WO2012126150A1 - Peptide ayant une activité analgésique et son procédé de préparation - Google Patents

Peptide ayant une activité analgésique et son procédé de préparation Download PDF

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Publication number
WO2012126150A1
WO2012126150A1 PCT/CN2011/001102 CN2011001102W WO2012126150A1 WO 2012126150 A1 WO2012126150 A1 WO 2012126150A1 CN 2011001102 W CN2011001102 W CN 2011001102W WO 2012126150 A1 WO2012126150 A1 WO 2012126150A1
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Prior art keywords
analgesic
vrd
active
peptide
active peptide
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PCT/CN2011/001102
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Chinese (zh)
Inventor
张景海
刘岩峰
王月秋
吴春福
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Shenyang Pharmaceutical University
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Shenyang Pharmaceutical University
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates

Definitions

  • the invention relates to the field of biomedical technology, relates to the structure, function and preparation method and application of the analgesic active peptide VRD, in particular, the invention relates to an analgesic active peptide VRD and its derivatives, analogues, active fragments, 54th The structure of conservative amino acid residues and a preparation method thereof, and the use as an analgesic drug in the field of medicine.
  • cockroach As a precious Chinese medicine, it is also called the whole worm and the whole cockroach. It is contained in "Kaibao Materia Medica”. The “Compendium of Materia Medica” is listed in the 40th volume of the worm department. It has a medicinal history of more than 2,000 years. The effect of attacking and dispersing, relieving pain and relieving pain. It is clinically used for the treatment of migraine, facial nerve spasm, rheumatic pain and cancer pain. The main active ingredient of cockroach is the toxin secreted by the tail gland, which contains a large number of active components with medicinal value. It is a natural active peptide library rich in multi-functional peptides.
  • analgesic anti-tumor ⁇ ⁇ ⁇ analgesic antibacterial activity peptide
  • the object of the present invention is to confirm by an in vivo biological activity experiment that a polypeptide derived from sputum has an analgesic activity (the active peptide is named an analgesic active peptide VRD).
  • the preparation method and application range of the analgesic active peptide VRD are also provided, specifically, the analgesic active peptide VRD gene is obtained from the ⁇ cDNA clone by using genetic engineering technology, and the active peptide with analgesic activity and its derivative are obtained.
  • Analogs, active fragments, mutants, analgesic active peptides VRD and derivatives, analogs, active fragments, mutants thereof are simple to prepare, and can be combined with any carrier to prepare a medically acceptable dosage form.
  • the second object of the present invention is to obtain an analgesic activity of the analgesic active peptide VRD, and obtain a series of analgesic active peptide VRD derivatives, analogs, active fragments and mutants by genetic engineering techniques, and obtain experimental products of a series of expression products.
  • analgesic activity to obtain an analgesic active peptide VRD and its derivatives, analogs, active fragments, mutants which can further develop analgesic drugs.
  • the third object of the present invention is to study the conservation of the analgesic activity of the 54th amino acid residue of the analgesic active peptide VRD, and use the genetic engineering technique to mutate the amino acid residue at the site to obtain a series of mutants.
  • the in vivo analgesic activity of the experimental animals expressing the series of mutants was determined to be conserved for the analgesic activity of the analgesic active peptide VRD.
  • the fourth object of the present invention is to solve the analgesic activity peptide VRD and its derivatives and the like by using genetic engineering technology or chemical synthesis technology for the preparation method of analgesic active peptide VRD and its derivatives, analogs, active fragments and mutants. , active fragments, mutants.
  • Analgesic active peptides VRD and its derivatives, analogs, active fragments, mutants, genetic engineering techniques include -
  • step ⁇ (2) transforming the recombinant expression vector obtained in step ⁇ into an appropriate host cell (prokaryotic or eukaryotic);
  • step (3) cultivating the transformed host cell of step (2) under suitable conditions for inducing expression
