Composition for treating pain

Abstract

The present invention relates to a pharmaceutical composition comprising, as an active ingredient, a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or 2, a salt of the peptide, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide. In particular, the present invention relates to a pharmaceutical composition for preventing or treating pain or inflammation, comprising the peptide.

Description

Composition for the treatment of pain

[0001] The present invention relates to a novel peptide and a pharmaceutical composition comprising the same. In particular, the present invention relates to a pharmaceutical composition comprising the novel peptide for preventing or treating pain.

[0002] Inflammation is a biological response to harmful stimuli, a protective response involving immune cells, blood vessels, and inflammatory mediators. While inflammation has a protective function, helping to heal infections and promote tissue regeneration, it can also result in tissue damage, disease, swelling, or pain.

[0003] In particular, since pain caused by inflammation significantly reduces the quality of life in daily life, the development of therapeutic agents to effectively treat inflammation or pain is necessary.

[0004] The present inventors discovered that a specific peptide can effectively treat pain or inflammatory diseases, and completed the present invention.

[0005] Accordingly, the present invention provides a novel peptide having a preventive or therapeutic effect on pain or inflammatory diseases and a pharmaceutical composition containing the same for preventing or treating pain or inflammatory diseases.

[0006] The present invention relates to a pharmaceutical composition for treating or preventing pain, comprising as an active ingredient a peptide comprising an amino acid sequence represented by the following sequence number 1 or 2, a salt of the peptide, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide:

[0007] Sequence number 1: YGQCY MYMQK W

[0008] Sequence number 2: YGQWY MWMQK W

[0009] In the present invention, the pain may be inflammatory pain (e.g., pain caused by arthritis, inflammatory arthritis, rheumatoid arthritis, colitis, or ulcerative colitis), but is not limited thereto.

[0010] In addition, the present invention relates to a pharmaceutical composition for treating or preventing inflammation, comprising as an active ingredient a peptide comprising an amino acid sequence represented by the above sequence number 1 or 2, a salt of the peptide, a polynucleotide encoding the peptide, or an expression vector comprising the above polynucleotide.

[0011] In the present invention, the inflammation may be, for example, arthritis, inflammatory arthritis, rheumatoid arthritis, colitis, or ulcerative colitis, but is not limited thereto.

[0012] In the present invention, the expression vector may be a viral vector or a non-viral vector.

[0013] Here, the viral vector may be, for example, an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a revector, or a lentivirus vector. Here, the serotype of the adeno-associated virus (AAV) vector may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, or AAV16.

[0014] Additionally, in the present invention, the non-viral vector may be a DNA vector, nanoparticle, cationic polymer, exosome, extracellular vesicle, or liposome. Here, the DNA vector may be, for example, a plasmid vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.

[0015] In addition, the present invention relates to a peptide comprising an amino acid sequence represented by the above sequence number 1 or 2, or a pharmaceutically acceptable salt thereof.

[0016] Here, the salt of the peptide is preferably a peptide acid addition salt, and may be, for example, a hydrochloride, acetate or sulfate salt of the peptide.

[0017] In addition, the present invention relates to a peptide comprising the amino acid sequence represented by the above sequence number 1 or 2 or a pharmaceutically acceptable salt thereof, which is pegylated, and to a pegylated peptide or a pharmaceutically acceptable salt thereof.

[0018] A pharmaceutical composition comprising the peptide according to the present invention or a pharmaceutically acceptable salt thereof or a pegylated form thereof may be formulated for oral administration or parenteral administration (e.g., intravenous injection or subcutaneous injection, etc.).

[0019] In particular, the pegylated peptide or a pharmaceutically acceptable salt thereof according to the present invention can be administered orally to exert a pharmacological effect.

[0020] In addition, the present invention relates to a polynucleotide encoding the above peptide.

[0021] The composition of the present invention can be formulated into a conventional dosage form (e.g., oral solid preparation, oral liquid preparation, injection, etc.) using a pharmaceutically acceptable carrier or excipient.

[0022] The dosage of the effective ingredient according to the present invention can be appropriately selected by a person skilled in the art according to the weight of the subject of administration, the type and condition of the disease, etc., and for example, the daily dosage can be used in a range of 0.001 mg / kg to 1000 mg / kg, but is not limited thereto.

[0023] The amino acid sequence of the peptide according to the present invention can be configured to be protected from protease enzymes by capping the N-terminus with lipoic acid and / or capping the C-terminus with p-nitroanilide.

[0024] Additionally, the amino acid sequence of the peptide according to the present invention can be configured in a cyclic form by linking the N-terminus and C-terminus with each other via a peptide bond. For example, the N-terminus and C-terminus of the amino acid sequence of SEQ ID NO: 8 above can be synthesized in a cyclic form by linking each other via a peptide bond.

[0025] The peptide according to the present invention exhibits excellent anti-inflammatory and analgesic effects. Therefore, the pharmaceutical composition of the present invention can be used as a medicament for the treatment or prevention of inflammation or pain.

[0026] Figure 1 shows the change in body weight over time when DSS was administered to experimental mice.

[0027] Figures 2 and 3 illustrate the results of the von Frey test on the abdomen and foot after administration of Apep_Acet on days 8 and 11 of DSS administration.

[0028] Figures 4 and 5 illustrate the results of the von Frey test on the abdomen and foot after administration of Apep_Acet on days 22 and 25 of DSS administration.

[0029] Figure 6 shows the results of the von Frey test in the abdomen after administration of pegylated Apep_PEG and Bpep_HCl on day 7 of DSS administration.

