TW200944236A - G-CSF conjugates modified by water-soluble polymers - Google Patents

Abstract

The present invention relates to G-CSF conjugates with a completely new structure of formula (I) which is modified by water-soluble polymers, or pharmaceutically acceptable salts thereof, their preparation method, and a pharmaceutical composition containing the compound of formula (I) or pharmaceutically acceptable salts thereof. water-soluble polymer-linkage-N terminal-G' (I).

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel structure, a water-soluble polymeric modified G-CSF conjugate of the formula (I) or a pharmaceutically acceptable salt thereof, And a process for the preparation thereof, and a pharmaceutical composition comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. Water Soluble Polymer-Linking Group-N-Terminal-G, (I) 先前 [Prior Art] Granulocyte Community Stimulating Factor (G-CSF) is produced by monocytes and fibroblasts and stimulates granulocyte community formation. It has a stimulating effect on the neutral ball. 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 swollen tumor cells [Metcalf, Blood 67: 257 (1986); Yan et.al &gt; Blood 84 (3): 795-799 (1994); Bensinger, et.al, Blood 81(11): 3158-3163 (1993); Neben, et.al, Blood 81(7): 1960-1967 (1993)], Therefore, recombinant granulocyte community stimulating factors are often used for adjuvant treatment of cancer patients receiving radiation therapy or chemotherapy and 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 [eg US5581476, US5214132, 5 94293 200944236). US5362853, US4904584] '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 due to its presence in humans Poor bioavailability, short half-life, and easy destruction by proteases in the body require frequent injections, making it difficult to achieve good clinical results. Studies have shown that proteins with therapeutic applications are modified by polyethylene glycol to form polyethylene glycol-protein conjugates, which greatly increases the likelihood of becoming a drug. This type of polyglycolethane modified protein has been fully used clinically [eg Katre 5 Advanced Drug Delivery Systems 5 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, due to the increase of molecular weight of the conjugate, its half-life in vivo is obtained. Prolonged, the possibility of antibody formation in the body has also decreased, coupled with @ reduced distribution volume, which can reduce toxicity. G-CSF or G-CSF variant proteins modified by polyethylene glycol have been disclosed in many documents, such as EP0335423, EP0401384, US5824778, US5985265, W00044785, 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 in vitro and in vivo biological activity, but is used for purification. The reaction is carried out with polyethylene glycol to modify the amino group with poor selectivity. Therefore, a mixture of polyethylene glycol modified at various positions is usually obtained, and separation and purification are required to obtain each monomer, and the yield is low, which is difficult to be 6 94293 200944236 ·&gt; Industrial production; in the US 5,824,784, the macromolecular polyethylene glycol aldehyde is used to directly modify G-CSF. By strictly controlling the pH of the reaction and selectively modifying the N-terminus of G-CSF, relatively specific n can be obtained. Terminal poly(diol) modified conjugates', 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 to prepare and control. ^ The content of aldehydes varies between batches of aldehydes, it is difficult, and it is difficult to control the ratio of macromolecular aldehydes to egg feeds, which will inevitably affect the reaction yield and production cost, and at the same time, due to the coupling of macromolecules with different amino groups. The bioactive conjugate of: in response to the final product quality and uniformity of activity.别别三衫 [Summary of the invention] • Insufficient new 4 pairs of techniques 'The object of the present invention is to provide a new type of U-funded, water-soluble polymer-modified G or a pharmaceutically acceptable compound thereof of the formula (1) Salt; a conjugate of conjugated „(1) or a pharmaceutically acceptable salt thereof according to the invention </ RTI> </ RTI> to provide a pharmaceutical composition containing a shaft conjugate or a salt thereof and use thereof. a salt which can be incinerated. The present invention relates to a conjugate of the formula (1) or a pharmaceutically acceptable hydropolymeric polymer-linking group - N-terminus-G, (I) wherein the water-soluble polymer Including polyethylene glycol polymerized amino acid, etc., preferably polyethylene glycol: alcohol, polylactic acid, 2KD to l〇〇KD, preferably 5KD to 4〇Κβ. The amount of the knife is selected from the group, the ethylene glycol may be Straight 94293 7 200944236 Linear or branched; The linking group is formed by a specific reaction between the functional group at the end of the water-soluble polymer and the functional group introduced into the N-terminus of the egg G'. · The shell G' is selected from the natural G-CSF, Gene recombinant G_CSF or a gene mutation product having g_csf function, preferably numbered as SEQ ID NO.] 〇-CSF derivative, more Is a naturally occurring human sequence of the sequence 2 SEQ ID NO: 1 represented by jyfet-G-CSF. 5 Scientifically acceptable, specifically, the chemical structure of the conjugate of the above formula (1) or its pharmaceutically acceptable salt is Formula (II) means:

