HK1149498B - Selenium containing modifying agents and conjugates - Google Patents

Description

Selenium-containing modifiers and conjugates

The present invention relates to a modifying agent comprising a water-soluble polymer, wherein the water-soluble polymer comprises at least one reactive selenium group, which is capable of reacting with a thiol, preferably a thiol (thio) group of a pharmaceutically active agent, thereby forming an-Se-S-bond. Furthermore, the invention relates to a method for producing said modifying agent and to the use of said modifying agent for the modification of pharmaceutically active agents, such as G-CSF. Furthermore, the invention relates to conjugates comprising a water-soluble polymer and a pharmaceutically active agent, wherein the water-soluble polymer is linked to the pharmaceutically active agent by an S-Se bond, to a process for the production of the conjugates and to their use as medicaments. Finally, the invention also relates to pharmaceutical compositions comprising the conjugates of the invention.

Over the last years, the number of pharmaceutically active agents, such as recombinant proteins and peptides, has increased dramatically. However, many of these proteins and peptides are unsuitable for therapeutic use due to their short half-life in vivo, immunogenicity, low resistance to proteolysis, or low solubility. To overcome these drawbacks, different solutions are used. One of them is the chemical modification of proteins or peptides by binding them to water-soluble polymers, in particular polyethylene glycol. This process, known as pegylation, has proven effective in improving protein properties. The conjugation of a protein to a PEG chain increases the molecular weight of the protein and prolongs the half-life in vivo. The PEG chain wrapped around the protein has a protective effect on the protein and thus reduces proteolytic degradation and immunogenicity of the conjugate.

PEG is most commonly conjugated to proteins and peptides through the amino group of a lysine residue and the amino group at the N-terminus of an amino acid. The number of lysines on the surface of proteins is usually high, and thus pegylation of the lysine amino groups results in the formation of complex mixtures comprising positional isomers and polyglycolylated forms.

Cysteine residues, which are often less frequent than lysine residues, provide a more desirable possibility for site-specific pegylation. Modification of free thiol residues can be performed using PEG reagents with different reactive groups. In the art, it is recommended to use PEG reagents with thiol or disulfide (protected thiol) reactive groups. However, these PEG reagents form disulfide bonds with sulfhydryl groups, which can be easily cleaved in a reducing environment. There are currently two commercially available PEG reagents that undergo protein modification by formation of disulfide bonds with the above-mentioned disadvantages. The reagents are based on PEG-thiol and PEG-o-pyridine disulfide reagents, described in US 2005/0014903A 1. Similarly, a method for PEGylation of Cys18 of G-CSF has been proposed in EP 1586334. However, this method results in conjugates containing-S-linkages, which include the disadvantages described above.

It is therefore an object of the present invention to provide advantageous reagents for modifying a pharmaceutically active agent comprising at least one thiol group, for example a polypeptide comprising at least one free cysteine group. Preferably, the modification should be reversible.

In particular, it is an object of the present invention to provide a modifier for modifying a pharmaceutically active agent comprising at least one sulfhydryl group, wherein the reaction time should be shortened. In addition, a low excess of modifier is necessary. Furthermore, site-specific pegylation should be achieved.

It is another object of the present invention to provide a simple and reliable method for producing a conjugate comprising a water-soluble polymer and a pharmaceutically active agent, wherein the water-soluble polymer should be attached via a thiol group of the pharmaceutically active agent.

Generally, commercially available thiol-reactive PEG reagents can only be used under basic conditions, however, unwanted side reactions such as formation of disulfide-linked protein dimers and multimers typically occur under basic conditions. It is therefore another object of the present invention to provide thiol-reactive PEG reagents that can also be used under acidic conditions, although the expected reaction rate is low compared to under basic conditions. Generally, proteins containing surface exposed cysteine residues can spontaneously oxidize to disulfide-linked dimers or multimers. This reaction is relatively slow under acidic conditions, and therefore it is advantageous that the modification reaction using the modifier of the invention can be carried out at an acidic pH.

Finally, it is an object of the present invention to provide pharmaceutical compositions comprising the conjugates of the invention.

The object of the present invention is surprisingly achieved by using modifiers containing selenium groups, especially selenium groups which are reactive with thiol groups.

A subject of the present invention is therefore a modifying agent comprising a water-soluble polymer, wherein said water-soluble polymer comprises at least one reactive selenium group, which is capable of reacting with a mercapto group (-SH) to form a selenium-sulfur bond (Se-S-bond).

The modifier comprises two essential features:

(1) a water-soluble polymer; and

(2) a reactive selenium group.

Optionally, the modifier further comprises

(3) A linking group, wherein typically the linking group links the water-soluble polymer and the reactive selenium group.

The specific description about the features (1), (2) and (3) is as follows:

the water-soluble polymer (1) is any polymer that is soluble in water at 25 ℃. Generally, the water-soluble polymer is at least about 40% by weight soluble in water, more preferably at least about 55% by weight soluble in water, still more preferably about 75% by weight soluble in water, and particularly preferably about 90% by weight soluble in water. Most preferably, the water-soluble polymer or segment is about 95% by weight soluble or completely soluble in water, wherein all solubility data refer to solubility at 25 ℃.

The water-soluble polymer (1) may have various geometric forms such as linear, branched, forked and multi-arm types. Water-soluble polymers having a linear structure are preferred. The water-soluble polymer may include monofunctional, homobifunctional, and heterobifunctional polymers.

The water-soluble polymer (1) is, for example, a polyethylene glycol, generally having an average molecular weight of 300-. In a preferred embodiment, the polymer has a molecular weight of 20000-. One preferred polymer has a molecular weight of about 20000 daltons.

Examples of suitable water-soluble polymers (1) are polyvinylpyrrolidone, polyvinyl alcohol, polyols (e.g. polyether polyols or polyester polyols), polyalkylene oxides such as polyethylene glycol (PEG), cellulose, sucrose, hydroxyalkyl starch (HAS) and hydroxyethyl starch (HES).

In a preferred embodiment, the water-soluble polymer is hydroxyalkyl starch (HAS) or particularly preferably hydroxyethyl starch (HES). In the context of the present invention, the term "hydroxyalkyl starch" is used to refer to starch derivatives substituted with hydroxyalkyl groups. In this case, the alkyl group may be substituted. Preferably, the hydroxyalkyl group contains 2 to 10 carbon atoms, more preferably 2 to 4 carbon atoms. Thus "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, of which hydroxyethyl starch and hydroxypropyl starch are preferred. One or more of the hydroxyalkyl groups of HAS contain at least one OH-group. The expression "hydroxyalkyl starch" also includes derivatives in which the alkyl group is mono-or polysubstituted. In this case, the alkyl group is preferably substituted by halogen, especially fluorine, or by aryl, as long as the HAS remains water-soluble. In addition, the terminal hydroxyl group of the hydroxyalkyl group may be esterified or etherified. Furthermore, the alkyl group of the hydroxyalkyl starch may be linear or branched. In addition, a straight or branched substituted or unsubstituted alkenyl group may be used in place of the alkyl group.

In the context of the present invention hydroxyethyl starch (HES) is the preferred HAS. Hydroxyethyl starch (HES) is a derivative of native amylopectin (amylopektin) and is degraded in vivo by alpha-amylase. The prior art describes the preparation of HES-protein conjugates (see for example HES-hemoglobin conjugates in DE 2616086 or DE 2646854). HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch in concentrations of up to 95% by weight. HES has advantageous biological properties and is used as a blood volume replacement agent and clinically in hemodilution therapy. Amylopectin consists of glucosyl groups, in which there are alpha-l, 4-glycosidic bonds in the backbone and alpha-l, 6-glycosidic bonds at the branching sites. The physico-chemical properties of such molecules depend mainly on the type of glycosidic bond. Due to the notched α -l, 4-glycosidic bond, a helix structure with about 6 glucose monomers per helix is formed.