[0030] Figure 7 shows the results of the von Frey test in the abdomen after administration of pegylated Apep_PEG and Bpep_HCl on day 21 of DSS administration.

[0031] Figures 8 and 9 illustrate the results of the von Frey test in the abdomen after administration of pegylated Apep_PEG and Bpep_PEG on days 8 and 22 of DSS administration.

[0032] Figure 10 illustrates the results of a mechanical allodynia test in a CFA animal model.

[0033] Figure 11 illustrates the results of the Rota-rod test in a CFA animal model.

[0034] Figures 12 and 13 illustrate the results of an open field test in a CFA animal model.

[0035] Figure 14 shows the results of paw thickness measurement in a CFA animal model.

[0036] Figure 15 shows the results of an allodynia evaluation to confirm pain induction after MIA administration in the OA model.

[0037] Figure 16 shows the results of the heterodynein evaluation after Bpep administration in the OA model.

[0038] Figure 17 shows the results of an allodynia evaluation to confirm pain induction after MIA administration in the OA model.

[0039] Figure 18 shows the results of the allodynia evaluation after Apep administration in the OA model.

[0040] Hereinafter, to facilitate understanding of the present invention, the present invention will be described in detail based on examples. However, the following examples are intended only to illustrate the content of the present invention and are not intended to limit the spirit or scope of the present invention in any way. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0041] AA. Production of peptides

[0042] A peptide having an amino acid sequence of SEQ ID NO: 1 in Table 1 below (hereinafter referred to as “peptide A”) and a peptide having an amino acid sequence of SEQ ID NO: 2 (hereinafter referred to as “peptide B”) were synthesized and purified using a solid-phase synthesis (SPPS) method.

[0043] [Table 1]

[0044]

[0045] BB. Preparation of peptide salts

[0046] A. Preparation of acetate of peptide A

[0047] After synthesis and purification, peptide A was obtained as a TFA salt. This was dissolved in water, acetic acid was added, and the mixture was stirred to replace the TFA counterion with acetate. Subsequently, peptide A acetate (Apep_Acet) was obtained by lyophilization.

[0048] B. Preparation of hydrochloride salt of peptide B

[0049] Peptide B was synthesized and purified, and obtained as a TFA salt. This was dissolved in water, hydrochloric acid was added, and the mixture was stirred to replace the TFA counterion with chloride. Subsequently, peptide B hydrochloride (Bpep_HCl) was obtained by lyophilization.

[0050] D. Preparation of PEGylated peptides

[0051] Peptide A acetate (Apep_Acet) was dissolved in water. 4-arm PEG 10K was added in an amount of 1 to 3 equivalents relative to Apep_Acet and stirred to perform a PEGylation reaction. The mixture was then left to stand for approximately 30 minutes to complete the reaction, and PEGylated peptide A (Apep_PEG) was obtained.

[0052] For peptide B, pegylated peptide B (Bpep_PEG) was obtained in a similar manner using peptide B hydrochloride (Bpep_Hcl).

[0053] Evaluation of analgesic efficacy using an animal model of CC. UC (ulcerative colitis)

[0054] 1. Test Purpose

[0055] This study was conducted to evaluate the analgesic effects of Bpep_HCl and Apep_Acet in ulcerative colitis (UC). UC was induced in a C57BL / 6N mouse model using 2% dextran sodium sulfate (DSS), and the analgesic effects were evaluated after single intraperitoneal and oral administration of the test substances. The DSS-induced colitis model has pathological characteristics and pain responses similar to those of human UC, and is widely recognized as an animal model for rapid and accurate assessment of the analgesic effects of drugs. This model is characterized by high reproducibility and a short induction period, enabling efficient and reliable assessment of analgesic effects. The primary objectives of this study were to confirm the analgesic effects of Bpep_HCl and Apep_Acet by intraperitoneal and oral administration in an UC animal model and to compare the analgesic effects according to the administration route.

[0056] 2. Test substance

[0057]

[0058] 3. Test system information and breeding environment

[0059]

[0060] Experimental animals were housed in cages of 5-6 animals per experimental group. Each cage was labeled and individuals within each cage were identified by tail marking.

[0061] Feed was placed on the wire mesh of the breeding cage, and purified tap water was provided in a water bottle so that the experimental animals could freely consume it.

[0062] 4. Test Preparation

[0063] After disinfecting the exterior of the animal transport box with 70% ethanol, the experimental animals were brought into the mouse breeding room of the animal laboratory. After visually checking for any abnormalities in the animals' health, they were allowed to acclimatize for at least three days.

[0064] Experimental animals were placed in 4-5 cages, 5-6 animals each, in 4x4 cages.

[0065] 5. Preparation and administration of test substances

[0066] A. Preparation of test substances

[0067] - For Apep_Acet ip (intraperitoneal) administration, dissolve 1 mg in 2 mL of DW (distilled water) and leave for 30 minutes.

[0068] - For Apep_Acet po (oral) administration, dissolve 12.5 mg in 2 mL of DW and leave for 30 minutes.

[0069] - For the Bpep_HCl ip administration substance, 7.5 mg was dissolved in 3 mL of DW and left for 30 minutes.

[0070] - For the Bpep_HCl po-administered substance, 9.4 mg was dissolved in 3 mL of DW and left for 30 minutes.

[0071] - For Apep_PEG po-administered material, 18.8 mg of Apep_Acet was dissolved in 1.5 mL of DW, then 60 mg of 4-arm PEG 10,000 was dissolved evenly in 1.5 mL of DW, mixed, and left for 30 minutes.

