Topical formulations comprising montelukast and combinations with mussel adhesive proteins

Abstract

There is provided topical pharmaceutical formulations comprising montelukast, or a pharmaceutical acceptable salt of solvate thereof, as well as combination products comprising (a) at least one mussel adhesive protein or a derivative thereof; and (b) montelukast, or a pharmaceutically-acceptable salt or solvate thereof. The formulations and combination products find particular utility in direct topical administration for the treatment of inflammation, of inflammatory disorders and/or of condition characterized by inflammation, including wounds, burns, psoriasis, acne and atopic dermatitis.

Claims

1. A method of promoting healing or recovery of a hemorrhoid-induced wound of a patient, the method comprising: topically administering a pharmaceutical formulation comprising a therapeutically effective amount of montelukast, or a pharmaceutically acceptable salt of solvate thereof, in admixture with a pharmaceutically-acceptable topical adjuvant, diluent or carrier, directly to a hemorrhoid-induced wound of a patient.

2. The method of claim 1, wherein the hemorrhoid-induced wound comprises damage to and/or below the epidermis or the dermis.

3. The method of claim 1, wherein said topically administering the pharmaceutical formulation further treats pain and/or itching associated with the hemorrhoid-induced wound or healing processes associated therewith.

4. The method of claim 1, wherein said topically administering the pharmaceutical formulation further prevents the exudation of body fluids and/or the risk of infection resulting from the hemorrhoid-induced wound, and/or further prevents physiological reactions that result from the hemorrhoid-induced wound healing processes.

5. The method of claim 1, wherein said topically administering is carried out on the skin.

6. The method of claim 1, wherein said topically administering is carried out on a mucosal surface.

7. The method of claim 1, wherein the pharmaceutical formulation is in the form of a gel, a cream or an ointment.

8. The method of claim 1, wherein the pharmaceutical formulation further comprises polyethylene glycol.

9. The method of claim 8, wherein the polyethylene glycol is polyethylene glycol 400.

10. The method of claim 1, wherein the pharmaceutical formulation further comprises at least one mussel adhesive protein or a derivative thereof.

11. The method of claim 1 further comprising: topically administering a second pharmaceutical formulation comprising at least one mussel adhesive protein or a derivative thereof, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, wherein the pharmaceutical formulation and the second pharmaceutical formulation are each suitable for administration in conjunction with the other.

12. The method of claim 11, wherein the pharmaceutical formulation and the second pharmaceutical formulation are suitable for sequential, separate and/or simultaneous administration in the treatment of the hemorrhoid-induced wound.

13. The method of claim 10, wherein the at least one mussel adhesive protein is selected from the group: mefp-1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, and combinations thereof.

14. The method of claim 10, wherein the at least one mussel adhesive protein comprises mefp-1.

15. The method of claim 10, wherein the derivative of a mussel adhesive protein is a peptide of the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (SEQ ID NO:1) or a salt thereof.

16. The method of claim 11, wherein the at least one mussel adhesive protein is selected from the group: mefp-1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, and combinations thereof.

17. The method of claim 11, wherein the at least one mussel adhesive protein comprises mefp-1.

18. The method of claim 11, wherein the derivative of a mussel adhesive protein is a peptide of the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (SEQ ID NO:1) or a salt thereof.

19. A method of promoting healing or recovery of a hemorrhoid-induced wound of a patient, the method comprising: providing a pharmaceutical formulation that is in the form of a gel, a cream or an ointment and comprises a therapeutically effective amount of montelukast, or a pharmaceutically acceptable salt of solvate thereof, in admixture with a pharmaceutically-acceptable topical adjuvant, diluent or carrier; topically applying the pharmaceutical formulation directly to a hemorrhoid-induced wound of a patient, wherein the hemorrhoid-induced wound comprises injury to the skin or mucosa, damage to and/or below the epidermis or the dermis, or damage to a subcutaneous tissue, a submucosal tissue or an internal organ, and wherein said topically applying promotes healing or recovery of the hemorrhoid-induced wound.

Description

(1) The invention is illustrated by the following examples, in which:

(2) FIGS. 1 and 2 and present ELISA test results for various inflammatory markers obtained from exudates from air pouches induced in mice according to Examples 1 and 2, respectively, tested with various test compounds;

(3) FIG. 3 illustrates burn scores for the healing over time of third degree burns induced in rats, treated with various test compounds;

(4) FIGS. 4 and 5 present ELISA test results for various inflammatory markers obtained from exudates from air pouches induced in mice according to Examples 4 and 5, respectively;

(5) FIG. 6 shows the effect of montelukast on wound healing in an acute wound model of Example 6;

(6) FIG. 7 shows wound hydroxyproline content in samples in the Example 6 model;

(7) FIG. 8 shows the effect of montelukast on wound healing in a diabetic wound model of Example 7;

(8) FIG. 9 shows wound vascular endothelial growth factor content (pg/g) in samples at Day 6 in the Example 7 model;

(9) FIG. 10 shows the results of a histopathological analysis in samples at Days 6 and 12 in the Example 7 model;

(10) FIG. 11 shows the effect of topically-applied montelukast on scald wound healing in an in vivo rat model of Example 16;

(11) FIGS. 12 and 13 show montelukast penetration in unharmed skin and scalded skin in a similar in vivo rat model (see Example 17), in the cases of montelukast being applied topically alone (FIG. 12) and after an initial application of MAP (FIG. 13);

(12) FIG. 14 shows the effect of topically applied montelukast in a patient with second degree burns compared to silver sulfadiazine treatment;

(13) FIG. 15 shows the effect of topically applied montelukast in a patient with a melanin-related skin condition (chloasma and malar rash); and

(14) FIG. 16 shows the effect of topically applied montelukast in a patient with steroid-dependent dermatitis.

EXAMPLES

Example 1

(15) Air Pouch Model I

(16) Healthy adult male C57BL/6 mice weighing between 20 and 30 g 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 or 22 t 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.

(17) 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.

(18) 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.

(19) 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 Provence, China) up to a total volume of 500 mL) and analyzed by histological embedding in paraffin wax, sectioning and staining.

(20) 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).

(21) Exudates were collected and volumes were quantified. The total number of cells was determined with a haematocytometer (ADVIA 2120 Hematology System, Siemens Healthineers). 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 (Biolegend™ from Dakewe Biotech Co., Beijing, China or Abcam™, Abcam (Shanghai) Trading Co. Ltd, China) and an ELISA reader (SH-1000 Hitachi, Japan) for tissue necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), prostaglandin E2 (PGE2), interferon gamma (IFN-γ) and 5-hydroxytryptamine (5-HT; serotonin).

(22) 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.

(23) In Table 1, MAP is a Mefp-1 solution, which is 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 were employed below were prepared by adding saline solution to this semi-finished product to obtain a concentration as described in Table 1. Montelukast sodium (“Mon”; MedChemExpress, Shanghai, China) and dexamethasone (MedChemExpress) were both obtained in powder form and dissolved in ultrapure water to obtain solutions with concentrations as described in Table 1. All the substances listed in Table 1 were administrated topically by directly injecting into the air pouches.

