T lymphocytes. It was also shown that Il-2 acts on a variety of other cells in the immune system.

There are three discrete forms of the receptor for interleukin-2, which includes two different receptor molecules, indicated or as IL-2r or IL-2r .

The receptor for IL-2 with the strongest affinity consists of two different receptors of IL-2. Both of these receptors have been cloned and characterized. The receptor for IL-2 with low affinity (IL-2r ) was cloned in 1984 and was described in detail /25/. The extracellular domain of this molecule had a molecular weight 24825 and had two N - linked glycosylation site. This molecule contains 11 cysteine, 10 of which are involved in intramolecular disulfide linkages. Alleged lomani bind IL-2 with this molecule was plotted on a map using matogenesis and epitope mapping. The receptor for IL-2 with intermediate affinity (IL-2r ) was cloned in 1989 and was not as fully characterized as IL-2r /26/. The extracellular domain of IL-2r has a molecular mass of 24693. The molecule contains 8 cysteines and 4 N-linked glycosylation site. The formation of disulfide bonds in this molecule is unknown. IL-2r is the cytoplasmic domain of 286 amino acids.

For IL-2 a IL-2r and 10-11M for the receptor with high affinity, which consists of a complex of IL-2r , IL-2r and IL-2. Existing models suggest that the formation of the complex with high affinity proceeds first by adding IL-2 to IL-2r , and then to IL-2r /27/.

The inhibitor of IL-2 may be valuable in the prevention of transplant rejection, and autoimmune diseases. Currently, monoclonal antibody that inhibits the binding of IL-2, experience with renal transplantation in man /28/. In one study of 15 patients it was shown that this antibody in combination with immunosuppressants, is equally effective in preventing rejection allograft, as in the control group treated with higher doses of immunosuppressants. In many diseases, some infections, as well as in transplantation and rejection of detected high blood levels of soluble IL-2r . This suggests about the participation of IL-2 in these diseases.

CRI is a protein, also called C3b/C4b receptor. CRI is present on erythrocytes and other cell types and specifically binds C3b, C4b and iC3b. CRI may also inhibit the classical and alternative pathway C3/C5 konvertieren circuit, and he essentially four allotypic forms. It is known that CRI contains repetitive coding sequences, and this fact is used to explain the existence of numerous allotypes /30/.

Reduced expression of CRI on erythrocytes was associated with lupus erythematosus, and it was also found that the number of CRI inversely proportional to the content of serum immune complexes. Himself CRI protein, CRI gene and methods of obtaining CRI described in /31/. As described above, were also described two-dimensional formations, containing CRI and the domains of the antibody /32/.

This invention relates to a method of modification of polypeptides and the resulting modified polypeptides.

The invention comprises essentially pure compounds covered by the formula, R1-X-R2in which R1and R2represent a biologically active group, and X is the ones polymeric spacer. R1and R2may be the same or different groups, and at least one of them, R1or R2is a peptide. In a preferred practical implementations of R1and R2represent or receptor antagonist interleukin-1; or 30 kDa of inhibi the p glycerol, or dextran, or acid rectum, or poly-amino acid, or carbohydrate polymers. Also includes pharmaceutical compositions containing essentially pure compounds in a mixture with a pharmaceutically acceptable carrier. Further included the treatment of patients in need of this, such therapeutic compositions. The compounds of formula R1-X-R2depicted on Fig. 19, hereinafter referred to as "dumbbells".

This invention also includes a method of obtaining essentially pure therapeutically valuable compounds covered by the formula, R1-X-R2that includes the interaction of the ones polymeric group having at least two reactive groups capable of forming covalent bonds with a biologically active group R; and the allocation of the specified product.

In another implementation, this invention includes a method of obtaining essentially pure therapeutically valuable compounds covered by the formula, R1-X-R2where R1and R2different, which is: the reactions nepeptidnoi polymer group, capable of forming a covalent bond upon reacting with a biologically active Krupki active group, R2with obtaining the specified connection; and the separation and purification of the specified connection.

In one implementation, this invention relates to site-specific tahilramani of TNF inhibitor and an inhibitor of IL-1,

To ensure the site-specificity of tahilramani, choose such philonous reagents that will react almost exclusively with free SH-groups of cysteine residues in polypeptides. An example of a reagent tahilramani, which is almost exclusively associated SH-groups of cysteine, is O-(2-maleimidomethyl)-O'-metropolitanpoker.

Site-specific tahilramani can either be natural "free" cysteine residues of a given polypeptide, either free cysteine contained on muteing natural polypeptides. Cysteine can be added or injected into the amino acid sequence of the natural polypeptide, and replace them with other amino acid residues in the selected location.

In one practical implementation of this invention, the polypeptides, which are subject to paglinawan get through recombinant DNA technology from a bacterial host cell. In most cases, the bacteria is biologicheskuyu activity. In some examples of the present invention the native polypeptide contains no free cysteine residues, but then get the modified polypeptide containing at least one free cysteine in a biologically active polypeptide. According to this method, the re-laying of bacterial expressed polypeptide facilitate accession, in turn, sulfhydryl containing such a compound as cysteine and disulfide containing such a compound as cysteine. After re-packing and cleaning this polypeptide handle a limited number of mild reducing agent such as dithiothreitol ("DTT"), to regenerate the sulfhydryl group of the new cysteine residue of the modified polypeptide. After dialysis in conditions calculated to prevent oxidation, the polypeptide can result in interaction with the agent specific tahilramani cysteine to site-specifically to obtain covalently modified polypeptide.

Preferred pegylated polypeptides of the present invention are site-specifically pegylated TNF inhibitors and inhibitors of IL-1. More specifically, the invention describes p is the new TNF inhibitors include 30 kDa inhibitor of TNF, in which the residue is aspartic amino acid at position 105, the native human protein is replaced by cysteine, using in vitro mutagenesis, and tahilramani occurs on the free cysteine at position 105. Other pegylated derivatives mutated 30 kDa of inhibitorof TNF include mutations, in which the cysteine was attached at positions 1, 14, 111 and 161. Except once pegylated Malinov, any and all combinations of different mutations can be included in one mutein to obtain modified 30 and TNF with more than one free cysteinemia balance, capable of tahilramani.

The most preferred pegylated IL-1ra includes natural or native IL-1ra, which contains four free cysteine. Monopegylated native IL-1ra lead to site-specific tahilramani when the cysteine at position 116. Other pegylated derivatives mutated IL-1r include mutiny with cysteine attached to the amino end of the polypeptide, cysteine, attached in positions 6, 8, 9, 84 or 141, and the substitution of cysteine at position 116 serine. Except once pegylated Malinov, any and all combinations of different mutations can be used in order to obtain izmenen the present invention will be apparent upon consideration of the subsequent detailed description of the invention, includes illustrative examples of the practical application of the invention.

Fig. 1 represents the amino acid sequence of native IL-1ra.

Fig. 2 represents the sequence of native 30 kDa inhibitor of TNF.

Fig. 3 represents the result of electrophoresis naegeliana and pegylated forms of IL-1ra and maleinovogo c84s116 IL-1ra in the system LTO-PAG )staining of Kumasi). Tracks 2, 3, 5, and 6 contain the reaction mixture tahilramani. Lanes 1 and 4 represent unmodified proteins:

Track 1 - IL-1ra

Track 2 - MPEG5000*IL-1ra

Track 3 - MPEG8500*IL-1ra

Track 4 - c84s116 IL-1ra

Track 5 - MPEG5000c84s116 IL-1ra

Track 6 - MPEG8500c84s116 IL-1ra

Fig. 4 represents the results of ion-exchange chromatography sorbent mono S: chromatogram A is the reaction mixture of tahilramani in obtaining MPEG5000*IL-1ra, peak 1 refers to modified, and the peak 2 - remotefilename IL-1ra; chromatogram B is cleared, MPEG5000*IL-1ra.

Fig. 5 depicts a chromatogram obtained by HPLC method, showing the elution profile of several substances - standards sizes, and MPEG8500*IL-1ra (Foy HPLC products tripticase splitting the alkylated MPEG5000*IL-1ra treated with tritium-labeled iodixanol acid in order to place free cysteine. Separation was carried out on a column however C8 (2.1 x 220 mm) at room temperature, and flow rate of 1000 μl/min with a linear gradient. Solvent A consisted of 0.1% solution of TFOC (triperoxonane acid) in water and solvent B - 0,085% solution of TFWC in a mixture of 80% acetonitrile and 20% water.

Fig. 7 represents the results of the fractionation method of reversed-phase HPLC chymotryptic cleavage of peptide 18 in Fig. 6. Conditions identical to the conditions for Fig 6. Peptides 5 and 8 contain tritium label, and peptide 5 has the amino acid sequence LCTAMEADQPVSL. Cysteine was identified as carboxymethylcysteine derived. This cycle was the only one who discovered radioactivity above the background level. Amino acid sequence of peptide 8 started with serine 103 IL-1ra. Re cleavage of this peptide with chymotrypsin allowed to eliminate all of tritium label from the polypeptide.

Fig. 8 represents the profile changes in the concentration of plasma IL-1ra in time for Mature IL-1ra, pegylated IL-1ra and several pegylated Malinov IL-1ra.

Fig. 10 represents the curves of changes of intravenous plasma concentration of IL-1ra in time for a number of samples once pegylated IL-1ra, twice pegylated IL-1ra and hoteleobrotowy varieties of IL-1ra PEG.

Fig. 11 represents the curves of changes of subcutaneous concentrations of plasma IL-1ra in time for the same number of connections, IL-1ra, as in Fig. 10.

Fig. 12 shows the plasma IL-G time after the introduction of the mouse TNF.

Fig. 13 is a comparison of the contents of IL-6 induced in the mouse with five ratios 105s30 kDa inhibitor of TNF to TNF (A) and five ratios connection 105s30 kDa inhibitor of TNF PEG2000(dumbbell) to TNF (B).

