NEW PHARMACEUTICAL USE

Abstract

There is provided a peptide of the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, or a salt thereof, for use as a pharmaceutical. The peptide is particularly useful in the treatment of conditions characterised by inflammation, including wounds, burns, hemorrhoids, psoriasis, acne, atopic dermatitis, COPD ulcerative colitis and IPF.

Claims

1. The compound Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, a regioisomer, a stereoisomer, or a pharmaceutically- or cosmetically-acceptable salt thereof.

2. The compound as claimed in claim 1, wherein the compound is: ##STR00002##

3. The compound as claimed in claim 1, which is not in the form of a salt.

4. A pharmaceutical formulation comprising compound as defined in claim 1, or a pharmaceutically- or cosmetically-acceptable salt thereof, and a pharmaceutically-acceptable adjuvant, diluent or carrier.

5. A pharmaceutical formulation including a compound as defined in claim 1, or a pharmaceutically- or cosmetically-acceptable salt thereof; another antiinflammatory agent; and a pharmaceutically-acceptable adjuvant, diluent or carrier.

6. A kit of parts comprising components: (A) a pharmaceutical formulation including a compound as defined in claim 1, or a pharmaceutically- or cosmetically-acceptable salt thereof, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and (B) a pharmaceutical formulation including another antiinflammatory agent in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (A) and (B) are each provided in a form that is suitable for administration in conjunction with the other.

7-8. (canceled)

9. A method of treatment of inflammation, an inflammatory disorder, and/or of a disorder characterised by inflammation, which method comprises the administration of a compound as defined in claim 1, or a pharmaceutically- or cosmetically-acceptable salt thereof, or a formulation comprising said compound or salt thereof, to a patient in need of such treatment.

10. The method as claimed in claim 9, wherein the inflammatory disorder is selected from psoriasis, acne, eczema, dermatitis, rhinitis, chronic obstructive pulmonary disease and ulcerative colitis.

11. The method as claimed in claim 10, wherein the dermatitis is atopic dermatitis or steroid-dependent dermatitis.

12. The method as claimed in claim 9, wherein the disorder is characterised by inflammation is a wound or a burn.

13. The method as claimed in claim 12, wherein the wound is an abrasion, a scratch, an incision, a laceration, a skin puncture, an avulsion, a bruise, a scar or a blister, or itching associated with any of the foregoing.

14. The method as claimed in claim 9, wherein the condition or disorder characterised by inflammation is hemorrhoids.

15. The method as claimed in claim 9, wherein the disorder characterised by inflammation is a viral infection.

16. The method as claimed in claim 15, wherein the viral infection is the common cold.

17-18. (canceled)

19. A method of treatment of idiopathic pulmonary fibrosis, which method comprises the administration of a compound as defined in claim 1, or a pharmaceutically- or cosmetically-acceptable salt thereof, to a patient in need of such treatment.

20. The claim 9, wherein the compound(s) or salt thereof, is administered topically in the form of a topical formulation.

21. The method as claimed in claim 20, wherein the administration is direct topical administration to a mucosal surface.

22. A method as claimed in claim 21, wherein the administration is direct topical administration to the lung.

23. The method as claimed in claim 20, wherein the administration is direct topical administration to the skin.

24. The method as claimed in claim 20, wherein the compound(s) or salt is administered with montelukast or a pharmaceutically-acceptable salt thereof, and the condition is a wound, a burn or hemorrhoids.

25. The method as claimed in claim 20, wherein the compound(s) or salt is administered with montelukast or a pharmaceutically-acceptable salt thereof, and the condition is chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis.

26. The method as claimed in claim 20, wherein the compound(s) or salt is administered with mefp-1, and the condition is ulcerative colitis.

27. The method as claimed in claim 20, wherein the compound(s) or salt is administered with trypsin and the condition characterized by inflammation is a viral infection.

28. The method as claimed in claim 27, wherein the viral infection is the common cold.

29. A process for the preparation of a pharmaceutical formulation, which process comprises bringing into association a compound as defined in claim 1 with one or more pharmaceutically-acceptable adjuvant, diluent or carrier.

30. A process for the preparation of a kit of parts as defined in claim 6, which process comprises bringing into association component (A) of the kit of parts with component (B) of the kit of parts.

31. The method as claimed in claim 9, wherein the inflammatory disorder is selected from conjunctivitis, keratitis, acute epithelial keratitis, nummular keratitis, interstitial keratitis, disciform keratitis, neurotrophic keratitis, mucous plaque keratitis, herpes simplex keratitis, herpes zoster keratitis, bacterial keratitis, fungal keratitis acanthamoebic keratitis, onchocercal keratitis, superficial punctate keratitis, ulcerative keratitis, exposure keratitis photokeratitis, contact lens acute red eye and optic neuritis.

32. The method as claimed in claim 19, wherein the compound(s) or salt thereof is administered topically in the form of a topical formulation.

33. The method as claimed in claim 32, wherein the administration is direct topical administration to the lung.

34. The method as claimed in claim 19, wherein the compound(s) or salt is administered with montelukast or a pharmaceutically-acceptable salt thereof.

Description

EXAMPLES

Example 1

[0131] Synthesis of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys

[0132] Fmoc-Lys-Boc-Wang resin (0.3 mmol/g; GLS180322-41301, GL Biochem, Shanghai, China) was loaded into a reaction column.

