WO2009079837A1 - Formulation pharmaceutique contenant une protéine de fusion sérum albumine humaine recombinante-interféron alpha - Google Patents

Formulation pharmaceutique contenant une protéine de fusion sérum albumine humaine recombinante-interféron alpha Download PDF

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Publication number
WO2009079837A1
WO2009079837A1 PCT/CN2007/003666 CN2007003666W WO2009079837A1 WO 2009079837 A1 WO2009079837 A1 WO 2009079837A1 CN 2007003666 W CN2007003666 W CN 2007003666W WO 2009079837 A1 WO2009079837 A1 WO 2009079837A1
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Prior art keywords
interferon
fusion protein
concentration
buffer
pharmaceutical preparation
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PCT/CN2007/003666
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English (en)
French (fr)
Inventor
Yanshan Huang
Guochang Ma
Tongying Wang
Feihu Xu
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Hangzhou Jiuyuan Gene Engineering Co., Ltd.
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Priority to PCT/CN2007/003666 priority Critical patent/WO2009079837A1/zh
Priority to US12/809,298 priority patent/US20100297081A1/en
Publication of WO2009079837A1 publication Critical patent/WO2009079837A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/21Interferons [IFN]
    • A61K38/212IFN-alpha
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38Albumins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02Inorganic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183Amino acids, e.g. glycine, EDTA or aspartame
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/08Solutions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/20Antivirals for DNA viruses
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Definitions

  • the present invention relates to a recombinant human serum albumin-interferon 0 fusion protein
  • the pharmaceutical preparation of (rHSA-IFN) is suitable for subcutaneous or intravenous administration as an immunomodulator for the treatment of viral infectious diseases, tumors and related diseases.
  • Interferon ct Interferon a, IFN
  • IFN Interferon a
  • IFN ot When IFN ot is used for hepatitis treatment, it is usually once a day or twice a week, but in most treatment time, the IFN ot concentration in the patient is lower than the effective concentration, and on the other hand, after administration, blood medicine When the concentration reaches a peak, the drug concentration is much higher than the effective concentration, which causes a significant side reaction.
  • the PEGi decoration method is now widely used, and IFN ot 2a decorated with PEG ⁇ has been around 40kD.
  • Human serum albumin (HS A) is a major component in human serum and plays a crucial role in maintaining osmotic pressure and plasma volume in the body.
  • Human serum albumin is a non-glycosylated protein with a molecular weight of 6615 kD.
  • the renal clearance rate is very low and the half-life in vivo is 14-20 days. It is also a natural carrier of factor and drug transport in the body.
  • Studies have shown that fusion proteins expressed by fusion of a therapeutic protein gene with an albumin gene can significantly reduce the rate of drug clearance in vivo and prolong the biological half-life.
  • HSA-CD4 fusion protein expressed by Kluyveromyces in the rabbit model was 140-fold longer than that of CD4 alone.
  • the half-life of the fusion protein of HSA-IFNa expressed by Kluyveromyces in the macaque is about 18 times longer than that of IFN a alone.
  • the human serum albumin gene is fused with the human interferon alpha gene, and the corresponding recombinant protein can be obtained by selecting a suitable recombinant expression method.
  • the C-terminus of human serum albumin can be linked to the end of human interferon alpha directly or via a flexible linker peptide sequence, or the C-terminus of human interferon alpha either directly or through a flexible linker sequence.
  • n is an integer from 1 to 10
  • n is an integer from 1 to 3
  • most preferably n is 1.
  • the interferon alpha is interferon oc 2a, interferon lb, interferon a 2b or interferon a con, preferably interferon a 2b.
  • the recombinant human serum albumin-interferon oc fusion protein formed after fusion overcomes the shortcomings of multiple administrations in the traditional interferon treatment process; and has the following advantages: 1) It can stimulate the body's immune response to each infection of the disease 2) prolong the retention time of interferon in the body; 3) maximize the therapeutic effect, reduce the potential side effects or toxicity of traditional interferon, and improve the efficacy.
  • rHSA-IFN o is not stable compared to conventional chemicals (Panayotatos; Nikos, 1998, US 5846935), and its activity is still affected by various environmental factors during long-term storage. For example, it is highly sensitive to temperature, oxygen and ultraviolet light.
  • It is an object of the present invention to provide a pharmaceutical preparation comprising a recombinant AJ6L albumin-interferon fusion protein capable of stably storing a recombinant jk albumin-interferon alpha fusion protein (rHSA-IFNoc) and suitable for practical clinical use.
  • a pharmaceutical preparation comprising a recombinant AJ6L albumin-interferon fusion protein capable of stably storing a recombinant jk albumin-interferon alpha fusion protein (rHSA-IFNoc) and suitable for practical clinical use.
  • the pharmaceutical preparation of the present invention contains, in addition to the recombinant human serum albumin-interferon alpha fusion protein (rHSA-IFN ot) as an active ingredient, a pharmaceutically acceptable maintenance system.
  • the pH of the agent in the aqueous solution is 5. 0-8. 0 buffer and a pharmaceutically acceptable excipient for enhancing the stability of the rHSA-IFN protein.
  • the invention has the advantages that the physicochemical and biological activity of the rHSA-IFN ct protein is stabilized by adding some components which can be accepted by the human body, thereby preparing a preparation suitable for clinical use, especially for injection. This preparation can prevent the active ingredient (rHSA-IFN a protein) from being damaged due to adsorption of the container or due to various factors such as degradation, oxidation, etc., thereby facilitating transportation, long-term preservation and clinical use.
  • the fusion protein can be obtained by fusing the albumin gene with the human interferon alpha gene and selecting a suitable recombinant expression method.
  • the C-terminus of AJ6L albumin can be linked to the N-terminus of human interferon alpha either directly or via a flexible linker sequence, or the C-terminus of human interferon alpha either directly or through a flexible linker sequence.
  • the terminal of human serum albumin is ligated, and a type of flexible linker peptide which can be selected has the formula [GlyGlyGlyGlySer]n, n is an integer from 1 to 10, preferably n is an integer from 1 to 3, and most preferably n is 1 .
  • the interferon ⁇ is interferon a 2a, interferon a lb, interferon o 2b or interferon a eon, preferably interferon ot 2b.
  • the concentration of the recombinant human serum albumin-interferon ot fusion protein is 0.1 to 5 mg/mL, preferably 0.5 to 2 mg/mL.
  • the stability adjuvant may be added as needed, such as an amino acid and a saccharide.
  • the preferred stability adjuvant is glycine or methionine, and the mass concentration (auxiliary shield volume/solution volume, w/v) is 1-4%; preferably the glycine mass concentration is 1-4 More preferably, the glycine mass concentration is 2.3%.
  • the buffer suitable for use in the present invention is any buffer capable of maintaining the pH of the preparation in an aqueous solution at a pH of 5.0 to 8.0, and may be selected from the group consisting of disodium hydrogen phosphate-citrate buffer, phosphate.
  • Buffer Tris-HCl buffer, Triacetate-Acetate buffer, Citrate buffer, Barbiturate buffer or Succinate buffer
  • the concentration is 5-10 Ommol/L, preferably 5-30 mmol/L, most preferably 10 mmol/L;
  • the buffer has a pH in the range of 5.0-8.0, preferably 6.0-7.0, most preferably pH 6.5.
