WO2021223422A1 - 一种经大分子量peg修饰的抗hiv多肽及其制备方法和用途 - Google Patents

一种经大分子量peg修饰的抗hiv多肽及其制备方法和用途 Download PDF

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WO2021223422A1
WO2021223422A1 PCT/CN2020/132063 CN2020132063W WO2021223422A1 WO 2021223422 A1 WO2021223422 A1 WO 2021223422A1 CN 2020132063 W CN2020132063 W CN 2020132063W WO 2021223422 A1 WO2021223422 A1 WO 2021223422A1
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peg
modified
formula
peptide
hiv
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PCT/CN2020/132063
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English (en)
French (fr)
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李学兵
程水红
李明莉
马丽英
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中国科学院微生物研究所
中国疾病预防控制中心性病艾滋病预防控制中心
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Publication of WO2021223422A1 publication Critical patent/WO2021223422A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This application belongs to the field of biopharmaceuticals, and relates to polyethylene glycol (PEG) modified anti-HIV polypeptides and preparation methods and uses thereof.
  • PEG polyethylene glycol
  • HIV human immunodeficiency virus
  • HIV can cause serious damage to the body's immune system and pose a great threat to human life and health. Since it was first discovered in 1981, its prevention has not been able to obtain an effective solution.
  • AIDS has shown a trend of an explosive epidemic on a global scale, and there are about 34 million people living with HIV worldwide. According to statistics from the Ministry of Health of my country, as of the end of 2011, the number of people living with HIV and PLHIV (PLHIV) in China has reached 780,000 (620,000 to 940,000). Due to the high mutation rate of the virus, no effective cure for AIDS has yet been found.
  • C34 molecules were used as the first generation of fusion inhibitors, which have problems such as poor water solubility and short half-life. Many researchers have modified C34 molecules with various groups to improve these problems, but there are few studies on the modification of macromolecules.
  • PEG modification of C34 peptide with larger molecular weight PEG can greatly improve its water solubility, significantly prolong the plasma half-life in SD rats, and prevent acute infections.
  • the rhesus monkey of the SHIV virus has a significant protective effect.
  • the present invention provides a modified C34 peptide having the structure of Formula I:
  • Z is cysteine, and the cysteine is connected to an amino acid in the peptide chain of formula I through an amide bond;
  • Y is a group of formula III:
  • m is a methoxy group
  • the molecular weight of the PEG is 10,000 or more.
  • the molecular weight of the PEG is about 10,000 to 100,000, preferably 10,000 to 50,000.
  • the present application provides a method for preparing the PEG-modified C34 peptide as described in the first aspect, the method comprising:
  • Z is cysteine, and the cysteine is connected to an amino acid in the peptide chain of formula VI through an amide bond; c is 0 or 1; d is 0 or 1; p is 0 or 1; q is 0 or 1; c and d are not at the same time as 0; p, q and d are not at the same time as 0; Ac means that the N-terminal of the peptide chain of formula VI has an acetylation modification; NH 2 means that the C-terminal of the peptide chain of formula VI has an amide Chemical modification
  • Y is a group of formula III:
  • m is a methoxy group, wherein the molecular weight of the PEG is 10,000 or more;
  • the application provides a pharmaceutical composition comprising the PEG-modified C34 peptide as described in the first aspect, and optionally a pharmaceutically acceptable carrier.
  • this application provides the use of the PEG-modified C34 peptide as described in the first aspect in the preparation of drugs for anti-HIV virus and/or inhibition of HIV virus activity and/or treatment and prevention of diseases related to HIV infection application.
  • the application provides the PEG-modified C34 peptide as described in the first aspect in the preparation of a pharmaceutical composition for anti-HIV virus and/or inhibition of HIV virus activity and/or treatment and prevention of diseases related to HIV infection In the application.
  • the present application provides a method for inhibiting the activity of HIV virus, the method comprising providing a therapeutically effective amount of the PEG-modified C34 as described in the first aspect of the present invention to a subject in need A peptide or a pharmaceutical composition comprising the PEG-modified C34 peptide.
  • the present application also provides a method for the treatment and/or prevention and/or adjuvant treatment of HIV infection-related diseases, the method comprising providing a subject in need with a therapeutically effective amount of the first aspect of the present invention
  • the PEG-modified C34 peptide or a pharmaceutical composition containing the PEG-modified C34 peptide comprising
  • C34 peptides modified with PEG with a larger molecular weight for example, PEG with a molecular weight of more than 10,000 have obvious advantages. Increased water solubility and half-life of rat plasma make it easier to enter the lymph nodes and have strong anti-HIV virus activity against rhesus monkeys acutely infected with SIV SF162P3.
  • FIG. 1 is a MALDI-TOF mass spectrum of PEG 10k NC prepared according to Example 1.
  • Figure 4 is a graph showing the dilution factor required to inhibit 50% of the virus using rat serum collected at various time points after administration.
  • Figure 5 is a graph showing the concentration of C34NC or PEG 40K NC in rat serum collected at various time points after administration.
  • Fig. 6 is a graph showing the plasma SIV RNA load of the PEG 40K NC experimental group and the normal saline control group at different time points after virus infection.
  • the anti-HIV virus activity can also be referred to as anti-virus activity.
  • it can be the inhibition of virus cell fusion and/or the inhibition of virus invasion into cells and/or the inhibition of virus replication.
  • C34 polypeptide is a polypeptide derived from the CHR (C-terminal heptad repeat) region of HIV-1 transmembrane protein gp41, By binding to the NHR (N-terminal heptad repeat) region of gp41, the formation of the 6-helix bundle is inhibited, thereby preventing the invasion of the virus.
  • C34 peptide is the first generation fusion inhibitor against HIV.
  • the C34 peptide commonly used in the art has the following amino acid sequence: Ac-WMEWD REINN YTSLI HSLIE ESQNQ QEKNE QELL-NH 2 (SEQ ID NO: 1), where Ac and NH 2 respectively represent acetylation at the N-terminus of the peptide chain Modification and amidation modification at the C-terminus.
  • the 10th amino acid N on C34 is a conservative natural glycosylation site on the C-terminal polypeptide of HIV transmembrane protein gp41.
  • one or more cysteines are added to the N-terminal, C-terminal or 10th position of the C34 peptide, and the maleamide-modified polyethylene glycol group is added through the Michael addition reaction. Coupling to this cysteine does not affect the anti-HIV activity of the C34 peptide.
  • PEG polyethylene glycol
  • PEG polyether compound widely used in industrial production and medicine.
  • PEG is prepared by polymerizing ethylene oxide and is commercially available in a wide molecular weight range of 300 g/mol to 10,000,000 g/mol.
  • PEGs with different molecular weights are used for different applications, and have different physical properties such as viscosity, due to the effect of chain length, their chemical properties are almost the same.
  • the most common PEG is monofunctional methyl ether PEG (methoxy polyethylene glycol), abbreviated as mPEG.
  • mPEG monofunctional methyl ether PEG
  • Branched or star-shaped PEG has about 3 to 100 PEG chains (arms) emanating from the central core group.
  • Comb PEG has multiple PEG chains that are usually grafted to the polymer backbone. Any such PEG polymer can be used for the PEG-modified C34 peptide of the present invention as defined above.
  • the term "PEG” or "PEG polymer” preferably encompasses any of the above-defined polymers.
  • the PEG/PEG polymer used for the PEG-modified C34 peptide of the present invention as defined above is preferably selected from the PEG polymer as defined above, preferably between about 10000 g/mol and 100000 g /mol, more preferably within a molecular weight of about 10,000 to 50,000 Da, etc., most preferably within a molecular weight of about 10k to 50kDa, for example, within a molecular weight of about 10k to about 40kDa, such as about 10kDa, about 20kDa, about 30kDa, about 40kDa, about 50kDa.
  • the PEG/PEG polymer used in the PEG-modified C34 peptide of the present invention as defined above can be selected from any PEG polymer as defined above, and can have different geometric shapes, for example, as herein A branched PEG having about 3 to 10 PEG chains emitted from a central core group or, for example, a star PEG having about 10 to 100 PEG chains emitted from a central core group is defined.
  • the PEG/PEG polymer used in the PEG-modified C34 peptide of the present invention as defined above can be selected from any such PEG polymer as defined herein, more preferably selected from having A PEG polymer of a defined molecular weight, and preferably, has a different geometric shape as defined herein, for example, having about 3 to about 10 PEG chains (arms) emitted from the central core group, preferably having A branched PEG of about 3 to about 5 PEG chains (arms) emitted from the central core group.
  • Particularly preferably used in the branched PEG of the present invention as defined above may be a PEG polymer as defined above, which has 3, 4, 5, 6, 7, 8, 9, 10 emanating from the central core group. Or more chains (arms).
  • the molecular weight of PEG means the average molecular weight of PEG.
  • the modified C34 peptide of the present invention has the structure of formula I:
  • Z is cysteine, and the cysteine is connected to an amino acid in the peptide chain of formula I through an amide bond;
  • Y is a group of formula III:
  • m is a methoxy group
  • the molecular weight of the PEG is 10,000 or more, preferably 10,000 to 100,000, more preferably 10,000 to 50,000.
