WO2019120112A1 - 一种模拟抗原化合物用于治疗hbv感染相关症状的用途 - Google Patents

一种模拟抗原化合物用于治疗hbv感染相关症状的用途 Download PDF

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WO2019120112A1
WO2019120112A1 PCT/CN2018/120491 CN2018120491W WO2019120112A1 WO 2019120112 A1 WO2019120112 A1 WO 2019120112A1 CN 2018120491 W CN2018120491 W CN 2018120491W WO 2019120112 A1 WO2019120112 A1 WO 2019120112A1
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drug
administered
compound
liposome
mimetic
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PCT/CN2018/120491
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WO2019120112A9 (zh
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吴玉章
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江苏孟德尔基因科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the use of a mimetic antigenic compound for the treatment of symptoms associated with HBV infection.
  • hepatitis B Chronic hepatitis B
  • HBV Hepatitis B virus
  • Hepatitis B is worldwide, but the intensity of the epidemic varies greatly from region to region. China is a high prevalence area of hepatitis B.
  • the positive rate of HBsAg in the general population is 9.09%. It is difficult to cure, has a poor prognosis, and is seriously harmful to health.
  • the treatment of hepatitis B mainly includes antiviral therapy, immunomodulatory therapy, anti-inflammatory therapy and anti-fibrotic therapy.
  • the effective anti-HBV drugs recognized at home and abroad mainly include interferons and nucleoside (acid) analogs, each having its own advantages and disadvantages.
  • the advantage of the former is that the course of treatment is relatively fixed, the HBeAg seroconversion rate is relatively high, the effect is relatively long-lasting, and the drug resistance variation is less;
  • the disadvantage is that it requires injection and administration, and the adverse reactions are obvious, and it is not suitable for liver function decompensation.
  • the latter has the advantages of oral administration, strong inhibition of the virus, and little adverse reaction, which can be used for liver function decompensation.
  • the disadvantage is that the course of treatment is relatively unfixed, the HBeAg seroconversion rate is low, and the curative effect is not long-lasting. Drug resistance mutations may occur, and the disease may worsen after stopping the drug. Therefore, there is a clinical need for a drug that can better treat symptoms associated with HBV infection.
  • CN1483736 (incorporated herein by reference) discloses a mimetic antigen polypeptide having a primary structure of CH 3 (CH 2 ) 14 COKSSQYIKANSK FIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPA, and discloses that the polypeptide can induce HBV neutralization in mice sexual antibodies and induce the production of cytokines and cytotoxic responses.
  • the mimetic antigen polypeptide for clinical treatment of symptoms associated with HBV infection.
  • a first aspect of the invention provides a method of treating a condition associated with HBV infection, the method comprising administering a therapeutically effective amount of a mimetic antigenic compound comprising the following two components to a subject having symptoms associated with HBV infection: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA, wherein treating symptoms associated with HBV infection includes increasing HBeAg/HBeAb seroconversion rate, increasing serum HBeAb positivity, and lowering serum HBV DNA Level, reduce serum ALT levels and / or improve liver damage.
  • a second aspect of the invention provides the use of a simulated antigenic compound comprising two parts for the preparation of a medicament for increasing the seroconversion rate of HBeAg/HBeAb: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)- KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA.
  • a third aspect of the invention provides the use of a simulated antigenic compound comprising two parts for the preparation of a medicament for increasing the HBeAb conversion rate of serum: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA.
  • a fourth aspect of the invention provides the use of a simulated antigenic compound comprising two parts for the preparation of a medicament for lowering serum HBV DNA levels: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and 2) AAAFLPSDFFPSVGGGDPRVRGLYFPA.
  • a fifth aspect of the invention provides the use of a simulated antigenic compound comprising two parts for the preparation of a medicament for lowering serum ALT levels: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2 )AAAFLPSDFFPSVGGGDPRVRGLYFPA.
  • a sixth aspect of the invention provides the use of a simulated antigenic compound comprising two parts for the preparation of a medicament for improving liver damage: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA.
  • the method of the invention can induce serum HBeAb positivity, HBeAg/HBeAb seroconversion in HBV infected patients, reduce serum HBV DNA levels, lower serum ALT levels, and repair damaged liver cells; therefore, it can be used as a treatment for symptoms related to HBV infection. Useful ways.
  • Figure 1 shows a chromatogram of a simulated antigen compound purified by reverse phase high performance liquid chromatography.
  • FIG. 2 shows the ultrastructure of the pseudo antigen compound-liposome prepared in Example 1 of the present invention observed by transmission electron microscopy (TEM) (FIG. 2a) and in Example 1 of the present invention detected by a laser particle size analyzer.
  • TEM transmission electron microscopy
  • FIG. 2b The prepared antigenic compound-liposome particle size distribution
  • Figure 3 shows liver tissue morphology (HE staining, x 200) of patients with chronic hepatitis B before treatment with simulated antigenic compounds (Figure 3a) and after treatment ( Figure 3b).
  • the present invention provides a method of treating a symptom associated with HBV infection, the method comprising administering a therapeutically effective amount of a mimetic antigen compound comprising the following two parts to a subject having symptoms associated with HBV infection: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA, wherein treating symptoms associated with HBV infection includes increasing HBeAg/HBeAb seroconversion rate, increasing serum HBeAb positivity, and lowering serum HBV DNA Level, reduce serum ALT levels and / or improve liver damage.
  • a mimetic antigen compound comprising the following two parts to a subject having symptoms associated with HBV infection: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA, wherein treating symptoms associated with HBV infection includes increasing HBeAg/HBeAb seroc
  • terapéuticaally effective amount refers to an amount that has a beneficial effect in a subject being treated, ie an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications.
  • the symptoms associated with the HBV infection are symptoms of one or more of the following diseases: chronic hepatitis B, cirrhosis, and hepatocellular carcinoma.
  • the subject refers to human and non-human mammals such as, but not limited to, non-human primates, cats, dogs, sheep, goats, horses, cows, pigs, and rodents (eg, But not limited to mice and rats; and non-mammals such as, but not limited to, birds, poultry, reptiles, amphibians.
  • the subject can be of any gender, any age.
  • the terms "subject” and "patient” are used interchangeably herein.
  • the subject is a human.
  • CH 3 (CH 2 ) 14 CO- in part (1) of the mimetic antigenic compound is replaced by CH 3 (CH 2 ) 16 CO-.
  • AAA and/or GGG in part (2) of the mimetic antigenic compound is replaced by SSS.
  • part (1) and part (2) of the mimetic antigenic compound are linked by a covalent bond.
  • the mimetic antigenic compound is encapsulated by a liposome to form a mimetic antigenic compound-liposome.
  • the mimetic antigen compound-liposome has a diameter of from 80 to 100 nm.