In the context of the present invention, the hydroxyethyl starch may have an average molecular weight (weight average) of 1-300kDa, with an average molecular weight of 5-100kDa being more preferred. Hydroxyethyl starch may also have a molar degree of substitution of 0.1-0.8 and a ratio between C2: C6 substitutions for hydroxyethyl groups of 2-20.

In a more preferred embodiment, the water-soluble polymer is a polyalkylene oxide, especially polyethylene glycol (hereinafter PEG). As used herein, "PEG" or "polyethylene glycol" comprises one of two structures: "- (CH)₂CH₂O)_n- "or" - (CH)₂CH₂O)_n-CH₂CH₂- ". The variable "n" is from 3 to 4000, preferably from 10 to 3500, more preferably from 55 to 2500, still more preferably from 80 to 1500, particularly preferably from 150-. If the PEG is branched, then- (CH)₂CH₂O)_nThe chain may be interrupted by one or more branching units.

The PEG residue may be, for example, monofunctional, difunctional, or multifunctional. When PEG is monofunctional (which is preferred), the PEG residue comprises a terminally capped group R, i.e., PEG is R- (CH)₂CH₂O)_n-. R may be, for example, hydrogen, hydroxy or C₁-C₂₀-alkoxy groups. Preferably, R is hydroxy, methoxy, ethoxy or benzyloxy, particularly preferably methoxy.

In addition, PEG can be linear and branched. If the PEG is branched, the linear structure is interrupted by a branching unit.

In general, the reactive selenium group (2) is any selenium group that reacts readily or at practical rates under conventional conditions. In the present invention, the reactive selenium group (2) is capable of reacting with a mercapto group (-SH) to form a Se-S bond.

In a preferred embodiment, the reactive selenium group (2) comprises a diselenide group (-Se-Se-), a selenium sulfite group (-SeSO-)₃ ^-) Selenol group (-SeH) or selenate group (-Se)^-). More preferably, the reactive alkenyl group (2) comprises a diselenide group (-Se-Se-) or a selenosulphite group (-SeSO)₃ ^-)。

Optionally, the modifier of the invention further comprises a linking group (3). The term "linking group" as used herein refers to an atom or group of atoms optionally used to link an interconnecting group, preferably used to link the water soluble polymer and the reactive selenium group. The linking group of the present invention is preferably hydrolytically stable.

Typically, the linking group comprises a bridge formed from 1 to 10 bridging atoms, preferably 2 to 6 bridging atoms, wherein the bridging atoms optionally may comprise side chains, such as alkyl or alkoxy residues.

An example of a suitable linking group is-CH₂-、-CH₂-CH₂-、-CH₂-CH₂-CH₂-、-CH₂-CH₂-CH₂-CH₂-、-O-CH₂-、-CH₂-O-、-O-CH₂-CH₂-、-CH₂-O-CH₂-、-CH₂-CH₂-O-、-O-CH₂-CH₂-CH₂-、-CH₂-O-CH₂-CH₂-、-CH₂-CH₂-O-CH₂-、-CH₂-CH₂-CH₂-O-、-O-CH₂-CH₂-CH₂-CH₂-、-CH₂-O-CH₂-CH₂-CH₂-、-CH₂-CH₂-O-CH₂-CH₂-、-CH₂-CH₂-CH₂-O-CH₂-、-CH₂-CH₂-CH₂-CH₂-O-、-CO-NH-CH₂-CH₂-CH₂-CH₂-、-CH₂-CO-NH-CH₂-CH₂-CH₂-、-CO-NH-CH₂-、-CO-NH-CH₂-CH₂-、-CH₂-CO-NH-CH₂-、-CH₂-CH₂-CO-NH-、-CO-NH-CH₂-CH₂-CH₂-、-CH₂-CO-NH-CH₂-CH₂-、-CH₂-CH₂-CO-NH-CH₂-、-CH₂-CH₂-CH₂-CO-NH-、-CH₂-CH₂-CO-NH-CH₂-CH₂-、-CH₂-CH₂-CH₂-CO-NH-CH₂-、-CH₂-CH₂-CH₂-CO-NH-CH₂-CH₂-、-CH₂-CH₂-CH₂-CH₂-CO-NH-、-CO-O-CH₂-、-CH₂-CO-O-CH₂-、-CH₂-CH₂-CO-O-CH₂-、-CO-O-CH₂-CH₂-、-O-CO-NH-CH₂-CH₂-、-NH-CH₂-、-NH-CH₂-CH₂-、-CH₂-NH-CH₂-、-CH₂-CH₂-NH-CH₂-、-CO-CH₂-、-CO-CH₂-CH₂-、-CH₂-CO-CH₂-、-CH₂-CH₂-CO-CH₂-、-CH₂-CH₂-CO-CH₂-CH₂-、-CH₂-CH₂-CO-、-NH-CO-CH₂-、-CH₂-NH-CO-CH₂-、-CH₂-CH₂-NH-CO-CH₂-、-NH-CO-CH₂-CH₂-、-CH₂-NH-CO-CH₂-CH₂、-CH₂-CH₂-NH-CO-CH₂-CH₂、-CO-NH-CH₂-、-CO-NH-CH₂-CH₂-、-O-CO-NH-CH₂-and combinations of two or more of any of the above groups.

Preferably, the linking group is-CO-NH-CHR-CH₂-, wherein R is hydrogen, carboxyl or C₁-C₆Alkyl, particularly preferably, R is hydrogen or carboxy.

A subject of a preferred embodiment of the present invention is a modifier having a structure as described in formula I or II,

wherein in the above formula P is a water-soluble polymer and L is a linking group. Preferably, P and L are each selected from the group consisting of water-soluble polymers and linking groups as described above. In particular, P is a PEG residue and L is-CO-NH-CHR-CH₂-, wherein R is hydrogen, carboxyl or C₁-C₆Alkyl, in particular R is hydrogen or carboxy.

As noted above, the water soluble polymer may be difunctional or polyfunctional. Thus, a subject of the invention is also a modifier having a structure as shown in formula Ia or IIa,

wherein in the above formula P is a water-soluble polymer, L is a linking group and n is a number from 2 to 10. Preferably, P and L are each selected from the group consisting of water-soluble polymers and linking groups as described above. Furthermore, n is preferably 2, 3 or 4, in particular 2. In particular, P is a PEG residue and L is-CO-NH-CHR-CH₂-, wherein R is hydrogen, carboxyl or C₁-C₆Alkyl, in particular R is hydrogen or carboxy.

A subject of a particularly preferred embodiment of the present invention is therefore a modifier having a structure as shown in formula III or IV,

wherein in the above formula PEG is polyethylene glycol and R is hydrogen, carboxyl or C₁-C₆An alkyl group. PThe above preferred embodiments of EG (e.g. molecular weight, terminal capping group) also apply to the PEGs of formulae III and IV.

A further subject of the invention is also a process for producing the abovementioned modifiers according to the invention.

Accordingly, the present invention comprises a process for producing a modifier according to the invention, comprising the steps of:

(i) providing a compound of formula V

Wherein A is a functional group and L is a linking group,

(ii) reacting a compound of formula V with an activated water-soluble polymer, and

(iii) (iii) optionally subjecting the product of step (ii) to a sulfitolysis reaction.

The product obtained in step (ii) corresponds to the compounds of formulae I, Ia and III. The products obtained in the additional reaction step (iii) correspond to the compounds of the formulae II, IIa and IV.

In step (i) of the process of the present invention there is provided a compound of formula V. The compound of formula V comprises one diselenide bond, two linking groups L and two functional groups a.

The linking group L refers to the preferred embodiments described above. Preferably, the linking group is-CHR-CH₂-, wherein R is hydrogen, carboxyl or C₁-C₆Alkyl, especially preferably R is hydrogen or carboxyl.

