TWI491410B - G-csf conjugates modified by water-soluble polymers - Google Patents

Description

Water soluble polymer modified G-CSF conjugate

The present invention relates to a novel structure, a water-soluble polymer modified G-CSF conjugate of the formula (I) or a pharmaceutically acceptable salt thereof, and a process for the preparation thereof, and a compound of the formula (I) or A pharmaceutical composition of a pharmaceutically acceptable salt thereof.

Water Soluble Polymer-Linking Group-N- T -G' (I)

Granulocyte community stimulating factor (G-CSF) is produced by monocytes and fibroblasts, which stimulates the formation of granulocyte colonies and stimulates the neutral sphere. G-CSF binds to target cell membrane receptors, mainly stimulates hematopoiesis in granulocyte lines, and also allows multi-functional hematopoietic stem cells to enter the cell cycle, promote proliferation, differentiation and maturation of myeloid hematopoietic cells, and drive the release of neutrophils to the bloodstream. Increases the number of peripheral neutrophils and enhances their functions, such as phagocytosis, antibody-dependent cellular cytotoxic activity against tumor cells, etc. [Metcalf, Blood 67: 257 (1986); Yan et. al, Bloom 84 (3) : 795-799 (1994); Bensinger, et. al, Blood 81 (11): 3158-3163 (1993); Neben, et. al, Blood 81 (7): 1960-1967 (1993)], thus recombined Granulocyte community stimulating factors are commonly used in cancer patients receiving radiation therapy or chemotherapy and adjuvant therapy for leukemia patients after bone marrow transplantation.

The human G-CSF commonly used in the market is Neupogen and Neutrogin, and the derivative of human G-CSF, Neu-wP, a derivative or variant protein of G-CSF, has also been reported in a large number of literatures [such as US5581476, US5214132, US 5,632,853, US 4,904, 584], these variant proteins contain multiple amino acid substitutions in order to find a more stable, more active, more clinically suitable G-CSF.

Commercially available recombinant human G-CSF is poorly bioavailable, has a short half-life, and is easily destroyed by proteases in the body. Therefore, it is necessary to administer frequent injections, and it is difficult to obtain a good clinical therapeutic effect. Studies have shown that proteins with therapeutic applications are modified by polyethylene glycol to form polyethylene glycol-protein conjugates, and the possibility of becoming a drug is greatly increased. This type of polyethylene glycol modified protein has been fully utilized clinically [eg Katre, Advanced Drug Delivery Systems, 10: 91 (1993); Inada, et. al, J. Bioact and Compatible Polymers, 5:343). (1990)]. The protein conjugate modified by polyethylene glycol has better physical and chemical stability, and also has better protease-mediated stability. In addition, the half-life of the conjugate is prolonged due to the increase in molecular weight of the conjugate. The possibility of forming antibodies in the body is also reduced, and the volume of distribution is reduced, which reduces toxicity. G-CSF or G-CSF variant proteins modified by polyethylene glycol have been disclosed in many documents, such as EP0335423, EP0401384, US5824778, US5985265, WO0044785, WO2001051510, US5824784, and the like. Specifically, in the polyethylene glycol-modified G-CSF conjugate disclosed in the patent US Pat. No. 5,985,265, the conjugate obtained by modifying the N-terminus of G-CSF has the best biological activity in vitro and in vivo, but with a deuteration reaction. The polyethylene glycol modified amino group has poor selectivity. Therefore, a mixture of polyethylene glycol modified at various positions is usually obtained, and separation and purification are required to obtain individual monomers, and the yield is low and difficult. Industrial production; in the US 5,824,784, the macromolecular polyethylene glycol aldehyde is directly used to modify G-CSF. By strictly controlling the pH of the reaction and selectively modifying the N-terminus of G-CSF, a relatively specific N-terminal polyg can be obtained. Ethylene glycol modified conjugate, but it is undeniable that the selectivity of this reaction is achieved by the difference in Pka between the amino group of the side chain and the N terminal group, which is quite difficult in preparation and quality control; Because the content of aldehydes in different batches of macromolecular aldehydes is different, it is difficult to control the ratio of macromolecular aldehydes to protein, which will inevitably affect the reaction yield and production cost, and the conjugates due to the coupling of macromolecular aldehydes with different amino groups. Differences in biological activity also affect the homogeneity and activity of the final product.

In view of the deficiencies of the prior art, it is an object of the present invention to provide a novel structure, a water-soluble polymer modified G-CSF conjugate of the formula (I) or a pharmaceutically acceptable salt thereof; A further object is to provide a process for preparing a conjugate of the formula (I) or a pharmaceutically acceptable salt thereof; a further object of the present invention is to provide a medicament comprising the conjugate or a pharmaceutically acceptable salt thereof Composition and its use.

