A HIGH-PURITY ADRENOCORTICOTROPIC HORMONE ANALOGUE AND A LARGE-SCALE PREPARATION METHOD THEREOF

Abstract

The invention belongs to the technical field of polypeptide preparation methods, and in particular relates to a high-purity ACTH (human sequence) or analogue and large-scale preparation method thereof. The main steps include: amino acids are coupled from the C-terminal to the N-terminal by Fmoc solid-phase synthesis method to obtain the crude ACTH (human sequence) or analogue peptidyl-resin with protective groups, wherein the reaction temperature of C-15 peptide synthesis is 40-60° C. After cleavage and precipitation, the crude product of ACTH (human sequence) or analogue is obtained, and then the high-purity product is obtained by liquid chromatography. The chromatographic purity of ACTH (human sequence) or analogue prepared by the invention is more than 99%, the stability is good, and the yield of the target peptide is ≥63%.

Claims

1. A method of treating spasm, multiple sclerosis, a rheumatic disorder, an allergic state, an edematous state or an immune disease, wherein the method comprises administering a composition comprising an adrenocorticotropic hormone (ACTH) or an analogue thereof, wherein the sequence of the adrenocorticotropic hormone or analogue thereof, from the N-terminal to the C-terminal, is TABLE-US-00017 (SEQ ID NO: 4) SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF.

2. The method of claim 1, wherein the composition further comprises one or more impurities, wherein the purity of the adrenocorticotropic hormone or analogue thereof is ≥99%, the content of the maximum single impurity is ≤0.5%, and the content of total impurities is ≤1%.

3. The method of claim 2, wherein the purity of the adrenocorticotropic hormone or analogue thereof is ≥99.5%, and the content of total impurity is ≤0.5%.

4. The method of claim 1, wherein the composition comprises a medicinal or pharmaceutically acceptable carrier.

5. The method of claim 1, wherein the spasm is infantile spasm.

6. The method of claim 1, wherein the composition is administered to a target animal.

7. The method of claim 6, wherein the target animal is a human.

8. The method of claim 7, wherein the target animal is an infant and/or a child of less than 2 years old.

9. The method of claim 6, wherein the target animal is a rat model of infantile spasm or neonatal spasm.

10. The method of claim 1, wherein the composition is administered intravenously, intraperitoneally, topically, dermally, intradermally, subcutaneously, via dermal infusion, via subcutaneous infusion, intraocularly, buccally, nasally via inhalation, intramuscularly, sublingually or orally.

11. The method of claim 10, wherein the composition is administered intraperitoneally.

12. The method of claim 1, wherein the composition is administered once daily or twice daily.

13. The method of claim 12, wherein the composition is administered twice daily.

14. The method of claim 1, wherein the composition is administered at a dose between 0.01 U/g to 10 U/g daily.

15. The method of claim 14, wherein the composition is administered at a dose between 0.02 U/g and 0.56 U/g daily.

16. The method of claim 15, wherein the composition is administered at a dose of 0.56 U/g daily.

17. The method of claim 1, wherein the composition is used to prepare formulation products, such as parenteral dosage form, oral dosage form, rectal dosage form or topical dosage form or other suitable dosage forms for the composition.

18. The method of claim 17, wherein the parenteral dosage form is liquid, lyophilized powder or other suitable dosage forms for injection.

19. The method of claim 18, wherein the liquid is sterile solutions, suspensions, emulsions or other suitable dosage forms.

20. The method of claim 17, wherein the oral dosage form is a tablet, a capsule, a granule, a pill, powder, sustained and controlled release preparation or other suitable dosage forms for oral administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1 is a flow chart of the stepwise condensation of solid-phase synthesis method of the present invention.

[0080] FIG. 2 is a liquid chromatogram of condensation of peptide 15 in example 4 at 20° C. for 20 h, using DIC/Oxyma Pure/urea as condensation agent.

[0081] FIG. 3 is a liquid chromatogram of condensation of peptide 15 in example 4 at 30° C. for 20 h, using DIC/HOBt/NaClO.sub.4 as condensation agent.

[0082] FIG. 4 is a liquid chromatogram of condensation of peptide 15 in example 4 at 35° C. for 20 h, using TBTU/DIPEA/HOBt as condensation agent.