(II) wherein, a straight chain or a branched chain selected from Cw, preferably a methyl group or an ethyl group, is a methyl group; and W2 is independently selected from hydrogen or a Cl_4 linear or branched alkyl group, preferably hydrogen, Methyl or ethyl; X is selected from 0, S, NH, 'from 吣 or meaning. 1 is selected from 1 to 20, more than an integer from i to 1G, more preferably an integer from 5 to 5; m An integer selected from 50 to 2500, preferably an integer from 1 〇〇 to 1 ;; 94293 8 200944236 η is selected from an integer of from 1 to 20, preferably from 1 to 10, more preferably from 1 to 5. Integer. - G' is selected from the group consisting of natural G-CSF, genetically recombinant G-CSF, or a gene mutation product having a 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 a 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:

N-G-CSF H m is an integer selected from the group consisting of 400 to 500;

m is an integer selected from 400 to 500. Further, the conjugate of the formula (I) and (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, trigastric acetic acid, propionic acid, butyric acid, Maleic acid or p-toluene acid or a mixture thereof, preferably acetic acid, trifluoroacetic acid; inorganic acid including hydrochloric acid, sulfuric acid, phosphoric acid or 9 94293 200944236 or a mixture thereof, preferably hydrochloric acid. In one aspect, the present invention provides a process for preparing a conjugate of the formula (Ι), (ΙΓ), comprising the steps of: reductive amination of a compound of formula (111) with G, ν terminal amino group to give a general formula 3 (IV): R2 R /S' 2

Η 0 (III) Η, Ν · -G1 _/S,

, G · R2 os R: deuterated, the substance (Iv) is deprotected from the thiol group to obtain the compound of the formula (V): R2, G, (IV) :: compound (V) and ^ hs^H-R2 G· (V) mPEG-MAL undergoes Michael addition coupling

Among them (II) 10 94293 200944236 Application of minor illness, AIDS and other immunodeficiency diseases, = ball reduction. In another aspect, the invention provides a pharmaceutical composition, an effective amount of a conjugate of the invention, or a pharmaceutically acceptable carrier. The present invention also relates to the preparation of a pharmaceutical composition for treating leukopenia, AIDS and other diseases such as leukopenia, AIDS and other diseases, diseases, diseases, bacterial infections and the like. Application in medicine. The invention discloses a new type of polyethylene-2 and its novel preparation method, and the following differences exist between the conventional conjugate and the method ω. 新 New: first; Lee T is introduced between polyethylene glycol and G_CSF The new core of the linking group ensures the specificity of the N-terminal reaction of the protein, because

= The protein obtained by molecular modification of the N-terminus is easy to separate and purify, ensuring the specificity of the decoration; U 'Because of the presence of the thiol group in the linking group, by controlling the reaction body', it is ensured that the Michael Plus with high specificity In addition, due to the presence of the linking group, other functional groups may also be added to the introduction of 'such as amidino group and an ester group, which provides a prerequisite for releasing G-CSF from the conjugate in vivo. condition. 94293 11 200944236 The conjugate of the invention or a pharmaceutically acceptable salt thereof has a natural human origin: -CSF physiological activity, and has a longer in vivo half-life than g_csf and better granulocyte stimulating factor activity. Specifically, the structural formula of the conjugate disclosed herein is represented by (11):

R is selected from a linear or branched alkyl group of Cw, preferably a methyl group or an ethyl group, more preferably a fluorenyl group;