The present invention relates to a conjugate of the formula (I) or a pharmaceutically acceptable salt thereof.

Water-soluble polymer-linking group-N-terminus-G'(I) wherein the water-soluble polymer comprises polyethylene glycol, polypropylene glycol, polylactic acid, polymeric amino acid, etc., preferably polyethylene glycol, polyethylene glycol The molecular weight of the alcohol is selected from 2KD to 100KD, preferably 5KD to 40KD; the polyethylene glycol may be straight Linear or branched; the linking group is formed by a specific reaction of a functional group at the end of the water-soluble polymer with a functional group introduced at the N-terminus of the protein G'; G' is selected from natural G-CSF, recombinant G-CSF or A gene mutation product having G-CSF function, preferably a G-CSF derivative of SEQ ID NOS: 1 to 10, more preferably a sequence of a naturally occurring human G-CSF or a Met represented by SEQ ID NO: -G-CSF.

Specifically, the chemical structural formula of the conjugate of the above formula (I) or a pharmaceutically acceptable salt thereof is represented by the formula (II): Wherein R is selected from a linear or branched alkyl group of C _1-4 , preferably a methyl group or an ethyl group, more preferably a methyl group; and R ₁ and R ₂ are independently selected from hydrogen or C _1-4 straight. a chain or branched alkyl group, preferably hydrogen, methyl or ethyl; X is selected from O, S, NH, 1 is selected from an integer of 1 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5; m is selected from an integer of 50 to 2500, preferably an integer of 100 to 1000; n is selected from 1 to An integer of 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.

G' is selected from natural G-CSF, genetically recombinant G-CSF or a gene mutation product having G-CSF function.

Further, the present invention relates to a conjugate of the above formula (I), (II) or a pharmaceutically acceptable salt thereof, characterized in that G' has a sequence of naturally occurring human G-CSF.

Further, the present invention relates to a conjugate of the above formula (I), (II) or a pharmaceutically acceptable salt thereof, characterized in that G' is Met-G-CSF (SEQ ID NO: 1).

The above conjugates of the formula (I), (II) include: An integer selected from 400 to 500; m is an integer selected from 400 to 500.

Further, the conjugate of the formula (I), (II) of the present invention may form a salt with an acid selected from an organic acid or an inorganic acid, and the organic acid includes acetic acid, trifluoroacetic acid, propionic acid, butyric acid, Maleic acid or p-toluenesulfonic acid or a mixture thereof, preferably acetic acid, trifluoroacetic acid; inorganic acid including hydrochloric acid, sulfuric acid, phosphoric acid or Methanesulfonic acid or a mixture thereof is preferably hydrochloric acid.

In another aspect, the present invention provides a process for the preparation of a conjugate of the formula (I), (II) comprising the steps of: I) reductive amination of a compound of the formula (III) with an N-terminal amino group of G' The compound of the formula (IV) is obtained: 2) Deprotecting the compound of the formula (IV) to give a compound of the formula (V): 3) Michael addition of the general compound (V) to mPEG-MAL to obtain the conjugate (II): Wherein R, R ₁ , R ₂ , G', X, 1, m, n are as defined above; Z is a thiol protecting group selected from the group consisting of: methyl ketone, ethyl hydrazino, propyl fluorenyl, trityl Or a tert-butyl group, preferably an ethyl hydrazide group.

Further, the present invention relates to the use of the conjugate of the present invention or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating leukopenia, AIDS and other immunodeficiency diseases, bacterial infection diseases caused by radiotherapy or chemotherapy.

In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of a conjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Further, the present invention relates to the use of the pharmaceutical composition for the preparation of a medicament for treating diseases such as leukopenia, AIDS and other immunodeficiency diseases, bacterial infections, etc. due to radiotherapy and chemotherapy.

The invention discloses a novel class of polyethylene glycol modified G-CSF conjugates and a novel preparation method thereof. Compared with the conventional conjugates and methods, the following differences exist: firstly, in polyethylene glycol and G A new concept of introducing a linking group between CSFs can ensure the specificity of the N-terminal reaction of the protein, because the protein obtained by modifying the N-terminus with a small molecule is easy to separate and purify, and the specificity of the modification point is ensured. Secondly, The presence of a sulfhydryl group in the linking group ensures the progress of the Michael addition reaction with high specificity by controlling the pH of the reaction system. In addition, other functional groups may also be added due to the presence of a linking group, such as Aachen. The introduction of an amine group, an ester group, provides a prerequisite for the release of G-CSF from the conjugate in vivo.

