UA99454C2 - Methods of treatment using glycopegylated g-csf - Google Patents

Abstract

The present invention provides a glycopegylated G-CSF that is therapeutically active and which has pharmacokinetic parameters and properties that are improved relative to an identical, or closely analogous, G-CSF peptide that is not glycopegylated. Furthermore, the invention provides methods for mobilizing hematopoiesis in a subject, particularly a subject who has received or will receive radiation or chemotherapy treatment. The methods and compositions of the invention can further be used lo prevent, - alleviate and treat the myelosuppressive effects such therapies.

Description

A valid application claims priority according to the filing dates of the prior patent application

US Mo. 60/909917 filed Apr. 2007; US Patent Application No. 60/911788, filed April 13, 2007; and prior US patent application Ser. No. 60/986,240, filed Nov. 7, 2007, the entire contents of which are incorporated herein by reference for all purposes.

Granulocyte colony-stimulating factor (5-C5E) is a glycoprotein that stimulates the survival, proliferation, differentiation and function of progenitor cells of neutrophil granulocytes and mature neutrophils. Both forms of recombinant human 5-C5E used in the clinic are powerful stimulators of neutrophil granulopoiesis, effective in preventing infectious complications of some neutropenic conditions. They can be used to accelerate the recovery of the neutrophil population after myelosuppressive effects. a-C5E reduces the morbidity of anticancer chemotherapy by reducing the frequency of febrile neutropenia, the morbidity of high-dose chemotherapy with simultaneous bone marrow transplantation, and the frequency and duration of infectious diseases in patients with severe chronic neutropenia.

In addition, 5-C5E has been shown to have a therapeutic effect when administered to a patient after the onset of a myocardial infarction.

It is well known that acute myelosuppression due to cytotoxic chemotherapy is a dose-limiting factor in cancer therapy. Adverse effects are also observed in other normal tissues, but the bone marrow is most sensitive to treatments specific to proliferating cells, such as chemotherapy and radiation therapy. For some cancer patients, hematopoietic toxicity often limits the ability to increase the dose of chemotherapy. Repeated or high-dose courses of chemotherapy can deplete the pool of hematopoietic stem cells and their progeny.

Prevention and protection from the side effects of chemotherapy and radiation therapy can be of great benefit to cancer patients. O-C5BE and other growth factors have been shown to reduce the severity of such side effects by increasing the number of critical target cells, especially hematopoietic progenitor cells.

Human 5-C5E was cloned in 1986 by groups of researchers from Japan and the United States (see, for example, Madaya et al., Maishge 319, 415-418, 1986). There are two forms of natural human glycoprotein, one containing 175 and the other 178 amino acids. For the development of pharmaceutical products using recombinant DNA technology, a more active form, which is more common, containing 175 amino acids, was used.

Recombinant human (5-C5E, synthesized in the BE. soy expression system, is called filgrastim. The structure of filgrastim is slightly different from the natural glycoprotein. Another form of human recombinant 5-C5E is called lenograstim and is synthesized in Chinese hamster ovary cells (CHO). pa- C5E is a monomeric protein that dimerizes the 5-C5ZE receptor by forming a 2:2 complex of 2 O-C5BE molecules and 2 receptors (Nogap eii ai., Viospetivighu, 35(15): 4886-96 (1996)). X-ray crystallography method revealed the following residues in the composition of 5-C5E, which form the part of the molecule interacting with the receptor: (34, P5, Ab, 57, 58,19, P10, 011, 512,115,

K16, E19, 020,1108, 0109, 0112, 7115, T116, 9119, E122, E123 and 1124 (see, for example, Ashoti ei a!., (1999) Maishge 401: 713).

Commercially available forms of gps-C5E have a short-term pharmacological effect and often have to be administered to the patient more than once a day during the leukopenia state. A longer half-life of the molecule in circulation would reduce the number of injections needed to alleviate leukopenia and prevent infections. Another problem with commercially available hpe-C5E products is the occurrence of dose-dependent bone pain. Because bone pain experienced by patients is a significant side effect of hPO-C5E therapy, the development of a hPO-C5E product that does not cause bone pain, or the development of a product that does not have these effects by nature or efficacy at low doses, is desirable , insufficient for the development of bone pain. Thus, there is an obvious need to develop improved recombinant 5-05 molecules.

Artificially produced variants of pO-C5E have been described (see US Patents MoMo 5581476, 5214132, 5362853, 4904584 and Kiveapaag-Oiivop ei ai., Viospetivighu 35: 9034-9041, 1996). Modification of pO-C5E and other polypeptides by introducing at least one additional carbohydrate chain compared to the native polypeptide was also described (US patent Mo 5218092). In addition, polymeric modifications of native PO-C5E have been described and studied, including the addition of REOs groups (see, for example, Zaiake-I5pikama et al., (1992) Seyi Zigisishte apa ERipsip 17: 157; Vomep 6i et al., (1999) Echrgythepia!Netaioiodu 27: 425; US Patents MoMo 5824778, 5824784, MO 96/11953, MO 95/21629 and UMO 94/20069).

The joining of synthetic polymers to the polypeptide chain in order to improve the pharmacokinetic properties of glycoprotein drugs is known from the state of the art. A typical example of a polymer conjugated with peptides is polyethylene glycol (RES, PEG). It was shown that the use of RES for the derivatization of peptide drugs reduced the immunogenicity of the peptides.

For example, US patent No. 4179337 (Rami5 ei a.) discloses non-immunogenic polypeptides, such as enzymes and peptide hormones, bound to polyethylene glycol (PES) or polypropylene glycol. Such peptides, in addition to reduced immunogenicity, are characterized by an increased clearance time from the circulation due to an increase in the size of the conjugates of the peptides in question with REO.

One method of attachment of RES (and its derivatives) to peptides is non-specific binding through an amino acid residue of the peptide (see, for example, US Pat. No. 4,088,538, US Pat. No. 4,414,147, US Pat. No. 4,055,635, and PCT MO 87/00056). Another method of attaching RES to peptides is the non-specific oxidation of glycosylic residues on the glycopeptide (see, for example, M/O 94/05332).

When using these non-specific methods, polyethylene glycol is attached randomly and non-specifically to reactive residues in the peptide chain. It is obvious that the random joining of RES molecules is characterized by a number of disadvantages, including the heterogeneity of the final product and the possibility of a decrease in the biological or enzymatic activity of the peptide. Thus, for the production of medicinal peptides, the derivatization strategy is preferred, which leads to the receipt of a specifically labeled, easily characterized and practically homogeneous product. Such methods have been developed.

Specifically labeled, homogenous peptide drugs can be obtained using enzymes. In contrast to the typical non-specific methods of attaching a synthetic polymer or other label to a peptide, syntheses based on enzymes have the advantages of regioselectivity and stereoselectivity. Two major classes of enzymes useful for the synthesis of labeled peptides include glycosyltransferases (eg, sialyltransferases, oligosaccharyltransferases, M-acetylglucosaminyltransferases) and glycosidases. These enzymes can be used for the specific addition of sugars, which can then be modified to obtain a medicinal substance. Alternatively, glycosyltransferases and modified glycosidases can be used to directly transfer modified sugars to the polypeptide chain (see, e.g., U.S. Pat.

US Mo. 6399336 and US Patent Application Publications 20030040037, 20040132640, 20040126838 and 20040142856, each of which is incorporated herein by reference). There are also known methods that combine elements of both chemical and enzymatic synthesis (see, for example, Hatatoio ei a) I., Sagronuag. Transportation 305:415-422 (1998) and US Patent Application Publication 20040137557, incorporated herein by reference).

In response to the need for an improved medicinal 5-C5E, the present invention provides a glycopegylated 5-C5E that has therapeutic activity, as well as improved pharmacokinetic parameters and properties compared to non-glycopegylated identical peptide 5-C5E or its close analog. Further, this invention presents methods of enhancing hematopoiesis in a subject, especially in a subject who has received or will receive treatment in the form of radiation or chemotherapy.

Accordingly, in one aspect, the present invention provides methods and compositions that stimulate the production of stem cells in a bone marrow transplant recipient. These methods and compositions include the introduction of a bone marrow transplant recipient of a certain amount of peptide, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group. In one aspect, the polymeric modifying group is covalently attached to the peptide near a glycosyl or amino acid residue of the peptide via an intact glycosyl linking group.

In another aspect, the present invention provides methods and compositions that increase the number of granulocytes in a subject. This method includes the stage of introducing a certain amount of peptide to the subject, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group. In one aspect, the 5-C5E peptide has the amino acid sequence set forth in ZEO IO MO:1, and a polymeric modifying group is covalently attached to the C-C5E peptide in a region of the amino acid sequence that begins with glycine at position 126 and ends with serine at position 143.

In yet another aspect, the present invention provides methods and compositions that increase the production of stem cells in a subject. These methods include the stage of introducing a certain amount of peptide to the subject, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group. In a further aspect, the 5-C5E peptide includes a structure according to the formula b given oaimAst (vadu va PE where a is 0 or 1; and

Zia-RES has a structure according to the formula sob no on | 5 no. КК вы- о о т ; . J. sidvis ON

Nas Azy nu MM p su de p means an integer from 1 to 2000.

In one aspect, the present invention provides methods of preventing, treating, or attenuating myelosuppression, particularly myelosuppression due to anticancer therapy. In one aspect, this method includes administering to the recipient a certain amount of the peptide, which is a covalent conjugate of the C-SZE peptide and a polymeric modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the 5-C5E peptide near the glycosyl or amino acid residue of the C-C5E peptide with the help of an intact glycosyl linking group.

In another aspect, the present invention provides methods of treating a disease in a subject characterized by inhibition of white blood cell production. In one aspect, the method of treating the disease includes the step of administering to the subject a certain amount of the peptide, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the 5-C5E peptide near the glycosyl or amino acid residue of the 5-C5E peptide using an intact glycosyl linking group.

The amount of peptide administered to the subject is sufficient to effectively alleviate the subject's condition.

In yet another aspect, the present invention provides methods of treating neutropenia in a mammal. These methods include the step of introducing a pharmaceutically effective amount of the peptide, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the 5-C5E peptide near the glycosyl or amino acid residue of the C-C5E peptide with the help of an intact glycosyl linking group.

In yet another aspect, the present invention provides methods of treating thrombocytopenia in a mammal.

These methods include the step of introducing a pharmaceutically effective amount of the peptide, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the -C5E peptide near the glycosyl or amino acid residue of the 5-C5E peptide using an intact glycosyl binding group.

In one aspect, the present invention provides methods of increasing a population of hematopoietic stem cells in culture. These methods include the step of adding to the culture of stem cells an effective amount of the peptide, which is a covalent conjugate of the peptide O-C5BE and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the C5-C5E peptide near the glycosyl or amino acid residue of the 5-C5E peptide using an intact glycosyl binding group.

In another aspect, the present invention provides methods of stimulating hematopoiesis in a subject. These methods include the step of introducing an effective amount of the peptide to the subject, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the 5-C5E peptide near the glycosyl or amino acid residue of the C-C5E peptide with the help of an intact glycosyl linking group.

In yet another aspect, the present invention provides methods of increasing the number of hematopoietic progenitor cells in a subject. These methods include the step of introducing an effective amount of the peptide to the subject, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group.

The polymeric modifying group of the covalent conjugate can be covalently attached to the peptide O-

CE near the glycosyl or amino acid residue of the C-C5E peptide with the help of an intact glycosyl binding group.

In one aspect, the present invention provides methods of stimulating the production of stem cells in a donor. These methods include the step of introducing an effective amount of peptide to the donor, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the -C5E peptide near the glycosyl or amino acid residue of the 5-C5E peptide using an intact glycosyl binding group.

In another aspect, the present invention provides methods of increasing the long-term engraftment of a bone marrow transplant in a recipient. These methods include the stage of introducing the recipient into the bone marrow of the peptide, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the C-C5E peptide near the glycosyl or amino acid residue of the C-C5E peptide using an intact glycosyl binding group.

In yet another aspect, the present invention provides methods of stimulating hematopoietic progenitor cells in a subject. These methods include the stage of administering to the subject a first composition containing a compound of the formula 1,1-11,4-phenylenebis-(methylene)-bis-1,4,8,11-tetraazacyclotetradecane (AMOZ100), and a second composition containing peptide, which is a covalent conjugate of the peptide -

SZE of the polymer modifying group. The polymeric modifying group of the covalent conjugate can be covalently attached to the C5-C5E peptide near the glycosyl or amino acid residue of the 5-C5E peptide using an intact glycosyl binding group. The first and second compositions can be introduced to the subject sequentially in any order or simultaneously.

In one aspect, the present invention provides a dosage form for oral administration.

This dosage form for oral administration may contain the following components: (a) a peptide that is a covalent conjugate of the 5Z-C5E peptide and a polymeric modifying group. The polymeric modifying group can be covalently attached to the C-SZE peptide near the glycosyl or amino acid residue of the 5-C5E peptide with the help of an intact glycosyl binding group; (B) surface-active substance (substances); (c) fatty acid(s); and (4) enteric material. In one aspect, components (a), (b) and (c) are mixed in a liquid phase and lyophilized before combining with component (4).

In another aspect, the present invention presents a method of increasing the production of stem cells in a donor, where the method includes the steps of introducing a certain amount of peptide to the donor, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group. In a further aspect, the polymer modifying group of the covalent conjugate is attached to the peptide near the glycosyl or amino acid residue of the peptide using an intact glycosyl linking group. In yet another aspect of the invention, the amount of peptide administered to the donor ranges from 1 mg to about 20 mg.

Another aspect of the invention presents a method of increasing the production of stem cells in a donor, where the method includes the steps of introducing a certain amount of peptide into the donor, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group. In a further aspect, the polymeric modifying group is attached to the peptide near the glycosyl or amino acid residue of the peptide using an intact glycosyl linking group. In yet another aspect, the amount of peptide administered to the donor may be in the form of a unit dosage form. In one embodiment, a single dose is selected from 25, 50, 100 and 200 mcg/kg.

In fig. 1 shows data relating to the absolute number of neutrophils (AMC) in response to XM22 (25 μg/kg; 50 μg/kg; 100 μg/kg) and neylast (100 μg/kg).

In fig. 2 shows the data relating to the number of POP4 cells in response to XM22 (25 μg/kg; 50 μg/kg; 100 μg/kg) and Neylast (100 μg/kg).

Fig. C is a table of data relating to pharmacokinetic parameters for four different study groups.

In fig. 4 shows data relating to the absolute number of neutrophils (AMC) in response to XM22 (6 mg) and neylast (6 mg).

In fig. 5 shows the data relating to the number of SHUOZ4 cells in response to XM22 (6 mg) and neylast (6 mg).

In fig. 6 shows the data of pharmacodynamics, which relate to the number of neutrophils in response to o-

C5E, glycoRES-C-C5BE, neilasta and control composition in Javan macaques.

In fig. 7 shows pharmacokinetic data, which relate to the concentration of the specified compounds in the plasma of Javan macaques.

In fig. 8 shows a schematic model of the structure of glycoRES-(4-C5E) and its receptor.

In fig. 9 shows the data relating to the concentration of XM22 and neilasta in blood serum after the administration of three different doses of XM22 and 100 μg/kg of neilasta.

In fig. 10 shows the data relating to the concentration of XM22 and neilasta in blood serum after administration of 6 mg of XM22 and 6 mg of neilasta.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Abbreviation

RES, PEG - polyethylene glycol; PPO, PPG - polypropylene glycol; Aha - arabinosyl; Eggs - fructosyl;

Rice o - fucosyl; ba! - galactoside; saiMAS-M-acetylgalactosaminyl; (zIsC - glucoside; (ISMAcS-M- acetylglucosaminyl; Map - mannosyl; MapAs - mannosaminyl acetate; Hu! - xylosyl; MecAs - sialyl (M- acetylneuraminyl); MbR - mannose-6-phosphate; 5ia - sialic acid, M- acetylneuraminil and their derivatives and analogues. "α-C5E" means granulocyte colony-stimulating factor.

Definition

Unless otherwise defined, the meaning of all technical and scientific terms used in this application generally coincides with the generally accepted meanings known to a person skilled in the art to which this invention belongs. In general, the nomenclature and laboratory procedures used in this application relating to cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are well known and widely used in the art. Standard techniques are used for the synthesis of peptides and nucleic acids. In general, the techniques and procedures are carried out in accordance with generally accepted methods known in the art and various general guidelines (see ZatyuogokK ei aiY., MoiIesshiag SiIopipd: A Garogaiogu Mapiai, 2 ed., (1989) Soda 5rgipd Nagbrog

And arogafog Rgez55, Soiy 5rgipd Nagrog, MU, included in this application by reference), references to which are given in the text of this document. The nomenclature and laboratory procedures used in this application relating to analytical chemistry and organic synthesis described below are well known and widely used in the art. Standard techniques or their modifications are used for chemical syntheses and chemical analyses.

All oligosaccharides described in this application are described by the full or abbreviated name of the non-reducing saccharide (i.e. saccharide), followed by the configuration of the glycosidic bond (c or VD), the bond with the ring (1 or 2), the position in the ring of the reducing saccharide, involved in the bond (2, 3, 4, 6 or 8), and the full or abbreviated name of the reducing saccharide (eg, SISMAc). Each saccharide is preferably a pyranose. In the review of E55epiaie oi siusoriodu, ed. Magki ei aiI,, S5NI Rhev55 (1999) considered the generally accepted glycobiological nomenclature.

It is assumed that oligosaccharides have reducing and non-reducing ends, regardless of whether the saccharide at the reducing end is a reducing saccharide. In accordance with accepted nomenclature, in this application oligosaccharides are depicted with a non-reducing end on the left and a reducing end on the right.

The term "sialic acid" refers to any member of the family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is M-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-O-glycero-O-galactonulopyranos-1-anoic acid (often abbreviated to Meib5Ac, MetAc or MAMA). The second representative of the family is M-glycolylyl euraminic acid (Men5Os or Meiss), where the M-acetyl group of MecAs is hydroxylated. The third representative of the sialic acid family is 2-keto-3-deoxynonulosonic acid (KOM) (Madapo eyi ai. (1986), 9. Vioi. Spet. 261:11550-11557;

Kapatogi eyi ai!., u. Vioi Spent 265: 21811-21819 (1990)). Also included here are 9-substituted sialic acids such as 9-O-Ci1-Svacyl-Mei5Ac, such as 9-O-lactyl-Mei5Ac or 9-O-acetyl-Mei5Ac, 9-deoxy-9-fluoro-Mei5Ac, and 9- azido-9-deoxy-Mey5Ac. See Mackey's review of the sialic acid family,

Journal of Biochemistry 2:25-40 (1992), Ziaius Asiae: Spettivigu, Meigaboiust apa Eipsiop, eds. K. 5spapceg (Zrgipdeg-

Mepad, Mem HogKk (1992)). The synthesis and use of sialic acid compounds in the sialylation procedure is disclosed in International Patent Application UMO 92/16640, published on October 1, 1992.

The term "peptide" means a polymer consisting of amino acid monomers linked by amide bonds, also called a polypeptide. In addition, unnatural amino acids such as DV-alanine, phenylglycine and homoarginine are included. Amino acids not coded by genes can also be used in the present invention. Further, the present invention can use modified amino acids that contain reactive groups, glycosylation sites, polymers, medicinal substances, biomolecules, etc. All amino acids used in the present invention can be both O- and I-isomers. In general, the G-isomer is preferred. In addition, other peptidomimetics may be used in the present invention. As used herein, "peptide" refers to both glycosylated and non-glycosylated peptides. This also includes peptides that are not fully glycosylated in the peptide expression system. See the review of Zraiyoia ei aiYi.,, in Spetivig apa

Viospetivigu oi Atipo Asiav, Reriides apa Rgoieip5, ed. V. UMeip5ieiip, Mags! OeckKeg, Mem MogK, p. 267 (1983).

An amino acid or nucleotide sequence of a peptide is "homologous" to another sequence if they are somewhat identical. Preferably, the homologous sequence is at least about 85 95 identical to the reference sequence, preferably about 90-100 95 identical, more preferably at least about 91 95 identical, at least about 92 95 identical, at least about 93 95 identical, at least about 95 identical by 94 95, even more preferably identical by at least about 95-99 95, preferably identical by at least about 96 95, identical by at least about 97 95, identical by at least about 98 95, even more preferably identical by at least about 99 95, and identical on approximately 100 95 reference amino acid or nucleotide sequence.

The term "peptide conjugate" refers to a variant of the invention where the peptide is conjugated to a modified sugar as described in this application.

The term "amino acid" refers to natural and synthetic amino acids, as well as derivatives and mimetics of amino acids that have a similar function to natural amino acids. Natural amino acids are amino acids encoded by the genetic code, as well as amino acids that are subject to further modification, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Analogues of amino acids mean compounds that have a basic chemical structure identical to natural amino acids, that is, they contain an α-carbon atom with attached hydrogen, a carboxyl group,

amino group and K-group, i.e. homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.

Such analogues have modified K-groups (for example, norleucine) or modified peptide chains, but retain the basic chemical structure identical to natural amino acids. Amino acid mimetics mean chemical compounds that have a structure different from the basic chemical structure of amino acids, but whose function is similar to that of natural amino acids.

As used in this application, the term "modified sugar" means a natural or non-natural sugar attached enzymatically to an amino acid or glycosyl residue of a peptide in the method of this invention. The modified sugar is selected from enzyme substrates, including but not limited to sugar nucleotides (mono-, di-, and triphosphates), activated sugars (eg, glycosylhalides, glycosylmesylates), and sugars that are neither activated nor nucleotide sugars. A "modified sugar" is covalently functionalized with a "modifying group". Applicable modifying groups include, but are not limited to, moieties

RES, medicinal fragments, diagnostic fragments, biomolecules, etc. The modifying group is mainly an unnatural or non-unmodified sugar. The point of functionalization by the modifying group is chosen in such a way that it does not interfere with the attachment of the "modified sugar" enzymatically to the peptide.

The term "water-soluble" refers to moieties having some detectable solubility in water.

Methods for detecting and/or measuring solubility in water are well known in the art. Typical water-soluble polymers include peptides, saccharides, polyesters, polyamines, polycarboxylic acids, etc. Peptides can have shifted sequences or consist of a single amino acid, such as polylysine. A typical polysaccharide is polysialic acid. A typical polymer is polyethylene glycol.

Polyethyleneimine is a typical polyamine, and polyacrylic acid is a typical polycarboxylic acid.

The polymer chain of the water-soluble polymer can be polyethylene glycol (ie REF).

However, it should be understood that other related polymers are also applicable in this invention and that in this connection, the term RES or polyethylene glycol is inclusive and not exclusive. The term REO includes polyethylene glycol in any form, including alkoxy-RES, difunctional RES, multi-armed RES, bifurcated RES, branched RES, suspended RES (ie, RES or related polymers in which one or more functional groups are suspended from the polymer chain) or RES, containing destructible links.

The polymer chain can be linear or branched. Branched polymer chains are known from the prior art. Usually, a branched polymer consists of a central part and a set of linear polymer chains connected to the central part. Branched forms of RES obtained by adding ethylene oxide to various polyols, such as glycerol, pentaerythritol, and sorbitol, are commonly used. The central part can also consist of several amino acids, such as lysine. Branched polyethylene glycol can generally be represented by the formula K-(RES-OH)t, where K represents the central part, such as glycerol or pentaerythritol, and t represents the number of branches.

Multi-armed RES molecules, such as those described in US Pat. No. 5,932,462, fully incorporated herein by reference, can also be used as a polymer chain.

Many other polymers may be used in the present invention. Particularly applicable in this invention are non-peptide water-soluble polymer chains having from 2 to 300 ends. Examples of applicable polymers include, but are not limited to, other polyalkylene glycols such as polypropylene glycol ("PPO"), copolymers of ethylene glycol and propylene glycol and the like, polyoxyethylated polyol, polyolefin alcohol, polyvinylpyrrolidone, polyhydroxypropylmethacrylamide, poly-c-hydroxy acid, polyvinyl alcohol, polyphosphazene, polyoxazoline , poly-h-acryloylmorpholine, such as described in US Pat. No. 5,629,384, fully incorporated herein by reference, as well as their copolymers, terpolymers, and mixtures thereof. Although the molecular weight of the polymer chains can vary, it is usually in the range of about 100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da.

As used herein in the context of administration of a peptide drug to a patient, the "area under the curve" ("AC") is defined as the total area under the curve that describes the concentration of the drug in the patient's systemic bloodstream as a function of time from zero to infinity.

When used in this application in the context of administering a peptide medicinal product to a patient, the term "half-life" or "1/2" is defined as the time required to halve the concentration of the medicinal product in the patient's blood plasma. The same peptide drug may have several different half-lives depending on multiple mechanisms of clearance, redistribution, and other mechanisms well known in the art. Usually, the sa half-life and D half-life are determined in such a way that the sa phase is associated with redistribution, and the B phase - with clearance. However, for protein drugs that largely do not leave the bloodstream, there may be at least two half-lives associated with clearance. For some glycosylated peptides, rapid clearance during B-phase may be mediated by receptors on macrophages or endothelial cells that recognize terminal galactose, M-acetylgalactosamine, M-acetylglucosamine, mannose, or fucose. Slower B-phase elimination may be due to renal glomerular filtration of molecules with an effective radius of 2 nm (approximately 68 kDa) and/or specific or nonspecific tissue uptake and metabolism. Glycopegylation can cap terminal sugars (eg, galactose or M-acetylgalactosamine) and thus block rapid α-phase clearance by receptors recognizing these sugars. Also, it can increase the effective radius of the molecule and thus weaken its distribution and absorption by tissues, which leads to a prolongation of the late B-phase. Thus, the exact mechanism of effect of glycopegylation on the half-life in α-phase and D-phase may vary depending on the size, degree of glycosylation and other parameters, as is well known in the art. For a more detailed explanation of the term "half-life" see Rpaptaseshiisai

Viotesppoiodu (1997, ed. BOTH Stotteiiip and KO 5ipaeiag, Naguiosa Rybiizpeg5, Ateegaat, pp. 101-120).

As used herein, the term "glycoconjugation" refers to the enzymatic conjugation of a modified sugar with an amino acid or glycosyl residue of a polypeptide, for example, a 5-C5E peptide according to the present invention. A subspecies of "glycoconjugation" is "glycopegylation", where the modifying group of the modified sugar is polyethylene glycol and its alkyl (for example, t-RES) or reactive (for example, NagM-RESi, HOOS-RES) derivatives.