[0133] 2 litres of methylene chloride (DCM; Shandong Jinling Chemical Industry Inc. Co., Shandong, China) was added to the column and allowed to soak the resin for about half an hour. The DCM was then drawn off and 2 litres of N,N-dimethylformamide (DMF; Shandong Shitaifeng Fertilizer Industry Inc. Co., Shandong, China) was used to wash the column three times.

[0134] 200 mL of piperidine (Shanghai Li Ming Industry and Trade Co., Ltd., China] was mixed with 1 litre of DMF and was used as deprotection solution. The liquid was drained after 15 minutes and the column was washed with DMF six times.

[0135] 69 g of Fmoc-Tyr(tBu)-OH (GLS170916-36901, GL Biochem) and 48 g of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU; GL Biochem) were dissolved in 300 mL of DMF and were added to the reaction column. 53 mL of N,N-diisopropylethylamine (DIPEA; Suzhou Highfine Biotech Co. Ltd, Jiangsu, China) was then added. The reaction time was one hour.

[0136] A sample was taken, and ninhydrin (Shanghai Shanpu Chemical Co. Ltd, China) was used to detect when the reaction was complete. At this point, the liquid was drained and the residue was washed three times with DMF.

[0137] The above coupling steps were repeated to couple the remaining amino acids in the same amounts: Fmoc-Thr(tBu)-OH, Fmoc-4-Hyp(tBu)-OH, Fmoc-4-Hyp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH and Fmoc-Ala-OH.

[0138] At the end of the reaction sequence, 20% piperidine in DMF was added as a deprotection solution in the manner described above. The liquid was then drained after 15 minutes and the column washed with DMF, DCM and methanol, 3 times each, respectively.

[0139] The liquid was drained to obtain the resin-bound polypeptide.

[0140] An appropriate amount of lysate, which was comprised of 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (Tis), was added to immerse the resin-bounded polypeptide. The mixture was put on a shaker and incubated for 2 hours at 30 to 35° C. The resin was then removed by filtration.

[0141] Anhydrous ice ether was added into the filtrate, which was centrifuged and the supernatant discarded. The resultant was washed with anhydrous ice ether three times. The isolated peptide was dried by desiccation to obtain 128 g of crude polypeptide. The crude compound was desalted using an anion exchange resin, analysed and freeze-dried. Approximately 70.7 g of purified peptide was obtained after purification, which was re-tested for confirmation.

[0142] 1 mg of crude product was dissolved in 1 mL of an acetonitrile and water mixture (1:3) and detected using a P3000A HPLC pump and LC3000 semi-preparation equipment (preparation column model: GS-120-10-C18-AP 30 mm; Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China). The appropriate gradient for elution was calculated and the target peak was detected at 14.351 min with LCMS (analysis column model: GS-120-5-C18-BIO, 4.6*250 mm; detection: UV at 220 nm; solvent A: 0.1% TFA in MeCN, solvent A: 0.1% TFA in water; flow rate 1.0 mL/min.; volume: 10 μL).

[0143] m/z 592.65 [M+2H].sup.2+ (97.89%).

Example 2

[0144] Air Pouch Model

[0145] Healthy adult male C57BL/6 mice weighing between 20 and 30 g were supplied by Nanjing Biomedical Research Institute of Nanjing University (NBRI). Prior to any experiments being conducted, mice were housed under standardized conditions (at a constant temperature of 22±2° C., with alternating 12 hour periods of light and darkness), and were fed on a standard mouse diet with water, for about a week.

[0146] General anesthesia was induced using intraperitoneal 3% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; 1 mL/10 g of body weight). The hair of the entire dorsum was shaved and depilated 1 day before sterile air injection.

[0147] Air pouches were produced by subcutaneous injection of sterile air (5 mL) into the intrascapular area of the mice. After three days, another injection of air (3 mL) was performed to maintain the pouches. In order to induce acute inflammation, three days after this final injection, animals received an injection of sterile carrageenan solution (CP Kelco, Taixing, Jiangsu Province, China. 1%, 0.5 mL; produced by adding 0.1 g of carrageenan powder into a beaker containing 10 mL of 0.9% saline solution and stirring). Mice were pre-treated with test samples or vehicle 1 hour before and 23 hours after the carrageenan injection into the subcutaneous air pouch. Animals were sacrificed 24 hours after the carrageenan injection.

[0148] Skin biopsies were taken from the air pouches. A part of the biopsy was fixed in formalin (produced by adding ultra pure water to 50 mL of a 40% formaldehyde solution (Nanchang Rain Dew Experimental Equipment Co., Ltd., Nanchang, Hubei Province, China) up to a total volume of 500 mL) and analyzed by histological embedding in paraffin wax, sectioning and staining.

[0149] The cavity was washed with 4 mL of sterile phosphate buffer solution (pH 7.4; prepared by dissolving 4 g of NaCl, 0.1 g of KCl, 1.749 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.1 g of KH.sub.2PO.sub.4 in ultrapure water, adjusting the pH to 7.4 with HCl and diluting with water to a total volume of 500 mL).