The conjugate of the present invention, or a pharmaceutically acceptable salt thereof, has natural human G-CSF physiological activity and has a longer in vivo loop half-life and better granulocyte stimulating factor activity than G-CSF.

In particular, the structural formula of the conjugate disclosed herein is represented by (II): Wherein R is selected from a linear or branched alkyl group of C _1-4 , preferably a methyl group or an ethyl group, more preferably a methyl group; and R ₁ and R ₂ are independently selected from hydrogen or C _1-4 straight. a chain or branched alkyl group, preferably hydrogen, methyl or ethyl; X is selected from O, S, NH, 1 is selected from an integer of 1 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5; m is selected from an integer of 50 to 2500, preferably an integer of 100 to 1000; and n is selected from 1 to An integer of 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.

G' is selected from natural G-CSF, genetically recombinant G-CSF or a gene mutation product having G-CSF function.

When X is O, S, there is an ester bond in the linking group between polyethylene glycol and G-CSF which is easily hydrolyzed by esterase in the body. At this time, G-CSF can be freely formd with the acceptor. Acting to reflect biological activity; when X is NH, At the time, its biological activity is mainly achieved by the conjugate itself. Of course, since the sulfhydryl bond is also hydrolyzed in the body to release G-CSF, the possibility of free G-CSF is not ruled out.

In the production method disclosed in the present invention, the thiol-containing linking group is first reacted with the N-terminal amino group of G-CSF by a reductive amination reaction to obtain an intermediate (IV). In fact, the linking group can be linked to the amino group in G-CSF by various reaction methods (such as the reaction of various activated esters with an amino group), but in view of the high selectivity of the reaction of the aldehyde group with the N-terminal amino group, in the present invention A linking group is introduced by a reductive amination reaction of an aldehyde group with an N-terminal amino group in G-CSF. The reducing agent used is selected from various reducing agents well known to those skilled in the art, and is preferably sodium cyanoborohydride or sodium triethoxysulfonate.

In the preparation of the intermediate (Y), different reaction conditions are selected depending on the thiol protecting group. When the protecting group is a trityl group or a tert-butyl group, the group is deprotected using acidic conditions (such as trifluoroacetic acid, hydrochloric acid, methanesulfonic acid, etc.); when the protecting group is an ethyl group, a propyl group, etc. The group is deprotected using various methods and reagents well known to those skilled in the art, and it is preferred to deprotect the group with hydroxylamine hydrochloride at pH = 5 to 7.

In the preparation of the compound of the formula (II), the reaction is carried out by controlling the pH of the reaction, using a classical Michael addition, which has a very good selectivity and can reach 99% or more. After the reaction, the reverse phase HPLC system can be used. Separate and purify by preparative column, ion exchange column or gel column.

In the present invention, "C _1-4 alkyl" means a linear or branched saturated aliphatic hydrocarbon group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group or the like.

In the present invention, it is preferred that the human G-CSF has no internal sequence change, thereby reducing the in vivo antigenicity of the protein, minimizing the formation of neutralizing antibodies in vivo, and improving the drug efficacy. Variant proteins of G-CSF and G-CSF can be obtained by methods reported in the literature by conventional gene expression methods including, but not limited to, Met-G-CSF, Trp-G-CSF, Asp-G-CSF, Glu-G. -CSF, preferably Met-G-CSF derived from E. coli expression (see SEQ ID NO: 1 for the sequence).

The compounds obtained in the present invention are usually administered in dosage units. The dosage unit can be better described as a pharmaceutical composition obtained by mixing the active compound with a pharmaceutical excipient.

In the pharmaceutical compositions provided herein, the conjugates of formula (I), (II) or pharmaceutically acceptable salts thereof are the active compounds. The pharmaceutical composition provided by the invention can be used as a adjuvant therapy for cancer patients undergoing chemotherapy or radiotherapy and bone marrow transplantation to prevent infection due to decreased immunity in vivo; and can also be used for treatment of chronic or relative leukopenia patients. It can also be used for the treatment of patients with acute myeloid leukemia, AIDS or other deficient diseases; it can also be used for antifungal treatment, especially for systemic or invasive Candida infections.

The dose of the conjugate of the formula (I), (II) or a pharmaceutically acceptable salt thereof is from 5 to 500 ug/kg, wherein "ug" means a conjugate of the formula (I), (II) or A unit of measurement for a pharmaceutically acceptable salt, "kg" refers to the measurement of the weight of a mammal unit. Of course, as we know, the dose administered and the frequency of administration depend on many factors, such as the sex and age of the patient, the type of disease the patient is required to prevent or treat.