The functional group a is capable of reacting with the activated water-soluble polymer, preferably capable of forming a stable linkage. The functional group a is preferably selected from amino, hydroxyl and carboxyl. Most preferably, the functional group A is a primary amino group, i.e. A is-NH₂。

The compound of formula V is reacted with the "activated water soluble polymer" in step (ii). The term activated water-soluble polymer comprises any water-soluble polymer as defined above comprising one or more groups reactive with the functional group a of formula V as defined above. Examples of reactive groups are N-hydroxy-succinimide ester, dichlorotriazine, monomethoxy (tresylate) trifluoroethylsulfonate, succinimide carbonate, benzotriazole carbonate, p-nitrophenyl carbonate, trichlorophenyl carbonate, N' -carbonyldiimidazole (carboylidazole), isocyanate, isothiocyanate and aldehyde reactive groups.

Preferably, activated PEG, i.e. PEG reagents bearing N-hydroxysuccinimide ester, dichlorotriazine, mono-methoxy trifluoroethylsulfonate, succinimide carbonate, benzotriazole carbonate, p-nitrophenyl carbonate, trichlorophenyl carbonate, N' -carbonyldiimidazole and aldehyde reactive groups are used in step (ii). Specifically, polyethylene glycol activated with an N-hydroxysuccinimide ester residue (hereinafter referred to as PEG-NHS) was used.

For the preferred embodiment, it is noted that: if the functional group "A" of formula V is an amino group, then the "activated water-soluble polymer" (preferably activated PEG) contains one or more amino-reactive groups. If "A" is a carboxyl group, then the "activated water-soluble polymer" contains one or more hydrazide groups. If "A" is a hydroxyl group, then the "activated water-soluble polymer" contains one or more isocyanate groups. Alternatively, the reverse is also possible. That is, the activated water-soluble polymer (preferably activated PEG) has an amino group, a hydroxyl group or a carboxyl group, and "A" is the above-mentioned functional group suitable for the activated water-soluble polymer.

Thus, in a preferred embodiment of the process of the present invention, in step (i) a compound of formula VI is provided.

Wherein in the above formula, R is hydrogen or carboxyl,

and in step (ii), the compound of formula VI is reacted with PEG-NHS. The compounds of formula VI are more specific embodiments of the compounds of formula V.

Particularly preferably, selenocysteine (R ═ COOH) or selenocysteine (R ═ H) is used as compound of formula VI.

The coupling reaction (ii) may be carried out in aqueous or non-aqueous solution. Wherein the pH is 6-10, preferably 7.5-8.5. The reaction can be carried out, for example, at from 10 to 50 ℃ and preferably from 20 to 30 ℃. The reaction time may be, for example, 1 to 20 hours.

Cysteamine is readily soluble in aqueous solutions, whereas the slow dissolution rate of selenocysteine is preferably accelerated by alkaline solutions such as 1M KOH. However, for reaction with PEG-NHS, the pH of the solution must be lowered. When the coupling reaction is complete, the remaining selenocysteine can be removed from the mixture by ultrafiltration or by organic phase extraction. Organic phase extraction can isolate PEG mixtures in solid form. Typically, it is not necessary to remove unreacted PEG-NHS, as it will hydrolyze over time to a non-reactive form. The amount of disubstituted selenocysteine or selenocysteine can be determined quantitatively by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or by reverse phase chromatography (RP-HPLC). Both methods can be used because the reaction product of step (ii) (hereinafter also referred to as (PEG-Se)₂) Twice as much as the starting material (PEG-NHS).

The reaction scheme shown below illustrates a preferred embodiment comprising reaction steps (i) and (ii):

wherein R is preferably H or COOH.

In an additional reaction step (iii), the modifying agent comprising a diselenide group may be reacted to form a modifying agent comprising a selenium sulphite group.

The reaction (iii) is a so-called sulfitolysis reaction. To carry out the reaction step (iii), a sulfitolysis reagent may be used. Suitable sulfitolysis agents may be prepared by mixing Na₂SO₃And Na₂S₄O₆And then the preparation. The reaction may be carried out in aqueous solution. The solution is preferably a buffer solution. The pH can be from 7 to 10, preferably from 8 to 9. The reaction can be carried out, for example, at from 10 to 50 ℃ and preferably from 20 to 30 ℃. The reaction time may be, for example, 1 to 20 hours.

The monomeric form (═ PEG-SeSO) can be monitored, for example, by SDS-PAGE₃ ^-) Is performed. When the reaction is complete, the low molecular weight reagents may be removed, for example, by ultrafiltration.

The reaction scheme shown below illustrates a preferred embodiment comprising reaction steps (i) to (iii):

wherein R is preferably H or COOH.

Both forms of the modifier (dimeric form, i.e., the reaction product of step (ii); and monomeric form, i.e., the reaction product of step (iii)) may contain impurities associated with unreacted activated water-soluble polymers such as PEG-NHS reagents. These impurities need not be removed because they do not interfere with the conjugation reaction, e.g., the pegylation reaction of thiol-containing active agents, e.g., thiol-containing proteins.

Optionally, the unreacted activated water-soluble polymer may be removed using a different chromatographic technique selected from, for example, ion exchange chromatography, molecular sieve chromatography, or reverse phase chromatography. For dimeric Se-PEG reagents prepared from selenocysteine and monomeric Se-PEG reagents, ion exchange chromatography can be used due to the presence of negative charges. The dimeric Se-PEG reagent prepared from selenocysteine can be efficiently purified by molecular sieve chromatography or reverse phase chromatography.

The modifying agent of the invention is useful for attaching a water-soluble polymer to a pharmaceutically active agent comprising a sulfhydryl group, preferably to a cysteine residue of a polypeptide. When the water-soluble polymer is PEG, the modifying agent may be used to pegylate a pharmaceutically active agent comprising a sulfhydryl group, in particular for pegylating a cysteine residue of a polypeptide. Preferably, the modifier of the invention is used for the pegylation of G-CSF, wherein in particular Cys18 of G-CSF is pegylated, more in particular mono-pegylated.

Thus, a subject of the present invention is also a conjugate comprising a water-soluble polymer and a pharmaceutically active agent comprising a thiol group, wherein the water-soluble polymer is linked to the pharmaceutically active agent by an S-Se bond.

Therefore, another subject of the present invention is a process for the preparation of a conjugate according to the invention, comprising the following steps:

(i) providing a pharmaceutically active agent comprising at least one free sulfhydryl group, and

(ii) reacting the pharmaceutically active agent with the modifying agent of the invention.

That is, a pharmaceutically active agent comprising a sulfhydryl group, such as a protein, peptide or amino acid having a free sulfhydryl group, may be reacted with a modifying agent of the invention. Covalent linkage is formed by this reaction via a selenosulfide bond (selenosulfide), as shown in the scheme below.

A is a pharmaceutically active agent, preferably a polypeptide, containing a free cysteine residue.

Similar reactions can occur when the water-soluble polymer is di-or polyfunctional, see reaction scheme below.

A is a pharmaceutically active agent, preferably a polypeptide, containing a free cysteine residue, and n is a natural number, preferably 2-10, more preferably 2, 3 or 4.

In general, the term "pharmaceutically active agent" refers to any agent, drug, compound, composition, or mixture that is capable of providing a pharmaceutical effect, preferably a beneficial effect, that is demonstrated in vivo or in vitro.

Common examples of pharmaceutically active agents are peptides, polypeptides, proteins, antibodies and antibody derivatives, polysaccharides, steroids, nucleotides, oligonucleotides, polynucleotides, fats, electrolytes and mixtures thereof.

