TWI522111B - Peptide for preparing a pharmaceutical composition for alleviating pain - Google Patents

Description

Use of a peptide for the preparation of a pharmaceutical composition for relieving pain

The present invention relates to the use of a peptide for the preparation of a pharmaceutical composition for relieving pain, in particular the use of a guavacin (PCD) peptide for pain relief.

Marine organisms can survive in the aquatic environment rich in pathogenic microorganisms, relying on the advantages of their own powerful innate immune system to prevent microbial attack. Therefore, antimicrobial peptides in marine organisms are the first line of defense against pathogen invasion and are involved in the regulation of host signaling mechanisms in immune responses. Previous studies have revealed that several antibacterial peptides inhibit nitric oxide by acting on each other or affecting nitric oxide synthase. An α-helical antibacterial peptide, which is named as piscidin-1 (PCD-1), has a cation in nature and is an amphipathic molecule. PCD-1 peptide has antibacterial properties against ectoparasites, bacterial and fungal pathogens. Therefore, there is no literature on the efficacy of antibacterial peptides in anti-pain.

Chronic pain currently affects 1.5 billion people worldwide, and in 2009 the global pain market was over $50 billion. In addition, previous studies have shown that about 20% of people in the world suffer from chronic pain, and the prevalence of neuropathic pain is 6.9%. However, the specific mechanism of neuropathic pain is still unclear. Neuropathic pain is a kind of A general health problem associated with nerve damage, long-term tissue damage, or injury on the periphery or central nervous system (CNS); the resulting pain is the result of varying degrees of complex changes in the pain pathway. Patients with neuropathic pain generally exhibit hyperalgesia (increased response to painful stimuli), allodynia (pain induced by non-painful stimuli) and spontaneous pain, and opioid ( Opioids) and other analgesics (such as non-steroidal anti-inflammatory drugs) are tolerant. Current research reports indicate that no medication available can alleviate all neuropathic pain. Therefore, due to its complex natural history, unclear etiology, and poor response to drugs, the current treatment of neurological symptoms remains challenging. Gabapentin in anti-epileptic drugs is widely used to treat neuropathic pain and to effectively relieve hyperalgesia, burning pain, shooting pain and hyperalgesia. However, Jiaba Bentin has side effects that can cause withdrawals such as dizziness, lethargy, peripheral neuropathy, and gait disturbances. Therefore, recent studies in the treatment of pain have focused on screening for a safe, specific and effective analgesic compound from nature to relieve neuropathic pain.

Therefore, previous studies or reports did not explore the relationship between PCD-1 peptide and neuropathic pain.

In the present invention, it was observed that the PCD-1 peptide of the guavadin (PCD) peptide significantly inhibited the proinflammatory protein in macrophages (RAW264.7 cells) and microglia cells (BV2 cells). The inducible nitric oxide synthase (iNOS) and the up-regulation of epoxidase-2 (COX-2), so that the compound containing the peptide has an analgesic use. The potential for adjuvant therapy; and the use of antibacterial peptides (such as PCD-1 peptide) is unlikely to induce addiction because of the short half-life of the peptide in serum. In addition, PCD-1 peptide has the nature of anesthetic drugs, such as injection of PCD-1 peptide can significantly inhibit the pain behavior induced by chronic contraction injury (CCI), such as thermal hyperalgesia, mechanical pain (mechanical Allodynia), cold allodynia, and weight bearing. More importantly, treatment with PCD-1 peptide did not affect the original motor function of the rat (as shown in Figure 13) and did not result in any significant change in external behavior. In summary, the present invention demonstrates the mechanism by which itadministration of PCD-1 peptides under the anti-pain effects of neuropathy, which may include reduction of spinal neuroinflammation (eg, microglia and stars). Activation of astrocyte and upregulation of interleukin-1β (IL-1β); reduction of phosphorylation of astrocytes - upregulation of mammalian phosphatase (p-mTOR); And down-regulation of tumor growth factor-β1 (TGF-β1) which reduces astrocytes and neurons. The present invention demonstrates that both PCD-1 peptides exhibit anti-inflammatory and analgesic effects in both in vitro and in vivo modes. Therefore, the PCD-1 peptide is the first antibacterial peptide identified as a potential candidate compound that can be developed into an anesthetic drug in the future.

The articles "a" or "an" are used herein to describe the elements and compositions of the invention. This terminology is only for convenience of description and the basic idea of the invention. This description is to be construed as inclusive of the singular When used in conjunction with the word "comprising", the term "a" may mean one or more than one.

The present invention provides a use of a peptide for the preparation of a pharmaceutical composition for relieving pain, wherein the peptide is a Picosidin (PCD) peptide.

The Piscidin peptide is an antibacterial peptide. The term "Piscidin peptide" as used herein may be referred to the contents of Jorge A. Masso-Silva et al. ("Antimicrobial Peptides from Fish", Pharmaceuticals 2014, 7, 265-310); The entire contents of the above-mentioned documents are incorporated herein. In a preferred embodiment, the PCD peptide is a PCD-1 peptide. In a more preferred embodiment, the amino acid sequence of the PCD-1 peptide is SEQ ID NO: 1.

As used herein, "alleviating pain" means pharmacologically improving the symptoms. This pain relief is sufficient to eliminate or significantly reduce the effects of pain or pain. More specifically, the pharmacological means herein includes administering a mixture comprising the peptide to a body, such as topical or systemic administration, or oral administration to alleviate or alleviate pain.

The invention may be used to treat, alleviate, ameliorate, ameliorate, delay onset, inhibit progression, reduce severity, and/or reduce the occurrence of one or more symptoms or features of pain and/or pain. In a preferred embodiment, the invention is further used to treat pain.