The terms "large scale" and "industrial scale" are used interchangeably and mean a reaction cycle that yields at least about 250 mg, preferably at least about 500 mg, and more preferably at least about 1 g of glycoconjugate upon completion of one reaction cycle.

As used in this application, the term "glycosyl linking group" means a glycosyl residue to which a modifying group is covalently attached (for example, a RES molecule, drug substance, biomolecule); the glycosyl linking group links the modifying group to the rest of the conjugate. In the methods according to the invention, a "glycosyl linking group" is covalently attached to a glycosylated or non-glycosylated peptide, thus linking the agent to an amino acid and/or glycosyl residue in the peptide. A "glycosyl linking group" is usually derived from a "modified sugar" by enzymatically linking the "modified sugar" to an amino acid and/or glycosyl residue in the peptide. The glycosyl linking group may be a structure derived from a saccharide that breaks down in the process of cassette formation, the modifying group is a modified sugar (eg, oxidation -» Schiff base formation -» reduction), or the glycosyl linking group may remain intact. "Intact glycosyl linking group" means a linking group derived from a glycosyl groups, where the saccharide monomer connecting the modifying group and the other part of the conjugate is not destroyed, for example, by oxidation, for example, with sodium metaperiodate. "Intact glycosyl linking groups" according to the invention can be obtained from a natural oligosaccharide by addition of glycosyl unit(s) or cleavage of one or more glycosidic units from the original polysaccharide structure .

As used herein, the term "targeting fragment" means a fragment that selectively localizes to a specific tissue or body region. Localization is mediated by specific recognition of molecular determinants, molecular size of the targeting agent or conjugate, ionic interactions, hydrophobic interactions, etc. Other mechanisms of targeting an agent to a specific tissue or body part are known to those skilled in the art. Typical targeting fragments include antibodies, antibody fragments, transferrin, NoZ-glycoprotein, coagulation factors, serum proteins, VD-glycoprotein, 5-C5E, CM-C5E, M-C5BE, EPO and others.

As used in this application, the term "drug fragment" means any fragment suitable for therapy, including but not limited to antibodies, anti-inflammatory agents, antitumor agents, cytotoxins, and radioactive substances. "Drug fragment" includes prodrugs of bioactive substances, constructs in which several drug fragments are connected to a carrier, for example, multivalent agents. A medicinal fragment also includes proteins and constructs comprising proteins. Typical bilirubins include, but are not limited to, granulocyte colony-stimulating factor (05-C5E), granulocyte-macrophage colony-stimulating factor (ZM-C5BE), interferon (e.g., interferon a, B, y), interleukin (e.g., interleukin II), serum proteins (e.g., factors MII, MPa, MII, IX and X), human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), as well as proteins fused to antibodies (for example, tumor necrosis factor receptor (TNF) fused with domain Es).

As used in this application, the term "Pharmaceutically acceptable carrier" includes any material with which the conjugate retains activity and does not react with the subject's immune system.

Examples include, but are not limited to, any conventional pharmaceutical carrier such as phosphate-buffered saline, water, emulsions such as water-in-oil emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, including coated tablets, and capsules. Usually such carriers contain excipients such as starch, milk, sugar, some types of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols or other known excipients.

Such carriers may also include flavoring or coloring agents and other ingredients. Compositions containing such carriers can be formulated using widely known conventional methods.

As used in this application, the term "administration" means oral administration, suppository administration, topical contact, intravenous, intraperitoneal, intramuscular administration, intralesional administration, intranasal or subcutaneous administration, or implantation in a subject of a slow release device, e.g. mini-osmotic pump. Administration can be by any route, including parenteral and transmucosal (ie, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intragastric and intracranial administration. Further, when the injection is performed for the purpose of tumor therapy, that is, capable of causing apoptosis, the injection can be performed directly into the tumor and/or into the tissues surrounding the tumor. Other methods of delivery include, but are not limited to, the use of liposomal preparations, intravenous infusion, transdermal patches, etc.

As used in this application, the term "reduce the intensity" or "reduce the intensity" means any indication of the success of the treatment of the pathology or disease, including any objective or subjective parameters such as attenuation, reduction or reduction of symptoms or improvement of physical or mental health of the patient.

The term "therapy" means the "treatment" of a disease or condition, including preventing the occurrence of a disease or condition in an animal that may be susceptible to the disease but does not yet have or show symptoms of the disease (prophylactic treatment), suppression of the disease (slowing or stopping development), relief of symptoms or side effects of the disease (including palliative therapy) and treatment of the disease (regression of the disease).

The term "effective amount" or "amount effective for" or "therapeutically effective amount" or any grammatical equivalent means an amount which, when administered to an animal for the treatment of a disease, is sufficient to effectively treat the disease.

The term "separated" refers to material that is substantially or completely free of the components used to produce that material. For peptide conjugates according to the invention, the term "isolated" refers to material that is sufficiently or completely free of components that are usually adjacent to the material in the mixture used to prepare the peptide conjugate.

The terms "isolated" and "pure" are used interchangeably. Usually isolated peptide conjugates according to the invention have a level of purity, which is expressed mainly in the form of an interval.

Of course, the lower purity range of the peptide conjugates is about 60 95, about 70 95, or about 80 95 and the upper purity range is about 70 95, about 80 95, or greater than about 90 95.

If the purity of the peptide conjugates is greater than about 90 95, their purity is also preferably expressed by the interval. Of course, the lower value of the interval is about 90 95, about 92 95, about 94 95, about 96 956, or about 98 95. The upper value of the purity interval is about 92 95, about 94 95, about 96 95, about 98 95, or about 100 Fo .

Purity is determined using analytical methods known in the art (eg, band intensity on a silver-stained gel, polyacrylamide gel electrophoresis, HPLC, or similar methods).

As used in this application, "virtually every representative of the population" describes a characteristic of a population of peptide conjugates according to the invention in which a defined percentage of modified sugars attached to the peptide are attached to multiple identical acceptor sites in the peptide. "Substantially every member of the population" refers to the "homogeneity" of the sites within the peptide conjugated to the modified sugar, and means conjugates according to the invention that are homogeneous by at least about 80 95 , preferably by at least about 90 95 , and more preferably by at least about 95 95. "Homogeneity" means constancy of structure in the population of acceptor groups with which modified sugars are conjugated. Thus, if in a peptide conjugate according to the invention each modified sugar is conjugated to an acceptor site having the same structure as the acceptor sites to which all other modified sugars are conjugated, such a conjugate is said to homogeneous by 100 95. Homogeneity is usually expressed as an interval. The lower value of the homogeneity interval of the peptide conjugates is about 6095, about 7095, or about 80 95 and the upper value of the homogeneity interval is about 70 95, about 80 95, or greater than about 90 95.

If the homogeneity of the peptide conjugates is at least approximately 90 95, their purity is also preferably expressed by the interval. Of course, the lower value of the interval is about 90 95, about 92 95, about 94 95, about 96 956, or about 98 95. The upper value of the purity interval is about 92 95, about 94 95, about 96 95, about 98 95, or about 100 95 The purity of peptide conjugates is usually determined using one or more methods known to those skilled in the art (for example, liquid chromatography-mass spectrometry (LC-M5) or matrix laser desorption ionization time-lapse mass spectrometry ( oi Pidni zresigoteigu, MAGOITORE), capillary electrophoresis, etc.). "Sufficiently uniform glycoform" or "sufficiently uniform glycosylation pattern" when describing a glycopeptide means the percentage of acceptor groups glycosylated by the glycosyltransferase under consideration (eg, fucosyltransferase). For example, in the case of (1,2-fucosyltransferase), a sufficiently uniform pattern of fucosylation is observed, if in the peptide conjugate according to the invention, practically all (as defined below) molecules of CZaIIr1,4-cisMAs-K and their sialylated analogues are fucosylated. In fucosylated structures , described below, the EBys-SISMAs connection is usually an a1,6 or a1,3 connection, and in the general case the "1,6" connection is preferred. A specialist in the field of technology should understand that the source material may contain glycosylated acceptor groups (e.g., fucosylated groups of CyYBr1,4-CysMAs-K).This means that the calculated percentage of glycosylation will include both acceptor groups glycosylated by the methods of the present invention and acceptor groups that are in the glycosylated state in the starting material .

The term "sufficient" in the above definitions of "sufficiently homogeneous" generally means that at least about 40 95, at least about 70 95, at least about 8095, or more preferably at least about 9095 and even more preferably at least about 95 95 of the acceptor groups of a given glycosyltransferase are glycosylated.

When describing substituent groups using generally accepted chemical formulas, formulas written from left to right also apply to chemically identical substituent groups obtained when writing the formula from right to left, that is, for example, -СНгО- can also be written as -ОСН»-.

Unless otherwise specified, the term "alkyl" alone or as part of another substituent means a straight or branched chain or cyclic hydrocarbon radical, or a combination thereof, which may be fully saturated, mono- or polyunsaturated and may include di- and multivalent radicals, containing the specified number of carbon atoms (ie C1i-Cio means from one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, cyclohexylmethyl, cyclopropylmethyl, homologues and isomers such as n -pentyl, n-hexyl, n-heptyl, n-octyl, etc. An unsaturated alkyl group is a group that contains one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-butadienyl, 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, Z-butynyl and higher homologues and isomers. Unless otherwise indicated, the term "alkyl" also includes alkyl derivatives described in more detail below, such as "heteroalkyls." Alkyl groups limited to hydrocarbon groups are called "homoalkyls".

The term "alkylene" alone or as part of another substituent means a bivalent radical derived from an alkane, as can be seen from the non-limiting example -СНаСН».СНоСН»о-, and further includes the "heteroalkylene" groups described below. In general, an alkyl (or alkylene) group contains from 1 to 24 carbon atoms, with groups containing 10 or fewer carbon atoms being preferred for this invention. "Lower alkyl" or "lower alkylene" means an alkyl or alkylene group with a shorter chain, usually containing 8 or fewer carbon atoms.

The terms "alkyl", "alkylamino" and "alkylthio" (or thioalkyl) are used in their conventional sense and mean alkyl groups attached to another part of the molecule through an oxygen atom, an amino group or a sulfur atom, respectively.

The term "heteroalkyl" alone or in combination with another term means, unless otherwise specified, a stable straight or branched chain or cyclic hydrocarbon radical, or a combination thereof, which contains the specified number of carbon atoms and at least one heteroatom selected from the group consisting of with 0, M, 5i and 5, where the nitrogen and sulfur atoms may optionally be in the oxidized form, and the nitrogen heteroatom may optionally be in the quaternary form. Heteroatom(s) O, M, 5 and 5i can be in any internal position of the heteroalkyl group or in the position in which the alkyl group is attached to another part of the molecule. Examples include, but are not limited to -СН2»-СН2-О-СН3, -СН»-СНо-МН-СН3, -СН»-СНо-М(СН3)-СН3, -СНе-5-

Сно-СН3, -СН»-СНо-5(0)-СН3, -СН.-СНо-5(0)2-СН3, -СН-СН-О-СН3, -5(СН3)3, -СН- ASN-M-OSN: and -

SnH-SnH-M(CH3)-CH»z. Up to two heteroatoms can be arranged sequentially, for example, -СН2-МН-

ОСН3 ии -СНо-0-54(СН3)з. Similarly, the term "heteroalkylene" alone or as part of another substituent means a bivalent radical derived from a heteroalkyl, as can be seen from the non-limiting example of -СН2-СНг-5-СН»-СНе- and -СН2-5-СН»-СНо- СНе-. In heteroalkylene groups, heteroatoms can also occupy one or both extreme positions (for example, alkyleneoxy,

alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Further, for alkylene and heteroalkylene linking groups, the direction of writing the linking group formula does not determine the orientation of the linking group. For example, the formula "С(О)2Б"- means both -С(О)2НА- and -АЭЕ(О) 2-.

The terms "cycloalkyl" and "heterocycloalkyl" alone or in combination with other terms mean, unless otherwise indicated, the cyclic forms of "alkyl" and "heteroalkyl", respectively. In addition, in cycloalkyl, the heteroatom can occupy a position in which the heterocycle is attached to another part of the molecule.

Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to /-1-(1,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, Z-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl and etc.

The term "halo" or "halogen" alone or as part of another substituent means, unless otherwise indicated, an atom of fluorine, chlorine, bromine or iodine. In addition, terms such as "haloalkyl" include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C1)alkyl" includes, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

The term "aryl" means, unless otherwise specified, a polyunsaturated aromatic substituent, which may consist of one or more rings (preferably 1 to 3 rings), fused or covalently bonded. The term "heteroaryl" means aryl groups (or rings) containing from 1 to 4 heteroatoms selected from M, O and 5, where the nitrogen and sulfur atoms are optionally in the oxidized state, and the nitrogen atom(s) are optionally are in the quaternary state.

A heteroaryl group can be attached to another part of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl , 4-oxazolyl, 2-phenyl- 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2- furyl, 3-furyl, 2 -thienyl, Z-thienyl, 2-pyridyl, Z-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2 -quinoxalinyl, 5-quinoxalinyl, Z-quinolyl, tetrazolyl, benzo|furanyl, benzo|bi|hyenyl, 2,3-dihydrobenzo|1,4|dioxin-6-yl, benzo|1,Z|dioxol-5-yl and b-quinolyl. Substituents for each of these ring systems are selected from the group of acceptable substituents described below.

For brevity, when the term "aryl" is used in combination with other terms (eg, aryloxy, arylthiooxy, arylalkyl), the term includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" includes radicals where the aryl group is attached to an alkyl group (ie, benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (eg, a methylene group) is replaced by, for example, oxygen (for example, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

All of the terms above (ie, "alkyl," "heteroalkyl," "aryl," and "heteroaryl") include both substituted and unsubstituted forms of said radical. Preferred substituent groups for each of the radicals are listed below.

Substituents for aryl and heteroaryl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are collectively referred to as "alkyl substituents" and may be one or more different groups, selected from a non-limiting list: -ОВ', -0, -МВ" -М-ОВ', -МВА'В", - halogen, -5IV'В'В", -ОС(О)В', -С(О )B -СО2Н8, -SOMA'B", -OS(O)MA B" -MA'E(O)B -MA-C(O)MA B, -

MA'E(O)28", -MA-S(MA'VYA")-MA"" -MA-S(MA'B")-MA", -5(O)B -5(0) 28, -5(0)2MA B", -"MAZO"B", -SM and -

MO", in an amount varying from zero to (2!t'-1), where t is the total number of carbon atoms in each radical. B, K", KK" and K"", each independently preferably means hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkyl or thioalkyl groups, or arylalkyl groups . When the substance according to the invention includes more than one K-group, for example, each of the K-groups is selected independently, as well as each group from EK, K", Ki A", if more than one such group is present. If K. and

E" are bound to the same nitrogen atom, they can combine with the nitrogen atom in a 5-, 6- or 7-membered ring. For example, -MAE'K" means, but is not limited to, 1-pyrrolidinyl and 4- morpholinil. From the above discussion of substituents, it will be clear to one skilled in the art that the term "alkyl" includes groups that contain carbon atoms bonded to groups other than hydrogen atoms, such as haloalkyl (eg, -C3 and -CH2C3) and acyl (for example, -C(O)CH3, -C(O)CE", -

С(О)СНоОС НН: etc.).

Just as the substituents described for the alkyl radical, the substituents for aryl and heteroaryl groups have the general name "aryl substituents".

Substituent groups are selected from, for example, halogen, -OB, -0, -MA, -M-OB, -MA'B", -58", - halogen, -5IV'B'B", -OS(О)B -С(О)В -СО2Н8 -"СОМА' В", -ОС(О)МА' В", -MA'Е(О)В", -MAАС(О)МА В, -

MA'E(O)28", -"MA-S(MA'VYA")-MA" -MA-S(MA'V") MA", -5(O)8", -5(0 )28", -5(O)2MA B", -"MAZO"B", -SM and -

MO», -B», -Mz, -CH(PH)2, fluorine (Ci-Ca) alkoxy and fluorine (C:i-Ca) alkyl, in an amount varying from zero to the total number of free valences of the aromatic ring, and where KK, K", K" and K" each independently preferably means hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. When a substance according to the invention includes more than one K-group, for example, each of the K-groups is selected independently, as well as each group from KK, K", "its KE", if more than one such group is present. In the schemes below , the symbol X stands for "K" as described above.

Two substituent groups on adjacent atoms of an aryl or heteroaryl ring can optionally be replaced by a substituent of the form -Т-С(О)-(САА)-О-, where T and O independently represent -МВ-, -О-, - СВВ'- or a single bond, and и is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by substituents of the form -А-(СНег)-В- , where A and B independently represent -СВВ'-, -О-, -МВ-, -5-, -5(0)-, -5(0)2-, -50)2MA'- or a single bond bond, and r means an integer from 1 to 4. One of the single bonds of the newly formed ring may optionally be replaced by a double bond. Alternatively, two substituent groups on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by substituents of the form - (САБА) -Х-(СВ'В)а-, where 7 and y are independently integers from 0 to 3, and X stands for -O-, -MA", -5-, -

C(0)-, -50)2- or -5(0)2MA"-. Substituent groups K, KK, K" and K" are preferably selected independently from hydrogen or substituted or unsubstituted (C1-C8)alkyl.

As used herein, the term "heteroatom" includes oxygen (0), nitrogen (M), sulfur (5), and silicon (51). "Stem cells" means "multipotent" or undifferentiated cells capable of unlimited self-copying and differentiation into different tissues of the body. Stem cells can differentiate into normal blood components, including red blood cells, white blood cells, and platelets. Stem cells are usually found in the bone marrow and blood and can be extracted for transplantation.

The term "hematopoietic cell" means a cell associated with the formation of blood cells. The term can be used interchangeably with the term "stem cells" defined above.

As used in this application, the term "stimulation of the production of stem cells" includes all processes that increase the number of ip mimo or ip migo stem cells. A higher number of stem cells can be due to an increase in the number of progenitor cells. The term also includes the processes of stem cell transport into and out of the bone marrow.

Similarly, the term "stimulation of the production of hematopoietic stem cells" includes all processes that increase the number of hematopoietic cells ip mimo or ip migo. A greater number of hematopoietic cells may be due to a large number of progenitor cells, acceleration of the maturation of pluripotent stem cells into hematopoietic cells, or a combination thereof. The term also includes the processes of transport of hematopoietic cells into and out of the bone marrow.

The term "granulocytes" means white blood cells, which are characterized by the presence of granules in the cytoplasm.

Introduction

The present invention covers methods of administration of glycopegylated 5-C5E for the prevention, relief of symptoms or treatment of diseases and conditions associated with hematopoietic deficiency, often caused by chemotherapy, radiation therapy and thrombocytopenia. 5-C5E mainly acts on the bone marrow, stimulating the production of inflammatory leukocytes, and further acts as an endocrine hormone that initiates the replacement of neutrophils produced during inflammatory functions. 5-C5E is also used in the clinic for bone marrow replacement after chemotherapy.

This invention presents a conjugate containing granulocyte colony-stimulating factor (5-C5E). The present invention also extends to conjugates containing glycosylated and non-glycosylated peptides that have stimulating activity of granulocyte colony-stimulating factor. Conjugates can be further modified by further conjugation with various molecules, such as medicinal substances, diagnostic substances, targeting substances, etc. In the 5-C5E conjugates described in this application, a polymeric modifying group can be covalently attached to the glycosyl or amino acid residue in the 5-SZE peptide, preferably by means of a glycosyl binding group. In an exemplary embodiment, the polymer modifying group is a water-soluble polymer. In a further preferred embodiment, the water-soluble polymer is polyethylene glycol.

In exemplary embodiments, the 5-C5E peptide according to the invention can be administered to a patient for the purpose of preventing infection in cancer patients undergoing radiation therapy, chemotherapy, and bone marrow transplantation, for the purpose of mobilizing progenitor cells for collection during transplantation of progenitor cells from peripheral blood, for treatment of severe chronic or relative leukopenia, regardless of its cause, and for the maintenance therapy of patients with acute myeloid leukemia. In addition, polypeptide conjugates or compositions according to the invention can be used for the therapy of AIDS or other immunodeficiency diseases, as well as bacterial infections, heart disease and hepatitis A, B and C.

In one embodiment, the c-C5E peptide conjugate according to the invention can be administered to a subject to increase hematopoiesis. Hematopoiesis is the process by which progenitor cells develop into mature blood cells, including red blood cells, white blood cells, and platelets. Normal hematopoiesis is coordinated by the action of various regulators, including glycoproteins such as colony-stimulating factors. These regulators modulate the survival, proliferation and differentiation of stem and progenitor cells and the activation state of mature cells. Violation of hematopoiesis leads to a decrease in the production of blood cells and platelets, which leads to a decrease in immunity and the inability to heal wounds and fight infections.

The present invention presents methods and compositions for stimulating hematopoiesis in a subject.

Methods according to the invention include the stage of introducing the subject of an effective amount of the peptide, which is a covalent conjugate of the C-C5E peptide and a polymer modifying group.

In one aspect, stimulation of hematopoiesis includes increasing the number of hematopoietic progenitor cells in the subject, which also leads to an increase in mature hematopoietic cells (blood cells).

Hematopoietic progenitor cells move to and remain in the bone marrow, where they mature and turn into red and white blood cells. Methods of stimulating the number of hematopoietic progenitor cells according to the invention include the stage of introducing an effective amount of peptide to the subject, which is a covalent conjugate of the 5-C5E peptide and a polymer modifying group. In one embodiment, the use of the peptide increases the number of hematopoietic progenitor cells, which are POP4 cells.

Myelosuppression

Myelosuppression is a decrease in the production of blood cells. Normal blood contains a large number of cells, including red blood cells that carry oxygen and white blood cells that fight infection. Normal blood also contains platelets, tiny fragments of cells that start the blood clotting process. These cells and cell fragments come from bone marrow, the substance found inside some bones. Healthy bone marrow produces large numbers of red blood cells, white blood cells, and platelets every day. With myelosuppression, the bone marrow produces too few of these cells. The present invention provides methods and compositions for the treatment, relief of symptoms and prevention of myelosuppression.

One of the characteristic features of myelosuppression is suppression of the production of white blood cells in the subject.

This suppression of white blood cell production can be caused by certain types of therapy, especially anticancer therapy, such as chemotherapy and radiation therapy. Suppression of the production of white blood cells can also be caused by diseases such as idiopathic thrombocytopenic purpura. In one aspect, the conjugates according to the invention are used to treat and alleviate diseases characterized by impaired production of white blood cells.

Conditions due to myelosuppression include neutropenia (including febrile neutropenia) and thrombocytopenia. Neutropenia is a disease characterized by an abnormal decrease in the number of neutrophils in the blood (the most common type of white blood cells). The decrease can be relative or absolute. In one aspect, the present invention provides methods of treating neutropenia in a mammal. These methods include the steps of introducing a pharmaceutically effective amount of conjugates (3-C5E according to the invention. It was shown that o-

C5E affects febrile neutropenia and mortality among adult cancer patients (Kyadegeg ei ai., U. SiIiy. Ope. (2007), 25(21):3158-67).

Thrombocytopenia is a disease in which the number of platelets in the blood is abnormally low; this disease is often characterized by abnormal bleeding. Methods according to the invention include the therapy of thrombocytopenia in mammals. These methods include the steps of introducing a pharmaceutically effective amount of 5-C5E conjugates according to the invention. As used in this application, the term "thrombocytopenia" covers both diseases of known etiology and idiopathic thrombocytopenia. Thrombocytopenia and idiopathic thrombocytopenia are also referred to in this application as "thrombocytopenic purpura" and "idiopathic thrombocytopenic purpura".

Stimulation of stem cells

One way to overcome myelosuppression is to stimulate the production of stem cells.

Stimulation of stem cell production includes an increase in the number of stem cells, including the number of hematopoietic progenitor cells, and the number of granulocytes, including neutrophils and eosinophils.

Stimulation of stem cell production also includes enhanced transport of stem cells from the bone marrow into the peripheral blood. Such stimulation facilitates the collection of stem cells from the donor, since peripheral blood is more readily available than bone marrow. In one aspect, the present invention provides methods of stimulating the production of stem cells in a subject.

In another aspect, the prevention, relief of symptoms and treatment of myelosuppression is achieved through the use of methods and compositions according to the invention by stimulating hematopoietic progenitor cells in the subject. Stimulation of hematopoietic progenitor cells includes both an increase in the number of hematopoietic progenitor cells and enhanced transport of hematopoietic progenitor cells into and out of the bone marrow.

Hematopoietic progenitor cells, such as SOZ4zh cells, mature and differentiate into blood components, namely red and white blood cells. The present invention provides methods of stimulating hematopoietic progenitor cells in a subject, which include the step of administering to the subject: (1) a first composition containing a component having the formula 1,1-P1,4-phenylenebis-(methylene)- bis-1,4,8,11-tetraazacyclotetradecane (AMOZ100), and (2) a second composition containing a peptide that is a covalent conjugate of the 5-C5E peptide and a polymeric modifying group. In one embodiment, the first and second compositions are administered to the subject sequentially in any order. In another embodiment, the first and second compositions are administered to the subject simultaneously. In one embodiment, both compositions are administered subcutaneously to the subject.

АМОЗ3100 is a bicyclam derivative capable of mobilizing a large number of SOZ4zh cells into the bloodstream both in healthy subjects and in cancer patients, as was shown in the works of Iev Yei Ai.,

Viosa, (2003), 102:2728-30; Oemipe eyi ai., u. SiIip. Opsoi, (2004), 22:1095-1102. Also, studies have shown that the combination of AMOZ3100 and the non-glycosylated form of 5-C5E is able to mobilize a greater number of COZ4 cells into the bloodstream than 5-C5E alone (Riotepbego et al., Viosad, (2005), 106(5): 1867-1874) .

In one embodiment, the production of stem cells is stimulated in a subject who serves as a bone marrow or hematopoietic cell donor. The donor receives the peptide conjugate described above.

The number of stem cells in the donor increases, and these stem cells leave the bone marrow and enter the peripheral blood. Such cells are then easily isolated from the blood of the donor using methods known in the art. In such embodiments, the donor and recipient of the bone marrow or hematopoietic cells may be the same person (autologous donor) or the donor may be a subject who is not the recipient (allogeneic donor).

In another aspect, peptide conjugates according to the invention are combined with at least one chemotherapeutic agent.