Fig. 14 represents the content of plasma IL-6 induced in mice one TNF and mixtures 1: 1 with TNF handleoversize compounds 105s30 kDa inhibitor of TNF PEG3500and PEG10000.

Fig. 15 represents the percentage of neutrophils induced for different ratios of TNF to 105s30 kDa to TNF (A); 105s30 30 kDa to TNF PEG3500(dumbbell) (B); 105s30 kDa to TNF PEG10000(dumbbell) (C); and 105s30 kDa to TNF PEG20000(dumbbell) (D).

Fig. 16 represents a dependency changes, the 105s30 kDa inhibitor of TNF PEG8500and PEG10000and hoteleobrotowy structures 30 kDa inhibitor of TNF PEG3500, PEG10000and PEG20000.

Fig. 17 represents the dependence of the changes of subcutaneous plasma concentration of 30 kDa inhibitor of TNF from time to time for the same number of connections 30 kDa inhibitor of TNF as in Fig. 16.

Fig. 18 represents the solubility for the three solutions of native IL-1ra and s IL-1ra PEG8500the change in optical density at 450 nm in time.

Fig. 19 represents the basic structure of compounds in accordance with the present invention, having the General formula R1-X-R2that is called connection weights.

This invention involves the selective modification of pharmaceutically useful polypeptides, in particular, inhibitors of factor necrophila ("TNF") and inhibitors of interleukin-1 ("IL-1"). More specifically, this invention describes a selective modification of the 30 kDa of TNF inhibitor and receptor antagonist IL-1 ("IL-1ra") Selective modification serve both to enhance the pharmacokinetic properties of the polypeptides, and to obtain homogeneous compositions for therapeutic use in application to man.

Additional polypeptides that could is. the CE links to the receptor of interleukin-2 should be understood so that they include both the-and - chain of IL-2r, if not explicitly and separately stipulated.

In a preferred practical implementations of the present invention modified polypeptides and DNA sequences are human. However, to the extent that there is sufficient homology between the DNA and peptide sequences of animals and the same form of person, it can be included in the scope of this invention.

In one specific implementation, the modification according to the present invention includes site-specific covalent binding of long-chain polymers with interest polypeptides. The selected polypeptides can be native or interest in natural polypeptides, or they may represent a biologically active mutiny polypeptides, which were obtained with the aim to strengthen the modification process described here. This method according to the present invention includes the selection, acquisition and selection of the desired Malinov that meet the purposes of the present invention. In other practical implementations of this invention, this method for modification of a polyp which was an essentially pure form, as that term is defined in the invention.

In some practical implementations of the invention, the modified polypeptides in accordance with the present invention associated with long-chain polymers at specific points in amino acid sequence. Such modified polypeptides in accordance with the present invention retain a significant part of their biological activity. In a preferred practical implementations of the modified polypeptides retain at least one-tenth of the biological activity of the native polypeptide when tested for binding to the receptor. In the preferred specific implementation, the modified polypeptide will retain at least one-fifth of the biological activity of the native polypeptide, and in the preferred specific implementation will retain at least one-fourth of the activity. In addition, the modified polypeptide will serve to improve the pharmacokinetic characteristics of the native polypeptide in at least one of the following areas:

1) increase the apparent molecular mass of the native polypeptide and, therefore, the reduction in the rate of excretion after subcutaneous or decrease the antigenicity of the native polypeptide.

In many practical implementations of the present invention achieves each of these goals. In a preferred practical implementations of this invention, the long chain polymer is a polyethylene glycol or monometoksipolietilenglikolya. Structural unit of polyethylene glycol designated here as PEG, and a structural unit monometoksipolietilenglikolya - as MPEG. The approximate molecular mass of these structural units indicated about their signs down. For example, the unit monometoksipolietilenglikolya with an approximate molecular weight of 5000 will be designated as MPEG5000or PEG5000. Other long-chain polymers that are included in the scope of this invention are polypropylenglycol ("BCP"), polyoxyethylene glycerol (PEG), dextran, intestinal acid or other polymers based on carbohydrates, and polymers of amino acids and derivatives of Biotin.

In another practical implementation of the present invention, the unit of donniebrasco polymer is dihydroxyphenylglycol, or HO-(CH2CH2O)n-H. Being activated for subsequent covalent joining the polypeptides or other biologically AK is x2">

In a preferred practical implementations of the present invention, long-chain polymer units linked to the polypeptide via covalent joining of sulfhydryl group (-SH) of cysteine residue. To achieve the selectivity of the reaction and the homogeneity of the reaction mixture, it is useful to use functional polymeric units that will react specifically with sulfhydryl groups. Functional or reactive group that is connected with long-chain polymer, denoted here as the activating group. Activating groups include maleimido group, sulfhydryl group, thiol, triflate, TResult, aziridine, oxirane and 5-pyridyl. Preferred activating groups are maleimide.

Activated dihydroxyphenylglycol because of the physical distance between the ends of the polymer chain are almost equally reactive at each end of the molecule. With appropriate choice of reaction conditions and polypeptides, activated dihydroxyphenylglycol or any other mnogokriterialnaya unit long-chain polymer will react with polypeptides with education hoteleobrotowy complexes, in which two of the polyp is discovered in the speed of reaction between the activated polymer linked group and various containstyrosine polypeptides, and features of the kinetics of these reactions, experimental research it is easy to get handleoversize complexes, which essentially pure compounds can be formed by the inclusion of two different polypeptide groups, or by inclusion of one polypeptide of the group and a great biologically active group. Examples of such hetero-dumbbell compounds are given below.

Limits and availability for reaction cysteine changes dramatically from polypeptide to the polypeptide. Therefore, in biologically active form many polypeptides are not "free" cysteine, or cysteine not associated with another cysteine. Moreover, the existence of "free" cysteine does not mean that cysteine available for bonding with the reactive reagents. Since the modification is usually carried out on an active or a three-dimensional Packed the polypeptide, the reaction is slightly or not at all in those cases, when the free cysteine is inside the folded structure. A further limitation in the modification of polypeptides is the potential impact that the modification may have on the active site of the polypeptide. Modification of cysteine, the relatively close proximity of the active site is if not impossible, to accurately predict what cysteine residues can be effectively modified.

The same factors also exist, when you get mutated polypeptides containing an additional cysteine residues. When such a polypeptide recombinante get through bacterial expression, negativnye proteins can interfere with the proper packing of the polypeptide. In addition, the cysteine must be available for agent tahilramani, and pegylated cysteine should not substantially interfere with the active site of the polypeptide.

The choice in this polypeptide potential sites for the introduction of ranatunga cysteine can be influenced based on information from various sources. For example, a good place to mutations through the inclusion of a free cysteine can be the sites of glycosylation. In this degree, which includes information about the binding or active site of the polypeptide, it can also be used to identify potential Mutanov. Connection or replacement of a cysteine residue at the amino or carboxyl end of the polypeptide may also be promising because of its location. And, finally, can be considered a mutation of the lysine ostad what about the polypeptide.

Although for any given polypeptide can choose a lot of potential Mutanov, which can satisfy the desired characteristics, only after synthesis, tahilramani and testing these modified Mutanov will know which one will meet the purposes of the present invention. In the light of the present invention and with the experience and knowledge of professionals, such synthesis, tahilramani and testing can be carried out without undue experimentation. It should be noted that even if tahilramani polypeptide causes a decrease, to some extent, the biological activity of the polypeptide, the improvement of the pharmacokinetic action of this polypeptide can significantly increase the value of the native polypeptide in various therapeutic applications.

When choosing Malinov target the preferred way to obtain these Mutanov is recombinant ekspressirovannoj gene encoding for mutina. Assuming that the gene coding for the native polypeptide is known, the gene can create or standard methods of site-specific mutagenesis of the native gene, or building altered gene by standard methods of gene synthesis. This those who xpression in many expressive systems, including animals, insects and bacteria. Although such systems were tested for the expression of native polypeptides, to a certain extent, the same system can be used to Malinov target. In the preferred conversion of the implementation of the present invention, genes encoding for Malinov target obtained by site-specific mutagenesis of the native gene, and the gene encoding this mutein, downregulation of bacterial expressive system. The gene encoding native IL-1ra, and the way to ekspressirovali specified gene in E. Coli is described in detail in /33/.

The gene encoding native 30 kDa inhibitor of TNF, and the way ekspressirovali specified gene in E. Coli is described in detail in the application /34/. Each of the applications included here with this link,

Mutiny and pegylated substances according to the present invention include allelic variations in protein sequence variations sequence due to natural variability from individual to individual) and essentially equivalent proteins. "Essentially equivalent", as that term is used in the description and the claims, denotes that mean the presence of a very high degree of homology of aminomalonate. The scope of this invention also includes mutiny and pegylated polypeptides, which are partially truncated version of the native polypeptides.

In one preferred specific implementation of the method of the present invention in which mutein target is produced by recombinant DNA technology in bacterial expressive system, perform the following steps:

1) Encoding gene for muteena target create by site-directed mutagenesis of the coding gene for the native polypeptide;

2) Encoding the gene for muteena target Express in bacterial expressive system;

3) is Separated from the bacteria and purified mutein target;

4) Mutein target is subjected to re-laying in the presence of cysteine or other sulfhydryl-containing group connection;

5) Re-laid mutein target isolate and purify;

6) Cleaned and re-laid mutein target handle a mild reducing agent;

7), the Reaction mixture was subjected to dialysis in the absence of oxygen; and

8) Detalizirovannoi the reaction mixture is treated with long-chain polymer containing an activating group.

In the preferred concretejob the dithiothreitol ("DTT"). In another specific implementation, the modification may occur before re-laying the expressed protein or mutein.

In the preferred specific implementation of the present invention pegylated mutiny and pegylated native polypeptides can be purified and introduced into pharmaceutical compositions in the usual ways. In another implementation, peeled mutiny you can also type in pharmaceutical compositions.