As used herein, the term "pain" includes, but is not limited to, acute or chronic pain, which includes pain caused by trauma or inflammation, such as back pain, toothache, headache, menstrual cramps, Sore throat, fever, rheumatic pain (such as joint pain, gouty arthritis, ankylosing spondylitis, and rheumatoid arthritis) and systemic connective tissue disease , cancer, neuropathy and pain associated with referred pain. In a preferred embodiment, the pain is a chronic pain.

In another embodiment, the pain is a neuropathic pain. In a preferred embodiment, the pain is caused by neuroinflammation.

This neuropathic pain is caused by inflammation of the nerves. In the pattern of neuropathy, the expression levels of iNOS and COX-2 of microglia and astrocyte are upregulated. The induction of iNOS in small neuroglial cells, astrocytes, and neuronal cells in the spinal cord is associated with thermal hyperalgesia. Thus, the PCD peptide (or PCD-1 peptide) inhibits the performance of inflammatory proteins to reduce pain. Such inflammatory proteins include, but are not limited to, COX-2 and iNOS. In a specific embodiment, the PCD peptide further inhibits neuroinflammation. In another specific embodiment, the PCD peptide inhibits the performance of an inflammatory protein. In a preferred embodiment, the PCD peptide inhibits the expression of a epoxidase-2 (COX-2). In a more preferred embodiment, the PCD peptide inhibits the expression of an inducible nitric oxide synthase (iNOS).

The term "performance" as used herein refers to the amount of expression of a gene, RNA or protein.

The pharmaceutical composition of the present invention may further comprise a PCD peptide (or PCD-1 peptide) of the present invention or a variant thereof having the same functional activity, and a pharmaceutically acceptable carrier. As used herein, the term "variant with the same functional activity" refers to a peptide that is essentially homologous to the original peptide, but which results in a peptide with the present invention due to one or more deletions, insertions or substitutions. Different amino acid sequences. The substitution may include a sequence of retention substitutions, meaning that the amino acid group is known to be substituted with a group having similar physiochemical properties. Suitable retention of amino acids in peptides or proteins, well known to those of ordinary skill in the art, and without altering the resulting molecules The biological activity is completed. Preferably, the steric structure of this variant must be identical to the original protein peptide.

The pharmaceutical compositions of the present invention comprise an effective amount of a PCD peptide (or PCD-1 peptide). As used herein, "effective amount" refers to an effective dose that prevents, reduces, stops, or reverses the development of an existing condition that is desired to be treated, or that partially or completely alleviates the condition. In a particular embodiment, the effective dose ranges from about 0.1 [mu]g to 100 [mu]g. In a preferred embodiment, the effective dose ranges from about 1 [mu]g to 50 [mu]g. In a more preferred embodiment, the effective dose ranges from about 10 [mu]g to 30 [mu]g.

The term "subject" as used herein refers to an animal. In a preferred embodiment, the system refers to a mammal. In a more preferred embodiment, the system refers to a human.

The term "pharmaceutically acceptable carrier" as used herein is to some extent determined by the particular composition being administered, as well as the particular method of administering the composition. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonics. And isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids and the like. The use of such media and agents for the use of pharmaceutically active ingredients is well known to those skilled in the art. In addition to any conventional media or agents that are incompatible with the active ingredients, their use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not cause an allergic or similar adverse reaction when administered to a subject. And the preparation of a water-soluble composition comprising a protein which is an active ingredient is a skill Well known to those skilled in the art. Typically, such compositions are prepared in injectable form, such as liquid solutions or suspensions; or solid forms suitable for dissolving or suspending in liquids, which may be prepared prior to injection.

Administration of the pharmaceutically acceptable carrier and PCD peptide can be administered in a variety of ways/paths and under various institutional specifications well known to those skilled in the art. In some embodiments, the pharmaceutically acceptable carrier and the PCD peptide are administered by intravenous, intramuscular, subcutaneous, topical, oral, or by inhalation; the primary is through the digestive system and the circulatory system. To deliver to the target location.

The pharmaceutically acceptable carrier and PCD peptide for administration can be formulated via sterile water, dispersion, aqueous suspension, oil emulsion, aqueous emulsion in oil, site-specific emulsion, long-term retention emulsion (long-residence emulsion), colloidal emulsion, microemulsion, nanoemulsion, microlipid, microparticles, microspheres, nanospheres, nanoparticles, micro-pumps, or various natural or synthetic compounds with sustained release capability polymer. The pharmaceutically acceptable carrier and PCD peptide can be formulated into an aerosol, a tablet, a pill, a sterile powder, a suppository, a lotion, an emulsion, an ointment, a paste, a gel, a hydrogel, and a continuous Release device, or other formulation for drug delivery.

Individuals with pain develop activation of glia on the nervous system to regulate pain. The activation of glial cells is accompanied by a number of cellular responses, including the production and release of substances that sensitize the pain pathways of the nervous system (such as the so-called "proinflammatory cytokines"). Types of glial cells include microglia and macroglia, which contain, for example, astrocyte, oligodendrocyte. And Schwann cells. In a specific embodiment, the PCD peptide reduces the activation of a glia cell (glia). In a preferred embodiment, the PCD peptide reduces the activation of a microglia. In a more preferred embodiment, the PCD peptide reduces the activation of an astrocyte.

The neuropathy-induced pain allergy is the activation of the mammalian target of rapamycin (mTOR); the phosphorylation of serine-2448 (Ser-2448) on mTOR is the marker of mTOR activation. . In general, inhibition of mTOR reduces neuroinflammation. In addition, interleukin-1β (IL-1β) is a proinflammatory factor. Thus, the inflammatory factors that are elevated by the inflammatory response include IL-1β and phosphorylated-mammalian-deficient proteins (phosph-mTOR, p-mTOR). Therefore, the PCD-1 achieves analgesic or anti-pain by suppressing the inflammatory reaction. In a specific embodiment, the PCD peptide inhibits up-regulation of IL-1β or phospho-mTOR expression. In a preferred embodiment, the PCD peptide reduces the amount of monophosphorylation-mammalian-deficient protein (phospho-mTOR). In a more preferred embodiment, the PCD peptide reduces the amount of interleukin-1β (IL-1β) expression.