[0072] B. Setting the test substance administration dose

[0073] Apep_Acet

[0074] - For IP, Apep_Acet was selected as 4mg / kg.

[0075] - For po, 50mg / kg was selected.

[0076] Bpep_HCl

[0077] - For IP, 20mg / kg was selected.

[0078] - For po, it was set at 25 mg / kg.

[0079] Apep_PEG

[0080] - Apep_Acet po was set to 50mg / kg as Apep_Acet, which is the same as the setting dose.

[0081] D. Selection of administration route of test substance

[0082] Oral administration is a convenient and non-invasive route for patients, minimizing discomfort during treatment. Furthermore, oral administration facilitates local diffusion of the substance's effects within the target organ, making it essential for observing the effects on colonic inflammation and pain. Intraperitoneal administration is suitable for observing the substance's systemic absorption. Therefore, oral and intraperitoneal administration were selected to evaluate the analgesic effects of the test substance.

[0083] A. Test substance administration method

[0084] - ip: After anesthesia with 2% isoflurane through a respiratory anesthesia machine, it was administered intraperitoneally using a 1 mL syringe and a 26-gauge needle.

[0085] - po: After anesthesia with 2% isoflurane through a respiratory anesthesia machine, it was administered orally using a 1 mL syringe and an oral zonde.

[0086] B. Number of times the test substance was administered

[0087] - Apep_Acet: Administered once each on the 8th and 11th days of the acute phase, and once each on the 22nd and 25th days of the chronic phase, for a total of 4 administrations.

[0088] - Apep_PEG and Bpep_HCl: Administered once on the 8th day of the acute phase and once on the 22nd day of the chronic phase, for a total of 2 administrations.

[0089] 6. Composition of the test group

[0090] - Apep test

[0091]

[0092] - Bpep test

[0093]

[0094] 7. Test method

[0095] A. Measurement of baseline pain response value

[0096] - Mechanical allodynia test: After acclimatization for 3 days for more than 30 minutes per day, the Von Frey test was performed to measure the basic pain response value before consuming drinking water containing 2% (w / v) DSS.

[0097] B. UC model production: Acute ulcerative colitis was induced in mice by allowing free access to drinking water containing 2% (w / v) DSS for 7 days. Afterwards, the drinking water was replaced with normal drinking water.

[0098] d. Confirmation of UC model induction: After ingesting drinking water containing 2% DSS, body weight and clinical signs were measured three times a week from day 0 until the end of the experiment to confirm the induction of ulcerative colitis.

[0099] A. Evaluation of drug analgesic efficacy in the UC model

[0100] - Mechanical allodynia test: Performed 1, 3, 5, and 7 hours after drug administration and on days 1, 2, and 3 to measure pain response and confirm analgesia.

[0101] 8. Test results

[0102] A. Evaluation of Apep_Acet induction and analgesic efficacy

[0103] - Confirmation of UC induction: Ulcerative colitis was induced in C57BL / 6 mice by feeding them drinking water containing 2% DSS for 7 days. Body weight was monitored during the DSS administration period, and weight loss was observed from day 7 (see Figure 1). Clinical signs included decreased hair luster, decreased activity, and changes in stool quality. Based on these clinical symptoms, it was confirmed that ulcerative colitis was induced on day 7 of DSS administration.

[0104] - Acute Von frey test (abdomen, foot)

[0105] Apep_Acet was administered at 4 mg / kg ip or 50 mg / kg po on days 8 and 11 of DSS administration, respectively, and the results of the von Frey test on the abdomen and foot are shown in Figures 2 and 3.

[0106] On the 8th day, Von Frey test was performed on the abdomen. The 4 mg / kg ip administration group showed the maximum analgesic effect compared to the control group on ulcerative colitis at 1 hour (UC, 2-way ANOVA, *p<0.05), and the analgesic effect gradually decreased until the 5th hour. A similar trend was observed in the 60 mg / kg po administration group, and in the 4 mg / kg ip and 60 mg / kg po administration groups on the 11th day.

[0107] In the Von Frey test on the foot on days 8 and 11, the IP administration group showed a significant analgesic effect for up to 5 hours, which gradually decreased until 24 hours. The PO administration group also showed an analgesic effect for up to 24 hours.

[0108] - Chronic Von frey test (abdomen, foot)

[0109] After DSS administration on days 7 and 11, Apep_Acet was administered again on days 22 and 25 to evaluate the chronic analgesic efficacy. Similar results to the acute experiment were observed in the chronic phase (Figs. 4 and 5), and the analgesic effects in the abdomen and feet were observed similarly to the acute experiment results (UC, 2-way ANOVA, *p<0.05).

[0110] B. Evaluation of the analgesic efficacy of Apep_PEG and Bpep_HCl

[0111] - Acute Von frey test (abdomen)

[0112] The acute analgesic efficacy of PEGylated Apep 50 mg / kg po (50 mg / kg as Apep_Acet) and Bpep_HCl 20 mg / kg ip and 25 mg / kg po was evaluated on the 7th day of DSS administration. The PEGylated Apep_PEG administration group showed the maximum analgesic effect at 5 hours compared to the ulcerative colitis control group (UC, 2-way ANOVA, **p<0.01), and the analgesic effect gradually decreased until 72 hours (3 days). The analgesic effect of the Bpep_HCl administration group disappeared after 7 hours (see Fig. 6).