The conjugate obtained by the present invention or a pharmaceutically acceptable salt thereof can be prepared in various unit forms by mixing with a conventional pharmaceutical carrier, and administered in various manners such as subcutaneous, intramuscular, and intravenous.

In order to explain the present invention in more detail, the following examples are given. However, the scope of the invention is not limited thereto.

Embodiment 1

Reductive amination of Met-G-CSF

The Met-G-CSF stock solution was dialyzed and converted into a 0.1 M acetic acid/sodium acetate solution, and 140 ml was obtained, which contained about 40 mg of protein. A small molecule aldehyde (7.0 mg) was dissolved in acetonitrile (300 ml), added to a protein solution, and sodium cyanoborohydride (176 mg) was added thereto, and the mixture was stirred at room temperature for 3 hours.

The reaction solution was dialyzed against a 2 M EDTA in 0.1 M PBS solution (pH = 6.2) at 4 °C.

Embodiment 2

Deprotecting group - ethyl hydrazide The protein solution prepared in Example 1 was added with 10 ml of 0.1 M hydroxylamine hydrochloride (pH = 6.3) to a concentration of 50 mM, and the reaction was stirred at room temperature for 30 minutes to remove The acetamino group frees the sulfhydryl group.

Embodiment 3

Preparation of mPEG-Met-G-CSF (39KD) The protein solution prepared in Example 2 was added to mPEG-MAL (400 mg, 20 KD), and the reaction was stirred at room temperature for 60 minutes. After the end of the HPLC reaction, the reaction was carried out directly. Prepared and purified, the obtained compound is the compound 1 of the present invention, and the structure is confirmed by MALDI-TOF-TOF, see Fig. 1.

Control conditions: analytical column: Jupiter C4, 5u, 300 , 150*4.6 mobile phase: A: 0.05% TFA / H ₂ OB: 0.05% TFA / CH ₃ CN Separation and purification conditions: packed column with SP Sepharose HP, 1.6cmx12cm, volume about 24ml, flow rate: 4ml/min, pretreatment of the column: first wash 5 times volume with 0.5M NaOH solution; then wash with purified water to neutral; Wash 5 times volume to equilibrium with 20 mM HAc/NaAc, pH 4.0. Loading: directly pump the desalted sample into the column with a pump; elution: first elute 10 times column volume with 20 mM HAc/NaAc, pH 4.0 to remove excess PEG; then set the salt gradient: 0 to 50%, At 50 min, 4 ml/min, impurities, products, and unreacted proteins were sequentially eluted and collected.

Embodiment 4

The preparation, isolation and purification methods of mPEG-Met-G-CSF (59KD) are the same as those in the third embodiment except that mPEG-MAL (400 mg, 20 KD) is replaced by mPEG-MAL (800 mg, 40 KD), and the obtained compound is Compound 2 of the present invention, MALDI-TOF-TOF, is shown in Figure 2.

Embodiment 5

Preparation of mPEG-Met-G-CSF (39KD), Compound 1 Injection

In the sterile ingredient room, weigh sodium acetate (0.12 g), polysorbate 20 (35 mg), sorbitol (50 g), add water for injection (1000 ml), stir to dissolve, add compound 1 (10克) Stir well, add water for injection to 3000 ml, under sterile conditions, filter with 0.22μm microporous membrane and then dispense, add sterile plug and roll the outer cover, that is.

Test Example 1

Comparison of the effects of PEG-G-CSF and G-CSF on peripheral blood leukocyte counts in mice Note: PEG-G-CSF refers to Compound 1 of the present invention.

1 Objective To evaluate and compare the efficacy of PEG-G-CSF and G-CSF in increasing peripheral blood leukocyte counts in mice treated with cyclophosphamide.

2 Materials and methods: PEG-G-CSF, G-CSF, cyclophosphamide (CTX), provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; diluted with physiological saline before use.

Kunming mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, weighing 18 to 22 g, and the number of animals in each group was 10 .

After the animals were adapted, cyclophosphamide was intraperitoneally injected, and PEG-G-CSF, G-CSF was injected subcutaneously on the second day. PEG-G-CSFO.5, 1.0 mg/kg was injected subcutaneously once; G-CSF was injected subcutaneously once a day for 4 times at doses of 0.1 and 0.2 mg/kg, respectively. At the end of the administration, the mice were sacrificed by cervical dislocation and blood was taken. Blood cell counts were counted using an ABC automated blood cell counter.

3 Results Intraperitoneal injection of CTX significantly reduced peripheral blood leukocytes, red blood cells, and platelet counts (both P<0.01 vs. control, Figures 3 to 5), indicating that CTX is a strong myelosuppressive agent.