[0083] FIG. 5 is a liquid chromatogram of condensation of peptide 15 in example 4 at 40° C. for 3 h, using DIC/Oxyma Pure as condensation agent.

[0084] FIG. 6 is a liquid chromatogram of condensation of peptide 15 in example 4 at 45° C. for 3 h, using DIC/HOBt as condensation agent.

[0085] FIG. 7 is a liquid chromatogram of condensation of peptide 15 in example 4 at 50° C. for 3 h, using DIC/HOBt as condensation agent.

[0086] FIG. 8 is a liquid chromatogram of condensation of peptide 15 in example 4 at 50° C. for 3 h, using DIC/HOBt/urea as condensation agent.

[0087] FIG. 9 is a liquid chromatogram of condensation of peptide 15 in example 4 at 50° C. for 3 h, using DIC/HOBt/NaClO.sub.4 as condensation agent.

[0088] FIG. 10 is a liquid chromatogram of condensation of peptide 15 in example 4 at 60° C. for 3 h, using DIC/HOBt as condensation agent.

[0089] FIG. 11 is a liquid chromatogram of the crude peptide of ACTH (human sequence) in Example 7.

[0090] FIG. 12 is a liquid chromatogram of the pure ACTH (human sequence) in example 8.

[0091] FIG. 13 is a liquid chromatogram of the pure ACTH (human sequence) analogue in example 9.

[0092] FIG. 14 is a liquid chromatogram of ACTH (human sequence) obtained by the method of U.S. Pat. No. 3,953,415A in comparative example.

[0093] FIG. 15 shows the effect of SEQ ID NO. 4 on the incubation period of spasticity in infant spasticity (IS) rat model.

DESCRIPTION OF THE EMBODIMENTS

[0094] In order to facilitate those skilled in the art to understand the content of the present invention, the technical solutions of the invention will be further described below in conjunction with the examples, but the following contents should not limit the scope of the invention claimed by the appended claims in any way.

[0095] The materials and reagents used in the following examples can be obtained from commercial channels without special instructions. Amino acids are L-type amino acids without special instructions

Example 1. Preparation of Fmoc-Phe-Resin

[0096] 500 g of 2-triphenylmethylchloromethane resin was added to 5 L DCM and was swelled for 1 h, 4.25 mol DIPEA and 0.75 mol Fmoc-Phe-OH were added to react at room temperature for 4 h to obtain Fmoc-Phe-resin. 1.25 L DIPEA/methanol (volume ratio 1:9) solution was added to react for 0.5 h to cap the unreacted sites. The Fmoc-Phe-resin was filtrated and washed one time with 5 L DCM, then the resin was dried and washed three times with methanol, the amount of methanol used for each washing was 5 L. The Fmoc-Phe-resin was dried under vacuum to constant weight at room temperature, and the substitution was determined to be 0.7 mmol/g.

Example 2. Preparation of Fmoc-Glu(OtBu)-Phe-Resin

[0097] 1) Resin swelling: 500 g of Fmoc-Phe-resin with substitution of 0.7 mmol/g prepared in example 1, namely, the molar amount of Fmoc-Phe was 0.35 mol, was added into the reactor, and 5 L DMF solution containing 50 g HOBt was added to fully swell Fmoc-Phe-resin.

[0098] 2) Deprotection reaction: 5 L of deprotection reagent was added (20% piperidine/DMF solution, by volume ratio) to step 1), the mixture was stirred and reacted for 20 min, the resin was dried, and the deprotection reaction was repeated for 3 times until the deprotection reaction was complete. After the deprotection reaction, 5 L DMF solution once time was used to wash for 8 times in total to obtain Fmoc-Phe-resin.

[0099] 3) Activation: 1.75 mol of Fmoc-Glu(OtBu)-OH was added into 5 L DMF solution, then 1.75 mol of Oxyma Pure and 1.75 mol of DIC were added respectively, and the mixture was reacted for 30 mins at room temperature to obtain the activated solution of Fmoc-Glu(OtBu)-OH;

[0100] 4) Condensation reaction: the activated solution of Fmoc-Glu(OtBu)-OH was added to the Phe-resin obtained in step 2) to react for 3 h at 30° C., the resin was filtered and washed with DMF for 8 times to obtain Fmoc-Glu(OtBu)-Phe-resin. The reaction was monitored by the method of Ninhydrin Test. If the test result was negative, the next reaction was conducted. If the test result was positive, the above steps 3) to 4) condensation reaction was repeated until the test result was negative.