  • the compound liposomes further comprise a pharmaceutically acceptable adjuvant, carrier or adjuvant.
  • the mimetic antigenic compound-liposome is in any pharmaceutically acceptable dosage form.
  • the mimetic antigen compound-liposome is in a liquid dosage form or a lyophilized dosage form, more preferably in a lyophilized dosage form.
  • the formulation of the mimetic antigenic compound is an injection, a transdermal agent, an oral, an inhalant or a suppository. In a preferred embodiment, the formulation of the mimetic antigenic compound is an injection.
  • the mimetic antigenic compound can be administered by any route of administration including, but not limited to, oral, rectal, nasal, pulmonary, epidural, ocular, aural, intraarterial. , intracardiac, intradermal, intravenous, intramuscular, intraperitoneal, intraosseous, intravesical, subcutaneous, topical, transdermal and transmucosal, for example by sublingual, buccal, vaginal and inhalation routes of administration.
  • the mimetic antigen compound is administered as an injection by the subcutaneous route.
  • the mimetic antigenic compound is administered to an adult patient by subcutaneous injection into the upper extremity or abdomen.
  • the mimetic antigenic compound is administered to a pediatric patient by subcutaneous injection of the anterolateral thigh.
  • the mimetic antigen compound is administered as an injection at a 45 degree needle depth to the subcutaneous fat of the subject. In a specific embodiment, the mimetic antigen compound is administered to a subject as an injection at a point of injection of from 1 to 3, and administered 3 to 21 times, preferably 6 to 21 times.
  • the mimetic antigen compound is administered in two stages, wherein the first phase is administered 6 times (the first 4 times each interval of 3-4 weeks, the fifth and sixth times each time) At intervals of 7-8 weeks), the second phase was administered 15 times (the first 9 times were separated by 3 weeks, and the last 6 times were separated by 4 weeks).
  • the mimetic antigen compound is administered at a dose of 300-1800 ⁇ g.
  • the mimetic antigen compound is administered at a dose of 600 or 900 ⁇ g.
  • the mimetic antigen compound is administered at a dose of 900 ⁇ g.
  • the invention also provides the use of a simulated antigenic compound comprising two parts for the preparation of a medicament for treating symptoms associated with HBV infection: (1) ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITE and (2) AAAFLPSDFFPSVGGGDPRVRGLYFPA
  • the treatment of symptoms associated with HBV infection includes increasing HBeAg/HBeAb seroconversion, increasing serum HBeAb conversion, lowering serum HBV DNA levels, and/or lowering serum ALT levels.
  • the symptoms associated with the HBV infection are symptoms of one or more of the following diseases: chronic hepatitis B, cirrhosis, and hepatocellular carcinoma.
  • CH 3 (CH 2 ) 14 CO- in part (1) of the mimetic antigenic compound is replaced by CH 3 (CH 2 ) 16 CO-.
  • AAA and/or GGG in part (2) of the mimetic antigenic compound is replaced by SSS.
  • part (1) and part (2) of the mimetic antigenic compound are linked by a covalent bond.
  • the compound is formulated as a compound-liposome formed by liposome encapsulation.
  • the mimetic antigen compound-liposome has a diameter of from 80 to 100 nm.
  • the mimetic antigenic compound-liposome is in any pharmaceutically acceptable dosage form.
  • the mimetic antigen compound-liposome is in a liquid dosage form or a lyophilized dosage form, more preferably in a lyophilized dosage form.
  • the medicament further comprises a pharmaceutically acceptable adjuvant, carrier or adjuvant.
  • the medicament is an injection, a transdermal, an oral, an inhalant or a suppository.
  • the drug is an injection.
  • the medicament is for use in humans and non-human mammals as well as non-mammals, preferably in humans.
  • the medicament may be administered by any route of administration including, but not limited to, oral, rectal, nasal, pulmonary, epidural, ocular, aural, intraarterial, intracardiac. , intradermal, intravenous, intramuscular, intraperitoneal, intraosseous, intravesical, subcutaneous, topical, transdermal and transmucosal, for example by sublingual, buccal, vaginal and inhalation routes of administration.
  • the medicament is administered as an injection by the subcutaneous route.
  • the drug is administered to an adult patient by subcutaneous injection of the upper limb or abdomen.
  • the drug is administered to a child patient by subcutaneous injection of the anterolateral thigh.
  • the drug is administered as an injection at a 45 degree needle depth to the subcutaneous fat of the subject. In a specific embodiment, the drug is administered as an injection to a subject at an injection point of 1-3, and administered 3-21 times, preferably 6-21 times.
  • the drug is administered in two stages, wherein the first phase is administered 6 times (the first 4 times each interval of 3-4 weeks, the fifth and sixth times each interval of 7) -8 weeks), the second phase was administered 15 times (the first 9 times were separated by 3 weeks, and the last 6 times were separated by 4 weeks).
  • the medicament is administered at a dose of 300-1800 ⁇ g.
  • the drug is administered at a dose of 600 or 900 ⁇ g.
  • the drug is administered at a dose of 900 ⁇ g.
  • the sequence of the mimetic antigen compound of the present invention is ⁇ -CH 3 (CH 2 ) 14 CO-(NH)-KSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPA, which is synthesized by Fmoc solid phase synthesis.
  • the fixed carboxy terminus extends from the C-terminus to the N-terminus.
  • Fmoc amino acid was used as raw material, the loading amount was 1 mM, and the side chain protection was: Ser(tBu), Thr(tBu), Tyr(tBu), His(Trt), Gln(Trt), ASP(OtBu), Glu(OtBu), Arg(Pmc).
  • HMP-resin was selected as the solid phase carrier, the sample loading was 0.25 mM, and the raw material/resin ratio was 4:1.
  • the amino acid coupled to the resin (the first amino acid at the carboxy terminus of the polypeptide) was activated by a symmetric acid anhydride activation method, and the activation of the remaining amino acids and palmitic acid was carried out by HOBt/DCC activation.
  • asparagine, arginine and palmitic acid are double coupled, and there is no de-Fmoc protecting group step after palmitic acid coupling.
  • TFA lysate (0.75 g phenol, 0.25 mL ethanedithiol, 0.5 mL thioanisole, 0.5 mL deionized water, 10.0 mL TFA) for cleavage minimizes the occurrence of side reactions.
  • TFA lysate 0.75 g phenol, 0.25 mL ethanedithiol, 0.5 mL thioanisole, 0.5 mL deionized water, 10.0 mL TFA
  • the concentration of the mimetic antigen compound-resin in the cleavage reaction was determined to be 40 mg/mL.
  • the cleavage reaction time was controlled at 1-2 h, and after the cleavage, the homozygous and the yield of the finally obtained mimetic antigen compound were both high, so the cleavage reaction time was determined to be 1.5 hours.