Ri R2 is independently of one another selected from hydrogen or c. 4 straight or branched chain, preferably hydrogen, methyl or ethyl; ❹ X is selected from 0, S, NH, or; 1 is selected from 1 to 20 The integer, preferably! An integer of 1〇, more preferably an integer of 1 to _5; m is selected from an integer of 5〇 to 2500, preferably an integer of 1〇〇 to 1〇〇〇; n is selected from an integer of 1 to 20' It is preferably an integer of 1 to 1 ,, 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 0, S, the linking group between the polyethylene glycol and g-CSF 12 94293 200944236 has a g-key which is easily hydrolyzed by the enzyme in the body. At this time, G_csf can be The biological activity is manifested by its free form and receptor action; when X is NH, /ΝΜΝ\ or its biological activity is mainly achieved by the conjugate itself 'of course, since the sulfhydryl bond is also hydrolyzed in the body The ability to release G_CSF does not rule out the possibility of free G-CSF. In the preparation method disclosed in the present invention, the intermediate group (IV) is obtained by first reacting a linking group containing a silk with a hydroxyl group of a quinone by a reductive amination reaction. It can be linked to the amino group in G-CSF by more than 35 kinds of reaction modes (such as the reaction of various activated esters with an amino group), but considering the high selectivity of the reaction between the aldehyde group and the N-terminal amino group, In the invention, a linking group is introduced by a reductive amination reaction of a chelating group with an N-terminal amino group in G-CSF. The reducing agent to be used is selected from various reducing agents well known to those skilled in the art, and is preferably sodium cyanoborohydride or sodium triethoxysulfonate. ❹ In the preparation of the intermediate (V), 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. The reaction has a good selectivity and can reach 99% or more. After the reaction, it can be separated and purified by reverse-phase high-performance liquid phase 94293 13 200944236 preparative column, ion exchange column or gel column. . . . in the present invention 'ci-4 alkyl, means a straight or branched saturated aliphatic hydrocarbon group such as a decyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group or the like. In the present invention 'Preferred to human G-CSF, because there is no change in its internal sequence, this will reduce the in vivo antigenicity of the protein, and minimally reduce the formation of neutralizing antibodies in the body' to improve the efficacy. G_CSF and variants of g_cSF The protein can be known by methods reported in the literature by conventional methods of gene expression 'including but not limited to Met-G-CSF, Trp-G-CSF, Asp-G-CSF, G CSF preferably derived from E. coli]y [et-G-CSF (sequence see SEQ ID NO: 1). The compound obtained in the present invention is usually administered in a dosage unit. The dosage unit can be better expressed as a drug obtained by mixing the active compound with a pharmaceutical excipient. In the pharmaceutical composition provided by the present invention, the conjugate of the formula (j), (π.) or a pharmaceutically acceptable salt thereof is an active compound. The bismuth pharmaceutical composition provided by the present invention can be used as Cancer patients undergo chemotherapy or radiotherapy and bone marrow transplant patients Adjuvant therapy to prevent infection due to decreased immunity in the body; can also be used for the treatment of patients with chronic or relative leukopenia; also for the treatment of patients with acute myeloid leukemia, AIDS or other deficient diseases; For the treatment of antifungal, in particular for systemic or invasive Candida infections. The agent of formula (I), (II) conjugate or a pharmaceutically acceptable salt thereof, is from 5 to 500 ug/kg, Wherein "Ug" refers to the unit of measurement of the conjugates of formula (1), (11) or their musically acceptable salts, "kg," which refers to the measurement of mammalian body weight 14 94293 200944236 units. Of course, as we know, the dose and frequency of administration will depend on a number of factors, such as the patient's gender, age, and 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 different manners such as subcutaneous, intramuscular, and intravenous. [Embodiment] In order to explain the present invention in more detail, the following examples are given. However, the scope of the present invention is not limited thereto. Embodiment 1

Reductive amination of Met-G-CSF

Met-G-CSF (pH=5.0) : 40mg/140ml

7.0 mg sodium cyano hydride: 176 mg The Met-G-CSF stock solution was dialyzed and converted to 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 0.1 M PBS solution (pH = 6.2) containing 2 mM EDTA at 4 °C. Example 2 Deprotection group - Ethyl group The protein solution prepared in Example 1 was added to 1 Oml of 15 94293 200944236 0.1 M hydroxylamine hydrochloride (ΡΗ=6·3)' to a concentration of 5 〇π1μ, the reaction was stirred at room temperature for 30 minutes, and the thiol group was freed by deacetylation. Example 3 * mPEG-Met-G-CSFH9KD) tjj§_

The protein solution prepared in the second embodiment was added with mPEG_MAL (400 mg, 20 KD), and the reaction was stirred at room temperature for 6 minutes. After the completion of the HPLC controlled reaction, the preparation was directly purified, and the obtained compound was the compound 1 of the present invention, and was subjected to MALDI. -TOF-TOF confirms that the structure is correct, see Figure 1. 〇Control conditions: Analytical column: Jupiter C4, 5u, 300 A, 150*4.6