(24) TABLE-US-00001 TABLE 1 Timing of Treatment No. Drug concn. (before carrageenan Group Mice (mg/mL) Dose/mouse injection) Control (air 6 normal 4.5 mg (of NaCl) n/a injection only) saline Model 6 normal 4.5 mg (of NaCl) 1 hour (carrageenan saline injection) Positive 6 0.4 200 μg 1 hour (dexamethasone) MAP 6 3 1.5 mg 1 hour Mon (oral) 6 1 200 μg 1 hour Mon (topical) 6 0.4 200 μg 1 hour MAP + 6 3 (MAP) 1.5 mg (MAP) 1 hour (MAP) Mon (oral) 1 (Mon) 200 μg (Mon) 1.5 hour (Mon) MAP + 6 6 (MAP) 1.5 mg (MAP) 1 hour (MAP) Mon (topical) 0.8 (Mon) 200 μg (Mon) 1.5 hour (Mon) MAP 0.25 mg 3 0.5 0.25 mg 1 hour MAP 2.5 mg 3 5 2.5 mg 1 hour MAP 5 mg 3 10 5 mg 1 hour MAP 7.5 mg 3 15 7.5 mg 1 hour

(25) The volume of the exudate and the weight of air pouch wall (±SD) for each group are tabulated in Table 2 below.

(26) TABLE-US-00002 TABLE 2 Group Exudate volume (mL) Pouch Wall Weight (g) Control 3.33 ± 0.10 0.58 ± 0.06 Model 3.73 ± 0.12 0.81 ± 0.14 Positive 3.60 ± 0.14 0.72 ± 0.09 MAP 3.62 ± 0.12 0.66 ± 0.06 Mon (oral) 3.58 ± 0.15 0.67 ± 0.10 Mon (topical) 3.68 ± 0.15 0.66 ± 0.13 MAP + 3.53 ± 0.08 0.61 ± 0.12 Mon (oral) MAP + 3.60 ± 0.09 0.66 ± 0.07 Mon (topical) MAP 0.25 mg 3.43 ± 0.12 0.85 ± 0.07 MAP 2.5 mg 3.60 ± 0.10 0.74 ± 0.04 MAP 5 mg 3.57 ± 0.15 0.69 ± 0.14 MAP 7.5 mg  3.6 ± 0.10 0.68 ± 0.15

(27) The total cell count and classification (×10.sup.8/mL) is tabulated in Table 3 below.

(28) TABLE-US-00003 TABLE 3 Group Leukocyte Neutrophil Monocyte Lymphocyte Control 0.04 0.01 0.00 0.01 Model 5.06 0.28 0.10 4.57 Positive 2.48 0.22 0.05 2.08 MAP 2.71 0.23 0.10 2.24 Mon (oral) 2.38 0.21 0.07 1.98 Mon (topical) 3.82 0.29 0.17 3.22 MAP + 1.53 0.21 0.06 1.16 Mon (oral) MAP + 1.65 0.19 0.11 1.27 Mon (topical) MAP 0.25 mg 0.40 0.09 0.01 0.26 MAP 2.5 mg 1.26 0.18 0.05 0.94 MAP 5 mg 1.19 0.17 0.04 0.92 MAP 7.5 mg 1.65 0.27 0.07 1.22

(29) The histological specimens were analysed 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 fibroplastic proliferation score were estimated as follows.

(30) 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.

(31) TABLE-US-00004 TABLE 4 Fibroplastic Inflammation Activity Edema proliferation Group score score Score Sum score Control 0.67 0.50 1.00 3.00 0.83 Model 3.00 2.00 1.50 6.67 0.17 Positive 1.50 0.83 1.67 4.00 0.00 MAP 2.67 1.50 1.67 6.00 0.17 Mon (oral) 1.83 1.00 1.00 4.17 0.33 Mon (topical) 2.17 1.00 1.50 5.50 0.83 MAP + 1.83 1.00 0.67 4.83 1.33 Mon (oral) MAP + 2.00 1.17 0.83 4.33 0.33 Mon (topical) MAP 0.25 mg 2.00 1.33 1.00 5.00 0.67 MAP 2.5 mg 1.67 1.00 1.67 4.67 0.33 MAP 5 mg 2.00 1.00 1.67 5.00 0.33 MAP 7.5 mg 2.00 1.00 1.00 5.00 1.00

(32) In the Control group, the pouch wall showed small amount of scattered inflammatory cell infiltration, rare neutrophil infiltration, mild edema, and mild fibrous tissue proliferation.

(33) In the Model group, the pouch wall showed severe inflammatory cell infiltration in the whole layer (a large number of neutrophils), severe interstitial edema, and occasionally fibrous tissue proliferation.

(34) In the Positive control group, the pouch wall showed mild to moderate inflammatory cell infiltration, occasionally infiltration in the whole layer, severe interstitial edema, and no fibrous tissue formation.

(35) In the MAP (1.5 mg) group, the pouch wall showed severe inflammatory cell infiltration in the whole layer, moderate to severe interstitial edema, and occasionally fibrous tissue proliferation.

(36) In the Mon (oral) group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and occasionally fibrous tissue proliferation.

(37) In the Mon (topical) group, the pouch wall showed moderate to severe inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild to moderate interstitial edema, and lightly fibrous tissue proliferation.

(38) In the MAP+Mon (oral) group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and obvious fibrous tissue proliferation.

(39) In the MAP+Mon (topical) group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and occasionally fibrous tissue proliferation.

(40) In the MAP (0.25 mg) group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and mild fibrous tissue proliferation.

(41) In the MAP (2.5 mg) group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, obvious interstitial edema, and occasionally fibrous tissue proliferation.

(42) In the MAP (5 mg) group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild to moderate interstitial edema, and occasionally fibrous tissue proliferation.

(43) In the MAP (7.5 mg) group, the pouch wall showed moderate inflammatory cell infiltration, with mild to moderate neutrophil infiltration, mild interstitial edema, and mild fibrous tissue proliferation.

(44) The ELISA test results of the exudate showed PGE2 and 5-HT levels that were below detection ranges. The results for TNF-α, IL-6, IL-10 and IFN-γ (±SD) are tabulated in Table 5 below and are shown graphically in FIG. 1 (in respect of which, abbreviations are given in Table 5).

(45) TABLE-US-00005 TABLE 5 Group TNF-α IL-6 IL-10 IFN-γ Control  8.62 ± 0.10  2.3 ± 0.00 24.32 ± 0.00  −5.00 ± 3.00  Model  674.65 ± 244.26 34050 ± 13984 72.13 ± 33.13 .sup. 167 ± 44.48 Positive  71.31 ± 111.19 3649 ± 1764 52.33 ± 11.60 2879 ± 1267 MAP  55.45 ± 11.51 10411 ± 6948  65.79 ± 12.64 1842 ± 1129 Mon (oral)  39.41 ± 17.62 8453 ± 9758 52.59 ± 7.22  2495 ± 711  (MON-O) Mon (topical)  90.73 ± 109.02 2528 ± 1538 68.18 ± 15.81 1690 ± 725  (MON-T) MAP + 27.08 ± 6.14 2459 ± 681  37.06 ± 11.91 1534 ± 685  Mon (oral) (MMO) MAP + 27.87 ± 8.49  330 ± 0.00 56.24 ± 13.73 2362 ± 1233 Mon (topical) (MMT) MAP 0.25 mg 14.95 ± 4.54  98.7 ± 73.55 70.50 ± 23.86 1778 ± 1430 (0.25 mg) MAP 2.5 mg 21.53 ± 2.69 5289 ± 5575 56.64 ± 17.56 379.91 ± 138.01 (2.5 mg) MAP 5 mg 21.76 ± 3.84   811 ± 224.56 65.50 ± 9.33  575.66 ± 289.83 (5 mg) MAP 7.5 mg 26.23 ± 2.02   757 ± 331.78 56.51 ± 7.15  688.76 ± 374.80 (7.5 mg)

Example 2

(46) Air Pouch Model II

(47) Following the same protocol as described in Example 1 above, a further comparative experiment was conducted in which mice (Comparative Medicine Centre, Yangzhou University, Jiangsu Province, China) were treated by administering test samples or vehicle in accordance with Table 6 below.