  • the buffer is preferably a rock salt buffer, the concentration is 5-100 mmol/L, preferably 5-30 mmol/L, and the most preferred concentration is 10 mmol/L;
  • the pH of the solution is between 5.0-8.0, preferably
  • the pharmaceutical preparation is obtained by dissolving the recombinant Ajk albumin-interferon ot2b fusion protein and glycine in a concentration of 5 to 100 mmol/L phosphate buffer having a pH of 5.0 to 8.0, and the recombinant albumin-interferon ct2b
  • the fusion protein is a fusion protein in which jk albumin is directly fused with an interferon or a fusion peptide of the formula [GlyGlyGlyGlySer] n is used as a linker peptide, n is an integer of 1-10, a fusion protein concentration is 0.1 to 5 mg/mL, and the glycine is a glycine.
  • the mass concentration is 1 ⁇ 4%.
  • the above pharmaceutical preparation is obtained by dissolving a recombinant human serum albumin-interferon 2b fusion protein and glycine in a concentration of 5 to 30 mmol of phosphate buffer at a pH of 6.0 to 7.0, and the recombinant Ajk albumin-interference A 2b
  • a fusion protein obtained by direct fusion with interferon or fusion with a peptide of the formula [GlyGlyGlyGlySer] n , wherein n is an integer of 1-3, the fusion protein concentration is 0.5-2 mg/mL, and the glycine concentration is 1 to 4 %.
  • the above pharmaceutical preparation is obtained by dissolving the recombinant AJ6L albumin-interferon ot2b fusion protein and glycine in a concentration of 10 mmol/L phosphate buffer at a pH of 6.5.
  • the recombinant human serum albumin-interferon cc 2b fusion protein is a fusion protein obtained by directly fusing human serum albumin with an interferon or a fusion peptide of the formula [GlyGlyGlyGlySer], and the fusion protein concentration is 0.5 mg/mL.
  • the amount of glycine was: 2.3%.
  • the above pharmaceutical preparation can also be prepared as a lyophilized powder, and the liquid preparation before lyophilization is substantially isotonic or isotonic, so that an appropriate amount of water for injection can be reduced to form an isotonic layer after lyophilization. Or isotonic solution.
  • the present invention also discloses the use of an aqueous injection containing a recombinant human serum albumin-interferon o fusion protein for the preparation of a medicament for the treatment of viral hepatitis such as hepatitis B and hepatitis C.
  • the duck hepatitis B model experiment proved that the recombinant albumin-interferon ⁇ fusion protein has good anti-hepatitis B virus effect.
  • Figure 1 shows the sample in a different pH buffer, at 40. Under C conditions, the electropherogram obtained by SDS-PAGE was sampled for two weeks (a) and four weeks (b);
  • Figure 2 is an electropherogram obtained by sampling SDS-PAGE at different concentrations of samples at 40 °C for two weeks (a) and four weeks (b);
  • Figure 3 shows the electropherograms obtained by sampling SDS-PAGE after storage for one year at different concentrations of samples at 4 °C.
  • rHSA-IFN ct-containing agents In order to improve the stability of rHSA-IFN ct-containing agents, intensive studies have been conducted and it has been found that at least one of the appropriate excipients (such as sugars, amino acids and their derivatives, surfactants, etc.) and inorganic salts are selected. This can be effectively achieved by selecting the appropriate pH and rHSA-IFN o protein concentration in the formulation.
  • the clarity test, SDS-PAGE electrophoresis, protein concentration, reversed-phase high performance liquid chromatography (HPLC), and biological activity assay were used as indicators to observe the rHSA-IFN ct agent. Changes. These methods are available from the Chinese Pharmacopoeia 2005 Edition.
  • the rHSA-IFN a 2b described in the following examples refers to a fusion protein prepared by the method disclosed in Chinese Patent No. 01124110.1, in which human serum albumin and interferon a 2b contain a linker peptide of GlyGlyGlyGlySer. Since the linker peptide is very short, the effect on the properties of the fusion protein is very small.
  • the following test results are naturally applicable to the fusion protein produced by direct fusion of human serum albumin and interferon. ⁇
  • Example 1 Effect of pH on the stability of rHSA-IFN a 2b
  • pH is an important factor affecting the stability of protein preparations in injectable pharmaceutical preparations.
  • the pH of the formulation is maintained by i ⁇ p into a suitable buffer salt.
  • a suitable buffer salt such as the addition of calate, acetate, citrate, barbiturate, trishydroxymethylaminodecane, borate, succinate and the like.
  • Example 2 Effect of adding different preparation excipients on the stability of rHSA-IFN a2b
  • Excipients suitable for protein preparation applications include albumin, sugars, acids, surfactants, metal chelators and the like.
  • the present invention selects some excipients suitable for human body use for screening.
  • Human serum albumin is a potential source of blood contamination, and our preparations contain albumin, so they are not considered.
  • the sugars suitable for use in the present invention may be selected from a variety of monosaccharides, oligosaccharides and polysaccharides, and daunting and nucleoside derivatives.
  • monosaccharides oligosaccharides and polysaccharides
  • oligosaccharides and polysaccharides and mundane and nucleoside derivatives.
  • glycerin, mannitol, sucrose, and the like may be added singly or in combination.
  • Polypeptides, amino acids and derivatives suitable for use in the present invention can be selected from the group consisting of glycine, alanine, serine, aspartic acid, glutamic acid, threonine, tryptophan, and lysine. Amino acid, hydroxylysine, histidine, arginine, cystine, cysteine, purine, phenylalanine, leucine, isoleucine, etc., and derivatives thereof.
  • Table 1 The formulation of the excipients screened by the present invention is shown in Table 1 (the percentage concentration in the table is the mass percentage concentration).
  • Tables 2 and 3 show that the samples were stored at 4 ° C and 40 ° C for 4 months without any defects. Turbidity occurs. Table 2: The sample is stored at 4 ° C for 4 months, and the clarity of the sample is tested every other month.
  • Table 3 The sample is stored at 40 °C for 4 months, and the clarity of the sample is tested every other month. T-0 month T-1 month T-2 month T-3 month T-4 month
  • Table 5 The sample is at 40. Stored under C conditions for 4 months, every other month by SDS-PAGE
  • the sample containing glycine or methionine is the most stable, so only the sample containing glycine or methionine is evaluated for potency.
  • Samples were stored at 4 °C and 40 °C for 4 months and samples were taken for biological activity testing.
  • the results (Table 7) show that the potency of all formulations is within the specified range, indicating that the chemical and physical degradation processes did not significantly alter the potency activity of the protein, with the effect of glycine being better, and different concentrations of glycine at The effect of the preparation on the biological activity of the protein is almost the same.
  • Protein concentration in injectable pharmaceutical preparations is also an important factor in the stability of protein preparations. Factors. If the concentration is too low, not only the dosage is increased, but also the wall is easily adsorbed; if the protein concentration is too high, the polymer is easily formed.
  • the present invention carries out the effects of different protein concentration conditions on the stability of the product under the following conditions:
  • Detection method SDS-PAGE non-reduction electrophoresis method (10 ⁇ g electrophoresis analysis per sample) The results are shown in Fig. 2, Fig. 3.
  • the refrigerator was placed at 4 °C for 1 year, which was stable.
  • the accelerated test at 40 °C showed that the higher the protein concentration, the more likely the polymer would be, which may affect its physical and chemical properties.