[0150] Exudates were collected and volumes were quantified. The exudates were centrifuged at 3000 rpm for 10 minutes at 4° C., and the supernatants were collected and stored at −20° C. for ELISA analysis using standard ELISA test kits from Beijing 4A Biotech Co., Ltd. (Beijing) and an ELISA reader (SH-1000 Hitachi, Japan) for tissue necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6).

[0151] After conducting some preliminary experiments to validate the model, an experiment was conducted in which mice were treated by administering test samples or vehicle in accordance with Table 1 below.

[0152] In Table 1, MAP solution was prepared as follows. Blue mussels were harvested in the coastal area of Shandong Province, China. Mussel feet were collected, cut into small pieces and homogenized in an extraction buffer comprising 5% acetic acid in 4 mol/L of aqueous urea. The crude extracts were collected after centrifuging and then purified by liquid chromatography. The purified protein (semi-finished product; concentration 8 mg/mL; purity as measured by HPLC 91.72%; pH 4.2) was stored at 0° C. The solutions that was employed below was prepared by adding saline solution to this semi-finished product to obtain the concentrations described in Table 1.

[0153] An isolated compound consisting of the mefp-1 decapeptide sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (hereinafter “Compound A”) was purchased from Innovagen (Innovagen AB, Lund, Sweden). It was stored as a powder at −20° C. and dissolved in saline water at a concentration of 0.6 mg/ml, pH 5.5. 0.5 mL of the solution was injected.

[0154] The compound of the invention (Compound B) was synthesized by GL Biochem (Shanghai) Ltd. according to Example 1 above, and was also stored as a powder at −20° C. and was dissolved in saline water at a concentration of 0.6 mg/ml, pH 5.5. 0.5 mL of the solution was injected.

[0155] All the substances listed in Table 1 were administrated topically by directly injecting into the air pouches.

TABLE-US-00001 TABLE 1 Timing of Treatment No. Drug concn. Dose/ (before carrageenan Group Mice (mg/mL) mouse injection) Control (air 5 Normal 4.5 mg n/a injection only) saline (of NaCl) Model 5 Normal 4.5 mg 1 hour (carrageenan saline (of NaCl) injection) MAP 5 0.6 300 μg 1 hour Compound A 5 0.6 300 μg 1 hour Compound B 5 0.6 300 μg 1 hour

[0156] The histological specimens were analyzed and an inflammation score, an activity score (i.e. the number and density of neutrophils shown in pathological slides, indicating the degree of inflammation and, in the case of open wound and infective diseases, the degree of infection), an edema score and a fibroblast proliferation score were estimated as follows.

[0157] The HE stained slices were observed under an optical microscope and were scored (1, 2 or 3 points) according to the perceived inflammation level (in cases showing only a small amount of inflammatory cells scattered in the area—1 point (mild); in cases where many inflammatory cells were observed—2 points (moderate); and, in cases with diffuse infiltration—3 points (severe). A similar scoring system was employed for edema levels (3 points for most severe and 1 point for mild) after overall observation. Scores for neutrophils employed the same methodology as that employed for inflammatory cells.

TABLE-US-00002 TABLE 2 Fibroplastic Inflammation Edema proliferation Group score Score score Sum Control 1 1.2 0.8 2.2 Model 2.2 1.8 0.6 4 MAP 2 1.2 0.4 3.2 Compound A 1.6 1.8 0.2 3.4 Compound B 1.6 1.2 1.00 2.8

[0158] The ELISA test results of the exudate for TNF-α, IL-6 and IL-1β are shown graphically in FIG. 1. The results showed that Compound B had very strong anti-inflammatory effects.

Example 3

[0159] Acute Wound Model

[0160] 6-8 weeks old male C57BL/6 mice were supplied by Changzhou Cvens Experimental Animal Co. Ltd (Changzhou, Jiangsu Province, China). Prior to any experiments being conducted, mice were housed under standardized conditions (at a constant temperature of 22±2° C., with alternating 12 hour periods of light and darkness), and were fed on a standard mouse diet with water, for about a week.

[0161] General anesthesia was induced using intraperitoneal 3% chloral hydrate (1 mL/10 g of body weight). The hair on the back was shaved by a baby hair shaver and depilated with cream. The skin area was wiped and sterilized with 75% alcohol twice.

[0162] An EMS skin biopsy punch (Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, Pa., USA) with a 18 mm diameter was used to make a round wound on the back. The full thickness of skin was removed and the depth of the wound reached the fascia. Wounds were left open without a suture.

[0163] Different drugs were administrated topically at 50 μL/wound, once daily from Day 0 to Day 12, as shown in Table 3 below. The control group did not have a wound inflicted. The model group was given same amount of normal saline. There were 8 mice in each group.

[0164] Recombinant Human Epidermal Growth Factor (rhEGF, Shanghai Haohai Biological Technology Co. Ltd, 23/F, Shanghai, China) was purchased and prepared according to the manufacturer's instructions. The lyophilized rhEGF powder (100000 IU/vial) was dissolved in 20 mL of normal saline to make a solution with a concentration of 5000 IU/mL. The working dose of rhEGF for this experiment was 1285 IU/wound.

[0165] For this experiment, Compound A was also obtained from GL Biochem (Shanghai) Ltd. The peptide powders were stored at −20° C. and dissolved in saline at concentrations of 61.8 μg/mL (Compound A), and 61 μg/mL (Compound B). 50 μL of the solutions were applied onto the wound surfaces.