  • Another object of the present invention is to provide a method for isolating and purifying an expression product of the above analgesic active peptide VRD and derivatives, analogs, active fragments thereof and mutants thereof.
  • the method can separate and purify the desired expression product from the cell lysate and the culture solution by means of salting out precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel filtration.
  • sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) or Western blotting (WESTERN) can be used. The presence of the expressed product and the corresponding molecular size.
  • the present invention is the first to carry out an analgesic biological activity test of the above analgesic active peptide VRD and its derivatives, analogs, active fragments and mutants in mice.
  • the present invention also provides the use of the above analgesic active peptide VRD and derivatives, analogs, active fragments thereof, mutants in the field of biopharmaceuticals, in particular, analgesic biological activity.
  • a further object of the invention is to provide a pharmaceutical composition comprising a protein as defined above and one or more pharmaceutically acceptable carriers or excipients.
  • Pharmaceutical compositions suitable for parenteral administration can be prepared according to the basic principles and methods known in the pharmaceutical industry (see, for example, Remington's Pharmaceutical Science, 15th., Mack Publishing Company, 1980).
  • the pharmaceutical compositions of the present invention can be administered by various routes of administration, particularly intravenous, intramuscular, intraarticular, intraperitoneal, intranasal, intradermal, subcutaneous, and the like.
  • the protein of the present invention or a pharmaceutical composition containing the same can be used as a therapeutic agent for treating various pain-related diseases of a specific type of human body.
  • the therapeutically effective dose of the pharmaceutical composition of the present invention should generally be determined by the clinician in accordance with individualized principles depending on the nature, severity, and sensitivity to the drug, as well as the route of administration.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells, and examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the analgesic active peptide VRD and its derivatives, analogs, active fragments and mutants of the invention have good analgesic activity, and the obtaining method is simple, simple and high in yield, and has important guiding significance and practical value. It also laid the foundation for industrial production.
  • N-terminal and C-terminal amino acid sequences of WCIKLPDSVPIRVPGKCHR are designed with corresponding oligonucleotide primers, and at the 5' end of the above two oligonucleotide primers, respectively, a restriction endonuclease and hydrolysis are added.
  • the positive recombinant plasmid was heat-transformed into E. coli BL21 (DE3), and then single bacteria were picked from the LB solid plate and inoculated to 3 ml LB (containing Kana antibiotic, 50 g/ml) at 37 'C, 200 r/ Min shake overnight culture.
  • the overnight culture was inoculated to 400 ml of fresh LB medium containing the corresponding antibiotics in a 1% inoculum, and cultured at 37 ° C, 200 r/min to an OD600 of 0.6-0.8, and the final concentration was 0.166 mmol.
  • the inducer of L, isopropyl ⁇ -D-thiogalactoside (IPTG) was cultured for 4 h.
  • the fermentation was terminated, centrifuged at 3000 g, and centrifuged at 4 'C for 20 min to collect the cells.
  • the cells were resuspended in 40 ml of lysis buffer (0.1 M PBS, 0.15 M NaCl, 50 mM imidazole), sonicated, and after centrifugation, centrifuged at 12,000 g for 4 min at 4 Torr to obtain a supernatant.
  • the crushing was repeated once according to the above procedure, and the supernatant was combined twice, and directly applied to a metal ion chelate chromatography column pre-equilibrated in 0.1 M PBS (pH 8.0), and washed thoroughly by two different pH buffers.
  • the purity of the expression product or the analgesic active peptide VRD obtained in Example 1 does not meet the desired purity requirement, and may be performed by hydrophobic chromatography, ion exchange chromatography, gel filtration chromatography, reversed phase chromatography, respectively, or in combination. Second for further separation and purification to the desired purity requirements.
  • Hydrophobic chromatography medium selected phenyl-hydrophobic chromatography additive or n-octyl-hydrophobic chromatography additive- or hexane-hydrophobic chromatography additive- or butyl-hydrophobic chromatography Adding; 2. Neutral salt selection (HN 3 ) 2 S0 4 or Na 2 S0 4 or NaCl; 3. The pH of the buffer is selected at pH 2 - pH 12, which does not affect the biological activity of the analgesic active ingredient, Does not affect the separation of the active ingredients; 4. The concentration of the buffer is selected at lm - 100 mM, which does not affect the biological activity of the analgesic active ingredient, and does not affect the separation of the active ingredient.