  • Biological activity From the viewpoint of stability of the protein and ease of use of the preparation, it is considered that the concentration of the rHSA-IFN a 2b protein is preferably between 0.5 and 2 mg/ml.
  • composition of the agent is desirable: 0.1-5 mg/ml of rHSA-IFN a 2b protein, preferably 0.5-2 mg/ml; a suitable concentration of glycine or methionine, preferably The most preferred concentration is 1-4% glycine, 2.3%.
  • the buffer selected is phosphate buffer, Tris-HCl buffer, acetic acid-sodium acetate buffer, preferably phosphate buffer at a concentration of 5-100 mmol/L, and the most preferred concentration is 10 mmol/L.
  • the osmotic pressure is between 250 and 500 mOsm and the pH is between 5 and 8, most preferably 6.5.
  • Example 4 Preparation of an aqueous injection for injection containing recombinant jk albumin-interferon oc 2b fusion protein
  • the final formulation consisted of: recombinant human serum albumin-interferon o 2b fusion protein concentration 0.5 mg / ml, phosphate buffer concentration 10 mmol / L, pH 6.5, glycine content 2.3% (weight ratio).
  • Example 5 Investigation of the stability of water for injection containing recombinant AJ6L albumin-interferon o 2b fusion protein
  • 1 contains 0.5 mg/ml recombinant Ajfe albumin-interferon o 2b fusion protein; 10 mM Na 2 HP0 4 -NaH 2 P0 4 pH 6.5 buffer; 2.3% glycine injection.
  • the solutions prepared according to the above four preferred prescriptions were stored at 4 ° C and 40 ° C for 4 months, respectively. Samples were taken for RP-HPLC every month, and samples were taken after 4 months. Learning activity detection.
  • Table 9 Biological activity testing of samples after storage at 4 ° C and 40 ° C for 4 months
  • the rHSA-IFNa2b protein drug formulation disclosed in the present invention has good stability.
  • Example 6 Injectable water containing recombinant human serum albumin-interferon a 2b fusion protein for the treatment of hepatitis B
  • DHBV duck hepatitis B virus
  • PCR reaction mixture lOxPCR buffer 5 ⁇ 1, 2.5mM MgCl 2 3 ⁇ 1, 2mM dNTP 5 ⁇ 1, 20pmol of upstream and downstream primers, Taq DNA Polymerase 1. 25u plus water to a total volume of 45 ⁇ 1, mixed with the above pre-denatured template 5 ⁇ 1, add a drop of paraffin oil, and put on the machine.
  • the PCR program was predenatured at 95 °C for 2 minutes, denatured at 94 °C for 30 seconds, annealed at 56 °C for 30 seconds, and extended at 72 °C for 45 seconds. A total of 30 cycles, the last 72 ° C extension for 10 minutes.
  • the negative control contained all of the components required for RT-PCR, but no template was added.
  • the PCR results were confirmed by gel electrophoresis: the PCR reaction product was taken from ⁇ and the loading buffer 2 ⁇ 1, and added to the 5% buffer (90 mM tr i s-boric acid; 2 mM EDTA pH 8. 0). Gel agarose in the comb hole. After 3 hours of 40V electrophoresis, the results were observed on a UV detector (wavelength was 300 nm). The PCR amplification results of the three Shaoxing ducks were all positive, and 3 positive bands appeared. No positive bands were found in the negative control. The serum of No. 1 Shaoxing Ma duck with the strongest strip was taken as positive serum.
  • the positive cherry valley ducks were randomly divided into five groups (60/group), and the preparation of the recombinant Ajk albumin-interferon ct 2b fusion protein was prepared by the method disclosed in Example 4, and the dosages were respectively small doses.