Bone marrow transplants

In some aspects, the present invention provides methods and compositions for stimulating the production of stem cells in a recipient of a bone marrow transplant. These methods include the stage of introducing a certain amount of peptide to the recipient, which is a conjugate of the 5-C5BE peptide and a polymer modifying group. In one embodiment, the polymeric modifying group is a water-soluble polymer that can be covalently linked to the C-C5E peptide near a glycosyl or amino acid residue of the peptide (-C5E, preferably via an intact glycosyl linking group. The peptide can be administered to a recipient prior to bone marrow transplantation brain, after transplantation or simultaneously with transplantation.

The success of transplantation determines the long-term engraftment of the graft. The term "engraftment" refers to the process by which infused or transplanted donor stem cells are directed into the recipient's bone marrow and produce all types of blood cells. The first sign of engraftment is the appearance of new white cells, red cells and platelets in the blood of the recipient after stem cell transplantation. Long-term engraftment refers to the process by which infused or transplanted donor stem cells remain in the recipient's bone marrow and produce blood cells for a long period of time without being rejected by the recipient's immune system. When middle and late progenitor cells are included in the transplant, the production of mature cells of donor origin is accelerated and the engraftment process is supported.

In accordance with the above, the present invention provides methods and compositions that increase the long-term engraftment of a bone marrow transplant received by the recipient. In one exemplary embodiment, the peptide according to the invention is administered to a donor prior to bone marrow transplantation. The peptide stimulates hematopoiesis in the donor, in particular by increasing the number of progenitor cells, which increases the probability of successful and long-term engraftment when transplanting the recipient's bone marrow and/or hematopoietic cells. The transplanted bone marrow can belong to the recipient himself (autologous transplant) or the bone marrow can be transplanted from a donor of the same biological species (allogeneic transplant).

In another embodiment, the peptide according to the invention is administered to a bone marrow recipient in order to increase the long-term engraftment of a donor bone marrow transplant obtained from the recipient or another subject. Receiving the peptide by the recipient can stimulate hematopoiesis in the recipient, which increases the probability of graft engraftment by stimulating the production of hematopoietic progenitor cells by the donor bone marrow.

Transplants of hematopoietic cells

Transplantation of hematopoietic cells is a generally accepted method of treatment of various hereditary or malignant diseases. While some transplant procedures use the entire population of bone marrow cells, other procedures use narrower populations enriched with stem cells. In addition to bone marrow, such cells can be obtained from other sources, such as peripheral blood and umbilical cord blood of infants. One of the advantages of using peripheral blood stem cells is that such cells are much easier to extract from peripheral blood than from bone marrow. However, the low number of pluripotent cells/progenitor cells in the bloodstream is a limiting factor for peripheral blood stem cell transplantation. Thus, for use in transplantation, it is necessary to increase the number of ex mimo stem cells.

Accordingly, the present invention provides methods for increasing the number of stem cells in culture. In an exemplary embodiment, such methods include adding an effective amount of the peptide according to the invention to the hematopoietic cell culture. In an exemplary embodiment, such a peptide is a conjugate of the 5-C5ZE peptide and a polymeric modifying group. In one embodiment, the polymer modifying group is a water-soluble polymer that can be covalently attached to the 5-C5E peptide near a glycosyl or amino acid residue of the 5-C5E peptide, preferably with an intact glycosyl linking group. In one embodiment, the present invention provides methods of obtaining increased populations of stem cells and progenitor cells that can be used for transplantation. These methods include the step of adding to the stem cell culture an effective amount of the peptide according to the invention.

Organ transplants

Just like bone marrow transplants, solid organ transplants such as liver, kidney, heart and lung can cause different immune reactions in the recipient. Such immune reactions can lead to acute rejection of these transplants. 5-C5E and other hematopoietic growth factors can be used to treat such responses (see US Pat. No. 5,718,893). Accordingly, the methods and compositions of the present invention can be used to prevent or reduce the likelihood of developing acute rejection of organ transplants in a patient.

Heart disease

In one embodiment, the methods and compositions of the present invention can be used to alleviate the course of heart disease and improve heart function. In one embodiment, methods and compositions according to the invention are used to stimulate the release of stem cells that form vessels. 5-C5E therapy can reduce angina in patients with heart disease, including patients after multiple surgeries and patients taking maximum doses of standard medications (see, for example, Medisa! Memuz Todau, Uipe 4, 2007, "Bemege Neagi Oizease Raiiyepiz OPyegei Mempi Nore"). Other studies have shown that 5-C5E is able to protect the heart muscle, preventing its death, even if the muscle has been damaged by heart disease (see, for example, Zippaau TeIedharpy Mem/5, AchChdivi 5, 2007, "OSyg UMopa-

Tigwi Neapiz Iyai Reraig Tnetvze/mev"). (4-C5ZE alone or in combination with mature stem cells from patients can be used during treatment to replace dead heart tissue and form new blood vessels.

Neurological diseases

In one embodiment, the methods and compositions of the invention can be used to treat neurological diseases, including but not limited to Alzheimer's disease and other degenerative brain diseases. Studies of Alzheimer's disease in mouse models have shown that in these models 5-C5E can reverse Alzheimer's-like symptoms (see Tzai et al., (2007), 9. Ehr. Mea. 204(6): 1273-80). These studies showed that injection of 5-C5E into the bloodstream stimulates the release of hematopoietic cells from the bone marrow. These stem cells travel from the bloodstream to the brain, where they attach to sites of damage and differentiate into new cells.

The use of 5-C5E leads to the growth of new cells in the places of the greatest damage to neurons.

C-СО5E conjugates

According to the methods mentioned above, the peptide is a covalent conjugate of the C-C5E peptide and a polymer modifying group. The polymer modifying group can be covalently attached to the 5-C5E peptide near the glycosyl or amino acid residue of the 5-C5E peptide, preferably with the help of a glycosyl linking group. In one embodiment, the glycosyl linking group is an intact glycosyl linking group. In preferred embodiments, the polymer modifying group and the glycosyl binding group are covalently attached by means of a linker. In an exemplary embodiment, the polymeric modifying group is a water-soluble polymer, such as polyethylene glycol. α-C5E was cloned and sequenced. In an exemplary embodiment, the amino acid sequence 5-C5E corresponds to ZEO IYUO MO:1. It will be apparent to one skilled in the art that the present invention is not limited to the sequences set forth in this application.

The present invention also extends to variants of 5-C5BE known from the prior art. As an example, which does not limit the present invention in any way, it is possible to cite the variant 5-C5E described in US patent Mo 6166183, which describes 5-C5E, which consists of a natural set of lysine residues, further linked to one or two molecules of polyethylene glycol. In addition, US Patent Nos. 6,004,548, 5,580,755, 5,582,823, and 5,676,941 describe a variant of 5-C5E in which one or more cysteine residues at positions 17, 36, 42, 64, and 74 are replaced by alanine or serine. US Patent No. 5,416,195 describes an O-C5BE molecule in which cysteine at position 17, aspartate at position 27, and serines at positions 565 and 66 are replaced by serine, serine, proline, and proline, respectively. Other variants are known from the state of the art, described, for example, in US patent Mo 5399345. Additional variants have an amino acid sequence selected from ZEO IU MO:3-11.

The expression and activity of a modified 5-C5E molecule according to the present invention can be determined using methods well known in the art, as described, for example, in U.S. Pat.

US Mo 4810643. For example, activity can be measured using a radiolabeled thymidine incorporation assay. Briefly, bone marrow from healthy donors is separated by density in RisoP-Nuradce (1.077 g/ml, Rpappasia, Rizsaiazhau, MU), and low-density cells are suspended in Issome medium (SIVSO, Ia KhdoPa, CA), which contains 10,956 fetal calf serum, glutamine and antibiotics. Approximately 2x1027 human bone marrow cells are incubated in control medium or supplemented with O-C5BE according to the present invention in 96-well flat-bottomed plates at approximately 37°C, 5 95 CO" in air for approximately 2 days. Cultures are pulsed with in the form of 0.5 μCcuries per well of ZN-thymidine for about 4 hours (Mem/Epdiapa Misieag,

Vo5iop, Ma55.) and measure the inclusion of the label, as described, for example, in the work of Mepitz, her a!. (1983, Viosa 61:781). Stronger incorporation of ZH-thymidine by human bone marrow cells compared to bone marrow cells in the presence of a control substance is an indicator of the activity and viability of the substance 5-C5E.

Conjugates according to the invention are formed by enzymatic addition of a modified sugar to a glycosylated or non-glycosylated 5-C5E peptide. The modified sugar located between the 5-C5E peptide and the modifying group on the sugar in this application may be referred to, for example, as an "intact glycosyl linkage group." Using the exceptional selectivity of enzymes such as glycosyltransferases, the present invention allows to obtain peptides that carry the desired group in one or more defined positions. Thus, according to this invention, the modified sugar is attached directly to the selected locus on the C-C5E peptide chain or, alternatively, the modified sugar is added to the carbohydrate group of the polypeptide. Peptides in which modified sugars are attached both to the carbohydrate in the glycopeptide and directly to the amino acid residue included in the 5-C5E polypeptide chain are also covered by this invention.

In contrast to known chemical and enzymatic peptide improvement strategies, methods according to the invention can be used to assemble peptides and glycopeptides that have a practically homogeneous derivatization pattern; enzymes used in accordance with this invention are generally selective for a specific amino acid residue or combination of amino acid residues in the 5-C5E peptide. Such methods can also be applied to large-scale production of modified peptides and glycopeptides. Thus, the methods according to the invention provide a practical means for large-scale production of glycopeptides with preselected derivatization patterns. These methods are particularly well suited for the modification of medicinal peptides, including but not limited to glycopeptides that are incompletely glycosylated during their production in a cellular system (eg, mammalian, insect, plant, fungal, yeast, or prokaryotic cells) or in transgenic plants or animals. .

The present invention also provides conjugates of glycosylated or non-glycosylated peptides with

C5E with an increased therapeutic half-life due to, for example, a reduced rate of clearance or a reduced rate of absorption by the immune or reticuloendothelial system (KE5). In addition, methods according to the invention provide the possibility of masking antigenic determinants on peptides, which weakens or cancels the host's immune response to the peptide. Selective attachment of targeting agents can also be used to target a peptide to a specific tissue or to a specific receptor on the cell surface specific for a given targeting agent.

In one embodiment, the present invention provides a conjugate of the selected modifying group and the 5-C5E peptide. The bridge between the peptide and the modifying group includes a glycosyl linking group located between the peptide and the selected group. As discussed herein, the selected modifying group is generally any group that can be attached to a saccharide unit to form a "modified sugar" recognized by the appropriate transferase enzyme that attaches the modified sugar to a peptide or glycosyl residue on a peptide. When inserted between the peptide and the selected group, the saccharide component of the modified sugar becomes a "glycosyl linking group", for example, an "intact glycosyl linking group". A glycosyl binding group is formed from any mono- or oligosaccharide, which, after modification by a modifying group, is a substrate for an enzyme that adds a modified sugar to an amino acid or glycosyl residue in a peptide.

The glycosidic linking group can be or include a saccharide group that is modified with a structural disorder before or during the addition of the modifying group. For example, a glycosyl linking group can be derived from a saccharide residue obtained by oxidative degradation of an intact saccharide to the corresponding aldehyde, e.g., using a metaperiodate, and further converted to a Schiff base with an appropriate amine, which is then reduced to the appropriate amine.

Conjugates according to the invention usually have the following general structure: / x x x

X; where the symbols a, b, c, and 5 mean positive integers other than zero; and i means zero or a positive integer. The "agent" is usually a water-soluble group, for example, an REO group.

The linker can be any of a number of linking groups (see below). Alternatively, the linker may be a single bond or a "zero-order linker".

Modifying groups can be, as discussed further in this application, any group attached to a saccharide unit. Such groups include polymers, including water-soluble and water-insoluble polymers, and may also include medicinal substances, diagnostic substances, directing substances, toxic substances, etc. In an exemplary embodiment, the modifying group is a water-soluble polymer, for example, t-RES. The water-soluble polymer is covalently attached to the C-C5E peptide by means of a glycosyl binding group covalently bound to an amino acid or glycosyl residue in the C-C5E peptide. The present invention also provides conjugates in which the amino acid residue and the glycosyl residue are modified with a glycosyl linking group.

Peptides according to the present invention contain at least one M- or O-linked glycosylation site. In addition to the presentation of conjugates formed by enzymatic addition of a glycosyl binding group, the present invention presents conjugates with a highly homogeneous pattern of substitutions. Using methods according to the invention, it is possible to form peptide conjugates in which almost all modified sugars in the population of conjugates according to the invention are attached to multiple copies of an amino acid or glycosyl residue with an identical structure. Thus, in one aspect, the present invention provides a peptide conjugate containing a population of water-soluble polymer molecules covalently linked to the 5-C5E peptide by means of an intact glycosyl linking group. In an exemplary embodiment of the conjugate according to the invention, almost every representative of the population of water-soluble polymer molecules is connected by means of a glycosyl binding group to a glycosyl residue in the composition of the 5-С9РЕ peptide, and all glycosyl residues in the composition of the 5-С5Е peptide, to which are attached glycosyl linking groups, have the same structures.

A peptide conjugate is also presented, in which a population of water-soluble polymer molecules is connected to it by means of a glycosidic linking group. In one embodiment, almost every representative of the population of water-soluble polymer molecules is connected with the help of a glycosyl binding group to an amino acid residue in the composition of the О-С5БЕ peptide, and all amino acid residues in the composition of the С-С5Е peptide, to which glycosyl binding groups are attached, have the same structures.

The present invention also provides conjugates similar to those described above, in which the 5-C5E peptide is conjugated to a medicinal fragment, a diagnostic fragment, a targeting fragment, a toxin fragment, etc. through an intact glycosyl binding group. Each of the above fragments can be a small molecule, a natural polymer (for example, a polypeptide) or a synthetic polymer.

Modified sugars

The present invention provides modified sugars, modified nucleotide sugars, and conjugates of modified sugars. In modified sugars according to the invention, the sugar group is preferably a saccharide, deoxysaccharide, aminosaccharide or M-acylsaccharide. The term "saccharide" and its equivalent terms "saccharyl", "sugar" and "glycosyl" means monomers, dimers, oligomers and polymers. The sugar group is also functionalized with a modifying group. The modifying group is conjugated to a sugar group, usually by conjugation with an amino, sulfhydryl, or hydroxyl, for example, primary hydroxyl, group on a carbohydrate residue. In an exemplary embodiment, the modifying group is attached by means of an amino group in the sugar composition, for example, through an amide, urethane, or urea formed as a result of the reaction of an amine with a reactive derivative of the modifying group.

Any sugar can be used as the sugar backbone of conjugates according to the invention.

Typical sugar backbones applicable to the compositions of the invention include, but are not limited to, glucose, galactose, mannose, fucose, and sialic acid. Other useful sugars include amino sugars such as glucosamine, galactosamine, mannosamine, the 5-amino analog of sialic acid, and others. The sugar backbone can be a structure that occurs in nature, or it can be modified and contain a site for conjugation with a modifying group. For example, in one embodiment, the present invention provides a peptide conjugate that contains a sialic acid derivative in which the 9-hydroxyl group is replaced by an amine. The amine is easily derivatized with an activated analogue of the selected modifying group.

Glycosyl binding groups

According to the peptide conjugates according to each of the methods described above, some embodiments of the peptide conjugates include a glycosyl linking group that is a sialic acid residue.

The bridge between the 5-C5E peptide and the selected substance, such as a water-soluble polymer, includes an intact glycosyl linking group located between the peptide and the selected substance. As discussed herein, the selected substance is virtually any molecule that attaches to a saccharide unit resulting in a "modified sugar" that is recognized by the appropriate transferase enzyme that attaches the modified sugar to the α-C5B peptide. The saccharide component of the modified sugar located between the O-C5BE peptide and the selected substance is called an "intact glycosyl binding group." The glycosyl binding group can be formed by any mono- or oligosaccharide, which after modification by the selected substance is a substrate of the corresponding transferase.

In one embodiment, the glycosyl linking group is a sialic acid residue, the structure of which is given by the formula:

НО чи он о в--нмы ОН where K means a water-soluble polymer, and the water-soluble polymer is attached to the sialic acid residue by means of the aforementioned linker.

In another embodiment, the glycosyl binding group is a sialic acid residue, the structure of which is shown in the formula:

SOU no on 9 ped nn ; ! "on ! as I o : r nn n. as Zo x nu p (6: where p means an integer from 1 to 2000.

In yet another embodiment, the glycosyl linking group is a modified sialic acid residue, the structure of which is shown in the formula:

He still

ON. me too Because I'm sorry o v A x what "MN" yes di!i-x4 di de Kg? is Н, СНгОВ", СООВ" or ОБ", where

B" means H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;

BZ and E" are independently selected from H, substituted or unsubstituted alkyl, OB8, МНОС(О) РУ, where

BB and E? independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or sialic acid;

Ia means a linker selected from the bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

V'by K"7 mean independently selected polymer "arms";

Xg and X" mean independently selected binding fragments that connect polymer groups K' and

In" izs;i

X? is a non-reactive group.

In a further embodiment, the amino acid residue is selected from serine or threonine. In yet another further embodiment, the amino acid residue is threonine at position 133 of ZEO IO MO:1.

In one embodiment, the glycosyl linking group comprises a substructure selected from: 2----bamdo--- banya banyu baduvvnvy and . where KE" means a modified sialic acid residue; and p means an integer from 1 to 10.

In a further embodiment, the glycosyl linking group has a formula selected from:

May day.

Her yakisu eat"

MtchemAETTVNAeTT la Map-nya (bismMAientoayutt, t (Env), r (bismAetttoav it U mon

A Mapenn (Fiomas--Ohaid

Map-t (SioMAyettoVuV t (EBvsh

GO. ! Mr. Ply | . to

Mine (ZiyumAE- Enter tya

M.A. (ViSMAs-Oayu VU

GT (gns),

AA---SismAs--ZibmAE--Map-(SiEMAs wai? la | SHY 7 (SMAstt byte (OismAst byte? where

AA means the amino acid residue of the indicated peptide;

Y means an integer that is equal to 0 or 1; p means an integer from 1 to 10; and

B'" is selected from H, OH, sialic acid, the indicated modified sialyl residue and 5ia-siae, where Biar means the indicated sialyl residue, where at least one K"" is selected from the indicated modified sialyl residue and Zia-

Ziar. In one embodiment, the amino acid residue is an asparagine residue.

In another embodiment, the indicated peptide 5-C5E has the structure represented by the formula: and 2-tvar reno pamAst (ranuttyaREv where 4 is 0 or 1; and Zia-RES has the structure shown by the formula:

CO

BUT : he 7 to... nn 8. o I oophorit nt p ! iv; where n means an integer from 1 to 2000. In a further embodiment, n means an integer from 400 to 500.

In yet another further embodiment, the 5-C5E peptide has the amino acid sequence shown in 5EO IU

MO:1.

In the discussion below, the invention is illustrated by reference to the use of selected sialic acid derivatives. One skilled in the art will appreciate that the purpose of the discussion is for clarity of illustration and that the proposed structures and compositions are generally applicable to the entire class of saccharide groups, modified saccharide groups, activated modified saccharide groups, and conjugates of modified saccharide groups.

In an exemplary embodiment, the present invention presents a peptide conjugate containing a modified sugar amine, the structure of which is given by the formula: (

MnNA- NV where C means a glycosyl group, Y means a connection or linker, and KE! means a modifying group.

Typical connections are connections between the MNeo group of the glycosyl group and a group with complementary reactivity on the modifying group. Thus, exemplary relationships include, but are not limited to

MNA", OV", BE: etc. For example, if K!' contains a carboxyl group, this group can be activated and connected to the МН» group on the glycosyl residue, which gives a connection to the МНС(ОК') structure. Similarly, OH- and 5Н-groups can be transformed into the corresponding ether or thioester derivatives .

Exemplary linkers include alkyl and heteroalkyl groups. Linkers include linking groups, for example, acyl-based linking groups, for example -C(O)MH-, -OC(O)MH-, etc. Linking groups are bonds between components , belonging to different groups according to the invention, for example, between a glycosyl residue and a linker (I) or between a linker and a modifying group (K').

Other linking groups are ethers, thioethers and amines. For example, in one embodiment, the linker is an amino acid residue, such as a glycine residue. The glycine carboxyl group is converted to the corresponding amide by reaction with an amine on the glycosyl residue, and the glycine amine is converted to the corresponding amide or urethane by reaction with an activated carboxylic acid or carbonate modifying group.

Another exemplary linker is an REO group or a RES group functionalized with an amino acid residue. RES binds to the glycosyl group through an amino acid residue at one end of the RES molecule and to EC" through the other end of the RES molecule. Alternatively, the amino acid residue can bond to K', and the end of RES, not linked to an amino acid residue, - with a glycosyl group.

An example of a molecule МН-И -В' has the formula: -чинс(охонНгаамммнюс(О ЧН ОосСНнсСНг)О(СНг)заМниВ", where the indices 5 and и are independently equal to 0 or 1. The indices a, br and 4 mean independent integers from 0 to 20, and c means an integer from 1 to 2500. Other similar linkers are based on molecules in which the MH group is replaced by, for example, -5, -O, and -CH."

In particular, this invention presents a peptide conjugate containing compounds in which МН-Й -

A" represents

Neo сНнгаМнеКоснгОосСнНесСНн»г)О(СНг)аМНВ,

MNessFhsNnaOosSnNesSnN»g)O(SNg)aMNA!,

МН(СнгаМнехсзозуснагОоснНесСнН»г)О(СНг)аМмн!,

MNeTeskhsng)amne!,

MH(CHg)aMNA. and

MN.

In these formulas, indices a, B and a are chosen independently from integers from 0 to 20, preferably from 1 to 5. Index c means an integer from 1 to 2500.

In an exemplary embodiment, C represents sialic acid, and selected compounds according to the invention have the formulas:

OK. ; De di ZVYUMUJchenyuchOm nboye. by. 0 VNNisichevisnoV what no sya x

ET vk EN tochi zd

Nosyk, where SNFNISNYUOVSTYOV zh he no Ne nNeЙOT en neo snmO sn uyu sm ; "maisnlchne ov; ok; no. Okddrya SPIVNIVIDNICHNYUN no, 0 m sNiSTZONIOMISNSM

Mi | who will throw his own update; kneosneninoennu nev on . He

BUT snonenonisnyuv dy and M ONIENYUMOSNIONI "Umneyu con; zone "MNSOZHneSN MOSS. I don't know yet. Ba.

NS NO NYUNONISNIOV

Not in: in

Ov

It will be apparent to one skilled in the art that the sialic acid residue in the exemplary compounds above may be replaced by any other aminosaccharide, including but not limited to glucosamine, galactosamine, mannosamine, their M-acetyl derivatives, etc.

In another exemplary embodiment, the primary hydroxyl group of the sugar is functionalized with a modifying group. For example, the 9-hydroxyl of sialic acid can be converted into the corresponding amine and functionalized to give a compound according to the invention. The formulas according to this embodiment include:

V eh ok: : nose Kolsnom onene vne nose s ichNv o T i kneyun

SI posa, s syanenovekmnevyoksna es not and Te enoesn; he oh I, DiVNYUNYUNONNISnina (okSnuchNyu NO eb di SNONIYUNONINUchNKN MN" not. "g goshi you knegosnu he. ok .

But there is a new boat

He: carries. b we SNO NOYU sny yum uch posy" DONYU NENyuNN no G no :

Zh "Mneyun" shk on; Xia .

In a further exemplary embodiment, the present invention provides a peptide conjugate that includes modified sugars, where the hydroxyl group in position 6 is converted into a corresponding amino group carrying a cassette linker-modifying group, as described above. Exemplary saccharyl groups useful as the basis of these modified sugars include sai, szaiIMAs, sis, SISMAs, Rys, Hui, Map, etc. A typical modified sugar according to this embodiment has the formula:

where is KZ-V? and E" are independently selected from Н, ОН, С(О)СН", МН and МНС(О)СнНЗ. В? means ОВ", МНЕ" or МН-

I-B", as described above.

Selected conjugates according to the invention are based on mannose, galactose or glucose, or on molecules having the stereochemistry of mannose, galactose or glucose. The general formulas of these conjugates are presented below: in in ve. y Kk shWd in TON van - On she now ni iz. ate

In another exemplary embodiment, the present invention provides compounds as described above, which are activated by their conversion to the corresponding nucleotide sugars. Exemplary nucleotide sugars used in the present invention in their modified form include nucleotide mono-, di- and triphosphates or their analogues. In one embodiment, the modified nucleotide sugar is selected from SHOR-glycoside, CMP-glycoside or ZOR-glycoside. Even more preferably, the nucleotide part of the modified nucleotide sugar is selected from OOR-galactose, OOR-galactosamine, OOR-glucose,

OorR-glucosamine, ZOR-mannose, COR-fucose, CMP-sialic acid or CMP-MetAc. In an exemplary embodiment, the nucleotide phosphate is bound to C-1.

Thus, in an exemplary embodiment in which the glycosyl group is sialic acid, the present invention provides peptide conjugates formed by compounds having the formulas:

But I didn't dream about it -0 and re oda h nn shk ne! in-x sn and OYA I am not and

Not FMOVYUNIOVEVVMNot but » may Kh

In ut h t kneensnya

Kk - Oh, I'm here, where is I-V! corresponds to that discussed above, and Y7-Sh' means a linker connected to a modifying group.

Like I, exemplary linkers according to G" include a bond, alkyl and heteroalkyl groups.

In another exemplary embodiment, the present invention presents a conjugate formed by a modified sugar according to the invention and a substrate, for example, a peptide, lipid, aglycon, etc., in particular, a modified sugar and a glycosyl residue of a glycopeptide or glycolipid. In this embodiment, the sugar group of the modified sugar is converted into a glycosyl linking group located between the substrate and the modifying group. An exemplary glycosyl linking group is an intact glycosyl linking group in which the glycosyl group or groups are not destroyed by chemical (eg, exposure to sodium metaperiodate) or enzymatic (eg, exposure to oxidase) processes. Selected conjugates according to the invention include a modifying group attached to the amino group of an aminosaccharide, for example, mannosamine, glucosamine, galactosamine, sialic acid, etc. An exemplary cassette modifying group-glycosyl linking group according to this motif is based on the structure of sialic acid acid, such as given in the formulas:

OM ON no Visn m-n o. oon ovo ION that ; but c 0-9 a- o

The actions of i and snhsoMchn s І on and on , dec, і12 are similar to those described above.