  • the purpose of the ultrafiltration treatment is to remove the salt or the impurity protein in the analgesic active ingredient solution or to change the buffer, and further, to concentrate the analgesic active ingredient solution.
  • Ultrafiltration membranes capable of permeating the analgesic active peptide VRD or its derivatives, analogs, and active fragments are selected for ultrafiltration treatment, at which time the ultrafiltration permeate is collected, and the heteroprotein is retained for separation.
  • Ultrafiltration membranes capable of retaining the analgesic active peptide VRD or its derivatives, analogs, and active fragments are selected for ultrafiltration treatment. At this time, the ultrafiltration concentrate (analgesic active peptide VRD or its derivatives, analogs) is collected.
  • the active fragment is trapped), and the buffer pair of the salt and the buffer, the impurity protein that can pass through the ultrafiltration membrane, and the water are permeated to achieve the purpose of removing the salt, the impurity protein, or replacing the buffer or concentrating.
  • Characteristics of ultrafiltration treatment 1.
  • the choice of ultrafiltration membrane is molecular weight of lOkDa or 20kDa or 30kDa Or 40kDa or 50kDa ultrafiltration membrane, preferably 30kDa ultrafiltration membrane, ultrafiltration membrane greater than or less than 30kDa, the yield or ultrafiltration efficiency is affected; 2.
  • the interception ultrafiltration membrane selection is molecular weight IkDa or 2kDa or Ultrafiltration membranes of 3kDa or 5kDa, preferably ultrafiltration membranes of IkDa, ultrafiltration membranes larger or smaller than IkDa, the yield or ultrafiltration efficiency are affected; 3.
  • the temperature of ultrafiltration treatment is selected from 0°C to 45' C, this condition neither destroys the physical and chemical properties of the ultrafiltration membrane nor affects the activity of the target.
  • the solution of the analgesic active ingredient treated by ultrafiltration was further subjected to desalting, buffer exchange and concentration using a buffer of ion exchange chromatography. 5M, 0. 1M, 0. 15M, respectively, the sample liquid to be treated is loaded on a well-balanced ion exchange chromatography column, and the buffer is used to wash the non-adsorbed eggs. 0. 2M, 0. 25M, 0. 3M, 0.4 M, 0.5 M salt solution was subjected to stage elution, and the eluate containing analgesic activity was combined. Characteristics of ion exchange chromatography separation: 1.
  • Chromatography medium selection cation exchange chromatography addition such as CM-ion exchange chromatography or SP-ion exchange chromatography or S-ion exchange chromatography and other cations Exchange chromatography addition, at which time the pH of the buffer is selected at pH 2 - pH 7; 2.
  • the chromatographic medium is selected for anion exchange chromatography, such as Q-ion exchange chromatography or DEAE-ion exchange chromatography or QAE - Ion exchange chromatography addition ion exchange chromatography addition, at this time the pH of the buffer is selected at pH 7-pH12; 3.
  • the concentration of the buffer is selected at lraM-50 mM, which does not affect the biological activity of the analgesic active ingredient, Does not affect the separation of the active ingredients; 4.
  • the stage eluted salt solution is selected to be the desired concentration of buffer or a neutral salt in 10 mM buffer to the required concentration; 5.
  • the analgesic active ingredient is further purified according to the basic framework of the hydrophobic chromatography separation method.
  • the analgesic active ingredient solution was added to a neutral salt to a concentration of 2 M, and was applied to a hydrophobic chromatography column previously equilibrated with a 2 M neutral salt-buffer solution to conduct a neutral salt concentration gradient (2.0 M to 0. 0 M).
  • the eluted peaks containing analgesic activity were collected and subjected to ultrafiltration to remove salt and concentrate.
  • Hydrophobic chromatography purification features 1.
  • Hydrophobic chromatography medium selected phenyl-hydrophobic chromatography additive or n-octyl-hydrophobic chromatography additive- or hexane-hydrophobic chromatography additive- or butane-hydrophobic chromatography Addition; 2. Neutral salt selection (HN 3 ) 2 S0 4 or N3 ⁇ 4S0 4 or NaCl; 3. The pH of the buffer is selected at pH 2 - pH 12; 4. The concentration of the buffer is selected at lmM - 100 mM.