[0113] - Chronic Von frey test (abdomen)

[0114] On day 21 of DSS administration, PEGylated Apep_PEG and Bpep_HCl were administered to evaluate the chronic analgesic efficacy. The PEGylated Apep_PEG group exhibited a significant analgesic effect in the abdomen (UC, 2-way ANOVA, ****p<0.0001), which persisted for up to 72 hours before gradually dissipating. Chronic analgesic effects were also observed in the intraperitoneal Bpep_HCl group (see Figure 7).

[0115] 9. Conclusion

[0116] Our results demonstrated that Apep and Bpep exhibited analgesic effects following intraperitoneal (ip) and oral (po) administration. In particular, PEGylated Apep demonstrated sustained analgesic effects over a prolonged period of time even after oral administration, with significant analgesic effects observed in both the acute and chronic phases. This sustained analgesic effect is highly beneficial in alleviating chronic pain in UC patients.

[0117] DD. Evaluation of the analgesic efficacy of PEGylated Apep and Bpep in an animal model of UC.

[0118] 1. Test Purpose

[0119] The objective of this study was to evaluate the analgesic efficacy of PEGylated Bpep and Apep in a DSS-induced ulcerative colitis (UC) mouse model.

[0120] 2. Test substance: Same as the test substance in Example CC.

[0121] 3. Information about the test system

[0122]

[0123] Experimental animals were housed in cages, with 7 to 8 animals per experimental group. Each cage was labeled, and individuals within each cage were identified by tail marking.

[0124] Feed was placed on the wire mesh of the breeding cage, and purified tap water was provided in a water bottle so that the experimental animals could freely consume it.

[0125] 4. Test Preparation

[0126] After disinfecting the exterior of the animal transport box with 70% ethanol, the experimental animals were brought into the mouse breeding room of the animal laboratory. After visually checking for any abnormalities in the animals' health, they were allowed to acclimatize for at least three days.

[0127] The experimental animals were placed in 7 cages, 7 to 8 animals each, in 4x4 cages.

[0128] 5. Composition of the test group

[0129]

[0130] 6. Test method

[0131] A. Preparation of ulcerative colitis-inducing substances and test substances

[0132] For DSS, tap water was used to prepare 20 g / L and then used as drinking water for one week.

[0133] After dissolving 18.8 mg of Bpep_HCl or Apep_Acet in 3 mL of DW, separately, 120 mg of 4-arm PEG 10,000 was uniformly dissolved in 3 mL of PBS, mixed at a ratio of 1:1 (v / v), and reacted for 30 minutes.

[0134] B. Method of inducing ulcerative colitis

[0135] Acute ulcerative colitis was induced in mice by allowing free access to 2% (w / v) DSS for 7 days. Afterwards, the diet was replaced with normal drinking water.

[0136] D. Setting the test substance administration dose

[0137] Apep_PEG and Bpep_PEG: Set to 25 mg / kg and 12.5 mg / kg.

[0138] A. Test substance administration method: Oral administration

[0139] After anesthesia with 2% isoflurane through a respiratory anesthesia machine, oral administration was performed using a 1 mL syringe and oral zonde.

[0140] B. Drug efficacy evaluation

[0141] Clinical score: Posture, hair condition, stool condition, and weight are measured and scored daily to determine whether colitis has occurred.

[0142] Mechanical allodynia test: Baseline is measured before DSS induction and again on day 5 during the induction period. After DSS induction, the pain response is measured at 1, 3, 5, 7, 24, 48, and 72 hours after single administration of the test substance during the acute period (day 8 abdominal) and chronic period (day 22 abdominal).

[0143] 7. Test results

[0144] A. Acute Von frey test (abdomen)

[0145] On the 8th day after DSS administration, Apep_PEG and Bpep_PEG were administered orally, and the analgesic efficacy was evaluated using the Von Frey test in the acute phase. (See Figure 8)

[0146] In the case of Apep_PEG, analgesic effect was observed at 1 hour in the 25 mg / kg administration group and disappeared on the 2nd day. In addition, analgesic effect was observed at 3 hours in the 12.5 mg / kg administration group and disappeared on the 1st day.

[0147] Bpep_PEG also showed an analgesic effect.

[0148] B. Chronic Von frey test (abdomen)

[0149] On the 22nd day after DSS administration, Apep_PEG and Bpep_PEG were administered orally in the same manner as in the acute phase, and the chronic analgesic efficacy was evaluated using the Von Frey test. (See Figure 9)

[0150] In the case of Apep_PEG, an analgesic effect reaching the Naive (normal) level by 7 hours was observed in the 25 mg / kg administration group, indicating that high efficacy was maintained even in the chronic phase (UC, 2-way ANOVA, **p<0.01).

[0151] Bpep_PEG showed an analgesic effect at 1 and 3 hours in the 12.5 mg / kg administration group, and this effect was maintained until 7 hours.

[0152] These results show that Apep_PEG and Bpep_PEG exhibit significant analgesic efficacy even in the chronic phase.

[0153] EE. Creation of a CFA (Completed Freund's Adjuvant) Animal Model and Evaluation of Bpep Efficacy

[0154] 1. Test Purpose

[0155] This study was conducted to establish a rheumatoid arthritis (RA) model and to investigate the efficacy of Bpep_HCl on inflammatory pain and its anti-inflammatory effect, and to evaluate its efficacy in this model.

[0156] The Complete Freund's Adjuvant (CFA) model is one of the inflammatory models primarily used to study inflammatory diseases, especially arthritis diseases such as rheumatoid arthritis (RA). It is recognized as an appropriate model for evaluating the efficacy through animal experiments by observing inflammatory pain responses. Therefore, in this study, a CFA model was created and the inflammatory pain and motor ability of Bpep_HCl were evaluated to confirm the efficacy of Bpep_HCl, such as analgesic and inflammation-reducing effects on inflammatory pain.