  • the sum of p, q, and d is 1.
  • c is 0 and d is 1. In a preferred embodiment, c is 0, d is 1, and both p and q are 0.
  • c is 1 and d is 0; and one of p and q is 1, and the other is 0.
  • c is 1, d is 0; and p is 1, q is 0.
  • c is 1 and d is 0; and p is 0 and q is 1.
  • Y in Formula II may be one or more of PEG or PEG polymers shown in the following formula:
  • n is an integer, the value of n depends on the required molecular weight of PEG, and in the above formula, n can be the same or different from each other.
  • the PEG-modified C34 peptide of the present invention has any structure selected from the following:
  • the PEG-modified C34 peptide of the present invention has any structure selected from the following:
  • PEG 10000 , PEG 20000 , PEG 40000 and/or PEG 10K , PEG 20K , PEG 40K refer to polyethylene glycols with average molecular weights of 10,000, 20,000 and 40,000, respectively.
  • the present application provides a method for preparing the PEG-modified C34 peptide as described above, the method comprising:
  • Z is cysteine, and the cysteine is connected to an amino acid in the peptide chain of formula VI through an amide bond; c is 0 or 1; d is 0 or 1; p is 0 or 1; q is 0 or 1; c and d are not at the same time as 0; p, q and d are not at the same time as 0; Ac means that the N-terminal of the peptide chain of formula VI has an acetylation modification; NH 2 means that the C-terminal of the peptide chain of formula VI has an amide Chemical modification
  • Y is a group of formula III:
  • m is a methoxy group, wherein the molecular weight of the PEG is 10,000 or more;
  • the PEG shown in Y (ie, formula III) is as defined above. PEGs of different molecular weights can be obtained commercially according to requirements.
  • the compound represented by formula V can be synthesized based on the prior art.
  • the compound of formula VI has any of the following sequences:
  • formula VI can be obtained by chemical synthesis of peptides, or can also be produced by genetic engineering.
  • the method of peptide synthesis can be carried out by methods known in the art.
  • the purification of peptides is well known in the art, and HPLC can be used, for example. Descriptions of useful peptide synthesis methods and purification methods can be easily obtained in the art.
  • methods such as modification of peptides can be performed by methods known in the art.
  • the addition reaction is carried out at a pH of 8.0-9.0.
  • the addition reaction is performed at a pH of 8.0, 8.5, or 9.0.
  • the addition reaction is carried out at a pH of 8.5.
  • the addition reaction is carried out at room temperature (for example, 20°C-30°C).
  • the addition reaction is carried out in a buffer.
  • the buffer is a weak acid and/or weak base salt buffer, for example, a phosphate buffer.
  • the phosphate buffer is an aqueous solution of 50 mM sodium dihydrogen phosphate and 50 mM disodium hydrogen phosphate.
  • the PEG-modified C34 peptide of the present invention has anti-HIV virus (HIV-1 NL4-3 type virus) activity.
  • the PEG-modified C34 peptide of the present invention can be used to treat and/or prevent AIDS.
  • the PEG-modified C34 peptide of the present invention can be used as an active ingredient of medicines or vaccines.
  • the present invention also provides the application of the PEG-modified C34 polypeptide according to the present invention in the preparation of an anti-HIV virus pharmaceutical composition, wherein the PEG-modified C34 peptide is used as an active ingredient.
  • the present application provides a method for inhibiting the activity of the HIV virus, the method comprising providing a therapeutically effective amount of the PEG-modified C34 peptide according to the present invention to a subject in need.
  • the present application also provides a method for the treatment and/or prevention and/or adjuvant treatment of HIV virus infection-related diseases, the method comprising providing a subject in need with a therapeutically effective amount of the compound described in the present invention C34 peptide modified by PEG.
  • the PEG-modified C34 peptide is administered in a pure form, or in a substantially pure form, but can also be administered as a pharmaceutical composition, formulation, or preparation.
  • the PEG-modified C34 peptide described herein can be provided in the form of a pharmaceutically acceptable (e.g., sterile) composition, preparation, or preparation. Therefore, another aspect described herein is a pharmaceutical composition comprising a PEG-modified C34 peptide and optionally a pharmaceutically acceptable carrier. These pharmaceutically acceptable compositions contain an effective amount of one or more of the PEG-modified C34 peptides described herein, the PEG-modified C34 peptides and one or more pharmaceutically acceptable carriers (additives). ) And/or diluent.
  • the pharmaceutical composition is directly administered to the subject as a drug, or is mixed with a pharmaceutically acceptable carrier or excipient and then administered to the subject, so as to achieve the purpose of treating and/or preventing HIV infection or HIV infection-related diseases.
  • the carrier material herein can be widely used in various carriers known in the art, for example, including but not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose). , Cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred.
  • the use of these materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Buccal tablets, suppositories, freeze-dried powder injections, etc. It can be general preparations, sustained-release preparations, controlled-release preparations, and various particulate drug delivery systems. In addition, if necessary, coloring agents, preservatives, flavors, flavors, sweeteners, or other materials can also be added to the pharmaceutical preparations.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavity injection, etc.; cavity administration, such as rectum and vagina; respiratory administration, such as nasal cavity; mucosal administration.
  • the above-mentioned administration route is preferably injection administration.
  • administering means using any of a variety of methods and delivery systems known to those skilled in the art to physically introduce a composition containing a therapeutic agent to a subject.
  • the administration route of the PEG-modified C34 peptide and its pharmaceutical composition of the present invention includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and local administration, which are usually performed by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, and intralymphatic , Intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions , And electroporation in vivo.
  • the PEG-modified C34 peptides and pharmaceutical compositions thereof are administered by non-parenteral routes, and in some embodiments, they are administered by injection.
  • Other non-parenteral routes include topical, epidermal or mucosal administration routes, for example, intranasal, vaginal, rectal, sublingual, or topical. Administration can also be performed, for example, once, multiple times, and/or over one or more extended periods of time.
  • the term "pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. Without excessive toxicity, irritation, allergic reactions or other problems or complications, it is commensurate with a reasonable benefit/risk ratio.
  • the term “subject” refers to mammals, such as rodents, felines, canines, and primates (such as monkeys, apes, etc.). Preferably, the subject according to the present application is a human.
  • the terms “subject” and “patient” are used interchangeably in certain contexts herein.
  • the "therapeutically effective amount" of a drug or therapeutic agent described herein is any amount of a drug that protects a subject from HIV infection or promotes the regression of HIV infection-related diseases when used alone or in combination with another therapeutic agent, so The regression of the disease is evidenced by a reduction in the severity of the disease symptoms, an increase in the frequency and duration of the symptom-free phase, or the prevention of injury or disability caused by the torture of the disease.
  • the ability of therapeutic agents to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by in vitro In the assay method, the activity of the agent is determined.
  • the therapeutically effective amount of a drug includes a "prophylactically effective amount", which when administered to a subject at risk of developing HIV infection or a subject at risk of HIV infection-related diseases, alone or in combination with other therapeutic agents, inhibits Any amount of medication for the occurrence of HIV infection or the onset of HIV infection-related diseases.
  • the prophylactically effective amount completely prevents the occurrence of HIV infection or the onset of HIV infection-related diseases.
  • the dosage of the PEG-modified C34 peptide and its pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight and individual response of the patient or animal, and the Specific active ingredients, route of administration and frequency of administration, etc.
  • the above-mentioned dosage can be administered in a single dosage form or divided into several, for example, two, three or four dosage forms.
  • the PEG-modified C34 peptide and its pharmaceutical composition of the present invention can be directly used alone for the treatment and prevention of HIV-infected persons, or can be used in combination with one or more anti-HIV drugs to achieve the purpose of improving the overall therapeutic effect.
  • anti-HIV drugs include but are not limited to reverse transcriptase inhibitors, protease inhibitors, invasion inhibitors, integration inhibitors and maturation inhibitors.
  • the above-mentioned reverse transcriptase inhibitor can be one or more of AZT, 3TC, ddI, d4T, ddT, TDF, Abacavir, Nevirapine, Efavirenz and Delavirdine;
  • the above-mentioned protease inhibitor can be Saquinavir mesylate, Idinavir, Ritonavir, One or more of Amprenavir, Kaletra, and Nelfinavir mesylate;
  • the above-mentioned invasion inhibitor can be Maraviroc, TAK-779, T-20, T2635, Sifuvirtide, Abvirtide, VIRIP (VIR-576), etc.
  • One or more of the above-mentioned integration inhibitors can be one or more of Raltegravir, Dolutegravir and Elvitegravi.
  • the specific therapeutically effective dose level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient used; the specific composition used The age, weight, general health status, gender and diet of the patient; the administration time, route of administration and excretion rate of the specific active ingredient used; the duration of treatment; the combination or simultaneous use with the specific active ingredient used Drugs; and similar factors well known in the medical field.
  • the practice in the art is to start the dosage of the active ingredient from a level lower than the level required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained.
  • the dosage of the PEG-modified C34 peptide and its pharmaceutical composition of the present invention for mammals, especially humans, can be between 0.001-1000 mg/kg body weight/day, for example, between 0.01-100 mg/kg body weight/day , For example, between 0.1-10mg/kg body weight/day.
