CN113456799A

CN113456799A - Application of p75ECD in preparing medicine for regulating pain

Info

Publication number: CN113456799A
Application number: CN202011562153.7A
Authority: CN
Inventors: 周意; 丁海峰; 王滢; 温波; 申付文; 叶欣; 李永涛
Original assignee: Suzhou Auzone Biological Technology Co ltd
Current assignee: Suzhou Auzone Biological Technology Co ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-10-01

Abstract

The p75 extracellular domain (p75ECD) or a functional fragment, variant, analog or derivative thereof can relieve, inhibit or treat different types of pain.

Description

Application of p75ECD in preparing medicine for regulating pain

Technical Field

The invention belongs to the technical field of biological pharmacy, and relates to a method for relieving, inhibiting or treating pain by using p75 ECD. More particularly, the invention relates to the use of p75ECD in the manufacture of a medicament for the relief, inhibition or treatment of pain. .

Background

Pain is a painful sensation from tissue damage or potential tissue damage, occurring simultaneously with human origin, and is a frequent, commonly encountered problem during the life activities of everyone from birth to death. However, since the human experience of "pain", the puzzle of pain has not yet been fully explained for the long centuries. Physicians remain unsure about many chronic, intractable pains.

Analgesics can selectively relieve pain and can be classified into strong analgesics and weak analgesics. The strong analgesic is analgesic for inhibiting central pain area, such as opioid analgesic (such as morphine, methylmorphine, ethylmorphine, etc.), synthetic analgesic (such as pethidine, annong pain, tebuconazole, etc.), has strong analgesic effect, but has certain addiction, and serious morbidity (withdrawal symptom) can occur after the drug is stopped after the drug is taken for many times. Weak analgesics refer to analgesics acting on peripheral pain receptors, often called antipyretic analgesics, and although such drugs are not addictive, the analgesic effect is to be improved.

p75NTR is a member of the tumor necrosis factor receptor superfamily, is a low affinity neurotrophin receptor (NTR) that can bind to brain-derived neurotrophic factor (BDNF), Nerve Growth Factor (NGF), neurotrophin-3/4 (NT-3/4), and their precursors, and mediate neuronal survival and apoptosis. During metabolism, p75NTR can be cleaved by metalloprotease and tumor necrosis factor α converting enzyme (TACE) to produce the soluble extracellular domain p75 ECD. The p75ECD has been advantageously recognized as a neuroprotective factor that can be used to diagnose, prevent or treat neurological disorders and/or protect neurons from apoptosis and neurodegeneration, but its effects on pain are not mentioned.

Disclosure of Invention

The present invention generally provides a p75ECD for use in the preparation of a medicament for modulating pain, wherein the p75 extracellular domain (p75ECD) or a functional fragment, variant, analog or derivative thereof of the present invention can alleviate, inhibit or treat various types of pain. The technical scheme is as follows:

use of a p75ECD in the manufacture of a medicament for modulating pain in the preparation of a medicament for alleviating, inhibiting or treating pain.

Further, the pain includes inflammatory pain or surgical pain.

Further, the p75ECD is p75 extracellular domain or a functional fragment, variant, analog or derivative thereof; the p75ECD is a p75ECD-Fc fusion.

Further, the p75ECD has the amino acid sequence provided by SEQ ID No. 1; the p75ECD-Fc fusion has the amino acid sequence provided by SEQ ID NO. 2.

Further, the p75ECD-Fc fusion is administered as a p75ECD-Fc fusion adeno-associated virus (AAV) vector.

Further, the p75ECD-Fc fusion AAV vector is administered at a dose of between 1x106 and 1x1013 viral genomes/kg body weight of the subject.

Further, the p75ECD-Fc fusion is a recombinant peptide.

Further, the p75ECD-Fc fusion peptide recombinant peptide is administered at a dosage between 0.1mg/kg body weight of the subject and 250mg/kg body weight of the subject.