For individuals with peripheral neuropathy, the PCD peptide (or PCD-1 peptide) provides anti-neuroinflammatory effects. The PCD peptide can reduce the down-regulation of a factor having anti-neuroinflammatory effects such as tumor growth factor-β1 (TGF-β1), for example, the factor inhibits the activation of microglia cells and astrocytes. In a specific embodiment, the PCD peptide increases the amount of expression of a tumor growth factor-β1 (TGF-β1).

The invention is further useful for the preparation of an anesthetic, analgesic or anti-inflammatory agent. The pain and / or inflammation are available Examples of chronic diseases include, for example, rheumatoid arthritis, osteoarthritis, spinal disc disease (such as sciatica), carpal/tarsal tunnel syndrome, low back pain, lower limb pain, upper limbs. Pain, cancer, tissue pain, and cervical, thoracic and/or lumbar or intervertebral discs, rotator tendons, joints, ankle joint disorders (TMJ), tendons, ligaments, muscles, spondilothesis, stenosis Stenosis), discogenic back pain, joint pain or similar symptoms of pain or repair related pain. The analgesic means an agent having an anesthetic effect that relieves pain but does not reduce the sensation. An analgesic is an agent or compound that reduces, reduces or eliminates pain. The term "anti-inflammatory agent" means an agent or compound having an anti-inflammatory effect. These agents reduce inflammation and reduce pain.

Figure 1 shows the expression of inflammatory proteins iNOS and COX-2 in RAW264.7 cells induced by PCD-1 peptide-lowering ester polysaccharide (LPS). (A) Survival of RAW264.7 cells treated with different doses of PCD-1 peptide for 24 hours, wherein the survival rate was analyzed by Alamar blue assay. (B) The results of Western immunological dot method for inhibiting LPS-stimulated RAW264.7 cells expressing iNOS and COX-2 by PCD-1 peptide and β-actin in the internal control group. (C) Results of the amount of iNOS measured by Western immunological dot method in RAW264.7 cells under different treatments. (D) Results of the amount of COX-2 measured by Western immunoblotting in RAW264.7 cells under different treatments. PCD-1 peptide significantly inhibited the expression of iNOS and COX-2 in RAW264.7 cells (repeated three times, compared with the group in which LPS alone was added, P < 0.05 is indicated by *).

Figure 2 shows that PCD-1 peptide reduces the expression of inflammatory proteins iNOS and COX-2 in BV2 cells induced by LPS. (A) Survival of BV2 cells treated with different doses of PCD-1 peptide for 24 hours, wherein the survival rate was analyzed by Alamar blue assay. (B) The results of Western immunological dot method for inhibiting LPS-stimulated BV2 cells expressing iNOS and COX-2 by PCD-1 peptide and β-actin in the internal control group. (C) Results of the amount of iNOS measured by Western immunological dot method in BV2 cells under different treatments. (D) Results of the amount of COX-2 measured by Western immunoblotting in BV2 cells under different treatments. PCD-1 peptide significantly inhibited the expression of iNOS and COX-2 in BV2 cells (repeated three times, compared with the group in which LPS alone was added, P < 0.05 is indicated by *).

Figure 3 shows the effect of PCD-1 peptide on thermal hyperalgesia similar to gabapentin in rats undergoing chronic compact injury (CCI) surgery. (A) Percentage of the maximum possible effect of anti-thermal hyperalgesia induced by different doses (0, 0.1, 1, 5, 10, 20 μg) of PCD-1 peptide in rats subjected to CCI surgery (MPE) ;%) dose response curve (the number of experimental animals in each group is 6). (B) Percentage of the greatest possible effect of anti-hyperalgesia (JE; %) exhibited by different doses (0, 1, 5, 10, 20 μg) of Jiaba Bentin in rats subjected to CCI surgery The dose response curve (the number of experimental animals in each group is 6). (C) is to determine the effective dose (ED50) of PCD-1 peptide and Jiaba Bentin from the standard curve, PCD-1 peptide and Jiaba Bentin for heat hyperalgesia The effective doses of 50 were 9.5 ± 3.2 μg and 2.3 ± 1.5 μg, respectively (compared with the group receiving CCI surgery and given blank solvent at the indicated time points, P < 0.05 is indicated by *).

Figure 4 shows that PCD-1 peptide has anti-mechanical and anti-cold hyperalgesia effects on rats undergoing CCI surgery. (A) and (B) are the effects of PCD-1 peptide (20 μg) on mechanical pain induced by rats undergoing CCI surgery. (C) and (D) are the effects of PCD-1 peptide (20 μg) on cold hyperalgesia induced by rats undergoing CCI surgery. Among them, PCD-1 was injected into the spinal canal. Anti-pain data (A and C) after peptides are expressed as a percentage (%) of the maximum possible effect (B and D). In rats receiving CCI surgery, the effect of anti-mechanical pain was observed in the 30th to 240th minute after injection of PCD-1 peptide into the spinal canal, and the anti-cold hyperalgesia effect was 30th to 210th minutes after injection. Occurred internally (the number of experimental animals in each group was 6; and the group receiving CCI surgery and given blank solvent was compared at the specified time point, P < 0.05 is indicated by *).

Figure 5 shows that PCD-1 peptide has anti-biological weight-bearing efficacy in rats undergoing CCI surgery. In rats receiving CCI surgery, PCD-1 peptide (20 μg) was injected into the spinal canal with a weight-bearing effect within 30 to 210 minutes (the number of experimental animals in each group was 6; and CCI surgery was given and given) The groups of blank solvents were compared at the indicated time points, P < 0.05 in *).