Example 3. Preparation of Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin

[0101] Refer to the method described in step 2)-4) of example 2, that was, each coupling amino acid was subjected to deprotection reaction, activation reaction and condensation reaction to the Fmoc-Glu(OtBu)-Phe-resin prepared in example 2. The amino acids were coupled according to the amino acid sequence of ACTH (human sequence) from the C-terminal to the N-terminal in the order of 3-14, the sequence of coupling amino acids was as follows: Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH.

Example 4a. Preparation of Fmoc-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin

[0102] 1) 50 g of Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-resin prepared in example 3 was taken, 500 mL of deprotection reagent (20% piperidine/DMF solution, by volume ratio) was added to the resin. The mixture was stirred and reacted for 20 min, the resin was dried, and the deprotection reaction was repeated for 3 times until the deprotection reaction was complete. After the deprotection reaction, 500 mL DMF solution each time was used to wash for 8 times in total. The reaction product Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-resin was evenly divided into 21 parts and respectively added into independent peptide reactor.

[0103] 2) Preparation of Fmoc-Asn(Trt)-OH activated solution: several activated solutions were respectively prepared, 4.2 mmol Fmoc-Asn(Trt)-OH was added to 25 mL DMF solution for each solution, and then 4.2 mmol condensation reagent as shown in Table 1 was added to react at room temperature for 30 mins.

[0104] 3) Each activated solution of Fmoc-Asn(Trt)-OH was added into the polypeptide reactor respectively for condensation reaction. The reaction temperature, type of condensation reagent and reaction time were shown in Table 1, wherein the amount of urea or sodium perchlorate was 0.1 Kg. After the reaction, the resin was filtered and washed with DMF for 8 times. During the reaction, the method of Ninhydrin Test was used to directly qualitatively detect whether the reaction was completed or not. Fmoc-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Ph e-Pro-Leu-Glu(OtBu)-Phe-resin was cut with 85% trifluoroacetic acid solution, the resulting solution was precipitated with ether, and the precipitate was dissolved with water. The chromatographic purity of 15 peptide, the residue of substrate 14 peptide and the racemic impurity of D-Asn-15 peptide were determined by HPLC, the details were shown in Table 1:

TABLE-US-00012 TABLE 1 Condensation reactions under different reaction conditions Racemic impurity Type of Time of Detection Chromatographic Residue of of Temperature condensation reaction/ of purity of 15 substrate D-Asn-15 Appended # of reaction reagent h ninhydrin peptides 14 peptide peptide drawings 1. 20° C. DIC/Oxyma Pure 3 positive — — — — 2. DIC/Oxyma Pure 8 positive — — — — 3. DIC/Oxyma Pure 20 positive — — — — 4. DIC/Oxyma Pure/ 3 positive — — — — urea 5. DIC/Oxyma Pure/ 8 positive — — — — urea 6. DIC/Oxyma Pure/ 20 positive 69.51% 5.19% not FIG. 2 urea detected 7. 30° C. DIC/HOBt 3 positive — — — — 8. DIC/HOBt 8 positive — — — — 9. DIC/HOBt 20 positive — — — — 10. DIC/HOBt/NaClO.sub.4 3 positive — — — — 11. DIC/HOBt/NaClO.sub.4 8 positive — — — — 12. DIC/HOBt/NaClO.sub.4 20 positive 69.81% 4.19% not FIG. 3 detected 13. 35° C. TBTU/DIPEA/HOBt 3 positive — — — — 14. TBTU/DIPEA/HOBt 8 positive — — — — 15. TBTU/DIPEA/HOBt 20 positive 68.75% 4.16% not FIG. 4 detected 16. 40° C. DIC/Oxyma Pure 3 — 69.45% 3.42% not FIG. 5 detected 17. 45° C. DIC/HOBt 3 — 69.80% 3.03% not FIG. 6 detected 18. 50° C. DIC/HOBt 3 — 71.06% 2.28% not FIG. 7 detected 19. DIC/HOBt/urea 3 — 68.00% 0.20% not FIG. 8 detected 20. DIC/HOBt/NaClO.sub.4 3 — 69.16% 1.03% not FIG. 9 detected 21. 60° C. DIC/HOBt 3 — 65.75% 0.30% 1.11% FIG. 10