Mobile phase: A: 0.05% TFA/H20 B: 0.05% TFA/CH3CN Gradient conditions: 0, 305 35, 369 A 60% 20% 20% 60% B 40% 80% 80% 40% @Isolation and purification conditions: use SP Sepharose HP packed column, 1.6cmxl2cm, volume about 24ml, flow rate: 4ml/min, pretreatment of the column: first wash 5 times volume with 〇.5M NaOH solution; then wash with purified water to neutral; then use 20mM HAc/NaAc , pH 4.0 wash 5 times the volume to balance. Loading: directly draw the desalted sample into the column with a pump; elution: first elute 10 times column volume with 20 mM HAc/NaAc, pH 4.0, 16 94293 200944236 to remove excess PEG; then set the salt gradient: 0 To 50%, 50 min, 4 ml/min, impurities, *products, and unreacted proteins were sequentially eluted and collected. The fourth example of mPEG-Met-G-CSFf59Km was prepared and isolated and purified in the same manner as in the third embodiment except that mPEG-MAL (400 mg, 20 KD) was replaced by mPEG-MAL (800 mg, 40 KD). The compound is the compound 2 of the present invention, and the MALDI-TOF-TOF is shown in Fig. 2. Example 5 mPEG-Met-G-CSF (39KD), a compound 1 injection, was prepared by "sodium acetate: 0.12 g", polysorbate 20: 35 mg sorbitol: 50 g of compound 1: 10 g in a sterile ingredient room. Weigh sodium acetate (0.12 g), polysorbate 20 (35 mg), sorbitol (50 g), add water for injection (1 ml), stir to dissolve 'Add compound 1 (10 g) Stir well, add water for injection to 3000 ml, filter under sterile conditions with 〇.22μιη microporous membrane, and then add the sterilized plug and roll the outer cover, which is obtained. Test Example 1 PEG-G-CSF and G- The effect of CSF on the peripheral blood white ball count of mice is higher than that of the vehicle: PEG-G-CSF refers to the compound of the invention 1. 17 94293 200944236 1 Test purpose, evaluation and comparison of PEG_G_CSF and g-csf increase cyclophosphamide Treatment of peripheral blood white blood cell counts in mice. * 2 Materials and methods: PEG-G-CSF, G-CSF, cyclophosphamide (CTX), provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; Dilution. Kunming mice, purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, weighing 18 to 22g Early, the number of animals in each group: 1 〇. ❹ After the animal was adapted, intraperitoneal injection of cyclophosphamide, subcutaneous injection of PEG-G-CSF, G_CSF on day 2, PEG_G_CSF 〇5, i 〇mg/kg subcutaneous injection once G-CSF was injected subcutaneously every day for 4 times, and the doses were 〇.1, 〇.2 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 blood stasis in the mice = count (all Ρ &lt; 0·01 vs. control ratios 'Fig. 3 to 5), indicating that CTX is a strong myelosuppressant . (4) The single white subcutaneous injection of rrcsF increased the white blood cell count of the mice after the treatment with the amphoteric amine. When the drug was administered at 5 mg/kg, the blood cells were ten times old: to the normal level; when the g was administered, the peripheral blood white blood level was _ Compared with the control, Figure 3). However, there was no significant increase in the stress, red blood cell and platelet counts, Fig. 3), indicating the specificity of the action of PEG-G-CSF. (The fourth round of continuous subcutaneous injection also increased the cycle of cyclohexane treatment of small gas week 94293 18 200944236 side blood white blood count. 0. lmg / kg administration, white blood cell count rose to the level, .G.2mg / kg dose At the time, the white blood cell count was higher than the normal level (iv) compared with the control (Fig. 3), and there was a significant dose dependency. ❻G-caffe has no significant effect on peripheral red blood cells and platelet counts (Fig. 4). 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 white blood cell count in mice. 〇4 Conclusion Both PEG-G-CSF and G-CSF significantly increased peripheral white blood cell counts in cyclophosphamide-treated mice; in the case of equivalent total doses, Compound 1 of this invention was injected subcutaneously with G_CSF Subcutaneous injections have comparable efficacy in 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; ❹ Fig. 2: MALDI-TOF-TOF chart of the compound 2 of the present invention; Fig. 3: PEG-G-CSF, G -CSF effect on the peripheral blood white count of mice treated with cyclophosphamide; Figure 4: PEG-G-CSF, the effect of G-CSF on the peripheral red blood cell count of cyclophosphamide-treated mice; Figure 5: Effect of PEG-G_CSF and G-CSF on peripheral blood platelet count in mice treated with cyclophosphamide. [Main component symbol description] Helmet 19 94293