In yet another exemplary embodiment, the conjugate is formed by the substrate and the 1st position of the saccharyl group, in which the modifying group is attached via a linker to the 6th carbon atom of the saccharyl group. Thus, exemplary conjugates according to this embodiment have the formulas: m-n ! tive

With nn; N. in? In vp v and ha where the radicals are similar to those described above. A person skilled in the art will understand that the modified saccharyl groups described above can also be conjugated to the substrate at the 2, 3, 4 or 5 carbon atom position.

Exemplary compounds according to this embodiment include compounds having the formulas:

AH

In in o sno ken all news la p. o k suznisnznoknsn m ornisnI young 2

Ve i ko shyne KONUMUA louse and - . Hey « according to the oral education, it is the same as the first;

Ihu; - and t m an

Also, where E-groups and indexes are similar to those described above.

The present invention also presents nucleotide sugars modified by Y-Sh8' at the carbon in the 6th position. Exemplary molecules according to this embodiment include: mn t ; with 8 4 There is o 9 The base and | about HER | And about |7o (oh ' you io

In no OH where the P-groups and Y are the groups discussed above. The index "y" is equal to 0, 1 or 2.

Another exemplary sugar nucleotide according to the invention is based on molecules having the stereochemistry of ZOR-mannose. An exemplary molecule according to this embodiment has the structure:

Yi Ka cha - Plural

In what time; Yi g!

And there is | and where at 7 H ut Ch M a pe p a i. Wash and wash everything - and wax

Y z h ze ni Zv i no on

In yet another exemplary embodiment, the present invention provides a conjugate based on the stereochemistry of SHOR-galactose. An exemplary compound according to this embodiment has the structure: o . eV? with! . AJ Mn i

NK g; | H a ; :

Vshinn shields are WE about X Who and I Z everything st t: H u chen reach yo v ee

GK 5 and o o sh- no

The modifying group K' is any compound selected from, but not limited to, water-soluble polymers, water-insoluble polymers, medicinal substances, diagnostic substances, etc. The nature of exemplary modifying groups is discussed in more detail below.

Water-soluble polymers

In some embodiments, the polymer modifying groups of the Z-C5E conjugates according to the invention are water-soluble polymers. These water-soluble polymers can be linear or branched. In one embodiment, it is a water-soluble polymer with a practically homodisperse molecular weight distribution.

In some embodiments, the conjugates according to the invention contain water-soluble polymers that are polyethylene glycol, for example, methoxy polyethylene glycol. The polyethylene glycol used in the present invention is not limited to a specific form or molecular weight range. For unbranched polyethylene glycol molecules, the molecular weight is preferably in the range of 500 to 100,000. It is preferred to use a molecular weight in the range of 2,000 to 60,000 and more preferably in the range of about 5,000 to about 30,000.

In another embodiment, the polyethylene glycol is a branched RES with more than one RES group attached. Examples of branched RES are described in US patent Mo 5932462; in the patent

USA Mo 5342940; in US patent Mo 5643575; in US patent Mo 5919455; in US patent Mo 6113906; in US patent Mo 5183660; in UMO 02/09766; and in the works of Codeg U., Viosopiddasge Spetivigu 5: 283-288 (1994); and Matavzaki ei ai., Adgis. Vioi Spet., 52: 2125-2127, 1998. This application also discloses other applicable branched structures of REb.

In an exemplary embodiment, the molecular weight of each polyethylene glycol branched RES is equal to or greater than 2000, 5000, 10000, 15000, 20000, 40000 or 60000 Daltons.

Many water-soluble polymers useful in the present invention are known to those skilled in the art.

The term "water-soluble polymer" extends to molecules such as saccharides (eg, dextran, amylose, hyaluronic acid, polysialic acid, heparans, heparins, etc.), polyamino acids, eg, polyaspartate and polyglutamate, nucleic acids, synthetic polymers (eg , polyacrylic acid, polyesters, for example, polyethylene glycol); peptides, proteins, etc. This invention can be carried out with the participation of any water-soluble polymer with the only limitation, which is that the polymer must contain a site for attaching another part of the conjugate.

Methods for activating polymers can also be found in US Pat. No. 94/17039, US Pat.

MO 5281698, and further in MO 93/15189, as well as in works that consider the conjugation of activated polymers with peptides, for example, blood growth factor MI (MO 94/15625), hemoglobin. (M/O 94/09027), oxygen carrier molecule (US patent No. 4412989), ribonuclease (and superoxide dismutase (Muegopeze ai a!., Arr. Viospet. Vioitesp. 11: 141-45(1985)).

In one embodiment of this invention, water-soluble polymers are used, in which a significant part of the polymer molecules in the polymer sample have approximately the same molecular weight; such polymers are called "homodisperse".

The present invention is further illustrated with reference to a polyethylene glycol conjugate. There are several reviews and monographs on the functionalization and conjugation of RES. See, for example, Naiti5, Masgopo).

Spent Ripped C25: 325-373 (1985); Zsociep, Meijodv ip Eplutiodu 135: 30-65 (1987); M/opa ei ai.,

Epghute Mistorb. Thespians 14: 866-874 (1992); Oeidado ei ai., Styisa! Kemieyem5 in Tpegareshiis Ogyd Sagtieg

Zuziet5 9: 249-304 (1992); 7aiirzKu, Viosopiidaie Spet. 6: 150-165 (1995); and Vnadga, ei ai!., Rpaptaliye, 57:5-29 (2002). Methods of obtaining reactive RES molecules and formation of conjugates using reactive molecules are known from the state of the art. For example, US patent No. 5,672,662 discloses a water-soluble and secretable conjugate of an active ester of a polymeric acid selected from linear or branched polyalkylene oxides, polyoxyethylated polyols, polyolefin alcohols, and polyacrylomorpholine.

US patent Mo 6376604 describes a method of preparing a water-soluble ester of 1-benzotriazolyl carbonate and a water-soluble non-peptide polymer by reacting the terminal hydroxyl group of the polymer with di-1-benzotriazolyl carbonate in an organic solvent. The active ester is used to form conjugates with a biologically active agent, such as a protein or peptide.

UMO 99/45964 describes a conjugate consisting of a biologically active substance and an activated water-soluble polymer containing a polymer chain in which at least one end is linked to the polymer chain by a stable bond, where at least one end contains a branched molecule, whose proximal reactive groups are connected to a branched molecule in which the biologically active substance is connected to at least one proximal reactive group. Other branched polyethylene glycols are described in UMO 96/21469, in US patent Mo 5932462 describes a conjugate formed by a branched molecule of RES, which includes a branched end that includes reactive functional groups. Free reactive groups can react with biologically active molecules, such as protein or peptide, forming conjugates between polyethylene glycol and the biologically active molecule. US patent No. 5,446,090 describes a bifunctional PES linker and its use in the formation of conjugates that include peptides at both ends of the PES linker.

Conjugates containing degradable RES bonds are described in UMO 99/34833; ii UMO 99/14259, as well as in US patent Mo 6348558. Such degradable connections are applicable in this invention.

The above-described methods of polymer activation, known from the state of the art, are applicable in the context of this invention in the formation of branched polymers described in this application, as well as for conjugation of these branched polymers with other molecules, such as sugars, nucleotide sugars, etc.

Exemplary polyethylene glycol molecules useful in the present invention include, but are not limited to, molecules having the formula:

XX

2700 t (Snanek (snasen (СН) ВВ where КЗ means Н, ОН, МН», substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl, for example, acetal, ОНС -, NaM-(CHg)a-, H5-(CHg)a or -(CHg)aC(M i.

Index "e" means an integer from 1 to 2500. Indices b, a and d independently mean integers from 0 to 20. Symbols 7 and 77 independently mean OH, MH", leaving groups such as imidazole, p-nitrophenyl, NOVT , tetrazole, halide, 5-H?, alcohol part of activated esters; -(CHg)pC(U)M or - (CHg)p(CHg)C(U)U. Symbol U stands for H(2), 50, -5, M-BO. Symbols Х, МУ, У, А" and У independently mean groups О, 5, М-В". The symbol M stands for OH, MH", halogen, 5-82, the alcohol part of activated esters, the amine part of activated amides, nucleotide sugars and proteins. Indices p, 4, 5 and m are independently selected from integers from 0 to 20. Symbols КУ, ШЧВЕ, ДВ" and "2 independently mean H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and substituted or unsubstituted heteroaryl.

In other exemplary embodiments, the polyethylene glycol molecule is selected from the following options:

Metiosna What is the child? Meossnato и 0. 0. o mme-(osnosn) M 2 zni | men Snsn tm it whose me-(OSsnaSnav-o y, ptete no

Me-chOsnosSNg) -y . o y ) mme-(OSNaSNa)v. NM

Me-(SnaSnadtOo; And that them, oh

Me-(OSnNaSnNazdttetya ti N me-(SnНЬsSnayu-Mnia).

Polyethylene glycol, applicable for the formation of the conjugate according to the invention, can be linear or branched. Branched polyethylene glycol molecules suitable for use in the present invention include, but are not limited to, molecules described by the following formula:

YAM (OM where KZ and EU are chosen independently from the groups denoted above as KZ. A" and A? are independently chosen from the groups denoted above as A!'. The indices e, i, o and 4 correspond to those described above. 7 and U correspond to described above. X' and X' are independently selected from 5, ЗС(О)МН, НМС(О)5, 5С(ОО)0, 0, МН, МНСс(О), (FOSMN and МНОУ, "С(ОМН.

In other exemplary embodiments, the branched RES is based on a cysteine, serine, or dilysine base. Thus, further exemplary molecules of RES include: из .

Yii

A ra neo STSNOSNasN Osn o y

Mne ni rt t vyurusnsnsnsniyus about ; at

One game MNSYUYUNINYOSTSNUYUOEN yet, but not in OZHYNY

NM rt vorenenionenos, about ; o "3 we in --yunenrmn we ve yunosnokhone nNeoyuSNSNYAOSNasNayusnya kNSeyustenuOyeNnt yus

She is a student of uch-uchnvon ny in estyuvonoyun, nNSTSNSnyaOStSN yus, MyoiOyusnynYaoSNUsn Os, at 9 will pick up in utchNesOSNASNYIUN , NSS . is7ut-tenoya knechOon;

In yet another embodiment, the branched RES molecule is based on a trilysine peptide. Trilysine can be mono-, di-, tri- or tetrapegylated. Exemplary molecules according to this embodiment have the formulas:

o no x " and o

And o o iVnegoyusniniasnInyuenkh

Me " " ' nya! MN." ! t i emne(OyuYUSTSNYAOSNISNo Osn 9 F. and

Ge) ; - and MNO (SNSN OSY NIOEN no Ar

With Fr

And mouths

Mnn- : y

NA KK. VN and

ХК жнеосниенаоеснсткОсть о я и де е, Ги и are independently selected from integers from 1 to 2500, and d, d' and d" are independently selected integers from 1 to 20.

In exemplary embodiments of the present invention, the RES is a t-RES (5, 10 or 20 kDa). An exemplary molecule of branched PES is serine- or cysteine-(t-PES)", where t-PES is t-

RES with a mass of 20 kDa.

One skilled in the art will appreciate that the branched polymers useful in the present invention include variations on the above-described themes. For example, the conjugate of dilysine and RES, given above, can include three polymer subunits, where the third subunit is attached to the α-amine shown in the above structure in its unchanged form. Similarly, the present invention extends to the use of trilysine functionalized with three or four polymeric subunits.

Specific embodiments according to the present invention include:

It is in January I

With and talk V

Mat Chitou and Yi ya on

HNM b; oh and

Me fluor e i shk CH ne chi fo Z

It, as well as carbonates and active complex esters of these molecules, such as:

rhema in a schi)

Me" o) I'm that

NO"

Ma. 5 a. en A. about 0 ; x o c) E g and iy

E and meter o E e 4 th . nude Sat

Me m soi not A, but ; ate

IS

Other activating or leaving groups useful for activating linear RES for use in the preparation of compounds described herein include, but are not limited to, molecules:

Dili m. chh Mat 0000 nya h . ; and - ,/x koe she . chik, uh N

Mo and Kz

Circle M A br

Kry 7ya- tm i: 5 vd t x 5 y o. y ek Zh p she Oy tn i ; m; ih Z ry

E- and F-Y and I and Zn

because what

REbS molecules activated by these and other molecules, and methods of obtaining activated RES are described in UMO 04/083259.

One skilled in the art will appreciate that one or more "arms" of the branched polymer formed by t-RES can be replaced by a RES molecule with a different end group, e.g.

OH, COOH, MH", C2-Si0-alkyl, etc. Further, the structures above are easily modified by inserting alkyl linkers (or removing carbon atoms) between the c-carbon atom and the functional group of the side chain. Thus, "homo"-derivatives, as well as higher and lower homologues are considered as the basis for branched RES molecules for use in this invention.

The branched RES molecules described in this application can be easily prepared using methods such as those shown in the scheme below:

MN, what? ner - tey oz vv --ЖО Ж tyzh, ots

And eat. X y a - tet tab (Tulo tyny Keti dn ny ny ne z She de Ha means O or 5, and r means an integer from 1 to 5. The indices y and y independently mean integers from 1 to 2500.

Thus, according to this scheme, a natural or unnatural amino acid is brought into contact with an activated t-RES derivative, in this case tosylate, to form compound 1 by alkylation of the He heteroatom of the side chain. A multifunctional t-RES amino acid is acylated at the M-terminus by a reactive t-RES derivative under appropriate conditions, resulting in the assembly of branched t-RES 2. It will be apparent to one skilled in the art that the tosylate leaving group may be replaced by any applicable leaving group, e.g., halogen, mesylate, triflate, etc. Similarly, the reactive carbonate used to acylate the amine can be replaced by an active ester, e.g., M-hydroxysuccinimide, etc., or the acid can be activated by using a dehydrating agent. , such as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.

In an exemplary embodiment, the modifying group is a RES group, however, any modifying group, such as a water-soluble polymer, a water-insoluble polymer, a drug, and others may be incorporated into the glycosidic group through a suitable linkage. Modified sugar is produced by enzymatic, chemical, or a combination of these processes that result in modified sugar. In an exemplary embodiment, sugars are replaced by an active amine at any position to which a modifying molecule can then be attached, but the sugar must remain a substrate for the enzyme that attaches the modified sugar to the O-C5BE peptide. In an exemplary embodiment where the modified sugar is galactosamine, the amino group is attached to the carbon atom in the 6-position.

Also, with the help of RES molecules, such as polyethylene glycol (RES), it is possible to increase the half-life of medicinal glycopeptides. For example, the chemical modification of proteins by the REO molecule (pegylation) increases the size of their molecules and reduces the availability of surface and functional groups, which depends on the size of the RES molecule bound to the protein. This leads to an increase in the half-life time in the blood plasma and proteolytic stability, as well as reduces the immunogenicity of the substance and the rate of its assimilation by the liver (Spapee ei ai. y). Siiiip. Ipme5i. 89: 1643-1651 (1992); Ruaiak ey a. Not known

Sottype Spent Raino! Rpaptosai 29: 113-127 (1980)). It was shown that pegylation of interleukin-2 enhances its antitumor effect by mimo (Kaige ei aI. Rgos. Mai!. Asad. zZsi. OA. 84: 1487-1491 (1987)) and that pegylation of the E(ab)" fragment obtained from monoclonal antibody A7, increases its localization in the tumor (Kpatiga ei aI. Viospet. Viorpu5. Ke5. Sottyp. 28: 1387-1394 (1990)). Thus, in another embodiment, the half-life of the ip mimo peptide derivatized with the RES molecule according to the method according to the invention increases compared to the life time of the ip mimo non-derivatized peptide.

The increase in the half-life of the ip mimo peptide is best expressed using the percentage interval of the increase in this indicator. The lower limit of the percent increase interval is about 40 95, about 60 95, about 80 95, about 100 95, about 150 95, or greater than about 200 95. The upper limit of the interval is about 60 95, about 80 95, about 100 95, about 150 95 or more than about 250 95.

Peptide B-C5E

Virtually any peptide or granulocyte colony-stimulating factor substance of any sequence can be used as the peptide component of the conjugates of the present invention. Granulocyte colony-stimulating factor cloned and sequenced.

In an exemplary embodiment, the 5-C5E peptide has the sequence given in 5EO IU MO:1:

MIR SRBESROZBET EXTEOUKKOYUSOSAAGOBKOSATUK

GSNREVBSUTISNZSOIR'UDRGSVBSRVOAGOGASSVOG NGO,

EGO OAI VIZ RESYARTOTYOSOMADRATTRAIOOMEE

HOMARAGHORTOCHLAMRACAVAVOKWASOMEUAVNSOVE EM

ZKU ENIAOR (BEOTO MO: V.

In another exemplary embodiment, the 5-C5E peptide has the sequence shown in ZEO IU MO:2:

TRIORAZVBSROBEXVOUVKSOSALSOVKSATUKI

SNREBIUIL.ONYASOTRZHMARI 58 SRZODI CSO AOSI ZO NVO E

GOST OABESIVRESORTOTOSOUAOEATIUOOMEEI,

OMARAGORTOSAMRABAZABOV VASU IMABNGOBESEU5

UKUTENIAOR (5EO 10 MO: 2).

In other exemplary embodiments, the 5-C5E peptide has the sequence shown in SEQ ID NO:3-11 below.

MIRSOVABOSROVA KSO UKKOSOSAAGOVKSMBESA,

TUKESNREBI ALL ON OR ZHARIZVSREOATOGSASSI VO,

MOISTURE. OATESIZRESOYA RT OT OBOMAVEATTGUI

OMEBK SMARAGORTOSAMVABAVAROKKASISUSMAHNGO

FRIGEUVtUVMUTENIAOR (VEO 9 MO:3);

MASRATOVEMKOMA TL LK NVZALUTUOVATRESRAB,

WORK KSSEOUKKOSOSASASOVKISATUKOSNEEVSMU, aKNOSTPRKMARGZBSRYAUSA HOBASSTVOSNZOTE KOSI OA

GSEOIBREGORT OT OGSBVBUAOEATT UOOMEESIMARAGOR

TOSAMRABABAEOKWA SOU SUA OBEG VEU USENYA

MY EYE);

MAORATOBRMESMA HOM LUNVAILUTUOVATRIJIRA VV

ROBY KS VOUKKIOYUSOSALAGOVK UZESATUKOSNREVI,

ME.ONZSOTRZHAREV5OR BOAGOGA oso SNO SE OSY,

GOAGSEOIBRESOR TT OT OGOUALKATT AT OOMEB LOBSTER

HORTOSAMRABAZABOKVASOU MANI OVEGEUVUVUSE nNrAOo(ZEOT MOVU;

MUTRISRABBTSROZE S KSOPEOMVKOSOSLASOKKISATU

KGSNREESU TYSNY AHIPRUARYABSRIVASOBASOSTVOSN

SPREKOSIL.OASEVIZRESOR TO T OBO AOBATEKOM

EESOSMARAGHORTOVBAMRAKAZVAROKKASOUSMAVNYOVNYI,

EUZUKUT VNI LORISEO 10 MO),

MIR SRABUEROBLTA KSO EOUKKCHOSOVAAASOVKSATEK

GOSNREESMSTSONTS OEM ARY Z5SREOAGHOGASSTVOSNVYI,

VELO OVIESIZRESORT OT OGOKABEATTUOOMEE

HOMARATORTOSAMRABAZAKOKKASSUSMAVNIOBEEU

UC VNI. AORICHVO IV MO:

MUTRISRABZI ROBEILKSTEOUVKIOOPOAAATOVKGSATU

KISNREVESUGT.O5BSSOSTRUARY.ZBSRHOASOGASTVOG NVO

OSI OAGESORESOR TT OTO APRATT MSOME

ESOMARASORTOBAMEABGAZABOKKASSUSSUAZNIGOBESE

UBHKEUBKNSAORIZEO TOU MOV);

MOTVISRAVVIROBEL KSTEOUAKIOSVOAAATOVKGSATU

KOSNEEESLU TI NIA SOT MARSA GO ASOST ON aBESHOST OA EOIT5RESORTOTOSOOUAORATTKUSOM,

BEESOMARATORTOSAMRAVABAROKKASOSUMMAVNSOBE

EUBUVUTKNIAORIVEO 10 MO:9);

MTRIORABETROZEI KS KOUVKOSOSAATOVKISATUK

SSNREESU TL SNOM ARIZVSRZOAGOBASSVOBNVYOI,

ELOSTGOAGESIZREGO RT OT OHOMAORATPUSOMEE

HOMARATSORTOSAMRRABAVOVKASOSUGMAVNSOVETSEU

ZUKUTHANILORTHOSAMR (ZEO Yu MO:10) and

MIREIERABAGROBET KS GOUKKIOSVSAATOVKTSATUK.

IENREESU TL OB KhPRMARIZSRBOAGOBAOSTVOBNVSY.

EGOSTGOATESIVEE ORTOTOSOUAOVATTROUOMEB

IOMARTTTRIOTAMRABAVAK OBKASOUBUAZNYOVZESEU SUVUSENIAORIZKO TO MO,

The present invention is in no way limited to the above sequences.

In an exemplary embodiment, the 5-C5E peptides according to the invention contain at least one O-linked glycosylation site, which is glycosylated by a glycosyl residue that includes a PES group. REO is covalently attached to the 5-C5E peptide with the help of an intact glycosyl binding group.

The glycosyl binding group covalently binds to an amino acid or glycosyl residue in the C-C5P peptide. Alternatively, the glycosyl linking group is attached to one or more glycosyl units in the glycopeptide. The present invention also provides conjugates in which the glycosyl binding group is attached to both amino acid and glycosyl residues.

The RES molecule is attached to an intact glycosyl linker directly or through a non-glycosyl linker, for example, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl.

In an exemplary embodiment, the 5-C5E peptide contains a group having the formula shown in formula I

Formula Her, he and o. oon but ; iv s, -M m y

So de O is chosen from -OH and B'-I -MN; C is selected from K-1I - and -C(O)X(C:-Cv)alkyl; B' means a group containing a linear or branched polyethylene glycol residue; and Y. means a linker, which is a bond, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl, so if Y means OH, o means K'-I-, and if c means -C(O)(C1- Sv)alkyl, O means K'-I-MH-. In the modified sialic acid structures described in this application, COOH also means COO and/or its salt.

In one embodiment, K'-I. has the formula:

AND; where a is an integer from 0 to 20.

In the exemplary embodiment, K' has a structure selected from: 2 o

AND

| in--(bnenomn; 5 I in--isnenomsn,

I am 5 years old. kneyuensnyosnsndyusn o o

M. o-you exactly

MNFfiOsNNYAoSNIsNAyuSN" | Mneguyensnostsndyuen" where е and и mean independent integers from 1 to 2500; and d means an integer from 1 to 20. In other embodiments, E! has a structure selected from: o o

In o-tnechnoia In e-vnecholh cha chmnsipsnsn(OosN.snyusN,, chnssposninIOSsNN Osn, oh in!

AH A I e pd he SNAVSN,; 7 ZK o-enenoksv kneosnenaOsneNSN,. | chischoyusnienienosnenaOsn,; where e and i mean independent integers from 1 to 2500; and d means an integer from 1 to 20. In other embodiments, E! has a structure selected from:

It is with eyes "MNO(OYUSNSsNOSN,SNoVOSN.; y

Mn"

NM "arches - "nedyusnennosntsneyuen" o sho; o

ДУ ит инеюистьснюсныснво сн»

M;

MN chnesIsnsnHosnIsnayusn»U her 0 . and

Ts sh Droitya MnesusnsnoyetionvOSV customs

I

Mnegtyenasnaossno yut; : : !

NM. and in

ZY tnodisnisnnosnnosnkasn in: and and

Why do I care about this,

And I chisiyustsniasnun is nk; and that t "neyusnnyosninlosna

Ц 1. where е and и mean independent integers from 1 to 2500; and d and d' stand for independent integers from 1 to 20.

In yet another embodiment, the present invention presents a 5-C5E peptide conjugate, where K' has a structure selected from: MmiegyuenesnoosnnvOSsNya with t a also O0VnshOoyusNnesndosnIsnUyuUyeENH mb In

NM yan o tiy ezhne (viIyusNoVOsVsnenOSN, I and Fr

And : ; nNe(osnsniaeensnUvOSsnV and Fr

And I, you and ivistsnayusnisnaosn"

Mn and ha ; "more MN and ""Ueneibisnientsosnisnosn"

Г5 i de e, Those 7 mean independent integers from 1 to 2500; and d, d' and d" mean independent integers from 1 to 20.

In other incarnations of E! has a structure selected from: 8--С(О)СНаСНОСНоСно Osn and 8-7 С(Ф)ХСНасНоСНоСно юн where e and ii denote independent integers from 1 to 2500.

In another exemplary embodiment, the present invention provides a peptide that contains a group having the formula: : CH

She and o som no; oh--wan-5 thank you

That's it! can be attached to an amino acid or a glycosyl residue that is attached directly or indirectly (eg, via a glycosyl residue) to an amino acid.

In other embodiments, the group has the formula: den i a Son no | :

O--bain-nOVIMAan-5 bat NYA y

One cyMmAs can be attached to an amino acid or a glycosyl residue that is attached directly or indirectly (eg, through a glycosyl residue) to the amino acid.

In yet another exemplary embodiment, the peptide contains a group having the formula: where OH in b. ЖЖОЙН or nab ! po--oa-onMAAA, a--NM Veli dian where AA stands for the amino acid residue of the specified peptide, and in both of the above structures, the C lines are similar to those above.

An exemplary amino acid residue of the C-C5E peptide with which one or more of the above molecules can form a conjugate includes serine and threonine, for example, threonine at position 133 5EO IO MO:1.

In another exemplary embodiment, the present invention presents a 5-C5E conjugate that includes a glycosyl residue represented by the formula:

And pek As Ka. and Silas Ko); cut as you k ; and more | poem VO a (V 8-Ak kbisnas-sieMAs-Mai a s y I (vi SHCI toi

MORE: | TsePeEMAschO a (KI "TsoemAlooaimc (Viv)y (ВХ) ho that I dea, B, s, y, i, r, 5, 1 and y denote independent integers chosen from 0 and 1. The index d means 1. Indices e, Her, d and y are independently selected from integers from 0 to 6. Indices i, K, 1 and t are independently selected from integers from 0 to 100. Indices m, mu, x and y are independently selected from 0 and 1, and at least one of the indices m, mu, x and y means 1. The symbol AA means the amino acid residue of the 5-C5E peptide.