Specific examples of suitable pharmaceutically active agents are asparaginase, amdoxovir (DAPD), becaplamine (becaplmin), bisphosphonates, calcitonin, cyanobacterial antiviral protein, denileukin (denileukin), Erythropoietin (EPO), erythropoiesis stimulating protein (NESP), blood clotting factors such as factor V, factor VII, factor VIIa, factor VIII, factor IX, factor X, factor XII, factor VIII, von willebrand factor; cetilinase, Cerezase, alpha-glucosidase, collagen, cyclosporin, alpha defensin, beta defensin, sialoprotein 4(exedin-4), granulocyte colony stimulating factor (G-CSF), Thrombopoietin (TPO), alpha-1 protease inhibitor, elcatin (elcatonin), granulocyte-macrophage colony stimulating factor (GM-CSF), fibrinogen, Follicle Stimulating Hormone (FSH), human growth hormone (GHGH), growth hormone Releasing Hormone (RH), growth regulating oncogene beta (GRO-beta), GRO-beta antibody, acidic fibroblast growth factor, basic fibroblast growth factor, CD-40 ligand, heparin, human serum albumin, interferons such as interferon alpha, interferon beta, interferon gamma, interferon omega, interferon tau, interferon alpha-glucosidase, collagen, cyclosporin, alpha-1 protease inhibitor, elcatonin, granulocyte, beta-macrophage colony stimulating factor (GM-CSF), fibrinogen, FSH, human Growth Hormone Releasing Hormone (GHRH), growth regulating oncogene beta (GRO-beta), GRO-beta antibody, acidic fibroblast growth factor, basic fibroblast growth factor, CD, Consensus interferon (consensus interferon); interleukins and interleukin receptors such as the interleukin 1 receptor, interleukin 2 fusion protein, interleukin 1 receptor antagonist, interleukin-3, interleukin-4, interleukin 4 receptor, interleukin-6, interleukin-8, interleukin-12, interleukin 13 receptor, interleukin 17 receptor; insulin, Low Molecular Weight Heparin (LMWH), proinsulin, influenza vaccine, insulin-like growth factor (IGF), insulin opsonin, macrophage colony stimulating factor (M-CSF), monoclonal antibodies, plasminogen activators such as alteplase (alteplase), urokinase, reteplase, streptokinase, pamiprase (pamiteplase), lanoteplase (lanoteplase), and tenecteplase (tenetepase); nerve Growth Factor (NGF), osteoprotegerin, platelet derived growth factor, tissue growth factor, transforming growth factor-1, vascular endothelial growth factor, leukemia inhibitory factor, Keratinocyte Growth Factor (KGF), Glial Growth Factor (GGF), T cell receptor, CD molecules/antigens, Tumor Necrosis Factor (TNF), monocyte chemoattractant protein-1, vascular endothelial growth factor, parathyroid hormone (PTH), or mixtures thereof. Importantly, the active agents described above contain free sulfhydryl groups or are modified in some way to contain free sulfhydryl groups. A "free sulfhydryl group" refers to a sulfhydryl group (-SH) that is not linked to another sulfhydryl group by a disulfide bond.

Examples of antibodies are known, such as antibodies against HER2 (e.g. trastuzumab (trastuzumab)), antibodies against VEGF (e.g. bevacizumab), antibodies against EGF (e.g. cetuximab (cetuximab)), antibodies against CD20 (e.g. rituximab (rituximab)), antibodies against TNF (e.g. infliximab, adalimumab)). Examples of antibody derivatives are also known, including for example immunoglobulin Fc fusion proteins (e.g. etanercept) or Fab fragments (e.g. ranibizumab).

Preferably, the pharmaceutically active agent is a polypeptide comprising at least one free thiol group, preferably a free thiol group belonging to a cysteine residue of said polypeptide. That is, the sulfhydryl groups typically belong to naturally occurring unpaired cysteine residues, however they may also be introduced into the protein structure by chemical or genetic means. Alternatively, free sulfhydryl groups may also be obtained by reducing the disulfide bonds of the protein (if such disulfide bonds are not essential for the desired biological activity of the protein).

Examples of preferred polypeptides are EPO, IFN- [ alpha ], IFN- [ beta ], IFN- [ gamma ], complex IFNs, factor VII, factor VIII, factor IX, G-CSF, GM-CSF, hGH, insulin, FSH, FTH or mixtures thereof. G-CSF is particularly preferred. Alternatively, EPO is particularly preferred.

In general, the invention preferably employs purified and isolated polypeptides having some or all of a primary structural conformation (i.e., a contiguous sequence of amino acid residues) and one or more of biological properties (e.g., immunological properties and in vitro biological activity) and physical properties (e.g., molecular weight). These polypeptides may also be characterized in that they are the product of a chemical synthesis reaction, or the expression product of a prokaryotic or eukaryotic host (e.g., cultured bacterial, yeast, higher plant, insect or mammalian cells) of an exogenous DNA sequence obtained by genomic or cFNA cloning or by gene synthesis. The products of a representative yeast (e.g., Saccharomyces cerevisiae) or prokaryotic (e.g., Escherichia coli) host cell do not bind to any mammalian proteins the products expressed by microorganisms in vertebrate (e.g., non-human mammalian and avian) cells do not bind to any human proteins.

In particular, recombinant human granulocyte colony stimulating factor (G-CSF) produced by e. Preferably the amino acid sequence of G-CSF is set forth in SEQ ID NO: 1 in (c). Shown in SEQ ID NO: the amino acid sequence in 1 is preferably not glycosylated and is commercially available as Filgrastim. Alternatively, recombinant human granulocyte colony stimulating factor (G-CSF) produced by expression from a eukaryotic host is used. The amino acid sequence of this preferred embodiment is identical to SEQ ID NO: 1, but not Met 1. In addition, the polypeptides of this preferred embodiment are glycosylated and are commercially available as legungestin (lenogustim).

In another preferred embodiment, Erythropoietin (EPO) and derivatives thereof may also be used. EPO is well known in the art. Erythropoietin is an acidic glycoprotein hormone of about 34 kDa. Human erythropoietin is a 166 amino acid polypeptide which occurs naturally as a monomer (Lin et al, 1985, PNAS 82, 7580-7584, EP 148605B 2, EP 411678B 2).

The EPO used in the present invention may be derived from any human or other mammal and may be obtained by purification from a natural source such as human or animal kidney, human or animal embryonic liver, preferably monkey kidney. Preferably, the EPO is produced recombinantly. This includes production in eukaryotic or prokaryotic cells, preferably mammalian, insect, yeast, plant, bacterial cells or in any other cell type that facilitates recombinant production of EPO. Furthermore, the EPO can be expressed in transgenic animals (e.g., in body fluids such as milk, blood, etc.), in the eggs of transgenic birds, especially poultry (preferably chickens), or in transgenic plants or algae.

The EPO can comprise one or more (preferably 1-4, preferably 4) saccharide side chains bound to the EPO by N-and/or O-linked glycosylation, i.e. the EPO is glycosylated. Typically, when EPO is produced in a eukaryotic cell, the polypeptide is posttranslationally glycosylated. Thus, the sugar side chains may already be bound to the EPO during biosynthesis in mammalian, in particular human, insect, plant or yeast cells.

Recombinant production of polypeptides is known in the art. Typically, this involves transfecting a host cell with a suitable expression vector, culturing the host cell under conditions capable of producing the polypeptide, and purifying the polypeptide from the host cell.

In particular, the EPO may have the amino acid sequence of human EPO (see EP 148605B 2). Furthermore, the expression "erythropoietin" or "EPO" also comprises EPO variants in which one or more amino acids (e.g. 1 to 25, preferably 1 to 10, more preferably 1 to 5, most preferably 1 or 2) are replaced by another amino acid compared to the sequence of human EPO and wherein the EPO has erythropoietic activity (see EP 640619B 1).

The measurement of erythropoietic activity is described in the art (for in vitro activity measurements see, e.g., Fibi et al, 1991, Blood, 77, 1203 ff; for in vivo EPO activity measurements see, e.g., Fibi, Hermentin, Pally, Lauffer, Zettlmeissl, 1995, N-and O-glycosylation proteins of recombinant human erythropoietin secreted from BHK-21 cells, Blood, 85 (5)), 1229-36; (preparation of EPO and modified EPO forms into female NMRI mice on days 1, 2, and 3 (50 ng protein/mouse), collection of Blood samples and determination of reticulocytes on day four)).