[0157] 2. Test resources

[0158] a. Reagent or solution

[0159]

[0160] B. Testing apparatus and devices

[0161]

[0162] D. Test substance: Same as the test substance in Example CC

[0163] 3. Information about the test system

[0164]

[0165] Experimental animals were housed in cages of three per experimental group.

[0166] Each breeding cage was labeled and individuals within a cage were identified by tail marking.

[0167] Feed was placed on the wire mesh of the breeding cage, and purified tap water was provided in a water bottle so that the experimental animals could freely consume it.

[0168] 4. Exam Preparation

[0169] After disinfecting the exterior of the animal transport box with 70% ethanol, the experimental animals were brought into the mouse breeding room of the animal laboratory. After visually checking for any abnormalities in the animals' health, they were allowed to acclimatize for at least five days.

[0170] The experimental animals were placed in six cages, three animals each, in a 4x4 cage.

[0171] The excipient is a mixture of 90% normal saline and 10% PEG400.

[0172] 5. Preparation and administration of test substances

[0173] A. Test substance preparation solution

[0174] Bpep_HCl was prepared by dissolving it in PEG 400 10% + Normal saline 90%.

[0175] B. Setting the test substance administration dose

[0176] Bpep ip was set at 20 mg / kg and sc (subcutaneous injection) at 10, 40, and 100 mg / kg.

[0177] D. Route of administration

[0178] Unlike the MIA (Monosodium Iodoacetate)-induced OA (Osteoarthritis: degenerative arthritis) model, which is induced by direct injection into the knee joint cavity, the CFA-induced RA model requires measurement of efficacy through systemic absorption rather than local effects in the knee joint cavity. Therefore, two routes of systemic administration were selected: the intravenous (IP) route and the sc route.

[0179] A. Test substance administration method

[0180] After confirming model induction on the 14th day of CFA administration, for the G3 group, the mice were anesthetized with 2% isoflurane, and 20 mg / kg of Bpep_HCl was administered via intraperitoneal injection (ip) under the peritoneum using a 1 mL syringe.

[0181] After confirming model induction on the 14th day of CFA administration, the G4, G5, and G6 groups were anesthetized with 2% isoflurane, and Bpep_HCl was administered subcutaneously (sc) using a 1 mL syringe at 10 mg / kg in the G4 group, 40 mg / kg in the G5 group, and 100 mg / kg in the G6 group, respectively.

[0182] B. Number of times the test substance was administered

[0183] Bpep was administered once per animal to both ip and sc groups (G3-G6).

[0184] 6. Composition of the test group

[0185]

[0186] 7. Test method

[0187] A. Baseline measurement

[0188] - Mechanical allodynia test: After acclimatization for 3 days for more than 30 minutes per day, the Von Frey test was performed to measure the baseline before CFA administration.

[0189] - Rota-rod test: After acclimatizing by gradually increasing the RPM in the order of 10, 25, and 35 for 30 minutes per day for 3 days, the baseline was measured by performing the Rota-rod at 35 RPM.

[0190] - Paw thickness: After inhalation anesthesia with 2% isoflurane before CFA administration, the thickness of the upper and lower paws of the right paws was measured using a digital caliper to obtain the baseline.

[0191] B. CFA model creation

[0192] CFA (1 mg / mL) was administered subcutaneously (sc) to the right hindpaw of mice anesthetized with 2% isoflurane using a respiratory anesthesia machine, at a dose of 5 ul using a 30G needle and Hamilton syringe, and the mice were observed for 14 days.

[0193] D. Confirmation of CFA model derivation

[0194] On the 14th day after CFA administration, the induction of the CFA model was confirmed by measuring paw swelling using the Von Frey test, Rota-rod test, and digital caliper.

[0195] A. Evaluation of drug analgesic and anti-inflammatory efficacy in the CFA model

[0196] - Mechanical allodynia test: Performed on days 1, 2, 4, 7, 9, 11, 14, and 16 after drug administration to measure pain response and confirm analgesia.

[0197] - Rota-rod test: Performed on days 1, 2, 4, 7, 9, 11, 14, and 16 after drug administration to measure the time until falling and to check the effect on motility.

[0198] - Open field test: On days 3 and 7 after drug administration, mice were placed in a black box measuring 40 cm × 40 cm × 40 cm, and their movements were measured using a camera for 20 minutes, after which they were analyzed using the EthoVision XT 16 program.

[0199] 8. Test results

[0200] a. Pain assessment

[0201] (1) Mechanical allodynia test

[0202] In the case of mechanical allodynia test using von frey filament test, concentration-dependent analgesic efficacy was observed when administered sc on the first day after drug administration (see Figure 10).

[0203] (2) Rota-rod test

[0204] In the group in which CFA arthritis was induced, motility was generally lower than that of the naive group, and the groups administered 10 and 40 mg / kg sc after drug administration tended to have higher motility. (See Figure 11)

[0205] (3) Open field test

[0206] In the open field test, the drug-administered group (particularly the 40 mg / kg sc group) showed a tendency for increased movement distance compared to the CFA arthritis-induced group (see Figure 12). Furthermore, the drug-administered group (particularly the 40 mg / kg sc group) showed a significant decrease in the time spent in the central region compared to the CFA arthritis-induced group (see Figure 13).