  • the treatment group consisted of 42 rats in each group. Treatment begins at 2 weeks after infection.
  • the recombinant human serum albumin-interferon a 2b fusion protein was administered subcutaneously in an aqueous solution for injection and a saline blank, once every other week.
  • the lamivudine group pulverized the tablets into powder form, opened them with cold water, and evenly mixed with the feed to feed the cherry valley ducks. Twice a day, morning and evening, each The patients were continuously administered for 3 months, and continued to observe for 3 months after stopping the drug.
  • the PCR program was predenatured at 95 ° C for 2 minutes, denatured at 94 ° C for 30 seconds, annealed at 56 ° C for 30 seconds, and extended at 72 ° C for 45 seconds. A total of 30 cycles, the last 72 ° C extension for 10 minutes.
  • the positive serum of Shaoxing Ma duck was used as the positive control. There were 5 positive controls in each reaction. The negative control contained all the components required for RT-PCR, but no template was added.
  • reaction products were each mixed with ⁇ and 2 ⁇ l of the loading buffer, and added to a 1.5% gel agarose comb well immersed in ⁇ ⁇ buffer (90 mM tr i s-boric acid; 2 mM EDTA pH 8.0). After 3 hours of 40V electrophoresis, semi-quantitative analysis was performed by a gel imaging analysis system, and the optical density scan values of each strip were obtained for statistical analysis. The mean of the five positive controls was averaged between each image.
  • Duck hepatitis B model Zheng Zhengming recombinant human serum albumin-interferon a 2b fusion protein injection has a good anti-hepatitis B virus efficacy.

Description

含有重 a L清白蛋白-干扰素 a融合蛋白的药物制剂 技术领域
本发明涉及一种含有重组人血清白蛋白-干扰素 0融合蛋白
( rHSA-IFN ) 的药物制剂, 适用于皮下或静脉等途径给药, 作为免 疫调节剂用于治疗病毒感染性疾病, 肿瘤及相关疾病。 背景技术
干扰素 ct (interferon a , IFN )是临床上应用于抗病毒最为广泛的 药物, 可应用于治疗人的丙型肝炎、 乙型肝炎、 恶性肿瘤以及与艾滋 病相关的 Kaposi's 肉瘤等临床病症。 IFN cc能有效抑制乙型和丙型肝炎 病毒的复制和降低患者血浆转氨酶。 然而,作为一个小分子蛋白,IFN α从血浆中清除的速度较快,其清除相半衰期为注射后 3 ~ 8h , 24h后 在血浆中就已检测不到 IFN ct的存在,这对治疗是极为不利的。
IFN ot用于肝炎治疗时,通常为每天一次或每周二次注射,但是在 大部分治疗时间内, 患者体内 IFN ot浓度是低于有效浓度的, 而另一方 面,在给药后,血药浓度达到峰值时,其药物浓度又是远高于有效浓度, 这就会产生明显的副反应。 为了增加 IFN a在体内的半衰期, 现在广 泛采用的是 PEGi 饰法, 目前已有 40kD左右 PEG^饰的 IFN ot 2a
(Pegasys, Roche )和121^0左右 PEG修饰的 IFN a 2b ( PEG-Intron, Schering-Plough)应用于临床。 这两类产品均可明显延长 IFN ct在体内 的半衰期。 现有两种 ΡΕ(3 ^φ的干扰素都是修饰在蛋白分子的赖氨酸 残基上, IFN ot含有 10 ~ 1 1 个赖氨酸, 因此?5(3 饰可以形成不同的 异构体。 这种众多异构体混合物造成不同的生理反应。 虽然, 利用定 点基因突变的方法引入一个半胱氨酸 (Q5C) , 并在此半胱氨酸上进行 PEG修饰, 从而可以实现单价定位修饰。 但是, 这种突变的 IFN cc的安 全性和有效性需要进一步进行人体评价。
AJ6L清白蛋白( Human serum albumin, HS A)是人体血清中的主要 成分,对维持体内渗透压和血浆体积起着至关重要的作用。人血清白蛋 白是分子量为 6615kD 的非糖基化蛋白, 肾清除率非常低, 体内半衰期 为 14 ~ 20d。 它也是体内因子和药物转运的天然载体。 研究表明将治疗 性蛋白基因与 清白蛋白基因融合所表达的融合蛋白可明显降低体 内药物的清除速率, 延长生物半衰期。 Yeh等发现,克鲁维氏酵母表 达的 HSA-CD4 融合蛋白在以家兔为动物模型的实验中半衰期比单独 的 CD4延长了 140倍。而克鲁维氏酵母表达的 HSA- IFN a的融合蛋白 (albuferon)在猕猴体内的半衰期比单独的 IFN a延长了大约 18倍
(Blaire L.等, The Journal of Pharmacology And Experimental Therapeutics. 2002, 303: 540-548)。
rHSA-IFN cx的制备方法有许多文献报道 (吴军等: 寸国专利 01124110.1 ; Blaire L.等, The Journal of Pharmacology And Experimental Therapeutics. 2002, 303: 540-548; Fu Yan等: US20060051859 ), 通过将 人血清白蛋白的基因与人干扰素 α基因融合在一起, 选择合适重组表 达方法即可获得相应的融合蛋白。 该融合蛋白结构中, 人血清白蛋白 的 C末端可以直接或通过一柔性的连接肽序列与人干扰素 α的 Ν端相 连, 或者人干扰素 α的 C末端直接或通过一柔性的连接肽序列与人血 清白蛋白的 Ν 端相连, 可以选择的一类柔性连接肽序列通式为 [GlyGlyGlyGlySer]n, n为 1-10的整数, 优选的是 n为 1-3的整数, 最 优选 n为 1。 上述融合蛋白中, 所述的干扰素 α为干扰素 oc 2a、 干扰素 lb, 干扰素 a 2b或干扰素 a con, 优选干扰素 a 2b。