Generally, the above examples of polypeptides may also include analogs, agonists, antagonists, inhibitors, isomers, and pharmaceutically acceptable salt forms thereof. In addition, the above polypeptides also include synthetic, recombinant, natural, glycosylated and non-glycosylated forms, and biologically active fragments thereof, so long as at least one free sulfhydryl group is present.

The term "biologically active", in particular "biologically active fragment" is understood by the person skilled in the art. In particular, a fragment or derivative is considered to be biologically active if it retains the biologically active properties of its parent molecule, even if the activity is increased or decreased. More specifically, biologically active means that the fragment or derivative should be therapeutically active if administered in an appropriate dose.

Thus, a subject of a preferred embodiment of the present invention is a conjugate having the structure of formula VII

Wherein in the above formula, P is a water-soluble polymer, L is a linking group, Se is a selenium atom, Pept is a polypeptide and S is a sulfur atom belonging to a cysteine residue of the polypeptide. For the preferred embodiment, P, L and Pept are explained above.

In a particularly preferred embodiment, the invention relates to conjugates having the structure of formula VIII

Wherein in the above formula, PEG is polyethylene glycol; l is a linking group, preferably-CO-NH-CHR-CH₂-residue, wherein R is hydrogen, carboxyl or C₁-C₆An alkyl group; se is a selenium atom; G-CSF is a granulocyte colony stimulating factor and S is a sulfur atom belonging to the cystine residue of said granulocyte colony stimulating factor. In formula VIII, "G-CSF" is preferably recombinant human granulocyte colony stimulating factor (G-CSF) produced in E. The preferred amino acid sequence of G-CSF is shown in FIG. 8. Alternatively, a G-CSF known as lenograstin (Lenographim) may be used.

Particularly preferably, "S" in formula VIII is the sulfur atom of Cys18 residue of G-CSF. Thus, mono-glycolation of G-CSF at Cys18 is particularly preferred.

The conjugates of the invention are useful as pharmaceutical agents. Thus, another subject of the invention is a conjugate according to the invention for use as a medicament.

In a preferred embodiment, the conjugates of the invention are used for the treatment of neutropenia. In particular, the treatment is useful in patients with non-myeloid malignancies who receive myelosuppressive anti-cancer drugs.

As discussed above, the present invention also relates to a method of preparing a conjugate of the present invention, comprising the steps of:

(i) providing a pharmaceutically active agent comprising at least one free sulfhydryl group, and

(ii) the pharmaceutically active agent is reacted with the modifying agent of the invention (conjugation reaction).

The conjugation reaction may be carried out in aqueous solution. The solution is preferably buffered. The pH is 3 to 10, preferably 6 to 9. The reaction can be carried out, for example, at from 2 to 50 ℃ and preferably from 20 to 30 ℃. Typically, the conjugation reaction can proceed until substantially no more conjugation occurs, which can generally be determined by monitoring the progress of the reaction over time. The progress of the reaction can be monitored by taking aliquots from the reaction mixture at different time points and analyzing the reaction mixture by, for example, SDS-PAGE or MALDI-TOF mass spectrometry. The reaction time may be, for example, 1 to 50 hours, preferably 10 to 25 hours.

In the process of the present invention, all of the above descriptions regarding preferred embodiments of the water-soluble polymer and the pharmaceutically active agent apply. Preferably, the water soluble polymer is PEG and the active agent is G-CSF.

For pegylation of free thiol groups, the thiol groups should preferably be sufficiently exposed to allow reaction with the modifying agent of the invention. In G-CSF, Cys18 residues are typically only partially exposed to solvents and do not come into sufficient contact with the modifier, and therefore, it is preferred to reversibly denature G-CSF (preferably under mild conditions) and then to perform the pegylation reaction. The reversible denaturation can be achieved by the addition of various compounds (e.g., urea, GdHCl, DMSO, SDS, NLS, tween). Following the pegylation reaction, a renaturation step is preferably carried out. Renaturation can be brought about by ion exchange and/or dilution.

The conjugation reaction is typically carried out in a molar excess of the modifying agent. In a preferred embodiment, the molar ratio of the reactive selenium atoms to the reactive mercapto groups is from 0.9: 1 to 10: 1, more preferably from 1.0 to 5: 1, more preferably from 1.1: 1 to 3: 1.

The resulting conjugation product is preferably purified to isolate, for example, excess reagents, unconjugated reactants, unwanted multi-conjugated materials and/or free or unreacted polymers. The resulting conjugate can then be further characterized using analytical methods (e.g., MALDI, capillary electrophoresis, gel electrophoresis, and/or chromatography).

Finally, another subject of the invention is a pharmaceutical composition comprising:

(a) the conjugate of the present invention is a conjugate,

(b) one or more pharmaceutically acceptable excipients.

For conjugate (a), all the above description with respect to the preferred embodiments applies.

Generally, "pharmaceutically acceptable excipient" refers to an excipient that can be included in the compositions of the present invention and that does not cause a significant adverse toxicological effect to the patient.

Examples of suitable excipients are sugars, antimicrobial agents, surfactants, buffers, acids, bases, antioxidants, inorganic salts and mixtures thereof.

Examples of suitable sugar excipients are monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides such as lactose, sucrose, trehalose; polysaccharides, such as starch; and sugar alcohols such as mannitol, sorbitol. Examples of inorganic salts or buffers are citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monovalent sodium phosphate, divalent sodium phosphate and combinations thereof. Examples of suitable surfactants are polysorbates, such as "tween 20" and "tween 80", sorbitan esters; lipids such as phospholipids (e.g., lecithin), fatty acids, and fatty acid esters; steroids such as steroids; and chelating agents such as EDTA.

The pharmaceutical composition of the present invention comprises all formulations, of which formulations suitable for injection are preferred. The amount of conjugate in the composition will vary, but is preferably a therapeutically effective dose when the composition is stored in a unit dosage form (e.g., a vial).

The pharmaceutical formulations of the present invention are preferably administered by injection and are therefore typically liquid solutions or suspensions.

The invention will be illustrated by the following examples.

Examples

A) The method used in the invention comprises the following steps:

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)

SDS-PAGE was used to detect the formation of new Se-PEG reagents and to evaluate the coupling reaction between proteins and Se-PEG reagents. SDS-PAGE was performed using a commercially available 4% -12% Bis-Tris gel (Invitrogen). Two detection methods were used: iodine staining for revealing PEG and Simply Blue (Invitrogen) staining for revealing proteins.

Cation Exchange Chromatography (CEC)

CEC was used to isolate G-CSF pegylated at Cys18 residue from the pegylation mixture. The separation was performed using an 8ml column packed with SP-5PW TSK-Gel (TO-SOHAAS). The binding buffer was 25mM CH, pH 3.8₃COOH/NaOH. A gentle linear gradient of elution buffer (75mM CH) 0-100% of 35 column volumes was used₃COOH/NaOH, pH 8.0) to separate the pegylated and non-pegylated forms of G-CSF. The separation was carried out at a flow rate of 2.0 ml/min. CEC fractions were collected from SDS-PAGE analysis (Simply Blue and iodine staining) performed under non-reducing conditions.

Reversed phase high performance liquid chromatography (RP-HPLC).

RP-HPLC was used to analyze the Se-PEG reagent. By using Corona plus and Knauer UV detector in Waters Bondapak^TMRP-HPLC was carried out on Phenyl columns (3.9X150mm, particle size 10 μm). The combined eluent is 40% CH₃CN/H₂And O. CH with a linear gradient of 40-50%₃CN/H₂O is(ii) isolating the PEG moiety. The flow rate was 1 ml/min. By dissolving the solid PEG mixture in 40% CH₃CN/H₂Samples were prepared and injected at an injection volume of 10L in O.