[0105] In the above table, the 15 peptide was H-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH; The 14 peptide was H-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH; The racemic impurity of D-Asn-15 peptide was H-(D-Asn)-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH.

[0106] The results showed that the residue of 14 substrate peptide was always more than 4% by using a variety of condensation reagents, repeating condensation times and prolonging condensation time when the reaction temperature was controlled at 20-35° C., which indicating that the reaction was not complete. When the reaction temperature was 40-60° C., the residue of 14 substrate peptide was controlled to be less than 4%. Especially when the reaction temperature was 50° C., the condensation rate could be significantly improved by adding appropriate amount of urea or NaClO.sub.4 to DIC/HOBt condensation reagent. Because the urea added was more helpful to the improvement of condensation rate, and the residue of 14 substrate peptide was better controlled at 0.20%.

Example 4b

[0107] 950 g of Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-resin prepared in example 3 was condensed under the optimal condition (#19) in Table 1, that was, the condensation reaction was conducted with Fmoc-Asn(Trt)-OH in the presence of the condensation reagent of DIC/HOBt/urea, and the obtained intermediate was used as the upstream raw material of example 5.

Example 5. Preparation of Fmoc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala- Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin

[0108] Refer to the method described in steps 2)-4) of example 2, each coupling amino acid was subjected to deprotection reaction, activation reaction and condensation reaction in the polypeptide reactor of example 4b. The amino acids were coupled according to the amino acid sequence of ACTH (human sequence) from the C-terminal to the N-terminal in the order of 16-39, the sequence of coupling amino acids were as follows: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH. At the end of the coupling, the peptidyl-resin of ACTH (human sequence) with protective groups was obtained.

Example 6. Preparation of H-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala- Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-Resin

[0109] 5 L of deprotection reagent (20% piperidine/DMF solution) was added and stirred, the reaction was conducted for 20 min, the resin was dried, the deprotection reaction was repeated for 3 times until the deprotection reaction was complete. After deprotection reaction, the resin was washed 8 times with 5 L DMF solution each time. Then, 5 L methanol solution was added to wash for 8 times, and the resin was filtered and dried under vacuum at 40° C. to obtain 1.75 Kg of the peptidyl-resin of ACTH (human sequence) without the N-terminal protection by Fmoc.

Example 7. Preparation of Crude Peptide of ACTH (Human Sequence) H-Ser-Tyr-S er-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH

[0110] 1.75 Kg peptidyl-resin prepared in example 6 was added to 17.5 L cleavage cocktail which was precooled to −12° C. to react for 2 h (the cleavage cocktail was composed of 90% volume of trifluoroacetic acid and 10% volume of scavengers, in which the scavengers was composed of 1% phenol, 1% anisole, 1% dimethyl sulfide, 1% 1,2-ethanedithiol, 1% triethylsilane, 1% triisopropylsilane and 4% water in volume concentration), the temperature of the whole reaction was controlled not more than 40° C. After the reaction, the product was filtered and the filtrate was collected. The part of the cleavage cocktail was removed by reducing the pressure and concentrating the filtrate, and then the filtrate was slowly added to 17.5 L methyl tert-butyl ether which was precooled to −12° C. for precipitation. The wet solid was collected by centrifugation and washed with 87.5 L methyl tert-butyl ether. The solid was collected, dissolved with water and freeze-dried. 0.75 Kg solid crude peptide was obtained with purity of 68.96% and content of the target peptide of 48% (i.e. 0.36 Kg target peptide). As shown in FIG. 11, the peak time of the target peptide was 28.176 mins.

