Conjugates of montelukast and peptides

Abstract

There is provided a peptide-containing compound that comprises a peptide component which is an amino acid sequence of from 2 to 45 (e.g. from 6 to 15) amino acids, which peptide component is covalently bonded to one or more compounds of formula I: ##STR00001##
wherein: R.sup.1 is selected from the group consisting of —C(CH.sub.3).sub.2OH, —COCH.sub.3, —C(CH.sub.3)═CH.sub.2 and —C(CH.sub.3).sub.2H; and n is 0, 1 or 2,
as well as regioisomers, stereoisomers, and pharmaceutically- or cosmetically-acceptable salts of said peptide-containing compound. The compound of formula I is preferably montelukast, montelukast styrene or hydrogenated montelukast styrene. The peptide-containing compound is particularly useful in the treatment of conditions characterised by inflammation, including wounds, hemorrhoids, burns, psoriasis, acne, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, chronic obstructive pulmonary disease, inflammatory bowel disease (such as ulcerative colitis). Compounds of the invention are also useful in the treatment of idiopathic pulmonary fibrosis.

Claims

1. A peptide-containing compound that comprises: a peptide component comprising the amino acid sequence X-Pro-Y-Z (SEQ ID NO: 5) wherein: X represents 1 to 2 amino acid residues each independently selected from the group consisting of Ala and Lys; Y is selected from the group consisting of Ser and pSer; Z represents 1 to 7 amino acid residues each independently selected from the group consisting of Tyr, pTyr, 3Hyp, 4Hyp, Thr, pThr, DOPA and Lys; and at least one of the Ala and/or Lys residues is covalently bonded to one or more compounds of formula I by an amide bond formed by reaction of the carboxylic acid group in formula I and one or more free amino groups in the Ala and/or Lys residues: ##STR00006## wherein: the squiggly bond represents cis or trans orientation relative to the carbon-carbon double bond; R.sup.1 is selected from the group consisting of —C(CH.sub.3).sub.2OH, —COCH.sub.3, —C(CH.sub.3)═CH.sub.2 and —C(CH.sub.3).sub.2H; and n is 0, 1 or 2, as well as regioisomers, stereoisomers, and pharmaceutically- or cosmetically-acceptable salts of said peptide-containing compound.

2. The peptide-containing compound as claimed in claim 1, wherein the peptide component consists of a sequence of 6 to 12 amino acids.

3. The peptide-containing compound as claimed in claim 1, wherein the peptide component is covalently bonded to at least one compound of formula I through a primary amide linkage formed at the N-terminal of said peptide component.

4. The peptide-containing compound as claimed in claim 1, wherein the peptide component is covalently bonded to at least one compound of formula I through a primary amide linkage formed through at least one free —NH.sub.2 group in one or more amino acids in the sequence that is not the N-terminal amino acid.

5. The peptide-containing compound as claimed in claim 1, wherein at least one of the amino acids in the peptide component is lysine.

6. The peptide-containing compound as claimed in claim 5, wherein at least about 5% of the amino acids are lysine.

7. The peptide-containing compound as claimed in claim 1, wherein at least about 5% of the amino acids of the peptide component contain an aromatic group.

8. The peptide-containing compound as claimed in claim 7, wherein at least about 10% of the amino acids contain an aromatic group.

9. The peptide-containing compound as claimed in claim 7, wherein the amino acids that contain an aromatic group are selected from the group consisting of tyrosine and 3,4-dihydroxyphenylalanine.

10. The peptide-containing compound as claimed in claim 1, wherein the peptide component consists of the amino acid sequence X-Pro-Y-Z (SEQ ID NO: 5).

11. The peptide-containing compound as claimed in claim 1, wherein the peptide component comprises or consists of the amino acid sequence:
G.sup.1-Lys-Pro-G.sup.2-T-Hyp-G.sup.3-Lys  (SEQ ID NO: 7), wherein G.sup.1 is absent or represents Ala; G.sup.2 is selected from the group consisting of Ser and pSer; T is selected from the group consisting of DOPA, Tyr and pTyr; Hyp is selected from the group consisting of 3Hyp and 4Hyp; G.sup.3 represents 1 to 4 amino acid residues each independently selected from the group consisting of Tyr, pTyr, 3Hyp, 4Hyp, Thr, pThr and DOPA.

12. The peptide-containing compound as claimed in claim 11, wherein the peptide component comprises or consists of the amino acid sequence:
Lys-Pro-G.sup.2-T-Hyp-G.sup.3-Lys  (SEQ ID NO: 8).

13. The peptide-containing compound as claimed in claim 11, wherein G.sup.1 represents Ala.

14. The peptide-containing compound as claimed in claim 11, wherein the peptide component comprises or consists of the amino acid sequence:
Ala-Lys-Pro-G.sup.2-T-Hyp-Hyp-Thr-G.sup.4-Lys  (SEQ ID NO: 9), wherein G.sup.4 is selected from the group consisting of Tyr, pTyr, 3Hyp, 4Hyp, Thr, pThr and DOPA.

15. The peptide-containing compound as claimed in claim 11, wherein G.sup.2 represents Ser.

16. The peptide-containing compound as claimed in claim 11, wherein T represents Tyr.

17. The peptide-containing compound as claimed in claim 14, wherein G.sup.4 represents Tyr or DOPA.

18. The peptide-containing compound as claimed in claim 17, wherein the peptide component comprises or consists of the amino acid sequence:
Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys  (SEQ ID NO: 4);
Ala-Lys-Pro-pSer-Tyr-Hyp-Hyp-Thr-DOPA-Lys  (SEQ ID NO: 11);
Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys  (SEQ ID NO: 12); and
Ala-Lys-Pro-pSer-Tyr-Hyp-Hyp-Thr-Tyr-Lys  (SEQ ID NO: 13).

19. The peptide-containing compound as claimed in claim 18, wherein the peptide component comprises or consists of the amino acid sequence:
Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys  (SEQ ID NO: 12).

20. The peptide-containing compound as claimed in claim 11, wherein G.sup.3 represents -V.sup.1-Thr-Tyr-V.sup.2-, wherein V.sup.1 is covalently bonded to Hyp and V.sup.2 is covalently bonded to Lys, and V.sup.1 and V.sup.2 are, independently, either absent or represent one Hyp residue.

21. The peptide-containing compound as claimed in claim 20, wherein -V.sup.1-Thr-Tyr-V.sup.2- represents -Hyp-Thr-Tyr-, -Hyp-Thr-Tyr-Hyp-, or -Thr-Tyr-Hyp-.

22. The peptide-containing compound as claimed in claim 11, wherein the peptide component comprises or consists of the amino acid sequence:
Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Hyp-Lys  (SEQ ID NO: 18);
Ala-Lys-Pro-pSer-Tyr-Hyp-Hyp-Thr-DOPA-Hyp-Lys  (SEQ ID NO: 19);
Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Hyp-Lys  (SEQ ID NO: 20);
Ala-Lys-Pro-pSer-Tyr-Hyp-Hyp-Thr-Tyr-Hyp-Lys  (SEQ ID NO: 21);
Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-DOPA-Hyp-Lys  (SEQ ID NO: 22),
Ala-Lys-Pro-pSer-Tyr-Hyp-Thr-DOPA-Hyp-Lys  (SEQ ID NO: 23);
Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-Tyr-Hyp-Lys  (SEQ ID NO: 24); and
Ala-Lys-Pro-pSer-Tyr-Hyp-Thr-Tyr-Hyp-Lys  (SEQ ID NO: 25).

23. The peptide-containing compound as claimed in claim 22, wherein the peptide component comprises or consists of the amino acid sequence:
Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-Tyr-Hyp-Lys  (SEQ ID NO: 24).

24. The peptide-containing compound as claimed in claim 1, wherein one or more compounds of formula I is/are covalently bonded through an N-terminal Ala or Lys residue and/or a C-terminal Lys residue.

25. The peptide-containing compound as claimed in claim 1, wherein one or two compounds of formula I is/are covalently bonded to the peptide component.

26. The peptide-containing compound as claimed in claim 25, wherein one compound of formula I is covalently bonded to the peptide component.

27. The peptide-containing compound as claimed in claim 1, wherein R.sup.1 is selected from the group consisting of —C(CH.sub.3).sub.2OH, —C(CH.sub.3)═CH.sub.2 and —C(CH.sub.3).sub.2H; and/or n is 0 in the one or more compounds of formula I.

28. The peptide-containing compound as claimed in claim 1, wherein the one or more compounds of formula I is selected from the group consisting of montelukast, montelukast styrene, or hydrogenated montelukast styrene.

29. The peptide-containing compound according to claim 1, wherein the peptide component comprises the amino acid sequence of
Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-Tyr-Hyp-Lys  (SEQ ID NO: 24), the compound of formula I is montelukast, and wherein the montelukast is covalently bonded to the N-terminal Ala residue.

30. The peptide-containing compound as claimed in claim 12, wherein the peptide component comprises or consists of the amino acid sequence:
Lys-Pro-Ser-Tyr-Hyp-DOPA-Lys  (SEQ ID NO: 14); or
Lys-Pro-Ser-pTyr-Hyp-DOPA-Lys  (SEQ ID NO: 15).

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

32. The pharmaceutical formulation as claimed in claim 31 wherein the pharmaceutically- or cosmetically-acceptable adjuvant, diluent or carrier is a topical adjuvant, diluent or carrier for topical administration.

33. The pharmaceutical formulation as claimed in claim 32, which is in the form of a gel, a spray, a cream, an ointment or a dry powder.

34. The pharmaceutical formulation as claimed in claim 32, which further includes an antiinflammatory agent.

35. A kit of parts comprising components: (A) a pharmaceutical formulation as defined in claim 32; and (B) a pharmaceutical formulation including an antiinflammatory agent in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, wherein components (A) and (B) are each provided in a form for co-administration together or separately as different formulations.

36. A method of treatment of idiopathic pulmonary fibrosis, comprising administering the peptide-containing compound of claim 1, or a pharmaceutically- or cosmetically-acceptable salt thereof, or a formulation comprising said compound or salt, to a patient in need of such treatment.

37. The method as claimed in claim 36, wherein the method comprises administering compound(s) by oral, inhalation, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal, pulmonary or anorectal delivery.

38. The method as claimed in claim 37, wherein the method comprises pulmonary administering by way of a spray comprising a powder aerosol or an aqueous mist for atomization.