  • the lysate containing the mimetic antigen compound is obtained under the cleavage reaction conditions determined above, and then the simulated antigen compound is purified by a two-step method: first, preliminary purification by size exclusion chromatography, chromatography system: P-6000 pump and AKTAexplorer 100; column: diameter 10 mm, column length 250 mm, packing Sephadex LH-20; column temperature: 25 ° C; mobile phase: DMSO; flow rate: 0.4 ml / min.
  • the simulated antigen compound fractions were collected and then purified using reversed-phase high performance liquid chromatography.
  • a lipid component is dissolved in an ether solution, and then mixed with a concentrate of the simulated antigen compound obtained in Example 1 to form an emulsion (W/O).
  • W/O emulsion
  • the size of the simulated antigen compound-liposome was determined using a Malvern ZEN1690 laser particle size analyzer.
  • the prepared simulated antigen compound-liposome freeze-dried product was resuspended in 1 mL of sterile water for injection, and then the impurities were removed by filtration through a 0.22 ⁇ m polycarbonate membrane filter, and 100 ⁇ L of the simulated antigen compound-liposome solution was used. Dilute to 0.01 mL of 0.01 M PBS (pH 7.4), stir well, add 1.2 mL to the sample container, open the computer program, set the test parameters, preheat the machine for 30 min, add samples for testing, and the results are shown in Figure 2b. As can be seen from Fig. 2b, the results of the laser particle size analyzer are similar to those of the electron microscopy.
  • the liposome size distribution ranges from 30 to 250 nm, and the liposome diameter of 70-95% or more is in the range of 80-100 nm
  • Example 3 Therapeutic effect of mimic antigen compound-liposome in chronic hepatitis B patients
  • Example 1 the simulated antigenic compound-liposome prepared in Example 1 was used to treat the screened chronic hepatitis B patients, and the therapeutic effects and effects in the chronic hepatitis B disease were studied.
  • Stage 2 (76-144 weeks): Of the three groups of subjects who completed the 76-week study above, if there was a virological response after treatment but no serological response/serign response but no virological response/virological serology None of the respondents, but 183 subjects who were willing to continue the study (including 58 in the first phase of the 600 ⁇ g treatment group, 60 in the first phase of the 900 ⁇ g treatment group and 65 in the first phase of the control group) continued 900 ⁇ g of mock antigen compound-liposome was administered and followed up for 3 years (144 weeks).
  • the first stage subcutaneous injection of the upper arm or abdomen, 45 ° needle, deep and subcutaneous fat, injection points 1-3.
  • 600 ⁇ g of the treatment group was administered with 600 ⁇ g of the simulated antigen compound-liposome + 300 ⁇ g of empty liposome;
  • 900 ⁇ g of the treatment group was administered with 900 ⁇ g of the mimetic antigen compound-liposome;
  • the control group was administered with 900 ⁇ g of the empty liposome.
  • the administration time was 0, 4, 8, 12, 20, and 28 weeks, respectively.
  • Second stage subcutaneous injection of 900 ⁇ g of mimetic antigen compound-liposome at 80, 83, 86, 89, 92, 95, 98, 101, 104, 108, 112, 116, 120, 124 and 128 weeks, respectively, injection
  • the site and method were the same as the first phase, a total of 15 injections.
  • the patient's HBeAg/anti-HBe seroconversion was detected after the simulated antigen compound-liposome treatment prepared in Example 1 of the present invention. Rates, anti-HBe conversion rate, serum HBV DNA copy number, and serum alanine aminotransferase (ALT) levels.
  • a total of 354 subjects were included in the intention-to-treat (ITT) population (defined as: all randomized, at least once, and at least once)
  • the main outcome measures (HBeAg/anti-HBe) data were obtained from the following results; among them, 119 patients in the control group, 1 patient was excluded from the main efficacy index data after no drug use; 119 patients in the 600 ⁇ g treatment group, 1 person The main efficacy index data were excluded after no-drug treatment; 116 patients in the 900 ⁇ g treatment group, and 4 patients were excluded from the main efficacy index data after no medication.
  • 3 patients in the control group, 3 patients in the 600 ⁇ g treatment group, and 2 patients in the 900 ⁇ g treatment group were excluded from the main efficacy index data after treatment.
  • the final number of intentional treatments included in the control group were 62 in the control group, 55 in the 600 ⁇ g treatment group, and 900 ⁇ g in the treatment group.
  • HBeAg seroconversion ie, HBeAg disappeared, and anti-Hbe turned positive.
  • the HBeAg/anti-HBe seroconversion occurs as a satisfactory endpoint for chronic hepatitis B treatment, marking a long-term prognosis improvement, such as a reduced incidence of cirrhosis and a slowing of disease progression.
  • the HBeAg/anti-HBe serological conversion rate of the control group was 20.2% (24/119).
  • the serological conversion rates of the patients in the original group were significantly improved after receiving 900 ⁇ g of the simulated antigen compound-liposome again.
  • the proportion of subjects with seroconversion in the control group increased from 17.7% (11/62) at 76 weeks to 38.7% (24/62) at the end of 144 weeks; 20.0% at 76 weeks for the 600 ⁇ g treatment group (11/55) increased to 40.0% (22/55) at the end of 144 weeks, and the 900 ⁇ g treatment group increased from 29.3% (17/58) at 76 weeks to 34.5% at the end of 144 weeks (20/58) )(Table 4).
  • the HBeAg/anti-HBe serological conversion rate after receiving the mimetic antigen compound-liposome treatment of the present invention is superior to that of the nucleoside analog drug (A Defovir, lamivudine and entecavir) and long-acting interferon.
  • the HBe antibody turns positive, that is, serum HBe antibody appears.
  • HBe antibody can reflect the host immune response;
  • HBe antibody transduction positive indicates that the host has obtained a relatively stable HBV-specific immune response, which is beneficial to the control of HBV infection.
  • the anti-HBe conversion rate was 29.4% (35/119) in the control group, and 95 of the 235 subjects (ITT population) who mimicked the antigen compound-liposome drug.
  • the tester developed anti-HBe conversion, and the conversion rate was 40.4%.
  • the anti-HBe conversion rates of the 600 ⁇ g treatment group and the 900 ⁇ g treatment group were 37.0% (44/119) and 44.0% (51/116), respectively (Table 6).
  • the control group and the 600 ⁇ g treatment group received 900 ⁇ g of simulated antigen compound-liposome treatment again after anti-HBe conversion compared to the serological conversion rate at 76 weeks of enrollment.
  • the seroconversion rate of the yang was improved.