TABLE-US-00003 TABLE 3 GROUP SAMPLE DOSAGE Control Saline / Model Model + saline / rhEGF Model + rhEGF 1285 IU/mouse Compound A Model + Compound A 3.09 μg/mouse Compound B Model + Compound B 3.05 μg/mouse

[0166] Wounds were dressed with gauze and transparent dresser after drug administration. Photographs were taken for each wound every other day from Day 0. Photos were scanned into a computer, and wound areas calculated using ImageJ image analysis software (National Institutes of Health, China).

[0167] The unhealed wound area was expressed as a percentage of the original wound area:

A.sub.t/A.sub.0×100%,

[0168] where A.sub.0 and A.sub.t refer to the initial area at Day 0 and the wound area at the date of measurement (time t), respectively.

[0169] Samples were taken at Day 4 and Day 7 post-wound infliction. The mice were sacrificed and the wound tissue taken by the same biopsy punch used to create the wound. Then 5 mm of tissue was cut off from the center of the sample and was preserved in 10% neutral buffered formalin (Nanchang Rain Dew Experimental Equipment Co., Ltd., Nanchang, Hubei Province, China), and analyzed by histological embedding (HE) in paraffin wax, sectioning and staining.

[0170] HE and Masson stained paraffin sections were analyzed under an optical microscope. Skin regeneration, fibroblastic proliferation, collagen regeneration scores and inflammation scores were estimated.

[0171] The rest of the samples were stored at −80° C. for further analysis. Tissue was cut into small pieces and liquid nitrogen was added to increase brittleness. 9 mL of normal saline was added to 1 g of tissue, which was then ground using a Tissuelyser (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) at 55 Hz for 60 seconds, followed by centrifuging at 8000 rpm for 10 minutes at 4° C.

[0172] The supernatant was collected, and the extracted protein was used for ELISA analysis using standard ELISA test kits and an ELISA reader (SH-1000 Hitachi, Japan). Vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-β1) and hydroxyproline (Hyp) were assayed. The ELISA kits were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

[0173] The effects of Compound B on wound healing are show in Table 4, and in FIG. 2, which show the ratio of remaining wound area of initial wound in different groups (±SD in the case of Table 4).

TABLE-US-00004 TABLE 4 Group Model rhEGF Compound A Compound B Day 0 86.4 ± 5.4 82.0 ± 2.2 77.2 ± 4.1  79.4 ± 3.4 Day 1  100 ± 7.2  100 ± 9.9  100 ± 17.1  100 ± 4.5 Day 2 96.7 ± 3.6 94.1 ± 4.3 81.2 ± 11.2 96.7 ± 5.6 Day 3 89.6 ± 3.1 78.6 ± 7.3 74.2 ± 0.9  76.7 ± 4.8 Day 4 73.9 ± 3.8 61.5 ± 3.3 60.9 ± 7.3  66.5 ± 1.4 Day 5 66.2 ± 5.9 53.0 ± 1.0 50.5 ± 11.3 55.8 ± 1.3 Day 6 56.2 ± 5.3 47.2 ± 5.8 45.8 ± 4.7  51.9 ± 1.7

[0174] The above data show that a low dose of Compound B improves wound healing. The improvement rate was defined as (remaining wound ratio in treatment group/remaining wound ratio in model group)×100%. At Day 4, the improvement rate in the Compound B group was about 10%.

[0175] The wound Hyp (μg/mg) content, which is an indicator of collagen regeneration, is shown in Table 5 below and in FIG. 3.

TABLE-US-00005 TABLE 5 control model rhEGF Compound A Compound B Mean Day 4 4.070 1.551 1.808 3.676 2.039 Day 7 4.070 2.355 2.679 2.694 2.810 SD Day 4 0.980 1.100 0.790 0.951 0.965 Day 7 0.980 1.021 0.594 0.215 1.067

[0176] The result for VEGF content (μg/g) are tabulated in Table 6 below and are shown graphically in FIG. 4.

TABLE-US-00006 TABLE 6 control Model rhEGF Compound A Compound B Mean Day 4 9091.3 6598.0 7614.2 5709.7 10076.3 Day 7 9091.3 6500.1 5710.2 7949.5 8117.9 SD Day 4 1575.9 897.4 3362.9 3229.8 976.1 Day 7 1575.9 2375.4 2306.1 1734.8 1163.6

[0177] The result showed that Compound B has increased the VEGF production in the wounded tissue for about 56.7% in comparison with that in model group.

[0178] The result for TGF-β1 content (μg/g) are tabulated in Table 7 below and are shown graphically in FIG. 5.

TABLE-US-00007 TABLE 7 control Model rhEGF Compound A Compound B Mean Day 4 29832.2 15764.4 18190.6 11866.2 20571.8 Day 7 29832.2 15639.4 15443.1 18074.8 18659.6 SD Day 4 6554.9 1946.1 8564.5 7216.3 2310.6 Day 7 6554.9 5141.7 5482.1 3477.0 1123.0

Example 4

[0179] Liquid Spray to Treat Allergic Dermatitis.