Figure 6 shows the activation of microglia induced by PCI-1 peptide treatment to inhibit CCI. Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Activation of microglia cells on the spinal cord can be observed by immunostaining markers with OX42 antibody. (A) is the control group (no treatment is given); (B) is the CCI group (accepting CCI surgery and given a blank solvent); (C) is 30 minutes after CCI + PCD-1 peptide (20 μg); (D) ) is the 90th minute after CCI + PCD-1 peptide (20 μg) and (E) is 180 minutes after CCI + PCD-1 peptide (20 μg). (F) Quantitative statistical results of immunofluorescence of OX42 antibody, which showed significant but not complete inhibition of CCI induced activation of microglia cells in the dorsal horn of the spinal cord after administration of PCD-1 peptide (number of experimental animals in each group) 6; compared with the control group, P < 0.05 is indicated by #; compared with the CCI group, P < 0.05 is indicated by *; scale bar = 50 μm).

Figure 7 shows the activation of astrocyte induced by CCI by PCD-1 peptide. Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Activation of stellate cells on the spinal cord can be observed by immunostaining markers with GFAP antibodies. (A) is the control group (no treatment is given); (B) is the CCI group (accepting CCI surgery and given a blank solvent); (C) is 30 minutes after CCI + PCD-1 peptide (20 μg); (D) ) is the 90th minute after CCI + PCD-1 peptide (20 μg) and (E) is 180 minutes after CCI + PCD-1 peptide (20 μg). (F) is a quantitative statistical result of immunofluorescence of GFAP antibody, which shows that activation of stellate cells on the dorsal horn of the spinal cord induced by CCI is significantly inhibited after administration of PCD-1 peptide (the number of experimental animals in each group is 6; The control group was compared as a comparison, P < 0.05 is indicated by #; compared with the CCI group, P < 0.05 is indicated by *; scale bar = 50 μm).

Figure 8 shows the activation of interleukin-1β (IL-1β) induced by PCD-1 peptide inhibition of CCI. Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Activation of IL-1β on the spinal cord can be observed by immunostaining markers with anti-IL-1β antibodies. (A) is the control group (no treatment is given); (B) is the CCI group (accepting CCI surgery and given a blank solvent); (C) is 30 minutes after CCI + PCD-1 peptide (20 μg); (D) ) is the 90th minute after CCI + PCD-1 peptide (20 μg) and (E) is 180 minutes after CCI + PCD-1 peptide (20 μg). (F) is a quantitative statistical result of immunofluorescence of IL-1β, which shows that IL-β is up-regulated in the dorsal horn of the spinal cord induced by CCI after administration of PCD-1 peptide (groups) The number of experimental animals was 6; compared with the control group, P < 0.05 is indicated by #; compared with the CCI group, P < 0.05 is indicated by *; scale bar = 50 μm).

Figure 9 shows the up-regulation of the phosphorylation-mammalian-suppressed protein (p-mTOR) induced by CCI by PCD-1 peptide. Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Activation of p-mTOR on the spinal cord can be observed by immunostaining with an anti-p-mTOR antibody. (A) is the control group (no treatment is given); (B) is the CCI group (CCI surgery and blank solvent); (C) 30 minutes after CCI+PCD-1 peptide (20 μg); (D) 90 minutes after CCI+PCD-1 peptide (20 μg) and (E) ) is the 180th minute after CCI + PCD-1 peptide (20 μg). (F) is a quantitative statistical result of immunofluorescence of p-mTOR, which shows that the inhibition of CCI induced a significant increase in p-mTOR in the dorsal horn of the spinal cord after administration of the PCD-1 peptide (the number of experimental animals in each group is 6). Compared with the control group, P < 0.05 is indicated by #; compared with the CCI group, P < 0.05 is indicated by *; scale bar = 50 μm).

Figure 10 shows that PCD-1 peptide treatment reduces CCI-induced upregulation of p-mTOR in small neuroglial cells, stellate cells, and neuronal cells (neuron). Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Spinal cord sections at 180 minutes after PCD-1 peptide injection to analyze the co-localization of p-mTOR with specific markers for microglia, stellate or neuronal cells. (A), (D) and (G) are the control group (no treatment is given); (B), (E) and (H) are the CCI group (accepting CCI surgery and giving blank solvent); (C), ( E) and (I) are spinal cord sections at 180 minutes after CCI + PCD-1 peptide (20 μg). Double immunofluorescence staining showed that PCD-1 peptide significantly inhibited the up-regulation of p-mTOR in CCI-induced stellate cells. The arrow marks the colocalization of GFAP antibodies (markers of stellate cells) with p-mTOR; such cases are compared with rats treated with CCI and given PCD-1 peptide (F) compared to CCI (E) It is reduced (scale bar = 25 μm).

Figure 11 shows the downregulation of tumor growth factor-β1 (TGF-β1) induced by PCD-1 peptide in CCI regulation. Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Activation of TGF-β1 on the spinal cord can be observed by immunostaining with anti-TGF-β1 antibody. (A) is the control group (no treatment is given); (B) is the CCI group (accepting CCI surgery and given a blank solvent); (C) is 30 minutes after CCI + PCD-1 peptide (20 μg); (D) ) It was the 90th minute after CCI+PCD-1 peptide (20 μg) and (E) the 180th minute after CCI+PCD-1 peptide (20 μg). (F) is a quantitative statistical result of immunofluorescence of TGF-β1, which shows that down-regulation of TGF-β1 in the dorsal horn of the spinal cord induced by CCI is significantly inhibited after administration of PCD-1 peptide (groups) The number of experimental animals was 6; compared with the control group, P < 0.05 is indicated by #; compared with the CCI group, P < 0.05 is indicated by *; scale bar = 50 μm).