Example 8. Purification of Crude Peptide of ACTH (Human Sequence) H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH

[0111] 1. Treatment of sample: 0.75 Kg of crude peptide of ACTH (human sequence) obtained in example 7 was taken to dissolve in 37.5 L acetonitrile water solution with the volume ratio of acetonitrile:water=30:70, the crude peptide was stirred to dissolve completely and filtered with 0.45 μm filter membrane, and then the filtrate was collected for use.

[0112] 2. The First Purification:

[0113] Conditions of Purification:

[0114] Chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane chemically bonded silica as stationary phase;

[0115] Column diameter and packing length: 20*25 cm;

[0116] Mobile phase A: Tris aqueous solution with molar concentration of 0.1 mol/L, pH was adjusted to 8.0 by ammonia water;

[0117] Mobile phase B: acetonitrile;

[0118] Flow rate: 80 mL/min;

[0119] The detection wavelength: 280 nm.

[0120] Gradient: B %: 30-60% (50 mins), the injection volume was 20 g.

[0121] Process of purification: the chromatographic column was equilibrated with mobile phase A and then loaded with 5 L sample solution. Linear gradient elution was conducted for 50 mins and the target peptide solution with purity of more than 90% was collected. The yield of target peptide was 79%.

[0122] 3. The Second Purification:

[0123] Conditions of Purification:

[0124] Chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane chemically bonded silica as stationary phase;

[0125] Column diameter and packing length: 20*25 cm;

[0126] Mobile phase A: Ammonium sulfate aqueous solution with molar concentration of 0.1 mol/L, pH was adjusted to 3.0 by sulfuric acid;

[0127] Mobile phase B: acetonitrile;

[0128] Flow rate: 80 mL/min;

[0129] The detection wavelength: 280 nm.

[0130] Gradient: B %: 30-60% (50 mins), the injection volume was 20 g.

[0131] Process of purification: the chromatographic column was equilibrated with mobile phase A and then loaded with 1 L sample solution. Linear gradient elution was conducted for 50 mins and the target peptide solution with purity of more than 99% was collected. The yield of target peptide was 86%.

[0132] 4. Desalting and Ion Control:

[0133] Conditions of Desalting:

[0134] Chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane chemically bonded silica as stationary phase;

[0135] Column diameter and packing length: 20*25 cm;

[0136] Mobile phase A: Acetic acid aqueous solution with concentration of 0.1%;

[0137] Mobile phase B: acetonitrile;

[0138] Flow rate: 80 mL/min;

[0139] The detection wavelength: 280 nm.

[0140] Gradient: B %: 30-60% (50 mins), the injection volume was 20 g.

[0141] Process of desalting: the chromatographic column was equilibrated with mobile phase A and then loaded with 1 L sample solution. Linear gradient elution for was conducted 50 mins and the target peptide solution was collected. The yield of target peptide was 95%.

[0142] 5. Freeze Drying

[0143] The target peptide solution obtained in the previous step was transferred to a stainless steel tray with appropriate size, and then detected by HPLC after freeze drying. The purity of ACTH acetate (human sequence, SEQ ID NO: 3) was 99.88% (as shown in FIG. 12, the peak time of main peak was 27.930 min, the content of maximum single impurity was 0.07%, and the content of total impurities was 0.13%). The target peptide collected was 258 g, the content was 90%, and the molar yield was 65%. The accurate molecular weight detected by high-resolution mass spectrometry was 4538.232 Da, which was consistent with the theoretical accurate molecular weight of ACTH (human sequence).

Example 9

[0144] Referring to the method of example 4b-8, the amino acid of Asp(Trt) at position 15 was replaced with Asn(Trt) to obtain the ACTH (human sequence) analogue (SEQ ID NO: 4, H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asp-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH) with the purity of 99.58% (as shown in FIG. 13, the peak time of the main peak was 28.213 min, the content of maximum single impurity was 0.16%, and the content of total impurities was 0.42%). The target peptide collected was 255 g, the content was 89%, and the molar yield of the target peptide was 63%. The accurate molecular weight detected by high-resolution mass spectrometry was 4537.232 Da, which was consistent with the theoretical accurate molecular weight of ACTH (human sequence) analogue.