[0180] Compound B (see Example 1 above) was dissolved in Water for Injection (WFI; prepared from a TC-RO-0.25/h-2 Water Treatment System, Yangzhou Tiancheng Water Treatment Devices & Engineering Co., Ltd., Yangzhou, China). The concentration was 0.3 mg/m L.

[0181] The enrolled subjects in the study had allergic skin. Prior to treatment, the patients presented symptoms including skin redness, itching and swelling. Subjects were required to use the liquid spray in the morning and the evening after facial cleansing.

[0182] The subjects all felt that itching was relieved within 8 minutes of the first use. Symptoms of capillary congestion disappeared and the patients' faces were no longer red and swollen after 1 day of use.

[0183] This experiments showed that a liquid spray comprising a compound of the invention relieves itching quickly and reduce redness, swelling and other symptoms caused by allergy.

Example 5

[0184] Cream to Treat Closed Comedones

[0185] Compound B (10 mg; see Example 1 above) was first dissolved in WFI (10 g).

[0186] A mixture was then made comprising sorbitan stearate (2 g), cetyl alcohol (4 g), octanoic/capric glyceride (6 g) and glyceryl monostearate (3 g) (all Sinopharm Chemical Reagent Co. Ltd.), which was stirred and heated to 85° C. to melt the mixture completely.

[0187] Glycerin (5 g; Sinopharm Chemical Reagent Co. Ltd.) and ammonium acryloyldimethyltaurate/VP copolymer (0.13 g; Clariant Chemical (Guangzhou) Co., Ltd., China) were mixed with 68.86 g of WFI. This mixture was stirred and heated to 85° C. to provide a homogeneous colloidal suspension.

[0188] The copolymer/water mixture was added to the sorbitan stearate-containing mixture, which was then quickly stirred using emulsification equipment for 5 minutes. The resultant emulsion was cooled to room temperature.

[0189] The Compound B solution was then added to the resultant mixture with stirring to obtain the finished cream. The concentration of Compound B in the final cream was 0.1 mg/g.

[0190] Subjects with closed comedones (blocked pores) and in an acute attacking period of acne were enrolled in the study. Symptoms included sebaceous glands blocked by keratinocytes, forming a slightly hard lump and a protruding white head.

[0191] The cream was evenly smeared in the morning and evening after facial cleansing. The lumps disappeared after 1 day of use, demonstrating that a cream comprising a compound of the invention can be used to treat closed comedones.

Example 6

[0192] Determination of Antioxidant Capacity of Compound of the Invention

[0193] The antioxidant capacity of Compound B was determined using a 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay.

[0194] Compounds A and B were obtained as described in Example 3 above. The peptide powders were stored at −20° C. and dissolved in saline at concentrations as indicated below. DPPH was purchased from Sigma-Aldrich China, Shanghai. Other reagents employed and identified below were purchased from Shanghai Aladdin Bio-Chem Technology Co. Ltd., China.

[0195] The DPPH method is one of the most commonly used methods in determination of antioxidant capacity. DPPH is a stable, nitrogen-centred free radical with a dark purple solution and a maximum absorption peak at 517 nm. When a free radical scavenger is present in the reaction system, it can be paired with a single electron of DPPH., and the absorption peak at 517 nm is gradually dissipated.

[0196] The degree of colour change bears a stoichiometric relationship to the number of paired electrons. Thus, antioxidant activity can be measured according to the change of absorbance. The greater the inhibition rate, the stronger the antioxidant capacity.

[0197] 6 mg of DPPH was weighed and placed into a 100 mL volumetric flask. This was made up to volume with 80% ethanol to prepare a solution with a 0.06 mg/mL concentration. This was stored at 4° C. in the dark.

[0198] Peptide solutions were prepared by dissolving 0.6 g of peptide powder in 1 mL of distilled water.

[0199] 200 μL of peptide solution was added to 3.8 mL of the DPPH solution. The solution was kept at room temperature for 1.5 hours in the dark. The absorbance at 517 nm was measured and was denoted as A1. The absorbance of the blank control sample was denoted as A0. Thus, the percentage of the DPPH remaining is calculated as: Clearance rate (%)=[A0−A1]/A0×100

[0200] The DPPH clearance rate for Compound A was 52.11%. That for Compound B was much less, at 8.9%, representing a significant improvement in antioxidant capacity.

Example 7

[0201] Stability of Compound of the Invention

[0202] Stabilities of Compounds A and B (obtained as described in Examples 1 and 3 above) were tested as follows.

[0203] 10 mg of the compounds were each dissolved in 1 mL of distilled water to make a stock solution with a concentration of 10 mg/mL. Then, 200 μL of the stock solutions were diluted with 20 mM phosphate buffer (PB; Sigma-Aldrich China) with different pHs as indicated in Table 8 below.

TABLE-US-00008 TABLE 8 No. pH value Buffer volume Final pH tested Compound A 1 5.3 1800 μL 5.3 2 7.2 1800 μL 7.2 3 8.1 1800 μL 8.1 Compound B 4 5.3 1800 μL 5.3 5 7.2 1800 μL 7.2 6 8.2 1800 μL 8.1

[0204] All of the samples above were filtered through a 0.22 μm membrane (SLGV033RS; Sigma-Aldrich China) to remove potential bacteria in the solution on a super-clean bench after being prepared.