  • kits comprising the PEG-modified C34 peptide described herein, the pharmaceutical composition containing the PEG-modified C34 peptide described herein, or for the manufacture of the PEG-modified C34 peptide described herein A component of the PEG-modified C34 peptide or a component for the manufacture of a pharmaceutical composition comprising the PEG-modified C34 peptide described herein.
  • the kit may also contain information materials.
  • the information materials may be explanatory materials, instructional materials, sales materials or other materials related to the methods described herein and/or the use of the PEG-modified C34 peptides in the methods described herein.
  • the information material describes a method of administering the PEG-modified C34 peptide to a subject.
  • the kit may also include a delivery device.
  • This application provides the application of the PEG-modified C34 peptide as described herein in the preparation of drugs for anti-HIV virus and/or inhibiting the activity of HIV virus and/or treating and preventing diseases related to HIV virus infection.
  • This application provides the application of the PEG-modified C34 peptide as described herein in the preparation of a pharmaceutical composition for anti-HIV virus and/or inhibition of HIV virus activity and/or treatment and prevention of diseases related to HIV virus infection.
  • the PEG-modified C34 peptide is used as an active ingredient.
  • Z is cysteine, and the cysteine is connected to an amino acid in the peptide chain of formula I through an amide bond;
  • Y is a group of formula III:
  • m is a methoxy group
  • the molecular weight of the PEG is 10,000 or more.
  • n is an integer other than zero.
  • Z is cysteine, and the cysteine is connected to an amino acid in the peptide chain of formula VI through an amide bond; c is 0 or 1; d is 0 or 1; p is 0 or 1; q is 0 or 1; c and d are not at the same time as 0; p, q and d are not at the same time as 0; Ac means that the N-terminal of the peptide chain of formula VI has an acetylation modification; NH 2 means that the C-terminal of the peptide chain of formula VI has an amide Chemical modification
  • Y is a group of formula III:
  • m is a methoxy group, wherein the molecular weight of the PEG is 10,000 or more;
  • n is an integer greater than zero.
  • a pharmaceutical composition comprising the PEG-modified C34 peptide according to any one of paragraphs 1-10 and optionally a pharmaceutically acceptable carrier and/or excipient.
  • the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources or can be prepared by reference to literature.
  • the reagents used in the following examples, unless otherwise specified, are of analytical grade.
  • the room temperature reaction specifically refers to controlling the reaction temperature within the range of 20°C to 30°C, including 20°C and 30°C.
  • the compounds mPEG 10k Mal, mPEG 20k Mal and mPEG 40k Mal were purchased from Sinobanger Biotechnology Co., Ltd., with a purity of 95%.
  • n is an integer greater than zero, so that the molecular weight of the PEG part is 10,000, 20,000, and 40,000, respectively.
  • Sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution 50 mM sodium dihydrogen phosphate, 50 mM disodium hydrogen phosphate, soluble in water, pH 8.0-9.0.
  • the mass spectrometer adopts MALDI-TOF mass spectrometer mass spectrometer.
  • PEG 10k NC was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to verify its correctness.
  • MALDI-TOF-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • the mass spectrum is shown in Figure 1.
  • An Agilent 1200 analytical high performance liquid chromatograph was used to detect the purity of PEG 10k NC.
  • Eluent mobile phase A (aqueous solution containing 0.1% trifluoroacetic acid by volume) and mobile phase B (an acetonitrile solution containing 0.1% trifluoroacetic acid by volume); elution conditions: 10%A to 100%A linear gradient elution, time 25min, elution flow rate is 1mL/min, UV detection wavelength is 220nm. Analytical high performance liquid chromatograph test results show that the purity of the obtained PEG 10k NC is 96%.
  • Example 2 Except that the mPEG 10k Mal in Example 1 was replaced with mPEG 20k Mal, the preparation, purification, and characterization of the method in Example 1 were followed to obtain PEG 20k NC (with a purity of 97.5%). The mass spectrum result is shown in FIG. 2.
  • Example 3 Except that the mPEG 10k Mal in Example 1 was replaced with mPEG 40k Mal, the preparation, purification, and characterization of the method in Example 1 were followed to obtain PEG 40k NC (purity 98%).
  • the mass spectrum result is shown in FIG. 3.
  • Example 1 The PEG-modified C34 peptides obtained in Example 1 to Example 3 were evaluated for water solubility.
  • the water solubility of C34 modified with large molecular weight PEG (molecular weight above 10,000) is significantly greater than that of unmodified C34, and it is also greater than C34 modified with a smaller molecular weight (for example, molecular weight less than 10,000, including 2000, 5000 etc.).
  • Effect example 2 In vitro inhibitory effect of the PEG-modified polypeptide of the present invention on HIV-1 NL4-3 type virus
  • HIV-1 NL4-3 type virus was obtained from the NIH AIDS Research and Reference Reagent Program.
  • the TZM-bl cells (NIH AIDS Research and Reference Reagent Program ) at 104 / well in 96 well plates, placed 37 °C, 5% CO 2 incubator overnight.
  • the medium is DMEM medium.
  • the peptide samples ie PEG 10K NC, PEG 20K NC and PEG 40K NC prepared in Example 1 to Example 3, as well as C34 and C34NC
  • DMEM medium 8 dilutions (concentrations of 64, 32, and 16 respectively).
  • 8, 4, 2, 1, 0.5nM Dilute the virus to be tested (ie HIV-1 NL4-3 type virus) into 2000 TICD 50 /mL with DEAE culture medium (containing 20 ⁇ g/mL DEAE-dextran).
  • a virus control group (VC, only adding 100 ⁇ L of DMEM medium and 100 ⁇ L of diluted virus) and a cell control group (CC, only adding 200 ⁇ L of DMEM medium) were established.
  • Table 1 EC 50 value and EC 90 value of each sample
  • Peptide sample EC 50 (nM) EC 90 (nM) C34 1.71 ⁇ 0.34 11.01 ⁇ 4.09 C34NC 0.67 ⁇ 0.29 3.80 ⁇ 0.62 PEG 10K NC 7.10 ⁇ 4.42 57.97 ⁇ 20.64 PEG 20K NC 22.20 ⁇ 13.42 101.42 ⁇ 24.04 PEG 40K NC 18.51 ⁇ 10.87 132.49 ⁇ 31.59
  • mice were SD rats (7 weeks old) from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., weighing 180-200 grams.
  • the 4 SD rats were randomly divided into 2 groups, 2 in each group (1 male and 1 female), which were used to collect plasma. Rats in each group were injected subcutaneously with a single subcutaneous injection of C34NC and PEG 40K NC at a dose of 1.7 ⁇ mol/kg (a small amount of borax buffer with pH 9.5 was added to physiological saline as a solubilizer).
  • Sample collection mark the time before administration as zero time, collect whole blood (300 ⁇ L) from the tail vein at zero time and at different time points after administration.
  • the time points in the C34NC group are 0.5h, 1.0h, 2.0h, 4.0h, 6.0h, 8.0h, 10.0h; the time points of the PEG 40K NC group were 3h, 6h, 9h, 20h, 23h, 29h, 48h, 72h.
  • Each whole blood sample was added to an EP tube containing 50 ⁇ L heparin sodium (83U/Ml) and 10 ⁇ L aprotinin (5ug/mL), centrifuged at 4500g at 4°C for 10 min, and the upper serum sample was collected and stored in a refrigerator at -80°C.
  • the concentration of the samples at different time points was estimated by measuring the in vitro inhibition of the collected serum samples against HIV NL4-3 virus.
  • Specific experimental procedure is as follows: The well grown digestion TZM-bl cells plated 96 well plates (10 4 cells / well), 37 °C, CO 2 incubator overnight culture; The next day, cells were diluted serum DMEM complete medium, The final dilution of the first well of serum is 10-fold dilution, and then sequential 2-fold dilutions, a total of 8 dilution gradients; aspirate the cell culture medium, add 100 ⁇ L of diluted serum to each well, and add 100 ⁇ l of virus containing 200TCID 50 to each well.
  • the drug concentration at each time point was obtained according to the standard curve, and the pharmacokinetic parameters were calculated by the non-compartmental model in the Winnonlin pharmacokinetic software.
  • the treatment group was injected subcutaneously with 1.38 ⁇ mol/kg PEG 40K NC ( Physiological saline solution) for 28 consecutive days; the control group was injected with 0.9% physiologic saline solution every day for 28 days starting from the 7th day after virus inoculation.
  • PEG 40K NC Physiological saline solution
  • RNA samples of rhesus monkeys were collected on the 1, 4, 7, 11, 14, 18, 21, 25, 28, 32, 36, 42, 49 and 70 days after virus inoculation.
  • a QIAmp viral RNA minikit (Qiagen, Valencia, CA) was used to extract and purify viral RNA from cell-free plasma, and then the RNA was eluted with an elution buffer (Qiagen, Valencia, CA) and analyzed immediately.
  • RNA standards (10 3 -10 13 copies/ml) are prepared and stored in the laboratory.