39. The method as claimed in claim 38, wherein the spray is liquid comprising a water (aerosol) spray and excipients comprise one or more of a viscosity modifier, a sugar, an emulsifier, a buffering agent, an alcohol and a preservative.

40. The method as claimed in claim 38, comprising an inhalation device selected from the group consisting of a pressurized metered-dose inhaler, a dry powder inhaler, a soft mist inhaler, and a nebulizer.

Description

(1) The invention is illustrated by the following examples, in which, for various compounds, including compounds of the invention,

(2) FIG. 1 shows the swelling rates in a mouse ear swelling model;

(3) FIG. 2 shows Hyp content (and therefore level of recovery), and

(4) FIG. 3 shows vascular endothelial growth factor and transforming growth factor-beta 1 levels in wound tissues, in an acute wound mouse model;

(5) FIG. 4 shows unhealed wound rate,

(6) FIG. 5 shows skin regeneration, and

(7) FIG. 6 shows fibroblast proliferation scores, in a diabetic wound mouse model;

(8) FIG. 7 shows the ratio of remaining wound area compared to the initial wound,

(9) FIGS. 8 to 11 show the results of histopathological analyses in terms of various markers of wound healing (skin regeneration, fibroplastic proliferation, inflammation and Masson stain, respectively), and

(10) FIG. 12 shows levels of edema, in different groups in a further diabetic wound model;

(11) FIGS. 13 and 17 both show the effect of compounds of the invention on edema caused by acute inflammation in a further mouse ear swelling models;

(12) FIG. 14 shows the unhealed wound rate in a further acute wound mouse model;

(13) FIG. 15 shows a comparison between compounds of the invention and known antiinflammatory steroids in a mouse ear swelling model; and

(14) FIG. 16 shows FIG. 1 IL-1β content in lung tissues for different compounds of the invention in a mouse lung injury model.

EXAMPLES

Example 1

(15) Synthesis of Montelukast Styrene-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys (i.e. Montelukast Styrene Covalently Bonded to Amino Acid SEQ ID No: 12 at the N-Terminus)

(16) To synthesise 3 mmol of peptide SEQ ID No: 12, the following procedure was followed.

(17) Fmoc-Lys-Boc-Wang resin (9.15 g, GLS180322-41301, GL Biochem, Shanghai, China) was loaded into a glass reaction column.

(18) Methylene chloride (DCM, 200 mL; Shandong Jinling Chemical Industry Co Ltd, Shandong, China) was added to the column and allowed to soak the resin for about half an hour. The DCM was then removed by vacuum filtration.

(19) The resin was washed 3 times with N,N-dimethylformamide (DMF, 200 mL; Shandong Shitaifeng Fertilizer Industry Co Ltd, Shandong, China).

(20) A 20% piperidine solution in DMF (200 mL; Shandong Shitaifeng Fertilizer Industry Co Ltd, Shandong, China) and was added as deprotection solution and reacted for 20 minutes. The solution was then removed by vacuum filtration and the column was washed with DMF six times.

(21) Fmoc-Tyr(tBu)-OH (4.14 g; GLS170916-36901, GL Biochem, Shanghai, China) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, 2.89 g; GLS170805-00705, GL Biochem, Shanghai, China) were added to the resin. DMF (150 mL) was added to the reaction column, followed by N,N-diisopropylethylamine (DIPEA, 2.33 g; Suzhou Highfine Biotech Co. Ltd, Jiangsu, China). A colour reaction was detected in the resin after 30 minutes, indicating the reaction was complete. The solvent was removed by vacuum filtration.

(22) The above coupling steps were repeated to couple the remaining amino acids in the same amounts (by mols): 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.

(23) Finally, montelukast (5.47 g; MedChemExpress, MCE China, Shanghai, China) was added to the resin. The liquid was then drained after 15 minutes and the column washed with DMF, DCM and methanol, 3 times each, respectively.

(24) 91.5 mL (i.e. 10 mL per gram of resin) 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 peptide-containing compound. The side chains were also deprotected during cleavage. After cleavage the solid support was removed by filtration and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether and lyophilized to yield 600 mg of crude title compound.

(25) 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 11.035 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).

(26) The crude compound was desalted using an anion exchange resin, analysed and freeze-dried. Approximately 50 mg of purified peptide was obtained after purification, which was re-tested for confirmation.

(27) MS: m/z 866.90 [M+2H].sup.2+.

(28) Based on the characterising data available and presented herein, it is understood that the compound prepared by way of this example is that identified above as the title compound. Otherwise, the compound that is prepared in Example 1 is a compound of the invention in which, in the compound of formula I, n is 0 and the compound of formula I is covalently bonded to amino acid SEQ ID No: 12 at the N-terminus. In any event, the compound of Example 1 is referred to hereinafter as “Compound A”.

Example 2

(29) Synthesis of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys(Montelukast Styrene) (i.e. Montelukast Styrene Covalently Bonded to Amino Acid SEQ ID No: 4 on the C-Terminus

(30) A similar procedure to that described in Example 1 above was employed starting with Fmoc-Lys(Dde)-OH (CAS No.: 150629-67-7) on a Wang resin.

(31) A 25% piperidine solution in DMF (200 mL; Shandong Shitaifeng Fertilizer Industry Co Ltd, Shandong, China) was added to remove the protective Fmoc group.

(32) The second protected amino acid Fmoc-DOPA(acetonide)-OH was added, along with TBTU and DIPEA, until the reaction was completed.

(33) The above coupling steps were repeated to couple the remaining amino acids in the same amounts (by mols): 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.

(34) The peptidyl-resin was placed in a flask and treated with 2% hydrazine monohydrate in DMF (25 mL/g). The flask was stoppered, and mixture was left to stand at room temperature for 3 minutes. The resin was then washed with DMF. Montelukast (5.47 g; MedChemExpress, MCE China, Shanghai, China) was added to the resin, along with TBTU and DIPEA and the mixture was reacted for 1 hour.

(35) The protected peptidyl-resin was treated with 91.5 mL of lysate (a mixture of 95% TFA, 2.5% water, and 2.5% Tis) for 1 hour. After cleavage the solid support was removed by filtration and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether and lyophilized to yield approximately 600 mg of crude title compound.

(36) After purification, 50 mg of pure product was obtained.

(37) MS: m/z 875.75 [M+2H].sup.2+.

(38) Based on the characterising data available and presented herein it is understood that the compound prepared by way of this example is that identified above as the title compound. Otherwise, the compound that is prepared in Example 2 is a compound of the invention in which, in the compound of formula I, n is 0 and the compound of formula I is covalently bonded to amino acid SEQ ID No: 4 on the C-terminus Lys. In any event, the compound of Example 2 is referred to hereinafter as “Compound B”.

Example 3

(39) Synthesis of Montelukast Styrene-Ala-Lys-Pro-pSer-Tyr-Hyp-Hyp-Thr-Tyr-Lys (i.e. Montelukast Styrene Covalently Bonded to Amino Acid SEQ ID No: 13 at the N-Terminus)

(40) A similar procedure to that described in Example 4 below was employed starting with Fmoc-Lys(Dde)-OH (CAS No.: 150629-67-7) on a Wang resin.

(41) A 25% piperidine solution in DMF (200 mL; Shandong Shitaifeng Fertilizer Industry Co Ltd, Shandong, China) was added to remove the protective Fmoc group.

(42) The second protected amino acid Fmoc-Tyr(tBu)-OH (GLS170916-36901, GL Biochem, Shanghai, China) was added, along with ByBOP and DIPEA, until the reaction was completed.

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

(44) The peptidyl-resin was placed in a flask and treated with 2% hydrazine monohydrate in DMF (25 mL/g). The flask was stoppered, and the mixture was left to stand at room temperature for 3 minutes. The resin was then washed with DMF. Montelukast (1.8 g;

(45) MedChemExpress, MCE China, Shanghai, China) was added to the resin, along with TBTU and DIPEA and the mixture was reacted for 1 hour.

(46) Lysate (10 mL per gram of resin), which was comprised of 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (Tis), was added to immerse the resin-bounded peptide-containing compound. The side chains were also deprotected during cleavage. After cleavage the solid support was removed by filtration and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether and lyophilized to yield 1.4 g of crude peptide.

(47) 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 with LCMS (analysis column model: GS-120-5-C18-BIO, 4.6*250 mm).

(48) The crude compound was desalted using an anion exchange resin, analysed and freeze-dried, which was re-tested for confirmation.

(49) After purification, 98 mg of pure product was obtained (a yield rate of approximately 7% from the crude product).

(50) MS: m/z 907.3 [M+2H].sup.2+.

(51) Based on the characterising data available and presented herein it is understood that the compound prepared by way of this example is that identified above as the title compound. Otherwise, the compound that is prepared in Example 3 is a compound of the invention in which, in the compound of formula I, n is 0 and the compound of formula I is covalently bonded to amino acid SEQ ID No: 13 at the N-terminus. In any event, the compound of Example 3 is referred to hereinafter as “Compound C”.

Example 4

(52) Synthesis of Ala-Lys(montelukast styrene)-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys(Montelukast Styrene) (i.e. Two Molecules of Montelukast Styrene Covalently Bonded Via the Lys Residues to Amino Acid SEQ ID No: 12)

(53) To synthesise 1 mmol peptide SEQ ID No: 12, the following procedure was followed.

(54) Fmoc-Lys(Dde)-OH (CAS No.: 150629-67-7) on a Wang resin (3 g, GL Biochem, Shanghai, China) was loaded into a glass reaction column.

(55) Methylene chloride (DCM, 60 mL; Shandong Jinling Chemical Industry Co Ltd, Shandong, China) was added to the column and allowed to soak the resin for about half an hour. The DCM was then removed by vacuum filtration.

(56) The resin was washed 3 times with N,N-dimethylformamide (DMF, 60 mL; Shandong Shitaifeng Fertilizer Industry Co Ltd, Shandong, China).

(57) A 20% piperidine solution in DMF (30 mL; Shandong Shitaifeng Fertilizer Industry Co Ltd, Shandong, China) and was added as deprotection solution and reacted for 20 minutes. The solution was then removed by vacuum filtration and the column was washed with DMF six times.

(58) Fmoc-Tyr(tBu)-OH (1.4 g; GLS170916-36901, GL Biochem, Shanghai, China) and benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (ByBOP, 1.56 g; Suzhou Highfine Biotech Co. Ltd, Jiangsu, China) were added to the resin, followed by DIPEA (1 mL; Suzhou Highfine Biotech Co. Ltd, Jiangsu, China). A colour reaction was detected in the resin after 30 minutes, indicating the reaction was complete. The solvent was removed by vacuum filtration.