(48) In Table 6, “Dex” is an abbreviation for dexamethasone and “Dopamine” is a 4 mg/mL aqueous solution of dopamine hydrochloride (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China).

(49) TABLE-US-00006 TABLE 6 Timing of Treatment No. Drug concn. (before carrageenan Group Mice (mg/mL) Dose/mouse injection) Control 6 normal 4.5 mg (of NaCl) n/a saline Model 6 normal 4.5 mg (of NaCl) 1 hour saline Dex 6 0.8 400 μg 1 hour Dopamine 6 4 2 mg 1 hour MAP 6 0.5 250 μg 1 hour Mon + 6 0.4 (Mon) 100 μg (Mon) 1.5 hour (Mon) MAP 1 (MAP) 250 μg (MAP) 1 hour (MAP) Dex + 6 1.6 (Dex) 400 μg (Dex) 1.5 hour (Dex) MAP 1 (MAP) 250 μg (MAP) 1 hour (MAP) Mon 6 0.2 100 μg 1 hour

(50) The volume of the exudate and the weight of air pouch wall (means±SD) for each group are tabulated in Table 7 below.

(51) TABLE-US-00007 TABLE 7 Group Exudate volume (mL) Pouch Wall Weight (g) Control 2.57 ± 0.05 0.44 ± 0.02 Model 3.10 ± 0.21 0.62 ± 0.09 Dex 2.87 ± 0.06 0.45 ± 0.01 Dopamine 2.93 ± 0.06 0.44 ± 0.07 MAP 3.00 ± 0.10 0.49 ± 0.04 Mon + MAP 2.90 ± 0.10 0.53 ± 0.04 Dex + MAP 3.03 ± 0.06 0.48 ± 0.05 Mon 2.83 ± 0.15 0.52 ± 0.03

(52) The total cell count and classification (×10.sup.6/mL) is tabulated in Table 8 below.

(53) TABLE-US-00008 TABLE 8 Group Leukocyte Neutrophil Monocyte Lymphocyte Control 0.07 0.02 0.03 0.01 Model 2.33 0.14 2.08 0.05 Dex 0.88 0.13 0.67 0.03 Dopamine 1.91 0.19 1.58 0.05 MAP 1.74 0.19 1.40 0.06 Mon + MAP 1.00 0.18 0.69 0.05 Dex + MAP 1.06 0.16 0.77 0.05 Mon 1.58 0.26 1.15 0.07

(54) The histological specimens were analysed and an inflammation score, an activity score and a fibroplastic proliferation score were estimated as in Example 1 above and are tabulated in Table 9 below.

(55) TABLE-US-00009 TABLE 9 Fibroplastic Inflammation Activity Edema proliferation Group score score Score Sum score Control 0.50 0.00 1.00 1.50 0.67 Model 2.17 1.83 2.00 6.00 0.00 Dex 0.83 0.33 0.83 2.00 0.00 Dopamine 2.00 1.67 2.33 6.00 0.00 MAP 1.33 1.17 1.67 4.17 0.00 Mon + MAP 1.67 1.17 1.17 4.00 0.00 Dex + MAP 1.00 0.17 1.17 2.33 0.00 Mon 1.83 1.50 1.50 4.83 0.00

(56) In the Control group, the pouch wall showed small amount of lymph cells infiltration, no neutrophil infiltration, partial mild to moderate edema, and mild fibrous tissue proliferation.

(57) In the Model group, the pouch wall showed severe inflammatory cell infiltration in the whole layer, severe interstitial edema, and no fibrous tissue proliferation.

(58) In the Dex group, the pouch wall showed small amount of lymph cells infiltration, occasionally light neutrophil infiltration, mild interstitial edema, and no fibrous tissue formation.

(59) In the Dopamine group, the pouch wall showed a lot of inflammatory cell infiltration, mild to moderate interstitial edema, and no fibrous tissue proliferation.

(60) In the MAP group, the pouch wall showed a lot of inflammatory cell infiltration, mild to moderate interstitial edema, and no fibrous tissue proliferation.

(61) In the Mon+MAP group, the pouch wall showed moderate inflammatory cell infiltration, mild interstitial edema, and no fibrous tissue proliferation.

(62) In the Dex+MAP group, the pouch wall showed small amount of scattered lymph cell infiltration, occasionally neutrophil infiltration, mild interstitial edema, and no fibrous tissue proliferation.

(63) In the Mon group, the pouch wall showed a lot of inflammatory cell infiltration, mild to moderate interstitial edema, and no fibrous tissue proliferation.

(64) The ELISA test results of the exudate for TNF-α, IL-6, IL-10 and IFN-γ always showed stable, high responses with a similar variation tendency. The results for IL-6 and IFN-γ (±SD) are tabulated in Table 10 below and shown graphically in FIG. 2.

(65) TABLE-US-00010 TABLE 10 Group IL-6 IFN-γ Control 1083.94 ± 433.74 −3.74 ± 1.3  Model 1213.54 ± 790.71 855.32 ± 570.36 Dex  3697.30 ± 5112.52 238.91 ± 272.27 Dopamine 1740.11 ± 765.02 5153.02 ± 5773.45 MAP 1686.11 ± 632.33 282.01 ± 192.24 Mon + MAP 1674.80 ± 696.85 1487.61 ± 815.47  Dex + MAP  8352.25 ± 6162.60 48.44 ± 35.58 Mon 1493.42 ± 564.77 896.90 ± 633.60

Example 3

(66) Burn Injury Model

(67) Healthy adult male Sprague-Dawley rats weighing between 200 and 300 g were supplied by Beijing Vital River Laboratory Animal Technology Co. Ltd, Beijing, China.

(68) Prior to any experiments being conducted, rats were housed under standardized conditions (at a constant temperature or 22 t 2° C., with alternating 12 hour periods of light and darkness) and were fed on a standard rat diet with water.

(69) Rats were anaesthetised with 3% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (1 mL/100 g of body weight) administered by intraperitoneal injection. The dorsal trunk was of each rat was shaved with an electric razor and surgically prepared with 70% alcohol scrubs. A constant temperature and constant pressure scald instrument (YLS-5Q, Shandong Academy of Medical Sciences, China) was employed, as this allows for a precise measurement of pressure applied to the skin surface. Thermal injury was induced by heating a stylus (with a surface area of 4 cm.sup.2) to 80° C. and applying the stylus to the surface of the skin for a contact time of 18 seconds. 500 grams of pressure was applied during the operation. In this way, four two-degree shallow to deep burn wounds were created on each rat. Test compounds or vehicle were applied to the surface of the wound.

(70) Rats were kept in separate cages but were allowed to move freely throughout the test period. In the first treatment, 500 μL of the relevant solutions was applied about 3 to 4 hours after the surgery (Day 0) and then once daily for the next 7 days. Gross appearances of the scald wounds were observed every day. Photographs were taken at 48 hours (Day 2), and on the 4.sup.th (Day 4) and 7.sup.th (Day 7) days with a digital camera to compare wound healing. The skin lesions were evaluated for the following criteria: blistering, swelling, redness, crust, bleeding, secretion, granulation tissue and scar tissue. The degree of healing was expressed as the wound contraction ratio (WCR):
WCR=(A.sub.0−A.sub.t)/A.sub.0×100%
in which A.sub.0 and A.sub.t refer to the initial area and the wound area at time t, respectively.