  • the one-step PrimeScript RT-PCR Kit provided by TaKaRa was used in the experiment, and the Perkin-Elmer ABI 7500 was used as the experimental instrument.
  • the C34 peptide modified with the large molecular weight PEG provided by the present invention can more significantly increase the water solubility of the C34 peptide, and has a significantly prolonged plasma half-life in rats, and has better anti-HIV activity in vivo.
  • the C34 peptide modified with large molecular weight PEG (molecular weight above 10,000) provided by the present invention is expected to become a good water-soluble and long-acting anti-HIV polypeptide drug.

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Abstract

经PEG修饰的抗HIV多肽。具体而言，在C34多肽的N端、C端或第10位添加半胱氨酸，并通过Michael加成反应将具有马来酰胺修饰的聚乙二醇基团偶联至所述半胱氨酸，对C34多肽进行PEG化修饰，其中，PEG部分的分子量为10000以上，优选为10000-100000，更优选为10000-50000。该经修饰的C34肽具有明显增加的水溶性和大鼠血浆半衰期，更易进入淋巴结且对急性感染SHIV SF162P3的恒河猴具有较强的抗HIV病毒活性。还涉及该经PEG修饰的抗HIV多肽的制备方法及用途。

Description

一种经大分子量PEG修饰的抗HIV多肽及其制备方法和用途
相关申请的交叉引用
本申请要求于2020年05月07日向中国国家知识产权局提交的第202010375950.8号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请属于生物制药领域，涉及经聚乙二醇(PEG)修饰的抗HIV多肽及其制备方法和用途。
背景技术
艾滋病是由人类免疫缺陷病毒(HIV)感染引起的具有极高致死率的疾病，HIV会导致全身免疫系统的严重损害，给人类生命健康安全造成极大威胁。自1981年首次被发现以来，其防治一直没能获得有效的解决。目前在全球范围内艾滋病已经呈现出了爆发性流行的趋势，全世界HIV携带者已达到3400万人左右。据我国卫生部统计估算，截至2011年底，中国存活艾滋病病毒携带者和艾滋病病人(PLHIV)已达到78万人(62～94万人)。由于该病毒的高突变率，目前尚未寻找到有效的治愈艾滋病的方法。
目前上市的HIV的治疗性药物多为针对病毒逆转录酶、整合酶、蛋白酶等的一些小分子酶抑制剂。由于HIV的超高突变性，使得这些小分子特效药物正在逐渐退化为常效、甚至无效药物。近年来，随着HIV入侵宿主细胞分子机制的逐步阐明，抑制该过程的多肽药物逐渐成为艾滋病防治领域的研究热点。这类多肽药物例如第一代侵入抑制剂C34和T20，其作用机理在于可特异性结合HIV囊膜上的融合蛋白，从而抑制病毒进入宿主细胞。
C34分子被用作第一代融合抑制剂，其存在水溶性差和半衰期短等问题，许多学者曾对C34分子进行多种基团修饰来改善这些问题，但关于大分子修饰的研究较少。
本课题组在专利申请201810547641.7公开了经修饰的C34肽，其中，经PEG化修饰的C34肽的水溶性由≤1mg/mL提高至3mg/mL以上，从而延长了C34分子在大鼠体内的血浆半衰期。该研究主要集中在PEG分子量为700-5000的修饰。
本发明人经过进一步的研究发现，以更大分子量的PEG对C34肽进行PEG修饰，可使其水溶性获得很大程度的改善，在SD大鼠体内的血浆半衰期得到明显延长，且对急性感染SHIV病毒的恒河猴有显著的保护效果。
发明内容
在第一方面，本发明提供了一种经修饰的C34肽，所述经修饰的C34肽具有式I的结构：
Ac-B pWMEWDREINN cB dYTSLIHSLIEESQNQQEKNEQELLB q-NH 2  式I；
其中，c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式I肽链的N端带有乙酰化修饰；NH 2表示式I肽链的C端带有酰胺化修饰；B为式II的基团
Figure PCTCN2020132063-appb-000001
式II中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式I肽链中的氨基酸相连；Y为式III的基团：
mPEG                    式III；
式III中，m为甲氧基，
其中，所述PEG的分子量为10000以上。
在优选的实施方式中，所述PEG的分子量为约10000-100000，优选为10000-50000。
在第二方面，本申请提供了制备如第一方面所述的经PEG修饰的C34肽的方法，所述方法包括：
(1)提供式V以及式VI的化合物
Figure PCTCN2020132063-appb-000002
Ac-Z pWMEWDREINN cZ dYTSLIHSLIEESQNQQEKNEQELLZ q-NH 2      式VI；
式VI中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式VI肽链中的氨基酸相连；c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式VI肽链的N端带有乙酰化修饰；NH 2表示式VI肽链的C端带有酰胺化修饰；
式V中，Y为式III的基团：
mPEG                       式III；
式III中，m为甲氧基，其中，所述PEG的分子量为10000以上；以及
(2)使得式VI化合物中Z的巯基与式V中的马来酰胺发生加成反应，获得所述经PEG修饰的C34肽。
在第三方面，本申请提供了一种药物组合物，所述药物组合物包含如第一方面所述的经PEG修饰的C34肽，并任选地包含药学上可接受的载体。
在第四方面，本申请提供了如第一方面所述的经PEG修饰的C34肽在制备抗HIV病毒和/或抑制HIV病毒活性和/或治疗和预防HIV病毒感染相关的疾病的药物中的应用。
在第五方面，本申请提供了如第一方面所述的经PEG修饰的C34肽在制备抗HIV病毒和/或抑制HIV病毒活性和/或治疗和预防HIV病毒感染相关的疾病的药物组合物中的应用。
在本申请进一步的方面，本申请提供了一种抑制HIV病毒活性的方法，所述方法包括向有需要的受试者提供治疗有效量的如本发明第一方面所述的经PEG修饰的C34肽或包含所述经PEG修饰的C34肽的药物组合物。
此外，本申请还提供了一种治疗和/或预防和/或辅助治疗HIV病毒感染相关的疾病的方法，所述方法包括向有需要的受试者提供治疗有效量的如本发明第一方面所述的经PEG修饰的C34肽或包含所述经PEG修饰的C34肽的药物组合物。
有益效果
本发明人发现相比现有技术中报道的PEG修饰的C34肽(其中PEG的分子量为700-5000)，采用分子量更大的PEG(例如，分子量在10000以上的PEG)修饰的C34肽具有明显增加的水溶性和大鼠血浆半衰期，更易进入淋巴结且对急性感染SHIV SF162P3的恒河猴具有较强的抗HIV病毒活性。尤其是PEG 40k修饰的C34肽，相比具有更小分子量的PEG(PEG2000、PEG5000等)修饰的C34肽，其水溶性以及体内半衰期得到明显改善，这使得其更易于成药，并且使得C34肽的体内长效稳定性得到显著提升。
附图说明
图1为根据实施例1制备的PEG 10kNC的MALDI-TOF质谱图。
图2为根据实施例2制备的PEG 20kNC的MALDI-TOF质谱图。
图3为根据实施例3制备的PEG 40kNC的MALDI-TOF质谱图。
图4为示出了使用给药后各个时间点采集的大鼠血清抑制50％病毒所需的稀释倍数的图。
图5为示出了给药后各个时间点采集的大鼠血清中C34NC或PEG 40KNC的浓度的图。
图6为示出了病毒感染后不同时间点PEG 40KNC实验组和生理盐水对照组的血浆SHIV RNA载量的图。
具体实施方式
在下面详细描述本发明之前，应理解的是，本发明并不局限于本文所述的特定的方法、方案和试剂，因为它们还可有所变化。还应理解的是，本文使用的术语仅出于描述具体实施方式的目的，而不旨在限制本发明的范围，本发明的范围仅为所附的权利要求所限。
不论在上文或下文中，本文引用的各文献(包括全部专利、专利申请、科学出版物、制造商说明书、使用说明等)均通过引用的方式整体并入本文。
除非另有定义，本文使用的所有技术术语和科学术语具有与本领域普通技术人员通常所理解的含义相同的含义。
在本发明中，所述抗HIV病毒活性也可称为抑制病毒活性，例如，可为抑制病毒进行细胞融合和/或抑制病毒侵入细胞和/或抑制病毒复制。
本文使用的术语“C34多肽”、“C34肽”或“C34”是由HIV-1跨膜蛋白gp41的CHR(C-端七价重复序列，C-terminal heptad repeat)区衍生而来的多肽，通过结合到gp41的NHR(N-端七价重复序列，N-terminal heptad repeat)区而抑制6-螺旋束的形成，从而阻止病毒的入侵。C34肽是抗HIV的第一代融合抑制剂。本领域中常用的C34肽具有如下的氨基酸序列：Ac-WMEWD REINN YTSLI HSLIE ESQNQ QEKNE QELL-NH 2(SEQ ID NO:1)，其中，Ac和NH 2分别表示在肽链的N端进行乙酰化修饰和在C端进行酰胺化修饰。