(59) The above coupling steps were repeated to couple the remaining amino acids in the same amounts (by mols): 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(Dde)-OH and Boc-Ala-OH.

(60) The peptidyl-resin was placed in a flask and treated with 2% hydrazine monohydrate in DMF (25 mL/g). The flask was stoppered, and the mixture was left to stand at room temperature for 3 minutes. The resin was then washed with DMF. Montelukast (3.6 g; MedChemExpress, MCE China, Shanghai, China) was added to the resin, along with TBTU and DIPEA and the mixture was reacted for 1 hour.

(61) Lysate (30 mL; 10 mL per gram of resin), which was comprised of 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (Tis), was added to immerse the resin-bounded peptide-containing compound. The side chains were also deprotected during cleavage. After cleavage the solid support was removed by filtration and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether and lyophilized to yield 1.6 g of crude peptide.

(62) 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 with LCMS (analysis column model: GS-120-5-C18-BIO, 4.6*250 mm).

(63) The crude compound was desalted using an anion exchange resin, analysed and freeze-dried, which was re-tested for confirmation. 20 mg of purified peptide (90% to 95% purity) was obtained from 1.6 g of crude peptide.

(64) MS: m/z 762.2 [M+3H].sup.3+.

(65) Based on the characterising data available and presented herein it is understood that the compound prepared by way of this example is that identified above as the title compound. Otherwise, the compound that is prepared in Example 4 is a compound of the invention in which, in the compound of formula I, n is 0 and the compound of formula I is covalently bonded to amino acid SEQ ID No: 12 via the Lys residues. In any event, the compound of Example 4 is referred to hereinafter as “Compound D”.

Example 5

(66) Mouse Ear Swelling Model I

(67) 35 healthy male BALB/c mice of 6-8 weeks of age and average body weight of 18-25 g supplied by Changzhou Cvens Experimental Animal Co. Ltd. were housed and cared for about for 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 7 groups as described in Table 1 below, with 5 mice in each group

(68) The left ear of each mouse was used as autologous control. The right ear of each mouse was treated by various different treatments, as summarised in Table 1 below. 20 μL of xylene (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China) was applied to the right ear of each mouse, both inside and outside. The ear started to swell in about 4 minutes. Then, 0.08 g of each study treatments or vehicles were applied to the right ears in each group. The mice were put back into their cages.

(69) A cream based on montelukast sodium was made (Mon), 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 acryloyldimethyltaurate/VP 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).

(70) The stearic acid, glycerin monostearate and hexadecanol were mixed and heated to 85° C. with stirring until the mixture melted completely. The ammonium acryloyldimethyltaurate/VP copolymer, purified water and sodium hydroxide were mixed with stirring at 85° C. to form a homogenous colloidal suspension. Montelukast sodium, glycerin, phenoxyethanol and ethylhexyl glycerin were then combined with stirring until the montelukast completely dissolved.

(71) The copolymer/water mixture was added to the stearic acid-containing mixture, which was emulsified by stirring quickly for five minutes using emulsification 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.

(72) Dexamethasone cream (DEX) was made using the same procedure, except that montelukast was replaced by 0.4 mg of dexamethasone (Shanghai Aladdin Bio-Chem Technology Co., LTD, Shanghai, China).

(73) A gel including Compound A (“A gel”) was made, which consisted of the following components: 0.5 g of Compound A powder (obtained from GL Biochem, Shanghai, China; prepared as described in Example 1 above), methyl cellulose (2.2 g; Shandong Guangda Technology Development Co., Ltd., ShanDong, China), glycerin (11 g) and propanediol 11 g (both Sinopharm Chemical Reagent Co. Ltd.), and purified water (75.3 g).

(74) The methyl cellulose and water were mixed together and stirred until to a homogeneous colloidal suspension was formed. Then, the Compound A powder, glycerin and propanediol were added to the methyl cellulose/water mixture, and the resultant mixture quickly stirred for 5 minutes to obtain the finished product.

(75) A gel based on Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys (Compound 1 (SEQ ID No: 12); 1.5 g; obtained as a powder from GL Biochem, Shanghai, China, and made by essentially the same process as that described in Example 1 above, without coupling montelukast at the end) was made by the same process as described above for Compound A (“1 gel”).

(76) Vehicle-1 in Table 1 below is the cream base without active ingredients. Vehicle-2 in Table 1 below is the gel base without active ingredients. Both were made using the same procedure as described above, without adding active ingredient.

(77) TABLE-US-00016 TABLE 1 Total Drug amount of Drug administration drugs Group concentration on right ear (μg/mouse) Model / Xylene / Vehicle-1 / xylene + cream / without API Vehicle-2 / xylene + gel / without API DEX 10 μg/μL xylene + Dex 400 cream Mon 5 mg/g xylene + Mon 500 cream 1 gel 1.5 mg/g xylene + 120 Compound 1 gel A gel 0.5 mg/g xylene +  40 Compound A gel

(78) 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 2 below and FIG. 1.

(79) TABLE-US-00017 TABLE 2 Vehicle Vehicle Dex Mon Compound Compound Model 1 2 Cream Cream 1 A 97.4  95%   97%    45%    38%    61.5%  55%     0.03  0.2     0.35     0.19     0.25     0.17     0.06   

(80) The above results show that both test compounds reduce the xylene induced swelling.

Example 6

(81) Acute Wound Model I

(82) 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.

(83) General anesthesia was induced using intraperitoneal 3% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd.; 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.

(84) EMS skin biopsy punch (Electron Microscopy Sciences, P.O. Box 550, 1560 Industry Road, Hatfield, Pa. 19440) with an 18 mm diameter was used to make a round wound on the midline of the back. Full thickness skin was removed, and the depth reached the fascia. The wounds left open without suture.

(85) Different drugs were administrated topically at 50 μL/wound, once daily from Day 0 to Day 7. The model group was given same amount of normal saline. There were 7 groups including 56 mice in this experiment, as shown in Table 3 below.

(86) 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 1285 IU/wound.

(87) Compound B was obtained as a powder from GL Biochem, and was prepared as described in Example 2 above). Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (Compound 2; SEQ ID No: 4) was obtained as a powder from GL Biochem, and was prepared essentially as described in Example 2 above, but without coupling montelukast at the end. Powders were stored at −20° C. and dissolved in saline at the concentrations indicated in Table 3 below (L and H indicate low and high doses, respectively).

(88) TABLE-US-00018 TABLE 3 Dose per Group Meaning Number wound per day Control C57 mice without / / wounds Model C57 mice with 8 Normal wounds/normal Saline saline rhEGF C57 mice with 8 1285 IU wounds/EGF 2 H C57 mice with 8 77.15 μg wounds/Compound 2 high dose 2 L C57 mice with 8 3.09 μg wounds/Compound 2 low dose B H C57 mice with 8 112.53 μg wounds/Compound B high dose B L C57 mice with 8 4.50 μg wounds/Compound B low dose

(89) Hydroxyproline (Hyp) is a nonproteinogenic amino acid, found in collagen, containing approximately 12-14% Hyp by mass. Hyp content in tissue hydrolysates is thus a direct measure of the amount of collagen present, and the Hyp content presented in wound tissue indicates directly the level of recovery. The Hyp content in each group and Day 4 and Day 7 after commencement of treatment are shown in FIG. 2.

(90) The results showed that test compounds improve Hyp content in all treatment groups. The lower dose of Compound 2 and Compound B both show effects at early stage (Day 4 (D4)). The higher doses of both test compounds also show accelerated effects on Hyp production at Day 7 (D7).

(91) Vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGF-β1) play prominent roles in wound healing process. VEGF and TGF-β1 are often co-expressed in tissues in which angiogenesis occurs. The content of these two factors in wound tissues were also detected and are shown in FIG. 3.

(92) The results showed that the two peptides could stimulate the production of VEGF and TGF-β1.

Example 7

(93) Diabetic Wound Model I

(94) 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 (Changzhou Cvens Experimental Animal Co. Ltd.).

(95) An EMS skin biopsy punch with a 18 mm diameter was used to make wounds.

(96) Different drugs were administrated topically at 50 μL/wound, once daily from Day 0 to Day 12. The model group was given same amount of normal saline. There were 7 groups including 52 mice in this experiment shown in Table 4 below.

(97) TABLE-US-00019 TABLE 4 Group Meaning Dose/day Concentration Control mice without wounds / / Model mice with wounds treated Normal / with normal saline Saline rhEGF mice with wounds treated 1285 IU 25.7 IU/μL with rhEGF 1 L mice with wounds treated 3.05 μg 0.061 μg/μL with Compound 1 (low dose) A M mice with wounds treated 22.3 μg 0.446 μg/μL with Compound A (medium dose) Mon L mice with wounds treated 40 μg 0.8 μg/μL with montelukast (low dose) 1 + mice with wounds treated 1.525 μg + 25 μg/μL + Mon with half dose of Compound 1 20 μg 25 μg/μL and half dose of montelukast

(98) 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 Institute of Health, USA).

(99) 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.

(100) The unhealed wound rate was showed in FIG. 4. The result showed that Compound A had the best effect on improving the wound recovery, and was better that of the combination of Compound 1 with montelukast.

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

(102) 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.

(103) The skin regeneration scores were showed in FIG. 5.

(104) Fibroblast proliferation was scored as the following criteria, and are presented in FIG. 6.

(105) TABLE-US-00020 Fibroblast proliferation score Collagen fiber hyperplasia Score Myofibroblastic proliferation 1 proliferation of fibrous tissue 2 Collagen appeared between the 3 fibrous tissues

(106) The pathological analysis results showed that the Compound A and Compound 1 could promote the skin regeneration and fibroblast proliferation. The conjugate Compound A was slightly better, especially in relation to the fibroblast proliferation score.

Example 8

(107) Diabetic Wound Model II

(108) A similar experiment with essentially the same protocol to that described in Example 7 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 (Changzhou Cvens Experimental Animal Co. Ltd.).

(109) Different concentrations of Compound A and Compound 1 (“A” and “1” respectively, as indicated in the Table 5 below) were prepared in substantially the same way as described in Examples 6 and 7 above. Medium and low dosages of montelukast sodium (“Mon” in Table 5 below; MedChemExpress, MCE China, Shanghai, China) were dissolved in ultrapure water to obtain solutions with concentrations as described in Table 5 below (L, M and H indicate low, medium and high doses, respectively). In view of the low solubility of montelukast in water, the high dose montelukast test sample was prepared by dissolving montelukast in 100% ethanol, and then adding ultrapure water to form a solution with a concentration of 20 μg/μL in 20% ethanol.