Pegylated polypeptides in accordance with the present invention formed by the reaction deactivated long-chain polymer, have additional useful properties. These handleoversize molecule may contain two interest polypeptide associated one polymer segment. This structure gives a certain degree of linearity of the polymer molecule and reduces the number of steric hindrance inherent in the use of such large hydrophilic polymers like polyethylene glycol. Achieving the goal of obtaining molecules with increased apparent molecular weight while maintaining high biological activity. In the scope of the present invention is particularly included bidentate molecules, where two of polypeptide associated with a single polymer chain, i.e. one bidentate molecule consists of two parts: an inhibitor of TNF and IL-1ra-part.

Native IL-1ra (Fig. 1) and numerous mutiny IL-1ra was periliously according to the present invention. Tahilramani wild type IL-1ra free sulfhydryl groups by the methods described below in the examples, leads to the takeover of MPEG to the cysteine residue at position 116 of IL-1ra (s). The other three cysteine in a fully native molecule is not available for tahilramani. In order to attach molecules of MPEG to different sites of IL-1ra and get the products you attach MPEG containing more than one molecule of MPEG, on the terminal amino groups of the protein added IL-1ra, in which the native amino acids in IL-1ra were replaced by cysteine, or additional cysteine. In order to obtain the addition products in which the residue 116 is not pagalilauan, s not replaced serine in a large number of Malinov. Below is a list of Malinov that were obtained for the interaction with MPEG (residue numbering is based on the sequence given in Fig. 1; "C" refers to a cysteine, and s - to Serino):

c0s116 - c0c116

c84s116 - c84c116

c6s116 - c6c116

c8s116 - c8c116

c9s116 - c9c116

c141s116 - c141c116

Native 30 kDa inhibitor of TNF (Fig. 2) is s based on the sequence shown in Fig. 2; "C" refers to cysteine):

c105 30 kDa inhibitor of TNF

c1 30 kDa inhibitor of TNF

c14 30 kDa inhibitor of TNF

c111 30 kDa inhibitor of TNF

c161 30 kDa inhibitor of TNF

The scope of this invention includes a class of compounds shown in Fig. 19, which can be represented by the formula, R1-X-R2in which R1and R2are biologically active groups, and at least one of the groups R1or R2is a polypeptide, and X represents ones polymeric sacer or the linker. R1and R2may be the same group or different groups. In these cases, when R1and R2are a diverse group, as R1and R2can be a polypeptide groups, or R1may be polypeptide, and R2can be any biologically active group. Compounds having such a structure, which were named "dumbbells" compounds, are those that are essentially pure. "Essentially pure" in this context means to be homogeneous in composition.

Homogeneous in composition suggests the presence of one molecule of linker X, one molecule of R1and odnoy group R1and R2attached to the linker at exactly the same place these groups in each molecule, such connection. In some practical implementations of the present invention are biologically active groups associated with the linker site-specific. For example, in connection c105 inhibitor of TNF PEG3000(dumbbell) two groups c105 30 kDa inhibitor of TNF is associated with linker-PEG3000at 105 cysteine residue.

When people talk about "homogeneous composition", it should be understood that at the level of molecule-to-molecule dumbbell connection does not necessarily also homogeneous with respect to the exact length of the group of the spacer. Professionals it is clear that any process of production, in which the use of PEG in the data within the molecular weight or other high molecular weight polymer, begins with a solution which contains the polymer of medium molecular weight. Therefore, when the link PEG with two reactive groups react with the polypeptide group is a PEG link by definition is polydisperse, and the resulting dumbbell connection is heterogeneous to the extent that it is subject to change the length of the linker what the professionals know. In the end, "essentially pure" to R1or R2or 2) which are interconnected by more than one linker X.

R1and R2define a biologically active group. Biologically active groups include any compound that can have a biological effect when interacting with the natural biological molecule. Biologically active groups include proteins, polypeptides, steroids, carbohydrates, such organic substances such as heparin, metal-containing agents, vitamins or any other biological active substances. At least one of the groups R1or R2is a polypeptide. In a preferred practical implementation as R1and R2are polypeptide.

Polypeptide is defined as any compound which is essentially a protein in nature. However, the polypeptide group may contain some ones components. For example, in this definition include glycosylated polypeptides or synthetic modified proteins.

Biologically active group, R1and R2include the linking group and the target group. Linking group is determined by their affinity to this Biologicheskaya on their ability to direct the location of the complex within the biological system. R1and R2may have affinity for the same ligand, in this case, the "dumbbell" can increase the affinity for this ligand. R1and R2may have affinity for different ligands, with R1used to guide this complex situation, where the ligand for R2will be dominant.

Preferred polypeptide groups are receptors, the extracellular part of the receptor molecules to the cell surface, extracellular matrix molecules, binding proteins and receptor antagonists. In the polypeptide group, which can be used as R1or R2also include the following polypeptides and any fragments thereof: receptor antagonist IL-1, 30 kDa inhibitor of TNF, 40 kDa inhibitor of TNF, IL-2 receptors is, (all references to W1 include a single or a combination of consensus repetitive sequences, CR1), PDCF-receptor, IL-2, MCSF-receptor, EGF-receptor, the receptor for IL-5 receptor, IL-3, GMCSF receptor, T-cell receptor, HLA-I, HLA-II, NGF-receptor, IgG (VHVI), CD40, CD27, the receptor for IL-6, integrins CR3, VLA4, ICAM and VCAM, CR2, GMP 140 Lec domain, a laminin-binding protein, fragments of laminin, mannose-binding protein, exon 6 peptide PDGF, and protease (2 catalytic desli they exist. In a preferred practical implementations of the group R1and R2represent or receptor antagonist IL-1, or 30 kDa inhibitor of TNF, or GRj, or the receptor for IL-2 (as-and - chain).

In a preferred practical implementation ones polymeric spacer can be further defined as follows: X = -Y1- (Z)n- Y2- where Y1and Y2represents the remains of activating groups, which react with R1and R2so, to link the spacer with groups of R1and R2and (Z)nrepresents the main polymer group. In accordance with the present invention, n is greater than 6 and preferably greater than 10.

Ones called polymer group, which is essentially not a peptide in nature. When enabled, less than 50 wt.% - amino acid residues as an integral part of Y1, Y2and Z consider ones by nature, and consider them as ones. In a preferred practical implementation ones linker X is non-immunogenic, biologically inert and hydrophilic. In addition, preferred linkers are able to give biologically active polypeptide groups such teleologism, without a significant reduction in affinity data R1and R2groups to their ligand. In the most preferred practical implementations connection R1-X-R2(where R1-R2and R1and R2are linking groups) has an affinity for its ligand, greater than the affinity of unmodified binder group to this ligand. For example, the essentially pure compound s 30 kDa inhibitor of TNF PEG3400the dumbbell has inhibitory activity against TNF 20 times greater than the inhibitory activity of showing respect to TNF s 30 kDa inhibitor of TNF.

The activating group Y1and Y2that are part of a polymeric linker X may include any of the activating groups, including maleimido group, sulfhydryl group, thiol, triflate, trizila, aziridine, oxirane and 5-pyridyl. Preferred activating groups are maleimide.

Polymer group (Z)nprimarily represents a group comprising polyethylene glycol, polypropyleneglycol, polyoxyethylene glycerol, dextran, poly-amino acids, acids of the colon or other carbohydrate polymers and polymers derived Biotin. the bretania gets any ones polymeric group, which would perform the functions described here.

One of the advantages of the present invention consists in the possibility to change the distance between the groups R1and R2by changing the length of the polymer group connecting the two linking groups. Suppose, though not justifying this theoretically that an increase in biological activity observed for multidimensional compounds of the present invention, can be attributed to the multidimensional nature of cellular receptors and ligands in vivo.

For this reason, the professional can easily determine, by changing the size of the spacer X, the optimal distance between the links R1and R2(which is usually directly proportional to the length of the polymeric link (Z)n).

In one practical implementation of the present invention, the group R1R2are the same. However, in another practical implementation of R1and R2are a diverse group. Such compounds can intend to create heterodimer, where R1and R2operate within the same biological systems. For example, it is believed that as the receptor antagonist IL-1 and TNF inhibitors interrupt the cascade of inflammation. You can plan Plex in a specific position by means of its affinity for binding with a specific substrate, and opposing the linking group has the desired activity in relation to this localized website.

An example of heterodimer, which has great potential to be an effective inhibitor of IL-2 is a dimer, in which R1represents the IL-2r , and R2IL-2r . This heterodimer simulates the complex of the receptor, which has high affinity for IL-2 (see example XVII). Additional heterodimers, which can act as an inhibitor of complement, is heterodimer, in which R1represents a binding domain of C3b CRI, and R2represents a binding domain C4b from CR1. Cm. example XVIII. Additional heterodimer R1represents exon 6 PDGF and R2represents IL-1rd. Cm. example XIX.

In a preferred practical implementation of the invention, the method for obtaining a bifunctional complexes R1-X-R2are essentially the same as the methods used for site-selective reactions of the polypeptides as described above. The synthesis of compounds: s 30 kDa inhibitor of TNF PEG3400-dumbbell - described below in example XIII. Bis-reactive polymer group reacts with CONTAINSTABLE with selected free cysteine residue. As described above, the cysteine can be a free cysteine of natural origin on this polypeptide group, or negativly cysteine, which was added or substituted in a natural sequence.

The preferred bis-reactive polymeric compound in accordance with this invention represents - (2-maleimido) - multimediali(oksietilenom) or bis-maleimide PEG. Synthesis of bis-maleimide PEG described in example XII. In accordance with the preferred method, bis-maleimido connection is obtained from bis-hydroxyl PEG through bis-amino intermediate connection.

An overview of several ways of turning the terminal hydroxyl PEG in the corresponding amino group was made by Harris /36/. This is implemented by obtaining a reactive intermediate compounds either by halogenation or oxidation of hydroxyl, followed by substitution of the activated terminal group of the nucleophile.