融合后形成的重组人血清白蛋白 -干扰素 oc融合蛋白克服了传统 的干扰素治疗过程中多次给药的缺点; 并具有以下几个优点: 1 )能刺 激机体对病每感染的免疫应答; 2 )延长干扰素在体内的存留时间; 3 ) 起最大的治疗作用、 减小传统干扰素的潜在副作用或毒性、 还可提高 疗效。 但是, 作为蛋白药物, rHSA-IFN o其稳定性还是无法与常规化学 药物相比( Panayotatos; Nikos, 1998, US 5846935 ), 其活性在长期贮 存时还是会受到多种环境因素的影响。 例如对温度, 氧和紫外线高度 敏感。 由于这些因子的作用, 可能发生多种物理或化学变化, 例如结 合, 聚合和氧化, 从而大大丧失活性。 因为如果贮存期间 rHSA-IFN oc 的稳定性不能保证的话, 会导致给药剂量的变化从而影响疗效。
因此, 研究出一种能稳定保存 rHSA-IFN ot蛋白, 并适合于实际临 床使用的药物制剂是极其有意义的。 但是, 到目前为止, 尚未有这方 面的研究报道。
发明内容
本发明的目的是提供一种能稳定保存重组 jk清白蛋白-干扰素 α 融合蛋白(rHSA-IFN oc ),并适合于实际临床使用的含有重组 AJ6L清白 蛋白-干扰素 融合蛋白的药物制剂。
本发明的药物制剂除了重组人血清白蛋白-干扰素 α融合蛋白 ( rHSA-IFN ot )作为有效成分外, 还含有药学上可接受的能够维持制 剂在水溶液状态下 pH值为 5. 0-8. 0 緩冲液及药学上可接受的增强 rHSA-IFN 蛋白稳定性的辅料。 本发明的优点在于通过添加一些能被 人体接受的成分, 增强了 rHSA-IFN ct蛋白的理化与生物学活性的稳 定, 从而制备出一种适合于临床使用, 特别是注射使用的制剂。 这种 制剂可以防止有效成分(rHSA-IFN a蛋白)由于容器吸附, 或因降解、 氧化等多种因素而导致失效, 从而方便运输、 长期保存和临床使用。
各类重组人血清白蛋白-干扰素 a融合蛋白的制备方法可参考相关 的文献(吴军等: 中国专利 01124110.1; Blaire L.等, The Journal of Pharmacology And Experimental Therapeutics. 2002, 303: 540-548; Fu Yan等: US20060051859 ),通过将 清白蛋白的基因与人干扰素 α基 因融合在一起, 选择合适重组表达方法即可获得相应的融合蛋白。 该 融合蛋白结构中, AJ6L清白蛋白的 C末端可以直接或通过一柔性的连 接肽序列与人干扰素 α的 N端相连, 或者人干扰素 α的 C末端直接或 通过一柔性的连接肽序列与人血清白蛋白的 Ν端相连, 可以选择的一 类柔性连接肽序列通式为 [GlyGlyGlyGlySer]n, n为 1-10的整数, 优选 的是 n为 1-3的整数, 最优选 n为 1。 上述融合蛋白中, 所述的干扰素 α为千扰素 a 2a、 干扰素 a lb、 干扰素 o 2b或干扰素 a eon, 优选干扰 素 ot 2b。
上述制剂中 , 所述重组人血清白蛋白-干扰素 ot融合蛋白浓度为 0.1 ~ 5mg/mL, 优选 0.5 ~ 2mg/mL。
上述制剂中, 所述稳定性辅料可根据需要酌情添加, 如氨基酸类 和糖类等。 本发明中优选稳定性辅料为甘氨酸或曱硫氨酸, 质量浓度 (辅料盾量 /溶液体积, w/v )为 1 - 4 %; 优选甘氨酸质量浓度为 1 ~ 4 %, 更优选甘氨酸质量浓度为 2.3 %。
上述制剂中, 适用于本发明的緩冲液为能够维持制剂在水溶液状 态下 pH值为 5.0-8.0的任何一种緩冲液, 可任选自磷酸氢二钠 -柠檬 酸緩沖液、 磷酸盐緩沖液、 三羟曱基氨基曱烷盐酸盐( Tris-HCl )緩冲 液、 醋酸-醋酸钠緩冲液、 柠檬酸緩沖液、 巴比妥盐緩沖液或琥珀酸盐 緩冲液,, 浓度为 5-10 Ommol/L, 优选 5-30mmol/L, 最优选 10mmol/L; 緩冲液的 pH范围在 5.0-8.0, 优选 6.0-7.0, 最优选 pH为 6.5。 其中, 緩冲液优选磯酸盐緩冲液, 浓度为 5- 100mmol/L, 优选 5- 30mmol/L, 最优选的浓度为 10mmol/L; 溶液的 pH 范围在 5.0-8.0之间, 优选
6.0-7.0, 最优选 pH为 6.5。
优选的,上述药物制剂由重组 Ajk清白蛋白-干扰素 ot2b融合蛋白 与甘氨酸溶于 pH为 5.0 ~ 8.0的浓度为 5 ~ 100mmol/L磷酸盐緩沖液中 得到,所述重组 清白蛋白-干扰素 ct2b融合蛋白为 jk清白蛋白与 干扰素直接融合或以通式为 [GlyGlyGlyGlySer]n为连接肽融合得到的 融合蛋白, n为 1-10的整数, 融合蛋白浓度为 0.1~5mg/mL, 所述甘 氨酸质量浓度为 1~4%。
更优选的,上述药物制剂由重组人血清白蛋白-干扰素 2b融合蛋 白与甘氨酸溶于 pH为 6.0 ~ 7.0的浓度为 5 ~ 30mmolL磷酸盐緩冲液 中得到,所述重组 Ajk清白蛋白-干扰素 a 2b
Figure imgf000006_0001
与干扰素直接融合或以通式为 [GlyGlyGlyGlySer] n为连接肽融合得到 的融合蛋白, n为 1-3的整数, 融合蛋白浓度为 0.5~2mg/mL, 所述甘 氨酸质量浓度为 1~4%。
更优选的,上述药物制剂由重组 AJ6L清白蛋白-干扰素 ot2b融合蛋 白与甘氨酸溶于 pH为 6.5的浓度 为 10mmol/L磷酸盐緩沖液中得到, 所述重组人血清白蛋白 -干扰素 cc 2b 融合蛋白为人血清白蛋白与干扰 素直接融合或以通式为 [GlyGlyGlyGlySer]为连接肽融合得到的融合蛋 白, 融合蛋白浓度为 0.5mg/mL, 所述甘氨^:量浓度为 2.3%。
才艮据需要, 还可将上述的药物制剂制备为冻干粉针, 冻干前的液 体制剂基本上为等张和或等渗的, 因此冻干后加入适量的注射用水能 够还原形成等张或等渗溶液。 本发明还公开了含有重组人血清白蛋白 -干扰素 o融合蛋白的注 射用水剂在制备用于治疗,乙型肝炎、 丙型肝炎等病毒性肝炎的药物中 的应用。 鸭乙肝模型实验证明重组 清白蛋白-干扰素 α融合蛋白的 注射用水剂具有良好的抗乙肝病毒疗效。 附图说明
图 1为样品在不同 ρΗ緩沖液中, 在 40。C条件下, 存放二周 (a ) 、 四周 (b ) 时取样进行 SDS-PAGE检测而得到的电泳图;
图 2为不同浓度的样品, 在 40°C条件下, 存放二周(a )、 四周(b ) 时取样进行 SDS-PAGE检测而得到的电泳图;
图 3 为不同浓度的样品, 在 4°C条件下, 存放一年后取样进行 SDS-PAGE检测而得到的电泳图。
实施方式
为了提高含有 rHSA-IFN ct药剂的稳定性,进行了深入的研究并且 发现如果从合适的辅料(如糖类, 氨基酸及其衍生物、 表面活性剂等) 和无机盐类中挑选至少一种加入制剂中, 并通过选择合适的 pH及 rHSA-IFN o蛋白浓度, 则可以有效达到这个目的。 在 rHSA-IFN 药剂稳定性研究过程中, 选取了澄清度检测, SDS-PAGE 电泳检测, 蛋白浓度, 反相高效液相色谱分析, 生物活性 检测等作为考察指标, 来观察 rHSA-IFN ct药剂的变化情况。 这些方法 均可从《中国药典 2005年版》获得。 下面结合具体实施例对本发明进行进一步描述。 下述实施例中所 述的 rHSA-IFN a 2b指按中国专利 01124110.1公开的方法制备的、 人 血清白蛋白和干扰素 a 2b中间含有连接肽为 GlyGlyGlyGlySer的融合 蛋白。 由于连接肽很短, 对于融合蛋白的性质影响甚小, 对本领域技 术人员来说, 下述试验结果也自然适用于人血清白蛋白和干扰素直接 融合产生的融合蛋白。〜