[0205] All the samples above were stored at room temperature and, three days later, were transferred to a 45° C. oven after initial sample analysis by HPLC (Column: Angilent ZORBAX Eclipse XDB-C18 (4.6×250 mm; 5 μm) (SN:USNH008244); buffer: A: 0.1% TFA in Water; B: 0.1% TFA in ACN; gradient: 0-25 minutes: 5%-30% B; 25-30 min: 100% B; flow rate: 1 mL/min; detection wavelength: 220 nm; sample volume: 20 μL).

[0206] The stability of the above samples was studied by observing the change of colour (Table 9), HPLC peak areas (Table 10) and purity (Table 11). The pH values for all samples were measured as being stable during the testing period.

TABLE-US-00009 TABLE 9 Compound A Compound B Day No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 0 NO NO NO NO NO NO 1 NO + ++ NO NO NO 3 NO + ++ NO NO NO (Transfer to 45° C.) 6 (45° C.) NO ++ ++ NO NO NO

[0207] NO means no colour changed was observed. + means a slight colour change is observed. ++ means a significant colour change is observed.

[0208] As shown in Table 9, the Compound A solutions became darker with time, particularly at higher pHs. The colour for rest of samples remained unchanged.

TABLE-US-00010 TABLE 10 Compound A Compound B Day No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 0   100%  100%  100%   100%  100% 1 100.1% 89.0% 64.8% 100.0% 98.9% 3 100.3% 41.7% 21.7%  99.9%   100% 99.2% (Transfer to 45° C.) 6 (45° C.)  99.6% 11.5%  3.8% 102.4% 100.6% 97.0%

[0209] The peak area presented in Table 10 above as an area % of Day 0's result represents the peptide concentration. As shown in Table 10, peak areas of three samples of Compound B were almost unchanged. For Compound B, the peak area at pH 5 was unchanged, but at pH 7.2 and pH 8.1, the peak areas decreased significantly, which indicates that the concentration of peptide decreased with time.

TABLE-US-00011 TABLE 11 Compound A Compound B Day No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 0 99.4% 98.9% 96.3% 99.2% 99.6% 99.5% 1 99.2% 92.1% 61.0% 99.2% 99.3% 99.4% 3 99.4% 94.8% 52.5% 98.4% 98.7% 98.4% (Transfer to 45° C.) 6 (45° C.) 98.0% 13.8% 3.9% 99.0% 98.2% 96.4%

[0210] When the peptides are oxidized, the HPLC peak shifts and the purity of peptide decreases. As shown in Table 11 above, the results show that most of Compound A was oxidized at pH 7.2 and pH 8.3.

[0211] It can be seem from the above results that the compound of the invention is stable at all pH values, but Compound A is stable only at pH 5.3.

Example 8

[0212] Compound B/Montelukast Dressing

[0213] An ointment was made by first dissolving montelukast sodium (200 mg; Arromax Pharmatech Co., Ltd, Suzhou, China) in polyethylene glycol 400 (20.0 g; Sinopharm Chemical Reagent Co. Ltd) with stirring. Compound B (16 mg; see Example 1 above) was then added to this solution.

[0214] Polyethylene glycol 3350 (21.3 g; Sinopharm Chemical Reagent Co. Ltd) was dissolved in in polyethylene glycol 400 (58.5 g) by heating to 60° C. with stirring. After this solution had cooled to 40-50° C., the solution containing Compound B and montelukast was added to it with stirring, followed by mixing for 5 to 10 minutes. Cooling to room temperature gave the finished product.

[0215] The ointment was coated evenly on a gauze with a flat plate. Cooling to room temperature gave the finished dressing.

Example 9

[0216] Compound B/Trypsin Spray

[0217] Compound B (30 mg; see Example 1 above) was dissolved in 10 mL of water. Trypsin (30 mg; Sichuan Deebio Pharmaceutical Co. Ltd., Guanhan, Sichuan, China) was dissolved in another 10 mL of water.

[0218] Calcium chloride (0.1 g), ethanol (0.5 g), water-soluble menthol (0.01 g), lactic acid 0.01 g and glycerol (30 g) (all Sinopharm Chemical Reagent Co. Ltd), were mixed together in 49.32 mL of water.

[0219] The solutions containing Compound B and trypsin were added to the mixture with stirring to provide a liquid for spraying.

Example 10

[0220] Compound B/Trypsin Aerosol for Inhalation

[0221] A aerosol formulation was prepared essentially as described in Example 9 above, from 20 mg of Compound B and 30 mg of trypsin, this time without glycerol and in a total of 99.32 mL of water.

Example 11

[0222] Common Cold Study

[0223] The subject that was enrolled in the study had a viral infection that was self-diagnosed as the common cold. The subject presented symptoms, including cough, sore throat and loss of voice for 2 days. Other anti-cold drugs had not been used prior to treatment.

[0224] The subject was required to use the liquid spray of Example 10 above in the morning and the evening.

[0225] On each use, 3 mL of the spray was poured into an atomizer storage tank which was attached to an atomizer. The atomizer was then opened and a mask was placed over the subject's mouth and nose.

[0226] The subject felt that throat pain was relieved after the first use. After the second use, the subject no longer felt pain in the throat and could speak, showing a liquid spray comprising a compound of the invention can relieve cough and sore throat symptoms caused by the common cold.