C34上的第10位氨基酸N是HIV跨膜蛋白gp41的C端多肽上的一个保守的天然糖基化位点。根据专利申请201810547641.7所证实的，在C34肽的N端、C端或第10位添加一个或多个半胱氨酸，并通过Michael加成反应将具有马来酰胺修饰的聚乙二醇基团偶联至该半胱氨酸，不影响C34肽的抗HIV活性。
如本文中所定义，将PEG定义为聚乙二醇(PEG)，一种广泛应用于工业生产和医学的聚醚化合物。PEG通过聚合环氧乙烷制备，并且在300g/mol至10,000,000g/mol的宽分子量范围都是可市售的。尽管具有不同分子量的PEG用于不同的应用，并且具有不同的物理特性例如粘度，归因于链长作用，其化学特性几乎是相同的。取决于用于聚合方法的引发剂，不同形式的PEG也是可得到的，最常见的PEG是单功能甲醚PEG(甲氧基聚乙二醇)，缩写为mPEG。还可以得到具有不同几何形状的PEG。分枝状或星状PEG具有从中央核心基团散发的约3至100个PEG链(臂)。梳状PEG具有通常接枝到聚合物骨架的多个PEG链。可以将任一此类PEG聚合物用于如上所定义的本发明的经PEG修饰的C34肽。在本发明上下文中，术语“PEG”或“PEG聚合物”优选地包含任一以上定义的聚合物。
因此，在本发明上下文中，用于如上所定义的本发明的经PEG修饰的C34肽的PEG/PEG聚合物优选地选自如上所定义的PEG聚合物，优选地在约10000g/mol至100000g/mol的分子量内，更优选地在约10000至50000Da的分子量内等，最优选地在约10k至50kDa的分子量内，例如在约10k至约40kDa的分子量内，例如约10kDa、约20kDa、约30kDa、约40kDa、约50kDa。更优选地，用于如上所定义的本发明的经PEG修饰的C34肽的PEG/PEG聚合物可选自如上所定义的任一PEG聚合物，并可具有不同的几何形状，例如如本文中所定义的具有从中央核心基团散发的约3至10个PEG链的分枝状PEG或者例如具有从中央核心基团散发的约10至100个PEG链的星状PEG。甚至更优选地，用于如上所定义的本发明的经PEG修饰的C34肽的PEG/PEG聚合物可选自如在本文中所定义的任一此类PEG聚合物，更优选地选自具有如上所定义的分子量的PEG聚合物，并且优选地，具有如本文中所定义的不同的几何形状，例如如具有从中央核心基团散发的约3至约10个PEG链(臂)，优选地具有从中央核心基团散发的约3至约5个PEG链(臂)的分枝状PEG。特别优选地用于如上所定义的本发明的分枝状PEG可以是如上所定义的PEG聚合物，其具有从中央核心基团散发的3、4、5、6、7、8、9、10或者更多的链(臂)。
在本发明中，除非另有明确说明，PEG的分子量意指PEG的平均分子量。
本发明的所述经修饰的C34肽具有式I的结构：
Ac-B pWMEWDREINN cB dYTSLIHSLIEESQNQQEKNEQELLB q-NH 2  式I；
其中，c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式I肽链的N端带有乙酰化修饰；NH 2表示式I肽链的C端带有酰胺化修饰；B为式II的基团
Figure PCTCN2020132063-appb-000003
式II中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式I肽链中的氨基酸相连；Y为式III的基团：
mPEG                         式III；
式III中，m为甲氧基，
其中，所述PEG的分子量为10000以上、优选10000-100000、更优选10000-50000。
在优选的实施方式中，p、q和d之和为1。
在优选的实施方式中，c为0，d为1。在优选的实施方式中，c为0，d为1，并且p和q均为0。
在优选的实施方式中，c为1，d为0；并且p和q之一为1，另一个为0。例如，c为1，d为0；并且p为1，q为0。例如，c为1，d为0；并且p为0，q为1。
在本发明中，当所述PEG为如上所述的分枝状PEG时，在连接有N-取代的马来酰胺之外的所有PEG链末端均具有甲氧基。
例如，在一个实施方式中，式II中的Y可为下式所示的PEG或PEG聚合物中的一种或多种：
Figure PCTCN2020132063-appb-000004
其中，n为整数，n的取值取决于所需的PEG的分子量，并且在上述式中，n可彼此相同或不同。
在优选的实施方式中，本发明所述的经PEG修饰的C34肽具有选自于如下的任一种结构：
Figure PCTCN2020132063-appb-000005
Figure PCTCN2020132063-appb-000006
在更优选的实施方式中，本发明所述的经PEG修饰的C34肽具有选自于如下的任一种结构：
Figure PCTCN2020132063-appb-000007
在上述结构中，PEG 10000、PEG 20000、PEG 40000和/或PEG 10K、PEG 20K、PEG 40K分别指平均分子量为10000、20000和40000的聚乙二醇。
在另一方面，本申请提供了制备如上所述的经PEG修饰的C34肽的方法，所述方法包括：
(1)提供式V以及式VI的化合物
Figure PCTCN2020132063-appb-000008
Ac-Z pWMEWDREINN cZ dYTSLIHSLIEESQNQQEKNEQELLZ q-NH 2    式VI；
式VI中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式VI肽链中的氨基酸相连；c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式VI肽链的N端带有乙酰化修饰；NH 2表示式VI肽链的C端带有酰胺化修饰；
式V中，Y为式III的基团：
mPEG                       式III；
式III中，m为甲氧基，其中，所述PEG的分子量为10000以上；以及
(2)使得式VI化合物中Z的巯基与式V中的马来酰胺发生加成反应，获得所述经PEG修饰的C34肽。
在本发明中，Y(即式III)中所示的PEG如上所定义。可根据需求，通过商购获得不同分子量的PEG。在本发明中，可基于现有技术合成式V所示的化合物。
在本发明中，所述式VI的化合物具有如下的任一序列：
Ac-CWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2
(SEQ ID NO:2)；
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLC-NH 2
(SEQ ID NO:3)；
Ac-WMEWDREINCYTSLIHSLIEESQNQQEKNEQELL-NH 2
(SEQ ID NO:4)。
在本发明中，式VI可通过肽的化学合成获得，或者还可通过基因工程而产生。肽合成的方法可采用本领域已知的方法进行。肽的纯化是本领域公知的，并可以例如使用HPLC。有用的肽合成方法和纯化方法的记载可以在本领域中容易地的获得。同时，对肽进行修饰(例如乙酰化、酰胺化等)等方法均可以采用本领已知的方法进行。
优选地，所述加成反应在8.0-9.0的pH下进行。在一些方式中，所述加成反应在8.0、8.5或9.0的pH下进行。最优选地，所述加成反应在8.5的pH下进行。
优选地，所述加成反应在室温(例如20℃-30℃)下进行。
优选地，所述加成反应在缓冲液中进行。优选地，所述缓冲液为弱酸和/或弱碱盐缓冲液、例如为磷酸盐缓冲液。优选地，所述磷酸盐缓冲液为50mM磷酸二氢钠和50mM磷酸氢二钠混合后的水溶液。
本发明的经PEG修饰的C34肽具有抗HIV病毒(HIV-1 NL4-3型病毒)的活性。可将本发明的经PEG修饰的C34肽用于治疗和/或预防艾滋病。就这一点而言，本发明的经PEG修饰的C34肽可作为药物或疫苗的活性成分。
本发明还提供了根据本发明的经PEG修饰的C34多肽在制备抗HIV病毒的药物组合物中的应用，其中所述经PEG修饰的C34肽作为活性成分。
在本申请进一步的方面，本申请提供了一种抑制HIV病毒活性的方法，所述方法包括向有需要的受试者提供治疗有效量的如本发明所述的经PEG修饰的C34肽。
此外，本申请还提供了一种治疗和/或预防和/或辅助治疗HIV病毒感染相关的疾病的方法，所述方法包括向有需要的受试者提供治疗有效量的如本发明所述的经PEG修饰的C34肽。
在一些实施方式中，以纯的形式、或基本上纯的形式给予经PEG修饰的C34肽，但是也可以作为药物组合物、制剂或制品给予。
为了向受试者给药，可将本文所述的经PEG修饰的C34肽以药学上可接受的(例如，无菌的)组合物、制剂或制品的形式提供。因此，本文所述的另一方面是药物组合物，所述药物组合物包含经PEG修饰的C34肽和任选的药学上可接受的载体。这些药学上可接受的组合物包含有效量的一种或多种本文所述的经PEG修饰的C34肽，所述经PEG修饰的C34肽与一种或多种药学上可接受的载体(添加剂)和/或稀释剂共同配制。
所述药物组合物作为药物直接给予受试者、或者与药学上可接受的载体或赋形剂混合后给予受试者，以达到治疗和/或预防HIV感染或HIV感染相关疾病的目的。本文的载体材料可以广泛使用本领域公知的各种载体，例如包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。其中优选的是水溶性载体材料。使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。使用上述剂型可以经注射给予，包括皮下注射、静脉注射、肌肉注射和腔内注射等；腔道给予，如经直肠和阴道；呼吸道给药，如经鼻腔；粘膜给药。上述给予途径优选的是注射给予。