Claims (1)

200944236 X. Patent application scope: 1. A conjugate represented by the formula (1) or a pharmaceutically acceptable salt thereof: ^ Water-soluble polymer-linking group - N-terminus - G' J (I) wherein, water-soluble The polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, polylactic acid or polymeric amino acid; the linking group is formed by a specific reaction of the functional hydrazone at the end of the water-soluble polymer with the functional group introduced at the N-terminus of the protein ❹G'; G' It is selected from natural G-CSF, genetically recombinant G-CSF or a gene mutation product having G-CSF function. 2. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that the water-soluble polymer is polyethylene glycol, including linear and branched type, and has a molecular weight of 2 KD-100 KD. Preferably, it is 5KD-40KD. 3. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that the chemical structure of the conjugate of the formula (I) is represented by the general formula (II): Wherein, 20 94293 (II) 200944236 R is selected from the group consisting of c&quot;linear or A- orbital, more (tetra)methyl or methyl; more preferably Ri, I are independently selected from the group consisting of hydroquinone C, and the &amp;; milk 4 Μ 4 linear or branched alkyl, hydrogen, methyl or ethyl; Μ X is selected from Ο, S, NH, un, or 1 is an integer selected from 1 to 20, preferably i An integer from 1 to 5 to 1 ;; 彳 m is ❹ m is selected from 50 to 2500, and the whole η is selected from! To 2. An integer of 1 _; 扪正欢 is preferably an integer from 1 to 10, more specifically an integer from A 1 to 5; the text is: = , genetically recombinant G_CSF or a gene mutation product having G_CSF gong b. 4. The conjugate of the first item or the pharmaceutically acceptable enthalpy thereof, wherein G is selected from the group consisting of 〇_咖5 as shown in the SEQ ID paper: the conjugate of claim i or item 3 Or a pharmaceutically acceptable salt thereof, characterized in that said G is a sequence of G-CSF.卞你97人恭 6. := The range of the first or third item of the bond or its * can be as good as, 'characterized by the G' is the sequence number SEQ ID No · 001 Met-G-CSF. 7. The conjugate of item i or item 3 of the patent, or a pharmaceutically acceptable salt thereof, characterized in that the conjugate comprises: 94293 21 200944236 彳0 0 -N J. 0 〇I }/~~G-CSF is an integer selected from 400 to 500; and 〇 m is an integer selected from 400 to 500. ❹8. 9. ❹ 10 If you apply for a patent scope! The conjugate of item 3 or a pharmaceutically acceptable salt thereof is characterized in that the conjugate and the acid are salt-formed, and the acid used is selected from an organic acid or a mineral acid. The conjugate of claim 8 or a pharmaceutically acceptable salt thereof is characterized in that the organic acid is selected from the group consisting of acetic acid, helium acetic acid, propionic acid, butyric acid, maleic acid or p-toluene. Or a mixture thereof, preferably acetic acid, tri-I acetic acid; the inorganic acid being selected from the group consisting of hydrochloric acid, sulfuric acid, phytic acid or formamidine or a mixture thereof, preferably hydrochloric acid. A method