(110) Different drugs were administrated topically at 50 μL/wound, once daily from Day 0 to Day 12, as show in in Table 5 below. The control group did not have wound inflicted.

(111) TABLE-US-00021 TABLE 5 Drug Dose/day concentration Group Meaning (μg) (μg/μL) Control without wounds / / Model normal saline Normal / Saline 1 H Compound 1, 5× 76.25 1.525 higher dose 1 M Compound 1, 15.25 0.305 medium dose 1 L Compound 1, 5× 3.05 0.061 lower dose A H Compound A, 5× 111.51 2.2302 higher dose A M Compound A, 22.3 0.446 medium dose A L Compound A, 5× 4.46 0.0892 lower dose Mon H Mon, 5× higher 1000 20 dose (20% ethanol) Mon M Mon, medium dose 200 4 Mon L Mon, 5× lower 40 0.8 dose

(112) The model group was given same amount of normal saline. There were 8 mice in each group. 4 mice were in the control group. The skin pieces taken during wound creation were used as the samples at Day 7 for the control group.

(113) The effects of drugs on wound healing in the first 12 days were showed in Table 6 below and in FIG. 7, which show the ratio of remaining wound area of initial wound in different groups (±SD in the case of Table 6).

(114) TABLE-US-00022 TABLE 6 Model Mon H Mon M Mon L A H A M A L 1 H 1 M 1 L Mean D1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 D2 0.982 0.938 0.956 0.919 0.884 0.971 1.058 1.051 0.991 0.860 D4 0.958 0.845 0.891 0.802 0.829 0.952 0.864 0.821 0.895 0.753 D6 0.867 0.707 0.734 0.623 0.692 0.690 0.773 0.718 0.786 0.593 D8 0.614 0.508 0.543 0.449 0.508 0.479 0.527 0.486 0.552 0.467 D10 0.547 0.401 0.432 0.409 0.447 0.386 0.471 0.478 0.490 0.433 D12 0.429 0.277 0.359 0.292 0.361 0.238 0.368 0.368 0.344 0.306

(115) The data show that the low dose of Compound 1 prompted wound healing in the earlier stages after wound infliction and that the medium dose of Compound A prompted better effects at the later stages.

(116) Histological specimens were analyzed as described in Example 7 above, except that the 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.

(117) The results of histopathological analysis are shown in FIGS. 8 to 11 and show that all treatment groups accelerated wound healing, especially the medium dose of Compound A, which showed a significant promoting effect on collagen deposition.

(118) The levels of edema in the different groups were also evaluated by histopathological analysis and the results are shown in FIG. 12. The data show that the medium dose of Compound A gave the best result.

Example 9

(119) Mouse Ear Swelling Model II

(120) A similar experiment with essentially the same protocol to that described in Example 5 above was carried out on 35 healthy male BALB/c mice. The mice were randomly divided into 7 groups as described in Table 7 below, with 5 mice in each group.

(121) Compounds A, B, C and D were all obtained from GL Biochem Ltd.

(122) In Table 7 below, hydrogels of Compounds A, B, C and D were prepared consisting of the amounts of active ingredients described, along with methyl cellulose (2.5%), propanediol (11%), glycerol (11%), acetic acid (pH regulator; 0 to 0.5 g). All excipients were obtained from Sinopharm Chemical Reagent Co. Ltd.). The gels were made up with water for injection.

(123) Dexamethasone acetate cream (5 mg DEX in 10 g cream, Fuyuan Pharmaceutical Co. Ltd., Anhui, China) was used as positive control.

(124) 40 μL of the various treatments drugs were applied to the right ear of each group.

(125) TABLE-US-00023 TABLE 7 Drug Total amount Drug administration of drugs Group concentration on right ear (μg/mouse) Model / Xylene / Dex 10 μg/μL xylene + 400 cream dexamethasone cream A gel 0.5 mg/g xylene + 20 Compound A gel B gel 0.39 mg/g xylene + 16 Compound B gel C gel 0.52 mg/g xylene + 20.8 Compound C gel D gel 0.66 mg/g xylene + 26.4 Compound D gel

(126) The results are shown in FIG. 13. The conjugates all had a very good effect on eliminating the edema caused by acute inflammation.

Example 10

(127) Acute Wound Model II

(128) A similar experiment with essentially the same protocol to that described in Example 6 above was carried out on 6 to 8 week-old male C57BL/6 mice,

(129) EMS skin biopsy punch with a 12 mm diameter was used to make two round wounds on the midline of the back. The two circles were tangential to each other and the skin between the circles was cut along the upper and lower tangents. Scissors were used to trim the wound. The wound was an oval shape.

(130) Different drugs were administrated topically at 50 μL/wound, once daily from Day 0 to Day 7. The model group was given same amount of normal saline. There were 10 groups including 80 mice in this experiment shown in Table 8 below.

(131) TABLE-US-00024 TABLE 8 Dose Group Meaning Number (/wound/day) Control C57 mice without / / wounds Model C57 mice with 8 Normal Saline wounds/normal saline rhEGF C57 mice with 8 1285 IU wounds/EGF A C57 mice with 8 25 μg wounds/Compound A B C57 mice with 8 19.64 μg wounds/Compound B C C57 mice with 8 26.15 μg wounds/Compound C D C57 mice with 8 32.93 μg wounds/Compound D 1 C57 mice with 8 17 μg wounds/Compound 1 Mon C57 mice with 8 8.5 μg wounds/montelukast 1 + mon C57 mice with 8 17 μg + 8.5 μg wounds/Compound 1 and montelukast

(132) Photographs were taken for each wound every other day from Day 0, and the unhealed wound area expressed, as described in Example 6 above.

(133) The unhealed wound rate is shown in FIG. 14. The results show that all four conjugates (A, B, C and D) had comparable effects on promoting the wound healing compared to the other groups.

Example 11

(134) Cream Formulation

(135) Sorbitan stearate (0.6 g), polysorbate-80 (1 g), hexadecanol (2 g), octanoic acid/decanoic acid glyceride (5 g), liquid paraffin (4 g), monostearate glyceride (2 g) and vaseline (5 g) (all Sinopharm Chemical Reagent Co. Ltd.) were mixed together, with stirring and heating to 85° C. until the mixture completely melted.

(136) Methyl cellulose (0.5 g), glycerin (4 g), trehalose (0.5 g), polyethylene glycol 200 (4 g), phenoxyethanol (0.3 g) and ethylhexyl glycerol (0.1 g) (all Sinopharm Chemical Reagent Co. Ltd.) were mixed together with purified water (69.45 g), with stirring and heating to 85° C. to give a homogeneous colloidal suspension.

(137) The two mixtures obtained above where mixed together with silicone oil (0.5 g) with quick stirring using emulsification equipment over 5 minutes. The resultant emulsion was cooled to 55° C.

(138) Compound A (50 mg; see Example 1 above) was dissolved in purified water (1 g) and then combined with the emulsion mixture with stirring until it was uniform. The resultant mixture was allowed to cool to room temperature to obtain the finished product.

Example 12

(139) Spray Formulation I

(140) Hydroxypropyl methylcellulose (HPMC; 0.1 g), hydroxyethyl cellulose (0.1 g), glucose (5 g), phenoxy alcohol (0.5 g) (all Sinopharm Chemical Reagent Co., Ltd.) and purified water (93.25 g) were stirred together with heating to 85° C. to provide a homogeneous colloidal suspension. The mixture was then cooled to room temperature.

(141) Compound A (50 mg; see Example 1 above) was dissolved in 1 g of purified water. This solution was added to the colloidal mixture. Uniform mixing gave the finished product.

Example 13

(142) Spray Formulation II

(143) A second spray was prepared using substantially the same procedure as that described in Example 12 above by adding the same aqueous solution of Compound A to a colloidal mixture made from slightly more HPMC and hydroxyethyl cellulose (0.2 g of each), along with the other components in the same amounts and 94.05 g of purified water.

Example 14

(144) Gel Formulation I

(145) This formulation was obtained using essentially the same procedure as that described in Examples 12 and 13 above, by adding the same aqueous solution of Compound A to a colloidal mixture made from 1 g each of HPMC and hydroxyethyl cellulose, along with the other components in the same amounts and 91.45 g of purified water.

Example 15

(146) Gel Formulation II

(147) A second gel was obtained using essentially the same procedure as described in Examples 12 to 14 above by adding the same aqueous solution of Compound A to a colloidal mixture made from 0.5 g of HPMC and 1.5 g of hydroxyethyl cellulose, along with the other components in the same amounts and 91.45 g of purified water.

Example 16

(148) Gel Formulation III

(149) A third gel was obtained using substantially the same procedure as described in Examples 12 to 15 above. Methyl cellulose (2.2 g) and propanediol 11 g (both Sinopharm Chemical Reagent Co., Ltd.), and glycerol (11 g) were first mixed with 74.75 g of purified water. Adding in the same aqueous solution of Compound A to the resultant colloidal mixture provided the finished product.

Example 17

(150) Clinical Example I—Allergic Rhinitis Patient

(151) A 45 year old female patient with allergic rhinitis had periodic snivels and nasal obstruction.

(152) Spray formulation I (see Example 12 above), packed in a nasal spray bottle, was administered to each nostril separately, 2 to 3 times per day for 5 days.

(153) The patient was instructed not to use her existing medication (oral montelukast sodium and budesonide) from the first dose of the spray formulation.

(154) The snivels and nasal obstruction were apparently relieved as of the second administration. The patient found that she did not feel the need to take montelukast sodium orally over the course of the administration of the new formulation. The budesonide had been found to have lost efficacy within a couple of months of use.

Example 18

(155) Clinical Example II—Burns Patient Symptom Relief

(156) A male patent had a feeling of severe itch on his medial upper arm during the course of recovery from severe second degree burns with a VAS of 4 to 5.

(157) Spray formulation I (see Example 12 above) was administrated to the wound and itch was relieved within one minute.

Example 19

(158) Clinical Example III—Wounded Patient Symptom Relief

(159) A patient was operated on and had severe pain from the surgical incision afterwards.

(160) Spray formulation I (see Example 12 above) was administrated to the incision and pain was relieved within one minute.