[0207] B. Inflammation assessment

[0208] Paw thickness

[0209] In the case of paw swelling, the vehicle group showed worsening compared to the baseline measurement day (14 days after CFA administration), but in the substance administration group, the degree of swelling worsening was observed to be statistically significantly reduced based on the paw swelling before administration in all of the Bpep_HCl 20 mg / kg ip, 10 mg / kg sc, 40 mg / kg sc, and 100 mg / kg sc groups (see Figure 14).

[0210] FF. Evaluation of the efficacy of Bpep in an animal model of osteoarthritis (OA).

[0211] 1. Test Purpose

[0212] In this study, we aimed to evaluate the pain relief effect of intra-articular administration of Bpep in an animal model of osteoarthritis (OA).

[0213] 2. Test substance

[0214]

[0215] 3. Information about the test system

[0216]

[0217] Experimental animals were housed in cages of three each. Each cage was labeled to distinguish them, and individuals within a cage were identified by tail marking.

[0218] Feed was placed on the wire mesh of the breeding cage and purified tap water was provided in a water bottle so that the experimental animals could freely consume it.

[0219] 4. Test Preparation

[0220] After disinfecting the exterior of the animal transport box with 70% ethanol, the animals were brought into the animal laboratory breeding room. After visually checking for any health abnormalities, such as hair loss or movement, they were placed in cages of three and kept for at least five days.

[0221] Experimental animals were placed in 10 cages of 3 animals each in a 4x4 cage.

[0222] 5. Preparation and administration of test substances

[0223] a. Preparation of Monosodium iodoacetate (MIA)

[0224] (1) Preparation: MIA was uniformly dissolved in PBS at a concentration of 40 mg / ml.

[0225] (2) Administration method: MIA was administered into the knee joint cavity.

[0226] (3) Number of administrations: Single administration

[0227] B. Preparation and administration of control substances

[0228] (1) Preparation of negative control material: Use finished saline.

[0229] (2) Method of administering negative control substance: Intra-articular administration of the knee joint

[0230] (3) Number of times control substance is administered: single administration, same as test substance

[0231] (4) Preparation of positive control substance: 40 mg of TA (triamcinolone acetonide) was diluted 1 / 2 evenly in PBS and used.

[0232] (5) Positive control substance administration method: Intra-articular administration of the knee joint

[0233] (6) Number of times positive control substance was administered: single administration, same as the test substance

[0234] D. Preparation and administration of test substances

[0235] (1) Preparation of test substance: 1 mg of Bpep was uniformly dissolved in 1 ml of DW.

[0236] (3) Setting of administration dose: It was set considering that the general intra-articular administration dose for rats is 25-50㎕.

[0237] (3) Selection of administration route of test substance: Intra-articular administration

[0238] (4) Test substance administration method: The test substance was administered into the knee joint cavity using an insulin syringe.

[0239] (5) Number of test substance administrations: Single administration

[0240] 6. Composition of the test group

[0241]

[0242] 7. Test method

[0243] A. Adaptation of pain response measurement equipment and measurement of baseline pain response values

[0244] (1) Animals were acclimated to the chamber of the measuring equipment for more than 1 hour three times.

[0245] (2) After equipment adaptation, the Von Frey test was performed to measure the baseline pain response value.

[0246] B. Creation of an osteoarthritis (OA) model

[0247] (1) Selection of test animals

[0248] The basic pain response value was measured and test animals with a threshold value of 6 or higher were selected.

[0249] (2) Administration of chemotherapy drugs

[0250] After the rats were anesthetized, the hair on the left hind leg was removed.

[0251] The left hind leg of the rat was flexed at 90°, and the drug was administered 5 mm deep into the knee joint space next to the patellar ligament using a 31G insulin syringe. The drug was administered at a dose of 50 μl per rat (MIA dose per rat: 2 mg).

[0252] After administration, the knees were bent and straightened 50 times to ensure that the drug was evenly distributed.

[0253] D. Confirmation of pain induction

[0254] (1) The Von Frey test was performed on days 1, 7, and 14 after MIA administration to determine whether pain was induced.

[0255] (2) Individuals whose measured values ​​decreased by more than 40% from the baseline pain response measurement values ​​were considered to have been induced with pain.

[0256] A. Confirmation of drug administration and analgesic efficacy

[0257] (1) After confirming that pain was induced, the test drug of each group was administered into the knee joint cavity.

[0258] (2) Pain response was measured by performing the Von Frey test 1 hour, 2 hours, 8 hours, 1 day, 2 days, 7 days, and 14 days after administration of the test drug.

[0259] Ma. Statistical analysis processing

[0260] 1) Statistical analysis was performed using the SigmaPlot (version 12.5) program.

[0261] 2) Statistics were analyzed using t-test and 2-way ANOVA (Newmam-Keuls multiple comparisons test) and expressed as Mean ± SD.

[0262] 8. Test results

[0263] a. Confirmation of pain induction

[0264] (1) Allodynia response was evaluated before MIA administration and on days 7 and 14 after MIA administration using the OA model.

[0265] (2) In the evaluation of heterogeneity, the OA model group (MIA) showed a 51.76% improvement in pain on the 7th and 14th days after MIA administration compared to the control group (Control, t-test, *** p<0.001) and 93.71% (Control, t-test, *** A significant reduction in the threshold of pain response was observed (p<0.001). (See Figure 15)

[0266] B. Evaluation of Bpep efficacy

[0267] (1) Bpep was administered intra-articularly to the knee joint on the 14th day after MIA administration, and the allodynia response was evaluated at 1 hour, 2 hours, 8 hours, 1 day, 2 days, 7 days, and 14 days.