Figure 12 shows that PCD-1 peptide inhibits CCI-induced downregulation of TGF-β1 in small glial cells, stellate cells, and neuronal cells. Rats undergoing CCI surgery were injected with 20 μg of PCD-1 peptide or blank solvent in the spinal canal. Spinal cord sections at 180 minutes after PCD-1 peptide injection to analyze the colocalization of TGF-β1 with specific markers for small neuroglial cells, stellate cells or neuronal cells. (A), (D) and (G) are the control group (no treatment is given); (B), (E) and (H) are the CCI group (accepting CCI surgery and giving blank solvent); (C), ( E) and (I) are spinal cord sections at 180 minutes after CCI + PCD-1 peptide (20 μg). Double immunofluorescence staining showed that PCD-1 peptide significantly inhibited the down-regulation of TGF-β1 in CCI-induced stellate cells and neuronal cells. The arrow marks the colocalization of TGF-β1 with GFAP antibodies (markers of stellate cells) or NeuN antibodies (markers of neuronal cells); such cases are in rats subjected to CCI surgery and given PCD-1 peptide treatment ( F and I) were increased compared to the CCI group (E and H) (scale bar = 25 μm).

Figure 13 shows that PCD-1 peptide did not affect the motor function of the CCI-treated rats in the single-wood bridge test. Rats who underwent CCI surgery and were given a blank solvent were compared with the number of seconds that the rats who received CCI surgery and were given PCD-1 peptide walked with the behavior of the single-wood bridge; no difference was observed between the two groups in 480 minutes. Significant differences (6 animals in each group).

The invention may be implemented in different content and is not limited to the following And examples. The following examples are merely representative of the various aspects and features of the present invention.

Example 1 Preparation of peptide

The peptide used in the present invention is a guavamycin (Piscidin, PCD) peptide, and the PCD-1 peptide is further used for the following experiment, wherein the peptide sequence of the PCD-1 peptide is SEQ ID NO :1 (FFHHIFRGIVHVGKTIHRLVTG). The PCD-1 peptide was synthesized and purified by GL Biochemistry (Shanghai, China) to a degree greater than 95%. The molecular weight and purity of the peptide were determined to exceed 95% by high performance liquid chromatography (HPLC). In addition, synthetic peptides were dissolved in sterile deionized water or PBS buffer for experiments.

Data and statistical analysis

The data presented in all experiments were presented as mean ± mean standard error (SEM). For statistical analysis, the difference between the groups was calculated by one-way analysis of variance (ANOVA), and the difference between multiple recombinations was compared according to the Student-Newman-Keuls post hoc test to analyze the significant differences. . The invention defines that when the P value is less than 0.05, there is a significant difference.

Example 2 Anti-inflammatory and anti-neuroinflammatory activity analysis methods:

The present invention performs anti-inflammatory and anti-neuroinflammatory activity analysis through macrophage cells and microglia cells.

(1) Mouse macrophage cell line (RAW 264.7 cells) of the present invention was purchased from American Type Culture Collection (ATCC, No. TIB-71). RAW 264.7 cells were cultured in DMEM (Dulcbeccos Modified Eagle Medium) medium, and contained 10% fetal bovine serum (FBS) and penicillin G (100 U/ml) and streptomycin (100 μg/ml) antibiotics, cultured at 37 °C, 5% CO ₂ incubator, when the cells grow to eight minutes, drain the culture solution, wash it twice with phosphate buffered saline (PBS), and then attach it with trypsin. The cells on the culture dish are knocked down and subcultured. The RAW 264.7 cell line was subcultured in a 6 cm ulture dish, controlled at 3 x 10 ⁶ cells, and after 12 hours of culture, ester polysaccharide (LPS, 0.01 μg/ml; Sigma L2654) and PCD-1 were administered. The peptide was collected and the cells were collected for anti-inflammatory test after 16 hours. In the anti-inflammatory group, different concentrations of PCD-1 peptide (2.5, 5 and 10 μg/ml in sequence) were added to the culture dish for ten minutes. The cells were collected by LPS treatment and subjected to anti-inflammatory tests.

(2) The microglial cells of the present invention are murine microglia cell lines (BV2 cells). BV2 cells were cultured in DMEM (Dulcbeccos Modified Eagle Medium) medium, and contained 10% fetal bovine serum (FBS) and penicillin G (100 U/ml) and streptomycin (100 μg/ml) antibiotics, and cultured at 37 ° C. In a 5% CO ₂ incubator, when the cells are as long as eight minutes, the culture medium is drained, washed twice with PBS, and then trypsin is used to knock down the cells attached to the culture dish. , subculture. The BV2 cell line was subcultured in a 6 cm culture dish, controlled at 3 x 10 ⁶ cells, and after 12 hours of culture, ester polysaccharide (0.01 μg/ml; Sigma L2654) and PCD-1 were administered at 16 After an hour, the cells were collected for anti-inflammatory test; in the anti-inflammatory group, different concentrations of PCD-1 peptide (2.5, 5, and 10 μg/ml in sequence) were added to the culture dish for ten minutes, and then added to the LPS treatment. The cells were tested for anti-inflammatory.