(71) On Days 2, 4 and 7 after application of the burn, two rats were sacrificed and the burned skins excised. The freshly isolated skin was fixed in 10% neutral buffered formalin (which contained 4% of formaldehyde). This was used for histological embedding, staining and immunohistochemical studies.

(72) Rats were treated by administering test samples or vehicle in accordance with the Table 11 below. Administered solutions were made up substantially as described in Example 1 above.

(73) TABLE-US-00011 TABLE 11 Drug concn. Dosage/ Group (mg/mL) wound Control (i.e. — — untreated rats) Saline normal saline 4.5 mg NaCl Dexamethasone 0.8 0.4 mg MAP 0.5 0.25 mg Montelukast 0.5 0.25 mg MAP + 1 (MAP) 0.25 mg (MAP) dexamethasone 1.6 (dexamethasone) 0.4 mg (dexamethasone) MAP + montelukast 1 (for both) 0.25 mg of each drug

(74) Results

(75) Burn scores are shown in Table 12 below and are illustrated in FIG. 3. Skin samples were sent to the Department of Pathology of Nanjing Hospital of Chinese Medicine, China. Samples were fixed and embedded, and histological sections were cut and stained. The stained sections were examined under a light microscope and pictures were taken. All pictures were interpreted and scored according to the following criteria: first degree—the epidermis was damaged and the substrate remains intact (score 1 point); superficial second degree—the epidermis was completely damaged and the papillary layer was damaged (1.5 points): deep second degree—the dermis was injured into the reticular layer and retain some dermis (2.5 points); and third degree—the dermis was completely damaged and the muscles and/or bones affected (3 points).

(76) TABLE-US-00012 TABLE 12 Group Day 2 Day 4 Day 7 Control 0.00 0.00 0.00 Saline (Model) 2.70 2.94 1.88 Dexamethasone (Dex) 2.25 2.64 2.50 MAP 2.36 2.79 2.63 Montelukast (Mon) 1.50 2.38 2.00 MAP + dexamethasone 2.50 2.63 2.75 MAP + montelukast 1.80 2.00 2.00

Example 4

(77) Air Pouch Model: Topical Administration Versus Oral Administration

(78) An experiment was carried out, essentially as described in Example 1 (except that, in this case C57BL/16 mice were supplied by Nanjing Biomedical Research Institute of Nanjing University (NBRI)).

(79) The experiment was conducted in which mice were treated by administering test samples or vehicle in accordance with Table 13 below. All substances listed in Table 13 were administrated topically by directly injecting into the air pouches, except for Mon (oral) (Mon-O), which was intragastric administrated.

(80) TABLE-US-00013 TABLE 13 Timing of Treatment No. Drug concn. (before carrageenan Group Mice (mg/mL) Dose/mouse injection) Control (air 6 Normal 4.5 mg (of NaCl) n/a injection only) saline Model 8 Normal 4.5 mg (of NaCl) 1 hour (carrageenan saline injection) Positive 6 0.4 200 μg 1 hour (dexamethasone) Mon-O 8 0.4 200 μg 1 hour Mon-T 8 0.4 200 μg 1 hour

(81) The volume of the exudate and the weight of air pouch wall (means t SD) for each group are tabulated in Table 14 below.

(82) TABLE-US-00014 TABLE 14 Group Exudate volume (mL) Pouch Wall Weight (g) Control 3.48 ± 0.12 0.68 ± 0.07 Model 3.76 ± 0.05 0.72 ± 0.09 Positive  3.3 ± 0.15 0.63 ± 0.09 Mon-O 3.69 ± 0.14 0.68 ± 0.08 Mon-T 3.54 ± 0.09 0.65 ± 0.06

(83) The total cell count and classification (×10.sup.6/mL) is tabulated in Table 15 below.

(84) TABLE-US-00015 TABLE 15 Group Leukocyte Neutrophil Monocyte Control 0.09 8.75 0.00 Model 3.34 260.03 74.17 Positive 3.49 285.97 62.53 Mon-O 2.66 216.89 46.81 Mon-T 2.19 170.98 42.10

(85) Histological specimens were analyzed and an inflammation score, an activity score, an edema score and a fibroblast proliferation score were estimated largely as described in Example 1 above, and are shown in Table 16 below.

(86) TABLE-US-00016 TABLE 16 Inflammation Edema Activity Group Score Score score Sum Control 0.33 1.17 0.00 1.50 Model 2.17 2.67 1.17 6.00 Positive 1.83 1.67 0.67 4.17 Mon-O 2.33 2.17 1.67 6.17 Mon-T 2.00 1.50 1.00 4.50

(87) In the Control group, the pouch wall showed a small amount of scattered inflammatory cells infiltration, mild edema and no obvious neutrophil infiltration.

(88) In the Model group, the pouch wall showed moderate to severe chronic inflammatory cell infiltration in the whole layer, moderate to severe edema and scattered neutrophil infiltration.

(89) In the Positive control group, the pouch wall showed moderate chronic inflammatory cell infiltration, moderate interstitial edema and a small amount of neutrophil infiltration.

(90) In the Mon-O group, the pouch wall showed moderate to severe chronic inflammatory cell infiltration in the whole layer, and moderate to severe edema.

(91) In the Mon-T group, the pouch wall showed moderate chronic inflammatory cell infiltration, mild to moderate edema, and a small amount of neutrophil infiltration.

(92) The ELISA test results of the exudate for TNF-α, IL-6, IL-10 and IFN-γ always showed stable, high responses with a similar variation tendency. The results for TNF-α, IL-6 and IFN-γ (±SD) are tabulated in Table 17 below and shown graphically in FIG. 4.

(93) TABLE-US-00017 TABLE 17 Group TNF-α IL-6 IFN-γ Control 8.42 ± 5.65 36.95 ± 14.22 18.34 ± 33.65 Model 143.60 ± 65.56  487.97 ± 231.99 718.51 ± 331.12 Positive 65.08 ± 7.70  428.05 ± 267.27 388.83 ± 130.02 Mon-O 146.3 ± 70.74 500.25 ± 292.84 557.95 ± 231.86 Mon-T 80.37 ± 61.72 312.05 ± 189.23 568.19 ± 316.70

Example 5

(94) Air Pouch Model: Combination of Montelukast

(95) Following the same protocol as described in Example 1 above, a further comparative experiment was conducted in which mice (supplied by Changzhou Cvens Experimental Animal Co. Ltd.) were treated by administering test samples or vehicle in accordance with Table 18 below.

(96) TABLE-US-00018 TABLE 18 Timing of Treatment No. Drug concn. (before carrageenan Group Mice (mg/mL) Dose/mouse injection) Control (air 6 Normal 4.5 mg (of NaCl) n/a injection only) saline Model 6 Normal 4.5 mg (of NaCl) 1 hour (carrageenan saline injection) Positive 6 0.4 200 μg 1 hour (dexamethasone) MAP 6 3 1.5 mg 1 hour MON-O 6 1 200 μg 1 hour MON-T 6 0.4 200 μg 1 hour MAP + Mon 6 3 (MAP) 1.5 mg (MAP) 1 hour (MAP) (oral) 1 (Mon) 200 μg (Mon) 1.5 hour (Mon) (MMO) MAP + Mon 6 6 (MAP) 1.5 mg (MAP) 1 hour (MAP) (topical) 0.8 (Mon) 200 μg (Mon) 1.5 hour (Mon) (MMT)

(97) The volume of the exudate and the weight of air pouch wall (means±SD) for each group are tabulated in Table 19 below.