B) Description of the drawings

FIG. 1: SDS-PAGE analysis of Se-PEG reagents (iodine staining, non-reducing conditions).

Lane 1: PEG molecular weight standards (5kDa, 12kDa, 20kDa, 30kDa)

Lane 2: starting 10kDa mPEG-NHS reagent

Lane 3: prepared from selenocysteine and 10kDa PEG-NHS (PEG-Se)₂A reaction mixture of reagents.

Lane 4: prepared from selenocysteine and 10kDa PEG-NHS (PEG-Se)₂A reaction mixture of reagents.

Lane 5: prepared from selenocysteine and 20kDa PEG-NHS (PEG-Se)₂A reaction mixture of reagents.

Lane 6: prepared from selenocysteine and 20kDa PEG-NHS (PEG-Se)₂A reaction mixture of reagents.

Lane 7: PEG-SeSO prepared from selenocysteine and 20kDa PEG-NHS₃A reaction mixture of reagents.

Lane 8: PEG-SeSO prepared from selenocysteine and 20kDa PEG-NHS₃A reaction mixture of reagents.

Lanes 2, 3, 4, 5 and 6: higher molecular weight plaques correspond to (Se-PEG)₂The reagent, lower molecular weight plaques correspond to the starting PEG-NHS reagent.

Lanes 7 and 8: by corresponding to (PEG-Se)₂Disappearance of high molecular weight plaques of (2) to confirm PEG-SeSO₃Is performed.

FIG. 2: prepared in anhydrous DMF (PEG-Se)₂SDS-PAGE analysis of reagents (iodine staining, non-reducing conditions))。

Lane 1: PEG molecular weight standards (5kDa, 12kDa, 20kDa, 30kDa)

Lane 2: prepared from selenocysteine and 10kDa PEG-NHS (Se-PEG)₂A reaction mixture of reagents.

FIG. 3: SDS-PAGE analysis of a PEGylated mixture of G-CSF and different Se-PEG reagents (Simply Blue staining, non-reducing conditions).

Lane 1: protein molecular weight standards (2.5kDa, 3.5kDa, 6kDa, 14.4kDa, 21.5kDa, 31.0kDa, 36.5kDa, 55.4kDa, 66.3kDa, 97.4kDa, 116.3kDa, 200kDa)

Lane 2: G-CSF + Neulasta (Pegylated filgrastim)

Lane 3: G-CSF with a conjugate prepared from cysteamine selenosulfate and 10kDa PEG-NHS (PEG-Se)₂A pegylated mixture of reagents.

Lane 4: G-CSF with a conjugate prepared from selenocysteine and 10kDa PEG-NHS (PEG-Se)₂A pegylated mixture of reagents.

Lane 5: G-CSF with a conjugate prepared from cysteamine selenosulfate and 20kDa PEG-NHS (PEG-Se)₂A pegylated mixture of reagents.

Lane 6: G-CSF and PEG-SeSO prepared from selenocysteine and 20kDa PEG-NHS₃A pegylated mixture of reagents.

Lanes 3 and 4: the higher molecular weight plaques correspond to G-CSF conjugated to a 10kDa Se-PEG reagent, and the lower molecular weight plaques correspond to native G-CSF.

Lanes 5 and 6: the higher molecular weight plaques correspond to G-CSF conjugated to a 20kDa Se-PEG reagent, and the lower molecular weight plaques correspond to native G-CSF.

FIG. 4: use (PEG-Se)₂SDS-PAGE analysis (Simply Blue staining, reduced and non-reduced conditions) of reagent-prepared, purified G-CSF conjugates.

Lane 1: protein molecular weight standards (2.5kDa, 3.5kDa, 6kDa, 14.4kDa, 21.5kDa, 31.0kDa, 36.5Da, 55.4kDa, 66.3kDa, 97.4kDa, 116.3kDa, 200kDa)

Lane 2: G-CSF + Neulasta; under reducing conditions

Lane 3: G-CSF conjugate with 10kDa PEG conjugated to Cys18 (with (PEG-Se) from selenocysteine and 10kDa PEG-NHS)₂Preparation); under reducing conditions

Lane 4: G-CSF + Neulasta; under non-reducing conditions

Lane 5: G-CSF conjugate with 10kDa PEG conjugated to Cys18 (with (PEG-Se) from selenocysteine and 10kDa PEG-NHS)₂Preparation); under non-reducing conditions

Under non-reducing conditions a single high molecular weight band was visible, which corresponds to pegylated G-CSF. PEG was cleaved from the protein under reducing conditions and a single band corresponding to native G-CSF was seen.

FIG. 5: use (PEG-Se)₂Or PEG-SeSO₃SDS-PAGE analysis (Simply Blue staining, reduced and non-reduced conditions) of reagent-prepared, purified G-CSF conjugates.

Lane 1: protein molecular weight standards (15kDa, 21kDa, 31kDa, 50kDa, 66kDa, 100kDa)

Lane 2: G-CSF; under non-reducing conditions

Lane 3: neulasta; under non-reducing conditions

Lane 4: G-CSF conjugate with 20kDa PEG conjugated to Cys18 (with (PEG-Se) from selenocysteine and 20kDa PEG-NHS)₂Preparation); under non-reducing conditions

Lane 5: G-CSF conjugate with 20kDa PEG conjugated to Cys18 (with PEG-SeSO from cysteamine selenosulfate and 20kDa PEG-NHS)₃Preparation); under non-reducing conditions

Lane 6: G-CSF; under reducing conditions

Lane 7: neulasta; under reducing conditions

Lane 8: G-CSF conjugate with 20kDa PEG conjugated to Cys18 (with (PEG-Se) from selenocysteine and 20kDa PEG-NHS)₂Preparation); under reducing conditions

Lane 9: G-CSF conjugate with 20kDa PEG conjugated to Cys18 (with (PEG-Se) from selenocysteine and 20kDa PEG-NHS)₂Preparation); under reducing conditions

Under non-reducing conditions a single high molecular weight band was visible, which corresponds to pegylated G-CSF. PEG was cleaved from the protein under reducing conditions and a single band corresponding to native G-CSF was seen.

FIG. 6: prepared from selenocysteine and 20kDa PEG-NHS (PEG-Se)₂RP-HPLC chromatography (Corona detector) of the reaction mixture of reagents.

FIG. 7: prepared from selenocysteine (PEG-Se)₂RP-HPLC chromatography (Knauer uv detector, 215nm) of the reaction mixture of reagents. The signal at tr ═ 18min indicates the absorption of the polymeric dimeric Se-Se group, which shows that the process proceeds in quantitative yield (PEG-Se)₂And (4) synthesizing.

FIG. 8: G-CSF and (PEG-Se) at pH 4.0, 5.0, 6.0, 7.0 and 7.5₂SDS-PAGE analysis of the PEGylated mixtures (Simply Blue staining, non-reducing conditions).

Lane 1: protein molecular weight standards (2.5kDa, 3.5kDa, 6kDa, 14.4kDa, 21.5kDa, 31.0kDa, 36.5kDa, 55.4kDa, 66.3kDa, 97.4kDa, 116.3kDa, 200kDa)

Lane 2: G-CSF + Neula sta

Lane 3: pegylated mixtures at pH 4.0

Lane 4: pegylated mixtures at pH 5.0

Lane 5: pegylated mixtures at pH 6.0

Lane 6: pegylated mixtures at pH 7.0

Lane 7: pegylated mixtures at pH7.5

The high molecular weight plaques correspond to G-CSF conjugated with a 20kDa Se-PEG reagent, and the low molecular weight plaques correspond to native G-CSF. The position of the G-CSF dimer is marked by an arrow.

The results show that Se-PEG reagent can be used under acidic conditions and that conjugation yields are better at higher pH.

C) The reaction is carried out

Example 1: preparation from selenocysteine and 10kDa PEG-NHS (PEG-Se)₂And (3) a reagent.