Further, the p75ECD is administered by intraperitoneal, intracerebroventricular, intrathecal, or intramuscular injection. Induced expression and purification of p75ECD-Fc fusion protein:

E.col.BL-21 competent cells prepared by the CaCl2 method by transforming the correctly identified recombinant plasmid pet22b + p75ECD-Fc are picked up and inoculated into 10ml LB culture medium containing 50 ug/ml kanamycin sulfate and cultured with shaking at 37 ℃ overnight.

According to the following steps: transferring the overnight strain liquid into 200ml LB culture medium containing 50 mug/ml kanamycin sulfate at a ratio of 100, carrying out shaking culture at 37 ℃ until OD600 is approximately equal to 0.5-0.6, adding IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 0.1-0.5mmol/L, moving to 30 ℃ and continuing culturing for 4 hours.

And (4) centrifugally collecting thalli, wherein the thalli can be purified or frozen at-70 ℃. Suspending the thalli subjected to induced expression in PBS according to the volume of an original bacterial liquid 1/10, adding 15 mul of 50mmol/L PMSF, carrying out ultrasonic cell disruption (6X 10sec X40 hz), adding Triton X-100 with the final concentration of 1%, slightly oscillating for 20 minutes, centrifuging for 15 minutes at 4 ℃ at 12000r/m, collecting supernatant as a protein sample, and filtering by using a 0.45-micron-aperture filter membrane; the protein sample was applied to a Sepharose-SPA column and eluted at an equilibrium with 20mM PBS, pH7.4, and a flow rate of 0.5 ml/min.

The p75ECD-Fc is adsorbed by SPA on the column, and other proteins flow out along with the eluent; eluting with citric acid buffer solution with pH4.0 to obtain purified p75 ECD-Fc; and (3) measuring the protein content, activity and purity of the collected p75ECD-Fc liquid, desalting, concentrating, subpackaging, and storing at-20 ℃ for later use.

By adopting the scheme, the method has the following advantages:

1. the p75ECD of the present invention is effective in attenuating inflammatory pain.

2. The p75ECD of the present invention can reduce acetic acid-induced abdominal pain.

3. The p75ECD of the present invention is not addictive to pain treatment.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental procedures in the following examples are conventional procedures unless otherwise specified, and the experimental reagents and materials involved are conventional chemical reagents and materials unless otherwise specified.

Example 1

The invention adopts a classic formalin test to research the influence of p75ECD-Fc on acute inflammatory pain.

Plantar subcutaneous injection of formalin is a classic acute inflammatory pain model in which animals develop specific spontaneous pain behavior manifested as licking and biting of the foot, with longer licking or biting indicating greater pain. This spontaneous pain behaviour can generally be divided into 2 phases, the first phase occurring 0-5min after injection and the second phase occurring 15-60min after injection, with a pause period between the phases. The second phase is believed to be associated with pain sensitization (central sensitization) mechanisms.

The experiment was performed using male/female Kunming mice (6-8 years old) housed under constant temperature (25 ℃) and humidity (50%) for 12h light/dark cycle under standard conditions with any food and water. The experimental animals are divided into 12 animals, each half of the animals is male and female, and the animals are randomly divided into a p75ECD-Fc group and a blank control group, and each group comprises 6 animals.

The formalin inflammatory modeling method comprises the following steps: mice were first acclimated in a clear plexiglass box (30X 20cm) for 30min to eliminate the autonomous exploration behavior. When the test started, each mouse was gradually restricted, the experimental group mice were intraperitoneally injected with p75ECD-Fc having a concentration of 10. mu.g/ml of 0.2m, the blank control group mice were intraperitoneally injected with 0.9% physiological saline of the same volume, after 30 minutes, all the mice were subcutaneously injected with 50ul of 1% formalin solution in the right rear sole, immediately after injection, placed back in the box, and the time for licking and biting the injected feet was recorded for 1h after injection at the same time with 5min as a time period. The results are shown in Table 1.