Example 12

[0227] Idiopathic Pulmonary Fibrosis (IPF) Model

[0228] Experimental animals and grouping: 40 adult male Sprague Dawley rats, after 7 days of adaptive feeding, were divided into 4 groups: sham-operation group (Sham), IPF model group (no treatment; Model), test group (Compound B) and positive control group (Positive Control).

[0229] The test were administered Compound B, at a dose of 130 μg/rat, which was introduced by aerosolized inhalation. Oral administration of pirfenidone (Etuary®, Beijing Continent Pharmaceutical Co., Ltd., Beijing, China) as a 120 mg/kg single-bolus dose served as the positive control.

[0230] A pulmonary fibrosis model was established by intratracheal instillation of bleomycin. The rats were anaesthetised and placed on an operating table in the supine position, to expose the trachea. Bleomycin (5 mg/kg) saline solution was injected into the trachea through the gap between the tracheal cartilage rings.

[0231] The sham-operation group were given an equal volume of normal saline. Shortly after administration, the rats were lifted vertically and were rotated, in order to evenly disperse the drug. Once the rats had recovered, after approximately 5 days, they were administrated the different drugs according to the plan for 28 days, consecutively. The experimental plan was showed in Table 12.

TABLE-US-00012 TABLE 12 Group treatment administration dose Sham Saline Aerosolized inhalation / Model Saline Aerosolized inhalation / Positive Control Pirfenidone Gavage 250 mg/kg Compound B Compound B Aerosolized inhalation 130 μg/Rat

[0232] The exhaust volume of the atomizing device was 0.15 mL per minute. The inhalation time for each rat was 1 minute.

[0233] At Day 29 following drug administration, rats were anesthetized by i.p. injection of chloral hydrate. After orbital blood collection, the rats were sacrificed. The thoracic cavity was quickly opened and the lung was collected for further analysis.

[0234] The lung wet weight was measured, and the lung coefficient was calculated (lung wet weight/rat weight×1000). The tissue was stored in refrigerator at −80° C. for further use.

[0235] A piece of lung tissue was accurately weighed, and 9 times the weight of saline was added. Then, the tissue was homogenized and centrifuged for 10 minutes at 3000 rpm. The homogenate was used to detect the expression of the transformation growth factor (TGF-β1), tumor necrosis factor-α (TNF-α) and alpha-smooth muscle actin (α-SMA). Standard ELISA methods were used for the detection. The contents of malonaldehyde (MDA) and the activity of superoxide dismutase (SOD) in lung tissue were also detected.

[0236] The effect of Compound B on TNF-α, α-SMA, TGF-β1 content in tissues of bleomycin-induced pulmonary fibrosis in rats are shown in Table 13.

TABLE-US-00013 TABLE 13 TNF-α (pg/mg α-SMA TGF-β1(ng/mg protein) (pg/mg protein) protein) Sham 181.85 ± 27.34  9.37 ± 0.72 0.41 ± 0.06 Model 301.69 ± 28.97 13.71 ± 1.41 0.55 ± 0.05 Compound B 187.91 ± 12.20 10.85 ± 1.42 0.40 ± 0.02 Positive Control 208.41 ± 17.73 10.98 ± 0.93 0.41 ± 0.03

[0237] The results show that Compound B inhibits the production of TNF-α, α-SMA, and TGF-β1 in lung tissue of bleomycin-induced pulmonary fibrosis in rats. The effects of inhibition were almost equivalent to that of the known IPF drug, pirfenidone.

[0238] The effect of Compound B on the lung coefficient, MDA, and SOD contents are shown in Table 14.

TABLE-US-00014 TABLE 14 lung coefficient MDA (U/mg SOD (U/mg (%) protein) protein) Sham 4.85 ± 0.26 0.12 ± 0.02 51.22 ± 1.07 Model 6.49 ± 0.69 0.66 ± 0.08 41.89 ± 2.82 Compound B 5.14 ± 0.37 0.17 ± 0.04 48.64 ± 1.85 Positive Control 5.54 ± 0.51 0.24 ± 0.05 48.59 ± 2.36

[0239] The results showed that the lung coefficient value was lower in the Compound B group than that in the Model group, which indicates that Compound B can reduce edema. Compared to the Model group, the lower MDA value and higher SOD value in the Compound B group show that Compound B has an antioxidant effect, by increasing SOD production and reducing lipid oxidation.

Example 13

[0240] Antitussive Experiment—Ammonia Induced Cough Method in Mice.

[0241] 36 ICR mice were randomly divided into 3 groups according to their body weight, labelled a CMC-Na (Negative) control group, a dextromethorphan hydrobromide (Positive) control group and a Compound B (B) group. Each group contained 12 mice, with 6 males and 6 females in each.

[0242] Compound B was administered via atomization inhalation using a YLS-8A Multifunctional cough and sputum inducing device (Jinan Yiyan Science and Technology Co., Ltd.) (0.15 mL/min) for 1 minute, once daily for 5 days. And the positive control drug was administered once daily by intragastric administration at 10 mg/kg for 5 days.

[0243] The mice were placed in an inverted beaker 1, 2, and 4 hours after the last drug administration. 1 mL of aqueous ammonia (25.0 to 28.0%) was placed on top of a boiling water bath and was evaporated into the beaker.