Example 20

(161) Clinical Example IV—Allergic Rhinitis Patients

(162) 38 Subjects enrolled in this study with seasonal and/or persistent allergic rhinitis. A majority of the subjects suffered with the disease for years and tried treatment with several medications, including steroids. The subjects were instructed not to use their existing medication from the first dose of the spray formulation.

(163) Spray formulation I (see Example 12 above), packed in a nasal spray bottle, was administered to each nostril separately, 2 times per day for 7 days.

(164) 4 subjects did not complete the study. The feedback collected from the remaining 34 subjects is shown in Table 9 below.

(165) Symptom incidence rate equals the number of subjects with the particular symptom, divided by the total number of subjects. Effective rate equals the number of subjects whose symptom was relieved, divided by the total number of subjects with the particular symptom.

(166) TABLE-US-00025 TABLE 9 Symptom Starting incidence Effective time of Duration of Symptoms rate(%) rate(%) efficacy efficacy Stuffy nose 88.23 66.57 Within 2-5 hours (30/34) (20/30) 10 min Running nose 100 67.65 Within 2-4 hours (34/34) (23/34) 30 min Itchy nose 82.35 60.71 Within Half day (28/34) (17/28)  1 min Sneeze 94.12 62.5 Within Half day (32/34) (20/30)  1 min Itchy eyes 73.53 68 Within 2-4 hours (25/34) (17/25) 10 min

(167) The patient's found that the spray formulation was easy to administer and gave rise to no irritation. Nasal congestion was quickly relieved, along with the persistent sneezing and itchiness of the eyes. 16 of the subjects were checked by a clinician after using the spray for 7 days. 50% of the patients showed less turbinate swelling, 68.75% had less nasal secretions and 43% demonstrated reduced mucosal edema. No side effects were reported.

Example 21

(168) Clinical Example V—Sore Throat Relieved by Atomization Inhalation

(169) An 80-year-old Caucasian male had feelings associated with the onset of the common cold. Symptoms included an itchy and achy throat, and nasal congestion. 5 mL of spray formulation I (see Example 12 above) was loaded into a portable nebulizer (Feellife Medical INC, Sehnzhen, China). The suction nozzle of the nebulizer was placed in the mouth and the device turned on. The treatment lasted approximately 10 minutes. The inhalation was carried out only once. The following morning, all the symptoms of a cold had gone.

Example 22

(170) Clinical Example VI—Operation Pain Relief in Burn Patient

(171) A patient with large, deep second-degree burns covering the whole of his back was hospitalised in the burn department of Beijing Jishuitan Hospital. He was treated for severe burns and suffered from what he described as unbearable operation pain every time his dressing was changed, with a VAS of 7 to 9.

(172) Spray formulation I (see Example 12 above), packed in a spray bottle, was sprayed directly onto the surface of the burn wound. After 5 minutes, the dressing was removed and changed for a new one. After use of the spray, the operation pain was reduced by about two thirds, according to the clinician's evaluation.

Example 23

(173) Clinical Example VII—Operation Pain Relief in Laser Surgery Patients

(174) Two subjects were tested in this study. The subjects received skin pigmentation removal surgery by fractional laser treatment.

(175) Spray formulation I (see Example 12 above), packed in a spray bottle, was sprayed onto the surface of operation area. After 10 minutes, the laser operation started. Following use of the spray, the operating pain was reduced by about one third, according to the clinician's evaluation.

(176) It is also normal for subjects that receive such laser surgery experience a burning pain for approximately 30 minutes after the treatment. However, in this study, the subjects did not feel any burning pain afterwards.

Example 24

(177) Clinical Example VIII—Fever and Cough Relief

(178) A 5-year-old boy caught a cold and developed a bad cough. His body temperature reached 38° C. during the night and he complained of a sore throat.

(179) A spray formulation I (see Example 12 above), packed in a spray bottle, was administered as an oral spray, 4 times per day. The symptoms of fever and sore throat disappeared the following day. The cough disappeared after 3 days.

Example 25

(180) Clinical Example IX—Contact Dermatitis Relief

(181) A 53-year-old female had contact dermatitis on her neck. Rashes and an itchiness appeared upon the wearing a metal necklace.

(182) Spray formulation I (see Example 12 above), packed in a spray bottle, was sprayed on the affected area. The feeling of itchiness was relieved within 5 minutes. After 2 doses (one in the evening and one the following morning), all symptoms had disappeared.

Example 26

(183) Clinical Example X—Cold Relief

(184) The patients were a 42-year-old female and her 10-year-old son. They had both caught a cold, suffering from a sore throat and runny nose.

(185) Spray formulation I (see Example 12 above), packed in a spray bottle, was administered as an oral spray. After 2 doses (one in the evening and one in the next morning), all symptoms had disappeared.

Example 27

(186) Clinical Example XI—Allergic Skin Disorder

(187) A 27 years old female with sensitive skin had an acne-like allergic skin disorder with slight itchiness on her face. She also had patches of redness and swelling on her face.

(188) Spray formulation I (see Example 12 above), packed in a spray bottle, was administered directly on to the affected areas on the face, 2 sprays at a time, 3 times per day. The feeling of itchiness was relieved within 30 minutes. The lesions completely disappeared after two weeks.

Example 28

(189) Animal Model I—Idiopathic Pulmonary Fibrosis (IPF)

(190) Experimental animals and grouping: 72 adult male Sprague Dawley rats, after 7 days of adaptive feeding, were divided into 6 groups: sham-operation (no infection and no treatment) group, IPF model group (no treatment), test group of high drug dose, test group of medium drug dose, test group of low drug dose and positive control group.

(191) The dosages of Compound A (Example 1) were set at 0.5 mg/mL, 0.1 mg/mL and 0.02 mg/mL, as the high, medium and low doses, respectively. Oral administration of pirfenidone (Etuary®, Beijing Continent Pharmaceutical Co., Ltd., Beijing, China) as a 30 mg/kg single-bolus dose served as the positive control drug.

(192) Modelling and administration: A pulmonary fibrosis model is 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. The sham-operation group were given an equal volume of normal saline. Quickly after administration, the rates were lifted vertically and rotated to evenly disperse the drug. Once the rats had recovered, after approximately 5 days, they were administrated different drugs according to the model plan for 28 days, consecutively. The experimental plan is shown in Table 10 below.

(193) TABLE-US-00026 TABLE 10 Group Treatment Dose Sham-operation Saline 50 μl IPF model group Saline 50 μl Positive control pirfenidone 250 mg/kg Compound A high Compound A 975 μg/Rat Compound A medium Compound A 195 μg/Rat Compound A low Compound A 39 μg/Rat

(194) The following observation indicators were investigated. 1) Daily, general observation of the rats' activity, sensitivity to external stimuli, fur luster, hair colour, mouth, lip, nose, weight, diet, breathing and mortality. 2) Determination of the rats' lung organ coefficient and lung dry-wet weight ratio (i.e. the ratio of the animal's lung weight organ to the animal's body weight, i.e. the ratio of viscera to body weight). 3) During the formation of pulmonary fibrosis, the expression of growth factor (TGF-β), tumour necrosis factor-α (TNF-α) and other cytokines which are involved in the onset of fibrosis were measured. Standard ELISA methods were used to detect the contents of TGF-β, TNF-α, IL-1β, malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in lung tissue. 4) Detection of the content of collagen and fibrin metabolite (hydroxyproline) in the lungs, as specific indicators for evaluating the degree of pulmonary fibrosis. 5) Detection of histopathological changes in lung tissue, which is the most important and objective indicator for evaluating pulmonary fibrosis.

(195) The results show that Compound A inhibits the overproduction of TGF-β and inflammatory cytokines. The results also show that the test drug has an antioxidation effect, by increasing SOD production and reducing lipid oxidation.

Example 29

(196) Animal model II—Antitussive Experiment: Ammonia Induced Cough Method in Mice.

(197) 60 mice were randomly divided into 5 groups according to their body weight: CMC-Na negative control group, dextromethorphan hydrobromide positive control group and high, medium and low doses (of Compound A, Example 1) groups. Each group contained 12 mice, 6 males and 6 females.

(198) The test drug was administered via atomization inhalation (0.15 mL/min) for 1 min, once daily for 5 days. The positive control drug was administered once daily by intragastric administration at 10 mg/kg for 5 days.

(199) The mice were placed in an inverted beaker 1, 2, and 4 hours after the last drug administration (either Compound A or the positive control drug). 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. The mice were stimulated by ammonia vapor for a predetermined time of 63.1, 50.1, 39.8, 31.6, 25.1, 20.0, 15.9 or 12.6 seconds. The difference in the logarithm of two adjacent stimulating times was set at 0.1, and being 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2 and 1.1. The mice were then quickly moved to a bell jar. The number of coughs within 1 minute was detected with a stethoscope. Typical coughs occurring three or more times in one minute are called ‘cough’ and those occurring less than three times in one minute are ‘cough free’.

(200) The ammonia stimulation time for the next mouse was determined according to the principle of the sequential method, i.e. if the first mouse was ‘coughing’, the next mouse was stimulated for a shorter period of time. Conversely, if the first mouse was ‘coughless’, the next mouse was stimulated for a longer period of time.

(201) The EDT.sub.50 was defined as the ammonia stimulating time at which half mice developed a “cough” and calculated by the equation:
EDT.sub.50=lg.sup.−1c/n
(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). The results are shown in Table 11 below.

(202) TABLE-US-00027 TABLE 11 1 h after 2 h after 4 h after administration administration administration group dose EDT.sub.50/s R1/% EDT.sub.50/s R1/% EDT.sub.50/s R1/% control 20.7 21.5 22.4 positive  10 50.1 241.7 44.7 207.3 41.4 184.8 mg/kg Compound  39 28.2 135.9 29.3 135.9 26.1 116.6 A High μg/mouse Compound 195 34.1 164.7 39.8 184.8 39.8 177.8 A Medium μg/mouse Compound 975 43.0 207.3 46.4 215.4 43.0 192.0 A Low μg/mouse
Where R1=(EDT.sub.50 in treatment group/EDT.sub.50 in control group)×100%, R>130% indicates an antitussive effect. R>150% indicates a strong antitussive effect.

(203) The results show that Compound A has a positive effect on cough relief.

Example 30

(204) Animal Model III—Expectorant Experiment—Phenol Red Excretion Method in Mice.

(205) 50 Mice were randomly divided into 5 groups according to body weight: Normal saline, negative control group, ammonium chloride positive control group and high, medium and low dose of test drug (either Compound A, above, or Compound E, below). 10 mice in each group, 5 males and 5 females.