实施例 1: pH对 rHSA-IFN a 2b稳定性的影响
可注射药物制剂中 pH是影响蛋白制品稳定性的一个重要因素。通 i± p入合适的緩沖盐类, 来维持制剂 pH。 如加入磚酸盐、 醋酸盐、 柠 檬酸盐、 巴比妥盐、 三羟甲基氨基曱烷、 硼酸盐、 琥珀酸盐等。 为了 考察不同 pH条件下制剂的稳定性, 按如下条件进行了不同 pH条件对 制剂稳定性的影响实验:
实验条件: 1ml/瓶 (蛋白浓度为 lmg/ml )各组中含有不同 pH的 緩冲液
① pH=4.0 (醋酸 -醋酸钠緩冲液), 10mmol/L
② pH=5.0 (醋酸 -醋酸钠緩冲液), 10mmol/L
③ pH=6.5 (磷酸緩沖液), 5mmol/L
④ pH=6.5 (磷酸緩冲液), 10mmol/L
(5)pH=6.5 (磷酸緩沖液), 100mmol/L
⑥ pH=7.5 (磷酸緩冲液), 10mmol/L ⑦ pH=8.0 ( Tris-HCl緩冲液), lOmmol/L
避光放置于 40 °C的恒温箱中, 共进行四周, 每隔两周取样分析。 检测方法: SDS-PAGE非还原电泳方法
结果见图 1。
4(TC的加速试验表明, 低 pH可以抑制聚体的形成, 但会加速降解 的产生; 高 pH会加速聚体的形成, 也不能减少降解蛋白的生成; 在 pH为 5.0-6.5时, 样品相对比较稳定, 产生的聚体和降解条带都较少。 考虑到人体环境的 pH值为 7.0左右, 为了与人体条件相近, 最终选择 6.5为样品制剂的 pH条件。 而且, 在 5-100mmol/L浓度范围内的磷酸 緩冲液, 其结果基本一致, 但为了使制剂总体渗透压适于人体应用, 并且又能保持一定的緩沖能力, 最终控制其在制剂中的浓度为 10mmol/L„
实施例 2: 添加不同制剂辅料对 rHSA-IFN a2b稳定性的影响 一般适合蛋白制剂应用的辅料有白蛋白、 糖类、 ^酸类、 表面 活性剂、 金属螯合剂等。 本发明选择了一些适合于人体应用的辅料进 行了筛选。 人血清白蛋白因为存在潜在的血源性污染问题, 而且我们 的制剂本来就包含了白蛋白, 因此就不予以考虑。
适用于本发明的糖类可选择多种单糖, 寡糖和多糖及碑脂和核苷 酸衍生物。 例如甘油, 甘露糖醇, 蔗糖等。 这些糖类, 可以单独添加, 也可以联合使用。
适用于本发明的多肽、 氨基酸及衍生物可以从下述一组物质中选 择而来的: 甘氨酸、 丙氨酸、 丝氨酸、 天冬氨酸、 谷氨酸、 苏氨酸、 色氨酸、 赖氨酸、 羟赖氨酸、 组氨酸、 精氨酸、 胱氨酸、 半胱氨酸、 曱疏氨酸、 苯丙氨酸、 亮氨酸、 异亮氨酸等以及它们的衍生物。 为了考察不同辅料对制剂的稳定性的影响,按如下条件对不同辅料 进行了筛选。本发明所筛选的辅料配方见表 1 (表中百分比浓度为质量 百分比浓度)。称取所需量的辅料,或量取配制好的浓的辅料储备溶液。 将辅料固体或辅料储备溶液加入到合适的緩沖液(pH6.5 的磷酸緩冲 液) 中, 然后加入用适当緩沖液(pH6.5 的磷酸緩沖液) 配的高浓度 rHSA-IFN a 2b蛋白溶液, 再用 lmol/1 HCL或 10%Na0H调节至所需 pH值, 以获得一定体积含 10mmol/L pH6.5的磷酸緩沖液、 蛋白浓度 为 0.5mg/ml的样品溶液。将配好的样品溶液分成两批,每批有 12种样 品, 每种样品各 5瓶, 0.5ml/瓶。 一批用作初始 T=0分析, 然后于 4°C 保存。 另一批 40°C保存, 考察 4个月, 每个月做分析检测。
表 1 : 辅料组成表
Figure imgf000010_0001
以下为不同配方稳定性实验结果:
( 1 )样品的澄明度
表 2、 3均显示样品在 4°C和 40 °C条件下存放 4个月后, 没有浑 浊现象产生。 表 2: 样品在 4°C条件下存放 4个月, 每隔一个月检测样品的澄明度
Figure imgf000011_0001
表 3: 样品在 40 °C条件下存放 4个月, 每隔一个月检测样品的澄明度 组别 T-0月 T-1月 T-2月 T-3月 T-4月
1
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
2
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
3
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
4
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
5
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
6
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
7
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
8
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
9
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
10
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
11
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清
12
无色澄清 无色澄清 无色澄清 无色澄清 无色澄清 ( 2 ) 电泳杂质检测
利用 SDS-PAGE的方法,每隔一个月取样检测。表 4显示样品在
4°C条件下存放 4个月内没有降解和聚体产生;表 5显示样品在 40°C加 速条件下, 只有含甘氨酸和曱硫氨酸的样品相对较稳定, 一直到 4个 月时才有降解和聚体发生。
表 4: 样品在 4°C条件下存放 4个月, 每隔一个月用 SDS-PAGE检测
Figure imgf000012_0001
表 5: 样品在 40。C条件下存放 4个月, 每隔一个月用 SDS-PAGE检测
Figure imgf000012_0002
( 3 ) RP-HPLC检测
利用 RP-HPLC的方法, 每隔一个月取样检测。 表 5显示样品在 4°C条件下存放 4 个月内稳定性 较好, 样品纯度没有发生大的变 化; 而在 40°C加速条件下, 只有含有甘氨酸和曱疏氨酸的样
有发生大的变化, 其它样品的纯度都有较大的降低。 表 6: 样品在 40°C条件下存放 4个月, 每隔一个月用 RP-HPLC检测
Figure imgf000013_0001
( 4 )生物学活性(效价)
根据以上各项研究结果显示,含有甘氨酸或曱硫氨酸的样品最稳 定, 因此只对含有甘氨酸或曱硫氨酸的样品进行效价评估。 样品在 4 °C与 40 °C条件下存放 4个月后分别取样进行生物学活性检测。结果 (表 7 )显示所有制剂的效价都在规定范围内, 表明发生的化学和物理降解 过程没有显著改变该蛋白质的效价活性, 其中又以甘氨酸的效果较佳, 并且不同浓度的甘氨酸在制剂中对蛋白的生物学活性的影响作用几乎 是一样的。
2 表 7: 不同处方样品在 4°C与 40°C条件存放 4个月后进行生物学活性检测
Figure imgf000014_0001
综合以上实验结果可得出以下结论: 糖类(包括葡萄糖、 蔗糖、 甘露醇)的添加, 反而不利于 rHSA-IFNa2b蛋白的稳定; 表面活性剂 也不能减少其聚体的形成; EDTA对稳定性也没有什么影响; 而氨基酸 类 (甘氨酸、 曱硫氨酸)对制品的稳定性有较好的作用, 其中以甘氨 酸的效果最好; 因此最终选择甘氨酸或甲硫氨酸作为 rHSA-IFN a 2b 药剂的辅料。