  • the proportion of subjects with seroconversion in the control group increased from 35.3% (22/62) at 76 weeks to 41.9% (26/62) at the end of 144 weeks; the treatment group at 600°g was 27.3 at 76 weeks. %(15/55) increased to 47.3% (26/55) at the end of 144 weeks.
  • Peripheral blood HBV DNA is formed by the release of HBV-infected hepatocytes into the bloodstream and is the most direct indicator of the therapeutic effect of HBV infection.
  • a decrease in peripheral blood HBV DNA copy number means that HBV replication is inhibited, reflecting the effectiveness of the treatment.
  • subjects in the control group, the 600 ⁇ g treatment group, and the 900 ⁇ g treatment group experienced a virological response rate of ⁇ 2 log-level reduction in HBV DNA load, which was 40.3% (48/119), respectively. 48.7% (58/119) and 50.0% (58/116) (Table 8).
  • ALT is produced specifically by hepatocytes and is usually located inside hepatocytes.
  • liver cells When liver cells are damaged, a large amount of ALT enters the peripheral blood from the inside of the liver cells, and serum ALT is significantly increased. Therefore, serum ALT levels reflect hepatocyte injury: elevated serum ALT levels suggest liver damage; decreased serum ALT levels suggest an effective control of HBV infection and a reduction in liver damage.
  • the ratio of ALT levels in the control group, 600 ⁇ g treatment group, and 900 ⁇ g treatment group fell to the normal range, 34.5% (41/119), 37.8% (45/119), and 48.3, respectively. % (56/116) (Table 10).
  • liver pathology is the most intuitive evidence of liver damage.
  • the same chronic hepatitis B patient was selected, and liver tissue was obtained by liver puncture before and after treatment with simulated antigen compound-liposome.
  • the pathological changes of liver before and after treatment were observed.
  • the results of treatment are shown in Figure 3. After two stages of treatment, liver edema and balloon-like changes were significantly reduced; point and focal necrosis were significantly reduced; lymphocytic infiltration was significantly reduced. It can be seen that the drug can effectively improve the liver pathology of patients with chronic hepatitis B, reduce liver damage, and is beneficial to the effective control of hepatitis B infection.
  • the present invention demonstrates that the mimetic antigen compound-liposome can effectively induce serum HBeAb positivity, HBeAg/HBeAb seroconversion in HBV-infected patients, reduce serum HBV DNA levels, lower serum ALT levels, and repair damaged liver cells. Therefore, it can be used for the preparation of a medicament for treating the symptoms associated with the aforementioned HBV infection.

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Abstract

本发明涉及一种治疗HBV感染相关症状的方法，所述方法包括将治疗有效量的包含以下两部分的模拟抗原化合物给药至患有HBV感染相关症状的受试者： (1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和 (2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。本发明的方法可诱导HBV感染患者血清HBeAb阳转、HBeAg/HBeAb血清学转换，降低患者血清HBV DNA水平，降低血清ALT水平，修复损伤肝细胞；因此，可以作为一种治疗HBV感染相关症状的有效方法。

Description

一种模拟抗原化合物用于治疗HBV感染相关症状的用途 技术领域
本发明涉及一种模拟抗原化合物用于治疗HBV感染相关症状的用途。
背景技术