The symbol 5ia-(K) means a group having the formula:

BUT. in

NE and, ; and ON 8-9

She MmNned an, where O is chosen from -ОН and К'-И-МН-. The symbol C means K'-I- or -C(O)(C1-C1)alkyl. K! means a group containing a linear or branched polyethylene glycol residue. I. means a linker, which is a bond, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl.

Of course, if O means OH, c means K'-I-, and if C means -C(O)(Ci-Cv)alkyl, O means K-I -

MN-.

In another exemplary embodiment, the modified RES group of sialic acid in the composition of the conjugate according to the invention has the formula: an integer from 1 to 2500. In one embodiment, 5 is 1, and the t-RES group has a molecular weight of approximately 20 kDa.

In yet another exemplary embodiment, sialic acid modified by RES has the formula:

he chik sadn ni u OY nin t Ya y Sche

Hey and

IS

I te» i , where I means a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl linking group that connects a molecule of sialic acid and a molecule of REO.

In one embodiment, at least two, more preferably three, more preferably four of the aforementioned asparagine residues are functionalized by the M-linked glycan chain, given above.

Conjugates according to the invention include intact glycosyl binding groups, which can be mono- or multivalent (for example, antenna structures). Thus, conjugates according to the invention include molecules in which the selected group is attached to the peptide by means of both a monovalent glycosyl binding group and a multivalent binding group. The present invention also extends to conjugates in which more than one selected group is attached to the peptide by means of a multivalent linking group.

Water-insoluble polymers

In another embodiment, similar to those described above, the modified sugars include a water-insoluble polymer, as opposed to a water-soluble polymer. Conjugates according to the invention may also include one or more water-insoluble polymers. This embodiment of the present invention is illustrated by the use of a conjugate as a carrier that provides controlled delivery of a medicinal peptide. Polymeric drug delivery systems are known in the art. See for example

Bipp ei ai., ed. ROIGMMEKIS OKOS5 AMO OKO SEGIMEKU 5UZTEM5, AS5 Zutrozisht zZegie5 t. 469,

Ategisap Spetis! bosieiu, MMazpipdiop, 0. S. 1991. It will be apparent to one skilled in the art that virtually any known drug delivery system is applicable to the conjugates of the present invention.

The above variants of EC", 1-8", IV", B"" and other radicals are equally applicable to water-insoluble polymers, which can be incorporated without restrictions into linear and branched structures using chemical methods available to specialists in the field of technology.

Типові водонерозчинні полімери включають, але не обмежуються поліфосфазинами, полівініллолвими спиртами, поліамідами, полікарбонатами, поліалкіленами, поліакриламідами, поліалкіленгліколями, поліалкіленоксидами, поліалкілентерефталатами, полівініловими ефірами, полівініллвими складними ефірами, полівінілгалідами, полівінілпіролідоном, полігліколідами, полісилоксанами, поліуретанами, поліметилметакрилатом, поліетилметакрилатом, полібутилметакрилатом, поліззобутилметакрилатом, полігексетилметакрилатом, полііззодецилметакрилатом, полілаурилметакрилатом, поліфенілметакрилатом, поліметилакрилатом, поліїізопропілакрилатом, полізобутилакрилатом, поліоктадецилакрилатом, поліетиленом, поліпропіленом, поліетиленгліколем, поліетиленоксидом, поліетилентерефталатом, полівінілацетатом, полівінілхлоридом, полістиреном, полівінілліролідоном, плюроніками (рІ|і полівінілфенолом і їх співполімерами.

Synthetically modified natural polymers suitable for use in the conjugates of the present invention include, but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose esters, cellulose esters, and nitrocelluloses. Particularly preferred representatives of the major classes of synthetically modified natural polymers include, but are not limited to, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose polymer, cellulose triacetate, and sodium sulfates. and methacrylic esters of alginic acid.

These and other polymers discussed in this application can be readily obtained from commercial sources such as 5idta Spetisai! Co. (51. I otsi5, MO.), Roiuzsiepse5 (UMagtepiop, RA.), Aidgispy (Mim"aiKye, UM,

Rika (Kopkopkota, MU) and Viokai (Kisptopa, CA), or synthesized from monomers purchased from these suppliers using conventional techniques.

Typical biodegradable polymers for use in conjugates according to the invention include, but are not limited to, polylactides, polyglycolides and their copolymers, polyethylene terephthalate, polybutyrate, polyvalerianate, poly(lactide-co-caprolactone), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, as well as their mixtures and copolymers. Gel-forming compositions, such as those comprising collagen, pluronics, etc., are particularly useful.

Polymers applicable in this invention include "hybrid" polymers comprising water-insoluble materials that contain at least part of their structure a bio-absorbable molecule. An example of such a polymer can be a polymer that includes a water-insoluble copolymer that contains a bioabsorbable region, a hydrophilic region, and a set of functional groups that form crosslinks in the polymer chain.

For purposes of this invention, the term "water-insoluble materials" includes materials that are sufficiently insoluble in water or aqueous media. Thus, even though some regions or segments of the copolymer may be hydrophilic or even water-soluble, the polymer molecule as a whole is not sufficiently soluble in water.

For purposes of this invention, the term "bioabsorbable molecule" includes a region that can be metabolized, degraded, absorbed, and/or excreted through the body's normal excretory pathways. Such products of metabolism or decay are also mostly non-toxic enough for the body.

The bioabsorbable region can be hydrophobic or hydrophilic if the copolymer composition as a whole does not become water-absorbable. Thus, the bioresorbable region is chosen based on the advantage that the polymer as a whole remains water-insoluble. Accordingly, the relative properties, that is, the content of different types of functional groups and the relative proportions of the bioabsorbable region and the hydrophobic region, are selected so that the applicable bioabsorbable compositions remain water insoluble.

Typical resorbable polymers include, for example, synthetic resorbable block copolymers of poly-α-hydroxycarboxylic acid/polyoxyalkylene (see Sopp, et al., US Pat. No. 4,826,945). These copolymers are not cross-linked and are water-soluble, which allows the body to remove degraded block copolymer compositions. See Uotspez ei ai.,.) Viotei. Maieg. Kev. 21: 1301-1316 (1987); and Sopp ei ai., in Viotei. Maieg. Kev5. 22: 993-1009 (1988).

Currently preferred bioabsorbable polymers include one or more components selected from polyesters, polyhydroxy acids, polylactones, polyamides, polyetheramides, polyamino acids, polyanhydrides, polyorthoesters, polycarbonates, polyphosphazines, polyphosphoesters, polythioethers, polysaccharides, and mixtures thereof. Even more preferably, the bioresorbable polymer includes a polyhydroxy acid component. Of the polyhydroxy acids, polylactate, polyglycolate, polycapronate, polybutyrate, polyvalerianate and their copolymers and mixtures are preferred.

In addition to the constituent fragments absorbed by the mimo ("bioresorbable"), preferred polymer coatings for use in methods according to the invention may also form a fragment that is excreted and/or metabolized.

Higher order copolymers may also be used in the present invention. For example, in the work of Sazeu et al., US patent No. 4438253, issued on March 20, 1984, triblock copolymers obtained as a result of transesterification of polyglycolic acid and polyalkylene glycol with a hydroxyl end are disclosed. Such compositions are disclosed as absorbable monofilament suture material. The flexibility of such compositions is controlled by incorporating an aromatic orthocarbonate, such as tetra-p-tolyl orthocarbonate, into the copolymer structure.

Other copolymers based on lactic and/or glycolic acid can also be used. For example, in the work of Zripa ei ai., US patent No. 5202413, issued on April 13, 1993, biodegradable multiblock copolymers containing consecutive blocks of polylactide and/or polyglycolide obtained by ring-opening polymerization of lactide and/or glycolide on oligomeric diol or diamine residue, followed by chain extension with a bifunctional compound such as diisocyanate, diacyl chloride, or dichlorosilane.

The bioabsorbable regions of the coatings applicable in this invention can be designed so that they can be broken down hydrolytically and/or enzymatically. For purposes of this invention, "hydrolytically cleavable" refers to the susceptibility of the copolymer, especially the bioresorbable region, to hydrolysis in water or in an aqueous environment. Similarly, as used herein, "enzymatically cleavable" refers to the tendency of the copolymer, particularly the bioresorbable region, to be cleaved by endogenous or exogenous enzymes.

When placed inside the body, the hydrophilic region can be cleaved into fragments that are excreted and/or metabolized. Thus, the hydrophilic region may include, for example, polyesters, polyalkylene oxides, polyols, polyvinylpyrrolidine, polyvinyl alcohol, polyalkyloxazolines, polysaccharides, carbohydrates, peptides, proteins and their copolymers and mixtures. Further, the hydrophilic region can also be, for example, polyalkylene oxide. Such polyalkylene oxides may include, for example, polyethylene oxide, polypropylene oxide and their mixtures and copolymers.

Polymers that are components of hydrogels can also be used in this invention.

Hydrogels are polymeric materials capable of absorbing relatively large amounts of water.

Examples of substances that form hydrogels include, but are not limited to, polyacrylic acids, sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidine, gelatin, carrageenan and other polysaccharides, hydroxyethylene methacrylic acid (HEMA), as well as their derivatives, etc. The resulting hydrogels can be stable, biodegradable ("bioresorbable. In addition, hydrogel compositions may include subunits that have one or more of these properties.

Biocompatible hydrogel compositions are known in the art, the integrity of which can be controlled by cross-linking, and which are currently preferred for use in the methods according to the invention. For example, in the works of Nibbei! et al., US Patent Nos. 5,410,016, issued April 25, 1995, and 5,529,914, issued June 25, 1996, disclose water-soluble systems that are cross-linked block copolymers in which a water-soluble central block segment is sandwiched between two hydrolytically labile peripheral segments. The ends of such copolymers are further capped with photopolymerizable acrylate functional groups. After the formation of cross-links, such systems turn into hydrogels. The water-soluble central block of such copolymers may contain polyethylene glycol; while the hydrolytically labile peripheral segments may be a poly-α-hydroxy acid such as polyglycolate or polylactate. See Zau/ppeu et al., Mastotoieschiez 26: 581-587 (1993).

In another preferred embodiment, the gel is a thermoreversible gel. Currently preferred are thermoreversible gels that include components such as pluronics, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogel, polyurethaneurea hydrogel, and combinations thereof.

In yet another exemplary embodiment, the conjugate according to the invention includes a liposomal component.

Liposomes can be prepared by methods known to those skilled in the art, for example, as described in Errziep et al., US Patent No. 4,522,811. For example, liposomal preparations can be prepared by dissolving the appropriate lipid(s) (such as stearoyl phosphatidyl ethanol amine, stearoyl phosphatidylcholine, arachidoyl phosphatidylcholine and cholesterol) in an inorganic solvent, which is then evaporated, yielding a thin film of dry lipid on the surface of the container. Next, an aqueous solution of the active substance or its pharmaceutically acceptable salt is introduced into the container. Then the container is manually rotated to wash off the lipid material from the walls of the container and break up the lipid aggregates, which leads to a liposome suspension.

The above-described microparticles and methods of obtaining microparticles are given as an example and do not cover the entire range of microparticles applicable in this invention. A person skilled in the art will understand that a variety of microparticles obtained by various methods are applicable to the present invention.

The framework formats discussed above in the context of water-soluble polymers, both linear and branched, are generally applicable to water-insoluble polymers as well. For example, cysteine, serine, dilysine and trilysine branched bases can be functionalized by two water-insoluble polymer groups. Methods of obtaining such substances are similar to methods of obtaining water-soluble polymers.

Methods of obtaining 5-C5E conjugates

In addition to the conjugates discussed above, the present invention provides methods for preparing these and other conjugates. Thus, in one aspect, the present invention provides a method of forming a covalent conjugate between a selected group and a 5-C5P peptide. In addition, the invention presents methods of targeting conjugates according to the invention to a specific tissue or part of the body.

In exemplary embodiments, a conjugate is formed between a RES molecule (or an enzymatically transferred glycosylated molecule that contains a RES molecule) and a glycosylated or non-glycosylated peptide. RES is conjugated to the C-C5E peptide through an intact glycosyl linking group located between and covalently bound to the 5-C5BE peptide and to the RES molecule, or to a non-glycosyl linker structure of RES (for example, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl). The method includes bringing the 5-C5E peptide into contact with a mixture containing a modified sugar and a glycosyltransferase, the substrate of which is a modified sugar. The reaction is carried out under conditions suitable for the formation of a covalent bond between the modified sugar and the C-C5E peptide. The sugar group of the modified sugar is selected from nucleotide sugars, activated sugars, and non-nucleotide and non-activated sugars.

The acceptor peptide (glycosylated or non-glycosylated) is usually synthesized de pomo or recombinantly expressed in prokaryotic cells (eg, bacterial cells such as E. soy) or in eukaryotic cells such as mammalian, yeast, insect, fungal or plant cells. Peptide 2-C5E can be both a full-length peptide and its fragment. Further, the C-C5E peptide can be wild-type or mutant. In an exemplary embodiment, (3-C5E carries a mutation that adds one or more M- or O-linked glycosylation sites to the peptide sequence.

In an exemplary embodiment, factor IX undergoes O-glycosylation and is functionalized as follows. A peptide with an open amino acid glycosylation site is obtained, or, if the peptide is glycosylated, the glycosyl group is removed to expose the amino acid. For example, serine or threonine is subjected to 4-1 M-acetylaminogalactosylation ((samMAs), and the MAs-galactosylated peptide is sialylated with a sialic acid-modifying group cassette using ZtbsamtmMAs1. Alternatively, the MAs-galactosylated peptide is galactosylated using Soge-1-saiT-1 , and the resulting product is sialylated with a sialic acid-modifying group cassette using ztZoAiT1. An exemplary conjugate according to this method contains the following bonds: ", where ia" means a cassette sialic acid-modifying group.

In methods according to the invention, similar to those described above, which use multiple enzymes and saccharyl donors, the individual stages of glycosylation can be carried out separately or combined in the so-called "prompt swarms" of reactions. For example, in the three-enzyme reaction above,

CyMAS-transferase, cyt and zytate and their donors can be combined in one vessel. Alternatively, the reaction with (a!mMmAs) can be carried out separately, and the enzymes and suitable saccharyl donors can be added in one step. Another way to carry out the reactions involves adding each enzyme and the appropriate donors sequentially and running the reaction in a "fast swarm" fashion. Combinations of each of the above methods are applicable for obtaining compounds according to the invention.

In the conjugates according to the invention, especially in glycopegylated M-linked glycans, the cassette

Zia-modifying group can connect to Zai a-2,6 or a-2,3 bond.

Methods according to the invention also provide modification of incompletely glycosylated peptides obtained in the recombinant system. When using a modified sugar in the method according to the invention, the 5-C5E peptide can be simultaneously further glycosylated and derivatized, for example, by a RES molecule, a medicinal substance, etc. The sugar group of the modified sugar can be a residue that would be conjugated with an acceptor in a fully glycosylated peptide, or another carbohydrate group with desired properties.

Peptides 0-C5E modified by methods according to the present invention can be synthetic peptides, wild-type peptides or mutant peptides obtained by methods known in the art, such as directed mutagenesis. Glycosylation of peptides is usually M-linked or O-linked. An exemplary M-linkage is the attachment of a modified sugar to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate group to the side chain of asparagine. Thus, the presence of each of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a single sugar (eg, M-acetylgalactosamine, galactose, mannose, cisSMAs, glucose, fucose, or xylose) to the hydroxy amino acid side chain, preferably serine or threonine, although 5-hydroxyproline or 5- hydroxylysine.

In one exemplary embodiment, (3-C5E is expressed in a mammalian cell system and modified by treatment with sialidase to cleave terminal sialic acid residues and subsequent looping using 57305aiZ and a RES-sialic acid donor.

In another exemplary embodiment, 5-C5E expressed in mammalian cells is first treated with sialidase to cleave terminal sialic acid residues, then pegylated using 5t732zai3 and a RES-sialic acid donor, and further sialylated using 51735ai33 and a sialic acid donor.

C2-C5E, expressed in the mammalian cell system, can also be treated with sialidase and galactosidase to cleave terminal residues of sialic acid and galactose, and then galactosylated using a galactose donor and galactosyltransferase and further pegylated using 5735aiZ and a RES-sialic acid donor.

In yet another exemplary embodiment, the -C5E is not first treated with sialidase, but is glycopegylated by a sialic acid transfer reaction with a sialic acid modifying group cassette and an enzyme such as 5TZOaiZ3.

In a further exemplary embodiment, 5-C5E is expressed in insect cells and modified using the following procedure: first, M-acetylglucosamine is added to 5-C5E using an appropriate M-acetylglucosamine donor and one or more spt-Y, II, YM and M; then 5-C5E is pegylated using the donor RES-galactose and galactosyltransferase. Yeast-derived c2-C5E can also be glycopegylated. For example, 5-C5E is first treated with endoglycanase to remove glycosyl groups, galactosylated using a galactose donor and galactosyltransferase, and then pegylated using ZTZsaiZ and a RES-sialic acid donor.

Addition of glycosylation sites to a peptide or other structure is easily accomplished by altering the amino acid sequence so that it contains one or more glycosylation sites.

Addition of glycosylation sites can also be carried out by introducing one or more residues presenting an OH group, preferably serine or threonine residues, into the α-SZE peptide sequence (for O-linked glycosylation sites). Addition of glycosylation sites can be carried out by mutation or complete chemical synthesis of the 5-C5E peptide. The amino acid sequence of the a-C5E peptide is preferably changed by changes at the DNA level, especially by mutations of previously selected bases

DNA encoding the peptide, such that the resulting codons are translated into the desired amino acids.

Mutation(s) of DNA is preferably carried out using methods known from the state of the art.

In an exemplary embodiment, the glycosylation site is added by polynucleotide shuffling. Polynucleotides encoding a candidate peptide can be modulated by DNA shuffling protocols. DNA shuffling is a process of recursive recombination and mutation performed by randomly fragmenting a set of related genes and then reassembling the fragments using a process similar to the polymerase chain reaction. See, for example, Zietiteg, Rgos. May Asad si. BOTH 91:10747-10751 (1994); Sietteg,

Maishige 370:389-391 (1994); and US Patent Nos. 5,605,793, 5,837,458, 5,830,721, and 5,811,238.

The present invention also provides a method of attaching to the peptide (or removing from the peptide) one or more selected glycosyl residues, after which a modified sugar is conjugated to at least one selected glycosyl residue in the peptide. This embodiment is applicable, for example, if it is necessary to conjugate a modified sugar with a selected glycosyl residue, absent in the peptide or present in an insufficient amount. Thus, before attaching the modified sugar to the peptide, the selected glycosyl residue is conjugated with the 5-C5E peptide by enzymatic or chemical binding. In another embodiment, the glycosylation pattern of the glycopeptide is changed before conjugation of the modified sugar by removing the carbohydrate residue from the glycopeptide. See, for example, UMO 98/31826.

The addition or removal of carbohydrate groups from the glycopeptide is carried out chemically or enzymatically. Chemical deglycosylation is preferably carried out by exposure to the polypeptide variant of the substance trifluoromethanesulfonic acid or an equivalent substance. This effect leads to the cleavage of most or all of the sugars except the linking sugar (M-acetylglucosamine or M-acetylgalactosamine), while the peptide remains intact. Chemical deglycosylation is described in the works of NakKitiyaaip ei ai., Agsp. Viospet. Viorpuvz. 259:52 (1987) and Edde ei ai,, Apai. Viospet. 118: 131 (1981). Enzymatic cleavage of carbohydrate groups from polypeptide variants can be carried out using various endo- and exoglycosylases, as described in TpoiaKiga egap, May. Epgutoi 138: 350 (1987).

Chemical attachment of glycosyl groups is carried out by any method accepted in the state of the art.

Enzymatic attachment of carbohydrate groups is preferably carried out using modifications of the methods described in this application, with the replacement of native glycosyl units with modified sugars used in this invention. Other methods of attaching sugar groups are described in US patents

MoMo 5876980, 6030815, 5728554 and 5922577.

Exemplary attachment points of a selected glycosyl residue include, but are not limited to: (a) consensus M- and O-glycosylation sites; (b) terminal glycosyl groups, which are acceptors for glycosyltransferase; (c) arginine, asparagine and histidine; (4) free carboxyl groups; (e) free sulfhydryl groups, such as the sulfhydryl group of cysteine; () free hydroxyl groups, such as hydroxyl groups of serine, threonine or hydroxyproline; (4) aromatic residues, such as phenylalanine, tyrosine or tryptophan residues; or (n) amide group of glutamine. Exemplary methods applicable to the present invention are described in UMO 87/05330, published on September 11, 1987, and in the work of Ariip apa M/gietop, SKS SVT.

VEHM. VIOSNEM,., pp. 259-306 (1981).

In an exemplary embodiment, this invention presents a method of obtaining pegylated (-С5БЕ, containing the group: но ; : V nos Shche ts on " INN

I where O means -ОН or В-Й -НМ-; symbol C means B-I - or -C(O)(C:-Cv)alkyl. B' means a group containing a linear or branched polyethylene glycol residue. The symbol Y. means a linker selected from a bond, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. In the general case, if Y means OH, b means K'-I-, and if b means -C(O)(C1-Cv)alkyl, Y means B'-I -MH-. The method according to the invention includes (a) bringing the substrate - C-C5BE peptide - into contact with the REO-sialic acid donor and the enzyme that transfers the RES-sialic acid group to the substrate-peptide 5-C5E.

An exemplary REO-sialic acid donor is a nucleotide sugar having the formula:

and about

Ж с СООН ду и роб--- MA in-NM " і su 9) he iv; ; A

Mne and the enzyme that transfers REOS-sialic acid to the amino acid or glycosylic residue of the 5-C5BE peptide, under conditions suitable for transfer.

In one embodiment, before the formation of the conjugate according to the invention, the substrate - C-C5E peptide is expressed in the host cell. An exemplary host cell is a mammalian cell. In other embodiments, the host cell is an insect, plant, fungal, or bacterial cell.

The method presented in this application is applicable to each of the 5-C5E conjugates described in the sections above.

5-C5E peptides modified by methods according to the present invention can be synthetic peptides or wild-type peptides or they can be mutant peptides obtained by methods known in the art, such as site-directed mutagenesis. Glycosylation of peptides is usually M-linked or O-linked. An exemplary M-linkage is the attachment of a modified sugar to the side chain of an asparagine residue. The tripeptide sequence asparagine-X-serine or asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate group to the side chain of asparagine. Thus, the presence of each of these tripeptide sequences in a polypeptide indicates a potential glycosylation site. O-linked glycosylation refers to the attachment of a single sugar (eg M-acetylgalactosamine, galactose, mannose, SISMAs, glucose, fucose or xylose) to the hydroxyl side chain of a hydroxyamino acid, preferably serine or threonine, although unusual or non-natural amino acids can also be used, e.g. , 5-hydroxyproline or 5-hydroxylysine.

Addition of glycosylation sites to a peptide or other structure can be carried out in accordance with this invention by changing the amino acid sequence so that it contains one or more glycosylation sites. Addition of glycosylation sites can also be carried out by introducing one or more residues presenting an OH group, preferably serine or threonine residues, into the peptide sequence (for O-linked glycosylation sites). Addition of glycosylation sites can be carried out by mutation or complete chemical synthesis of the peptide.

The amino acid sequence of the peptide in some embodiments is changed by changes at the DNA level, especially mutations of preselected bases of the DNA encoding the peptide in such a way that the resulting codons are translated into the desired amino acids. DNA mutation(s) are carried out using methods known from the state of the art.

In an exemplary embodiment, the glycosylation site is added by polynucleotide shuffling. Polynucleotides encoding a candidate peptide can be modulated using DNA shuffling protocols. DNA shuffling is a process of recursive recombination and mutation performed by randomly fragmenting a set of related genes and then reassembling the fragments using a process similar to the polymerase chain reaction. See, for example, Zietiteg, Rgos. May Asad si. BOTH 91:10747-10751 (1994); Sietteg,

Maishige 370:389-391 (1994); and US Patent Nos. 5,605,793, 5,837,458, 5,830,721, and 5,811,238.

Exemplary methods of adding or removing glycosylation sites and removing glycosyl structures or substructures are described in detail in M/O 04/099231, UMO 03/031464 and related US and PCT patent applications.

The present invention also uses a method of attaching to the 5-C5E peptide (or removing from the 5-C5E peptide) one or more selected glycosyl residues, after which a modified sugar is conjugated to at least one selected glycosyl residue in the peptide. Such techniques are applicable, for example, if it is necessary to conjugate a modified sugar with a selected glycosyl residue that is absent from the peptide or present in insufficient amounts. Thus, before attaching the modified sugar to the peptide, the selected glycosyl residue is conjugated with the 5-C5ZE peptide by enzymatic or chemical binding. In another embodiment, the glycosylation pattern of the glycopeptide is changed before conjugation of the modified sugar by removing the carbohydrate residue from the glycopeptide. See, for example, UMO 98/31826.

Exemplary attachment points of the selected glycosyl residue include, but are not limited to: (a) consensus M-linked glycosylation sites and O-linked glycosylation sites; (B) terminal glycosyl groups, which are acceptors for glycosyltransferase; (c) arginine, asparagine and histidine; (4) free carboxyl groups; (e) free sulfhydryl groups, such as the sulfhydryl group of cysteine; (Its) free hydroxyl groups, such as hydroxyl groups of serine, threonine or hydroxyproline; (du) aromatic residues, such as phenylalanine, tyrosine or tryptophan residues; or (j) amide group of glutamine. Exemplary methods applicable to the present invention are described in MO 87/05330, published September 11, 1987 and in the work of Ariip apa Umgisiop, SKS SKIT. KEM. VIOSNEM,., p. 259-306 (1981).

According to the present invention, modified RES sugars are conjugated with a glycosylated or non-glycosylated peptide using a suitable conjugation enzyme.

Preferably, the concentration of the modified sugar donor(s), enzyme(s) and acceptor peptide(s) should be selected so that glycosylation is carried out until the desired degree of acceptor modification is achieved. It should be understood that although the subject of the discussion below is sialyltransferase, it is generally applicable to other glycosyltransferase reactions.

Several methods of using glycosyltransferases for the synthesis of the necessary oligosaccharide structures are known, which are generally applicable to this invention. Exemplary methods are described, for example, in UMO 96/32491, as well as in the work of Mohei Ai., Ryge Arri. Spent 65:753 (1993), US Pat

MoMo 5352670, 5374541, 5545553, and publicly owned U.S. patents MoMo 6399336 and 6440703 incorporated herein by reference.