After the above cells were collected, they were washed with cold PBS, and then dissolved in a solution (pH 7.5, 1 μg/ml aprotinin, 50 mM Tris, 150 mM NaCl, 100 μg/ml phenylmethylsulfonyl fluoride, 1% Triton X-100). The lysate was centrifuged at 20,000 xg for 60 minutes at a temperature of 4 ° C, and the supernatant fraction was taken out and analyzed for inducible nitric oxide synthase (iNOS) and epoxidase-2 by western blotting. The amount of performance of COX-2). The protein concentration of the supernatant was detected using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of sample buffer (2% 2-mercaptoethanol, 0.1% bromophenol bule, 50 mM Tris-HCl, 2% SDS, pH 7.2) was added to each protein sample. The protein was electrophoresed on a 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) for 90 minutes at 70 volts; the protein on SDS-PAGE was transfected with 135 mA current to have a transfer buffer. Liquid (transfer buffer; 380 mM glycine, 1% SDS, 50 mM Tris-HCl, 20% methanol) PVDF membrane (Polyvinylidene Difluoride membrane) (0.45 mm pore size, Immobilon-P, Millipore, Bedford, MA, USA), at 4 Place at °C overnight. The transfected PVDF membrane was immersed in a 5% solution of Tris-Tween buffer saline (Tris-HCl 20 mM, NaCl 137 mM, pH 7.4, 0.1% Tween 20) for 1 hour at room temperature; : 1000 dilution ratio of anti-iNOS antibody (BD Pharmingen, San Diego, CA, USA; catalog no. 6103322; polyclonal antibody) and anti-COX-2 antibody (Cayman Chemical, Ann Arbor, MI, USA; catalog no. 160106 ;polyclonal antibody) was allowed to react at room temperature for 180 minutes. The immunoreactive band of the protein of iNOS (about 135 kDa) and COX-2 (about 70 kDa) is profitable. The display was performed with an agent (enhanced chemiluminescence; ECL kit; Millipore), and image analysis was performed using an image analysis processing apparatus (UVP BioChemi imaging system; UVP LLC, Upland, CA, USA). The relative density of the immunoreactive bands was calculated using LabWorks 4.0 software (UVP LLC, Upland, CA, USA), and the relative differences between the different groups of immunoreactive bands were calculated using the same image map. . The intensity of each immunoreaction zone in the group simply added to LPS was set to 100%. Further, the present invention reacts a PVDF membrane with an anti-β-actin antibody (1:2500 dilution; catalog no. A5441; Sigma Co., Ltd., St Louis, MO, USA; monoclonal mouse antibody) to treat β-actin as Internal control group.

Analysis results: PCD-1 peptide inhibits the expression of iNOS and COX-2 in LPS-treated RAW264.7 cells and small glial cells

Inhibition of iNOS and COX-2 induces anti-nociceptive behavior. In order to investigate the regulation of iNOS and COX-2 by the PCD-1 peptide, the present invention treats the mouse macrophage cell (RAW264.7 cells) and immune cells with a non-toxic concentration of PCD-1 peptide. The effect of iNOS and COX-2 on the amount of expression was also examined. The DD264.7 cells treated with the PCD-1 peptide in the dosage range of 20 μg/ml of the present invention for 24 hours did not affect the growth of the cells (as shown in Fig. 1A); therefore, the present invention employs a non-toxic concentration (0). PCD-1 peptide up to 10 μg/ml) was subjected to subsequent anti-inflammatory activity analysis. The expression levels of iNOS and COX-2 were detected by Western immunoblotting method (as shown in Fig. 1B); and the relative intensity of the expression was shown in Fig. 1C and Fig. 1D. The amount of iNOS and COX-2 protein expressed in the LPS group alone was 100%, and other groups Otherwise, the PCD-1 peptide concentration was 2.5, 5, and 10 μg/ml, respectively. It can be seen that simply treating the cells with LPS leads to up-regulation of iNOS and COX-2 expression, and the PCD- The addition of the peptide 1 will also inhibit the expression of iNOS and COX-2 protein depending on the increase in concentration (as shown in Figures 1B, 1C and 1D).

In addition, the present invention also detects the effect of PCD-1 peptide on iNOS and COX-2 in small neuroglial cell lines (BV2 cells) to mimic the effects on immune cells located in the central nervous system (CNS). As observed on RAW264.7 cells, PCD-1 peptide inhibited the upregulation of iNOS and COX-2 expression on BV2 cells induced by LPS (as shown in Figure 2).

Example 3 Rat model of chronic constriction injury and its testing method:

(1) Chronic constriction injury (CCI) and placement of the spinal canal (intrathecal catheter)

Rats underwent spinal canal implantation with 2.5% isofurane anesthesia. First, after the head of the mouse was shaved to the back neck, the rats were fixed on the operating table and injected with antibiotics by intraperitoneal injection. Cut the muscles in the midline of the neck, separate the muscle layer from the skull, and do not expose the cisternal membrane. Use a needle to place the pool-like membrane (poke a small hole and open, at this time there will be cerebrospinal fluid outflow. Insert the spinal canal into the sheath (PE5 polyethylene tube, 9 cm long, inner diameter is 0.008 inches, outer diameter is 0.014 inches, Colorado Springs, CO, USA) for spinal canal injection and at least five days of healing for rats undergoing spinal canal implantation Induction surgery for chronic constriction injury (CCI).

Induction of Chronic Tightening Injury (CCI) Referring to the prior literature, the present invention uses a blunt dissection to expose the sciatic nerve about 5 mm long in the right thigh of the right lower extremity of the rat, with a 4-0 chrome gut pair. The sciatic nerve was subjected to 4 mild ligations with a spacing of 1 mm.

(2) Pain behavior assessment method:

(a) Thermal hyperalgesia

The thermal hyperalgesia of the present invention was tested using an IITC analgesiometer (IITC Inc., Woodland Hills, CA, USA). Place the rats in a plastic cage above the raised glass plate and irradiate the hind paws of the rats with a low-intensity radioactive heat source (25 in effect) until the rats show signs of positive pain (eg sputum or Shrink the foot). The paw withdrawal latency (PWL) is calculated in seconds and the irradiation of the radioactive heat source is set to an upper limit of 30 seconds. The present invention records PWL at 30, 60, 90, 120, 150 and 180 minutes after administration of PCD-1 peptide or gabapentin, respectively. The present invention further converts the PWL data into a drug effect and presents it as a percentage of the maximum possible effect (MPE; %) using the following formula: Percentage of the maximum possible effect = (administration) Value of post reaction time - reference value) / (upper limit value - reference value) x 100%

The value of the reaction time after administration is the time of the contraction reaction recorded at the 30th, 60th, 90th, 120th, 150th and 180th minutes after administration of the PCD-1 peptide, the gabapentine or the blank vehicle; Value refers to the immediate reaction time before spinal canal injection; the upper limit is 30 second.