(206) Compound A was administered via atomization inhalation (0.15 mL/min) for 1 min, once daily for 5 days and the positive control drug was administered by intragastric administration for 5 days.

(207) Half an hour after the last administration of Compound A, 5% phenol red solution was injected into the abdominal cavity. The mice were sacrificed after a further half an hour. The skin of the neck was removed and the trachea from the thyroid cartilage to the bifurcation was separated, soaked it in 5% sodium bicarbonate solution with constant shaking. The sodium bicarbonate solution was used to detect the phenol red content.

(208) The absorbance at 558 nm was detected by spectrophotometry (721G Spectrophotometer, Shanghai Jingke, Shanghai, China). The optical density value was used to calculate the phenol red content In the trachea by reference to the phenol red standard curve. The results of each group and the negative control group were tested for significant t-test. The results of the expectorant experiment are showed in Table 12 below.

(209) TABLE-US-00028 TABLE 12 Phenol red concentration Group Treatment Dose (μg/ml) Negative Normal saline 6.970 ± 0 .339.sup.  control Positive NH.sub.4Cl 0.15 g/kg 10.335 ± 0.337**  control High Compound A 39 μg/mouse 9.001 ± 0.637  Medium Compound A 195 μg/mouse 10.480 ± 0.550*** Low Compound A 975 μg/mouse 10.489 ± 0.610*** Comparison with the negative control: *P < 0.05 ,**P < 0.01,***P < 0.001

(210) The results show that Compound A reduces mucus production, and therefore, reduces sputum.

Example 31

(211) Effect of Compound a on the Activity of Human Herpes Simplex Virus, Type-II (HSV-II)

(212) A serum free 1640 medium was prepared using RPM11640 powder (1000 mL dosage; Thermo Fisher Scientific China), L-glutamine (0.29 g; Sinopharm Chemical Reagent Co. Ltd, Shanghai, China), sodium bicarbonate (2.2 g; Sinopharm Chemical Reagent Co.), HEPEs (2.39 g; Thermo Fisher Scientific China) and deionized water (1000 mL).

(213) The reagents were mixed until they dissolved, and the solution was sterilised by filtration. The mixture was formulated as either a complete medium containing 10% serum by adding 10% neonatal bovine serum before use or the mixture was formulated as a maintenance solution by adding 2% of neonatal bovine serum.

(214) 20 mg of Compound A (Example 1) was dissolved in 1 ml of 0.9% aqueous sodium chloride solution to prepare a 20 μg/μL stock solution. 0.05 mL of the stock solution was added to 1.95 mL of the complete (10%) medium to formulate a 500 μg/mL drug solution. The maintenance solution (2%) was used instead of the complete medium in antivirus tests Nos. 3 and 4, below.

(215) Working solutions with concentrations of 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9063, 1.9531 and 0.9766 μg/mL were prepared by double dilution.

(216) 20.34 mg of sodium lauryl sulfonate (SDS; manufactured by AMRESCO LLC, Solon, Ohio, USA and packed by Biosharp Company, Hefei, China; purity: 99%) was dissolved in 10.17 mL of the complete culture medium to produce a 2000 μg/mL stock solution. A similar stock solution was also prepared in the same way using the maintenance solution for the antivirus tests. Working solutions, with concentrations as described above, were then prepared by double dilution.

(217) 2.25 mg of acyclovir (ACV; Zhiyuan Pharmaceutical Co., Ltd, Wuxi City, China; purity: 99.3%) was dissolved in 2.25 mL of the complete culture medium to form a 1000 g/mL stock solution. A similar stock solution was also prepared in the same way using the maintenance solution for the antivirus tests. 0.8 mL of each stock solution was double diluted to provide working solutions with concentrations of 500, 250 and 125 μg/mL. 0.2 mL of each stock solution was added to 1.95 mL of the complete culture medium to provide a concentration of 100 μg/mL, which was then diluted provide to solutions with concentrations of 50, 25 and 12.5 μg/mL.

(218) 1. HSV-2 Viral Toxicity Test

(219) 0.5 mL of a suspension of human herpes simplex virus type-II (HSV-2; SAV strain; Shanghai Institute of Cell Biology) was inoculated into the monolayer culture of Vero cells (Shanghai Institute of Cell Biology) and the virus suspension was removed after 1 hour of adsorption.

(220) The maintenance solution was added and cultured at 37° C., under 5% CO.sub.2, until more than 95% of the cells showed obvious pathological changes under a microscope (Nikon ECLIPSE TS100 inverted phase control microscope, with imaging system). The cells were harvested, repeatedly frozen and thawed (3 cycles) and then centrifuged at 3000 rpm for 10 minutes in a Model 400C Medical Low Speed Centrifuge (Beijing Baiyang Centrifuge Co., Ltd.). The supernatant was collected as viral solution.

(221) The Vero cell suspension with a density of 2×10.sup.5 (cell number) was inoculated into a 96 well culture plate (Costar, Corning Inc., Oneonta, N.Y., USA) at 0.1 mL/well and cultured at 37° C., under 5% CO.sub.2 in a Thermo Scientific CO.sub.2 incubator for 18 hours, until a monolayer was visible under a microscope. The virus that was collected above was inoculated into the monolayer Vero cells with a 10-fold dilution in the maintenance solution in each 0.1 mL/well. The maintenance solution was replenished and cultured at 37° C., under 5% CO.sub.2. Pathological changes of the cells were observed under a microscope after culturing for 24 hours. Each dilution was repeated in 3 wells. Normal cells were used as a control for the experiment. The virus virulence test was repeated 3 times.

(222) Three visual fields were observed for each well. The average percentage of pathological cells (P) in the field of vision was determined.

(223) The median infectious dose (TCID.sub.50, 50% tissue culture infectious dose of a virus) of the virus was calculated according to the Reed and Muench conventional method, that is TCID.sub.50, which is the logarithm of dilution showing a mortality next above 50%−(difference of logarithms×logarithm of dilution factor). Generally, the following formula is used to calculate “difference of logarithms” (difference of logarithms is also known as “proportionate distance” or “interpolated value”): Difference of logarithms=[(mortality at dilution next above 50%)−50%]/[(mortality next above 50%)−(mortality next below 50%)].

(224) 2. Cytotoxicity of Compound A and Control Drugs

(225) Vero cells were inoculated on a 96-well culture plate and grew into monolayers. 0.2 mL of either Compound A solution (Example 1) or control drugs (20.34 mg of sodium lauryl sulfonate or 2.25 mg of acyclovir, as described above) was added to each well that contained a different concentration of complete medium correct (as described above). This was repeated in 3 wells for each concentration.

(226) The solvent and normal cell cultures were used as a negative control. Cells were cultured at 37° C., under 5% CO.sub.2, and growth and morphological changes of the cells were observed under the microscope for 2 days. Three visual fields under a microscope were selected for each well, the percentage of pathological cells was counted, and the average values were calculated. The evaluation time point of the test was set as 24 hours and the median toxic concentration (TC.sub.50) and maximum non-toxic concentration (TC.sub.0) were calculated. The experiment was repeated 3 times.

(227) Cells were inoculated as described above. The solvent and normal cell cultures were used as negative controls. 24 hours after adding Compound A or the control drug, 5 mg/mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Sigma-Aldrich (China), Shanghai, China) in PBS (diluted from a 10× stock solution, Sigma-Aldrich (China)) was introduced (20 μL/well), and cultivation continued for 4 hours. The supernatant in each well was then discarded and 150 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich (China)) was added, followed by 10 minutes of shaking in the dark at room temperature.

(228) The optical absorption value (OD.sub.550) at 550 nm was measured by an enzyme-linked immunosorbent meter (Multiskan Spectrum; Thermo Scientific, Shanghai, China).

(229) 3. Effect of Test Drug and SDS on the Cytopathic Effect of Viruses after Directly Acting on HSV-2

(230) HSV-2 virus, preserved at −80° C. (in a Haier DW-86L486 ultra-low temperature freezer) with a determined TCID.sub.50 was diluted to 200 TCID.sub.50 at the determined titer (the TCID.sub.50 value was initially determined each time, allowing the 200 TCID.sub.50 to be determined). The 200 TCID.sub.50 solutions were mixed with an equal volume of either Compound A or SDS liquid at which the viral titer was 100 TCID.sub.50. The mixed solution was incubated in a water bath (DK-8B constant temperature electrothermal water bath; Shanghai Jinghong Biotech Co., Ltd.) at 37° C. for 1 hour, and then inoculated in a 96-well culture plate containing monolayer Vero cells. To each well was added 0.1 mL of the mixed solution.

(231) The supernatant containing virus and drug was discarded after 1 hour of adsorption. The monolayer Vero cells were then washed twice with the maintenance solution. Finally, 0.2 mL of the maintenance solution was added to each well. The resultant mixture was continuously cultured at 37° C., under 5% CO.sub.2, until the cytopathic rate of the drug free culture reached 95% under the microscope. The evaluating time point of the test was set as 24 hours.

(232) Beside the experimental groups, three control groups were tested in parallel: solvent, no drug control (virus control) and normal cell control. Each group was made up of 3 wells and the experiment was repeated 4 times.

(233) The virus culture was diluted to 0.1, 1, 10, 100 and 1000 TCID.sub.50 and inoculated into monolayer cell cultures. Each dilution was conducted in triplicate. The cytopathic rates were observed for each well. There should no cytopathic effects at 0.1 TCID.sub.50, whereas a cytopathic effect should be seen at 100 TCID.sub.50; otherwise the neutralisation tests were not established.

(234) The evaluation indicators were the same as that of the virus toxicity test. Three visual fields under a microscope were observed for each well. The average percentage of pathological cells (P) in the field of vision was determined and the median infective dose (TCID.sub.50) of the virus was calculated according to the Reed and Muencl method (as described above).

(235) The drug toxicity to the Vero cells was determined by the cell morphology method, while antiviral tests were carried out at non-toxic concentrations. After incubation with different concentrations of test drug and SDS for 1 hour, 100 TCID.sub.50 HSV-2 (SAV strain) were inoculated into monolayer Vero cell culture.

(236) The results show that the cytopathic effect of the cells caused by viral infection was inhibited to varying degrees, suggesting that Compound A has an inhibitory effect on HSV-2.