(98) TABLE-US-00019 TABLE 19 Group Exudate volume (mL) Pouch Wall Weight (g) Control 3.33 ± 0.10 0.58 ± 0.06 Model 3.73 ± 0.12 0.81 ± 0.14 Positive 3.60 ± 0.14 0.72 ± 0.09 MAP 3.62 ± 0.12 0.66 ± 0.06 MON-O 3.58 ± 0.15 0.67 ± 0.10 MON-T 3.68 ± 0.15 0.66 ± 0.13 MMO 3.53 ± 0.08 0.61 ± 0.12 MMT 3.60 ± 0.09 0.66 ± 0.07

(99) The total cell count and classification (×10.sup.6/mL) is tabulated in Table 20 below.

(100) TABLE-US-00020 TABLE 20 Group Leukocyte Neutrophil Monocyte Lymphocyte Control 0.04 0.01 0.00 0.01 Model 5.06 0.28 0.10 4.57 Positive 2.48 0.22 0.05 2.08 MAP 2.71 0.23 0.10 2.24 MON-O 2.38 0.21 0.07 1.98 MON-T 3.82 0.29 0.17 3.22 MMO 1.53 0.21 0.06 1.16 MMT 1.65 0.19 0.11 1.27

(101) Histological specimens were analyzed and an inflammation score, an activity to score, an edema score and a fibroblast proliferation score were estimated as described in Example 1 above, and are shown in Table 21 below.

(102) TABLE-US-00021 TABLE 21 Fibroblast Inflammation Activity Edema proliferation Group score score Score Sum score Control 0.67 0.50 1.00 3.00 0.83 Model 3.00 2.00 1.50 6.67 0.17 Positive 1.50 0.83 1.67 4.00 0.00 MAP 2.67 1.50 1.67 6.00 0.17 MON-O 1.83 1.00 1.00 4.17 0.33 MON-T 2.17 1.00 1.50 5.50 0.83 MMO 1.83 1.00 0.67 4.83 1.33 MMT 2.00 1.17 0.83 4.33 0.33

(103) In the Control group, the pouch wall showed small amount of scattered inflammatory cell infiltration, rare neutrophil infiltration, mild edema, and mild fibrous tissue proliferation.

(104) In the Model group, the pouch wall showed severe inflammatory cell infiltration in the whole layer (a large number of neutrophils), severe interstitial edema, and occasionally fibrous tissue proliferation.

(105) In the Positive control group, the pouch wall showed mild to moderate inflammatory cell infiltration, occasionally infiltration in the whole layer, severe interstitial edema, and no fibrous tissue formation.

(106) In the MAP (1.5 mg) group, the pouch wall showed severe inflammatory cell infiltration in the whole layer, moderate to severe interstitial edema, and occasionally fibrous tissue proliferation.

(107) In the MON-O group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and occasionally fibrous tissue proliferation.

(108) In the MON-T group, the pouch wall showed moderate to severe inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild to moderate interstitial edema, and lightly fibrous tissue proliferation.

(109) In the MMO group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and obvious fibrous tissue proliferation.

(110) In the MMT group, the pouch wall showed moderate inflammatory cell infiltration, with a small amount of neutrophil infiltration, mild interstitial edema, and occasionally fibrous tissue proliferation.

(111) The ELISA test results of the exudate showed PGE2 and 5-HT levels that were below detection ranges. The results for TNF-α. IL-6, IL-10 and IFN-γ (±SD) are tabulated in Table 22 below and are shown graphically in FIG. 5.

(112) TABLE-US-00022 TABLE 22 Group TNF-α IL-6 IL-10 IFN-γ Control 8.62 ± 0.10  2.3 ± 0.00 24.32 ± 0.00  −5.00 ± 3.00  Model 674.65 ± 244.26 34050 ± 13984 72.13 ± 33.13 .sup. 167 ± 44.48 Positive  71.31 ± 111.19 3649 ± 1764 52.33 ± 11.60 2879 ± 1267 MAP 55.45 ± 11.51 10411 ± 6948  65.79 ± 12.64 1842 ± 1129 Mon (oral) 39.41 ± 17.62 8453 ± 9758 52.59 ± 7.22  2495 ± 711  Mon (topical)  90.73 ± 109.02 2528 ± 1538 68.18 ± 15.81 1690 ± 725  MMO 27.08 ± 6.14  2459 ± 681  37.06 ± 11.91 1534 ± 685  MMT 27.87 ± 8.49   330 ± 0.00 56.24 ± 13.73 2362 ± 1233

Example 6

(113) Acute Wound Model

(114) 6-8 weeks old male C57BL/6 mice were supplied by Changzhou Cvens Experimental Animal Co. Ltd. Prior to any experiments being conducted, mice were housed under standardized conditions (at a constant temperature or 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.

(115) 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 on the back was shaved by a baby hair shaver and depilated with cream. Wiped the skin area and sterilized with 75% alcohol 2 times;

(116) EMS skin biopsy punch (Electron Microscopy Sciences, P.O. Box 550, 1560 industry Road, Hatfield, Pa. 19440) with 0.6 mm diameter was used to make two round wounds on each side of the midline of the back. Full thickness skin was removed and the depth reached the fascia. The wounds left open without suture.

(117) Different drugs were administrated topically at 20 μL/wound, once daily from Day 0 to Day 12. The model group was given same amount of normal saline. There were 6 groups including 54 mice in this experiment shown in Table 23. Recombinant Human Epidermal Growth Factor (rhEGF, Shanghai Haohai Biological Technology Co. Ltd, Shanghai, China) was purchased and prepared according to the manufacturer's instructions. Lyophilized rhEGF powder (100000 IU/vial) was dissolved in 20 mL of normal saline to make a solution with a 5000 IU/mL concentration. The working dose of rhEGF for this experiment was 100 IU/wound. Montelukast sodium (“Mon”; MedChemExpress, MCE China, Shanghai, China) was obtained in a powder form and was dissolved in ultrapure water to obtain solutions with concentrations as described in Table 22 (L, M and H indicate low, medium and high montelukast doses, respectively)

(118) TABLE-US-00023 TABLE 23 GROUP Day 3 Day 7 Day 12 Total MEANING DOSAGE Control 3 4 2 9 saline / Model 3 4 2 9 Model + saline / rhEGF 3 4 2 9 Model + rhEGF 100 IU/wound Mon L 3 4 2 9 Model + Mon L 50 μg/wound Mon M 3 4 2 9 Model + Mon M 100 μg/wound Mon H 3 4 2 9 Model + Mon H 200 μg/wound

(119) Wounds were wrapped up with transparent dresser after drug administration.

(120) 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).

(121) The unhealed wound area was expressed as a percentage of the original wound area:
A.sub.t/A.sub.0×100%,
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.