Step 1 dissolution of selenocysteine

8.5mg of selenocysteine were dissolved in 101.6l of 1M KOH. By adding 1.017ml of 0.2M K₂B₄O₇The pH was adjusted to about 9.8.

Step 2, coupling reaction of PEG and selenocysteine:

924mg (0.092mmol) of 10kDa PEG-NHS reagent was dissolved in 2mL of 0.2M sodium phosphate pH 8.5. To this solution was added 0.411mL (0.0092mmol) of dissolved selenocysteine (step 1). The reaction was allowed to proceed at room temperature for 18 hours.

And 3, removing low-molecular-weight impurities:

ultrafiltration was performed using Amicon Ultra-15 at 10 MWCO. The buffer was replaced with 0.2M sodium phosphate pH 7.5. The volume after buffer replacement was 4 mL.

Example 2: preparation from selenocysteine and 10kDa PEG-NHS in anhydrous DMF (PEG-Se)₂And (3) a reagent.

Step 1, coupling reaction of PEG and selenocysteine:

3.3mg (0.01mmol) of selenocysteine were suspended in 3mL of a suspension containing 69.1mg (0.5mmol) of K₂CO₃In anhydrous DMF. After 1 hour, 200mg (0.02mmol) of 10kDa PEG-NHS reagent was added and the resulting reaction mixture was stirred at room temperature under an inert argon atmosphere for 5 days. To this solution was added 10mL of water, and the resulting mixture was stirred for an additional 1 day (the PEG-NHS reagent was quenched by hydrolysis).

And 2, removing low-molecular-weight impurities:

the resulting polymer mixture was filtered and the resulting filtrate was washed 3 times with dichloromethane. The combined organic phases were concentrated under reduced pressure and the polymer mixture was then precipitated by dropwise addition of diethyl ether. After centrifugation, the precipitate was dried under high vacuum at room temperature for 12 hours.

Example 3: preparation from cysteamine seleno and 10kDa PEG-NHS (PEG-Se)₂And (3) a reagent.

Step 1, preparation of selenocysteine stock solution:

6.2mg of selenocysteine was dissolved in 0.31mL of 0.2M sodium phosphate pH 8.5.

Step 2, coupling reaction of PEG and selenocysteine:

939mg (0.094mmol) of 10kDa PEG-NHS reagent was dissolved in 4.26mL of 0.2M sodium phosphate pH 8.5. To this solution was added 0.15mL (0.0094mmol) of dissolved selenocysteine (step 1). The reaction was allowed to proceed at room temperature for 18 hours.

And 3, removing low-molecular-weight impurities:

ultrafiltration was performed using Amicon Ultra-15 at 10 MWCO. The buffer was replaced with 0.2M sodium phosphate pH 7.5. The volume after buffer replacement was 4.775 mL.

Example 4: preparation from selenocysteine and 20kDa PEG-NHS (PEG-Se)₂And PEG-SeSO₃And (3) a reagent.

Step 1, dissolving selenocysteine:

23.3mg of selenocysteine were dissolved in 140l of 1M KOH. By adding 0.4ml of 0.2M K₂B₄O₇The pH was adjusted to about 9.9.

Step 2, coupling reaction of PEG and selenocysteine:

610mg (0.03mmol) of 20kDa PEG-NHS reagent was dissolved in 2mL of 0.2M sodium phosphate pH 8.5. To this solution was added 27.5l (0.003mmol) of dissolved selenocysteine (step 1). The reaction was allowed to proceed at room temperature for 18 hours.

And 3, removing low-molecular-weight impurities:

ultrafiltration was performed using Amicon Ultra-15 at 10 MWCO. The buffer was replaced with 0.2M sodium phosphate pH 7.5. The volume after buffer exchange was 13.6 mL.

Step 4, preparation of a sulfitolysis reagent:

by mixing 0.315g of Na₂SO₃And 0.121g of Na₂S₄O₆The sulfitolysis reagent was prepared by dissolving in 1mL of 1M TRIS/HCl solution at pH 8.0 and diluting to 25mL with water.

Step 5 (PEG-Se)₂Sulfitolysis of reagents:

6.25mL of sulfitolysis reagent was added (100-fold molar excess of bisulphite to bisseleno groups) to 10.2mL (PEG-Se)₂Reagent (step 3). The reaction was allowed to proceed at room temperature for 18 hours.

And 6, removing low-molecular-weight impurities:

ultrafiltration was performed using Amicon Ultra-15 at 10 MWCO. The buffer was replaced with 0.2M sodium phosphate pH 7.5. The volume after buffer replacement was 8.1 mL.

Example 5: preparation from cysteamine seleno and 20kDa PEG-NHS (PEG-Se)₂And PEG-SeSO₃And (3) a reagent.

Step 1, preparation of selenocysteine stock solution:

12.4mg of selenocysteine was dissolved in 0.341mL of 0.2M sodium phosphate pH 8.5.

Step 2, coupling reaction of PEG and selenocysteine:

583mg (0.03mmol) of 20kDa PEG-NHS reagent was dissolved in 1.906mL of 0.2M sodium phosphate pH 8.5. To this solution was added 27.5l (0.003mmol) of dissolved selenocysteine (step 1). The reaction was allowed to proceed at room temperature for 18 hours.

And 3, removing low-molecular-weight impurities:

ultrafiltration was performed using Amicon Ultra-15 at 10 MWCO. The buffer was replaced with 0.2M sodium phosphate pH 7.5. The volume after buffer replacement was 13.0 mL.

Step 4 (PEG-Se)₂Sulfitolysis of reagents:

6.25mL of sulfitolysis reagent (example 4, step 4) was added (100-fold molar excess of sulfurous acid over the bisselenium group) to 9.75mL (PEG-Se)₂Reagent (step 3). The reaction was allowed to proceed at room temperature for 18 hours.

And 5, removing low-molecular-weight impurities:

ultrafiltration was performed using Amicon Ultra-15 at 10 MWCO. The buffer was replaced with 0.2M sodium phosphate pH 7.5. The volume after buffer replacement was 9.0 mL.

Example 6: preparation from cysteamine seleno and 20kDa PEG-NHS (PEG-Se)₂。

Step 1, preparation of selenocysteine stock solution:

22.2mg of selenocysteine was dissolved in 1.48mL of 1M K pH 8.0₂B₄O₇In (1).

Step 2, coupling reaction of PEG and selenocysteine:

321mg (0.016mmol) of 20kDa PEG-NHS reagent are dissolved in 1.075mL of 0.1M K pH 8.0₂B₄O₇In (1). To this solution was added 0.114(0.0054mmol) of dissolved selenocysteine (step 1). The reaction was allowed to proceed for 24 hours at room temperature in the absence of light.

Step 3 removal of Low molecular weight impurities and solids (PEG-Se)₂Separation of (2):

the pH of the reaction mixture was adjusted to 3.0 by the addition of oxalic acid and diluted with water to a final volume of 20 mL. The buffer solution was then extracted three times with dichloromethane. With Na₂SO₄The combined organic fractions were dried. The organic solution was concentrated by rotary evaporation and precipitated by dropwise addition of diethyl ether (PEG-Se)₂. After centrifugation, the precipitate was dried under high vacuum at room temperature for 12 hours.

Example 7: G-CSF with a conjugate prepared from selenocysteine and 10kDa PEG-NHS (PEG-Se)₂And (4) conjugation of the reagent.

Step 1. pegylation of G-CSF on Cys 18:

to adjust the pH of the G-CSF solution to 7.5, 3.636mL of 0.2M sodium phosphate pH8.5 were added to 11.2mL of the total solution of G-CSF (20 mg). Then 2.18mL (PEG-Se) prepared from selenocysteine was added₂Reagent (example 1, step 3) and 0.172mL of 10% SDS. The pegylation reaction was allowed to proceed for 24 hours at room temperature in the absence of light. The pegylated mixtures were analyzed by SDS-PAGE.