Example 10 (Comparative Example)

[0145] ACTH (human sequence) (SEQ ID NO: 3) was prepared strictly according to the specific steps from step 1 to step 14 in example 4 of U.S. Pat. No. 3,953,415. The total yield was 17%, and the purity of ACTH (human sequence) was only 75.72%, as shown in FIG. 14.

Example 11 Study of Stability

[0146] The samples prepared in example 8 were packed in double-layer medical low-density polyethylene bags and a layer of aluminum-plastic composite bags and stored at 2-8° C. for evaluation at 1, 2, 3 and 6 months respectively. The stability results of the samples after acceleration for 6 months were shown in Table 2:

TABLE-US-00013 TABLE 2 Stability results of acceleration for 6 months Accelerated test Inspection Detection Start 1 month 2 months 3 months 6 months items method Result Conclusion Character Visual White White White White White Qualified powder powder powder powder powder Related HPLC 0.07% 0.17% 0.17% 0.16% 0.11% Qualified substances (maximum single impurity) Related HPLC 0.13% 0.29% 0.29% 0.38% 0.39% substances (total impurities) The purity of HPLC 99.88% 99.71% 99.71% 99.62% 99.61% Qualified chromatographic Moisture Karl 5.0% 7.3% 7.5% 8.4% 8.3% Qualified Fischer Polymer HPLC 0.03% 0.05% 0.03% 0.06% 0.04% Qualified

[0147] The results showed that the character, related substances, purity of chromatographic, moisture and polymer of ACTH (human sequence SEQ ID NO: 3) had no obvious change trend at 2-8° C. It was suggested that the ACTH (human sequence SEQ ID NO: 3) was stable under this storage condition. The polymer refers to an impurity with a molecular weight greater than that of ACTH (human sequence SEQ ID NO: 3).

[0148] The samples prepared in example 9 were packed in double-layer medical low-density polyethylene bags and a layer of aluminum-plastic composite bags and stored at 2-8° C. for evaluation at 1, 2, 3 and 6 months respectively. The stability results of the samples after acceleration for 6 months were shown in Table 3:

TABLE-US-00014 TABLE 3 Stability results of acceleration for 6 months Accelerated test Inspection Detection Start 1 month 2 months 3 months 6 months items method Result Conclusion Character Visual White White White White White Qualified powder powder powder powder powder Related HPLC 0.16% 0.24% 0.17% 0.27% 0.10% Qualified substances (maximum single impurity) Related HPLC 0.42% 0.42% 0.43% 0.46% 0.49% substances (total impurities) The purity of HPLC 99.58% 99.58% 99.57% 99.54% 99.51% Qualified chromatographic Moisture Karl 5.2% 7.5% 7.6% 8.5% 8.6% Qualified Fischer Polymer HPLC 0.03% 0.04% 0.05% 0.06% 0.06% Qualified

[0149] The results showed that the character, related substances, purity of chromatographic, moisture and polymer of ACTH analogue (human sequence SEQ ID NO: 4) had no obvious change trend at 2-8° C. It was suggested that the ACTH analogue (human sequence SEQ ID NO: 4) was stable under this storage condition. The polymer refers to an impurity with a molecular weight greater than that of ACTH analogue (human sequence SEQ ID NO: 4).

Example 12 Data of Security

[0150] (1) Study of Activity In Vitro

[0151] ACTH was a receptor agonist of melanocortin receptor 2 (MC2R). The combination of ACTH and MC2R could stimulate the signal pathway of the downstream and change the signal factors such as calcium ions and cAMP. The binding ability of ACTH and MC2R was determined by detecting calcium ion signals, and then the cell activity of ACTH in vitro was judged.

[0152] The samples in example 8 and example 10 were taken for activity study in vitro. CHO-K1 cells expressing MC2R were cultured in 10 cm vessels and stored at 37° C. and 5% CO.sub.2.