There are other practical alternatives to the synthesis of bis-maleimide PEG, shown in example XII. Reactive intermediate compound in the transformation of this into the corresponding hydroxyl amine may be halogenated derivative e.g. the com /38/ or intermediate substance aldehydic character (see /36/). Bis-maleimide PEG is not the only sulfhydryl-specific reagent that can be used. Glass and colleagues have developed another way to attach the PEG to sulfhydryl /39/. However, with thiols this reaction is reversible. Another way to attach the PEG to cysteinsulfinic is a derivative of bis-4-vinyl-pyridine PEG.

Harris /36/ also provides an overview of the methods of synthesis of various electrophilic derivatives of PEG that can be used to modify proteins. These reagents include chlorocarbonate, isocyanate, epoxide, Succinimidyl succinate, acid chloride cyanuric acid, a mixed anhydride, carbodiimide and sulfonates. The last group includes TResult, toilet and mesylates. Some of the reagents react selectively with amines (e.g., acid chloride cyanuric acid and carbodiimide), while others react with sulfhydryl, and amines (e.g., epoxide and cresylate). Some of these reagents have been used to modify proteins, and this could lead to loss of activity in varying degrees.

Preferred obtaining complexes of R1-X-R2in which R1and R2different demands Duhs , and then with R2. Obtaining such heterodimers can be done by a professional without undue experimentation. In some cases, intermediate connection R1-X, you must first select and clear before perform the reaction with R2and in other cases, purification of the intermediate compounds may be unnecessary.

The extracellular domains as IL-2r , and IL-2r can be cloned using PCR and cloned into a vector capable of directing expression in E. coli. These proteins can be re-packaged and clean from E. coli and their ability to inhibit IL-2 activity measured in the bioassay. Can be used in vitro mutagenesis to replace the native cysteine residues in the molecule to provide site-directed attachment of PEG. Mutiny as IL-2r , and IL-2r can then be identified that provides an effective connection PEG without loss of activity of pegylated substances. Linked through the PEG heterodimer can be obtained by philliopines first IL-2r in the presence of an excess of bis-maleimide PEG. Once pegylated IL-2r can be cleaned and to add to it IL-2r to interact with active maleimide group and education heterodimer. This molecule has Imiterere R1represents IL-2, and R2is IL-2r , may also be useful as a receptor antagonist of IL-2.

Example I. Synthesis reagents to attach poliatilenglikola.

The three reagent, to show the different tools that can be used for modification of polypeptides. The structure of intermediates and reagents described below, see the Appendix of example I.

A. Synthesis of reagent 1: MPEGxan ester-maleimide

Ester derivative of succinic acid, MPEGx(intermediate compound 1) was obtained according to the method described in /40/. The resulting product was weighed and dissolved in a minimum volume of dry dioxane at 60oC. After cooling the solution to room temperature was added an equimolar amount of tri-n-butylamine and isobutyl ether of Harborview acid. The reaction was conducted under stirring for 30 minutes At this time was preparing borate buffer, pH 8,8, titration of 0.5 M solution of boric acid 1,6-hexanediamine. The solution containing the mixed anhydride was dropwise added to the aliquot of borate buffer containing 10-fold excess (molar ratio) of 1,6-hexanediamine to mix any the Wali. This intermediate polymeric compound (intermediate compound 2) acted in excess of 2.5:1 (molar ratio) ester of sulfosuccinimidyl 4-(-maleimidomethyl)cyclohexane-1 carboxylic acid (sulfo-SMCC, production Pierce Chemical Co, Rockford III) in 50 mm buffer of phosphate or HEPES Buffer, pH 7.0, for two hours at room temperature. The resulting polymer was purified by GPC method, passing the reaction mixture through a Sephadex G-25, using 50 mm sodium phosphate (or HEPES), pH 7.0, for elution at 4oC. Maleimidomethyl went blank when the volume of the column, and it was determined by recording the absorbance at 260 nm. The reagent was used for alkylation of polypeptides within one hour after cleaning. Since MPEG attached in this reaction, can be easily removed in the primary hydrolysis, this reagent is useful for identifying the point of attachment of MPEG to the protein.

B. Synthesis of reagent 2: MPEGx-amid maleimid

MPEGx-toilet (intermediate compound 3) was obtained as described in /4/. The output of the sulfonated intermediate product was assessed spectrophotometrically as described in /42/. This product turned in the production of phthalimide (intermediate compound 4) I/. The content of amino groups in equivalents per gram of the product was determined by micromicrofarad hydrochloric acid. Spent the interaction of MPEGx-NH2with sulfo-SMCC in HEPES or phosphate buffer with a pH of 7.2 at room temperature for two hours. Were made of samples with molar ratios of MPEGx-Amin to sulfo-SMCC from 5: 1 to 1:5.

To determine the optimal conditions in reactions tahilramani used the final reagent (reagent 2), and the quantity and quality of the results obtained in this reaction MPEGxIL-Ira was assessed by electrophoresis in the system LTO-polyacrylamide gel (EPAG) (designation of MPEG IL-1ra will be used for product tahilramani IL-1ra, obtained in the reaction with the reagent 2, and for product tahilramani IL-1ra, obtained in the reaction with reagent 3 described below, the designation of MPEGxIL-1ra). The best result was observed with respect SMCC to MPEGx- NH21:1. A higher ratio SMCC caused the emergence of many more high molecular weight derivatives of IL-1ra, registered APAG, and many peaks in the analytical ion exchange chromatography, and a lower ratio leads to a decrease in output of the pegylated protein. Reagent 2 was purified metal is AGx-H2(intermediate compound 5) can be modified further to obtain other maleimide-derived (reagent 3). Last performed by the interaction of MPEG-NH2with maleic anhydride by the method of Butler and Hartley /43/, suited to this occasion, and cyclization of this intermediate compound (intermediate compound 6) to the corresponding O-(2-maleimidomethyl)-O-metropolitanpoker using the method described in /44/.

The application for example I

Synthesis of reagent 1

Patterns of source materials, intermediates and reagents synthesis of 1.

Starting material:

The General formula monometoksipolietilenglikolya (MPEGx):

CH3O-(CH2CH2O)n-H,

where x denotes the average molecular weight in kilodalton, and n means the average number of repeating oxyethylene groups.

The intermediate connection 1:

< / BR>
Intermediate compound 2:

< / BR>
Reagent 1:

< / BR>
Synthesis of reagent 1

Patterns of source materials, intermediates and reagents synthesis of 2.

Starting material:

The General formula monometoksipolietilenglikolya (MPEGx):

CH3O-(CH2CH

Intermediate compound 3:

< / BR>
Intermediate compound 4:

< / BR>
The intermediate compound 5 (MPEGx- NH2:

CH3O-(CH2CH2O)n-1-(CH2CH2) -NH2< / BR>
Reagent 2:

< / BR>
Synthesis of reagent 3

Patterns of source materials, intermediates and reagents synthesis of 3.

Starting material:

The intermediate compound 5 (MPEGx-NH2):

CH3O-(CH2CH2O)n-(CH2CH2) -NH2< / BR>
The intermediate compound 6:

< / BR>
Reagent 3:

O-(2-maleimidomethyl)-O'-metropolitanpoker

< / BR>
Example II: Receiving pegylated native IL-1ra

To optimize the reaction tahilramani native were verified on various parameters, and successful tahilramani was determined by visual observation according to one narrow band at 22 kilodalton during electrophoresis system LTO-page with staining of Kumassi, and one sharp peak in the case of analytical ion exchange chromatography. If not specified otherwise, the reaction tahilramani was carried out at a concentration of 1 mg/ml of native IL-1ra at room temperature in HEPES buffer with a pH of 7.2 with a ratio of MPEG-reagent to IL-1ra 2:1. Reagent, isoparxIL-1ra, but these results apply to all three reagents.

A. Time

Analyzed the reaction tahilramani, which lasted from 0.5 to 24 hours at room temperature. Conversion of IL-1ra in pegylated form is completed (80%-90%) for 2 to 4 hours, and the total number IL-1ra does not increase or decrease after longer periods of incubation. Quality IL-1ra, which is determined by the electrophoresis system of the LTO-SDS page, decreases with time, as evidenced by the appearance of additional bands and spots on colored gel in the field of higher molecular masses.

B. Temperature

The reaction mixture for tahilramani incubated at 4, 25, 37 and 50oC, and then analyzed after 0,5; 1; 2; 4 and 17 hours of Reaction at 25 and 37oC led to the formation of a large number (about 50 - 80%) pegylated protein within one to two hours, but the response at 4 and 50oC resulted in significantly lower yield (10 - 20%), even with a longer duration of response. Quality IL-1ra, apparently, does not significantly change with temperature.

C. the Concentration of protein

The reaction tahilramani conducted at concentrations of the protein (native IL-1ra) in the range of 50 mcg/P> D. pH

Native IL-1ra was periliously under the reaction conditions that have been set higher at pH's between a 5.5 and 7.5. Quality IL-1ra slightly better at a lower pH (5,5), as established methods of electrophoresis in the system LTO-PAG and ion exchange, but the percent conversion at this the same.

E. the Ratio of MPEG-amido-maleimid to native

Were tested relations MPEG-amido-maleimid to native IL-1ra from 0.5:1 to 20:1. Relations higher than about 2:1 lead to effective transformation in pegylated form of IL-1ra (50 - 90%). However, the relationship is higher than 5:1 lead to a decrease in quality IL-1ra, resulting in the increase in the number of bands in the region of very high molecular masses during electrophoresis system LTO-PAG and many peaks in ion-exchange chromatography.