Example 8: G-CSF with a conjugate prepared from cysteamine selenosulfate and 10kDa PEG-NHS (PEG-Se)₂Preparation of conjugates of the reagents.

Step 1. pegylation of G-CSF on Cys 18:

to adjust the pH of the G-CSF solution to 7.5, 3.246mL of 0.2M sodium phosphate pH8.5 were added to 10.8mL of the total solution of G-CSF (19.2 mg). Then 3.65mL of 10kDa (PEG-Se) prepared from cysteamine selenide was added₂Reagent (example 3, step 3)) And 0.2mL of 10% SDS. The pegylation reaction was allowed to proceed for 26 hours at room temperature in the absence of light.

Step 2. termination of pegylation:

the buffer was replaced with 25mMCH at pH 3.8 using a Sephadex G-25 column₃COOH/NaOH and 1+1 dilution with the same buffer. The resulting sample was then left at 4 ℃ for 18 hours. The pegylated mixtures were analyzed by SDS-PAGE under non-reducing conditions.

Step 3. isolation of G-CSF pegylated on Cys18 by CEC:

the fraction containing G-CSF pegylated on Cys18 was eluted with between 63% and 71% elution buffer. The collected fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions.

Example 9: G-CSF with a conjugate prepared from cysteamine selenosulfate and 20kDa PEG-NHS (PEG-Se)₂Preparation of conjugates of the reagents.

Step 1. pegylation of G-CSF on Cys 18:

to adjust the pH of the G-CSF solution to 7.5, 0.462mL of 0.2M sodium phosphate pH8.5 was added to 1.4mL of the total solution of G-CSF (2.5 mg). Then 2.6mL (PEG-Se) prepared from selenocysteine was added₂Reagent (example 5, step 3) and 0.045mL of 10% SDS. The pegylation reaction was allowed to proceed for 24 hours at room temperature in the absence of light.

Step 2. termination of pegylation:

the buffer was replaced with 25mMCH at pH 3.8 using a Sephadex G-25 column₃COOH/NaOH and 1+1 dilution with the same buffer. The resulting sample was then left at 4 ℃ for 24 hours.

Step 3. isolation of G-CSF pegylated on Cys18 by CEC:

the fraction containing G-CSF pegylated on Cys18 was eluted with between 63% and 67% elution buffer. The collected fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions.

Example 10: preparation of G-CSF and PEG-SeSO prepared from cysteamine selenide and 20kDa PEG-NHS₃A conjugate of an agent.

Step 1. pegylation of G-CSF on Cys 18:

to adjust the pH of the G-CSF solution to 7.5, 2.039mL of 0.2M sodium phosphate pH8.5 was added to 6.179mL of the total solution of G-CSF (11 mg). Then 7.92mL of 20kDa PEG-SeSO prepared from cysteamine selenide was added₃Reagent (example 5, step 5) and 0.163mL of 10% SDS. The pegylation reaction was allowed to proceed for 24 hours at room temperature in the absence of light.

Step 2. termination of pegylation:

the buffer was replaced with 25mMCH at pH 3.8 using a Sephadex G-25 column₃COOH/NaOH and 1+1 dilution with the same buffer. The resulting sample was then left at 4 ℃ for 24 hours.

Step 3. isolation of G-CSF pegylated on Cys18 by CEC:

the fraction containing G-CSF pegylated on Cys18 was eluted with between 59% and 67% elution buffer. The collected fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions.

Example 11: G-CSF with a conjugate prepared from cysteamine selenosulfate and 20kDa PEG-NHS (PEG-Se)₂Conjugation of reagents at different pH values.

Step 1. Pegylation of G-CSF on Cys18 in buffers pH 4.0, 5.0, 6.0, 7.0 and 7.5

The pH of the G-CSF solution (140. mu.l/0.25 mg of G-CSF) was adjusted to 4.0, 5.0, 6.0, 7.0 and 7.5 by adding 0. mu.l, 4.1. mu.l, 6.6. mu.l, 15.3. mu.l and 7.2. mu.l of 0.2M sodium phosphate, respectively, and then G-CSF and (PEG-Se) were performed₂Pegylation of the reagent. To obtain the same concentration in all samples, addA suitable volume of water. With a 10-fold molar excess of 20kDa (PEG-Se) at room temperature in the absence of light and in the presence of 0.1% SDS₂Reagents G-CSF was pegylated for 24 hours. The pegylated mixtures were analyzed by SDS-PAGE.

Claims (15)

1. A modifier comprising a water soluble polymer, wherein the water soluble polymer is selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, a polyol, a polyalkylene oxide, cellulose, sucrose and hydroxyalkyl starch (HAS) and comprises at least one reactive selenium group capable of reacting with a thiol group to form an-Se-S-bond; and the modifier has a structure as shown in formula I or II,

wherein in the above formula P is a water-soluble polymer and L is a linking group.

2. The modifying agent of claim 1, wherein the water soluble polymer is polyethylene glycol (PEG) or hydroxyethyl starch (HES).

3. The modifier of claim 1 or 2, having a structure as shown in formula III or IV,

wherein in the above formula PEG is polyethylene glycol and R is hydrogen, carboxyl or C₁-C₆An alkyl group.

4. A process for producing the modifier of any one of claims 1 to 3, comprising the steps of:

(i) providing a compound of formula V

Wherein A is a functional group and L is a linking group,

(ii) reacting the compound of formula V with an activated water-soluble polymer, and

(iii) (iii) optionally subjecting the product of step (ii) to a sulfitolysis reaction.

5. The method of claim 4, wherein in step (i) a compound of formula VI is provided

Wherein in the above formula, R is hydrogen or carboxylRadical or C₁-C₆An alkyl group, a carboxyl group,

and in step (ii), the compound of formula VI is reacted with PEG-NHS.

6. Use of a modifying agent according to any one of claims 1 to 3 for binding a water-soluble polymer to a cysteine residue of a polypeptide.

7. The use of claim 6, wherein the water soluble polymer is polyethylene glycol, the polypeptide is G-CSF, the cysteine residue is Cys18, and Cys18 of the G-CSF is pegylated.

8. A conjugate comprising a water-soluble polymer and a pharmaceutically active agent comprising a thiol group, wherein the water-soluble polymer is selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, a polyol, a polyalkylene oxide, cellulose, sucrose and hydroxyalkyl starch (HAS), the water-soluble polymer being linked to the pharmaceutically active agent via an S-Se bond.

9. The conjugate of claim 8, having the structure of formula VII:

wherein in the above formula, P is a water-soluble polymer, L is a linking group, Se is a selenium atom, Pept is a polypeptide and S is a sulfur atom belonging to a cysteine residue of the polypeptide.

10. The conjugate of claim 8 or 9, having the structure of formula VIII:

wherein in the above formula, PEG is polyethylene glycol; l is-CO-NH-CHR-CH₂-residue, wherein R is hydrogen, carboxyl or C₁-C₆An alkyl group; se is a selenium atom; G-CSF is a granulocyte colony stimulating factor and S is a sulfur atom belonging to a cysteine residue of the granulocyte colony stimulating factor.

11. The conjugate of claim 10, wherein the polyethylene glycol residue specifically binds to Cys18 of the G-CSF.

12. Use of a conjugate according to claim 10 or 11 in the manufacture of a medicament for the treatment of neutropenia.

13. The use of claim 12, wherein the treatment is in a patient with non-myeloid hematological malignancies receiving a myelosuppressive anti-cancer drug.

14. A process for the preparation of a conjugate according to any of claims 8 to 11, comprising the steps of:

(i) providing a pharmaceutically active agent comprising at least one free thiol group, and

(ii) reacting the pharmaceutically active agent with the modifying agent of any one of claims 1-3.

15. A pharmaceutical composition comprising:

(a) the conjugate of any one of claims 8-11, and

(b) one or more pharmaceutically acceptable excipients.