(237) 4. Effect of Test Drug and ACV on HSV-2 (Direct Method)

(238) The virus was diluted to 100 TCID.sub.50 and inoculated into monolayer Vero cells culture at 0.1 mL in each well. The supernatant was discarded after 1 hour of adsorption and the culture was washed 2 times with the maintenance solution. Solutions different concentrations of Compound A or control drug (acyclovir) were then added at 0.2 mL/well. The cultures were continuously cultivated at 37° C., under 5% CO.sub.2. Each concentration was repeated in triplicate.

(239) Beside the experimental groups, three control groups were tested in parallel: solvent, no drug control (virus control) and normal cell control.

(240) During the culture period, the pathological changes were observed under a microscope and the tests were terminated when the cytopathic rate of the virus control reached >95%. The evaluation time point of the test was 24 hours and the experiments were repeated 3 times.

(241) The judgment criteria were the same as those used in the virus toxicity test, i.e. three visual fields were selected for the microscopic examination of each well, the average percentage of pathological cells (P) in the field of vision was determined, taking the average of the three visual fields.

(242) The linear regression equation was calculated according to the percentage of cytopathic effect for each reagent concentration group towards the drug concentration. The IC.sub.50 values were calculated and the significance test of correlation coefficient was also calculated.

(243) The results show that Compound A has an anti-virus effect.

Example 32

(244) Synthesis of Montelukast-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys (i.e. Montelukast Covalently Bonded to Amino Acid SEQ ID No: 12 at the N-Terminus)

(245) The procedure as described in Example 1 was repeated. A second product peak was detected at 5.813 minutes by 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) and the compound.

(246) MS: m/z 875.90 [M+2H].sup.2+.

(247) Based on the characterising data available and presented herein, it is understood that the compound isolated by way of this example is that identified above as the title compound. The compound of Example 32 is referred to hereinafter as “Compound E”.

(248) The yield ratio of Compound E to Compound A was 1:9.

Example 33

(249) Synthesis of Hydrogenated Montelukast Styrene-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys (i.e. Hydrogenated Montelukast Styrene Covalently Bonded to Amino Acid SEQ ID No: 12 at the N-Terminus)

(250) The synthesis of the above-mentioned compound was exactly the same as the procedure for Compound A as described in Example 1, except that montelukast styrene was used as a reagent in place of montelukast.

(251) MS: m/z 867.91 [M+2H].sup.2+.

(252) Based on the characterising data available and presented herein, it is understood that the compound prepared by way of this example is that identified above as the title compound. Otherwise, the compound that is prepared in Example 33 is a compound of the invention in which, in the compound of formula I, and n is 0 and the compound of formula I is covalently bonded to amino acid SEQ ID No: 12 at the N-terminus. In any event, the compound of Example 33 is referred to hereinafter as “Compound F”.

Example 34

(253) Clinical Example XII—Fever Relief

(254) An 11 year old boy showed symptoms of a fever, with a temperature at 39° C. at 2100 hours. The subject was also coughing intermittently and had a runny nose.

(255) A spray formulation of Compound E (2 mg; see Example 32 above) in normal saline solution (5 mL) was administered to each nostril as a mist by atomisation (device: handheld nebulizer, Lifetrons Beaute NS-400) at 2200 hours, over a period of 5 minutes.

(256) At 2215 hours the subject fell asleep. Around midnight, the subject began to sweat and his temperature fell a little. A second dose of Compound E (1 mg) in normal saline solution (2.5 mL) was administered in the same way, at 0030.

(257) By 0330, the subject's temperature decreased to 37.0° C. By 0800, the subject had a normal temperature. Thus, between 2215 hours the previous day and 0800 hours the following day, a cough was only observed for half a minute in total. During sleep that night, there was no observable nasal obstruction. In the morning, although a runny nose had returned, it had improved significantly compared to 11 hours previously.

(258) The subject received a third dose of Compound E (1 mg) in normal saline solution (2.5 mL), which was administered in the same way, at 0830 hours the same morning. Two hours later, his nose had stopped running. After that and up until 1500 hours on the same day, the subject had no fever, cough or runny nose.

(259) At 1530 hours on the second day, the subject received a forth dose of Compound E (1 mg) in normal saline solution (10 mL) by atomisation (apparatus: Yuyue, Air-compressing Nebulizer, 403 M). At 2045 hours, the subject had a temperature of 37.1° C. At 2100 hours, the subject received a fifth dose of Compound E (1 mg) in normal saline solution (10 mL) by atomisation. By 2215 hours, the subject's temperature was 36.8° C.

Example 35

(260) Comparison of Compounds A and E in a Mouse Ear Swelling Model (III)

(261) A similar experiment with essentially the same protocol to that described in Example 5 above was carried out on 30 healthy male BALB/c mice. The mice were randomly divided into 6 groups as described in Table 13 below, with 5 mice in each group.

(262) TABLE-US-00029 TABLE 13 Drug Drug administration Total amount Group concentration on right ear of drug Model / Xylene / Dexamethasone 5 mg/g xylene + 0.8 g cream Dexamethasone cream Budesonide 0.64 mg/ml xylene + 40 μl Nasal spray Budesonide Nasal Spray Fluticasone 50 μg/100 ml xylene + 40 μl Propionate Fluticasone Nasal Spray Propionate Nasal Spray Compound A 0.5 mg/ml xylene + 40 μl Compound A Compound E 0.5 mg/ml xylene + 40 μl Compound E

(263) Compounds A and E were obtained from GL Biochem Ltd and synthesized as described in Examples 1 and 32, respectively. Aqueous solutions of Compounds A and E were prepared in by dissolving 0.5 mg of powder in 1 mL normal saline (0.9% w/v NaCl solution). 40 μL of the prepared solution was applied to the right ear of each group.

(264) Dexamethasone acetate cream (5 mg DEX in10 g cream, Fuyuan Pharmaceutical Co. Ltd., Anhui, China), Budesonide Nasal Spray (32 μg/spray×120 spray, 0.64 mg/ml, AstraZeneca AB, SE-151 85, Södertälje, Sweden) and fluticasone propionate nasal spray (50 mcg/spray, 0.05% w/w, Glaxo Wellcome, S.A., Avenida de Extremadura n° 3-09400, Aranda de Duero, Burgos, Spain) were used as positive control. The cream was put into a 1 mL syringe to measure the dose based on calibration of weight and volume. The bottles of the spray were opened and 40 μL of the liquid was pipetted and applied to the right ear of each group.

(265) The results are shown in FIG. 15.

(266) The conjugates all had a very good effect on eliminating the edema caused by acute inflammation. The anti-inflammatory effects of Compounds A and E were equivalent.

Example 36

(267) Synthesis of Montelukast-Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-Tyr-Hyp-Lys (i.e. montelukast covalently bonded to amino acid SEQ ID No: 24 at the N-terminus) and Montelukast Styrene-Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-Tyr-Hyp-Lys (i.e. montelukast styrene covalently bonded to amino acid SEQ ID No: 24 at the N-terminus)

(268) Essentially the same procedure as that described in Example 1 above, but in which the order of the coupling steps were adjusted to provide according to the above amino acid sequence, was employed to synthesise a modified peptide with SEQ ID No: 24.

(269) Following this, montelukast was coupled onto the N-terminal of Ala. The two title compounds were separated and thereby purified by LCMS, in a similar manner to that described in Example 32 above. The compounds are referred to hereinafter as Compound G (that comprising montelukast) and Compound H (that comprising montelukast styrene), respectively.

(270) The ratio for the yield of Compounds G:H 1:7

(271) MS (Compound G): m/z 876.6[M+2H]2.sup.+

(272) MS (Compound H): m/z 867.6 [M+2H]2.sup.+

Example 37

(273) In Vitro CysLTR1 FLIPR Antagonist Test

(274) The in vitro antagonist effect of Compounds A and E (see Examples 1 and 32 above, respectively) on a CYSLTR1 cell line was measured using Fluo-4 Direct™ Calcium Assay Kit [Cat #F10471, Thermo Fisher Scientific]. As a comparison, montelukast sodium and montelukast styrene were also tested and pranlukast was used as a positive control. 10 different concentrations were tested in duplicate for each compound.

(275) A CYSLTR1/HEK293 cell line was used. The cells were prepared and 20 μL of the cell suspension were added to the 384-well plates (20K/well; poly-D-Lysine protein coating plate, Greiner #781946). The plate was placed at 37° C. in a 5% CO.sub.2 incubator overnight.

(276) A probenecid in FLIPR assay buffer was prepared from the relevant starter pack (Cat. no. F10471): A 250 mM stock solution of water-soluble probenecid was prepared by adding 1 mL of Fluo-4 Direct™ calcium assay buffer to 77 mg vials containing probenecid (Component B for Cat. nos. F10471). 10 mL of Fluo-4 Direct™ calcium assay buffer and 200 μL of the 250 mM probenecid stock solution was added to one bottle of Fluo-4 Direct™ calcium reagent (Component A). This 2× Fluo-4 Direct™ calcium reagent loading solution was sufficient for two microplates. The solution was vortexed allowed to sit for 5 minutes (protected from light), to ensure that the reagent was completely dissolved. The reagent was prepared fresh each day

(277) All of the compounds were dissolved and serially diluted in Fluo-4 Direct™ calcium assay buffer (without probenecid). The cell plate was removed from the incubator and the medium was decanted gently. 20 μL of the compounds were transferred to the cell plate, and 20 μL of 2× Fluo-4 Direct™ No-wash Loading Buffer was added. The final concentrations of each compound were 100, 30, 10, 3, 1, 0.3, 0.1 and 0.03 μM. The plate was incubated for 50 minutes at 37° C. in a 5% CO.sub.2 incubator for 10 minutes at room temperature. Fluorescence was measured using the instrument settings appropriate for excitation at 494 nm and emission at 516 nm.

(278) The data were analysed using Prism (GraphPad Software, USA) and IC50s calculated for each compound. The results are shown in Table 14 below.

(279) TABLE-US-00030 TABLE 14 Compound ID IC50 (nM) Max Dose (nM) Compound A 474.7 100000 montelukast 158.5 100000 styrene Compound E 90.55 100000 montelukast 13.74 100000 sodium Pranlukast 0.2178 1000

(280) The results show that montelukast has 11 times the affinity to CysLTR1 than montelukast styrene. Therefore, Compound E had 5 times higher affinity than that of Compound A and the affinity for the tested compounds to CysLTR1 was largely determined by the structure of the compound of formula I.

Example 38

(281) In Vitro CysLTR1 FLIPR Antagonist Test

(282) The assay procedure described in Example 37 above was repeated for Compound G (see Example 36 above), and further Compound E (see Example 32 above) as a comparison. The results are shown in Table 15.