for preparing a conjugate of the first or third aspect of the patent range 1) a compound of the formula (ΙΠ) and a reductive amination of G, a hydrazone amino group to give a compound of the formula (IV): R2 (III) 2) Deprotecting the compound of formula (IV) to give a protecting group (v): 94293 22 200944236 R2 (V) Kelga Chenghua (IV) ) The compound (V) is synthesized with mPEG-MAL (11): Wherein R, Ri, R, π, and γ 1 ® are as defined in Patent Application No. 3; triphenylf- or tert-butyl, preferably ethylidene. Propionyl, patent Scope 1 or conjugate or its autistically acceptable salt in the preparation of treatment due to radiation:: schizophrenia, AIDS and other immunodeficiency diseases, white blood drugs Applications. A pharmaceutical composition comprising a conjugate of the susceptibility disease, which comprises a conjugate as described in the pharmaceutical laboratory or a pharmaceutically acceptable carrier thereof. Further salt, and Yue 94293 23 200944236 13. The pharmaceutical composition according to claim 12, in the preparation of a medicament for treating leukopenia, AIDS and other immunodeficiency diseases, bacterial infection diseases caused by radiotherapy or chemotherapy application. ❹ 24 94293 200944236 ❹ ❹ Order &lt;110&gt; Jiangsu Hengrui Pharmaceutical Co., Ltd. &lt;120&gt; Water-soluble polymer modified G-CSF &lt;130> &lt;160&gt; 10 &lt;170&gt; PatentIn version 3.3 &lt;210&gt ; 1 &lt;211&gt; 175 &lt;212> PRT &lt;213&gt; Unknown &lt;220&gt; MOD-RES &lt;400&gt; 1 Met Thr Pro Leu Gly Pro Ala Ser 1 5 Lys Cys Leu Glu Gin Val Arg 20 Lys Gin Glu Lys Leu Cys Ala Thr Tyr 35 40 Val Leu Leu Gly His Ser Leu Gly 50 55 Cys Pro Ser Gin Ala Leu Gin Leu 65 70 Ser Gly Leu Phe Leu Tyr Gin 85 Gly Ser Pro Glu Leu Gly Pro Thr 100 Leu Asp Phe Ala Thr Thr lie Trp Gin 115 120 Pro Ala Leu Gin Pro Thr Gin Gly 130 135 Phe Gin Arg Arg Ala Gly Gly Val 145 150 Phe Leu Glu Val Ser Tyr Arg 165 Val &lt;210〉 2 25 105 10 15 90 170 45 30 60 75 80 125 110 140 155 95 160 175 94293 200944236 Summary &lt;211&gt; 174 &lt;212&gt; PRT &lt;213> Unknown* &lt;220> MOD-RES &lt;400〉 2 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Se r Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu .Gin 20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 ❹ Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser 85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 100 105 110 Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala Leu Gin Pro* Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser Hi s Leu Gin Ser Phe 145 150 155 160 ❹ Leu Glu Val Ser Tyr Arg Val Leu Arg'His Leu Ala Gin Pro 165-170 <210> 3 &lt;211&gt; 175 &lt;212> PRT &lt;213> Unknown &lt;220> MOD-RES &lt;400&gt; 3 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 1 5 10 15 Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu 20 25 30 2 94293 200944236 ❹ Gin Glu Lys Leu Cy s Ala Thr Tyr Lys Leu Cys His Pro Glu 35 40 45 Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu 50 55 60 Cy s Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin 65 70 75 Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu 85 90 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp 100 105 110 Asp Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly 115 120 125 Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala 130 135 140 Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 145 150 155 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin 165 170 &lt;210&gt; 4 <211> 174 <212> PRT <213> Unknown &lt;220> 1 MOD-RES <400> 4 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu 1 5 10 Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala 20 25 30 Glu Lys Leu Cy s Ala Thr Tyr Lys Leu Cys His Pro Glu Glu 35 40 45 Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser 50 55 60 Pro Ser Gin Ala Leu Gin Leu. Ala Gly Cys Leu Ser Gin Leu 65 70 75 Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly 85 90 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val 95 175 15 95 3 Leu Ser His 80 lie Ala Ala Ala Ser 160 Lys Gin Val. Cy s Ser 80 Ser Asp 94293 200944236 m 100 105 110 Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Λ —Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe . 130 135 140 Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 165 170 &lt;21G&gt; 5 &lt;211> 175 &lt;212> PRT &lt;213&gt; Unknown <220 MOD-RES &lt;400〉 5 Called Ser Leu Pro 10 lie Gin Gly 25 Lys Leu Cys Gin Ser Phe Leu Leu 15 Asp Gly Ala Ala Leu 30 His Pro Glu Glu Leu 45 Ala Pro Leu Ser Ser 60 Leu Ser Gin Leu His 80 Ala Leu Glu Gly lie 95 Gin Leu Asp Val Ala 110 Glu Leu Gly Met Ala 125 Ala Phe Ala Ser Ala 140 Ser His Leu Gin Ser 160 Leu Ala Gin Pro 175 Trp Thr Pro Leu Gly Pro Ala Ser 1 5 Lys Cys Leu Glu Gin Val Arg Lys 20 Gin Glu Lys Leu Cys Ala Thr Tyr 35 40 Val Leu Leu Gly His Ser Leu Gly lie Pro Trp 50 55 Ala Gly Cys 75 Leu Leu Gin 90 Asp Thr Leu 105 Gin Met Glu Cys Pro Ser Gin Ala Leu Gin Leu ❹ 65 70 Ser Gly Leu Phe Leu Tyr Gin Gly 85 Ser Pro Glu Leu Gly Pro Thr Leu 100 Asp Phe Ala Thr Thr lie Trp Gin 115 120 Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro 130 135 Leu Val Ala 155 Leu Arg His 170 Phe Gin Arg Arg Ala Gly Gly Val 145 150 Phe Leu Glu Val Ser Tyr Arg Val 165 4 94293 200944236 &lt;210&gt; 6 ^ &lt;211&gt; 175 ' &lt;212> PRT _ &lt;213>Unknown # 〈220 〉 MOD-RES &lt;400&gt; 6 Tr p Thr Pro Leu 1 Lys Ser Leu Glu 20 Gin Glu Lys Leu 35 e Val Leu Leu Gly 50 Cys Pro Ser Gin 65 Ser Gly Leu Phe Ser Pro Glu Leu 100 Asp Phe Ala Thr 115 Pro Ala Leu Gin 130 ❹ Phe Gin Arg Arg 145 Phe Leu Glu Val Gly Pro Ala Ser 5 Gin Val Arg Lys Cys Ala Thr Tyr 40 His Ser Leu Gly 55 Ala Leu Gin Leu 70 Leu Tyr Gin Gly 85 Gly Pro Thr Leu Thr lie Trp Gin 120 Pro Thr Gin Gly 135 Ala Gly Gly Val 150 Ser Tyr Arg Val 165 Ser Leu Pro Gin 10 lie Gin Gly Asp 25 Lys Leu Cys His lie Pro Trp Ala 60 Ala Gly Cys Leu 75 Leu Leu Gin Ala 90 Asp Thr Leu Gin 105 Gin Met'Glu Glu Ala Met Pro Ala 140 Leu Val Ala Ser 155 Ser Phe Leu Leu 15 Gly Ala Ala Leu 30 Pro Glu Glu Leu 45 Pro Leu Ser Ser Ser Gin Leu His 80 Leu Glu Gly lie 95 Leu Asp Val Ala 110 Leu Gly Met Ala 125 Phe Ala Ser Ala His Leu Gin Ser 160 Leu Arg His Leu Ala Gin Pro 170 175 &lt;210> 7 <211> 175 <212> PRT &lt;213&gt; Unknown &lt;220> MOD-RES &lt;400&gt; 7 5 94293 200944236 Asp 1 Ly s Gin Val Cy s 65 Ser Ser Asp Pro Phe 145 Phe Thr Pro Cys Leu Glu Lys 35 Leu Leu 50 Pro Ser Gly Leu Pro Glu Phe Ala 115 Ala Leu 130 Gin Arg Leu Glu Leu Gly Pro 5 Glu Gin Val 20 Leu Cys Ala Gly His Ser Gin