从以上实验数据中还可看到, 甘氨酸浓度在 1-4% (重量比)时对制 剂中蛋白的稳定作用几乎差不多, 但考虑到注射给药制剂的渗透压应 与人体生理渗透压相近, 从而选择 2.3% (重量比)的浓度作为制剂中 甘氨酸最优用量。
实施例 3: rHSA-IFNa2b蛋白浓度对其稳定性的影响
可注射药物制剂中蛋白浓度也是影响蛋白制品稳定性的一个重要 因素。 浓度过低, 不仅使给药体积增大, 而且容易被器壁吸附; 蛋白 浓度太高则易形成聚体。 为了使制剂的装量适宜便于使用、 并使 rHSA-IFN oc 2b蛋白在制剂中保持相对稳定 , 本发明按如下条件进行了 不同蛋白浓度条件对制品稳定性的影响实验:
实验条件: lml/ ( 10mmol/L磷酸缓冲液 pH=6.5, 2.3%甘氨酸) 各组中含有不同浓度的 rHSA-IFN cc 2b蛋白
① 0.1mg/ml
② 0.5mg/ml
③ 1.0mg/ml
@2.0mg/ml
⑤ 5.0mg/ml
避光放置于 4°C冰箱和 40°C的恒温箱中, 进行稳定性考察, 定时 取样分析。
检测方法: SDS-PAGE非还原电泳方法 (每个样取 10 μ g电泳分析) 结果见图 2、 图 3。
在 0.1-5.0mg/ml的浓度范围内, 4°C冰箱放置 1年, 均比较稳定; 40°C的加速试验表明, 蛋白浓度较高会容易产生聚体, 从而可能影响 到其理化性质、 生物学活性。 从蛋白的稳定性及制剂使用方便性来考 虑, rHSA-IFN a 2b蛋白的浓度在 0.5-2mg/ml之间是最合适的。
综合实施例 1 ~ 3 , 如下的药剂组成是理想的: 0.1-5mg/ml 的 rHSA-IFN a 2b蛋白, 优选的为 0.5-2mg/ml; 合适浓度的甘氨酸或曱硫 氨酸, 优选的是 1-4%甘氨酸, 最优选的浓度为 2.3%。 所选緩沖液有磷 酸緩沖液、 Tris-HCl緩沖液、 醋酸-醋酸钠緩冲液, 优选的是磷酸緩冲 液, 浓度为 5-100mmol/L, 最优选的浓度为 10mmol/L。 最终使得制剂 的渗透压在 250-500mOsm, pH在 5-8之间, 最优的是 6.5。
实施例 4: 含有重组 jk清白蛋白 -干扰素 oc 2b融合蛋白的注射用水剂 的制备
取 500毫升人血清白蛋白-干扰素 o 2b融合蛋白原液,蛋白浓度为 2.5毫克 /毫升, 并含 10mmol/L磷酸缓冲液(pH 6.5 ), 称取 57.5克甘 氨酸加入到原液中完全溶解, 再加入 40毫升 pH为 6.5的 0.5mol/L的 磷酸緩沖液, 用 10%NaOH调节 pH至 6.5, 最后加入适量的注射用水 稀释使制剂最终体积为 2500毫升,混合均匀后用 0.22微米的滤膜对该 制剂进行无菌过滤并分装于安瓿瓶中。 最终制剂组成为: 重组人血清 白蛋白-干扰素 o 2b融合蛋白浓度为 0.5毫克 /毫升,磷酸緩冲液的浓度 为 10mmol/L, pH 6.5, 甘氨酸含量为 2.3% (重量比)。
实施例 5: 含有重组 AJ6L清白蛋白-干扰素 o 2b融合蛋白的注射用水剂 稳定性的考察
根据实施例 4公开的方法, 配制如下几种优选处方:
①含 0.5mg/ml 重组 Ajfe清白蛋白-干扰素 o 2b 融合蛋白; 10mM Na2HP04-NaH2P04 pH6.5緩冲液; 2.3%甘氨酸 的注射水剂。
②含 2.0mg/ml 重组 清白蛋白-干扰素 a 2b 融合蛋白; 10mM Na2HP04-NaH2P04 pH6.5緩沖液; 2.3%甘氨酸的注射水剂。
③含 0.5mg/ml 重组 J6L清白蛋白-干扰素 oc 2b 融合蛋白; 10mM Na2HP04-NaH2P04 pH6.5緩沖液; 2.3% 曱硫氨酸的注射水剂。
④含 2.0mg/ml 重组 Ajk清白蛋白-干扰素 a 2b 融合蛋白; 10mM Na2HP04-NaH2P04 pH6.5緩沖液; 2.3%曱硫氨酸的注射水剂。
按照上述四种优选处方配制的溶液, 分别于 4°C和 40°C条件下存 放 4个月, 每月取样进行 RP-HPLC检测, 并与 4个月后取样进行生物 学活性检测。
表 8: 样品在 4°C和 40°C条件下存放用 RP-HPLC检测
Figure imgf000017_0001
表 9: 样品在 4°C与 40°C条件存放 4个月后进行生物学活性检测
Figure imgf000017_0002
从以上实验数据可以看出,本发明公开的 rHSA-IFNa2b蛋白药物 制剂具有较好的稳定性。
实施例 6: 含有重组人血清白蛋白-干扰素 a 2b融合蛋白的注射用水剂 在治疗乙型肝炎中的应用
6.1、 鸭乙肝模型制备 ' (1) 筛选鸭乙肝阳性血清
根据鸭乙肝病毒 (DHBV)序列,设计一对引物用于扩增 DHBV P序列。 上游引物: 5, atg ccc caa cca ttg aag ca 3', 下游引物: 5 ttc caa ttt egg gaa ggg ca 3,。 取 3只绍兴麻鸭, 在无菌条件下抽血, 分离 血清。 取 5μ1血清加 50μ1裂解液, 100°C煮沸 10分钟, 快速离心后置 于水上, 作为模板待用。 配制 PCR反应混合液: lOxPCR buffer 5μ1, 2.5mM MgCl23μ1, 2mM dNTP 5μ1, 上、 下游引物各 20pmol, Taq DNA 聚合酶 1. 25u加水至总体积 45μ1, 与上述预变性模板 5μ1混匀后, 加 滴一滴石蜡油, 上机。 PCR程序为 95 °C预变性 2分钟, 以 94°C变性 30 秒, 56°C退火 30秒, 72°C延伸 45秒为一循环。共 30个循环,最后 72°C 延伸 10分钟。 阴性对照含有 RT- PCR所需的所有成分, 但不加模板。
PCR结果用凝胶电泳验证:将 PCR反应产物各取 Ι ΟμΙ和上样緩沖液 2μ1 混匀, 加至浸泡于 ΙχΤΒΕ buffer ( 90mM tr i s-硼酸; 2mM EDTA pH8. 0 ) 的 1. 5%凝胶琼脂糖梳孔中。 40V电泳 3小时后, 在紫外检测仪上观察 结果 (波长为 300nm) , 3个绍兴麻鸭 PCR扩增结果均为阳性, 出现 3 条阳性条带, 阴性对照未出现阳性条带。 取条带最浓的 1 号绍兴麻鸭 血清作为阳性血清。
( 2 ) 制备鸭乙肝模型: 于孵化当日挑选健康樱桃谷鸭雏鸭, 每只 经腿静脉注射 lOOul阳性鸭血清。 另取 10只 1 日龄雉鸭作为正常对照 组。取阳性鸭血清感染 1 曰龄北京鸭樱桃谷鸭。感染后第 2周腿静脉 每只 0. 3ml ,用 PCR法检测, PCR方法及凝散电泳检测方法同上。 筛选阳性樱桃谷鸭为鸭乙肝模型。
6. 2、 抗乙肝病毒治疗
( 1 )将阳性樱桃谷鸭按照随机分五组( 60只 /组), 重组 Ajk清白蛋白 -干扰素 ct 2b融合蛋白的注射用水剂的制备如实施例 4公开的方法,用 量分别为小剂量组( 3微克 /公斤), 中剂量组( 10微克 /公斤), 大剂 量组( 40微克 /公斤); 拉米夫丁( 5mg /天)为阳性对照组, 以及生理 盐水空白对照组。 治疗组每组 42只。 于感染后第 2周开始治疗。 重组 人血清白蛋白-干扰素 a 2b 融合蛋白的注射用水剂和生理盐水空白组 为皮下给药, 每隔一周一次。 拉米夫丁组将药片磨成粉状, 用冷水化 开, 与饲料搅拌均匀, 给樱桃谷鸭喂服。 每天两次, 早晚各一次, 各 组均连续给药 3个月, 停药后继续观察 3个月。
( 2 )模型组于感染后于治疗前(感染后第 2周)、 治疗 1个月 (即感 染后第 6周)、 治疗 3个月结束时(即感染后第 14周)、 停药后第 1个 月 (即感染后第 18周)、 停药后第 2个月 (即感染后第 22 周)、 停药 后第 3个月 (即感染后第 26周),每组各取 10只樱桃谷鸭静脉采血并 处死。
6. 3、 半定量 PCR法检测鸭乙肝病毒滴度