(122) Samples were taken at Day 3, Day 7 and Day 12 post wound-wound infliction. The mice were sacrificed and wound tissue of an area 1.8 cm×2.5 cm was removed. Half of the tissue was preserved in 10% neutral buffered formalin (Nanchang Rain Dew Experimental Equipment Co., Ltd., Nanchang, Hubei Provence, China), and analyzed by histological embedding in paraffin wax, sectioning and staining. HE and Masson stained paraffin sections were analyzed under an optical microscope. Skin regeneration, fibroblastic proliferation, collagen regeneration scores and inflammation scores were estimated.

(123) 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.

(124) 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). Tissue necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), vascular endothelial growth factor (VEGF) and hydroxyproline (Hyp) were assayed. The ELISA kits were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

(125) The effect of montelukast on wound healing were show in Table 24 below (where the numbers indicate the percentage of remaining wound area versus the initial wound at various time points in the different groups (±SD; n=5), and in FIG. 6.

(126) TABLE-US-00024 TABLE 24 Group Model rhEGF Mon L Mon M Mon H Day 2 90.58 ± 6.48 85.73 ± 10.29 78.41 ± 9.51  79.97 ± 11.79 76.11 ± 6.40 Day 4  79.97 ± 12.74 68.15 ± 16.47  57.59 ± 11.54 55.09 ± 8.83 48.60 ± 6.49 Day 6 48.79 ± 9.10 30.85 ± 1.87  27.53 ± 3.76 28.51 ± 1.03 29.13 ± 6.16 Day 8 24.76 ± 6.29 16.93 ± 4.03  14.08 ± 2.85 13.87 ± 3.51 14.59 ± 3.04 Day 12 10.66 ± 4.13 5.02 ± 3.03  1.15 ± 2.29  2.15 ± 3.73  2.48 ± 3.43

(127) The above data show that montelukast had a significant effect on wound healing. At Day 4, the improvement rate in the Mon H group was about 39%.

(128) The wound HYP (μg/mg) content in samples, which is an indicator of collagen regeneration, is shown in Table 25 below and in FIG. 7.

(129) TABLE-US-00025 TABLE 25 Mean Hyp content (μg/mg tissue weight) (n = 4) SD Control 4.43 5.06 5.50 5.03 5.01 0.44 Model 5.40 2.82 3.89 4.25 4.09 1.06 RhEGF 6.54 4.89 5.39 5.45 5.57 0.70 Mon L 4.65 4.41 6.71 6.05 5.46 1.11 Mon M 4.94 4.18 6.80 4.98 5.23 1.11 Mon H 4.62 5.83 3.99 4.64 4.77 0.77

Example 7

(130) Diabetic Wound Model

(131) A similar experiment with essentially the same protocol to that described in Example 6 above was carried out on 8 to 12 week-old male db/db mice (C57BL/KsJ-db/db, with a body weight of 35-45 g/mouse; supplied by Changzhou Cvens Experimental Animal Co. Ltd.).

(132) An EMS skin biopsy punch with a 0.8 mm diameter was used to make wounds.

(133) Different drugs were administrated topically at 20 μL/wound, once daily from Day 0 to Day 18. The model group was given same amount of normal saline. There were 3 groups including 39 mice in this experiment shown in Table 26 below.

(134) TABLE-US-00026 TABLE 26 GROUP Day 6 Day 12 Day 18 Tot. MEANING DOSAGE Model 4 8 1 13 Model + saline / rhEGF 4 8 1 13 Model + rhEGF 100 IU/each Mon 4 8 1 13 Model + Mon 100 μg/each

(135) Samples were taken on Day 6, Day 12 and Day 18 post-wound inflictions.

(136) The effect of montelukast on wound healing are show in FIG. 8.

(137) The result for VEGF content (pg/g) at Day 6 are tabulated in Table 27 below and are shown graphically in FIG. 9.

(138) TABLE-US-00027 TABLE 27 Model rhEGF Mon VEGF Mean 3.15 4.55 5.47 SD 0.97 1.60 1.31

(139) The result showed that montelukast has increased the VEGF production in the wounded tissue for about 74% In comparison with that in model group.

(140) Histological specimens were analyzed and skin regeneration, fibroblast proliferation, collagen regeneration scores (Masson score) and inflammation scores were estimated as follows.

(141) The HE and Masson stained slices were observed under an optical microscope and were scored (1, 2 or 3 points) according to the following criteria. Skin regeneration score was 1 point when the newly generated skin covered area was no more than one third of the wound area; the score was 2 points when the newly generated skin covered an area greater than one third but less than two thirds of the wound area; and the score was 3 points when the newly generated skin covered area was at least two thirds of the wound area.

(142) Fibroblast proliferation was scored as the following criteria:

(143) TABLE-US-00028 Fibroblast proliferation score Collagen fiber hyperplasia Score Myofibroblastic proliferation 1 proliferation of fibrous tissue 2 Collagen appeared between the fibrous tissues 3

(144) Inflammation was scored as the following criteria:

(145) TABLE-US-00029 Inflammation score Inflammatory cell infiltration Score Occasional 0 Sporadic 1 Manifest 2 Diffuse infiltration 3

(146) Collagen deposition score criterion for the Masson stained sample were as follows. A comparison was made with normal tissue. No clear blue staining was given 0 points; blue fiber appearing in a scattered pattern was scored as 1 points; if more blue fiber appeared, this was scored as 2 points, and a diffuse blue colour was given 3 points.

(147) The results of histopathological analysis are shown in FIG. 10.

(148) The results shown that montelukast had the effects on accelerating wound healing, especially at an early stage following wound infliction.

Example 8

(149) Montelukast Cream I

(150) A cream based on montelukast sodium was made, consisting of the following components: montelukast sodium (200 mg; Arromax Pharmatech Co., Ltd, Suzhou, China), stearic acid (2 g), glycerin monostearate (2 g), hexadecanol (2 g), glycerin (5 g) and sodium hydroxide (0.25 g) (all Sinopharm Chemical Reagent Co. Ltd, Shanghai, China); ammonium acryloyldlmethyltaurateNP copolymer (0.13 g; Clariant Chemical (Guangzhou) Co., Ltd., Guangzhou, China); phenoxyethanol (0.3 g) and ethylhexyl glycerin (0.1 g) (both Shanghai Rayson Chemicals Co., Ltd., Shanghai, China); and purified water (88.42 g).

(151) The stearic acid, glycerin monostearate and hexadecanol were mixed and heated to 85° C. with stirring until the mixture melted completely.

(152) The ammonium acryloyldimethyltaurate/VP copolymer, purified water and sodium hydroxide were mixed with stirring at 85° C. to form a homogenous colloidal suspension.

(153) Montelukast sodium, glycerin, phenoxyethanol and ethylhexyl glycerin were then combined with stirring until the montelukast completely dissolved.

(154) The copolymer/water mixture was added to the stearic acid-containing mixture, which was emulsified by stirring quickly for five minutes using emulfication equipment. The resultant emulsion was cooled to 55° C., the montelukast-containing mixture was added with mixing. The resultant mixture was allowed to cool to room temperature to obtain the finished product.

Example 9

(155) Montelukast Cream II

(156) A similar procedure to that described in Example 8 above was followed to produce a second cream.

(157) On this occasion, stearic acid (2 g) and glycerin monostearate (2 g) were heated first to 85° C. with stirring until the mixture melted completely; then polysorbate 80 (1 g; Sinopharm Chemical Reagent Co. Ltd) and sodium hyaluronate (0.4 g; Shandong Focuschem Biotech Co., Ltd, Shandong, China) were mixed together with purified water at 85° C. until the sodium hyaluronate was fully swelled.