Ala Leu 70 Phe Leu Tyr 85 Leu Gly Pro 100 Thr Thr lie Gin Pro Thr Arg Ala Gly 150 Val Ser Tyr 165 Ala Ser Arg Lys Thr Tyr 40 Leu Gly 55 Gin Leu Gin Gly Thr Leu Trp Gin 120 Gin Gly 135 Gly Val Arg Val Ser Leu Pro 10 lie Gin Gly 25 Lys Leu Cys lie Pro Trp Ala Gly Cys 75 Leu Leu Gin 90 Asp Thr Leu 105 Gin Met Glu Ala Met Pro Leu Val Ala 155 Leu Arg His 170 Gin Ser Asp Gly His Pro 45 Ala Pro 60 Leu Ser Ala Leu Gin Leu Glu Leu 125 Ala Phe 140 Ser His Leu Ala Phe Leu Leu 15 Ala Ala Leu 30 Glu Glu Leu Leu Ser Ser Gin Leu His 80 Glu Gly lie 95 Asp Val Ala 110 Gly Met Ala Ala Ser Ala Leu Gin Ser 160 Gin Pro 175 ❹ <210> 8 <211> 175 &lt;212> PRT &lt;213> Unknown &lt;220> MOD-RES &lt;400&gt; 8 Asp Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 1 5 10 15 Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu 20 25 30 Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 6 94293 200944236 Λ 65 70 Ser G1y Leu Phe ' Ser Pro G.lu Leu 100 Asp Phe Ala Thr 115 Pro Ala Leu Gin 130 Phe Gin Arg Arg 145 Phe Leu Glu Val ❹ <210> 9 &lt;211&gt; 175 &lt;212&gt; PRT &lt;213> Unknown &lt;220> MOD-RES &lt;400&gt; 9 Glu Thr Pro Leu 1 Lys Cys Leu Glu 20 Gin Glu Lys Leu ❹ 35 Val Leu Leu Gly 50 Cys Pro Ser Gin 65 Ser Gly Leu Phe Ser Pro Glu Leu 100 Asp Phe Ala Thr 115 · Pro Ala Leu Gin 130 Leu Tyr Gin Gly Leu 85 Gly Pro Thr Leu Asp 105 Thr lie Trp Gin Gin 120 Pro Thr Gin Gly Ala 135 Ala Gly Gly Val Leu 150 Ser Tyr Arg Val Leu 165 Gly Pro Ala Ser Ser 5 Gin Val Arg Lys lie 25 Cys Ala Thr Tyr Lys 40 His Ser Leu Gly lie 55 Ala Leu Gin Leu Ala 70 Leu Tyr Gin Gly L Eu 85 Gly Pro Thr Leu Asp 105 Thr lie Trp Gin Gin 120 Pro Thr Gin Gly Ala 135 75 80 Leu Gin Ala Leu Glu Gly lie 90 95 Thr Leu Gin Leu Asp Val Ala 110 Met Glu Glu Leu Gly Met Ala 125 Met Pro Ala Phe Ala Ser Ala 140 Val Ala Ser His Leu Gin Ser 155 160 Arg His Leu Ala Gin Pro 170 175 Leu Pro Gin Ser Phe Leu Leu 10 15 Gin Gly Asp Gly Ala Ala Leu 30 Leu Cys His Pro Glu Glu Leu 45 Pro Trp Ala Pro Leu Ser Ser 60 Gly Cys Leu Ser Gin Leu His 75 80 Leu Gin Ala Leu Glu Gly lie 90 95 Thr Leu Gin Leu Asp Val Ala 110 Met Glu Glu Leu Gly Met Ala 125 Met Pro Ala Phe Ala Ser Ala 140 7 94293 200944236 Phe Gin Arg Arg 145 Phe Leu Glu Val Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser 150 155 160 Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 165 170 175 <210> 10 <211> 175 &lt;212&gt; PRT <213> Unknown &lt;220> MOD-RES &lt;400&gt; 10 Glu Thr Pro Leu 1 p Lys Ser Leu Glu 20 Gin Glu Lys Leu 35 Val Leu Leu Gly 50 Cys Pro Ser Gin 65 Ser Gly Leu Phe Ser Pro Glu Leu 100 o Asp Phe Ala Thr 115 Pro Ala Leu Gin 130 Phe Gin Arg Arg 145 Phe Leu Glu Val Gly Pro Ala Ser Ser Leu 5 10 Gin Val Arg Lys lie Gin 25 Cys Ala Thr Tyr Lys Leu 40 His Ser Leu Gly lie Pro 55 Ala Leu Gin Leu Ala Gly 70 Leu Tyr Gin Gly Leu Leu 85 90 Gly Pro Thr Leu Asp Thr 105 Thr He Trp Gin Gin Met 120 - Pro Thr Gin Gly Ala Met 135 Ala Gly Gly Val Leu Val 150 Ser Tyr Arg Val Leu Arg 165 170 Pro Gin Ser Phe Leu Leu 15 Gly Asp Gly Ala Ala Leu 30 Cys His Pro Glu Glu Leu 45 Trp Ala Pro Leu Ser Ser 60 Cys Leu Ser Gin Leu His 75 80 Gin Ala Leu Glu Gly lie 95 Leu Gin Leu Asp Val Ala 110 Glu Glu Leu Gly Met Ala 125 Pro Ala. Phe Ala Ser Ala 140 Ala Ser His Leu Gin Ser 155 160 His Leu Ala Gin Pro 175 8 94293