慢性乙型肝炎(简称乙肝)是一种由乙型肝炎病毒(Hepatitis B virus，HBV)引起的、以肝脏炎性病变为主并可引起多器官损害的一种疾病。乙肝呈世界性流行，但在不同地区的流行强度差异很大。我国属乙肝高流行区，一般人群的HBsAg阳性率为9.09％，其治愈困难，预后差，严重危害健康。目前对于乙肝的治疗主要包括抗病毒治疗、免疫调节治疗、抗炎治疗和抗纤维化治疗。国内外公认有效的抗HBV药物主要包括干扰素类和核苷(酸)类似物，各有其优缺点。前者的优点是疗程相对固定，HBeAg血清转换率较高，疗效相对持久，耐药变异较少；其缺点是需要注射给药，不良反应较明显，不适于肝功能失代偿者。后者的优点是口服给药，抑制病毒作用强，不良反应少而轻微，可用于肝功能失代偿者，其缺点是疗程相对不固定，HBeAg血清转换率低，疗效不够持久，长期应用可产生耐药变异，停药后可出现病情恶化等。因此，目前临床上亟需一种能够更好地治疗HBV感染相关症状的药物。
CN1483736(A)(以引用的方式纳入本文)中公开了一种一级结构为CH 3(CH 2) 14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPA的模拟抗原多肽，并公开了所述多肽可在小鼠体内诱导产生HBV中和性抗体，并诱导产生细胞因子和细胞毒性反应。然而，目前并没有该模拟抗原多肽用于临床治疗HBV感染相关症状的报道。
发明内容
本发明的第一方面提供了一种治疗HBV感染相关症状的方法，所述方法包括将治疗有效量的包含以下两部分的模拟抗原化合物给药至具有HBV感染相关症状的受试者：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA，其中治疗HBV感染相关症状包括提高HBeAg/HBeAb血清学转换率、提高血清HBeAb转阳率、降低血清HBV DNA水平、降低血清ALT水平和/或改善肝脏损伤。
本发明的第二方面提供了包含以下两部分的模拟抗原化合物用于制备提高HBeAg/HBeAb血清学转换率的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
本发明的第三方面提供了包含以下两部分的模拟抗原化合物用于制备提高血清HBeAb转阳率的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
本发明的第四方面提供了包含以下两部分的模拟抗原化合物用于制备降低血清HBV DNA水平的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
本发明的第五方面提供了包含以下两部分的模拟抗原化合物用于制备降低血清ALT水平的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
本发明的第六方面提供了包含以下两部分的模拟抗原化合物用于制备改善肝脏损伤的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
本发明的方法可诱导HBV感染患者血清HBeAb阳转、HBeAg/HBeAb血清学转换，降低患者血清HBV DNA水平，降低血清ALT水平，修复损伤肝细胞；因此，可以作为一种治疗HBV感染相关症状的有效方法。
附图说明
根据以下参照附图进行的详细描述，本发明的上述和其他方面、特征和优点会变得更加清楚。
图1示出了模拟抗原化合物经过反相高效液相色谱法纯化后的层析图谱。
图2示出了通过透射电镜(TEM)观察的本发明实施例1中制备的模拟抗原化合物-脂质体的超微结构(图2a)和通过激光粒度分析仪检测的本发明实施例1中制备的模拟抗原化合物-脂质体的粒径分布(图2b)。
图3示出了慢性乙肝患者接受模拟抗原化合物治疗前(图3a)和治疗后(图3b)的肝脏组织形态(HE染色，×200)。
具体实施方式
如上所述，本发明提供了一种治疗HBV感染相关症状的方法，所述方法包括将治疗有效量的包含以下两部分的模拟抗原化合物给药至具有HBV感染相关症状的受试者：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA，其中治疗HBV感染相关症状包括提高HBeAg/HBeAb血清学转换率、提高血清HBeAb转阳率、降低血清HBV DNA水平、降低血清ALT水平和/或改善肝脏损伤。
本文所使用的术语“治疗有效量”是指在被治疗的受试者中具有有益效果的量，即足以治愈、减轻或部分阻止给定的疾病及其并发症的临床表现的量。
在本发明方法的一个实施方案中，所述HBV感染相关症状为以下一种或多种疾病的症状：慢性乙型肝炎、肝硬化和肝细胞癌。
在本发明的方法中，所述受试者是指人类和非人类哺乳动物，例如但不限于非人灵长类动物、猫、狗、绵羊、山羊、马、牛、猪和啮齿动物(例如但不限于小鼠和大鼠)；以及非哺乳动物，例如但不限于鸟类、家禽、爬行动物、两栖动物。所述受试者可以是任意性别、任何年龄。术语“受试者”和“患者”在本文中可交换使用。在一个优选的实施方案中，所述受试者是人类。
在本发明方法的一个实施方案中，所述模拟抗原化合物的部分(1)中的CH 3(CH 2) 14CO-被CH 3(CH 2) 16CO-替换。
在本发明方法的一个实施方案中，所述模拟抗原化合物的部分(2)中的AAA和/或GGG被SSS替换。
在本发明方法的另一个实施方案中，所述模拟抗原化合物的部分(1)和部分(2)通过共价键链接。
在本发明方法的一个具体的实施方案中，所述模拟抗原化合物被脂质体包裹以形成模拟抗原化合物-脂质体。在一个具体的实施方案中，所述模拟抗原化合物-脂质体的直径为80-100nm。在另一个具体的实施方案中，所述化合-物脂质体还包含药学上可接受的佐剂、载体或辅料。在一个实施方案中，所述模拟抗原化合物-脂质体为药学上可接受的任意剂型。在一个优选的实施方案中，所述模拟抗原化合物-脂质体为液体剂型或冻干剂型，更优选为冻干剂型。
在本发明方法的一个实施方案中，所述模拟抗原化合物的制剂为注射剂、透皮剂、口服剂、吸入剂或栓剂。在一个优选的实施方案中，所述模拟抗原化合物的制剂为注射剂。
在本发明的方法中，可将所述模拟抗原化合物以任何给药途径进行给药，所述给药途径包括但不限于口腔、直肠、鼻、肺、硬膜外、眼、耳、动脉内、心脏内、皮肤内、静脉内、肌肉内、腹腔内、骨内、膀胱内、皮下、局部、经皮和经黏膜，例如通过舌下、口腔、阴道和吸入给药途径。
在本发明的一个优选的实施方案中，将所述模拟抗原化合物作为注射剂以皮下途径给药。在一个具体的实施方案中，将所述模拟抗原化合物通过上肢或腹部皮下注射给药至成人患者。在另一个具体的实施方案中，将所述模拟抗原化合物通过股前外侧皮下注射给药至儿童患者。
在一个具体的实施方案中，将所述模拟抗原化合物作为注射剂以45度进针深度给药至受试者的皮下脂肪。在一个具体的实施方案中，将所述模拟抗原化合物作为注射剂给药至受试者时的注射点为1-3个，并给药3-21次，优选给药6-21次。
在本发明的一个实施方案中，将所述模拟抗原化合物分两个阶段进行给药，其中第一阶段给药6次(前4次每次间隔3-4周，第5、6次每次间隔7-8周)，第二阶段给药15次(前9次每次间隔3周，后6次每次间隔4周)。在本发明的实施方案中，以300-1800μg的剂量给药所述模拟抗原化合物。在一个优选的实施方案中，以600或900μg的剂量给药所述模拟抗原化合物。在一个更优选的实施方案中，以900μg的剂量给药所述模拟抗原化合物。