(158) These two mixtures were combined, emulsified and stirred, followed by cooling to 55° C., and then the same montelukast-containing mixture (as described in Example 8 above) was added with mixing. Cooling to room temperature gave the finished product.

Example 10

(159) Montelukast Cream III

(160) A similar procedure to that described in Example 8 above was followed to produce a third cream.

(161) On this occasion, sorbitan monostearate (0.6 g), polysorbate 80 (1 g), hexadecanol (2 g), caprylic/capric triglyceride (5 g), paraffin oil (4 g), glyceryl monostearate (2 g) and petroleum jelly (5 g) (all Sinopharm Chemical Reagent Co. Ltd) were mixed and heated to 85° C. with stirring until the mixture melted completely. Then, ammonium acryloyldimethyltaurate/VP copolymer (0.13 g), glycerin (4 g), trehalose (0.5 g; Sinopharm Chemical Reagent Co. Ltd) were mixed together with purified water (70.5 g) at 85° C. until the various components dissolved.

(162) A third mixture was made comprising montelukast sodium (200 mg), polyethylene glycol 200 (4 g; Sinopharm Chemical Reagent Co. Ltd), phenoxyethanol (0.3 g) and ethylhexyl glycerin (0.1 g), with stirring until the montelukast dissolved.

(163) The copolymer/water mixture was added to the sorbitan monostearate-containing mixture, followed by emulsification, stirring, and cooling to 55° C. Silicon oil (0.5 g; Sinopharm Chemical Reagent Co. Ltd) was then added, along with the montelukast-containing mixture, with mixing. Cooling to room temperature gave the finished product.

Example 11

(164) Montelukast Ointment I

(165) An ointment was made by first dissolving polyethylene glycol 3350 (21.3 g; Sinopharm Chemical Reagent Co. Ltd) in polyethylene glycol 400 (58.5 g; Sinopharm Chemical Reagent Co. Ltd) by heating to 60° C. with stirring. Montelukast sodium (200 mg) was dissolved in polyethylene glycol 400 (20.0 g) with stirring.

(166) After cooling the first solution to 40-50° C., the second solution was added to it with stirring and mixing for 5 to 10 minutes. Cooling to room temperature gave the finished product.

Example 12

(167) Montelukast Ointment II

(168) A further ointment was made as described in Example 11 with the exception that polyethylene glycol 4000 (21.3 g, Sinopharm Chemical Reagent Co. Ltd) was dissolved in the same amount of polyethylene glycol 400 (58.5 g).

Example 13

(169) Montelukast Aerosol Powder Spray

(170) An aerosol powder spray based was made by adding montelukast sodium (200 mg) to ethanol (70 g; Sinopharm Chemical Reagent Co. Ltd) with stirring until it dissolved completely.

(171) Porous starch (28 g; Sinopharm Chemical Reagent Co. Ltd) was added to the resultant solution to provide a suspension, which was subsequently filled into an aerosol spray bottle.

Example 14

(172) Montelukast Dressing

(173) A dressing was made by coating evenly the montelukast sodium ointment, prepared as described in Example 12 above, on a gauze (Shanghai Health Materials Factory Co., Ltd, Shanghai, China) with a flat plate and cooling to room temperature.

Example 15

(174) Swollen Ear Model

(175) 12 Health male BALB/c mice with 6-8 weeks age and average body weight of 18-25 g supplied by Changzhou Cvens Experimental Animal Co. Ltd. were housed and cared for about 1 week prior to the experiment. The housing temperature was 25-27° C. with 74% humidity, with alternating 12 hour periods of light and darkness, and free access to food and water. The mice were randomly divided into 4 groups as described in Table 28, with 3 mice in each group.

(176) TABLE-US-00030 TABLE 28 Total amount Drug administration of drugs Group Drug concentration on right ear (μg/mouse) Model / xylene / Blank / xylene + cream / without API Dex Cream 10 μg/μL xylene + Dex cream 400 Mon Cream 5 mg/g xylene + Mon cream 500

(177) The montelukast cream (Mon Cream) is the cream described in Example 8 above. Blank cream was made following the same procedure, with our adding montelukast to the mixture. Dexamethasone cream (Dex Cream) was also made using the same procedure, replacing montelukast with dexamethasone (160 mg; Shanghai Aladdin Bio-Chem Technology Co. Ltd.).

(178) The left ear of each mouse was used as an autologous control. The right ear of each mouse was treated with the various treatments. First 20 μL of xylene (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) was applied to the right ear of each mouse, both on the inside and the outside. The ear started to swell after about 4 minutes. Then, 40 μL of the various creams were applied to the right ear of each group. The mice were put back in their cages.

(179) The mice were sacrificed by cervical dislocation after 40 minutes. The left and right ears were cut off. A skin pouch (Electron Microscopy Sciences, Hatfield, Pa., USA) with a diameter of 8 mm was used to take a piece of the ear from the same site of both ears. The weights were recorded and the swelling rates were calculated as follows:
Swelling rate=(right ear weight−left ear weight)/left ear weight×100%
and the results showed in Table 29.

(180) TABLE-US-00031 TABLE 29 Weight (g) Group Left ear Right ear Difference Swelling rate Model 0.013 0.025 0.012 0.923 97.4% 0.012 0.021 0.009 0.750 0.012 0.027 0.015 1.250 Blank 0.013 0.025 0.012 0.923 83.5% 0.012 0.023 0.011 0.917 0.015 0.025 0.010 0.667 Dex 0.013 0.019 0.006 0.462 .sup. 48% Cream 0.013 0.022 0.009 0.692 0.014 0.018 0.004 0.286 Mon 0.012 0.018 0.006 0.500 38.1% Cream 0.014 0.020 0.006 0.429 0.014 0.017 0.003 0.214

(181) The results showed that montelukast cream significantly reduced xylene-induced swelling in mouse ears. The effect was even better than that of the well-known topical antiinflammatory medication, dexamethasone.

Example 16

(182) Rat Scald Model I

(183) 6-8 weeks old male Sprague Dawley rats with average body weights of between 250 and 300 g were supplied by Changzhou Cvens Experimental Animal Co. Ltd. Prior to any experiments being conducted, rats were housed under standardized conditions (at a constant temperature or 22 t 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.

(184) A self-made mould was used. A section of bamboo was taken, and the top ⅓ along its length was removed. An oval hole was made in the bottom of the bamboo with dimensions of 2.5×4.5 cm, allowing the back of rat to be exposed to hot water. The exposed skin area was about 10-13% of its total body surface area (TBSA):
TBSA (cm.sup.2)=K×W×⅔,
where K is the correction factor (surface area to body weight/shape) constant for the given species (9.1 for the rat), and W is the body weight (in g) of the rat.

(185) The hair on the back of the rats was shaved. 10% chloral hydrate were injected to anesthetize the rats (350 mg/kg; 3.5 mL/kg).

(186) 6 rats were divided into 2 groups of 3 rats. After anesthesia, the rats were fixed inside the mould. Adhesive tape was used to fix the limbs and the abdomen, and the bamboo was tightly attached to the back, exposing the skin through the hole. Boiling hot water at 100° C. was applied to the rats' skin for 12 seconds.

(187) Montelukast cream (prepared as described in Example 8 above) was subsequently administered to the scald wounds in the test group, and a blank cream (the same cream with no active ingredient) was administered in the control group. Treatments were thereafter administered once a day for ten days.