“给予”表示使用本领域技术人员已知的多种方法和递送系统中的任一种，向受试者物理引入包含治疗剂的组合物。本发明的经PEG修饰的C34肽及其药物组合物的给予途径包括静脉内、肌肉内、皮下、腹膜内、脊柱或其它胃肠外给予途径，例如通过注射或输注。本文中使用的短语“胃肠外给予”是指，通常通过注射进行的除了肠内和局部施用以外的给予模式，且包括但不限于，静脉内、肌肉内、动脉内、鞘内、淋巴管内、病灶内、囊内、眶内、心内、真皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、 蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注、以及体内电穿孔。在某些实施方式中，所述经PEG修饰的C34肽及其药物组合物通过非胃肠外途径给予，在某些实施方式中，注射给予。其它非胃肠外途径包括局部、表皮或粘膜给予途径，例如，鼻内地、阴道地、直肠地、舌下地或局部地。还可以执行给予，例如，一次、多次，和/或在一个或多个延长的时间段中。
如本文所用，术语“药学上可接受的”是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
如本文所用，术语“受试者”表示哺乳动物，诸如啮齿动物、猫科动物、犬科动物和灵长类动物(如猴、猿等)。优选地，根据本申请的受试者是人。术语“受试者”和“患者”在本文中的某些语境下可互换地使用。
本文所述的药物或治疗剂的“治疗有效量”是当单独使用或与另一种治疗剂联合使用时保护受试者免于HIV感染或促进HIV感染相关疾病消退的药物的任何量，所述疾病消退通过疾病征状的严重程度的降低、无疾病征状阶段的频率和持续时间的增加、或由疾病折磨引起的损伤或失能的预防来证明。使用熟练的从业人员已知的多种方法可以评价治疗剂的促进疾病消退的能力，诸如在临床试验期间在人受试者中，在预测对于人类的效力的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物的治疗有效量包括“预防有效量”，其为当单独地或与其它治疗剂联合给予给处于发生HIV感染的风险的受试者或具有HIV感染相关疾病的风险的受试者时，抑制HIV感染的发生或HIV感染相关疾病的发作的任何药物量。在某些实施方案中，预防有效量完全阻止HIV感染的发生或HIV感染相关疾病的发作。
本发明的经PEG修饰的C34肽及其药物组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重及个体反应，所用的具体活性成分，给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药。
本发明的经PEG修饰的C34肽及其药物组合物可以直接单独用于HIV感染者的治疗和预防，也可以与一种或多种抗HIV药物联合使用，以达到提高整体治疗效果的目的。这些抗HIV药物包括但不限于逆转录酶抑制剂、蛋白酶抑制剂、侵入抑制剂、整合抑制剂和成熟抑制剂等。上述的逆转录酶抑制剂可以是AZT、3TC、ddI、d4T、ddT、TDF、Abacavir、Nevirapine、Efavirenz和Delavirdine等的一种或几种；上述的蛋白酶抑制剂可以是Saquinavir mesylate、Idinavir、Ritonavir、Amprenavir、Kaletra和Nelfinavir mesylate等的一种或几种；上述的侵入抑制剂可以是Maraviroc、TAK-779、T-20、T2635、西夫韦肽、艾博韦肽、VIRIP(VIR-576)等的一种或几种；上述的整合抑制剂可以是Raltegravir、Dolutegravir和Elvitegravi等的一种或几种。
对于任何具体的患者，具体的治疗有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体活性成分的活性；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；所采用的具体活性成分的给药时间、给药途径和排泄率；治疗持续时间；与所采用的具体活性成分组合使用或同时使用的药物；及医疗领域公知的类似因素。例如，本领域的做法是，活性成分的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。一般说来，本发明的经PEG修饰的C34肽及其药物组合物用于哺乳动物特别是人的剂量可以介于0.001-1000mg/kg体重/天，例如介于0.01-100mg/kg体重/天，又例如介于0.1-10mg/kg体重/天。
本文所提供的另一方面涉及试剂盒，所述试剂盒包含本文所述的经PEG修饰的C34肽、本文所述的含有经PEG修饰的C34肽的药物组合物、或者用于制造本文所述的经PEG修饰的C34肽的组分或用于制造包含本文所述的经PEG修饰的C34肽的药物组合物的组分。
除了上述组分以外，所述试剂盒还可包含信息资料。所述信息资料可为说明性资料、指导性资料、销售资料或其它与本文描述的方法和/或将所述经PEG修饰的C34肽用于本文描述的方法有关的资料。例如，所述信息资料描述将所述经PEG修饰的C34肽给予受试者的方法。所述试剂盒还可包含递送装置。
本申请提供了如本文所述的经PEG修饰的C34肽在制备抗HIV病毒和/或抑制HIV病毒活性和/或治疗和预防HIV病毒感染相关的疾病的药物中的应用。
本申请提供了如本文所述的经PEG修饰的C34肽在制备抗HIV病毒和/或抑制HIV病毒活性和/或治疗和预防HIV病毒感染相关的疾病的药物组合物中的应用。其中，所述经PEG修饰的C34肽作为活性成分。
在本文中，除非另有说明，否则术语“包含、包括和含有(comprise、comprises和comprising)”或等同物为开放式表述，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它已确定的出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。
本文所述各方面的实施方式可由如下编号的段落说明：
1.一种经PEG修饰的C34肽，所述经PEG修饰的C34肽具有式I的结构：
Ac-B pWMEWDREINN cB dYTSLIHSLIEESQNQQEKNEQELLB q-NH 2    式I；
其中，c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式I肽链的N端带有乙酰化修饰；NH 2表示式I肽链的C端带有酰胺化修饰；B为式II的基团
Figure PCTCN2020132063-appb-000009
式II中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式I肽链中的氨基酸相连；Y为式III的基团：
mPEG                      式III；
式III中，m为甲氧基，
其中，所述PEG的分子量为10000以上。
2.如段落1所述的经PEG修饰的C34肽，其特征在于，式I中，p、q、d不同时为1。
3.如段落1或2所述的经PEG修饰的C34肽，其特征在于，式I中，c、d不同时为1。
4.如段落3所述的经PEG修饰的C34肽，其特征在于，式I中，d为1，c、p、q为0。
5.如段落1-4中任一项所述的经PEG修饰的C34肽，其特征在于，所述PEG分子量为约10000-100000。
6.如段落5所述的经PEG修饰的C34肽，其特征在于，所述PEG的分子量为10000-50000。
7.如段落6所述的经PEG修饰的C34肽，其特征在于，所述PEG的分子量为10000-40000。
8.如段落5所述的经PEG修饰的C34肽，所述经PEG修饰的C34肽具有如下的任一种结构：
Figure PCTCN2020132063-appb-000010
Figure PCTCN2020132063-appb-000011
Figure PCTCN2020132063-appb-000012
以及
Figure PCTCN2020132063-appb-000013
9.如段落1-4中任一项所述的经PEG修饰的C34肽，其特征在于，式II中，Y为以下中的一种或多种：
Figure PCTCN2020132063-appb-000014
其中，n为不为0的整数。
10.如段落9所述的经PEG修饰的C34肽，所述经PEG修饰的C34肽具有如下的任一种结构：
Figure PCTCN2020132063-appb-000015
11.制备如段落1-10中任一项所述的经PEG修饰的C34肽的方法，所述方法包括：
(1)提供式V以及式VI的化合物
Figure PCTCN2020132063-appb-000016
Ac-Z pWMEWDREINN cZ dYTSLIHSLIEESQNQQEKNEQELLZ q-NH 2    式VI；
式VI中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式VI肽链中的氨基酸相连；c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式VI肽链的N端带有乙酰化修饰；NH 2表示式VI肽链的C端带有酰胺化修饰；
式V中，Y为式III的基团：
mPEG                     式III；
式III中，m为甲氧基，其中，所述PEG的分子量为10000以上；以及
(2)使得式VI化合物中Z的巯基与式V中的马来酰胺发生加成反应，获得所述经PEG修饰的C34肽。
12.如段落11所述的方法，其特征在于，所述加成反应在7.2-9.5的pH下进行。
13.如段落12所述的方法，其特征在于，所述加成反应在7.2、8.0或9.5的pH下进行。
14.如段落11-13中任一项所述的方法，其特征在于，所述加成反应在20℃-30℃下进行。
15.如段落11-14中任一项所述的方法，其特征在于，所述加成反应在磷酸盐缓冲液中进行。
16.如段落11-15中任一项所述的方法，其特征在于，所述式VI的化合物具有如下的任一序列：
Ac-CWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2；