[0244] The time for mice to receive ammonia exposure was determined by a method characterized by analyzing data immediately after one test or a batch of tests. The results of the previous analysis determined how to conduct the next test or batch of tests. In this way, the process of experiment-analysis-experiment proceeded sequentially: [0245] (a) the time taken for the previous mouse to cough determined the time scheduled (which was shorter) for the next mouse to receive ammonia stimulation; or [0246] (b) if the former mouse did not cough, the subsequent one would be stimulated for a longer time.

[0247] The difference between the logarithmic value of two adjacent time periods was 0.1.

[0248] The number of coughs within 1 minute was detected using a stethoscope. If a mouse coughed three or more times over one minute, this was labelled as called ‘cough’, and less than three times in one minute was labelled ‘cough free’.

[0249] The EDT.sub.50 was defined as the ammonia stimulating time at which half mice developed a “cough”, calculated by the equation:

EDT.sub.50=Ig.sup.−1 c/n

[0250] (c equals the sum of r and x, r is the number of animals in each stimulating time group, x is the logarithm of stimulation time and n was the number of animals in each group). Ig is a base 10 logarithmic value. The results shown in Table 15 below.

TABLE-US-00015 TABLE 15 1 hour 2 hours 4 hours Time after admin. EDT.sub.50 R EDT.sub.50 R EDT.sub.50 R Dose (s) (%) (s) (%) (s) (%) Negative 20.7 21.5 22.4 Positive 10 mg/kg 50.1 241.7 44.7 207.3 41.4 184.8 B 195 μg/ 28.2 131.7 28.2 131.8 26.9 100 mouse [???]

[0251] In Table 15, R is the EDT.sub.50 in treatment group divided by the EDT.sub.50 in the Control group expressed as a percentage. An R value of >130% means that the compound has an antitussive effect and an R value of >150% indicates that it has obvious antitussive effect.

[0252] The results show that Compound B has a positive effect on cough relief at 1 and 2 hours.

Example 14

[0253] Liquid Spray to Treat Common Cold

[0254] Compound B was dissolved in Injection water (WFI; from a TC-RO-0.25T/h-2 water treatment system, Yangzhou Tiancheng Water Treatment Devices & Engineering Co., Ltd., Yangzhou, China). The concentration was 0.5 mg/mL. 5 mL was loaded into a plastic water spray bottle.

[0255] The enrolled subject in the study was inflicted with a common cold for 2 days prior to treatment. Other cold treatments were not used before treatment. The subject, who presented a cough, a sore throat and a loss of voice, was required to use the liquid spray (3 to 5 presses each time, in the morning and evening.

[0256] The subject felt that her throat pain was relieved after the first use. After the second use, the subject felt that her throat was no longer painful and could speak.

[0257] This experiment showed that a liquid spray comprising a compound of the invention relieves cough and sore throat symptoms caused by the common cold.

Example 15

[0258] Aerosol Inhalation to Treat Fever.

[0259] The same formulation as described in Example 14 above was administered to a subject who enrolled in the study having had got a fever (a body temperature over 38.5° C.) for 1 day. Other anti-fever medications were not used prior to treatment.

[0260] The subject was required to use the aerosol inhalation device in the morning and in the evening by pouring 3 mL of the formulation into an atomizer storage tank connected to an atomizer and a mask, which was worn over the mouth and nose.

[0261] The subject felt that within two hours, his body temperature had dropped to normal. Fever symptoms did not recur after 3 uses.

[0262] This experiment shows that an aerosol inhalation comprising a compound of the invention relieves fever.

Example 16

[0263] Liquid Spray to Relieve Operational Pain During Laser Surgery.

[0264] The enrolled subject was required to use 1 mL of a liquid spray comprising the same formulation as that described in Example 14 above in a water spray bottle on half of her face before and after a lattice laser operation to remove of facial melanin.

[0265] The subject reported that she did not feel pain during the operation on half of her face to which the spray was applied. Similarly, she felt no pain after the operation. Conversely, pain was felt during the operation on the half of her face that was not sprayed. Furthermore, after the operation, the pain lasted about 2 hours.

[0266] This experiment shows that a liquid spray comprising a compound of the invention relieves operation pain during laser surgery.

Example 17

[0267] Combination Treatment of Ulcerative Colitis

[0268] A gel comprising Compound B (obtained as described above) and mefp-1 (USUN Bio Co., Jiangyin, China), both at concentrations of 1 mg/g, were prepared by combining the active ingredients with methyl cellulose (2.5%), propanediol (11%), glycerol (11%) and acetic acid (pH regulator; up to 0.5 g) to give a gel precursor with a pH of 5.5. All excipients were obtained from Sinopharm Chemical Reagent Co. Ltd. The gel was made up with water for injection. The gel was packed into a disposable anal-intestinal drug delivery system.

[0269] Before treatment, the subject defecated more than 20 times per day and exhibited severe bleeding from, and ulceration within, the colon. The gel formulation was given by through the anus. After having been given one dose on the first day, the patient defecated just three time on the second day. The subject then received two further doses on the second day. By the third day, bleeding was controlled.

[0270] After 14 days of administration at two doses per day, the symptoms of the patient's ulcerative colitis had been alleviated and there was no further bleeding.