本发明还提供了包含以下两部分的模拟抗原化合物用于制备治疗HBV感染相关症状的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA，其中所述治疗HBV感染相关症状包括提高HBeAg/HBeAb血清学转换率、提高血清HBeAb转阳率、降低血清HBV DNA水平和/或降低血清ALT水平。
在一个实施方案中，所述HBV感染相关症状为以下一种或多种疾病的症状：慢性乙型肝炎、肝硬化和肝细胞癌。
在一个实施方案中，所述模拟抗原化合物的部分(1)中的CH 3(CH 2) 14CO-被CH 3(CH 2) 16CO-替换。在另一个实施方案中，所述模拟抗原化合物的部分(2)中的AAA和/ 或GGG被SSS替换。在另一个实施方案中，所述模拟抗原化合物的部分(1)和部分(2)通过共价键链接。
在一个具体的实施方案中，将所述化合物被配制为由脂质体包裹形成的化合物-脂质体。在一个具体的实施方案中，所述模拟抗原化合物-脂质体的直径为80-100nm。在一个实施方案中，所述模拟抗原化合物-脂质体为药学上可接受的任意剂型。在一个优选的实施方案中，所述模拟抗原化合物-脂质体为液体剂型或冻干剂型，更优选为冻干剂型。
在一个实施方案中，所述药物还包含药学上可接受的佐剂、载体或辅料。在另一个实施方案中，所述药物为注射剂、透皮剂、口服剂、吸入剂或栓剂。在一个优选的实施方案中，所述药物为注射剂。
在一个实施方案中，所述药物用于人类和非人类哺乳动物以及非哺乳类动物，优选用于人类。
在本发明的实施方案中，所述药物可以任何给药途径进行给药，所述给药途径包括但不限于口腔、直肠、鼻、肺、硬膜外、眼、耳、动脉内、心脏内、皮肤内、静脉内、肌肉内、腹腔内、骨内、膀胱内、皮下、局部、经皮和经黏膜，例如通过舌下、口腔、阴道和吸入给药途径。
在本发明的一个优选的实施方案中，将所述药物作为注射剂以皮下途径给药。在一个具体的实施方案中，将所述药物通过上肢或腹部皮下注射给药至成人患者。在另一个具体的实施方案中，将所述药物通过股前外侧皮下注射给药至儿童患者。
在一个具体的实施方案中，将所述药物作为注射剂以45度进针深度给药至受试者的皮下脂肪。在一个具体的实施方案中，将所述药物作为注射剂给药至受试者时的注射点为1-3个，并给药3-21次，优选给药6-21次。
在本发明的一个实施方案中，将所述药物分两个阶段进行给药，其中第一阶段给药6次(前4次每次间隔3-4周，第5、6次每次间隔7-8周)，第二阶段给药15次(前9次每次间隔3周，后6次每次间隔4周)。在本发明的实施方案中，以300-1800μg的剂量给药所述药物。在一个优选的实施方案中，以600或900μg的剂量给药所述药物。在一个更优选的实施方案中，以900μg的剂量给药所述药物。
以下通过具体实施例来说明本发明的内容。应理解，所述具体实施例仅为说明目的，并不意味着本发明的内容仅限于具体实施例。实施例中使用的模拟抗原化合物或试剂可通过商业途径购得，或者通过本领域技术人员已知的常规方法制备得到；所使用的实验仪器可通过商业途径购得。
实施例1：模拟抗原化合物的合成和纯化
1)模拟抗原化合物的合成
本发明的模拟抗原化合物的序列为ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPA，采用Fmoc固相合成法进行合成。
固定羧基端由C端向N端延伸肽链。在Applied Biosystems 431A多肽合成仪上进行。Fmoc氨基酸为原料，上样量1mM，侧链保护为：Ser(tBu)，Thr(tBu)，Tyr(tBu)、His(Trt)、Gln(Trt)、ASP(OtBu)、Glu(OtBu)、Arg(Pmc)。选用HMP-树脂为固相载体，上样量0.25mM，原料/树脂比4：1。偶联到树脂上的氨基酸(多肽羧基端第一个氨基酸)采用对称酸酐活化法活化，其余氨基酸和棕榈酸的活化采用HOBt/DCC活化法。其中，天冬酰胺、精氨酸和棕榈酸采用双偶联，棕榈酸偶联后无脱Fmoc保护基团步骤。
2)模拟抗原化合物-树脂的裂解
选用TFA裂解液(0.75g苯酚、0.25mL乙二硫醇、0.5mL苯甲硫醚、0.5mL去离子水、10.0mL TFA)进行裂解，可最大限度地抑制副反应的发生。首先在裂解液体积为1.5mL，反应温度为25℃且反应时间为2.0小时的条件下，测定模拟抗原化合物-树脂浓度对裂解反应分离效果的影响，结果如下表1所示：
表1.模拟抗原化合物-树脂浓度对裂解反应分离效果的影响
Figure PCTCN2018120491-appb-000001
※注：以“模拟抗原化合物/模拟抗原化合物-树脂((％)”间接反映出“收率”的变化。
从表1中可以看出，当模拟抗原化合物-树脂浓度过高(53.33mg/mL)时，裂解液加入DMSO可产生沉淀，分离的模拟抗原化合物占模拟抗原化合物-树脂的26.03﹪，收率较低；当模拟抗原化合物-树脂浓度为40mg/mL时，裂解液加入DMSO不产生沉淀，分离的模拟抗原化合物占模拟抗原化合物-树脂的29.80﹪，收率较高。因此，将裂解反应中模拟抗原化合物-树脂的浓度确定为40mg/mL。
接着进一步测定裂解反应时间对裂解反应分离效果的影响，结果如下表2所示：
表2裂解反应时间对模拟抗原化合物-树脂裂解液分离纯化的影响
Figure PCTCN2018120491-appb-000002
※注：以“模拟抗原化合物/模拟抗原化合物-树脂(％)”间接反映出“收率”的变化。
从表2中可以看出，将裂解反应时间控制在1-2h，经裂解后，最终获得的模拟抗原化合物的纯合和收率均较高，因此将裂解反应时间确定为1.5小时。
3)模拟抗原化合物的纯化
在上述确定的裂解反应条件下获得含有模拟抗原化合物的裂解液，接着采用两步法对模拟抗原化合物进行纯化：首先采用体积排阻层析法进行初步纯化，层析系统：P-6000泵及AKTAexplorer 100；层析柱：直径10mm，柱长250mm，填料Sephadex LH-20；柱温：25℃；流动相：DMSO；流速：0.4ml/min。收集模拟抗原化合物组分接着使用反相高效液相色谱法进行纯化，层析系统：AP-5 50/275 POROS 50R1柱和AKTA explorer 100；层析柱：直径50mm，柱长275mm，填料POROS 50R1；柱温：34℃；流动相A：30％乙醇-10mmol/L磷酸，流动相B：90％乙醇-10mmol/L磷酸；流速：100ml/min。图1示出了模拟抗原化合物经过反相高效液相色谱法纯化后的层析图谱。经测定，经过上述两步纯化，模拟抗原化合物的纯度达到98.66±0.14％，收率为86.89±0.43％，达到了较好的精制效果。
实施例2：模拟抗原化合物-脂质体的制备
1)采用二次乳化法制备模拟抗原化合物-脂质体：将类脂成分溶于乙醚溶液中，再与实施例1所获得的模拟抗原化合物的浓缩液混合，形成乳化液(W/O)，注入磷酸盐缓冲液(PB)或水中，同时控制温度和搅拌，二次形成乳化液(W/O/W)，随着乙醚的挥发逐步形成模拟抗原化合物-脂质体。通过超滤装置和透析(透析倍数须达到200倍以上)以及10μm微孔滤膜过滤，以去除液体中可能游离的和可能发生聚集沉淀的模拟抗原化合 物-脂质体，即获得模拟抗原化合物-脂质体浓缩液。然后将0.5L模拟抗原化合物-脂质体浓缩液中加入20％甘露醇水溶液140mL，20％人血白蛋白30mL，30mL磷酸盐缓冲液，充分混匀后，按1mL/瓶的规格进行分装。E-C Apparatus公司Supermodulyo型冻干机预冷至8℃后，将上述样品放入冻干机，降温至-39℃过夜；依次开启冷凝器、真空泵及加热器，升华样品中的有机溶剂和水分，最终得到白色疏松块状物；灭菌并包装得到成品。