(b) Mechanical allodynia

Mechanical pain was analyzed using a calibrated Von Frey filament (Stoelting, Wood Dale, IL, USA) to analyze the paw withdrawal threshold (PWT), which was calculated in grams. Rats were placed in plastic cages above the raised metal mesh floor and stimulated by Chaplan's Up-Down method with a series of Von Frey filaments with logarithmic strength to stimulate the middle of the hind paw of the rat. The reflex reaction of the foot after the period, if there is no reaction, gradually increases until the rat reacts and records the strength of the filament used to cause the reaction as the threshold of the contraction.

(c) cold allergy (cold allodynia)

The rats were placed in a plastic cage above the raised metal mesh plate, and the central region of the hind paw of the rat was stimulated with 25 μl of acetone, and the degree of cold hyperalgesia in the rat was monitored after 1 minute of stimulation with acetone ( According to the magnitude of the acetone reaction, the scores of the six levels of the present invention are: 0: repeated wiping and tapping the soles of the feet within 2 seconds after the stimulation; 1: long-term retraction within 2 seconds after the stimulation or Repeatedly tapping the soles of the feet; 2: Quickly and violently shrinking, tapping or slamming within 2 seconds after stimulation; 3: Quickly retracting, tapping or slamming within 2 seconds after stimulation; 4: 2 seconds after stimulation Retracting, tapping or footing; 5: No reaction.

The experiment was repeated four times with acetone (acetone) every five minutes, and the total score was added. Therefore the smallest possible total score is 0 points and the maximum possible total score is 20 points.

(d) Weight bearing test

Rat placed in the biped balance tester (incapacitance tester; Singa Technology Corporation, Taiwan), and the two hind feet of the rat were concentrated on the two force sensors on the biped balance pain tester, and the weight of the individual force applied to the left and right hind legs of the rats was tested. The weight of the hind foot is expressed in grams; and the calculation is performed by measuring the difference between the normal hind foot and the affected hind foot at the same time point.

(e) Narrow beam test

The wooden bridge used in the present invention is a wooden beam having a length of 80 cm and a width of 2.5 cm, and the ends thereof are supported by wooden brackets so as to be 100 cm from the ground. A foaming filling (1 metre wide, about 12 cm thick) was placed under the beam to prevent the rat from falling and causing injury. The upper limit of the time taken for the rats to walk the single-wood bridge was set to 15 seconds, and the rats were pre-trained to walk the wooden bridge before the test.

(3) Immunostaining fluorescence staining analysis

Rats from each group (including normal rats not given any treatment, rats receiving CCI surgery and given blank solvent, and 30, 90, 180 after receiving CCI surgery and giving PCD-1 peptide (20 μg) Spinal cord tissue was collected from the rats of the minute. Tissue samples were sliced at a thickness of 10 μm, and anti-OX42 antibody, anti-GFAP antibody, anti-phosphorylated-mammalian-deficient protein (phosph-mTOR; p-mTOR) antibody, anti-interleukin-1β (IL) at 4 °C The -1β) antibody or the anti-tumor growth factor-β1 (TGF-β1) antibody was reacted overnight. The identification of cell types is based on different biomarkers, such as microglia (OX42), astrocyte (GFAP) or neural cells (neun; NeuN). Sample sections were incubated with Alexa Fluor 488-labeled chicken anti-mouse IgG antibody at room temperature (1:400 ratio dilution, cat. 705-546-147; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; green fluorescence), DyLight 549-conjugated donkey anti-rabbit IgG antibody (1:400 ratio dilution, cat. 711-506-152; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; red fluorescence) or Alexa Fluor 488 conjugated donkey anti-goat IgG antibody (1:400 ratio dilution, cat. 705-546-147; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; green fluorescence) reaction for 40 minutes. This will enable detection of p-mTOR or TGF-β1 as a pseudo green signal. For dual immunofluorescence staining, spinal cord sections are reacted with the following antibody cocktails at 4 ° C to overnight anti-antises, such as anti-OX42 antibodies and anti-p-mTOR antibodies, anti-GFAP antibodies and anti-phosto-mTOR antibodies, anti-NeuN antibodies and antibodies p-mTOR antibody, anti-OX42 antibody and anti-TGF-β1 antibody, anti-GFAP antibody and anti-TGF-β1 antibody, anti-NeuN antibody and anti-TGF-β1 antibody; followed by conjugate with Alexa Fluor 488 and conjugated with DyLight 549 The mixture of grades of antibody was reacted at room temperature for 40 minutes. Four spinal cord sections were randomly selected and scanned with a Leica DM-6000 CS fluorescence microscope (Leica Instruments Inc., Wetzlar, Germany) followed by a SPOT digital image capture system (SPOT Xplorer Digital camera, Diagnostic Incstruments, Inc., Sterling) Heights, MI, USA) for image capture. In terms of quantitative fluorescence analysis, one continuous slice of spinal cord is taken every four slices, and four consecutive slices are calculated. Finally, the computational observer used the Image J (National Institutes of Health, Bethesda, Maryland, USA) imaging software to perform quantitative analysis of the image without knowing the conditions. The pixel value of the immunoreactive positive area of the dorsal horn of the spinal cord on the image was included in the calculation. The data for immunohistochemical staining were presented as a percentage change after 100% comparison with normal animal groups without any treatment.

result:

(1) PCD-1 peptide has a dose-dependent effect on the effects of heat-hyperalgesia in rats undergoing CCI surgery

The paw withdrawal reaction time (PWL) was performed at different time points from 0 to 180 minutes after injection of 0, 0.1, 1, 5, 10, 20 μg of PCD-1 peptide through the spinal canal. Treatment of PCD-1 peptide increased the percentage of the greatest possible effect (MPE; %) against the hyper-hyperalgesia (CPE) in rats undergoing CCI surgery, depending on the dose-dependent increase (Figure 3A)). In particular, the 20 μg dose of PCD-1 peptide showed a significant increase in heat-induced hyperalgesia at all time points of the experiment, so further experiments will be conducted using this dose. The effect of PCD-1 peptide on the anti-hyperalgesia in rats undergoing CCI surgery is similar to that in rats given gabapentin (a widely used anti-neuropathic drug). 3B)). A 50% effective dose (ED50) of PCD-1 peptide and Jiaba Bentin was calculated using a standard curve, while PCD-1 peptide and Jiaba Bentin were 50% resistant to thermal hyperalgesia. The effective dose (ED50) was 9.5 ± 3.2 μg and 2.3 ± 1.5 μg, respectively (as shown in Figure 3C).