[0153] The results showed that high-purity ACTH (human sequence SEQ ID NO: 3, 99.88%) and low-purity ACTH (human sequence SEQ ID NO: 3, 75.72%) had binding ability to melanocortin receptor 2 (MC2R) and showed biological activity. The half inhibitory concentration (EC.sub.50) of high-purity ACTH was 180.2 nM, the half inhibitory concentration of low-purity ACTH was 287.9 nM. The lower the half inhibitory concentration, the higher the activity. Therefore, the activity of high-purity ACTH was better than that of low-purity ACTH, and the activity of the former was 1.6 times that of the latter. The specific activity test datas were shown in Table 4:

TABLE-US-00015 TABLE 4 Activity test datas The source of sample Bottom Top HillSlope EC.sub.50(nM) Example 8 −2.739 100.9 0.9745 180.2 Example 10 −1.846 94.88 1.249 287.9

[0154] (2) Study of Toxicity In Vitro

[0155] The cardiac metabolic toxicity of drugs was judged by detecting the affinity between drugs and heart receptor protein (hERG) in vitro. The higher the affinity between the drug and the hERG receptor, the lower the half inhibitory concentration, which indicated that the lower the dose required for its toxicity to the heart, and the stronger the metabolic toxicity to the heart.

[0156] The samples in example 8, example 10 and positive control drug of dofetilide were taken for toxicity study in vitro. The results showed that the IC.sub.50 of high-purity ACTH and low-purity ACTH were more than 10000 nM, and the IC.sub.50 of positive control drug was 2.09 nM. Therefore, the toxic dose of high-purity ACTH and low-purity ACTH was much higher than that of the positive control drug, showing no cardiac metabolic toxicity. The specific metabolic toxicity datas were shown in Table 5:

TABLE-US-00016 TABLE 5 Datas of cardiac metabolic toxicity The source of sample IC.sub.50(nM) MaxDose(nM) % Inh @ MaxDose Exemple 8 >10000 10000 −1.28 Exemple 10 >10000 10000 12.47 positive control drug 2.090 10000 104.47

Example 13 Pharmacodynamics Research

[0157] (1) Method: To investigate the effect of SEQ ID NO. 4 on the incubation period of spasticity in infantile spasticity (IS) rat model.

[0158] Spasticity: The rats showed obvious frequent and powerful tail flick, and the hips twisted violently from side to side.

[0159] Incubation period: Time from NMDA injection to onset of spasticity (time is measured in units of seconds).

[0160] (2) Operation Steps:

[0161] Sham group: Sprague Dawley (SD) rats were intraperitoneally injected with saline (10 mL/kg each time) at 9:00 am and 19:00 pm respectively on the 15th day of pregnancy, and the neonatal rats (n=12) were intraperitoneally injected with saline of the same volume as the drug group on the 15th day after birth.

[0162] Model group (IS group): SD rats were intraperitoneally injected with betamethasone (0.4 mg/kg each time) at 9:00 am and 19:00 pm respectively on the 15th day of pregnancy. The neonatal rats (n=12) were intraperitoneally injected with N-methyl-D-aspartic acid (NMDA, 15 mg/kg) at 15:30 pm on the 15th day after birth to establish infant spasticity (neonatal spasticity) rat model, and the incubation period from NMDA injection to spasticity was recorded.

[0163] Drug group (SEQ ID NO. 4 group): SD rats were intraperitoneally injected with betamethasone (0.4 mg/kg each time) at 9:00 am and 19:00 pm respectively on the 15th day of pregnancy. The neonatal rats (Divided into 2 groups, n=12 animals in each group) were intraperitoneally injected with SEQ ID NO. 4 (0.01 U/g in low-dose-group and 0.28 U/g in high-dose group) at 9:00 am and 15:00 pm respectively on the 15th day, for drug pretreatment. After the second injection of SEQ ID NO. 4 for 30 minutes (i.e. 15:30 pm), NMDA (15 mg/kg) was intraperitoneally injected to establish infant spasticity rat model, and the incubation period from NMDA injection to spasticity was recorded.

[0164] (3) Results: The specific results are shown in FIG. 15. Compared with the sham group, the spasticity incubation period of neonatal rats in IS group is shortened, which indicates that the model of infantile spasticity rats has been successfully constructed. In addition, compared with the IS group, the drug group treated with SEQ ID NO. 4 can significantly prolong the incubation period of spasticity in neonatal rats (*** means p<0.001, in low-dose group; **** means P<0.0001, in high-dose group), implying a potential for antispasmodic application, such as infantile spasm or neonatal spasm.