Optimal reaction conditions as for the number of received IL-1ra, and for the quality of the obtained substances, within the used parameters are as follows: 2:1 MPEG-amido-maleimid /IL-1ra at 25oC for 2 to 4 hours, using MPEG-amido-maleimide obtained when the ratio of sulfo-SMCC to MPEG-amine 1:1. In these conditions, in pegylated form turn 80 - 90% of IL-1ra using reagent synthesized Libi IL-1ra PEG-dumbbell

Dumbbell complexes PEG containing IL-1ra, get in the same way as other pegylated variants of IL-1ra. Use 2 to 4 molar excess of bis-maleimide PEG-IL-1ra in HEPES buffer at pH 7.0. Used a variant of IL-1ra may be a wild-type molecule, which is free and accessible cysteine residue, or mutein obtained as described above. IL-1ra is present in a concentration of 2 to 5 mg/ml Mixture for tahilramani incubated at room temperature for 4 to 6 hours Connection IL-1ra PEG-dumbbell clean from naegeliana and once pegylated compounds cation-exchange chromatography on a MonoS column at pH 5.5 MES buffer concentration of 20 to 50 mm, using a gradient of NaCl concentration from 0 to 1000 mm. Further purification can be achieved by a GPC method using BioRad column or Superdex 75, as described below.

Example III: Purification of pegylated native IL-1ra

Cleanup MPEGxIL-1ra can be successfully perform the methods of cation-exchange or gel permeation chromatography. These methods are applied to pegylated IL-1ra obtained from all three reagents described above.

A. Cation-exchange chromatography

MPEGxIL-1ra can be cleared ispol gradient from 0 to 500 mm NaCl in the same buffer. For example, unmodified IL-1ra released from the column at 220 mm NaCl, while the purity test, using different methods, including analytical ion exchange chromatography and electrophoresis in the system LTO-PAG. MPEG5000IL-1ra released from the column at 160 mm (Fig. 4).

B. Gel permeation chromatography

MPEG5000IL-1ra, which has a length of about 52 kDa, and MPEG8500IL-1ra, which has a length of about 68 kDa (based on the calibration column for standards of known size), can be easily separated from unmodified IL-1ra (17 kDa) by GPC on a column with sorbent Superdex 75 (Pharmacia) standard chromatographic method (Fig. 5).

Example IV: feature

Cleaned MPEGxIL-1ra gives a single symmetrical peak with repeated chromatography sorbent MonoS shows himself pure in the electrophoresis system of the LTO-SDS page and gel permeation chromatography (Fig. 3 and 4). Comparison trypticase cards IL-1ra, MPEGxIL-1ra shows a single peak corresponding to the peptide containing C116 and C122, which is missing on the map conjugate, with the appearance of a new broad peak at this map. Further cleavage of this new peak conducted with chemotrypsin, and subsequent CLASS="ptx2">

Example V: getting Malinov IL-1ra

On single-stranded DNA of the gene IL-1ra, cloned in bacteriophage M13, carried out mutagenesis. Used mutagenic set BioRad, which was used in the way described by Cuncolim with TCS. in /45/. In short, generate a matrix of single-stranded DNA using E. coli strain that contains the dut and ung mutations, leading to the matrix, including uracil instead of thymidine. Mutagenic oligonucleotides ranging in length from 20 to 30 base pairs were senatoriable in the matrix and resentational the second strand using DNA polymerase and DNA ligase. Data reaction mixture was used for transformation of strain wild-type E. coli, in which the uracil-containing strand destroy under the action of the regenerative mechanisms of DNA and mutant strands allow you to play. Mutant phage were sorted and set the sequence of amino acid residues in standard ways. Then the fragment containing the mutated gene, was subcloned into the expression vector pT5T /44/ and was transformed into a strain expressing T7 system (E. coli B121DE3). You can also use other expression system of E. Coli.

Expressing clones grown at 37oC on Luria Broth with the addition of 15 μg/ml tetracycline. When the culture reached the opt is M, to induce the gene expression of IL-1ra. The total accumulation of the protein IL-1ra was maximum after 4 to 6 hours and did not change significantly up to 12 hours after induction.

Example VI: cleanup Malinov

Cell culture, induced as described above, collected by centrifugation at 10000 g for 10 min the Cells are again suspended in 20 - 50 ml of 30 mm buffer of sodium acetate at pH 5.2. Lysis was achieved by double passage through a French unit for filtering under pressure at 18,000 psi. The cell lysate was centrifuged at 10000 g for 10 min Soluble portion was placed in a column with sorbent S-sepharosa and washed with the same buffer containing 75 mm NaClIL-1ra mutein out from the column with buffer containing 200 mm NaCl. A single passage over ion exchange resins leads to a product of sufficient purity (>95%) to study tahilramani. Further purification successfully undertake using other ion-exchange resins, as well as Q-Sepharose or MonoQ. This method is equally well have been used for several Malinov IL-1ra. In some cases it was necessary to slightly modify the pH and/or concentration of NaCl to clear mutiny that have small differences in the charge of the protein due to changes in the individual, this method is widely applicable to all of the studied Malinov.

Example VII: tahilramani mutein IL-1ra

In addition to the native IL-1ra, were tahilramani mutiny c84s116, c84c116, c0s116 and c9s116. Using the same conditions that were applied to native IL-1ra, received and separated pure pegylated forms c84s116 and c84c116. As c84c116 contains two reactive cysteine, tahilramani leads to a protein with a higher molecular mass of approximately 40 kDa during electrophoresis system LTO-PAG. This protein can be cleaned by the method of cation-exchange or gel permeation chromatography, and at last, for the case when using PEG5000it comes with the expected molecular weight of about 68 kDa.

Example VIII: efficiency IL-1ra

Effectiveness of pegylated molecules of native IL-1ra was tested standard breakdown of the competitive binding of the receptor, using S35IL-1ra as a ligand. The mouse cells (EL4), containing the receptor of IL-1 murine type 1, or hamster cells (CHO) expressing the human receptor type 1 from the cloned gene, used 1106cells per well and 1105cells, respectively, in microeletronic links on 96 Luna the PM. In a series of dilutions from 28 mm to 13 PM added cold ligand, and left to incubate for 4 hours at 4oC. Then the cells were filtered on a filter with ml filter plate (Millipore, filter Durapore with a pore size of 0.5 μm), washed to remove radioactive contaminants, the filter with cells was removed and measured activity using the Ambis Radioanalytical Ivnaging Systein.

We calculated the equilibrium constant of dissociation (KD), and they were used for comparing pegylated and unmodified forms of IL-1ra. Unmodified wild type IL-1ra and c84s116 have equal KD murine receptor type 1, 150 - 300 PM in our experience, KD for pegylated forms of IL-1ra is about 400 - 800 PM, and for pegylated c84s116 - 500 - 10000, 2.5 and 3.5 times higher than the values for the corresponding unmodified protein. KD for all but one (c6s116) from naegeliana of Malinov are within 65 - 150% of the native protein, within the limits of error of the experiment (see table 1).

Example IX: the pharmacokinetics of pegylated native mutein IL-1ra

Was validated pharmacokinetic character of the molecules of several native and pegylated malinovich IL-1ra after intravenous injection of these molecules rats. Nati is Rob blood and analyzed on native or pegylated IL-1ra using tests enzyme-linked immunosorbent assay (PASIS). From the graph (Fig. 8) changes in the concentration of plasma IL-1ra in time, it is clear that tahilramani has a significant impact on the disappearance of IL-1ra from plasma after intravenous injection. Drop the contents in plasma IL-1ra and pegylated derivatives of IL-1ra is best described by three exponential components. These data indicate that tahilramani extend in rats, the half-time of retention of these exponential components up to 6 times (table 2). The half-time of retention of these exponential components increases with the molecular size of PEG (table 2). In addition, there is evidence that the increased duration of the half period of retention may be determined by the site-specificity of tahilramani. To interpret the data of Fig. 8 used the standard compartmental analysis. Extension of period of retention can be explained based on the adopted pharmacokinetic theory, which argues that the plasma half-time of retention for drugs inversely associated with plasma purification from drugs and is directly proportional linked to the apparent volume of distribution of drugs. Pharmacokinetic analysis of the disappearance of pegylated period of retention is inversely proportional to the decrease of plasma purification. The decrease in plasma purification coincides with the expectation associated with the volume, decreased glomerular filtration pegylated molecules through the kidneys. The extension of the half-period of philliopines also in proportion to the increase of distribution (Vd stable condition, table 2) pegylated molecules. The increase in volume of distribution indicates greater penetration of pegylated molecules in the extravascular system. Through this mechanism, tahilramani improves treatment with IL-1ra, increasing the extent to which the active molecules are moved from the systemic circulation into the extravascular system, system, which is expected to be detected by the receptors of IL-1ra. Because of the similarities between rats and humans in the mechanisms as purification and distribution for IL-1ra, it is obvious that tahilramani will improve in a similar way and pharmacokinetic properties in the human body.

I. Additional intravenous pharmacokinetics for pegylated IL-1ra

For eight additional pegylated Malinov IL-1ra, using previously described methods, has been described intravenous pharmacokinetics. In Fig. 10 shows the dependence of the change of intravenous plasma concentrations of IL-1ra in time for each g-units (single or double, tahilramani) there is a decrease of plasma treatment and, therefore, increasing the average time intravenous retention and increases the half-time of disappearance of IL-1ra from plasma. The site paglierani is important in determining the extent to which reduced plasma purification and increases the mean retention time. Adding two units of PEG-IL-1ra extends the average time intravenous holding 14-fold compared with wild type IL-1ra.

2. Subcutaneous pharmacokinetics for pegylated IL-1ra

Absorption the pharmacokinetics of pegylated Malinov IL-1ra described after subcutaneous injection of these molecules rats. From the tail vein took a series of blood samples and analyzed on native or pegylated IL-1ra using tests enzyme-linked immunosorbent assay (PASIS). Curves changes resulting subcutaneous plasma concentrations of IL-1ra in time shown in Fig. 11. Data subcutaneous pharmacokinetics (table 3) show for pegylated Malinov changeable system availability associated with a specific site and the size of this PEG-link, and associated with subcutaneous injection neoprimitivism dependencies. Table 3 also shows a positive and significant impact of tahilramani on average in the survival usually grows. It is likely that this increase is the result of slower absorption through the lymphatic circulation (longer average retention time), which is associated with the size of the molecules, as well as delayed purification after pegylated molecule has reached the systemic circulation (plasma). This is significant and will improve the pharmacokinetic character introduced subcutaneously with IL-1ra for a person.