TABLE 1 Effect of p75ECD on formalin diadocinol

The first phase of p75ECD-Fc licks the foot for a shorter time than that of the blank control group; in the second phase, the animal licking time of the p75ECD-Fc group is obviously reduced compared with that of the blank control group, which shows that the p75ECD-Fc of the invention has obvious analgesic effect on formalin-induced pain.

Example 2

An acetic acid induced abdominal pain experiment is carried out, and the influence of p75ECD-Fc on the abdominal pain caused by acetic acid injection is researched.

Mice mainly show the rolling reaction (writing Test) such as abdominal wall contraction, straightening and the like after the intraperitoneal injection of acetic acid, particularly show the most obvious rolling reaction 1 hour after the injection, and the rolling reaction becomes one of the common models for screening analgesics within 1 hour after the observation of the injection of the acetic acid.

20 Kunming mice, which are half male and half female, were randomly divided into experimental groups and blank control groups. Before the start of the experiment, each mouse was housed in an opaque cage for 30 minutes, and the p75ECD-Fc was administered in the same manner, at the same time and dose as in example 2, to a blank control group by intraperitoneal injection of 0.2ml of Phosphate Buffer Solution (PBS) having a concentration of 0.01mol/L, and after 30 minutes, all mice were intraperitoneally injected with 0.6% acetic acid to establish an abdominal pain model. The reversal reaction was observed within 1 hour after the injection of acetic acid. Abdominal pain intensity was assessed by the number of distinct abdominal flips with straightening of both hind limbs observed within 15 minutes immediately after injection. The results are shown in Table 2 below

TABLE 2 Effect of p75ECD on acetic acid induced abdominal pain

Compared with a blank control group, the mice in the p75ECD-Fc group have obviously reduced turnover reaction, which indicates that the pretreatment of the p75ECD-Fc provided by the invention can relieve the abdominal pain caused by acetic acid injection.

Sequence listing

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Claims

Use of a p75ECD in the manufacture of a medicament for modulating pain in the preparation of a medicament for alleviating, inhibiting or treating pain.
2. The use of the p75ECD of claim 1, in the preparation of a medicament for modulating pain, wherein: the pain includes inflammatory pain or surgical pain.
3. The use of the p75ECD of claim 1, in the preparation of a medicament for modulating pain, wherein: said p75ECD is the p75 extracellular domain or a functional fragment, variant, analog or derivative thereof; the p75ECD is a p75ECD-Fc fusion.
4. Use of the p75ECD of claim 1 or 3 in the manufacture of a medicament for modulating pain, wherein: the p75ECD has the amino acid sequence provided by SEQ ID No. 1; the p75ECD-Fc fusion has the amino acid sequence provided by SEQ ID NO. 2.
5. Use of the p75ECD of claim 3 in the preparation of a medicament for modulating pain, wherein: the p75ECD-Fc fusion is administered as a p75ECD-Fc fusion adeno-associated virus (AAV) vector.
6. Use of the p75ECD of claim 5 in the preparation of a medicament for modulating pain, wherein: the p75ECD-Fc fusion AAV vector was expressed at 1x10⁶To 1x10¹³Between doses of individual viral genomes/kg body weight of the subject.
7. Use of the p75ECD of claim 3 in the preparation of a medicament for modulating pain, wherein: the p75ECD-Fc fusion is a recombinant peptide.
8. Use of the p75ECD of claim 7 in the manufacture of a medicament for modulating pain, wherein: the p75ECD-Fc fusion recombinant peptide is administered at a dose between 0.1mg/kg body weight of the subject and 250mg/kg body weight of the subject.
9. The use of the p75ECD of claim 1, in the preparation of a medicament for modulating pain, wherein: the p75ECD is administered by intraperitoneal, intracerebroventricular, intrathecal or intramuscular injection.