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLC-NH 2；以及
Ac-WMEWDREINCYTSLIHSLIEESQNQQEKNEQELL-NH 2。
17.如段落11-16中任一项所述的方法，其特征在于，所述式V的化合物具有如下的结构中的一种或多种：
Figure PCTCN2020132063-appb-000017
以及
Figure PCTCN2020132063-appb-000018
其中，n为大于0的整数。
18.如段落1-10中任一项所述的经PEG修饰的C34肽在制备抗HIV的药物组合物中的用途。
19.如段落20所述的用途，其中，所述HIV为HIV-1NL4-3型病毒。
20.如段落1-10中任一项所述的经PEG修饰的C34肽在制备预防或治疗艾滋病的药物组合物中的用途。
21.一种药物组合物，所述药物组合物包含如段落1-10中任一项所述的经PEG修饰的C34肽以及任选的药学上可接受的载体和/或赋形剂。
22.如段落1-10中任一项所述的经PEG修饰的C34肽在制备预防或治疗艾滋病的疫苗中的用途。
实施例
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到或可参照文献制备的物质。下述实施例中所用试剂，如无特殊说明，均为分析纯级别。
下述实施例中如无特殊说明，所述室温反应具体为将反应温度控制在20℃-30℃范围内，包括20℃和30℃。
材料：
由北京中科亚光生物科技有限公司合成以下序列：C34以及【Ac-WMEWD REIN C YTSLI HSLIE ESQNQ QEKNE QELL-NH 2】(SEQ ID NO:4)(下文称为“多肽C34NC”)，纯度为98％。
化合物mPEG 10kMal、mPEG 20kMal和mPEG 40kMal购自赛诺邦格生物科技股份有限公司，纯度为95％。
mPEG 10kMal、mPEG 20kMal和mPEG 40kMal的分子式如下所示：
Figure PCTCN2020132063-appb-000019
其中，n为大于零的整数，使得PEG部分的分子量分别为10000、20000和40000。
磷酸二氢钠/磷酸氢二钠缓冲溶液：50mM磷酸二氢钠，50mM磷酸氢二钠，溶于水，pH为8.0-9.0。
质谱采用MALDI-TOF质谱仪质谱仪。
实施例1：PEG化多肽PEG 10kNC的制备
室温下，将10mg(0.005mmol)mPEG 10kMal与10mg(0.0025mmol)多肽C34NC的混合物溶于10mL的磷酸二氢钠/磷酸氢二钠缓冲溶液(50mM，pH8.0-9.0)中，通过HPLC监测反应，直至多肽C34NC反应完全。
采用安捷伦1200反相高效液相色谱仪对所获得的经PEG修饰的C34多肽进行纯化。色谱柱型号：Angilent Eclipse XDB-C8Semi-Prep，5μm，9.4×250mm；洗脱液组成：流动相A(含体积百分浓度为0.1％的三氟乙酸的水溶液)，流动相B(含体积百分浓度为0.1％的三氟乙酸的乙腈溶液)；洗脱条件：A从40％到70％线性梯度洗脱，时间11min，洗脱流速为2mL/min，紫外检测波长220nm。收集目标多肽的峰，冷冻干燥得到蓬松状态的PEG化多肽PEG 10kNC纯品。
所得PEG 10kNC的结构为：
Figure PCTCN2020132063-appb-000020
PEG 10kNC的结构经基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)表征验证其正确性，质谱图如图1所示。采用安捷伦1200分析型高效液相色谱仪检测PEG 10kNC的纯度。色谱柱的型号：Angilent Eclipse XDB-C18 Analytical，5μm，4.6×150mm。洗脱液：流动相A(含体积百分浓度为0.1％的三氟乙酸的水溶液)和流动相B(含体积百分浓度为0.1％的三氟乙酸的乙腈溶液)；洗脱条件：由10％A到100％A线性梯度洗脱，时间25min，洗脱流速为1mL/min，紫外检测波长220nm。分析型高效液相色谱仪检测结果显示，所得PEG 10kNC的纯度为96％。
实施例2：PEG化的多肽PEG 20kNC的制备
除将实施例1中的mPEG 10kMal换成mPEG 20kMal外，按照实施例1中的方法制备、纯化以及表征，得到PEG 20kNC(纯度为97.5％)，质谱结果如图2所示。
所得PEG 20kNC的结构为：
Figure PCTCN2020132063-appb-000021
实施例3：PEG化的多肽PEG 40kNC的制备
除将实施例1中的mPEG 10kMal换成mPEG 40kMal外，按照实施例1中的方法制备、纯化以及表征，得到PEG 40kNC(纯度为98％)，质谱结果如图3所示。
所得PEG 40kNC的结构为：
Figure PCTCN2020132063-appb-000022
效果例1：PEG修饰的C34肽的水溶性
对实施例1-实施例3获得的经PEG修饰的C34肽进行水溶性评价。
将1mg的C34溶解于1mL的蒸馏水中，溶液中见明显浑浊，超声后仍无变化，可见C34在水中的浓度<1mg/mL。
而分别将20mg的PEG 10KNC、PEG 20KNC、PEG 40KNC溶解于100μL的蒸馏水中，溶液澄清，可见本发明所制备的经大分子量PEG修饰的C34在水中的浓度可达200mg/mL。在相同的条件下，专利申请201810547641.7制备的C34NCPEG 2000(PEG分子量为2000)在水中的浓度最多可为10mg/mL。
因此，经大分子量PEG(分子量在10000以上)修饰的C34的水溶性明显大于未经修饰的C34，也大于以较小的分子量进行PEG化修饰的C34(例如，分子量低于10000，包括2000、5000等)。
效果例2：本发明的经PEG修饰的多肽对HIV-1 NL4-3型病毒的体外抑制作用
为了评估由实施例1-实施例3制备的抗HIV多肽对HIV病毒的抑制作用，在HIV-1 NL4-3型病毒中进行了测试。HIV-1 NL4-3型病毒从NIH AIDS Research and Reference Reagent Program获得。
将TZM-bl细胞(NIH AIDS Research and Reference Reagent Program)以10 4/孔接种于96孔板中，置于37℃，5％CO 2培养箱中培养过夜。培养基为DMEM培养基。用DMEM培养基将肽样品(即实施例1-实施例3制备的PEG 10KNC、PEG 20KNC和PEG 40KNC，以及C34和C34NC)稀释成8个稀释度(浓度分别为64、32、16、8、4、2、1、0.5nM)。用DEAE培养液(含20μg/mL的DEAE-dextran)将待测病毒(即HIV-1 NL4-3型病毒)稀释成2000TICD 50/mL。弃去96孔板中的培养基，依次缓慢加入稀释后的药物，每孔100μL并设3个复孔。接着将稀释好的HIV-1 NL4-3病毒缓慢加入各孔中，每孔100μL(即200TICD 50/孔)。同时设立病毒对照组(VC，仅加入100μL的DMEM培养基和100μL的稀释后的病毒)和细胞对照组(CC，仅加入200μL的DMEM培养基)。
将96孔板四周贴好封口膜，置于37℃，5％CO 2培养箱中培养48小时，然后取出96孔板，每孔吸取100μL培养液，加入100μL发光检测液(Perkin Elmer公司)，室温避光放置2min，吸取150μL上清转移至黑色96孔板中，于发光检测仪(1420VICTOR D Multilabel Counter，型号1420-020)中检测，获得各个样品的发光值。
根据Reed and Muench法计算药物的抑制率，其中发光值为使用3个复孔所测发光值的均值：
Figure PCTCN2020132063-appb-000023
然后根据获得的抑制率使用GraphPad Prism Software 5.0软件的非线性回归计算每种多肽样品的EC 50值和EC 90值。结果如表1所示。
表1：各样品的EC 50值和EC 90值
肽样品 EC 50(nM) EC 90(nM)
C34 1.71±0.34 11.01±4.09
C34NC 0.67±0.29 3.80±0.62
PEG 10KNC 7.10±4.42 57.97±20.64
PEG 20KNC 22.20±13.42 101.42±24.04
PEG 40KNC 18.51±10.87 132.49±31.59
表1的结果表明，因用于修饰的基团PEG的分子量(40K)大于C34的分子量(4K)，PEG化修饰C34之后，其体外抗HIV NL4-3活性有所下降，但从PEG 10KNC、PEG 20KNC、PEG 40KNC的活性的变化趋势来看，PEG 20KNC活性比PEG 10KNC下降了两倍，同等情况下，PEG 40KNC活性和PEG 20KNC相比理论上也应下降两倍，但实际上相差不大甚至略高。考虑到分子量更高的PEG更易进入动物淋巴结，所以选择PEG 40KNC研究其体内半衰期和抗病毒作用。
效果例3：PEG 40KNC在SD大鼠体内的血浆半衰期
实验方法：
动物实验：试验动物为来自北京维通利华实验动物技术有限公司的SD大鼠(7周龄)，体重180-200克。将4只SD大鼠随机分为2组，每组2只(1只雄性和1只雌性)，用于收集血浆。每组大鼠分别以1.7μmol/kg的剂量单次皮下注射C34NC和PEG 40KNC(生理盐水中加入少量pH 9.5的硼砂缓冲液作为增溶剂)。
样品采集：将给药前记为零时刻，分别在零时刻和给药后的不同时间点从尾静脉收集全血(300μL)，其中C34NC组的时间点为0.5h、1.0h、2.0h、4.0h、6.0h、8.0h、10.0h；PEG 40KNC组的时间点为3h、6h、9h、20h、23h、29h、48h、72h。将各全血样品加入含有50μL肝素钠(83U/Ml)和10μL抑肽酶(5ug/mL)的EP管中，4℃，4500g离心10min，收集上层血清样品并置于-80℃冰箱保存。