A single subcutaneous injection of PEG-G-CSF increased the peripheral blood white blood cell count of mice treated with cyclophosphamide, and the white blood cell count returned to normal level when administered at 0.5 mg/kg; when administered at 1.0 mg/kg, The peripheral blood white blood cell count was even higher than the normal level (P<0.01 vs. control ratio, Fig. 3). However, PEG-G-CSF did not significantly increase peripheral red blood cells and platelet counts (Fig. 4, Fig. 3), indicating the specificity of PEG-G-CSF action.

Continuous subcutaneous injection of G-CSF also increased the week of mice treated with cyclophosphamide Blood white blood cell count. When 0.1 mg/kg was administered, the white blood cell count rose to the normal level; when 0.2 mg/kg was administered, the white blood cell count was higher than the normal level (P<0.01 compared with the control, Fig. 3), and there was a significant dose dependency. However, G-CSF had no significant effect on peripheral red blood cells and platelet counts (Fig. 4, Fig. 5).

Based on the above results, the single subcutaneous injection of PEG-G-CSF and multiple subcutaneous injections of G-CSF were equivalent to the peripheral blood white blood cell count in mice.

4 Conclusions Both PEG-G-CSF and G-CSF significantly increased peripheral blood leukocyte counts in cyclophosphamide-treated mice; single subcutaneous injection of G-CSF of compound 1 of the present invention in the case of equivalent total doses Multiple subcutaneous injections have comparable effects on peripheral white blood cell counts in mice.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a MALDI-TOF-TOF chart of the compound 1 of the present invention; Fig. 2 is a MALDI-TOF-TOF chart of the compound 2 of the present invention; and Fig. 3: PEG-G-CSF, G- Effect of CSF on peripheral blood leukocyte counts in mice treated with cyclophosphamide; Figure 4: Effect of G-CSF on the peripheral red blood cell count of cyclophosphamide-treated mice; Figure: Effect of PEG-G-CSF and G-CSF on peripheral blood platelet counts in cyclophosphamide-treated mice

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Claims (17)

a conjugate of the formula (II) or a pharmaceutically acceptable salt thereof, Wherein R is selected from methyl or ethyl; R ₁ and R ₂ are independently selected from hydrogen, methyl and ethyl; X is l is selected from an integer from 1 to 20; m is selected from an integer from 50 to 2500; n is selected from an integer from 1 to 5; G' is selected from the group consisting of native G-CSF, genetically recombinant G-CSF. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein l is an integer of 1 to 10. The conjugate of claim 2 or a pharmaceutically acceptable salt thereof, wherein l is an integer of 1 to 5. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein m is an integer of from 100 to 1000. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein G' is selected from the group consisting of G-CSF shown in SEQ ID NOS: 1 to 10. derivative. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that said G' is a sequence of a naturally occurring human G-CSF. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein G' is Met-G-CSF represented by SEQ ID NO: 1. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that the conjugate comprises: m is an integer selected from 400 to 500; and m is an integer selected from 400 to 500. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that the conjugate and the acid are salted, and the acid used is selected from the group consisting of organic acids or inorganic acids. The conjugate of claim 9 or a pharmaceutically acceptable salt thereof, characterized in that the organic acid is selected from the group consisting of acetic acid, trifluoroacetic acid, propionic acid, butyric acid, maleic acid or p-toluenesulfonic acid. Or a mixture thereof; the inorganic acid is selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid or methanesulfonic acid or a mixture thereof. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein the organic acid is acetic acid or trifluoroacetic acid. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein the inorganic acid is hydrochloric acid. A process for the preparation of a conjugate according to claim 1, comprising: 1) reductive amination of a compound of the formula (III) with an N-terminal amino group of G' to give a compound of the formula (IV): 2) Deprotecting the compound of the formula (IV) to give a compound of the formula (V): 3) Michael addition of the compound of the formula (V) to mPEG-MAL to obtain the compound (II): Wherein R, R ₁ , R ₂ , G', X, 1, m, n are as defined in the first item of the patent application; Z is a thiol protecting group selected from the group consisting of: methyl ketone, ethyl fluorenyl, propyl hydrazine Base, trityl or tert-butyl. The method of claim 13, wherein Z is an ethyl group. Use of a conjugate of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of leukopenia caused by radiotherapy or chemotherapy. A pharmaceutical composition comprising a pharmaceutically effective amount of a conjugate as described in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Use of a pharmaceutical composition according to claim 16 for the preparation of a medicament for the treatment of leukopenia caused by radiotherapy or chemotherapy.