(2) PCD-1 peptide has anti-mechanical pain, anti-cold hyperalgesia and anti-biological weight-bearing effect on rats undergoing CCI surgery

Rats subjected to CCI surgery treated with blank vehicle had a paw withdrawal threshold (PWT) that remained unchanged at all experimental time points (as shown in Figure 4A). Compared with the CCI group (CCI surgery and blank solvent), rats receiving CCI surgery significantly increased the threshold of withdrawal at 30 minutes after injection of PCD-1 peptide; at the 60th minute, the threshold of contraction reached peak. And maintained significantly greater than the CCI group's paw withdrawal threshold within 240 minutes after receiving PCD-1 peptide treatment (as shown in Figures 4A and 4B). Next, the rats received cold stimulation of acetone to measure cold allodynia. Cold hyperalgesia in rats treated with blank solvent was close to the baseline value at all experimental time points, whereas PCD-1 peptide treated rats were 30 after injection. Minutes showed an increase in anti-cold hyperalgesia; this anti-cold hyperalgesia peaked at 60 minutes; and remained significantly greater than the CCI group within 210 minutes after receiving PCD-1 peptide treatment (Figure 4C) And 4D). In addition, in the 30th and 210th minute after receiving the PCD-1 peptide treatment, the rats were measured for the weight of the biped by the bipedal balance pain tester and found to be greatly reduced (as shown in Fig. 5).

(3) PCD-1 peptide reduces CCI-induced activation of microglia cells and stellate cells

In order to investigate the activation of glial cells of the central nervous system, the present invention stains spinal cord slices with a specific antibody such as OX42 antibody against microglial cells and GFAP antibodies against astrocyte. mark. Compared with the control group (normal rats that did not receive CCI surgery and drug treatment) (as shown in Figure 6A), rats that received CCI alone showed an increase in activation of microglial cells (stained with OX42 antibody). (As shown in Figure 6B). However, the CCI-operated rats also reduced the activation of the microglial cells at 30, 90, and 180 minutes after injection of the PCD-1 peptide (as shown in Figures 6C to 6F). Similarly, after the CCI operation, the stellate cells (stained by GFAP antibody) were also activated, but the activation of the stellate cells was reduced at 30, 90, and 180 minutes after injection of the PCD-1 peptide. (as shown in Figures 7A to 7F).

(4) PCD-1 peptide regulates CCI-mediated up-regulation of IL-1β and p-mTOR and down-regulated TGF-β1

In order to detect the regulation of IL-1β by PCD-1 peptide, the present invention utilizes immunohistochemical staining for IL in the control group (normal rats not subjected to CCI surgery and drug treatment) and spinal cord slices of rats exclusively subjected to CCI surgery. The amount of -1β was measured. Simply accepting CCI surgery Rat IL-1β was higher than the control group and PCD-1 peptide (20 μg) treated rats (as shown in Figure 8). However, the p-mTOR of stress-associated protein in rats subjected to CCI surgery was also elevated, but such a rise was inhibited by injection of PCD-1 peptide (Figure 9). ). It is worth noting that p-mTOR on stellate cells is also elevated by CCI surgery, but such a rise is inhibited by the injection of PCD-1 peptide (see Figure 10). Another protein TGF-β1 associated with the nerve conduction pathway, compared with the control group (normal rats that did not receive CCI surgery and drug treatment), the rats that received CCI surgery only reduced their performance, but this reduction The phenomenon was also improved by the injection of PCD-1 peptide (as shown in Figure 11). Finally, stellate cells and neuronal cells on rats subjected to CCI surgery can upregulate the expression of TGF-β1 by PCD-1 peptide treatment (as shown in Figure 12). Furthermore, the present invention treated with PCD-1 peptide did not affect the original motor function of the rat (as shown in Figure 13) and did not cause any significant change in external behavior.

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention, and it is intended to be The modifications are intended to be included in the scope of the patent application of the present invention.

<110> Academia Sinica; National Sun Yat-Sen University

<120> Use of a peptide for the preparation of a pharmaceutical composition for relieving pain

<130> 2448-AS-TW

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> PRT

<213> Morone chrysops

<400> 1

Claims (11)

A use of a peptide for the preparation of a pharmaceutical composition for relieving pain, wherein the peptide is a Picosidin (PCD) peptide, wherein the PCD peptide is a PCD-1 peptide, wherein the PCD The amino acid sequence of the -1 peptide is SEQ ID NO: 1. The use of claim 1, wherein the pain is a neuropathic pain. The use of claim 1, wherein the pain is pain caused by nerve inflammation. The use of claim 3, wherein the PCD peptide further inhibits neuroinflammation. The use according to claim 1, wherein the PCD peptide inhibits the expression of a epoxidase-2 (COX-2). The use according to claim 1, wherein the PCD peptide inhibits the expression level of an inducible nitric oxide synthase (iNOS). The use of claim 1, wherein the PCD peptide reduces the activation of a microglia. The use of claim 1, wherein the PCD peptide reduces the activation of astrocytc. The use according to claim 1, wherein the PCD peptide reduces the amount of expression of an interleukin-1β (IL-1β). The use of the first aspect of the invention, wherein the PCD peptide reduces the amount of monophosphorylation-mammalian-deficient protein (phospho-mTOR). The use according to claim 1, wherein the PCD peptide increases a tumor growth The amount of expression of factor-β1 (TGF-β1).