通过测定收集的血清样品对HIV NL4-3型病毒的体外抑制情况来推算不同时间点处样品的浓度。具体实验步骤如下：将生长良好的TZM-bl细胞消化后铺96孔板(10 4个/孔)，37℃，CO 2培养箱过夜培养；第二天，用细胞完全培养基DMEM稀释血清，第一孔血清终稀释倍数为10倍稀释，然后依次连续2倍稀释，共8个稀释梯度；吸弃细胞培养基，每孔加入100μL稀释好的血清，每孔加入100μl含200TCID 50的病毒，37℃，CO 2培养48h；每孔小心吸弃100μL培养上清，加入100μL发光检测液(Perkin Elmer公司)，室温避光裂解2min，混匀后吸取150μl上清转移至黑色96孔板中检测荧光表达情况。计算50％抑制病毒感染所需的血清稀释倍数。
另外，各时间点药物浓度依据标准曲线得到，药动力学参数由Winnonlin药动力学软件中的非房室模型计算得出。
同时，基于相同的实验条件，对基于专利申请201810547641.7制备的C34NCPEG 2000和C34NCPEG 5000(即，由PEG2000修饰的C34肽和由PEG5000修饰的C34肽)在SD大鼠体内的血浆半衰期进行了分析。
实验结果：各个时间点处50％抑制病毒感染所需的血清的稀释倍数以及C34NC或PEG 40KNC在血清中的浓度分别如图4和图5所示。PEG 40KNC的半衰期T 1/2(h)为10.39h，而C34NCPEG 2000和C34NCPEG 5000的半衰期仅为2.59h和2.21h。在4h时，C34NC在血浆中已不存在，而PEG 40KNC在70h后在血浆中仍存在。由此可知，多肽经PEG 40K修饰后，在SD大鼠体内的血浆半衰期得到特别显著的延长，进而说明其药效在SD大鼠体内能得到明显延长。
效果例4：PEG 40KNC对急性感染SHIV SF162P3的恒河猴的治疗作用
实验方法：
动物实验：筛选8只未感染SV、SIV、SRV-1或TB的成年中国恒河猴(3kg-4kg，购自中国医学科学院医学实验动物研究所)，之后以单次剂量100TCID 50对8只恒河猴静脉注射SHIV SF162P3型病毒。将上述8只恒河猴随机分为急性感染治疗组(n＝4)和对照组(n＝4)，治疗组自接种病毒后第7天开始每天皮下注射1.38μmol/kg的PEG 40KNC(生理盐水溶液)，连续注射28天；对照组自接种病毒后第7天开始每天用0.9％生理盐水溶液连续皮下注射28天。
样品采集：于接种病毒后的第1、4、7、11、14、18、21、25、28、32、36、42、49和70天采集恒河猴的血样。使用QIAmp病毒RNA minikit(Qiagen，Valencia，CA)从无细胞血浆中提取并纯化病毒RNA，之后用洗脱缓冲液(Qiagen，Valencia，CA)洗脱RNA并立即进行分析。通过实时定量逆转录PCR(qRT-PCR)测定法测量在不同时间点感染了SHIV的恒河猴的血浆病毒载量，对每个样品进行双重测试反应，结果以log 10 RNA copies/mL为指标。RNA标准品(10 3-10 13copies/ml)是实验室制备并保存的。实验中采用TaKaRa提供的一步PrimeScript RT-PCR Kit，实验仪器使用Perkin-Elmer ABI 7500。
实验结果：
如图6所示，图中3log 10 RNA copies/mL处的水平虚线表示测定的病毒载量的灵敏度极限。可见，在药物治疗过程中(病毒感染后7-36天)，PEG 40KNC实验组的血浆SHIV RNA载量明显低于生理盐水对照组(P＝0.0126)，特别是在第11天，生理盐水组血浆SHIV RNA载量达到峰值(急性感染的建立)，而此时PEG 40KNC实验组明显低于峰值，说明PEG 40KNC能够显著降低急性感染SHIV SF162P3的恒河猴血浆中的病毒载量，从而对SHIV SF162P3的急性感染有显著的保护作用。
因此，本发明所提供的经大分子量的PEG修饰的C34肽能够更显著地增加C34肽的水溶性，并且具有显著延长的大鼠体内的血浆半衰期，且有较好的体内抗HIV活性。本发明提供的经大分子量PEG(分子量在10000以上)修饰的C34肽有望成为水溶性好且长效的抗HIV多肽药物。

Claims (22)

  1. 一种经PEG修饰的C34肽，所述经PEG修饰的C34肽具有式I的结构：
    Ac-B pWMEWDREINN cB dYTSLIHSLIEESQNQQEKNEQELLB q-NH 2   式I；
    其中，c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式I肽链的N端带有乙酰化修饰；NH 2表示式I肽链的C端带有酰胺化修饰；B为式II的基团
    Figure PCTCN2020132063-appb-100001
    式II中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式I肽链中的氨基酸相连；Y为式III的基团：
    mPEG    式III；
    式III中，m为甲氧基，
    其中，所述PEG的分子量为10000以上。
  2. 如权利要求1所述的经PEG修饰的C34肽，其中，式I中，p、q、d不同时为1。
  3. 如权利要求1或2所述的经PEG修饰的C34肽，其中，式I中，c、d不同时为1。
  4. 如权利要求3所述的经PEG修饰的C34肽，其中，式I中，d为1，c、p、q为0。
  5. 如权利要求1-4中任一项所述的经PEG修饰的C34肽，其中，所述PEG分子量为约10000-100000。
  6. 如权利要求1-4中任一项所述的经PEG修饰的C34肽，其中，所述PEG的分子量为10000-50000。
  7. 如权利要求1-4中任一项所述的经PEG修饰的C34肽，其中，所述PEG的分子量为10000-40000。
  8. 如权利要求1-4中任一项所述的经PEG修饰的C34肽，其中，所述经PEG修饰的C34肽具有如下的任一种结构：
    Figure PCTCN2020132063-appb-100002
    Figure PCTCN2020132063-appb-100003
    Figure PCTCN2020132063-appb-100004
    以及
    Figure PCTCN2020132063-appb-100005
  9. 如权利要求1-4中任一项所述的经PEG修饰的C34肽，其中，式II中，Y为以下中的一种或多种：
    Figure PCTCN2020132063-appb-100006
    Figure PCTCN2020132063-appb-100007
    其中，n为不为0的整数。
  10. 如权利要求1-9中任一项所述的经PEG修饰的C34肽，其中，所述经PEG修饰的C34肽具有如下的任一种结构：
    Figure PCTCN2020132063-appb-100008
  11. 制备如权利要求1-10中任一项所述的经PEG修饰的C34肽的方法，所述方法包括：
    (1)提供式V以及式VI的化合物
    Figure PCTCN2020132063-appb-100009
    Ac-Z pWMEWDREINN cZ dYTSLIHSLIEESQNQQEKNEQELLZ q-NH 2    式VI；
    式VI中，Z为半胱氨酸，所述半胱氨酸通过酰胺键与式VI肽链中的氨基酸相连；c为0或1；d为0或1；p为0或1；q为0或1；c和d不同时为0；p、q和d不同时为0；Ac表示式VI肽链的N端带有乙酰化修饰；NH 2表示式VI肽链的C端带有酰胺化修饰；
    式V中，Y为式III的基团：
    mPEG      式III；
    式III中，m为甲氧基，其中，所述PEG的分子量为10000以上；以及
    (2)使得式VI化合物中Z的巯基与式V中的马来酰胺发生加成反应，获得所述经PEG修饰的C34肽。
  12. 如权利要求11所述的方法，其中，所述加成反应在7.2-9.5的pH下进行。
  13. 如权利要求12所述的方法，其中，所述加成反应在7.2、8.0或9.5的pH下进行。
  14. 如权利要求11-13中任一项所述的方法，其中，所述加成反应在室温下进行。
  15. 如权利要求11-14中任一项所述的方法，其中，所述加成反应在磷酸盐缓冲液中进行。
  16. 如权利要求11-15中任一项所述的方法，其中，所述式VI的化合物具有如下的任一序列：
    Ac-CWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2；
    Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLC-NH 2；以及
    Ac-WMEWDREINCYTSLIHSLIEESQNQQEKNEQELL-NH 2。
  17. 如权利要求11-16中任一项所述的方法，其中，所述式V的化合物具有如下的结构中的一种或多种：
    Figure PCTCN2020132063-appb-100010
    以及
    Figure PCTCN2020132063-appb-100011
    其中，n为大于0的整数。
  18. 如权利要求1-10中任一项所述的经PEG修饰的C34肽在制备抗HIV的药物组合物中的用途。
  19. 如权利要求20所述的用途，其中，所述HIV为HIV-1NL4-3型病毒。
  20. 如权利要求1-10中任一项所述的经PEG修饰的C34肽在制备预防或治疗艾滋病的药物组合物中的用途。
  21. 一种药物组合物，所述药物组合物包含如权利要求1-10中任一项所述的经PEG修饰的C34肽以及任选的药学上可接受的载体。
  22. 如权利要求1-10中任一项所述的经PEG修饰的C34肽在制备预防或治疗艾滋病的疫苗中的用途。
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