A single glycosyltransferase or a combination of glycosyltransferases may be used in the present invention. For example, it is possible to use a combination of sialyltransferase and galactosyltransferase. In embodiments where more than one enzyme is used, the enzymes and substrates are preferably combined in the initial reaction mixture, or the enzymes and reagents for the second enzymatic reaction are added to the reaction medium when the first enzymatic reaction is complete or nearly complete. When two enzymatic reactions are carried out sequentially in one vessel, the total yield of the product turns out to be greater compared to procedures that include the isolation of an intermediate product. In addition, cleaning and removal of excess solvents and by-products is facilitated.

In one embodiment, both the first and second enzymes are glycosyltransferases. In another preferred embodiment, one enzyme is an endoglycosidase. In another embodiment, more than two enzymes are used to assemble the modified glycoprotein according to the invention.

Enzymes are used to change the saccharide structure on the 5-C5E peptide at any time before or after the addition of the modified sugar to the peptide.

In yet another embodiment, methods according to the invention include the use of one or more exo- or endoglycosidases. A glycosidase can be mutant and/or variant, such an enzyme forms or is altered in order to form rather than break glycosyl bonds. Such a mutant glycanase usually contains a replacement of an amino acid residue in the active site with an acidic amino acid residue.

For example, if the endoglycanase is epdo-H, Ab5p at position 130, Siy at position 132, or a combination thereof are usually substituted. Amino acid residues are usually replaced by serine, alanine, asparagine or glutamine.

Such a mutant enzyme can catalyze the reaction using a synthesis step analogous to the reverse step of endoglycanase-catalyzed hydrolysis. In this embodiment, the glycosyl donor molecule (for example, the desired oligo- or monosaccharide structure) contains a leaving group, and the reaction proceeds with the addition of the donor molecule to the CiSMAs residue on the protein. For example, the leaving group can be a halogen, such as fluoride. In other embodiments, the leaving group is an Ap or an Azp-peptide molecule. In further embodiments, the (ISMAs) residue on the glycosyl donor molecule is modified. For example, the SISMAs residue may contain a 1,2-oxazoline group.

In one embodiment, the enzymes used to prepare the conjugate according to the invention are present in catalytic amounts. The catalytic amount of a particular enzyme varies depending on the concentration of the enzyme substrate as well as the reaction conditions such as temperature, time, and pH. Methods of determining the catalytic amount for a given enzyme for selected substrate concentrations and reaction conditions are well known to those skilled in the art.

The reaction temperature according to the invention can vary from slightly above zero to the denaturation temperature of the most sensitive enzyme. The preferred temperature range is from about 0 "C to about 55 "C and more preferably from about 20 "C to about 37 "C. In another exemplary embodiment, one or more stages of this method are carried out at an elevated temperature using a thermophilic enzyme.

In one aspect, the reaction mixture is stored for a period of time sufficient to glycosylate the acceptor, thereby producing the desired conjugate. Some amount of conjugate can often be detected after several hours, and the extractable amount is usually formed after 24 hours or earlier. One skilled in the art will appreciate that the reaction rate depends on several variables (eg, enzyme concentration, donor concentration, acceptor concentration, solvent volume) that can be optimized for the chosen system.

The present invention also presents the production of modified peptides on an industrial scale. As used herein, "on an industrial scale" generally refers to the production of at least one gram of the final purified conjugate.

In the discussion below, conjugation of modified sialic acid molecules with a glycosylated peptide is used to illustrate the invention. Exemplary modified sialic acid is indicated by RES. The following discussion focuses on the use of sialic acid, modified RES, and glycosylated peptides for clarity of presentation and does not imply that the present invention is limited to the conjugation of these two substances. It will be apparent to one of ordinary skill in the art that the discussion is generally applicable to the addition of modified glycosyl groups other than sialic acid. In addition, the discussion is also applicable to the modification of the glycosyl unit by agents other than RES, including other RES compounds, drugs and biomolecules.

An enzymatic approach can be used to selectively attach pegylated or ppgylated carbohydrates to a peptide or glycopeptide. In this method, modified sugars containing RES, PPO or a masked reactive functional group are used, which are combined with the appropriate glycosyltransferase or glycosynthase. By selecting a glycosyltransferase that will form the desired carbohydrate bond and using a modified sugar as a donor substrate, it is possible to attach RES or PPO directly to the 5-C5BE peptide chain, to existing glycopeptide carbohydrate residues, or to carbohydrate residues added to the peptide.

On the 5-C5E peptide, which is modified according to the methods described in this invention, there is a sialyltransferase acceptor, in the form of a natural structure or introduced into the peptide by recombinant, enzymatic or chemical means. Suitable acceptors include, for example, galactosyl acceptors such as ZaIV1,4cISMAs, CyIr1,4CaiIMAs, Ca!Ir1,3CaiIMAs, lacto-M-tetraose, CaIr1,3CiSMAs, ZaiIV1,3Aga, Ca!IV1,60ISMAs, sa!/p1, 4Obis(lactose) and other acceptors known to a person skilled in the art (see, for example, Racizop eyi ap, y. Vioi. Spet. 253: 5617-5624 (1978)).

In one embodiment, the sialyltransferase acceptor is present on the glycopeptide and is modified after glycopeptide synthesis by mimo. Such glycopeptides can be sialylated using the claimed methods without previously changing the glycosylation pattern of the glycopeptide. Alternatively, methods according to the invention can be used for sialylation of a peptide that does not contain a suitable acceptor; first modify the peptide to include an acceptor using methods known in the art. In an exemplary embodiment, the remaining zaiMmAs is added by exposure to saiMmAs transferase.

In an exemplary embodiment, the galactosyl acceptor is assembled by adding a galactose residue to a suitable acceptor bound to the 5-C5E peptide, for example, SisSMAs. This method involves incubating the modified 5-C5ZE peptide in a reaction mixture that contains a sufficient amount of galactosyltransferase (for example, saiIr!,3 or CaB1,) and a suitable galactosyl donor (for example, OOR-galactose). The reaction is carried out substantially to completion or, alternatively, the reaction is terminated upon addition of a preselected amount of galactose residue. Other methods of assembling the selected saccharide acceptor will be apparent to one skilled in the art.

In yet another embodiment, the oligosaccharides bound to the glycopeptide are first "trimmed" in whole or in part, which exposes a sialyltransferase acceptor or group to which one or more suitable residues can be attached to form a suitable acceptor. Enzymes such as glycosyltransferases and endoglycosylases can be used for sugar addition and trimming reactions (see, for example, US Pat. No. 5,716,812).

In the discussion below, the method of the invention is illustrated using modified sugars to which RES groups are attached. This method is chosen for clarity of presentation. It will be apparent to one skilled in the art that this discussion also applies to embodiments in which the modified sugar carries a drug substance, biomolecule, etc.

In an exemplary embodiment of the present invention, in which the carbohydrate residue is "trimmed" before the addition of the modified sugar, the peripheral mannose residues are trimmed to the first generation biantennal structure. A modified sugar bearing an REOS group is conjugated to one or more carbohydrate residues exposed by "trimming". In one example, the RES group joins through the SISMAs group, conjugation with the RES group. A modified SISMAc is attached to one or both terminal mannose residues of the biantennary structure. Alternatively, unmodified SISMAs can be attached to one or both ends of the branched group.

In another exemplary embodiment, the RES molecule is attached to one or both terminal mannose residues of the biantennary structure using a modified sugar containing a galactose residue conjugated to a SISMAc residue attached to the terminal mannose residues. Alternatively, unmodified Sai can bind to one or both terminal residues of SiISMAs.

In yet another example, a molecule of РЕСб attaches to a residue of Са! using modified sialic acid.

In another exemplary embodiment, the structure from the peripheral mannose residues is "trimmed" to the mannose from which the biantennal structure branches. In one example, the RES molecule is attached through

Polymer-modified SISMAs. Alternatively, unmodified mannose is added

SISMAs, and then Zai with an attached RES molecule. In yet another embodiment, unmodified residues

SISMAs ii Sa! are sequentially attached to mannose, and then sialic acid, modified by the REO molecule, is attached.

In a further exemplary embodiment, peripheral mannose residues are "trimmed" to SISMAs to which the first mannose is attached. This SISMAs is conjugated to the residue of cSai, which carries a PE molecule. Alternatively, unmodified sia followed by sialic acid modified with a water-soluble sugar is added to sisSMAs. In yet another further example, the terminal SISMAs is conjugated with vaii, and then SiasMMAs is fucosylated with a modified fucose carrying a REb molecule.

Peripheral mannose residues can also be "trimmed" to the first CisMas attached to

Azp in the peptide. In one example, SISMAs in the composition of the residue SISMAs-(Pys)a is conjugated with SISMAs carrying a water-soluble polymer. In another example, SISMAs in the composition of the SISMAs-(Pys)a residue is modified (bzai), which carries a water-soluble polymer. In yet another further embodiment, cISAs are modified with bSaI, followed by conjugation of cSai with a sialic acid residue modified by a REO molecule.

Other exemplary embodiments are described in public domain patent application publications, US MoM 20040132640; 20040063911; 20040137557; US patent applications MoMo: 10/369979; 10/410913; 10/360770; 10/410945 and PCT/О502/32263, each of which is included in this application by reference.

The above examples illustrate the capabilities of the methods described in this publication.

Using the methods described in this publication, it is possible to "trim" and "grow" a carbohydrate residue having almost any desired structure. The modified sugar can be attached to the ends of the carbohydrate group, as described above, or it can be between the peptide chain and the end of the carbohydrate.

In an exemplary embodiment, the existing sialic acid is cleaved from the 3Z-C5E glycopeptide with the help of sialidase, thereby exposing most of the corresponding galactosyl residues. Alternatively, a peptide or glycopeptide is labeled with a galactose residue or an oligosaccharide residue ending with a galactose unit. After exposure or addition of galactose residues, a suitable sialyltransferase is used to add modified sialic acid.

A schematic representation of this approach is shown in diagram 1.

Scheme 1

KN

With S o cho Glycoprotein ey z notre still in sh Za!

РО or RE ket ОН 9 and schyno Schalintranoverase

SM-VA- Z MNSOSNUMN-REO(VRO) 5BA-Z-MNSSSnMH-RES

Glycoprotein yes and (sa -vleknoosnineo ovi

ZA-BMNOVSNMRES

In another approach, depicted in Scheme 2, a masked reactive functional group is present on the sialic acid. This masked functional group is preferably not affected by the conditions used to attach the modified sialic acid to (C-C5E. After the covalent attachment of the modified sialic acid to the 5-C5E peptide, the masking group is cleaved off and the C-C5E peptide is conjugated with an agent such as RES The agent is conjugated to the peptide in a specific way by reacting it with an unmasked reactive group on the modified sugar residue.

Scheme 2 h Ca glycoprotein

Zn Sv x I and s Fo - VAV MNSOSNIZ-VE! 9 Call Saturday, Sunday, and Sunday, - . but. K-t o dn Sialyltranepherase zhi sha BY MNSOSNUVVE

BO age un no? what

BAB-MNSOSNIOSE

FA NNSOSnov RE

Glycoprotein She: with diiotrvit in ; schy Z veSalid yayu RRO: heads

DY t a-VA-MNSOSNUERES and a! ze

BA-V-CHNEOSNVRES

Any modified sugar described in this application can be used together with the appropriate glycosyltransferase, depending on the terminal sugars of the oligosaccharide side chains of the glycopeptide (Table 1). As discussed above, the terminal sugar of the glycopeptide required to introduce a pegylated structure can be introduced naturally during expression or can be attached after expression using the appropriate glycosidase(s), glycosyltransferase(s), or a mixture of glycosidase(s) and glycosyltransferase(s).

Table 1 about K3-th | h.-k.

KkU-u | MIN. (0)

K;-«2g E

MN

Ky Mn kA 7 o schu o o A R. II mo

KgA| and her m'mo island uo otutoo o. in "Ma suma a t ma no on no on o. . Sh is. Derivatives of SHOR-galactosamine (if A-MN, Ka

Derivatives of SHOR;-galactose can mean acetyl) o ХК ha) х.К их вх З o Na o - o 7 KE o o Se 2 o o Lo vyA| and I mo

KA yap mo Oto oo to vo o b'ma em sma fa a no on no on o. . so Derivatives of SHOR;-glucosamine (if A-MH, Ka

OOR-glucose derivatives can mean acetyl) o

Oh hey ХВ и и мн

A. ka o o o Zh vi Ii her mM nn, 37 o M o-R--3--R o 2.2 her MN 050077 2 9 o Ye A Oka doma

Her skeleton barked? k-X o A-Ka no on i - o Ma sg"ma 2-Vz no on vn

ZOR-mannose derivatives ZOR-fucose derivatives

X-O, MN, 5, SN", MA(K.-5)5.

UK - X AH; VAH.

O - No, O, 5, MN, M-K.

K, Ki-4-H, linker-M, M.

M "RES; for example, you-RES

In a further exemplary embodiment, SHOR-galactose-RES reacts with p1,4-galactosyltransferase from cow's milk, which transfers the modified galactose to the corresponding terminal structure of M-acetylglucosamine. Terminal SISMAs residues on a glycopeptide can be produced during its expression, if it occurs in systems such as mammalian, insect, plant or fungal systems, but can also be produced by treating the glycopeptide with the necessary sialidase and/or glycosidase, and/or glycosyltransferase .

In another exemplary embodiment, an (ISMAc transferase such as OMT1-5) is used to transfer the pegylated cICM to the terminal mannose residue on the polypeptide. In yet another exemplary embodiment, the M- and O-linked glycan structures are enzymatically removed from the glycopeptide to reveal amino acid or terminal glycosyl residue, which is then conjugated to a modified sugar.For example, endoglycanase is used to remove

M-linked structures of the glycopeptide to reveal the terminal siISMAc bound to Abp in the polypeptide. OOR-Sa!-RES and the corresponding galactosyltransferase are used to attach RES-galactose to the open cISMAs.

In an alternative embodiment, the modified sugar is added directly to the 5-C5E peptide chain using a glycosyltransferase that transfers sugar residues to the peptide chain. This exemplary embodiment is shown in Scheme 3. Exemplary glycosyltransferases applicable in the present invention include, but are not limited to, CZaIMAs transferases (CaiMAS T1-14), CisSMAs transferases, fucosyltransferases, glucosyltransferases, xylosyltransferases, mannosyltransferases, etc. This approach allows directly attach modified sugars to peptides that do not carry carbohydrates or, alternatively, to existing glycopeptides. In both cases, the attachment of the modified sugar takes place in a specific position of the peptide chain, which is determined by the substrate specificity of the glycosyltransferase, and not randomly, which occurs when the protein peptide chain is modified by chemical methods. Various substances can be introduced into proteins or glycopeptides that do not contain the peptide sequence of the glycosyltransferase substrate by incorporating the appropriate amino acid sequence into the polypeptide chain.

Scheme C on the eye and Protein and enicoprotein

Shchy o and Ko S SCHO sh saimneaso (stnkhnNEo

U teo --- s ma oka davl y me / my ass V bamn-ooenU:nneEy nn

RE

In each of the exemplary embodiments above, after conjugation of the modified sugar with the peptide, one or more additional steps of chemical or enzymatic modification may be performed. In an exemplary embodiment, an enzyme (eg, fucosyltransferase) is used to attach a glycosyl unit (eg, fucose) to a terminal modified sugar attached to the 5-C5E peptide. In another example, an enzymatic reaction is used to "cap" sites to which the modified sugar has failed to conjugate. As an alternative, a chemical reaction is used to change the structure of the conjugated modified sugar.

For example, a conjugated modified sugar reacts with agents that stabilize or destabilize its bond with the peptide component to which the peptide sugar is attached. In another example, the modified sugar component is deprotected after conjugation to the peptide. One skilled in the art will appreciate that there are a variety of enzymatic and chemical procedures applicable in the methods of the present invention in the step following the conjugation of the modified sugar to the 5-C5E peptide. This invention extends to the further complication of the conjugate of the modified sugar with the 5-C5E peptide.

Enzymes

In addition to the enzymes discussed above in the context of acyl-linked conjugate formation, the glycosylation pattern of the conjugate and parent substrates (eg, peptides, lipids) can be complicated, "truncated," or otherwise modified by methods used by other enzymes . Methods of restructuring peptides and lipids using enzymes that transfer a sugar donor to an acceptor are discussed in detail in OeRgeez5, UMO 03/031464 A2, published on April 17, 2003. A brief description of the enzymes selected for use in this method is provided below.

Glycosyltransferases

Glycosyltransferases catalyze the stepwise addition of activated sugars (donors are MOP or MMP sugars) to a protein, glycopeptide, lipid or glycolipid or to the non-reducing end of a growing oligosaccharide. M-linked glycopeptides are synthesized with the help of a transferase and a lipid-bound oligosaccharide donor YuoI-PP-MASi2bIszMapo by block transfer and subsequent cutting of the rod. In this case, the nature of the "rod" saccharide is slightly different from the following added sugars. A very large number of glycosyltransferases are known in the art.

The glycosyltransferases applicable in the present invention can be any, provided that they can use a modified sugar as a donor. Examples of such enzymes include glycosyltransferase of the Leloir pathway, such as galactosyltransferase, M-acetylglucosaminyltransferase, M-acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase, mannosyltransferase, xylosyltransferase, glucurononyltransferase, etc.

For enzymatic carbohydrate syntheses that involve glycosyltransferase reactions, the glycosyltransferase can be cloned or isolated from any source. Many cloned glycosyltransferases and their polynucleotide sequences are known. See, for example, "Te M/M/MU Skcide To Siopei Spiusozuigapegegaze5" (trans:/Lymly.msi.so.yK/HOIBI/Chi diyide.pit).in various public databases such as SepVapkK, bm/iz5- Glucose, EMVI and others, you can find amino acid sequences of glycosyltransferases and nucleotide sequences encoding glycosyltransferases, from which their amino acid sequences can be deduced.

Glycosyltransferases that can be used in the methods of the invention include, but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, M-acetylgalactosaminyltransferases, M-acetylglucosaminyltransferases, glucuronyltransferases, sialyltantransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid transferases, and oligosaccharyltransferases. Useful glycosyltransferases include glycosyltransferases derived from both eukaryotes and prokaryotes.

DNA encoding glycosyltransferases can be obtained by chemical synthesis, screening of mRNA reverse transcripts from appropriate cells or cell line cultures, screening of genomic libraries of appropriate cells, or combinations of these procedures. Screening of mRNA or genomic DNA can be carried out using oligonucleotide probes built on the basis of glycosyltransferase gene sequences. Samples can be labeled with a detectable group, such as a fluorescent group, a radioactive atom, or a chemiluminescent group, according to known procedures and used in conventional hybridization tests. Alternatively, glycosyltransferase gene sequences can be obtained by polymerase chain reaction (PCR), for which PCR oligonucleotide primers are derived from glycosyltransferase gene sequences. See patent

United States Mo 4683195 in the name of Myiiy5 ei ai. US patent No. 4683202 in the name of MiiYiyiv.

Glycosyltransferase can be synthesized in host cells transformed with vectors containing DNA encoding the glycosyltransferase enzyme. Vectors are used for amplification

DNA encoding a glycosyltransferase enzyme and/or for expression of DNA encoding a glycosyltransferase enzyme. The expression vector is a replicating DNA construct in which the DNA sequence encoding the glycosyltransferase enzyme is functionally linked to suitable regulatory sequences capable of regulating the expression of the glycosyltransferase enzyme in a suitable host. How necessary such regulatory sequences are depends on the choice of the host and the method of transformation. In general, regulatory sequences include a transcriptional promoter, an optional operator sequence, a transcription regulatory sequence, a sequence that encodes suitable ribosomal mRNA binding sites, and sequences that regulate transcription termination and translation. Amplification vectors do not require the expression of regulatory domains. All that is required is the ability to replicate in the host, which is usually achieved by an origin of replication, and a selection gene to recognize transformants.

In an exemplary embodiment of the present invention, a prokaryotic enzyme is used. Such glycosyltransferases include enzymes involved in the synthesis of lipooligosaccharides (GO5) produced by many gram-negative bacteria (Rheviop ei ai., Styisa! Kemiemu5 ip Misgoriiodu 23(3): 139-180 (1996)). Such enzymes include, but are not limited to proteins of the pa operon of species such as EE. soybeans and ZaitopeiMya iurpitigist, which include B1,6-galactosyltransferase and pB1,3-galactosyltransferase (see, for example, EMVI database numbers M80599 and M86935 (E. soybean); EMVI database no. 556361 (5. turpitigist)) , glucosyltransferase (database number Bm/i55-

Rti P25740 (E. soybean), B1,2-glucosyltransferase (psa) (number in the database bZm/iz5-Rgoi P27129 (E. soybean) and number in the database BZu/iz5-Rgoi P 19817 (5. urpitigist)) , Her B1,2-M-acetylglucosaminyltransferase (Pac) (EMVI database number 0000039 (E. soya). Other glycosyltransferases with known amino acid sequences include glycosyltransferases encoded by operons such as pav characterized in organisms such as KierziePa rpeshtopiae, E. soii, zaitopeia iurpitigish t, zaitopeia epiegisa, Megbvipia epiegosoiiisa, Musobrasiegicht Iergozit, and operon ti Rzeidotopav5 aegidipoza.

The present invention also applies galactosidases involved in the production of structures containing lacto-M-neotetraose, O-galactosyl-p-1,4-M-acetyl-O-glucosaminyl-D-1,3-O-galactosyl- p-1,4-0-glucose and the trisaccharide sequence of the RU blood group, O-galactosyl-a-1,4-0O-galactosyl-VD-1,4-O-glucose, identified in the composition of GO5 of the mucous pathogens Mei55zegia doppogpoeae and M .

U. May. MystorioI. 41: 236-243 (1994)) Genes of M. tepipuyidie and M. dopogppoeae encoding glycosyltransferases involved in the biosynthesis of these structures were identified in M. tepipdica and immunotypes III and 11 (Cheppipd5 ei aiY., Moi. Misgobioi . 18: 729-740 (1995)) and in the Eb62 mutant of M. dopogpoeae (SoivpiisN, U. Ehr. Mei. 180: 2181-2190 (1994)). In M. tepipoiidia, a locus consisting of three genes,

IA, IdIV and IEE, encode glycosyltransferase enzymes necessary for joining the last three sugars in the chain of lacto-M-neotetraose (Umakagspik eyi ai., 9. Vioi. Spet. 271: 19166-73 (1996)). Recently, the enzymatic activity of the products of the IdIV and IA genes was shown, which is the first evidence of the predicted glycosyltransferase function (UmMakagspikK ei ai., 9. Vioi. Spet. 271(45): 28271-276 (1996)). In M. dopogtpoeae there are two additional genes, ID, which joins B-Yu-szaiIMAs to the position C of the terminal galactose of the lacto-M-neotetraose structure, and IDS, which joins the terminal α-Yu-zaiY to the lactose element of the truncated GO5, which leads to the formation of the structure of the Re blood group antigen (Soivpsp (1994), see above). In M. tepipoiaiv, a separate immunotype I 1 also expresses the Pk blood group antigen, and this immunotype has been shown to carry the IdiC gene (deppipd5 ei ai.,, (1995), see above).

Glycosyltransferases of Meili5zegia and genes associated with them are also described in publication OBRM 5545553 (Soi5Ppiisp). The α1,2-fucosyltransferase and α1,3-fucosyltransferase genes were also characterized

Neiissorasieg ruiogi (Mapip eyi ai., 9U. Vioi. Snpet. 272: 21349-21356 (1997)). Glycosyltransferases Satruyuorasieg |e)npi are also applicable in this invention (see, for example, pEr:/aitr.spgv-tgv.Pp/"redgo/SAiM/diyi 42.pIti).

Fucosyltransferases

In some embodiments, the glycosyltransferase used in the methods of the invention is a fucosyltransferase. Fucosyltransferases are known to those skilled in the art. Exemplary fucosyltransferases include enzymes that transfer I-fucose from ZOR-fucose to the hydroxyl position of the acceptor sugar. Fucosyltransferases that transfer a non-nucleotide sugar to an acceptor are also applicable in this invention.

In some embodiments, the acceptor sugar is, for example, IsSMAs in the group sa!Iv(1--3,4)siSMAsB in the composition of the oligosaccharide glycoside. Fucosyltransferases suitable for carrying out this reaction include fucosyltransferase sa!p(1--3,4)cISMAsrB1-o(1--3,4) (ET ES Mo. 2.4.1.65), first characterized in the composition of human milk ( see Raisis, ei aiYi., Sagropuagaite

Transportation 190: 1-11 (1989); Riiievi!v, eyi ai., u. Vioi Snet 256: 10456-10463 (1981); and Mypeyal, ei ai., Sap. U. Spet. 59: 2086-2095 (1981)), and fucosyltransferases sair(1--4)cIsSMAsv-c (ETIM, ETM, ETMI) detected in human blood serum. ETMIII (E.S. Mo. 2.4.1.65), fucosyltransferase sialyl o(2--3)aIv(1--3)c1sMAsv, was also characterized. A recombinant form of fucosyltransferase Ca!/pB(1--3,4)cISMAsr-o(1--3,4) has also been characterized (see Yushiptakhv, ei ai., Vioogu. Mei. I eiCetg5 1: 425-428 (1991) and KiKomkhkach-i aiaiyo, eyi aI., Sepe5 apa Yuemeiortepi 4: 1288-1303 (1990)). Other exemplary fucosyltransferases include, for example, c 1,2-fucosyltransferase (E.S. Mo 2.4.1.69).

Enzymatic fucosylation can be carried out using the methods described in the work

Moiiisope, ei ai., Eig. U. Viospet. 191: 169-176 (1990) or in US patent No. 5374655. The cells used for the production of fucosyltransferase also contain an enzymatic system for the synthesis of SOUR-glucose.

Galactosyltransferases

In another group of embodiments, the glycosyltransferase is a galactosyltransferase. Exemplary galactosyltransferases include "(1,3)-galactosyltransferases (ES Mo. 2.4.1.151, see, for example, YuBarKomukhki et al., Thapzriapi Rhos. 25:2921 (1993) and Matatoyo et al. Maishge 345: 229- 233 (1990), bull (SepVapk 94989, doliazze ei ai., U. VioiYi, Spet. 264: 14290-14297 (1989)), mouse (SepVapkK t26925; I agzep ei ai., Rgos. Mai! Asai. si. OBA 86:8227-8231 (1989)), pork (SepVapk

IZ36152; Zpmanap eyi oai., Ittypo oepeiys5 41: 101-105 (1995). Another suitable a!1,3-galactosyltransferase is a galactosyltransferase involved in the synthesis of blood group B antigen (E.S. Mo. 2.4.1.37, Matatoiyo ei ai.,.U. Vio!. Spet. 265: 1146- 1151 (1990) (human)). Another exemplary galactosyltransferase is soge sai!1-T1.