Example X: obtaining Malinov 30 kDa inhibitor of TNF

Native residue was substituted with cysteine as when the terminal amino group and the terminal carboxyl group of the protein, as well as in all three glycosylation sites (residues 1; 14; 105; 111 and 161, as shown in Fig. 2). Mutagenesis was performed on single-stranded DNA of the gene 30 kDa inhibitor of TNF cloned into the bacteriophage M13. This gene is described in detail in /21/. Mutagenesis was performed as described in /43/ (see example V). Subjected to mutagenesis gene was isolated and subcloned into the expression vector pT5T /44/, and transformed into E. coli strain BL21DE3 with expressing T7 system. Mutiny 30 kDa inhibitor of TNF was purified and subjected to re-package as described for native 30 kDa inhibitor of TNF /21/. Repacking involves adding cysteine to the solution, soain in mateine.

Example XI: tahilramani of Malinov 30 kDa inhibitor of TNF

Mutein s 30 kDa inhibitor of TNF was treated with 6-fold molar excess of DTT in HEPES concentration of 50 mm, pH 7.0 for 30 min at room temperature to remove excess cysteine associated in the process of re-packaging. Then the protein was subjected to dialysis in degassed HEPES concentration of 50 mm, pH 7.0 for 2 hours to remove DTT. Then spent the interaction s 30 kDa inhibitor of TNF with 5-fold molar excess philonous reagent 1 (see example 1A) for 2 hours at room temperature in HEPES concentration of 50 mm, pH 7.0. In pegylated form becomes approximately 60% mutein.

The reaction mixture is tahilramani was loaded into a column for high-speed liquid chromatography of proteins (SJHP) sorbent Pharmacia, feeding 0.25 ml/min 50 mm Tris with pH 7.0, 100 mm NaCl. The fractions containing s-PEG-30 kDa-TNF collected and loaded in TSK-200 SW HPLC column (Bio-Rad), feeding of 0.2 ml/min in the same buffer. The fractions containing almost pure s-PEG-30 kDa-TNF, which was determined by electrophoresis system LTO-page with silver staining, collected and determined the concentration of protein by Bio-Rad protein proost

Example XII: obtaining bis-maleimide PEG

Synthesis of (2-amino-ethyl) -- minopoly(oksietilenom) derivative of PEG (designated here and in the following as bisamino-PEG) consists of three phases: 1) sulfonation hydroxyl group, using treilhard, as described by Nilsson and Mobicom /40/, 2) substitution trailervideo intermediate connection phthalimido /39/, and 3) reconstruction of intermediate phthalimide connection in the amine hydrazinehydrate /39/. The structure of the source compounds, intermediates and products are shown in Annex 1 to this example. Under optimal conditions obtained approximately 80% conversion of hydroxyl in the amine, as defined break with 2,4,6-trinitrobenzenesulfonic (TNBS). Bisamino PEG can be isolated in pure form from the reaction mixture by the method of ion exchange chromatography. This is a key stage for the removal of reactive by-products that may interfere with dimer formation.

Bisamino PEG was etilirovany maleic anhydride /41/, and the resulting intermediate compound was cyclically education - (2-maleimidomethyl -- maleimido(oksietilenom) a /42/. This compound reacts with sulfhydryls by the reaction of the accession of Michael from obrezala for polyethylene glycol PEGx< / BR>
HO-(CH2CH2O)p-H

where x denotes an average molecular weight in kilodalton, and n is the average number of repeating oxyethylene groups.

The intermediate connection 1

F3-CH2-SO2-O-(CH2CH2O)n-1- (CH2CH2)-O-SO2-CH2F3.

Intermediate compound 2

< / BR>
Intermediate compound 3

H2N-(CH2CH2O)n-1-(CH2CH2) -NH2< / BR>
Intermediate compound 4

O-(2-maleimidomethyl)-O-methyl-polyethylene glycol

< / BR>
Example XIII: results of in vivo complexes with s 30 kDa-TNF PEG

On two different stimulated TNF physiological actions tested in vivo inhibitory effect of four samples, pegylated s 30 kDa inhibitor of TNF. One result was the emergence of IL-6 in plasma of mice, which was intravenously injected recombinant TNF person. Another result was the increase of neutrophil migration into the peritoneal cavity after injection intraperitoneally with recombinant TNF person.

Experience one. Intravenous s 30 kDa inhibitor of TNF-alpha (PEG2000, PEG3500, PEG10000simultaneously with recombinant TNF person yanantin TNF person used BALB/C female mice weighing from 20 to 23, Preliminary experience has been obtained, the time dependence of the appearance of IL-6 in plasma after intravenous injection through the tail vein of two doses of recombinant human TNF (Fig. 12). Peak content of IL-6 was observed two hours after stimulation with 10 or 20 µg of recombinant TNF person on the mouse. In subsequent experiments used a lower dose.

Compared the impact s 30 kDa-TNF PEG2000-dumbbell and naegeliana s 30 kDa inhibitor of TNF. Recombinant human TNF was administered intravenous dose of 10 μg per mouse, one or simultaneously with TNF inhibitors. Were tested (Fig. 13) four different relationship to TNF inhibitors. The relationship was calculated based on the protein content. Each dose was tested on three mice. The blood was collected two hours after intravenous injection. Levels of IL-6 was determined using PACES.

As s 30 kDa-TNF and S103 30 kDa-TNF PEG-dumbbell almost completely inhibited the appearance with the introduction at the ratios of inhibitor to TNF 10: 1 and 5:1. With a ratio of 1:1 s 30 kDa-TNF PEG2000-dumbbell raises 95% decrease in the content of IL-6 stimulated one TNF, while naegeliana s 30 kDa-inhib the relationship, as s 30 kDa-TNF and s 30 kDa-TNF EVP2000the dumbbells are good inhibitors of this stimulated TNF, physiological parameter. With a ratio of 1:1, s 30 kDa inhibitor PEG2000the dumbbell has a stronger inhibitory effect than naegeliana inhibitor.

Tested other samples pegylated s 30 kDa-TNF. Inhibiting effects s 30 kDa-TNF PEG3500-dumbbell and s 30 kDa-TNF PEG10000the dumbbell was tested for the induction of IL-6 in plasma. Inhibitors were injected intravenously simultaneously with recombinant TNF person at a ratio of 1: 1 (s 30 kDa-TNF-dumbbell: TNF) (Fig. 14). In each of the two groups of mice treated with inhibitor, was 3 individuals. 10 mice received injections only TNF. With a ratio of 1:1, introduction or s 30 kDa-TNF PEG3500-dumbbell or s 30 kDa-TNF PEG10000-the dumbbell does not cause the appearance of IL-6 in plasma of mice, whereas after administration to mice of the same recombinant TNF person causes the appearance in plasma of a significant amount of IL-6.

The results of these two experiments showed that s 30 kDa-TNF PEG2000, PEG3500and PENG10000NII in low ratio (1:1) with respect to stimulus.

Experience two. Introduction subcutaneous s 30 kDa-TNF (PEG3500, PEG10000and PEG20000simultaneously with intraperitoneal injection of recombinant human TNF inhibits the migration of neutrophils into the peritoneal cavity.

For measurement of neutrophil migration into the peritoneal cavity after stimulation with recombinant TNF person, used BALB/c female mice weighing from 20 to 23, Used the same methodology as Kim McIntyre with TCS. /45/, which are briefly described here. Directly into the peritoneal cavity of mice were administered 0.1 ml of TNF. After 4 h, mice were killed and immediately after that washed the peritoneal cavity was injected into it 4 ml of balanced salt solution Hank (CPX) (not containing calcium and magnesium). The abdomen gently massaged. The peritoneal fluid was pulled with a needle and syringe. On the count of Coulter counted the total number of peritoneal cells. An aliquot of cell suspension was dried on glass and stained with the dye Diff-Kwik. These cells was calculated by the differential method by direct microscopic observation. One hundred cells were examined and classified as either neutrophils or lymphocytes, or macrophages.

In the preliminary experiment compared the physiological solution, or 7.5 ng of recombinant TNF person. TNF has caused an increase in the percentage of neutrophils and in the absolute number of neutrophils present in the peritoneal wash fluid. In mice exposed to a physiological solution of washing liquid collected 9,4 104neutrophils, which amounted to only 2.3% of the total number intraperitoneal cells. In mice, subjected to the action of TNF (7.5 ng), the total number of neutrophils increased to 12,9105and the percentage of neutrophils increased to 19.7%.

Also compared activity naegeliana s 30 kDa inhibitor of TNF and three pegylated samples s 30 kDa inhibitor of TNF (PEG3500, PEG10000and PEG2000dumbbells). Keeping the stimulus TNF constant of 7.5 ng per mouse, tested inhibitors at the ratio of 100:1; 10:1 and 1:1 (sample C 30 kDa-TNF : TNF). Relations are considered, based on the protein content. The mice were injected subcutaneously s 30 kDa-TNF simultaneously with intraperitoneal introduction of TNF. Each dose was tested on six mice. Four hours peritoneal wash fluid was collected and analyzed. The values shown in Fig. 15 represent the percentage of neutrophils in the br and s 30 kDa-TNF PEG3500-dumbbell significantly inhibit the migration of neutrophils, is 100: 1. s 30 kDa-TNF PEG10000and PEG20000-dumbbell significant inhibit the migration of neutrophils at the ratio of 10:1.

The results of this experiment show that s 30 kDa-TNF PEG3500- PEG10000and PEG20000the dumbbells are good inhibitors stimulated TNF migration of neutrophils in the peritoneal cavity. s 30 kDa-TNF inhibitors PEG10000and PEG20000-dumbbell are more active than naegeliana s 30 kDa-TNF and s 30 kDa-TNF PEG3500.