  • the analgesic active ingredient is progressively separated according to the basic framework of the ion exchange chromatography separation method.
  • the sample solution to be treated is applied to an ion exchange chromatography column which has been previously equilibrated with a buffer, and the non-adsorbed heteroprotein is firstly washed with a buffer to elute the salt concentration gradient (0. 0 M to 1. 0 M).
  • the combined elution peaks containing analgesic activity were collected.
  • Characteristics of ion exchange chromatography purification 1.
  • the chromatographic medium is selected from cation exchange chromatography, such as CM-ion exchange chromatography or SP-ion exchange chromatography or S-ion exchange chromatography.
  • the pH of the buffer is selected at PH2-PH7; 2.
  • the chromatographic medium is selected for anion exchange chromatography, such as Q-ion exchange chromatography or DEAE-ion exchange chromatography or QAE - Ion exchange chromatography addition ion exchange chromatography addition, at this time the buffer pH is selected at pH7-pH12; 3.
  • the buffer concentration is selected at lmM-50mM; 4.
  • Salt gradient elution salt solution selection is required concentration Buffer or neutral salt in lOmM buffer to the desired concentration; 5.
  • the chromatographic medium can be selected from Sephacryl S-100 HR or Sephacryl S-200 HR or Sephadex G-50 or Sephadex G- 75 or Sephadex G- 100 or Sephadex G-150 or Superose 12 prep grade or Superose 6 prep grade or Superdex. 30 prep grade or Superdex 75 prep grade or Superose 12 HR or Superose 6 HR or Superdex Peptide HR or Superdex75 HR or Superdex Pept ide PE and other gel chromatography addition; 2.
  • the pH of the eluent is selected at pH2- P H12; The concentration of the eluent is selected to be greater than 0.15 M and above.
  • the 1% trifluoroacetic acid was firstly loaded with a 0.1% trifluoroacetic acid, a 1:1 15% acetonitrile, a RP 15
  • the -20% acetonitrile aqueous solution was thoroughly washed, and then eluted with an acetonitrile concentration gradient (20% to 95%), and the eluted peak component-analgesic active peptide target having the largest elution peak area was collected.
  • the sequence encoding the analgesic active peptide gene is amplified using an oligonucleotide primer, and the 5' oligonucleotide primer sequence used in the PCR reaction contains an EcoR V restriction endonuclease cleavage site and an analgesic activity peptide.
  • the coding sequence of the amino acid sequence of the N-terminal region of VRD, the 3' primer sequence contains the coding sequence of the restriction endonuclease of EcoR I restriction endonuclease, a translation stop codon and the C-terminal region of the analgesic activity peptide VRD. .
  • the restriction endonuclease cleavage site on the primer corresponds to a restriction endonuclease site on the yeast expression vector PPIC9K (wherein the endonuclease cleavage of EcoR V and SnaB I is blunt), the plasmid
  • the vector encodes antibiotic resistance (Ampr and KarO, a replicon from the 2 ⁇ plasmid, an A0X1 promoter, which can be efficiently expressed by methanol in Pichia pastoris, an alpha-factor signal peptide sequence, a transcription termination signal, a HIS4 Selectable markers and integration sequences.
  • the pPIC9K vector was digested with SnaB I and EcoR I, and the PCR-amplified analgesic peptide VRD gene was digested with EcoR V and EcoR I, and then ligated into the PPIC9K vector, and the ligated product was heat-transformed into E. col i DH 5 ⁇ strain. Transformants were selected on LB culture dishes containing Amp, and clones containing the desired construct were cultured overnight in LB liquid medium containing Amp (100 g/ml). The plasmid was extracted and sequenced to verify that the insert IH was indeed inserted.
  • the plasmid After the plasmid is linearized, it is electrotransformed into yeast cells, and the linearized recombinant vector generates a stable yeast transformant by homologous recombination with the homologous sequence of the host genome.
  • High copy integrated transformants were screened using G418. The screened strain was inoculated into a 250 ml shake flask containing 25 ml of MGY, BMG or BMGY medium at 28 to 30.
  • the culture medium for inducing the expression of the engineered bacteria contains an expression product, and the structural characteristic of the expression product is: VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHRo for the i Xiu guide project 3 ⁇ 4 ⁇ towel; ⁇ , m 2 ⁇ m ⁇ ⁇
  • This example provides strategies and basic methods for obtaining derivatives, analogs, and active fragments of an analgesic active peptide VRD, similar to the strategies and basic methods of Example 1, Example 2, and Example 3.