(283) TABLE-US-00031 TABLE 15 Compound ID IC50 (nM) Max Dose (nM) Compound G 99.55 50000 Compound E 140.5 50000 montelukast 7.300 50000 sodium Pranlukast 0.2496 4000

(284) The results show that Compounds G and H had the same level of affinity to CysLTR1, which indicated that the change on amino acid sequence of the peptide had little impact on the affinity of the conjugate.

Example 38

(285) Lipopolysaccharide-Induced Lung Injury in Mice

(286) 36 BALB/c male mice, with a body weight of between 20 to 22 g, were housed in an animal facility at between 22 to 26° C. and between 55 and 75% relative humidity and a 12/12 hour day/night cycle with food and water ad libitum.

(287) Mice were randomly divided into 6 groups as indicated in Table 16 below.

(288) TABLE-US-00032 TABLE 16 Group Dose Administration Control / / Model Saline Inhalation Compound A 2 mg/kg Inhalation Compound G 10 mg/kg Inhalation (high dose) Compound G 2 mg/kg Inhalation (medium dose) Compound G 0.4 mg/kg Inhalation (low dose)

(289) Compound A was prepared analogously to the procedure described in Example 1 above and Compound G was prepared analogously to the procedure described in Example 36 above.

(290) The mice were anesthetized by intraperitoneal injection of 3% chloral hydrate (0.1 mL/10 g). Tongues were pulled aside with tweezers. Lipopolysaccharide (LPS, 1 mg/mL, 50 μL) administered by pipetted to the back wall of the pharynx. Tongues were released and noses pinched immediately for 30 seconds. Then, mice were released from retention and placed back into cages wake naturally. Mice in the control group were treated with the same volume of saline.

(291) Test compounds were administered via atomization/inhalation for 30 minutes after LPS induction. 24 hours later, mice were sacrificed.

(292) The thoracic cavity was quickly opened and whole lung was stripped. A piece of lung tissue weight was accurately weighed, and saline was added at a ratio of 9 mL saline to 1 g of lung tissue. Then, the tissue was homogenized and centrifuged for 10 minutes at 3000 rpm. The homogenate was used to detect TGF-β1 using ELISA kit (Beijing 4A Biotech Co., Ltd, China) and the results showed in FIG. 16.

(293) The results showed that both compounds of the invention reduce inflammatory cytokines in lung tissues. The IL-1β concentration in lung tissue in Compound A group was the same level as in low dose group for Compound G. Compound G also shows dose dependent efficacy in reducing inflammatory cytokines.

(294) Thus, although both compounds of the invention have been shown to be efficacious in treating LPS-induced acute lung injury in mice, the potency of Compound G was about 5 times higher than that of Compound A.

Example 39

(295) Idiopathic Pulmonary Fibrosis (IPF) Model in Rats

(296) 60 adult SD rats (30 male, 30 female) were purchased from Zhejiang Experimental Animal Center, China. Animals were housed at between 21 and 26° C. and at between 40 and 70% relative humidity with free access to food and water.

(297) After 7 days of adaptive feeding, rats were randomly divided into 6 groups as showed in Table 17 below.

(298) TABLE-US-00033 TABLE 17 Administration Group Drug Dose Method Sham / / inhalation Model normal saline 0.15 mL inhalation pirfenidone pirfenidone 240 mg/kg Gavage Compound A Compound A 2 mg/kg inhalation Compound E Compound E 2 mg/kg inhalation Compound H Compound H 2 mg/kg inhalation

(299) The rats were anaesthetized and placed on an operating table in the supine position, to expose the trachea. Bleomycin (5 mg/kg, Bleomycin Hydrochloride for Injection, Haizheng Pfizer Pharmaceutical Co., Ltd.) and saline solution were injected into the trachea through the gap between the tracheal cartilage rings.

(300) The sham-operation group was given an equal volume of normal saline instead of bleomycin. The rats were lifted vertically immediately after administration and were rotated to allow bleomycin to evenly disperse.

(301) Once the rats had recovered, after approximately 7 days, they were administrated different drugs according to the model plan. 6.5 mg of test compounds of the invention in powdered form was accurately dissolved in 5 mL saline to make a 1.3 mg/mL solution. 0.15 mL of the solution was atomized and inhaled by each rat. Inhalation was carried out once a day.

(302) For the pirfenidone group, 12 pirfenidone capsules (Beijing Contini Pharmaceutical Co., Ltd., Beijing, China; 100 mg) were opened and the contents were fully suspended in 25 mL of 0.5% CMC-Na solution to obtain 48 mg/mL suspension. The dosage of pirfenidone was 1.0 mL/200 g in rats, i.e. 240 mg/kg, and was given by oral gavage.

(303) After 28 days of administration, rats were anesthetized by intraperitoneal injection of chloral hydrate and then sacrificed. The thoracic cavity was quickly opened and whole lung tissue was removed. The lung wet weight was weighed, and the lung coefficient was calculated (lung wet weight/rat weight×1000) and are shown in Table 18 below.

(304) TABLE-US-00034 TABLE 18 Lung Group coefficient (%) Sham 6.272 ± 0.496 Model 13.484 ± 1.395  Compound A 8.771 ± 0.897 Compound E 9.462 ± 1.123 Compound H 9.825 ± 0.647 pirfenidone 10.218 ± 0.984 

(305) The results show that Compounds A, E and H reduce lung edema caused by bleomycin induction.

(306) The right bronchus was ligated, and the left lung was perfused with formalin solution in vitro. The left lung was cut and fixed in formalin solution for pathological examination. The remaining tissue was stored in a refrigerator at −80° C. for later use.

(307) The fixed lung tissue was embedded in paraffin and sequential 4 μm sections were stained with haematoxylin-eosin (HE) and modified Masson's trichrome. Fibrotic lung injury was assessed morphologically by semiquantitative parameters. All morphological changes were scored according to the severity of damages. The scores were given as 1-4 according to light, mild, moderate and severe degree, respectively. No lesion was scored as 0. Evaluation from HE stained sections were the sum of degree of fibrosis and inflammation. Evaluation from Masson stained sections were the degree of collagen deposition in pulmonary interstitium. The results are shown in Table 19 below.

(308) TABLE-US-00035 TABLE 19 HE evaluation Masson evaluation Group Mean SD Mean SD Sham 1.78 1.20 0.22 0.44 Model 5.50 0.84 2.67 0.52 pirfenidone 5.50 0.52 2.50 0.5 Compound A 4.00 0.45 2.25 0.4 Compound E 4.29 0.29 1.86 0.37 Compound H 4.00 0.14 1.88 0.13

(309) The results showed that, compared with the sham-operated group, pulmonary fibrosis and bronchial pneumonia in the model group were more serious. Compared with the Model group, the pathological changes in the drug-treated group were similar, and the degree of pathological changes were less. The order of pathological changes was as follows: Model, pirfenidone>Compound E>Compound A>Compound H>sham. These results indicated that Compounds A, E and H prevent bleomycin-induced lung fibrosis in mice and their efficacy was stronger than that of the pirfenidone.

Example 40

(310) Synthesis of Montelukast-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Hyp-Lys (i.e. Montelukast Styrene Covalently Bonded to Amino Acid SEQ ID No: 20 at the N-Terminus)

(311) Essentially the same procedure as that described in Example 1 above, but in which the order of the coupling steps were adjusted to provide according to the above amino acid sequence, was employed to synthesize a modified peptide with SEQ ID No: 20.

(312) Following this, montelukast was coupled onto the N-terminal of Ala. The compound was purified by LCMS, in a similar manner to that described in Example 32 above. The title compound is are referred to hereinafter as Compound J.

(313) MS: m/z 924.15 [M+2H]2+

Example 41

(314) Mouse Ear Swelling Model IV

(315) Essentially the same protocol to that described in Example 5 above was carried out on 15 healthy male BALB/c mice, using Compounds J (see Example 40 above) as test compound. The mice were randomly divided into 3 groups as described in Table 20 below, with 5 mice in each group.

(316) A hydrogel of Compound J was prepared comprising 0.5 mg/g of active ingredient and methyl cellulose (2.5%), propanediol (11%), glycerol (11%), acetic acid (pH regulator; 0 to 0.5 g). All excipients were obtained from Sinopharm Chemical Reagent Co. Ltd. The gel was made up with water for injection.

(317) Dexamethasone acetate cream (DEX cream; 5 mg of dexamethasone in 10 g of cream; Fuyuan Pharmaceutical Co. Ltd., Anhui, China) was used as a positive control. 40 μL of the various treatments drugs were applied to the right ear of each group.

(318) TABLE-US-00036 TABLE 20 Drug Total amount Drug administration of drugs Group concentration on right ear (μg/mouse) Model / Xylene / Dex cream 10 μg/μL xylene + 400 dexamethasone cream Compound J 0.5 mg/g xylene + 20 Compound J gel

(319) The results are shown in FIG. 17. Compound J showed a very good effect on eliminating the edema caused by acute inflammation.

Example 42

(320) Clinical Example—Allergic Conjunctivitis

(321) A 52 year old female patient was diagnosed with allergic conjunctivitis and experienced with swollen eyelids, itching, and a watery nose.

(322) 0.5 mg/mL of Compound G in saline solution was packed in a spray bottle. The spray was administered to each eye, 2 to 3 times per day for 7 days.

(323) The patient felt relief from itchy eyes after one treatment. At the second day of treatment, her eyelids were less swollen. Full recovery of all symptoms took place within a week.

Example 43

(324) Clinical Example—Ulcerative Colitis

(325) A hospitalized patient with ulcerative colitis suffered from severe symptoms including bad abdominal pain and cramping, frequent diarrhea (more than 20 time a day), and did not respond to over-the-counter medications. The patient experience rectal bleeding, passing small amounts of blood with stools, an urgency to defecate and fever.

(326) A hydrogels of Compound G was prepared consisting of 0.5 mg/g of active ingredient, methyl cellulose (2.5%), propanediol (11%), glycerol (11%), acetic acid (pH regulator; 0 to 0.5 g). All excipients were obtained from Sinopharm Chemical Reagent Co. Ltd. The gel was made up with water for injection.

(327) The patient was given 2 g of the above gel by anal administration, once daily. On the second day after administration, the diarrhea reduced to only 5-6 times daily, with less bleeding.

(328) The patient continued to use the gel with a view to determining long-term efficacy.