B1,4-galactosyltransferases are also suitable for use in the methods according to the invention, including, for example, E.S. Mo. 2.4.1.90 (I asMAs synthetase) and E.S. Mo. 2.4.1.22 (lactose synthetase) (bovine (r'Adoziago ei ai., Yeig. U. Viospet. 183: 211-217 (1989)), human (Mazvgi ei ai., Viospet. Viorpuv.

Transportation bot type 157: 657-663 (1988)), mouse (MaKagauma ei ai!., U. Viospet. 104: 165-168 (1988)), as well as

E.S. Mo. 2.4.1.38 and ceramide galactosyltransferase (ES Mo. 2.4.1.45, (ap ei ai., U. Meshigozsi. Kev5. 38: 234-242 (1994)). Other applicable galactosyltransferases include, for example, "a1,2 - galactosyltransferases (for example, with 5spi7osasspagotusez rotbre, SParei!i ei ai., Moi Vio! Seii 5: 519-528 (1994)).

Sialyltransferases

Sialyltransferases are another type of glycosyltransferase applicable to recombinant cells and reaction mixtures according to the invention. Cells producing recombinant sialyltransferases also produce CMP-sialic acid, which is a sialic acid donor for sialyltransferases. Examples of sialyltransferases useful in the present invention include ZTZoAi PI (e.g., rat or human ZTZ5aI II), 5TZaI IM, 5T3saI, 5tZaII II, 5T65aI, 5tTZaI M, ztvoAi II, 5Tb6osaIMas I, 5T6saIMas YIiIi II (Asum 5T6 this application uses the nomenclature of sialyltransferases given in the work of T5tsii ei ai., SiIusobBioyudu 6: m-chem (1996)). Exemplary "(2,3)- sialyltransferase, designated as da(2,3)-sialyltransferase (E.S. Mo. 2.4.99.6), transfers sialic acid to the non-reducing terminal Cα1 in the disaccharide or glycoside сайр1--30иС. See Map dep Eipaep ei ai!., U. Vioi. Snet 256: 31 59 (1981), Umeipvivip ei ai., 9. VioY. Spent 257: 13845 (1982) and UMep eyi ai., 9. Vioi. Spent 267: 21011 (1992). Another exemplary Ca2,3-sialyltransferase (E.S. Mo. 2.4.99.4) transfers sialic acid to the non-reducing terminal Ca! disaccharide or glycoside. See

Keagisk ei ai., 9. VioYi. Spent 254: 4444 (1979) and siShezrie ei ai., 9. Vioi. Spent 267: 21004 (1992). Further exemplary enzymes include sa!1I-p-1,4-1IsMAs sa-2,6-sialyltransferase (see Kygozama et al.

U. Viospet. 219: 375-381 (1994)).

Preferably, for the glycosylation of carbohydrates on glycopeptides, the sialyltransferase should be able to transfer sialic acid to the sequence (аИВ1,4сИМАс - as the most common penultimate sequence subject to terminal sialic acid on fully sialylated carbohydrate structures (see Table 2).

Table 2

Sialyltransferases that use the sequence of sai!p1,4cIsSMAs as an acceptor substrate (sequences) about v8tvbay | Mammals | MeiAso2bsari4sisMA.ІГ/-Z | 47 dopoitpoyeae 1) Soosnee eyi ai., Vio/Tesppoiodu 9: 1347-1355 (1991). 2) Matataoi ei ai!., U. Viospet. 120: 104-110 (1996).

Z) siiBen eia!., 9. VioYi. Spent 271: 28271-28276 (1996).

An example of a sialyltransferase applicable in the claimed methods is 5TZvai II, also designated a(2,3)-sialyltransferase (E.S. Mo. 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to sia in the glycoside CyIr1,3SISMAs or Sa!Ir1,4c1ISsMAs (see, for example, Umep et al., U. Viol. Sp. 267: 21011 (1992); Map aep Eippaep et al., 9. Physiol. 256: 3159 (1991)) and is responsible for sialylation of asparagine-linked oligosaccharides in glycopeptides. Sialic acid binds to sia to form a c-bond between the two saccharides. Linking (connection) of saccharides is carried out according to position 2 of MecAs and position Z of Zai. This particular enzyme can be isolated from the liver of rats (Uueipeieip ei ai., y. Vioiyi Spet. 257: 13845 (1982)); known sequences of human cDNA (Zazaki et al. (1993) 9. Vioi Spec. 268: 22782-22787; Kpadazha 4 Ratsibzop (1994) U. Vioi Spec. 269: 1394-1401) and genomic DNA (Kiadama et al. (1996) ) 9. Viol. Speth. 271: 931-938), which allows to obtain this enzyme by means of recombinant expression. In one embodiment, rat ZztTZeai PI is used in the claimed methods of sialylation.

Other exemplary sialyltransferases useful in the present invention include sialyltransferases

Satruiobasieg |e)ipi, including C5T-I and C5T-II and sialyltransferases that form a(2,3)-bonds.

See, for example, UMO 99/49051.

Sialyltransferases other than those listed in Table 2 are also applicable in the economic and large-scale process of sialylation of commercially important glycopeptides. As a simple test of the applicability of these other enzymes, different amounts of each enzyme (1-100 mU/mg protein) react with asialo-di AOR (at a concentration of 1-10 mg/ml) and compare the ability of the sialyltransferase in question to sialylate glycopeptides with bovine 5t6sba! I, 5T3baY Sh or both sialyltransferases. Alternatively, other glycopeptides can be used instead of asialo-4 ACP for this evaluation, or

M-linked oligosaccharides, enzymatically cleaved from the peptide chain. For the practical large-scale process of sialylation of peptides, sialyltransferases are applicable, which have the ability to sialylate M-linked oligosaccharides of glycopeptides more efficiently than ZztT6eai I. saIMmAs transferases

Also applicable in this invention are M-acetylgalactosaminyltransferases, especially for linking the molecule (szamMAc) to an amino acid in the O-glycosylation site of the peptide. Suitable M-acetylgalactosaminyltransferases include, but are not limited to, F(1,3)-M-acetylgalactosaminyltransferase, B(1, 4)-M-acetylgalactosaminyltransferases (Madaya ei ai., 9.

Axes Spent 267: 12082-12089 (1992) and Btiy ei ai., 9. Vioi Spet. 269: 15162 (1994)) and polypeptide M- acetylgalactosaminyltransferase (Nota ei ai!., 9. Wioi. Speth. 268: 12609 (1993)).

The production of proteins such as CyMAS Ti-xx using cloned genes through genetic engineering is well known. See, for example, US Pat. No. 4,761,371. One method involves collecting sufficient samples, and then the amino acid sequence is determined by sequencing from the M-end.

The obtained information is then used to isolate a cDNA clone encoding a full-length (membrane-bound) transferase, the expression of which in the insect cell line 519 allows the synthesis of a fully active enzyme. The acceptor specificity of the enzyme is further determined using semi-quantitative analysis of amino acids surrounding known glycosylation sites in 16 different proteins, and subsequent glycosylation analysis of synthetic peptides. This work showed that glycosylated segments of peptides have a higher proportion of specific amino acid residues, and that amino acid residues in specific positions surrounding glycosylated residues of serine and threonine can have a more pronounced effect on acceptor efficiency than other amino acids.

Cell-bound glycosyltransferases

In another embodiment, the enzymes used in the method according to the present invention are cell-bound glycosyltransferases. Although a large number of soluble glycosyltransferases are known (see, for example, US Pat. No. 5,032,519), cell-associated glycosyltransferases are usually in membrane-bound form. It is believed that many previously studied membrane-bound enzymes are proteins embedded in the membrane; that is, they are not separated from the membrane by sonication and require detergent exposure for solubilization. Surface glycosyltransferases have been identified on the surface of vertebrate and invertebrate cells, and it has also been established that such surface glycosyltransferases retain catalytic activity under physiological conditions. However, the more well-known role of glycosyltransferases on the cell surface is in cell recognition of each other. (Koy, MOGESTSOG AK ARRKOASNEZ 0

ZIRAACEI HER SHO AV RNEMOMEMA, 1990).

Methods have been developed to alter glycosylases expressed by cells. For example, in the work I akhep eyi aI., Rgos. May Asai. si. OBA 86: 8227-8231 (1989) describes a genetic approach to the isolation of cloned DNA sequences that determine the expression of oligosaccharide structures on the cell surface and the corresponding glycosyltransferases. It is known that SOR-galactose is expressed in the cDNA library obtained from mRNA isolated from a mouse cell line; B-O-galactosyl-1,4-M-acetyl-O-glucosaminide "41,3-galactosyltransferase was transfected into CO5-1 cells. The activity of "1,3-galactosyltransferase" was then determined in cultured transfected cells.

In the work of Egapsizso eyi aYi., Rhos. Maya). Asad si. OBA 89: 2713-2717 (1992) discloses a method of anchoring B-lactamase on the outer surface of EzsPegispia soybean. A three-part fusion protein is obtained, consisting of (i) the signal sequence of the protein of the outer surface of the membrane, (ii) the intramembrane domain of the protein of the outer surface of the membrane and (ii) the complete sequence of the mature pB-lactamase, which gives an active pD-lactamase molecule bound to membrane However, the Egapvibzso method applies only to prokaryotic cell systems and, as the authors admit, requires the presence of a complete three-part fusion product for proper functioning.

Sulfotransferases

The present invention also provides methods of obtaining peptides that include sulfated molecules, which include, for example, sulfated polysaccharides such as heparin, heparan sulfate, carrageenan, and related substances. Useful sulfotransferases include, for example, chondroitin-

β-sulfotransferase (chicken cDNA, described in the work of Riksh and A.I., 9. Viol. Spet. 270: 18575-18580 (1995); number in the SepVapk database 049915), glycosaminoglycan M-acetylglucosamine M-deacetylase/Mm-sulfotransferase 1 (Oihop et al., Sepotisv 26: 239-241 (1995); 0118918) and glycosaminoglycan M-acetylglucosamine M-deacetylase/M-sulfotransferase 2 (mouse cDNA described in OgeiYapa et al.,).

Vio Spent 269: 2270-2276 (1994) and Egike5op ei ai., 9. Vioi. Spent 269: 10438-10443 (1994); Human cDNA, described in SepVapk database number 02304).

Glycosidases

The present invention also extends to the use of wild-type and mutant glycosidases. It was shown that mutant B-galactosidase enzymes catalyze the formation of disaccharides by binding a-glycosyl fluoride to a galactosyl acceptor molecule (M/ipeg5, US patent Mo 6284494; issued September 4, 2001). Other glycosidases useful for use in the present invention include, for example, B-glucosidases, B-galactosidases, B-mannosidases, B-acetylglucosaminidase, D-M-acetylgalactosaminidase, B-xylosidases, DV-fucosidases, cellulases, xylanases, galactanases, mannanases , hemicellulases, amylases, glucoamylases, co-glucosidases, α-galactosidases, α-mannosidases, VD-M-acetylglucosaminidase, α-M-acetylgalactosaminidase, α-xylosidases, α-fucosidases and neuraminidase/sialidase.

Immobilized enzymes

The present invention also presents the use of enzymes on a solid and/or soluble basis.

In an exemplary embodiment, a glycosyltransferase is presented, conjugated to RES using an intact glycosyl linker according to the methods according to the invention. The RES-linker-enzyme conjugate is not necessarily attached to a solid base. The use of enzymes on a solid basis in the methods according to the invention simplifies the preparation of the reaction mixture and the purification of the reaction product, and also facilitates the isolation of the enzyme from the mixture. In the methods according to the invention, a glycosyltransferase conjugate is used. Other combinations of enzymes and bases will be apparent to one skilled in the art.

Fused proteins

In other exemplary embodiments, the methods according to the invention use fusion proteins that have more than one enzymatic activity involved in the synthesis of the desired glycopeptide conjugate. Fusion proteins can consist of 3, for example, a catalytically active glycosyltransferase domain connected to the catalytic domain of an auxiliary enzyme. The catalytic domain of an auxiliary enzyme can, for example, catalyze any stage of the formation of a nucleotide sugar, which is a donor for glycosyltransferase, or catalyze the reaction of the glycosyltransferase cycle. For example, a polynucleotide encoding a glycosyltransferase can be fused in the same reading frame to a polynucleotide encoding an enzyme involved in the synthesis of a nucleotide sugar.

The resulting fusion protein can catalyze not only the synthesis of a nucleotide sugar, but also the transfer of a sugar group to an acceptor molecule. The fusion protein can be two or more enzymes of the cycle bound into one expressed nucleotide sequence. In other embodiments, the fusion protein includes the catalytically active domains of two or more glycosyltransferases. See, for example, US Patent No. 5,641,668. Various applicable fusion bilayers facilitate the design and production of modified glycopeptides according to the invention (see, for example, patent application PCT/CA98/01180, published as UMO 99/31224 on June 24, 1999).

Production of modified sugars

In general, modified sugars are used in the methods according to the invention.

In one aspect, the sugar group or groups of the sugar group-linker cassette and the RES or cassettes of the RES-linker are linked to each other by means of reactive groups, which are usually converted during the binding process into a new organic functional group or a non-reactive group. The reactive functional group(s) of the sugar can be in any position of the sugar molecule. Reactive groups and classes of reactions applicable in this invention are usually groups and reactions well known in the art of bioconjugate chemistry. Currently, the predominant classes of reactions involve reactive sugar groups, which take place in relatively mild conditions. Such reactions include, but are not limited to, nucleophilic substitutions (i.e., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., the Michael reaction , joining Dils-

Alder). These and other applicable reactions are described, for example, in the publications of Magsy, AOMAMSEYUO

OMKSAMIS SNEMIZTAKU, Z ed., add UmMieu 5 bop5, Mem/ Moik, 1985; Neptapzopus, VIOSOMYOSATE

TESNMIOTSOE5, Asadetis Rge55, Zap Oyeedo, 1996; and Reepeu ei am, MOBIRISATIOM OE RAOTEIM5;

Admapsez ip Spetivigu Zegiev, Moi. 198, Ategisap Spetis! bosieiu, Mavpipdiop, 0.S., 1982.

Applicable reactive functional groups suspended from the sugar nucleus or modifying group include, but are not limited to: (a) carboxyl groups and various derivatives thereof, including but not limited to /N-hydroxysuccinimide esters, /N-hydroxybenzotriazole esters, acyl halides, acylimidazoles, thioethers, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;

(B) hydroxyl groups that can be converted into, for example, ethers, esters, aldehydes, etc.; (c) haloalkyl groups, where the halide can later be replaced by a nucleophilic group, such as, for example, an amine, carboxylate anion, thiol anion, carbanion or alkoxide ion, leading to the covalent attachment of a new group instead of a functional group of a halogen atom; (4) dienophilic groups capable of participating in Diels-Alder reactions, such as, for example, maleimido groups; (is) aldehyde or ketone groups, followed by possible derivatization by the formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or by mechanisms such as Grignard reactions or reactions with alkyllithium; () sulfonylhalide groups, for further reactions with amines, for example, with the formation of sulfonamides; (94) thiol groups, which can, for example, turn into disulfides or react with acyl halides; (n) amino groups or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized; () alkenes subject to, for example, cycloaddition, acylation, which participate in the Michael reaction, etc.; (I) epoxides that can react with, for example, amine and hydroxyl compounds.

Reactive functional groups can be chosen so that they do not participate in or interfere with the reactions necessary to assemble the reactive sugar nucleus or modifying group. Alternatively, the presence of a protecting group can protect a reactive functional group from participating in the reaction. Methods of protecting a particular functional group in a manner that does not affect the chosen reaction conditions will be apparent to one skilled in the art. For examples of suitable protecting groups, see, for example, the work of sgeepe ei am., RKOTESTEM SOKOSHRBZ IM OKSAMIS ZUMTNEbBI5, add

UMieu in Bop5, Mem HogKk, 1991.

The following discussion describes some specific examples of modified sugars useful in the present invention. In exemplary embodiments, a sialic acid derivative is used as the sugar core to which the modifying group is attached. The following discussion focuses on sialic acid derivatives solely for clarity of presentation and is not intended to limit the scope of this invention. It is obvious to those skilled in the art that other different sugar groups can be activated and derivatized in a similar way as described for the example of sialic acid.

For example, there are many ways to modify galactose, glucose, M-acetylgalactosamine and fucose and other carbohydrate substrates that are easily modified by methods known in the art. See, for example, EiPIPaiabi ei aI. satiated Honey. Spent 6:93 (1999); and zspageg ei ai., U. Ogd. Spent 65:24 (2000)).

In an exemplary embodiment, the C-C5E peptide, which is modified by the method according to the invention, is a glycopeptide produced by mammalian cells (for example, CHO cells) or in a transgenic animal and, thus, contains incompletely sialylated M- and/or O-zv linked oligosaccharide chains. Glycopeptide oligosaccharide chains that do not contain sialic acid and contain a terminal galactose residue can be pegylated, PPylated or otherwise modified with a modified sialic acid.

In scheme 4, aminoglycoside 1 is affected by an active ester - a derivative of a protected amino acid (for example, glycine), while the amine residue of the sugar is transformed into the corresponding amide adduct of the protected amino acid. Further, the adduct is affected by aldolase with the formation of α-hydroxycarboxylate 2. Compound 2 is converted into the corresponding CMP derivative by the action of CMP-5A synthetase, followed by catalytic hydrogenation of the CMP derivative to give compound 3. The amine introduced during the formation of the glycine adduct is used as a site addition of PES by reaction of compound C with an activated PES or PPO derivative (for example, PES-C(O)MH5, PES-OC(O)O-p-nitrophenyl), which leads to the preparation of compounds 4 and 5, respectively.

Scheme 4 -n I. EM BA synthetzza, STE no. si and i Z MURIE

MN 1 frame. and MO. he is I oh-! M MinsAye vyldonavya, piruvte nyu V o uche na nn "VIN VN pi arion schu y ichi a che and y sm yi S with Z sche ana u novn U t ROSY 0ovo vno Oka / y note tanfbno 0-th in no on

RE kna nach ben what 4 va Z a? :

SMRUNA-5-MNSOSNIMY--- In the world of kvsosyumy teo vesDsoyuchkie TASTE OF SOSNeMNU sMEVACH M ISOSVMN- SOKHNE

Table C shows examples of typical sugar monophosphates derivatized with the REO molecule.

Some of the compounds listed in Table 3 were prepared by the method shown in Scheme 4. Other derivatives were prepared by methods known in the art. See, e.g., Carrey et al., Biol. 11:11K (2001); and

SNAPEG EI A!., Siusobioiodu 10: 1049 (2000)). Other reactive amine analogs of RES and PPO are commercially available or can be prepared by methods readily available to those skilled in the art.

Table C

Mi» Mne her mo I mo 0-6 o. o-6-0-,0 no on o ma no on U ta no. Rye o "Ma no on k-o shSh-o Oma no on v-mn-yYa vomn-dnO o

CMP-BA-5-MH-B CMP-MetAs-9-0-8

MN" To me

CE 2 (p

M Sh mo o-B-o o o-k-o o no on o "Ma no on still "Ma ooZhv obust no on KM shSh-e-O "Ma no on p-O on o Aschn on o

СМР-КОМ-5-0-8 СМР-МешАс-9-МН-А

MN» Mne 5 C 5 E

I mo Y o mo t-O orto v-mn o-o on o Ma on Oma - - t- -zh no zh--9 Oma no on no 5-0 ro ma no on o b

AsMNA-"On AeMN-- on

СМР-МецАс-8-0-8 СМР-МешАс-8-МН-А мно МН» o Со С her mo her mo o--a o. | o-v-0 o no 0-k m de, no Mmn-v ee, no i (o) o "Ma no on no 2. s o Ma no on deMN no? demn--KZn oo

CMP-MeshAs-7-0-8 CMP-MeshAs-7-MN-B

MN. many; o S o S o--v-o oo v-v-9 oo no on s t C no on e t C

KE eTu "ma NO ON no IAKE A-o to "ma nO ON

AsMN O-v As MN-v

The modified sugar phosphates applicable in this invention may be substituted in other positions as well as in the above. The currently preferred sialic acid substitutions are given in the formula below:

Mne with S her i what gu in the CURRENT

VZUU Kha OO) sva

Like Go

At where X means a linking group preferably selected from -O-, -M(H)-, -5, СНе- and -М(В)", in which each K is independently selected from K'-H". Each of Y, 7, A, and B represents a group selected from the group given above for X. Each of X, Y, 7, A, and B is independently selected and thus may be identical or different. The symbols B', B, BZ, B" and 2" mean H, a RES molecule, drug, biomolecule or other substance. Alternatively, these symbols mean a linker bound to a RES molecule, drug, biomolecule or other substance.

Exemplary substances attached to the conjugates disclosed herein include, but are not limited to RES derivatives (eg, acyl-RES, acyl-alkyl-RES, alkyl-acyl-RES, carbamoyl-

PES, aryl-PES), PPO derivatives (for example, acyl-PPO, acyl-alkyl-PPO, alkyl-acyl-PPO, carbamoyl-

PPO, aryl-PPO), medicinal substances, diagnostic substances, mannose-6-phosphate, heparin, heparan, ZI Eh, mannose, mannose-b-phosphate, ziau! Hemi5 X, BOB, MESOBE, proteins, chondroitin, keratan, dermatan, albumin, integrins, antennal oligosaccharides, peptides and others. The methods of conjugation of various modifying groups to the saccharide group are easily available to specialists in the field of technology

VIOMEBVISAG ARRIISATIOM5, under the editorship. in). Miyop Nagti5, Riepyt Ryb. Sogr., 1992; ROI M (ETNMI EME

SI USOY) SNEMISAY AMO VIOGHOSISAY ARRIISATIOM5, under the editorship. U. Miyop Nagtikh, ASZ Zutrovisht zZepez Mo. 680, Ategisaps Spetis! Bossiviu, 1997; Nappapsop, VIOSOMIOSATE TESNMIOOE5, Asadetis

Rgez5, Zap Oiedo, 1996; and Yuipp ei aiI., Eaz. RO UMEKIS OKOSZ AMO OKO SE IMEKU 5UZTEM5, AS

Zutrovzisht 5egie5 Moi. 469, Ategisaps Spetis! Zosieiu, UMazpipdiop, 0. S 1991).

Linker groups (cross-linking groups) The preparation of modified sugars for use in the methods of the present invention involves the addition of a RES molecule to a sugar residue and preferential formation of a stable adduct that is a substrate for glycosyltransferase. Thus, it is often preferable to use a linker, for example, a linker formed by reacting the RES and a sugar group with a cross-linking agent to conjugate the RES and the sugar. Exemplary bifunctional compounds that can be used to attach modifying groups to carbohydrate groups include, but are not limited to, bifunctional polyethylene glycols, polyamides, polyesters, polyesters, and others. General approaches to linking carbohydrates with other molecules are known from the literature. See, for example, G ee ei ai!., Viospetivighu 28: 1856 (1989); Vpagya ei a!., Apai. Viospet. 178: 408 (1989); dapaa ei ai., U. At. Spent 5 obs. 112: 8886 (1990) and

Veapag»hki eyi a, M/O 92/18135. In the discussion below, it is assumed that the reactive groups do not impair the properties of the sugar group of the newly formed modified sugar. The topic of discussion is chosen for clarity of presentation. One skilled in the art will appreciate that the discussion also applies to reactive groups on a modifying group.

Various reagents are used to modify the components of the modified sugar with the help of intramolecular chemical cross-links (see reviews of reagents and procedures for the formation of cross-links UuoYiYa, K, Mei. Eplutoi. 25: 623-651, 1972; MeezaiI, N. N, apa

Soopeu, 0. A., in: EMAUME5 AZ OKIS, (ed. NoiIseprego and Kobregi5) p. 395-442, MMIeu, Mem MOiK, 1981; Her, T.N., Meiy. Epgutoi 91: 580-609, 1983; Myzhop ey a!., Moi. Cheers! Ner. 17: 167-183, 1993, each of which is incorporated herein by reference). Preferred cross-linking reagents are obtained from various zero-length cross-linking reagents and homobifunctional and heterobifunctional cross-linking reagents.

Zero-length cross-linking reagents involve the direct conjugation of two chemical groups present without the addition of additional material. This category includes agents that catalyze the formation of a disulfide bond. Another example is reagents inducing the condensation of a carboxyl group and a primary amino group with the formation of an amide bond, such as carbodiimides, ethyl chloroformate,

Woodward's K reagent (2-ethyl-5-phenylisoxazolium-3-sulfonate) and carbonyldiimidazole. In addition to these chemical reagents, the enzyme transglutaminase (glutamyl-peptide γ-glutamyltransferase; EC 2.3.2.13) can act as a zero-length reagent that forms cross-links. This enzyme catalyzes the acyl transfer reaction to the carboxamide groups of glutaminyl residues bound to the protein, usually using the primary amino group as a substrate. Preferred homo- and heterobifunctional reagents contain two identical or different sites, respectively, which may be reactive toward amino, sulfhydryl, guanidine, indole, or nonspecific groups.

Precipitation of insoluble C-C5E

Many recombinant proteins expressed in bacteria are expressed as insoluble aggregates in bacterial inclusion bodies. Inclusion bodies are protein deposits found in the cytoplasm and periplasm of bacteria (see, for example, Siagk, Sig. Or.

Vioyesn. 12:202-207 (2001)). Recombinant 5-C5E proteins are expressed in bacterial inclusion bodies, and this publication presents methods for the synthesis of such proteins, which allow obtaining active 5-C5E proteins.

Conditions for precipitation of active 5-C5E

To obtain active C-C5E proteins from bacterial cells, 5-C5E proteins are expressed in bacterial inclusion bodies. Bacteria are collected, subjected to destruction, and inclusion bodies are isolated and washed. In one embodiment, three washings are carried out: the first washing with a buffer with a pH of 6.0-9.0; containing a monovalent salt, for example, sodium chloride; nonionic detergent, e.g.