  • one or more amino acid residues are additionally added to the N-terminus or C-terminus of the analgesic active peptide VRD, and the obtained analgesic active peptide VRD derivative, analog, and active fragment still have analgesic biological activity. Its structural characteristics are:
  • n can be 1 or 2 or 3 or 4 or 5).
  • An analgesic active peptide of N is a structural feature of a VRD derivative, an analog, and an active fragment: MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHRSG GGGSGGGGSGGGG;
  • the strategy and basic method for obtaining the 54th series mutant of the analgesic active peptide VRD in this example are the same as the strategy and basic method of the first embodiment, the second embodiment, the third embodiment and the fourth embodiment.
  • the 54 S residues of the analgesic active peptide VRD were respectively mutated into A, Y, E, K, F, I, ⁇ , D, R residues by gene mutation technology.
  • the corresponding mutants were obtained by genetic engineering expression, and their structural features were:
  • a mutant of an analgesic active peptide VRD derivative, an analog, or an active fragment which has the structural characteristics of: MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPD (A or Y or E or K or F or I or T or D or R) VPIRVPGKCHR, MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPD (A or Y or E or K or F or I or T or D or R) VPIRVPGKCHR(SGGGG)n (n may be 1 or 2 or 3 or 4 or 5).
  • the in vivo bioactive-analgesic activity of the analgesic active peptide VRD and its derivatives, analogs, and active fragments was determined by an in vivo analgesic model in mice. In addition, it was also aimed to verify the effect of conserved amino acid residues in the analgesic active peptide VRD and its derivatives and analog active fragments on analgesic activity. Protein concentration was determined by the Lowry method.
  • Acetic acid writhing analgesia model in mice Injecting glacial acetic acid as a chemical stimulator into the peritoneal cavity of Kunming mice, which in turn causes deep, large-area and long-lasting painful stimuli, resulting in a "writhing" response in the mouse (abdominal The inner concave, the torso and the hind legs are stretched, and the buttocks are raised).
  • Inhibition rate number of blank group writhing X l 00 /o
  • Analgesic active peptide VRD sample group (0.5 mg / kg) of experimental animals writhing times (Mean SEM) was 22. 00 ⁇ 1. 79, writhing inhibition rate was 47. 30%
  • Analgesic active peptides VRD derivatives, analogs, and active fragments of each sample group were all inhibited by writhing of more than 45%.
  • the analgesic activity peptide VRD 54 S is mutated to E or K or Y or T or D or R mutant and their corresponding derivatives, analogues, active fragments have a writhing inhibition rate of more than 38%, relative
  • the analgesic biological activity of the pre-mutation (analgesic active peptide VRD) was not statistically significant;
  • the analgesic activity peptide VRD 54 S is mutated to A or F or I mutant and its corresponding derivatives, analogs, active fragments, the writhing inhibition rate is below 32%, before the relative mutation (analgesic activity)
  • the analgesic biological activity of the peptide VRD was shown to be reduced, and the decrease was statistically significant;
  • the 54th S or E or K or Y or T or D or R of the analgesic active peptide VRD is an analgesic activity of the analgesic active peptide VRD and its derivatives, analogs, and active fragments. base.

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Abstract

L'invention concerne une sorte de peptide issu du venin de scorpion ayant une activité analgésique et son procédé de préparation, et la séquence d'acides aminés dudit peptide est représentée en tant que SEQ ID No : 1. L'invention concerne également des dérivés et des fragments actifs dudit peptide et leurs procédés de préparation. Ledit peptide ayant une activité analgésique peut être mélangé à des vecteurs pharmaceutiquement acceptables pour préparer une injection, une formulation orale, une formulation pour une absorption transdermique, une formulation pour une absorption muqueuse, et similaires.
PCT/CN2011/001102 2011-03-22 2011-07-05 Peptide ayant une activité analgésique et son procédé de préparation Ceased WO2012126150A1 (fr)

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CN2011100690037A CN102690343A (zh) 2011-03-22 2011-03-22 镇痛活性肽vrd及其制备方法与应用
CN201110069003.7 2011-03-22

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CN101880319A (zh) * 2010-02-09 2010-11-10 沈阳药科大学 镇痛活性肽grr及其制备与应用
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DATABASE GENBANK 1 October 2008 (2008-10-01), Database accession no. ABL62806.1 *

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