取 5μ1樱桃谷鸭血清加 50μ1裂解液, 100°C煮沸 10分钟, 快速离 心后置于水上, 作为模板待用。 配制 PCR反应混合液: l OxPCR buffer 5μ1 , 2. 5mM MgCl2 3μ1 , 2mM dNTP 5μ1 , 待测细胞因子上、 下游引物各 20pmol , Taq DNA聚合酶 1. 25u加水至总体积 45μ1, 与上述预变性模 板 5μ1混匀后, 加滴一滴石蜡油, 上机。 PCR程序为 95°C预变性 2分 钟, 以 94°C变性 30秒, 56°C退火 30秒, 72°C延伸 45秒为一循环。 共 30个循环, 最后 72°C延伸 10分钟。 以 1号绍兴麻鸭阳性血清为阳性 对照,每次反应共设 5个阳性对照, 阴性对照含有 RT- PCR所需的所有 成分, 但不加模板。 将 PCR
反应产物各取 Ι ΟμΙ 和上样緩沖液 2μ1 混匀, 加至浸泡于 Ι χΤΒΕ buffer ( 90mM tr i s-硼酸; 2mM EDTA pH8. 0 )的 1. 5%凝胶琼脂糖梳孔 中。 40V电泳 3小时后, 经凝胶成像分析系统进行半定量分析,' 获 取每个条带的光密度扫描值, 进行统计分析。 每张图像之间以 5个阳性对照的'平均值均衡。
6. 4、 治疗后鸭血清乙肝病毒滴度变化情况
10中可见, 治疗 3个月刚刚结束时, 各治疗组鸭血清中乙肝 病毒滴 均有所下降, 以拉米夫丁治疗组最为明显; 停药 1 个月后, 重组人血清白蛋白-干扰素 cc 2b 融合蛋白的注射用水剂治疗组病毒滴 度继续下降, 至停药后 3个月, 重组 jk清白蛋白-干扰素 o 2b融合蛋 白的注射用水剂仍具有一定的病毒抑制作用, 未见有反弹现象。 而拉 米夫丁治疗组停药后 DHBV DNA滴度反弹明显。 重组人血清白蛋白 -干扰素 a 2b融合蛋白的注射用水剂治疗后 鸭乙肝病毒滴度变化情况
Figure imgf000020_0001
与模型组比较 : *ρ<0. 05 ; 与拉米夫丁组比较: Δρ<0. 05
鸭乙肝模型实^正明重组人血清白蛋白-干扰素 a 2b融合蛋白的注 射用水剂具有良好的抗乙肝病毒疗效。
9

Claims

权 利 要 求 书
1. 一种含有重组 清白蛋白-千扰素 c融合蛋白的药物制剂,其特征 在于: 该药物制剂除了重组人血清白蛋白 -干扰素 α融合蛋白
( rHSA-IFN a )作为有效成分外, 还含有药学上可接受的能够维持 制剂在水溶液状态下 pH值为 5. 0-8. 0的緩沖液及药学上可接受的 增强重组 jk清白蛋白-干扰素 a融合蛋白稳定性的辅料。
2. 根据权利要求 1所述的药物制剂, 其特征在于: 所述的重组人血清 白蛋白 -干扰素 a融合蛋白结构中, Ajk清白蛋白的 C末端可以直 接或通过一柔性的连接肽序列与人干扰素 o的 N端相连,或者人干 扰素 a的 C末端直接或通过一柔性的连接肽序列与 清白蛋白 的 N端相连。
3. 根据权利要求 2所述的药物制剂, 其特征在于: 所述的柔性连接肽 序列通式为 [GlyGlyGlyGlySer]n, n为 1-10的整数。
4. 根据权利要求 3所述的药物制剂, 其特征在于: 所述的柔性连接肽 序列通式为 [GlyGlyGlyGlySer]n, n为 1-3的整数。
5. 根据权利要求 4所述的药物制剂, 其特征在于: 所述的柔性连接肽 序列通式为 [GlyGlyGlyGlySer]n, n为 1。
6. 根据权利要求 1所述的药物制剂, 其特征在于: 所述的干扰素 c为 干扰素 a 2a、 干扰素 ot lb、 干扰素 a 2b或干扰素 a con。
7. 根据权利要求 6所述的药物制剂, 其特征在于: 所述的干扰素 cc为 干扰素 o 2b。
8. 根据权利要求 1所述的药物制剂, 其特征在于: 所述重组人血清白 蛋白-干扰素 融合蛋白浓度为 0.1 ~ 5mg/mL。
. 根据权利要求 8所述的药物制剂, 其特征在于: 所述重组 jk清白 蛋白 -干扰素 α融合蛋白浓度为 0.5 ~ 2mg/mL。
10.才艮据权利要求 1所述的药物制剂, 其特征在于: 所述的药学上可接 受的增强重组人血清白蛋白-干扰素 α融合蛋白稳定性的辅料为甘 氨酸或甲硫氨酸, 质量浓度为 1 ~ 4 %
11.根据权利要求 10所述的药物制剂, 其特征在于: 所述的药学上可 接受的增强重组人血清白蛋白 -干扰素 α融合蛋白稳定性的辅料为 甘氨酸, 质量浓度为 1 ~ 4 %
12. 居权利要求 11 所述的药物制剂, 其特征在于: 所述的药学上可 接受的增强重组人血清白蛋白-干扰素 α融合蛋白稳定性的辅料为 甘氨酸, 质量浓度为 2.3 %
13.根据权利要求 1所述的药物制剂, 其特征在于: 所述的药学上能接 受的能够维持制剂在水溶液状态下 ρΗ值为 5. 0-8. 0的緩沖液, 可 任选自磷酸氢二钠-杵檬酸緩沖液、 磷酸盐緩冲液、 三羟曱基氨基 曱烷盐酸盐緩冲液、醋酸-醋酸钠緩沖液、柠檬酸緩沖液、 巴比妥盐 緩冲液或琥珀酸盐緩冲液,浓度为 5- 100醒 ol/L,溶液的 pH为 5. 0 ~ 8. 0。
14. 据权利要求 13所述的药物制剂, 其特征在于: 所述的药学上能 接受的能够维持制剂在水溶液状态下 pH值为 5. 0-8. 0的緩冲液为 磷酸盐緩冲液, 浓度为 5-100mmol/L, 溶液的 pH为 5. 0 ~ 8. 0。
15.根据权利要求 14所述的药物制剂, 其特征在于: 所述的药学上能 接受的能够维持制剂在水溶液状态下 pH值为 5. 0-8. 0的緩冲液为 磷酸盐緩冲液, 浓度为 5-30mmol /L, 溶液的 pH为 6. 0 ~ 7. 0。
16.根据权利要求 15所述的药物制剂, 其特征在于: 所述的药学上能 接受的能够维持制剂在水溶液状态下 pH值为 5. 0-8. 0的缓冲液为 磷酸盐緩冲液, 浓度为 10mmol/L, 溶液的 pH为 6. 5。
17.根据权利要求 1所述的药物制剂, 其特征在于: 所述药物制剂由重 组人血清白蛋白-干扰素 a 2b融合蛋白与甘氨酸溶于 pH为 5.0 ~ 8.0 的浓度为 5 ~ 100mmol/L磷酸盐緩沖液中得到, 所述重组人血清白 蛋白 -干扰素 2b 融合蛋白为人血清白蛋白与干扰素直接融合或以 序列通式为
Figure imgf000023_0001
为连接肽融合得到的融合蛋白, n为 1-10的整数, 融合蛋白浓度为 0.1 - 5mg/mL, 所述甘氨^ "量浓度 为 1 ~ 4%。
18.根据权利要求 17所述的药物制剂, 其特征在于: 所述药物制剂由 重组 jk清白蛋白-干扰素 cc 2b融合蛋白与甘氨酸溶于 pH为 6.0 ~ 7.0的浓度为 5 - 30mmol/L磷酸盐緩冲液中得到, 所述重组人血清 白蛋白-干扰素 ot 2b 融合蛋白为 jk清白蛋白与干扰素直接融合或 以序列通式为 [GlyGlyGlyGlySer]n为连接肽融合得到的融合蛋白, n 为 1-3的整数, 融 蛋白浓度为 0.5 ~ 2mg/mL, 所述甘氨酸质量浓 度为 1 ~ 40/0
19.根据权利要求 18所述的药物制剂, 其特征在于: 所述药物制剂由 重组 AJDL清白蛋白-干扰素 a2b融合蛋白与甘氨酸溶于 pH为 6.5的 浓度为 10mmol/L磷酸盐緩沖液中得到, 所述重组人血清白蛋白- 干扰素 cc 2b 融合蛋白为 清白蛋白与干扰素直接融合或以序列 通式为 [GlyGlyGlyGlySer]为连接肽融合得到的融合蛋白,融合蛋白 浓度为 0.5mg/mL, 所述甘氨^ 量浓度为 2.3%。
20.权利要求 1-19任一项所述的药物制剂, 其特征在于: 所述药物制 剂可制备为冻干粉针。
21.重组人血清白蛋白-干扰素 ot融合蛋白用于制备权利要求 1-19任一 项所述的药物制剂的用途, 所述药物制剂用于治疗乙型肝炎或丙型 肝炎。
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