2)将制备的模拟抗原化合物-脂质体冻干成品用1mL无菌注射水重悬后,用0.22μm的Millipore聚碳酸酯膜滤器过滤除去杂质，取100μL的模拟抗原化合物-脂质体溶液，用无水乙醇稀释到2mL，重悬模拟抗原化合物-脂质体溶液，充分搅拌；用移液枪将制备的样品滴于支持膜(铜网)上，晾干；打开透射电镜，将载有样品的铜网放入透射电镜中进行观察，放大倍数分别为×160000，结果如图2a所示。从图2a中可以看出，模拟抗原化合物-脂质体成单个囊泡存在，大小相对均一，大部分小于100nm。
3)运用马尔文ZEN1690型激光粒度分析仪检测制备的模拟抗原化合物-脂质体粒径大小。首先将制备的模拟抗原化合物-脂质体冻干成品用1mL无菌注射水重悬后，用0.22μm的聚碳酸酯膜滤器过滤除去杂质，取100μL的模拟抗原化合物-脂质体溶液，用0.01M的PBS(pH7.4)稀释到2mL，充分搅拌，取1.2mL加入样品容器中，打开电脑程序，设定检测参数，预热机器30min，添加样品进行检测，结果如图2b所示。从图2b中可以看出，激光粒度仪分析结果与电镜结果类似，脂质体大小分布范围为30-250nm，70-95％以上脂质体直径在80-100nm范围内。
实施例3：模拟抗原化合物-脂质体在慢性乙肝患者中的治疗作用
本实施例利用实施例1中制备的模拟抗原化合物-脂质体成品，对筛选的慢性乙肝患者进行治疗，研究其在慢性乙肝疾病中的治疗作用及效果。
(1)受试对象的选择：
第一阶段(0-76周)，选择选择HBeAg阳性慢性乙肝患者360名作为治疗对象。分成以下3组，每组120例受试者：一组给予600μg模拟抗原化合物-脂质体+300μg空脂质体(600μg治疗组)；一组给予900μg模拟抗原化合物-脂质体(900μg治疗组)；一组给予900μg空脂质体(对照组)。
第二阶段(76-144周)：上述完成76周研究的三组受试者中，如果治疗后有病毒学应答但无血清学应答/有血清学应答但无病毒学应答/病毒学血清学均无应答，但愿意继续参加研究的受试者有183人(其中包含第一阶段600μg治疗组的58人，第一阶段900μg 治疗组的60人和第一阶段对照组的65人)，继续给予900μg模拟抗原化合物-脂质体治疗，并随访观察至3年(144周)。
(2)给药方式：
第一阶段：采用上臂或腹部皮下注射方式给药，45°进针，深及皮下脂肪，注射点为1-3个。600μg治疗组给予600μg模拟抗原化合物-脂质体+300μg空脂质体；900μg治疗组给予900μg模拟抗原化合物-脂质体；对照组给予900μg空脂质体。给药时间分别为第0、4、8、12、20、28周。
第二阶段：分别在第80、83、86、89、92、95、98、101、104、108、112、116、120、124和128周，皮下注射900μg模拟抗原化合物-脂质体，注射部位及方式同第一阶段，共注射15次。
(3)疗效评估：
观察在第一阶段(0-76周)及第二阶段(76-144周)中，检测经过本发明实施例1制备的模拟抗原化合物-脂质体治疗后，患者的HBeAg/抗HBe血清转换率、抗HBe转阳率、血清HBV DNA拷贝数和血清谷丙转氨酶(ALT)水平。具体地，在第一阶段治疗后，共有354例受试者被纳入意向性治疗(ITT)人群(被定义为：所有经随机化分组，至少使用了一次被研究药物、且至少有一次用药后主要疗效指标(HBeAg/抗-HBe)数据的受试者)进行下列结果统计；其中，对照组119例，1人因无用药后主要疗效指标数据被剔除；600μg治疗组119例，1人因无用药后主要疗效指标数据被剔除；900μg治疗组116例，4人因无用药后主要疗效指标数据被剔除。在第二阶段治疗后，
对照组3人，600μg治疗组3人，900μg治疗组2人因无用药后主要疗效指标数据被剔除；最后纳入统计的意向性治疗人数分别为对照组62人、600μg治疗组55人和900μg治疗组58人，共计175人。
①模拟抗原化合物-脂质体治疗后，患者发生较高HBeAg/抗HBe血清学转换率：
对于HBeAg阳性慢性乙型肝炎患者而言，HBeAg血清学转换即HBeAg消失、抗Hbe转为阳性。发生HBeAg/抗HBe血清学转换，作为慢性乙肝治疗的满意终点，标志着长期预后的改善，如肝硬化发生率降低和疾病进展减慢。在第一阶段研究结束时，对照组的HBeAg/抗HBe血清学转换率为20.2％(24/119)。整体模拟抗原化合物-脂质体药物的235例受试者(ITT人群)中，有79例受试者发生HBeAg/抗HBe血清学转换，转换率为33.6％；其中，600μg治疗组和900μg治疗组血清学转换率分别为28.6％(34/119)和38.8％(45/116)(表3)。
在第二阶段治疗结束时，相比于76周入组时的血清学转换率，原各组的患者再次接受900μg模拟抗原化合物-脂质体治疗后，血清学转换率均有较大幅度提高。其中对照组发生血清学转换的受试者比例从76周时的17.7％(11/62)提升到144周结束点时的38.7％(24/62)；600μg治疗组从76周时的20.0％(11/55)提升到144周结束点时的40.0％(22/55)，900μg治疗组从76周时的29.3％(17/58)提升到144周结束点时的34.5％(20/58)(表4)。
表3.第1阶段(0-76周)，HBeAg/抗HBe血清学转换率
Figure PCTCN2018120491-appb-000003
表4.第2阶段(76-144周)，HBeAg/抗HBe血清学转换率
Figure PCTCN2018120491-appb-000004
根据2016年美国肝病学会慢性乙型肝炎实践指南中的数据(表5)，接受本发明的模拟抗原化合物-脂质体治疗后HBeAg/抗HBe血清学转换率优于核苷类似物药物(阿德福韦、拉米夫定和恩替卡韦)和长效干扰素。
表5.临床治疗乙肝主要药物治疗后HBeAg/抗HBe血清学转换率*
Figure PCTCN2018120491-appb-000005
*数据源于“AASLD Guidelines for Treatment of Chronic Hepatitis B”，Hepatology.2016；63(1):261-83。
②模拟抗原化合物-脂质体治疗后，患者发生较高抗HBe转阳率：
HBe抗体转阳，即血清HBe抗体出现。HBe抗体能反映宿主免疫应答情况；HBe抗体转阳提示宿主体内获得了比较稳定的HBV特异性的免疫应答，有利于HBV感染的控制。在第一阶段研究结束时，对照组的抗HBe转阳率为29.4％(35/119)，整体模拟抗原化合物-脂质体药物的235例受试者(ITT人群)中，有95例受试者发生抗HBe转阳，转换率为40.4％。600μg治疗组和900μg治疗组的抗HBe转阳率分别为37.0％(44/119)和44.0％(51/116)(表6)。
在第二阶段治疗结束时(表7)，相比于76周入组时的血清学转换率，对照组和600μg治疗组的患者再次接受900μg模拟抗原化合物-脂质体治疗后，抗HBe转阳血清学转换率均有提高。其中，对照组发生血清学转换的受试者比例从76周时的35.3％(22/62)提升到144周结束点时的41.9％(26/62)；600μg治疗组从76周时的27.3％(15/55)提升到144周结束点时的47.3％(26/55)。