[0268] (2) The negative control group before drug administration, the Saline administration group, TA 20 mg / ml, Bpep 0.01 mg / ml, Bpep 0.1 mg / ml, and Bpep 1 mg / ml administration groups showed a significant pain response threshold reduction effect of 96.78% (Naive, 2way ANOVA, &&&p<0.001), 96.78% (Naive, 2way ANOVA, &&&p<0.001), 96.60% (Naive, 2way ANOVA, &&&p<0.001), 96.86% (Naive, 2way ANOVA, &&&p<0.001), and 96.86% (Naive, 2way ANOVA, &&&p<0.001), respectively, compared to the control group. (See Figure 16)

[0269] (3) The TA 20 mg / ml administration group showed a significant increase in the pain response threshold by 59.81% (Saline, 2-way ANOVA, $$$p<0.001) after 8 hours compared to the negative control group, the Saline administration group, and showed a 30.51%, 30.29%, and 14.65% increase in threshold after 1, 2, and 7 days, respectively.

[0270] (4) The Bpep 0.01 ㎎ / ㎖ administration group showed a significant increase in the pain response threshold by 61.71% (Saline, 2-way ANOVA, ***p<0.001), 60.20% (Saline, 2-way ANOVA, ***p<0.001), and 39.52% (Saline, 2-way ANOVA, *p<0.05) after 8 hours, 1 day, and 2 days, respectively, compared to the negative control group, the Saline administration group. After 7 days, the threshold was increased by 21.88%.

[0271] (5) The Bpep 0.1 ㎎ / ㎖ administration group showed a significant increase in the pain response threshold by 50.23% (Saline, 2-way ANOVA, ##p<0.01), 74.75% (Saline, 2-way ANOVA, ###p<0.001), and 48.54% (Saline, 2-way ANOVA, ##p<0.01) after 8 hours, 1 day, and 2 days, respectively, compared to the negative control group, the Saline administration group. After 7 days, the threshold was increased by 6.52%.

[0272] (6) The Bpep 1 mg / ml administration group showed a significant increase in the pain response threshold by 54.37% (Saline, 2way ANOVA, @@p<0.01), 49.84% (Saline, 2way ANOVA, @p<0.05), and 60.49% (Saline, 2way ANOVA, @@@p<0.001) after 8 hours, 1 day, and 2 days, respectively, compared to the negative control group, the Saline administration group. After 7 days, the threshold was increased by 27.24%.

[0273] (7) The EC50 value of Bpep at 8 hours after drug administration was estimated to be less than 0.01 mg / ml.

[0274] (8) It was observed that Bpep according to the present invention exhibits a pain suppression effect superior to TA, a drug used for osteoarthritis patients, at a concentration about 20-200 times lower than TA.

[0275] GG. Evaluation of the efficacy of Apep-Acetate in an animal model of osteoarthritis (OA).

[0276] 1. Test Purpose

[0277] In this study, we aimed to evaluate the pain relief effect of subcutaneous administration of Apep_Acetate.

[0278] 2. Test substance

[0279]

[0280] 3. Information about the test system

[0281]

[0282] Experimental animals were housed in cages of three each. Each cage was labeled to distinguish them, and individuals within a cage were identified by tail marking.

[0283] Feed was placed on the wire mesh of the breeding cage and purified tap water was provided in a water bottle so that the experimental animals could freely consume it.

[0284] 4. Test Preparation

[0285] After disinfecting the exterior of the animal transport box with 70% ethanol, the animals were brought into the animal laboratory breeding room. After visually checking for any health abnormalities, such as hair loss or movement, they were placed in cages of three and kept for at least five days.

[0286] Experimental animals were placed in 10 cages of 3 animals each in a 4x4 cage.

[0287] 5. Preparation and administration of test substances

[0288] a. Preparation of Monosodium iodoacetate (MIA)

[0289] (1) Preparation: MIA was uniformly dissolved in PBS at a concentration of 40 mg / ml.

[0290] (2) Administration method: MIA was administered into the knee joint cavity.

[0291] (3) Number of administrations: Single administration

[0292] B. Preparation and administration of control substances

[0293] (1) Preparation of negative control material: Use finished saline.

[0294] (2) Method of administering negative control substance: Subcutaneous administration

[0295] (3) Number of times control substance is administered: single administration, same as test substance

[0296] D. Preparation and administration of test substances

[0297] (1) Preparation of test substance:

[0298] - 1 mg of Apep_Acet was dissolved evenly in 10 ml of DW.

[0299] - 2 mg of Apep_Acet was dissolved evenly in 10 ml of DW.

[0300] - 4 mg of Apep_Acet was dissolved evenly in 10 ml of DW.

[0301] (3) Setting of administration dose: It was set considering that the general subcutaneous administration dose for rats is 5-10 ml / kg.

[0302] (3) Selection of administration route of test substance: Subcutaneous administration.

[0303] Subcutaneous administration can produce a faster effect than oral administration, but absorption is slower than intramuscular administration, so it is used to delay the onset of action. It is particularly suitable for patients who cannot take oral medication or who are unconscious.

[0304] (4) Test substance administration method: The test substance was administered subcutaneously using a 3 ml syringe.

[0305] (5) Number of test substance administrations: Single administration

[0306] 6. Composition of the test group

[0307]

[0308]

[0309] 7. Test method

[0310] A. Adaptation of pain response measurement equipment and measurement of baseline pain response values

[0311] (1) Animals were acclimated to the chamber of the measuring equipment for more than 1 hour three times.

[0312] (2) After equipment adaptation, the Von Frey test was performed to measure the baseline pain response value.