Example XIV: obtaining and bioactivity s 30 kDa-TNF PEG-dumbbell

Synthesis of

2-3 ml/g recombinant s 30 kDa-TNF treated with 4-fold molar excess of DTT for 2 h at room temperature. Then the TNF subjected to dialysis in degassed HEPES concentration of 50 mm, pH 7.0 for 3 h at 4oC. To obtain dumbbells, connected via PEG, TNF lead in the interaction with the bis-maleimide PEG, at different molar ratios, in the HEPES concentration of 50 mm, pH 7.0. The TNF inhibitor reacts with bis-maleimide PEG in equimolar aspect] is G the dumbbell of TNF was separated from naegeliana and once pegylated TNF, using SJHP sorbent MONO-S in a solution of acetic acid concentration of 50 mm with a pH of 4.0 using a stepped gradient of 260 mm 310 mm and 350 mm NaCl. Associated molecule PEG dumbbell of TNF released from the column with a gradient of 310 mm NaCl. The remaining naegeliana the TNF remove chromatographic sorbent Superdex 75.

Step-attaching reagent

After DTT treatment and dialysis in the HEPES concentration of 50 mm, pH 7.0 add equimolar amount of bis-maleimide PEG, after 1.5 h incubation, again add an equimolar amount of bis-maleimide PEG. The mixture is incubated for 1.5 h This leads to the optimal content of PEG-linked dumbbell connection. Then add 2-fold excess of PEG-reagent receiving end, the ratio of PEG /TNF 4:1. All this is incubated for 2 h, and then the mixture is subjected to dialysis in acetate concentration of 50 mm with a pH of 4.0 used for the chromatography was carried out on the sorbent Mono-S. a mixture is Formed which contains mainly dimer, which is connected through the PEG, and once pegylated TNF inhibitor. This makes it easier to clean the PEG-linked dumbbells, because it has more separation between once pegylated and imiserver education dumbbell connection and contributes to a more effective cleaning.

Step response:

After DTT treatment and dialysis in the HEPES concentration of 50 mm, pH 7.0 add 8-fold molar excess of bis-maleimide PEG. The mixture is incubated for 2 h at room temperature. This makes essentially the entire TNF once in pegylated form. Once pegylated TNF inhibitor is separated from the PEG-reagent and unreacted inhibitor of TNF using HPLC sorbent Mono-S in acetate concentration of 50 mm with a pH of 4.0 with a gradient of NaCl. Once paglinawan connection is subjected to dialysis HEPES concentration of 50 mm, pH 7.0, and concentrated to 2-4 mg/ml Then add DTT treated the TNF, in order to obtain a PEG-linked dumbbell. After 2 h of PEG-linked dumbbell connection purified using HPLC sorbent Mono-S. This method can be used to obtain a PEG-linked heterogeneo by adding a second, different protein compounds.

This method optimizes the formation of dumbbell connection and can be used to obtain geterokationnykh compounds. However, this method is somewhat time consuming.

The biological activity of PEG-linked Gentilini motociclete TNF was measured in a sample of cytotoxicity cells murine L-929. This allowed to determine for these molecules ED50which have the following meanings, ng/ml:

Wild type TNF r - 220

"Related dumbbell - 220

PEG1900-dumbbell - 4,1

PEG3500-dumbbell - 4,8

PEG10000-dumbbell - 4,6

PEG20000-dumbbell - 4.2V

Dumbbell connection of TNF have also greatly increased activity in the inhibition of the cytotoxicity of TNF in the bioassays L 929. The value of the ED50in relation to TNF are:

Wild type TNF r, ug/ml - 70

PEG3400dumbbells, ng/ml - 80

PEG2000dumbbells, ng/ml - 22

Example XV: the pharmacokinetics of pegylated 30 kDa inhibitor of TNF

1. Intravenous pharmacokinetics for pegylated 30 kDa inhibitor of TNF

Determined the pharmacokinetic behavior of several types of pegylated molecules 30 kDa inhibitor of TNF after intravenous administration to rats. Native or pegylated TNF inhibitor was injected intravenously as a bolus dose. Then the tail vein was collected a series of blood samples and analyzed for the presence naegeliana or pagliarulo of TNF breakdown by enzyme-linked immunosorbent assay (PASIS). The resulting temporary who has a significant effect on the disappearance of a TNF inhibitor from plasma after intravenous injection. To interpret the data of Fig. 16 used the statistical theory of time (area under the curve and the area under the first moment curve). These data show that tahilramani extends in the rat and the average time intravenous holding TNF almost 50 times (table 4). Average time intravenous retention increases with increasing size of the bound molecule of PEG (table 4). Although theoretically it is not confirmed, the extension of the average retention time can be explained on the basis of the usual pharmacokinetic theory, which asserts that the time of intravenous retention for drugs inversely associated with plasma purification for this medication, and directly proportionate to the apparent volume of distribution for the drug. Pharmacokinetic analysis of the disappearance of pegylated molecules of the TNF inhibitor from plasma shows that the extension of period of retention inversely associated with low plasma purification for pegylated molecules, compared with the non-pegylated TNF inhibitor (table 4). The decrease of plasma purification is consistent with the expected defined size, decrease in glomerular filtration pageliability TNF in rats and humans, it is obvious that tahilramani similarly improve the pharmacokinetic properties of TNF in humans.

2. Subcutaneous farmacocinetica for pegylated 30 kDa inhibitor of TNF

Studied the pharmacokinetics absorption pegylated TNF inhibitor after injecting it subcutaneously to rats. From the tail vein was collected a series of blood samples and analyzed, determining the change in concentration naegeliana and pagliarulo of TNF in time; the results are shown in Fig. 17. Data subcutaneous pharmacokinetics (table 4) show for Tagliavini molecules changeable system availability associated with the size of the PEG, and associated with subcutaneous injection neoprimitivism dependencies. Table 4 also shows a positive impact of tahilramani on the average retention time for subcutaneously injected TNF. With increasing amount of PEG usually increases the average retention time. Although theory is not confirmed, this increase is probably the result associated with the size of the molecules slow absorption through the lymphatic circulation system (longer average retention time), as well as delayed purification after pegylated moonage of TNF for a person.

Example XVI: solubility pegylated proteins IL-1 ra

The results of the study of solubility is shown in Fig. 18. Shows solubility curves for three different drugs

IL-1ra and s IL-1ra PEG8500. Experiments were performed at 37oC microeletronic cups with a protein concentration of 160 mg/ml Cup was closed with a lid and then viewed in a special device at 405 nm after a certain period of time. The increase in absorbance indicated on the planting of the polymer. There is a clear decrease in the number drop-down from a solution of protein for pegylated sample as compared to native IL-1ra.

30 kDa inhibitor

Native 30 kDa-TNF should not concentrate more than up to 5 mg/ml After tahilramani solubility increases at least 5 times.

Example XVII: getting heterogeneo inhibitor of IL-2

PEG-related heterogenen can be obtained by first Tagliavini IL-2ra in the presence of an excess of bismaleimide PEG. Danocrine pegylated IL-2ra fashionable peel and add to it IL-2ra for the reaction with the remaining reactive maleimide group with the formation of heterodimer.

Potential sites for peril is casinomania, as well as native free cysteine residue in the molecule. It was found that the cysteine residue 192 to the soluble extracellular domain is not involved in disulfide linking /46/. This cysteine residue is located in epitone anti-IL-2r monoclonal antibodies, which do not affect the binding of IL-2 to IL-2r /47/. This indicates that this residue is a likely candidate for tahilramani without having to interfere with the activity of IL-2r .

For IL-2r potential sites include amino - and carboxyl end groups, N-linked sites glycosylamine and region 108-118, which is similar to a biologically significant area in the receptor murine erythropoietin /48/. Analysis of point mutations of other residues in the receptors may also be given the opportunity to discover other sites tahilramani that will lead to optimal properties in heteropaternal molecule.

Example XVIII: getting heterogenea that inhibit classical pathway complement system

Have been identified and cloned many proteins that regulate the complement system. Some of them are membrane proteins. One of the membrane proteins called CR1 (complement receptor 1). A soluble form of CR1 was investigated in vivo in mo is passive Arthus reaction /50/ and the rejection of allograft /51/.

Soluble CR1 associated with C3b and C4b. It consists of 30 short consensus repetitive sequences (CPR). Most of the CPR contains one site of potential glycosylation sites and four cysteine. Probably all these cysteine participate in disulfide binding. It is established that the CPR 1-4 are involved in the binding of C4b. Two separate portions CR1, CAT 8-11 and 15-18 CAT involved in the binding of C3b /52/, /11/. according to this invention, it is possible to obtain heterogenen, which contains C4b-binding domain and C3b-binding domain of CR1. Using PCR, it is possible to clone a CAT, which contain C4b-binding and C3b-binding domains of CR1. This CAT will be a CAT 1 through 5 (C4b-binding) and transmission 8 through 12 (C3b-binding). The gene encoding these checkpoints, can be cloned into the expression vector in E. coli. E. coli-expressed proteins can be re-Packed and cleaned. The success of re-packaging, you can appreciate the ability to link C3b or C4b. You can spend in vitro mutagenesis of these genes to replace the native cysteine amino acid residues. These cysteine you can then use to link PEG-molecules. Possible sites for Tagliavini will be the site of glycosylation or terminal carboxyl group ostad the ENES, which contain an additional cysteine at the carboxyl end group of the residue. PEG-associated heteropentalene connection can be obtained in a two-stage method of examples XIV. Cleaning can be performed by the method of ion exchange chromatography.

Example XIX: the synthesis of IL-1ra bis(maleimide)-(platelet-derived growth factor) is a peptide PEG-heterogenen

Peptide platelet-derived growth factor (PDGF) YGRPRESGKKRKRKRLKPT described in a /53/. To make it possible for linking to maleimido added terminal C.