表6.第1阶段(0-76周)，抗HBe转阳的比例
Figure PCTCN2018120491-appb-000006
表7.第2阶段(76-144周)，抗HBe阳转的比例
Figure PCTCN2018120491-appb-000007
Figure PCTCN2018120491-appb-000008
③模拟抗原化合物-脂质体治疗后，受试者外周血HBV DNA拷贝数降低：
外周血HBV DNA是由被HBV感染的肝细胞释放到血液中形成，是反映HBV感染治疗效果的最直接的指标。外周血HBV DNA拷贝数的降低，则意味着HBV复制受到抑制，反映了治疗手段的有效性。在第一阶段研究结束时，对照组、600μg治疗组和900μg治疗组的受试者发生HBV DNA载量下降≥2个对数级的病毒学应答率，分别为40.3％(48/119)、48.7％(58/119)和50.0％(58/116)(表8)。
在第二阶段治疗结束时，对照组第76周时血清HBV DNA(IU/ml)载量下降≥2个对数级的患者的比例为27.4％(17/62)，第144周达到59.7％(37/62)。600μg治疗组第76周时血清HBV DNA(IU/ml)载量下降大于或等于2个对数级的患者比例为27.3％(15/55)，第144周达到56.4％(31/55)。900μg治疗组第76周时血清HBV DNA(IU/ml)载量下降大于或等于2个对数级的患者比例为37.9％(22/58)，第144周达到51.7％(30/58)(表9)。
表8.第1阶段(0-76周)，血清HBV DNA下降≥2个对数级的百分比
Figure PCTCN2018120491-appb-000009
表9.第2阶段(76-144周)，血清HBV DNA下降≥2个对数级的百分比
Figure PCTCN2018120491-appb-000010
④模拟抗原化合物-脂质体治疗后，受试者的血清ALT水平降低：
ALT是由肝细胞特异性产生，一般位于肝细胞内部。当肝细胞受到损伤，则大量的ALT由肝细胞内部进入到外周血，血清ALT显著升高。因此，血清ALT水平反映肝细胞损伤情况：血清ALT水平上升，提示肝脏损伤的发生；血清ALT水平的下降，则提示HBV感染的有效控制，肝脏损伤的降低。在第一阶段研究结束时，对照组、600μg治疗组和900μg治疗组的受试者ALT水平降至正常范围的比率，分别为34.5％(41/119)、37.8％(45/119)和48.3％(56/116)(表10)。
表10.第1阶段(0-76周)，ALT水平降至正常范围的比率
Figure PCTCN2018120491-appb-000011
⑤模拟抗原化合物-脂质体治疗后，受试者的肝脏病理得到改善：
肝脏病理切片是反映肝脏损伤情况的最直观的证据。选取同一名慢性乙肝患者，在模拟抗原化合物-脂质体治疗前后，以肝脏穿刺的方式获取肝脏组织，观察治疗前后肝脏病理变化情况。治疗结果如图3所示，经过两个阶段的治疗，肝脏水肿、气球样变程度明显减轻；点、灶状坏死明显减少；淋巴细胞浸润明显减少。由此可见，该药物具有可有效地改善慢性乙肝患者的肝脏病理，降低肝脏损伤，有利于乙肝感染的有效控制。
综上，本发明证明了所述模拟抗原化合物-脂质体可有效诱导HBV感染患者血清HBeAb阳转、HBeAg/HBeAb血清学转换，降低患者血清HBV DNA水平，降低血清ALT水平，修复损伤肝细胞；因此，可用于制备治疗上述HBV感染相关症状的药物。

Claims (10)

  1. 包含以下两部分的模拟抗原化合物用于制备提高HBeAg/HBeAb血清学转换率的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
  2. 包含以下两部分的模拟抗原化合物用于制备提高血清HBeAb转阳率的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
  3. 包含以下两部分的模拟抗原化合物用于制备降低血清HBV DNA水平的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
  4. 包含以下两部分的模拟抗原化合物用于制备降低血清ALT水平的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
  5. 包含以下两部分的模拟抗原化合物用于制备改善肝脏损伤的药物的用途：(1)ε-CH 3(CH 2) 14CO-(NH)-KSSQYIKANSKFIGITE和(2)AAAFLPSDFFPSVGGGDPRVRGLYFPA。
  6. 权利要求1-5中任一项的用途，其中所述模拟抗原化合物的部分(1)中的CH 3(CH 2) 14CO被CH 3(CH 2) 16CO替换，所述模拟抗原化合物的部分(2)中的AAA序列和/或GGG序列被SSS替换，或者其中所述模拟抗原化合物的部分(1)和部分(2)通过共价键链接。
  7. 权利要求1-5中任一项的用途，其中所述模拟抗原化合物被配制为由脂质体包裹形成的模拟抗原化合物-脂质体；优选地，所述模拟抗原化合物-脂质体的直径为80-100nm；或者其中所述模拟抗原化合物-脂质体为药学上可接受的任意剂型；优选地，所述模拟抗原化合物-脂质体为液体剂型或冻干剂型，更优选为冻干剂型。
  8. 权利要求1-5中任一项的用途，其中所述药物还包含药学上可接受的佐剂、载体或辅料；优选地，所述药物为注射剂、透皮剂、口服剂、吸入剂或栓剂；更优选地，所述药物为注射剂。
  9. 权利要求1-5中任一项的用途，其中所述药物以任何给药途径进行给药，所述给药途径包括但不限于口腔、直肠、鼻、肺、硬膜外、眼、耳、动脉内、心脏内、皮肤内、静脉内、肌肉内、腹腔内、骨内、膀胱内、皮下、局部、经皮和经黏膜，例如通过舌下、 口腔、阴道和吸入给药途径；优选地，将所述药物作为注射剂以皮下途径给药；更优选地，将所述药物通过上肢或腹部皮下注射给药至成人患者或将所述药物通过股前外侧皮下注射给药至儿童患者；并且其中将所述药物作为注射剂以45度进针深度给药至受试者的皮下脂肪；优选地，将所述药物作为注射剂给药至受试者时的注射点为1-3个，并给药3-21次，优选给药6-21次。
  10. 权利要求1-5中任一项的用途，其中将所述药物分两个阶段进行给药，在第一阶段给药6次，其中前4次每次间隔3-4周，第5、6次每次间隔7-8周；在第二阶段给药15次，其中前9次每次间隔3周，后6次每次间隔4周；并且其中将所述药物以300-1800μg的剂量进行给药所述药物；优选地，将所述药物以600或900μg的剂量进行给药所述药物；更优选地，将所述药物以900μg的剂量进行给药。
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CN101717433A (zh) * 2009-12-08 2010-06-02 张嘎 一种多肽免疫原及其制备方法和应用
CN107028887A (zh) * 2017-03-28 2017-08-11 江苏孟德尔基因科技有限公司 脂质体用于治疗慢性乙型病毒性肝炎的用途

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