[0313] B. Creation of an osteoarthritis (OA) model

[0314] (1) Selection of test animals

[0315] The basic pain response value was measured and test animals with a threshold value of 6 or higher were selected.

[0316] (2) Administration of chemotherapy drugs

[0317] After the rats were anesthetized, the hair on the left hind leg was removed.

[0318] The left hind leg of the rat was flexed at 90°, and the drug was administered 5 mm deep into the knee joint space next to the patellar ligament using a 31G insulin syringe. The drug was administered at a dose of 50 μl per rat (MIA dose per rat: 2 mg).

[0319] After administration, the knees were bent and straightened 50 times to ensure that the drug was evenly distributed.

[0320] D. Confirmation of pain induction

[0321] (1) The Von Frey test was performed on days 1, 7, and 14 after MIA administration to determine whether pain was induced.

[0322] (2) Individuals whose measured values ​​decreased by more than 40% from the baseline pain response measurement values ​​were considered to have been induced with pain.

[0323] A. Confirmation of drug administration and analgesic efficacy

[0324] (1) After confirming that pain was induced, the test drug of each group was administered into the knee joint cavity.

[0325] (2) Pain response was measured by performing the Von Frey test 1 hour, 3 hours, 5 hours, 7 hours, and 24 hours after administration of the test drug.

[0326] Ma. Statistical analysis processing

[0327] 1) Statistical analysis was performed using the SigmaPlot (version 12.5) program.

[0328] 2) Statistics were analyzed using t-test and 2-way ANOVA (Newmam-Keuls multiple comparisons test) and expressed as Mean ± SD.

[0329] 8. Test results

[0330] a. Confirmation of pain induction

[0331] (1) Allodynia response was evaluated before MIA administration and on days 7 and 14 after MIA administration using the OA model.

[0332] (2) In the evaluation of heterogeneity, the OA model group (MIA) showed a 30.18% threshold reduction effect on the 7th day after MIA administration compared to the control group (Control), and a 80.35% (Control, t-test, ***p<0.001) threshold reduction effect on the pain response on the 14th day. (See Figure 17)

[0333] B. Apep efficacy evaluation

[0334] (1) Apep_Acetate was administered subcutaneously on the 14th day after MIA administration, and the allodynia response was evaluated at 1 hour, 3 hours, 5 hours, 7 hours, and 24 hours.

[0335] (2) The negative control group before drug administration, the Saline administration group, Apep_Acetate 1 mg / kg, Apep_Acetate 2 mg / kg and Apep_Acetate 4 mg / kg administration groups, had an 80.43% (Naive, 2-way ANOVA, &&& p<0.001), 80.33% (Naive, 2way ANOVA, &&& p<0.001), 80.33% (Naive, 2way ANOVA,&&& p<0.001) and 80.33% (Naive, 2way ANOVA, &&& A significant reduction in the threshold of pain response (p<0.001) was observed (see Figure 18).

[0336] (3) The Apep_Acetate 1 mg / kg administration group showed a threshold increase effect of 0.22%, 1.36%, 5.82%, and 1.76%, respectively, compared to the negative control group, the saline administration group, after 1 hour, 3 hours, 5 hours, and 7 hours.

[0337] (4) The Apep_Acetate 2 mg / kg administration group showed a threshold increase effect of 3.91%, 37.58%, 35.51%, 28.73%, and 7.16%, respectively, compared to the negative control group, the Saline administration group, after 1 hour, 3 hours, 5 hours, 7 hours, and 24 hours.

[0338] (5) The Apep_Acetate 4 mg / kg administration group was 56.64% higher than the negative control group, the Saline administration group, at 1 hour, 3 hours, 5 hours, and 7 hours, respectively (Saline, 2-way ANOVA, ## p<0.01), 66.31% (Saline, 2way ANOVA, ### p<0.001), 80.33% (Saline, 2-way ANOVA, ### p<0.001) and 38.29% (Saline, 2way ANOVA, # The effect of increasing the threshold of pain response (p<0.05) was shown.

[0339] (6) EC of Apep_Acetate 5 hours after drug administration 50 The value was measured as 1.82 mg / kg.

[0340] The present invention can be usefully used as a composition for treating inflammation.

[0341] Sequence number 1: YGQCY MYMQK W

[0342] Sequence number 2: YGQWY MWMQK W

Claims

A pharmaceutical composition for treating or preventing pain, comprising as an active ingredient a peptide comprising an amino acid sequence represented by sequence number 1 or 2, a salt of the peptide, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide. A pharmaceutical composition in claim 1, wherein the expression vector is a viral vector or a non-viral vector. A pharmaceutical composition according to claim 1, wherein the pain is inflammatory pain. A pharmaceutical composition according to claim 1, wherein the inflammatory pain is pain caused by arthritis or colitis. A pharmaceutical composition for treating or preventing inflammation, comprising as an active ingredient a peptide comprising an amino acid sequence represented by sequence number 1 or 2, a salt of the peptide, a polynucleotide encoding the peptide, or an expression vector comprising the polynucleotide. A pharmaceutical composition according to claim 5, wherein the inflammation is arthritis or colitis. A peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or 2, or a pharmaceutically acceptable salt thereof. In claim 7, a pegylated peptide or a pharmaceutically acceptable salt thereof. A polynucleotide encoding the peptide of claim 7. A pharmaceutical composition for oral or parenteral use for the treatment or prevention of pain or inflammation, comprising the peptide of claim 7 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for subcutaneous injection in claim 10. A pharmaceutical composition for oral or parenteral use for the treatment or prevention of pain or inflammation, comprising the peptide of claim 8 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for subcutaneous injection in claim 12.

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