Heterogenen synthesized in two stages. In the first stage 1.6 nanomoles IL-1ra, suspended in 3 ml of HEPES buffer concentration of 0.05 m, pH 7.5, was mixed with 6.4-nanomolar bis-maleimide PEG1900dissolved in 11 μl of the same buffer. This reaction was carried out for 30 min at 20oC. In the second stage, to the product obtained in the first stage, added 32 nanomole peptide PDGF dissolved in 4 μl of 0.2 M buffer phosphate, pH 7.0. The reaction was carried out for 1 h at 20oC. Then the reaction was interrupted by the introduction of an equal volume of buffer for electrophoresis system LTO-PAG containing 30 mcmole 2-mercaptoethanol.

Samples of the products of the first stage of this reaction and the products are full of dochstader the e-ordinator-page on 15% polyacrylamide gel followed by staining the blue Kumasi. The product of the two-stage reaction gives an additional band corresponding dumbbell connection of the expected size. In heteropentalene connection by two-stage reaction makes approximately 33% of the original IL-1ra.

The products of the first reaction stage can be distinguished by cation-exchange chromatography on resin S-Sepharose. Heterodimer it is possible to select in using cation-exchange chromatography, due to the abundance of basic amino groups in the peptide.

It should be understood that the application of the recommendations of the present invention for expressing specific system or pugilism reagent, in the light of these recommendations, will be within the abilities of a specialist. Thus, it is obvious that various modifications and variations of the method and products of the present invention. It is implied that the present invention includes these modifications and variants, if they are in the volume formulae taking into account the possibilities for equivalent replacement of specified attributes.

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1. Essentially pure compound of the formula

the SCP, representing an inhibitor of tumor necrosis factor (TNF) or biologically active portion, which naturally contain or are modified so that they contain reactive sulfur;

X - ones therapeutically acceptable water-soluble polymer spacer.

2. Essentially, the net connection on p. 1, wherein R1and R2each is a 30 kDa inhibitor of TNF, or a scaled down version, or mutein, or 40 kDa inhibitor of TNF, or a scaled down version, or mutein.

3. Essentially, the net connection on p. 2, wherein R1and R2each is a 30 kDa inhibitor of TNF or its scaled down version and each modified so that it contains at least one negativly the residue is cysteine.

4. Essentially, the net connection on p. 3, wherein R1and R2each is a 30 kDa inhibitor of TNF or its scaled down version and these negativnye cysteine residues are sites of amino acid residues 1, 14, 105, 111, or 165.

5. Essentially, the net connection on p. 2, wherein R1and R2each is a 30 kDa inhibitor Fan as-Y-(Z)n-Y2- where Y1and Y2a balance of activating groups, (Z)nis polyethylene glycol, polypropyleneglycol, polyoxyethylene glycerin, dextran, acids colon, poly-amino acids or polymeric carbohydrates.

7. Essentially, the net connection on p. 6, wherein X is defined as Y1-(Z)n-Y2- where Y1and Y2a balance of activating groups, and (Z)nis polyethylene glycol.

8. Essentially, the net connection on p. 1, characterized in that the said peptide linking group, R1and R2covalently associated with the specified ones polymeric spacer thioester bonds.

9. Essentially, the net connection on p. 1, characterized in that the said peptide linking group associated with the specified ones polymeric spacer through native or negativly the residue is cysteine.

10. Essentially, the net connection on p. 4 or 5, wherein R1and R2each is a 30 kDa inhibitor of TNF or its scaled down version, X is defined as Y1-(Z)n-Y2- where Y1and Y2a balance of activating groups, (Z)nis polietilenglikolya formula R1-X-R2in which R1and R2each represents a receptor antagonist interleukin-1 receptor antagonist IL-1 or its biologically active portion, which naturally contain or are modified so that they contain reactive sulfur, X - ones therapeutically acceptable water-soluble polymer spacer.

12. Essentially, the net connection on p. 11, wherein the receptor antagonist IL-1 modified so that it contains at least one negativly the cysteine residue, which can be located in the sites of amino acid residues 0, 84, 6, 8, 9, or 141.

13. Pharmaceutical composition having activity of TNF and containing an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a compound according to any one of paragraphs.1 - 10 effective number.

14. The pharmaceutical composition according to p. 13, characterized in that it is intended for the treatment or prevention of TNF-mediated diseases.

15. The pharmaceutical composition according to p. 14, wherein the disease is selected from respiratory distress syndrome in adults, Legos is 16. A method of treating TNF-mediated disease, wherein the needy in the treatment of patients prescribed pharmaceutical composition under item 13.

17. The method according to p. 16, wherein the disease is selected from respiratory distress syndrome in adults, pulmonary fibrosis, rheumatoid arthritis, inflammatory bowel disease and septic shock.

18. The method of obtaining essentially pure compounds of formula

R1- X - R2,

where R1and R2each represents a polypeptide group selected from a TNF inhibitor, receptor antagonist IL-1 and biologically active parts, which naturally contain or are modified so that they contain reactive cysteine;

X - ones therapeutically acceptable water-soluble polymer spacer, which is biologically inert and having a General formula of Y1- (Z)n- Y2where Y1and Y2are those the remains of activating groups, which react with R1and R2to associate a spacer with groups of R1and R2and (Z)nrepresents the main polymer group, with n > 6,

characterized in that th is th, having at least two reactive groups capable of forming thioester linkages by reacting with a cysteine amino acid residues with formation of the specified R1- X - R2connection, and spend the isolation and purification of the compounds R1- X - R2.

19. The method according to p. 18, wherein R1and R2each is a 30 kDa inhibitor of TNF, or a scaled down version, or 40 kDa inhibitor of TNF, or a scaled down version.

20. The method according to p. 18, characterized in that (Z)nselected from polyethylene glycol, polypropyleneglycol, polyoxyethylene glycerol, dextran, acids rectum, poly-amino acids or carbohydrate polymers.

21. The method of obtaining essentially pure therapeutically valuable compounds of the formula

R1- X - R2,

where R1and R2each represents a polypeptide group selected from a TNF inhibitor, receptor antagonist IL-1 and biologically active parts, which naturally contain or are modified so that they contain reactive cysteine;

X - ones therapeutically acceptable water-soluble polymer spacer, avaudioplayer those remnants of activating groups, which react with R1and R2to associate a spacer with groups of R1and R2and (Z)nrepresents the main polymer group, with n > 6,

characterized in that interoperate R1with ones therapeutically acceptable polymeric group containing at least two reactive groups capable to form thioester communication when interacting with cysteine amino acid residues with formation of the complex R1X, then communicate complex R1- X with R2with the formation of the compounds R1- X - R2and spend the isolation and purification of the compounds R1- X - R2.

22. The method according to p. 21, wherein R1and R2each is a 30 kDa inhibitor of TNF, or a scaled down version, or 40 kDa inhibitor of TNF, or a scaled down version.

23. The method according to p. 21, characterized in that (Z)nselected from polyethylene glycol, polypropyleneglycol, polyoxyethylene glycerol, dextran, acids rectum, poly-amino acids or carbohydrate polymers.

24. Native receptor antagonist IL-1, characterized in that its cysteine OS is bound and has a General formula

Y1- (Z)n- Y2,

where Y1and Y2represent the remains of activating groups able to react with polypeptides containing reactive sulfur;

(Z)nrepresents the main polymer group, with n > 6.

25. Native receptor antagonist IL-1 on p. 24, wherein specified ones of the water-soluble polymer is a polyethylene glycol.

26. Mutein 30 kDa inhibitor of TNF or its truncated version containing advanced relative to the native polypeptide reactive sulfur, amino acid sequence which is at least one negativly the cysteine residue in position 1, 14, 105, 111, or 165.

27. Mutein under item 26, characterized in that the specified negativly the cysteine residue is located at position 105.

28. Mutein receptor antagonist IL-1 containing complementary to the native polypeptide reactive sulfur, amino acid sequence which is at least one negativly the cysteine residue in position 0, 84, 6, 8, 9, or 141.

29. Mutein under item 28, wherein the cysteine residue at position 116 NAT is which is a 30 kDa inhibitor of TNF or its scaled down version, containing at least one negativly the cysteine residue in position 1, 14, 105, 111 and 165, and at least one native or negativly the cysteine residue covalently attached to ones water-soluble polymer that is biologically inert and has a General formula

Y1- (Z)n- Y2,

where Y1and Y2represent the remains of activating groups able to react with polypeptides containing reactive sulfur; (Z)nrepresents the main polymer group, with n > 6.

31. Connection on p. 30, characterized in that the specified negativly the cysteine residue is located at position 105.

32. Connection on p. 30 or 31, characterized in that (Z)nselected from polypropylenglycol, polyoxyethylene glycerol, dextran, acids colon, poly-amino acids or polymeric carbohydrates.

33. The Union, representing the receptor antagonist IL-1, containing at least one negativly the cysteine residue in position 0, 84, 6, 8, 9, or 141, and at least one native or negativly the cysteine residue covalently attached to ones water-soluble polymer, which is biological which are the remains of an activating group, able to react with polypeptides containing reactive sulfur;

(Z)nrepresents the main polymer group, with n > 6.

34. Connection on p. 33, wherein the cysteine residue at position 116 native receptor antagonist IL-1 replaced by another amino acid residue.

35. The method of obtaining mutein polypeptide containing complementary to the native polypeptide reactive sulfur, comprising obtaining a DNA sequence that encodes a polypeptide of interest to us, the introduction of the published sequence using site-specific mutagenesis mutations, providing sequence that encodes mutein with at least one negativnym a cysteine residue, the expression of the modified DNA sequences in the bacterial cell, the separation, purification, and, if necessary, reactivation expressed polypeptide, wherein the site-specific mutagenesis is subjected to a DNA sequence encoding a polypeptide selected from a TNF inhibitor, receptor antagonist IL-1 and biologically active portions, and change is made in any site sequence, and forms with the target is, trichosis the fact that the TNF is a 30 kDa inhibitor of TNF, or a scaled down version, or 40 kDa inhibitor of TNF, or a scaled down version.

 

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