Tiyop X-100; ionic detergent, for example, sodium deoxycholate; and EDTA; a second wash with detergent-free buffer and a third wash with Neo. Next, the proteins found in the inclusion bodies are solubilized. Solubilization can be carried out using denatured substances, guanidyl chloride or urea; extreme pH values; or detergents, or any combination of the above effects. In one embodiment, 5-6 M guanidine NSI or urea are used for solubilization (3-C5ZE.

In another embodiment, OTT is added.

After solubilization, denaturants are removed from the protein mixture containing 5-C5E. Denaturants can be removed by a variety of methods, including transfer to precipitation buffer or buffer exchange. Methods of buffer replacement include dialysis, diafiltration, gel filtration, and immobilization of protein on a solid support (see, e.g., SiagK, Sig. Or. Viol. 12:202-207 (2001)). Any of the above methods can be combined to remove denaturants.

The formation of disulfide bonds in 5-C5E proteins is stimulated by the addition of a precipitation buffer that contains an oxidizing-reducing pair. Oxidant-reducing pairs include reduced and oxidized glutathione (-U5E-IZ550), cysteine/cystine, cysteamine/cystamine, YTT/Z55O and OTE/5555. (see, for example, SiagK, Sig. Or. Bioesp. 12:202-207 (2001)). In one embodiment, the oxidizing/reducing pair is 5H/Z550 in a 10:1 ratio.

Precipitation can be carried out in buffers with a pH in the range of, for example, 6.0 to 10.0. Buffers for precipitation may include other additives to facilitate precipitation, for example, I -arginine (0.4-1 M); RES; low concentrations of denaturants, such as urea (1-2 M) and guanidine chloride (0.5-1.5 M); and detergents (for example, Sparz, 505, STAV, laurylmaltoside, Tueep 80 and Tiyop X-100).

After sedimentation, the 5-C5E protein can be dialyzed to remove redox or other unwanted buffer components. In one embodiment, dialysis is performed using a buffer containing sodium acetate, glycerol, and a nonionic detergent, for example, Tmeep-80. After dialysis, protein c-

CE can undergo further purification and/or concentration using ion exchange chromatography. In one embodiment, the cation exchange resin 5P-sepharose is used.

One skilled in the art will understand that a protein has been digested correctly if the digested protein has detectable biological activity. For C-C5E protein, activity can be measured by various methods. For example, biologically active proteins 5-C5E are substrates of O-linked glycosylation, described in US patent applications 60/535284, filed January 8, 2004; 60/544411, filed on February 12, 2004; and Notary Register Mo 019957-01882005 filed on February 2, 2004, the contents of which are incorporated herein by reference for all purposes. 5-C5E protein activity can also be measured using a cell proliferation assay or a rat white blood cell assay (also described in US Patent Applications 60/535284, filed Jan. 8, 2004; 60/544411, filed Feb. 12, 2004; and Notary Register Me 019957-01882005, filed on February 2, 2004, the contents of which are incorporated into this publication by reference for all purposes). Analysis of cell proliferation and analysis of white blood cells can be performed before or after O-linked glycosylation of 5-C5E salted proteins.

Methods of isolation of conjugates according to the invention

Alternatively, the products obtained from the processes described above can be used without purification. However, product selection is of course preferable. Standard well-known methods for the isolation of glycosylated saccharides can be used, such as thin-layer or thick-layer chromatography, column chromatography, ion exchange chromatography or membrane filtration. Preferably, membrane filtration, more preferably using a reverse osmosis membrane, or one or more column chromatography techniques, as discussed below in this application and in the literature cited in this application, is used for isolation.

For example, membrane filtration can be used to remove proteins such as glycosyltransferases where the membranes have a molecular weight cut-off of about 3000 to about 10000. Further, nanofiltration or reverse osmosis can be used to remove salts and/or purify the saccharide product (see, e.g. UMO 98/15581). Nanofiltration membranes are a class of reverse osmosis membranes that pass monovalent salts but retain polyvalent salts and uncharged solutes greater than about 100 to about 2000 Daltons, depending on the membrane used. Thus, in normal use, the saccharides produced by the methods of the present invention will be retained by the membrane, and impurity salts will pass through the membrane.

If the modified glycoprotein is produced inside the cell, the first step is to remove debris, that is, host cells or lysed fragments, for example, by centrifugation or ultrafiltration; the protein may optionally be concentrated using a commercially available protein concentration filter followed by separation of the polypeptide variant from impurities during one or more steps selected from immunoaffinity chromatography, ion exchange column separation (e.g., with diethylaminoethyl (OEAE) or matrices that contain carboxymethyl or sulfopropyl groups), chromatography on Vipe-Zerpagose, SM Vise-zerpagose, MOMO-O, MOMO-5,

Iepiii Iesiip-Zerpagose, MUSA-Zerpagose, Sop A-Zerpagose, ESheg Toworeap, Vshu! Touoreai!, RNepui

Touoreagi, or rgoivip A Zerpagose, 5O5Z-RACE chromatography, silica chromatography, chromatographic focusing, reverse-phase HPLC (eg, silica gel with attached aliphatic groups), gel filtration using, for example, Berpradech molecular sieves or size-exclusion chromatography, column chromatography , which selectively bind the polypeptide, and precipitation with ethanol or ammonium sulfate.

Modified glycopeptides produced in culture are usually isolated by primary extraction from cells, enzymes, etc. followed by one or more steps of concentration, salting out, aqueous ion exchange or size exclusion chromatography. The modified glycoprotein can be further purified using affinity chromatography. Finally, HPLC can be used in the final stages of purification.

At each of the above stages, a protease inhibitor such as phenylmethylsulfonyl fluoride (PMP) can be added to inhibit proteolysis, as well as antibiotics to prevent the growth of adventitious contaminants.

In another embodiment, supernatants from systems producing a modified glycopeptide according to the invention are first concentrated using a commercially available protein concentration filter, such as an Artichoke or Miroghe Reisop ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable matrix for purification. For example, a suitable affinity matrix may contain a peptide ligand, lectin or antibody molecule linked to a suitable base. Alternatively, an anion exchange resin, such as a matrix or substrate with suspended OEAE groups, can be used. Suitable matrices include acrylamide, agarose, dextran, cellulose or others commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Sulfopropyl groups are especially preferred.

Finally, for further purification of the composition containing the polypeptide variant, one or more stages of HPLC using hydrophobic KR-NRI C media, such as silica gel with suspended methyl or other aliphatic groups, can be used. Some or all of the above purification steps, in various combinations, can also be used to obtain a homogeneous modified glycoprotein.

The modified glycoprotein according to the invention, obtained by large-scale fermentation, can be purified by methods similar to those described in the work of Ogaai ei ai., U. Spgotais. 296: 171 (1984). This reference describes two consecutive stages of CR-NRIS for the purification of recombinant human II-2 on a preparative HPLC column. Alternatively, techniques such as affinity chromatography can be used to purify the modified glycoprotein.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition. The pharmaceutical composition contains a pharmaceutically acceptable diluent and a covalent conjugate of an unnatural molecule of REES, a medicinal substance or biomolecule and a glycosylated or non-glycosylated peptide.

The polymer, medicinal substance or biomolecule is conjugated to the C-C5E peptide through an intact glycosyl linking group located between and covalently bound to the -C5BE peptide, and also to the polymer, medicinal substance or biomolecule.

Pharmaceutical compositions according to the invention are applicable in various drug delivery systems.

Preparations applicable in this invention are described in Keptipdiopye RNagtaseciisaI! Zsiepse5, Mase

Rybiyepipd Sotrapu, RPyaaei!rpia, RA, 171p ey. (1985). For a brief overview of drug delivery methods, see I apdeg, Zsiepse 249:1527-1533 (1990).

Pharmaceutical compositions can be formulated for any suitable route of administration, including, for example, local, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier is preferably water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate can be used. Suitable biodegradable microspheres (for example, polylactate, polyglycolate) can also be used as carriers of the pharmaceutical composition according to the invention. Suitable biodegradable microspheres are disclosed, for example, in US Patent Nos. 4,897,268 and 5,075,109.

Usually, pharmaceutical compositions are administered parenterally, for example, intravenously. Thus, the present invention provides compositions for parenteral administration that contain a substance dissolved or suspended in a suitable carrier, preferably an aqueous carrier, for example, in water, buffer, saline, PB5 and others. The compositions may contain pharmaceutically acceptable additional substances necessary to achieve conditions close to physiological ones, such as pH regulators and buffers, tonicity regulators, wetting agents, detergents, and others.

These compositions can be sterilized using conventional sterilization techniques or passed through a sterilizing filter. The resulting aqueous solutions may be packaged for use as is or in lyophilized form, in which case the lyophilized preparation is combined with a sterile aqueous vehicle prior to administration. The pH of the preparations is usually in the range from 3 to 11, more preferably from 5 to 9 and most preferably from 7 to 8.

In some embodiments, glycopeptides according to the invention can be included in liposomes formed by standard lipids that form vesicles. There are various methods of obtaining liposomes, described, for example, in the work of 520Ka ei ai, App. Kew. Viorpukh Vioepa. 9: 467 (1980) and U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. Targeting of liposomes using various targeting agents (eg, sialyl galactosides of the invention) is well known in the art (see, e.g., U.S. Pat.

US MoMo 4957773 and 4603044).

Standard methods can be used to bind targeting agents to liposomes. These methods typically include incorporation into liposomes of lipid components, such as phosphatidylethanolamine, which can be activated to attach targeting agents or derivatized lipophilic compounds, such as glycopeptides derivatized with lipids according to the invention.

For the functioning of targeting mechanisms, it is usually necessary that the targeting agents are on the surface of the liposome in such a way that the targeting groups can interact with their target, for example, with a receptor on the cell surface. Carbohydrates according to the invention can be attached to a lipid molecule to form a liposome using methods known to those skilled in the art (for example, alkylation or acylation of the hydroxyl group on the carbohydrate with a long-chain alkyl halide or fatty acid, respectively). Alternatively, the liposome can be formed in such a way that the connecting part is embedded in the membrane during the formation of the membrane. The connecting part should include a lipophilic part, which is embedded and rigidly fixed in the membrane. It should also include a reactive part that is chemically available on the surface of the liposome in contact with the aqueous medium. The reactive part is chosen in such a way that it is suitable from a chemical point of view to form a stable chemical bond with the targeting agent or the carbohydrate attached later. In some cases, direct attachment of the targeting agent to the linker molecule is possible, but in most cases it is more convenient to use a third molecule as a chemical bridge, thus linking the linker molecule located in the membrane to the targeting agent or carbohydrate in three-dimensional space, rather than on the surface of the vesicle.

Compounds obtained by methods according to the invention may also be applicable as diagnostic reagents. For example, labeled compounds can be used to localize a site of inflammation or tumor metastases in a patient suspected of having inflammation. For this application, compounds can be labeled with 7251, 14C or tritium.

The active ingredient in the pharmaceutical compositions according to the present invention is glycopegylated C-C5E and its derivatives having biological properties of follicle-stimulating hormone, for example, stimulating ovulation. Preferably, the composition according to the invention, which contains 5-C5E, is administered parenterally (for example, intravenously, intramuscularly, subcutaneously or intraperitoneally). Effective dosages are expected to vary greatly depending on the disease being treated and the route of administration, but will be in the range of 0.1 (approximately 7 U) to 1,000 (approximately 7,000 U) mcg/kg of body weight for the active ingredient.

Preferred dosages for the treatment of anemic conditions are from about 50 to about 300

Unit/kg three times a week. Since the present invention presents 5-C5E, which has an increased residence time, the indicated dosages can be reduced when the composition according to the invention is administered.

Dosage forms

In preferred aspects, according to any method described above, the peptide conjugates according to the invention, used to treat a disease related to hematopoiesis or myelosuppression, are provided in the form of dosage forms for oral administration. In preferred embodiments, these dosage forms for oral administration include the following components: (a) a peptide that is a covalent conjugate of the C-C5E peptide and a water-soluble polymer, wherein the water-soluble polymer is covalently attached to the 5-C5E peptide near a glycosyl or amino acid residue of the peptide 5-C5E using an intact glycosyl binding group; (B) one or more surfactants; (c) one or more fatty acids; and (4) enteric material. In particularly preferred embodiments, the peptide, surfactants, and fatty acids are mixed in a liquid phase and lyophilized prior to combining with the enteric material.

It should be understood that the examples and embodiments described in this publication are provided solely for the purpose of illustration and that, in light of this, various modifications and changes suggested to those skilled in the art are included within the spirit and scope of this application and are covered by the following claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

The following examples are provided to illustrate compositions and methods according to the present invention and are not intended to limit the claimed invention.

EXAMPLES

Example 1: Pharmacodynamics data on the example of neutrophil count in response to the 5-C5E peptide conjugate according to the invention

Studies of pharmacodynamics using the example of neutrophil counts in response to 5-C51E conjugates according to the invention and commercially available c-C5E neylast showed that the compositions according to the invention had a similar period of action to neylast (Fig. 1) at the same concentration. The number of neutrophils depended on the dose - increasing the concentration of conjugates (3-C5E according to the invention (glycoRES -

CE) led to an increase in the number of neutrophils at the peak of the action period. GlycORES 5-C5E induced a response approximately 30 95 higher compared to neilasta, indicating a 60 95 increased bioavailability of glycoRES O-C5BE compared to neilasta at comparable doses.

The study included 53 subjects. There were 20 subjects in each dose group, of which 15, determined randomly, received glycoRES O-C5BE, and five - neilasta. In general, glycoRES O-

C5E was well tolerated, with a side effect profile similar to that of neilasta. There was no study discontinuation due to side effects. There were also no cases of serious side effects. In addition, no antibodies to glycoORES 5-C5E were detected.

Table 4 shows representative data for three different concentrations of glycoRES-C5E for the time period from 24 to 168 hours after the introduction of glycoRES 5-C5E.

Table 4 96 17111110 36.9222 77111111 237667, | 124625 SHHD-BZHZH

Example 2: Data of pharmacodynamics on the example of the counting of SOZ34 cells in response to the 5-C5E peptide conjugate according to the invention

Study of pharmacodynamics using the example of SOC4 cell counting in response to conjugates of

SZE according to the invention (glycoORES O-C5E) and commercially available z-C5E neylast showed that the compositions according to the invention had a similar period of action to neylast (Fig. 2) at the same concentration. GlykoRES O-C5BE induced a significantly greater number of SOZ34 cells -- at the peak of its activity than neilasta at the same concentration.

The number of cells depended on the dose - increasing the concentration of glycoRES 5-C5E led to an increase in the number of SOZ4- cells at the peak of the action period.

Table 5 shows representative data for three different concentrations of glycoRES-C5E for the time period from 72 to 168 hours after the introduction of glycoRES 5-C5E.

Table 5 pi: C PO Teh POLYA PO SU TO POT i

Example 3: Pharmacodynamic data on the example of neutrophil count in response to the 5-C5E peptide conjugate according to the invention, fixed-dose study

Studies of pharmacodynamics using the example of neutrophil counts in response to fixed doses of 5-C5E conjugates according to the invention (glycoORES (-C5E) and commercially available 5-C5E neylast showed that the compositions according to the invention had a similar period of action to neylast (Fig. 4) at the same concentration.GlycoRESi 4-C5E induced a response approximately 30 95 higher compared to neilasta, which indicates a 60 95 increased bioavailability of glycoRES O-C5E compared to neilasta at the studied dose.

36 healthy subjects participated in the study. In general, GlycORES 5-C5E was well tolerated, side effects were similar to those of Neylast. There were no study discontinuations due to side effects, and there were no cases of serious side effects. In addition, no antibodies to glycoRESs (-C5E.

Example 4: Data of pharmacodynamics on the example of counting cells of SOC34-- in response to the peptide conjugate 5-C5E according to the invention, fixed dose study

Studies of pharmacodynamics using the example of SOC4 cell counts in response to fixed doses of 5-C5E conjugates according to the invention (glycoORES (-C5E) and commercially available 5-C5E neylast showed that the compositions according to the invention had a similar period of action to neylast (Fig. 5).

Example 5: Mobilization of allogeneic/autologous precursors of POP34 cells in peripheral blood

Bone marrow donors were administered 10-20 μg/kg of glycopegylated 5-C5E (glycoRES 0-C51) for 5 days to increase the level of SOZ34zh- cells from resting values (approximately 2 cells/μL) to approximately 10 cells/μL, which is enough to obtain 2-4x105 cells of sors3a4h/kg during a single apheresis. Bone marrow donors can be allogeneic (different from the recipient) or autologous (identical to the recipient).

LIST OF SEQUENCES

"-110" Viosepegih AC "120" Methods of treatment using glycopegylated 5-C51E "130" В 8829/:М -1505 05 60/909917 -151" 2007-04-03 -1505 05 60/911788 -151" 2007- 04-13 -1505 05 60/986240 -151" 2007-11-07 -1505 MO2008124406 -151" 2008-04-01

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Fig. 10

Claims (31)

1. A method of increasing the production of stem cells in a donor, where the specified method includes the introduction of a certain amount of peptide into the specified donor, which is a covalent conjugate of the О-C5E 1 peptide of a polymeric modifying group, where the specified polymeric modifying group is covalently attached to the specified peptide near the glycosyl or amino acid residue of the specified peptide with the help of a glycosyl binding group, where the specified amount is a single dosage form selected from 50 μg/kg, 100 μg/kg m1.200 μg/kg. 2. The method according to claim 1, where the specified polymer modifying group is a water-soluble polymer. 3. The method according to claim 2, where the specified water-soluble polymer is polyethylene glycol. 4. The method according to claim 2, where the specified water-soluble polymer is selected from a linear water-soluble polymer and a branched water-soluble polymer. 5. The method according to claim 1, where the indicated polymeric modifying group and the indicated glycosyl binding group are covalently connected by means of a linker. b. The method according to claim 5, where the specified glycosyl binding group includes a modified sialic acid residue, and where the specified modified sialic acid residue has a structure according to the formula: со: но ен но В о кК-- НМ он , where K means the specified polymeric modifier group, and said polymeric modifying group is attached to said sialic acid residue by means of said linker. 7. The method according to item b, where the specified modified sialic acid residue has a structure according to the formula: Со: но он о но: О НО Ах АХ Нм (c) п (c) SY on , (c) where п means an integer from 1 to 2000. 8. The method according to claim 1, where the specified polymer modifying group has an essentially homodisperse molecular weight distribution. 9. The method according to claim 1, where the indicated glycosyl binding group contains a modified sialic acid residue having the formula: on on on Kb u o ra v Her i o Z MN dz Tuesday yes and where is VE? is H, СН2ОВ, СООВ! or OB, where B" means H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl; БЕ" are independently selected from H, substituted or unsubstituted alkyl, ОВУ, МНС(ОК", where BZ and B? are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or sialic acid; I" means a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; Vic! means independently selected polymer "arms"; Xy X" means independently selected bonds binding fragments connecting polymer groups VIV with Si X" is a non-reactive group. 10. The method according to claim 1, where the specified amino acid residue is selected from serine 1 threonine. 11. The method according to claim 1, where the specified O-C5E peptide has the amino acid sequence 5EO IU MO: 1. 12. The method according to claim 11, where the specified amino acid residue is threonine at position 134 5EO Y MO: 1. 13. The method according to point b, where the specified glycosyl binding group contains a substructure selected from: i- (SaimAs); (ba), and- (Saimds),--(Sa),- bZia-- V/» where VE! means the specified modified sialic acid residue; and ri 4 stand for independent integers chosen from 0 or 1. 14. The method according to claim 13, where d is 0. 15. The method according to claim 6, where the specified glycosyl linking group has the formula selected by 3: shi g Map--(SisMAs - sa), - В» AA- -- 6|ISMAs--(|SMAs-- Map Map t (no), Map AA- -- SISMAs---S|ISMAs--- Map. (siSMAs -ba), SHU shi g Map--(SismAs --- ba), - Vy» AA- bISMAS---biSMAs-- Map-- -(SISMAs -- ba), - 15 Map -- -- ((6IsMAs --bai),-5 and (siSMAs --- bai)r-- V u g Map--(SismAs - Sa); - Vy» AA- biSMAS-- biSMAS-- Map --- (SisMAs -ba|);-B5 Map-- I -(SISMAs -- sa) -- 85 (siSMAs -- ba); -5 , where AA means the amino acid residue of the specified peptide; is means an integer that is equal to 0 or 1; p means an integer from I1 to 10; 1 BE" is selected from H, OH, sialic acid, the indicated modified sialyl residue and bi-5ia", where 5ia? means the indicated sialyl residue, where at least one BE" is selected from the indicated modified sialyl residue and 5ia-5ia". 16. The method according to claim 15, where the specified amino acid residue is an asparagine residue. 17. A method of increasing the number of granulocytes in a subject, where the specified subject is suitable for bone marrow transplantation, where the specified method includes the administration of a certain amount of peptide to the specified subject, which is a covalent conjugate of the О-С5Е 1 peptide of the polymer modifying group , where the indicated peptide (O-C5E has the amino acid sequence shown in 5EO I MO: 1, 1 where the indicated polymeric modifying group is covalently attached to the indicated O-C5E peptide in the region of the indicated O-C5E peptide, which begins with glycine at position 126 and ends serine at position 143, where the specified amount is a unit dosage form selected from 50 μg/kg, 100 μg/kg, and 200 μg/kg. 18. Method of treatment of the subject's disease. in need of treatment, where the specified disease is characterized by inhibition of the production of white blood cells in the specified subject, where the specified method includes the step of administering to the specified subject a certain amount of the peptide, which is a covalent conjugate of the O-C5E peptide and a polymeric modifying group , wherein said polymeric modifying group is covalently attached to said peptide near a glycosyl or amino acid residue of said peptide via a glycosyl linking group, wherein said amount is effective to alleviate said disease in said subject, wherein said amount is a unit dosage form selected with 50 μg/kg, 100 μg/kg and 200 μg/kg. 19. The method according to claim 18, where said inhibition of white blood cell production is due to chemotherapy, radiation therapy or idiopathic thrombocytopenic purpura. 20. A method of treating neutropenia or thrombocytopenia in a mammal, which includes the administration of a pharmaceutically effective amount of a peptide, which is a covalent conjugate of the O-C5E peptide and a polymeric modifying group, where the specified polymeric modifying group is covalently attached to the specified peptide near the glycosyl or amino acid residue of the specified peptide by means of a glycosyl binding group, where the specified amount is a unit dosage form selected from 50 μg/kg, 100 μg/kg and 200 μg/kg. 21. The method of increasing the population of hematopoietic stem cells in culture, where the specified method includes the stage of adding to the specified stem cells an effective amount of the peptide, which is a covalent conjugate of the О-С5Е 1 peptide of the polymer modifying group, where the specified polymeric modifying group is covalently attached to the specified peptide near the glycosyl or amino acid residue of the specified peptide by means of a glycosyl binding group, where the specified amount is a unit dosage form selected from 50 μg/kg, 100 μg/kg 1,200 μg/kg. 22. A method of increasing hematopoiesis or increasing the number of hematopoietic progenitor cells in a subject, where the specified method includes the stage of administering to the specified subject an effective amount of the peptide, which is a covalent conjugate of the O-C5E 1 peptide of the polymeric modifying group, where the specified polymeric a modifying group is covalently attached to said peptide near a glycosyl or amino acid residue of said peptide via a glycosyl linking group, wherein said amount is a unit dosage form selected from 50 μg/kg, 100 μg/kg, 1,200 μg/kg. 23. The method according to claim 22, where the indicated hematopoietic progenitor cells are cells of sor. 24. A method of ensuring long-term engraftment of a bone marrow transplant, where the specified method includes: (a) administration to the donor of the specified bone marrow peptide, which is a covalent conjugate of the О-С5Е 1 peptide of a polymeric modifying group, where the specified polymeric modifying group is covalently attached to the specified of the peptide near the glycosyl or amino acid residue of the specified peptide by means of a glycosyl linking group, where the specified peptide is administered in a single dosage form selected from 50 μg/kg, 100 μg/kg, and 200 μg/kg; (5) extracting said bone marrow from said donor; 1 (c) infusion of the indicated bone marrow to the recipient. 25. The method according to claim 24, where the donor is the same person as the recipient. 26. The method according to claim 25, where the donor is a person different from the recipient. 27. A method of increasing the number of hematopoietic progenitor cells in a subject, wherein said method includes administering to said subject: (a) a first composition containing a compound of formula (1) representing 1,1-01,4-phenylene- bis-(methylene)-bis-1,4,8,11-tetraazacyclotetradecane (AMO3100); 1 (5) of the second composition containing a peptide that is a covalent conjugate of the O-C5E 1 peptide of a polymeric modifying group, where the specified polymeric modifying group is covalently attached to the specified peptide near the glycosyl or amino acid residue of the specified peptide by means of a glycosyl binding groups where the specified peptide is administered in a single dosage form selected from 50 μg/kg, 100 μg/kg, 1,200 μg/kg. 28. The method according to claim 27, where the specified first composition and the specified second composition are introduced sequentially and in any order. 29. The method according to claim 27, where the specified first composition and the specified second composition are introduced simultaneously. 30. The method according to claim 27, where the indicated hematopoietic precursor cells are cells of the sor. 31. A dosage form for oral administration, which includes components: (a) a peptide that is a covalent conjugate of the C-C5E peptide 1 of a water-soluble polymer, where the specified water-soluble polymer is covalently attached to the specified O-C5E peptide near the glycosyl or amino acid residue of the specified peptide O-C5E using an intact glycosyl binding group, where this peptide is presented in a single dosage form selected from 50 μg/kg, 100 μg/kg, 1,200 μg/kg; (5) surface-active substance (substances); (c) fatty acid(s); (4) enteric material, where the specified components (a), (b) 1 (c) are mixed in a liquid phase 1 lyophilized before combining with component (4). TNe rgezepi ipueptsop rgohidez a rIusoreruiaiea O-S5E fai 15 ShegarepisaTsu asiue apia mission Bav rragitasoKipeis ragateieet5 apa rgoregtsev Fa age pirgoueyed geIaiue (0 ap i1depisai, og sioveIu apaIovoiv, O-S5E reride Shai 15 poi gIederuiYi. Eishpietgtoge, She ipueptsop rgohide5 tefodyv Tog torShlipe Petag(oroiieviv ip a vyb)esi, ragysshatpu a vybiesi mo Bav geseiuei og m teseihe tadpayop og sretofShegaru (geaypepi. Tre tefodv5 api sotrovyop5 oi She ipueptsop sap Tygiyeg be izey Io rgueepi, - aPeuia(e apa (geai Fe tuye Iovyrrgevvihe ePesiv visi (Pegariev.