(188) Ten days after scalding, hairs were observed to be growing in the scald area of the test group. There was no sign of hair growth in the scald area of the control group. This is shown in FIG. 11, which shows that montelukast cream helps to recover skin function after burning.

Example 17

(189) Rat Scald Model II

(190) 8 Sprague Dawley rats were divided into two groups of 4. Scald wounds were applied in essentially the same way as that described in Example 16 above, by applying (or not applying, as appropriate) hot water at the temperature, and for the times, shown in Table 30 below.

(191) In one of the groups, immediately after scalding, montelukast cream (only; prepared as described in Example 8 above was applied to the scald wounds. 1 g montelukast cream was used for each wound. For the other group, immediately after scalding, 0.5 mL of MAP saline solution with a concentration of 4.9 mg/mL was applied to the surface of the scald wound. After a short wait of a few minutes to allow the solvent to evaporate, a further 0.5 mL of the same MAP solution was applied, this was repeated 3 times in total. After this, 1 g of the same montelukast cream was applied on top of the MAP.

(192) TABLE-US-00032 TABLE 30 Group Meaning Treatment Drug administration Mon Montelukast Unscalded 1 g of montelukast cream 85° C. 15 s cream applied 85° C. 10 s to the surface 75° C. 15 s of the scald wound MM MAP coating, Unscalded MAP solution applied 3 times, then 85° C. 15 s then 1 g of montelukast montelukast 85° C. 10 s cream applied to the cream 75° C. 15 s surface of the scald wound

(193) Samples were taken from each rat at time zero, and then 1, 2, 3, 6 and 8 hours after scalding, using an EMS skin biopsy punch with an 8 mm diameter. The full thickness skin was removed and weighed.

(194) Then, tissue was cut into small pieces. 9 mL of normal saline was added to 1 g of tissue, which was then ground using a Tissuelyser at 55 Hz for 60 seconds, followed by centrifuging at 8000 rpm for 10 minutes at 4° C. and the supernatant collected for HPLC analysis.

(195) The HPLC conditions were as follows: column: Angilent ZORBAX Eclipse XDB-C8 (4.6×250 mm, 5 μm); buffers: A: 0.1% TFA (trifluoroacetic acid, (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) in water, B: 0.1% TFA in acetonitrile (Merck, Darmsladt, Germany); gradient: 0-20 minutes: 49% B; flow rate: 1 mL/min; detection wavelength: 388 nm; sample volume: 20 μL.

(196) The amount of montelukast contained in each of the samples was detected and calculated based on peak areas. A standard curve of peak area versus montelukast amount was used for the calculation.

(197) The results for the unscalded, intact skin samples are shown in Table 31 below and in FIG. 12.

(198) TABLE-US-00033 TABLE 31 Montelukast Sampling Peak Montelukast Wet concentrations Time (h) area amount (g) weight (g) (μg/g) 0 756 0 0.069 0 1 171262 2.53 0.069 36.69 2 381470 5.44 0.080 67.98 3 52374 0.89 0.066 13.44 6 53490 0.90 0.058 15.56 8 244228 3.54 0.064 55.32

(199) The results showed that on intact skin, the concentration peak was one hour later in MM group than that in Mon group. This indicated that MAP can delay the penetration of montelukast into the skin.

(200) The results for scald groups were showed in FIG. 13.

(201) Compared to intact skin, it seemed that the skin became harder to penetrate after scalding. In both Mon and MM groups, peaks of montelukast showed at 2 hours. However, the amount of montelukast contained in the samples decreased significantly over time in the MM group compared to the Mon group. These results confirm that MAP can the delay the penetration of montelukast, Irrespective of the skin conditions.

Example 18

(202) Clinical Example of Montelukast Cream to Treat Edema in a Burns Patient

(203) Montelukast cream was prepared according to the same method described in Example 8 above. The concentration of montelukast sodium in the cream was 5 mg/g.

(204) The enrolled subject in the study was a patient with second degree burns in the head and neck area (FIG. 14; photograph (a)). Montelukast cream was applied on the left side of the patient's face, starting 20 hours after the burn injuries were sustained. Silver sulfadiazine ointment, a well-recognized topical burns treatment, was applied to the right side of the face as a comparison. Both drugs were applied twice per day.

(205) As shown in FIG. 14, one day after treatment (photograph (b)), the edema in the left side of the face was clearly being reduced faster and more extensively than on the right side. This relative improvement continued over the next few days (2 days (photograph (c)), 3 days (photograph (d)), and 14 days (photograph (e)), after treatment).

(206) This experiment showed that montelukast cream effectively treats edema in acute inflammation stage of burns and was more effective that silver sulfazidine.

Example 19

(207) Clinical Example of Montelukast Cream to Treat Burns Scar Itch

(208) Montelukast cream (with a 5 mg/g concentration of montelukast sodium) was prepared according to the same method described in Example 8 above.

(209) The enrolled subjects in the study were burn patients with old cicatricial scars. Symptoms Included Irregular shaped hypertrophic scars with pain and itching. Ambient temperature changes or emotional agitation is known to aggravate pain and/or itching.

(210) Subjects were required to use the cream in the morning and the evening after cleansing the scar.

(211) All of the enrolled subjects felt that itching was relieved within 8 minutes of the first use, demonstrating that montelukast cream may be used to treat burn scar itch.

Example 20

(212) Clinical Example of Montelukast Cream to Treat Melanin Related Skin Diseases I

(213) Montelukast cream (with a 5 mg/g concentration of montelukast sodium) was prepared according to the same method described in Example 9 above.

(214) The enrolled subjects in the study were patients with melanin related skin diseases. Symptoms included pigmentation after burns or acne eruption. Subjects were required to use the cream in the morning and evening after facial cleansing.

(215) All of the enrolled subjects felt that the skin colour improved after 2 weeks of use, demonstrating that montelukast cream may be used to treat melanin pigmentation and/or melanin-related skin diseases.

Example 21

(216) Clinical Example of Montelukast Cream to Treat Melanin Related Skin Diseases II

(217) Montelukast cream (with a 5 mg/g concentration of montelukast sodium) was prepared according to the same method described in Example 9 above.

(218) The enrolled subject in the study was a patient with melanin related skin diseases. Symptoms included chloasma and malar rash.

(219) A laser pre-treatment was employed to attempt to remove the worst of the symptoms. The subject was required to use the cream in the morning and evening after facial cleansing.

(220) The subject felt that her skin colour improved after 2 weeks after 2 weeks of use. This is the case, as can be seen from FIG. 15, in which both sides of the face are seen before the laser treatment (photographs (a)), 7 days after treatment with montelukast cream commenced (photographs (b)), and 14 days after treatment with montelukast cream (photographs (c)). This demonstrates that montelukast cream may be used to treat melanin pigmentation and/or melanin-related skin diseases.

Example 22

(221) Clinical Example of Montelukast Cream to Treat Steroid Dependent Dermatitis

(222) Montelukast cream (with a 5 mg/g concentration of montelukast sodium) was prepared according to the same method described in Example 9 above.

(223) The enrolled subject in the study was a patient with steroid-dependent dermatitis. Symptoms included skin redness, papules, itching and swelling.

(224) The subject was required to use the cream in the morning and evening after facial cleansing.

(225) The use of the cream was observed to relieve itching in the subject within 5 minutes. As can be seen from FIG. 16 (before (photograph (a)), and after (photograph (b)), treatment), montelukast cream may be used as an adjuvant therapy for steroid-dependent dermatitis.