Immune 7P truncated peptide and application thereof

Abstract

The invention relates to a 7P truncated peptide of an immunogenic peptide of hepatitis C virus and application thereof in the prevention or treatment of liver damages. The invention also relates to a pharmaceutical composition containing the same, a preparation method thereof and pharmaceutical application thereof.

Description

technical field

[0001] The present invention relates to a truncated peptide of hepatitis C virus immunogenic peptide 7P and a method for preventing or treating liver injury using it. The invention also relates to a pharmaceutical composition containing the peptide, a preparation method and a pharmaceutical application. Background technique

[0002] The peptide whose sequence is GQTYTSG (abbreviated as 7P herein) is an immunogenic peptide originally designed according to hepatitis C virus. Peptide 7P and its derivatives. In addition, in PCT application WO / 2007 / 137456, the inventors also found that 7P and its derivatives can also be used for the prevention and treatment of liver damage, especially for the prevention or treatment of immune liver damage and hepatotoxic chemical substances. of liver damage. However, there is no indication of the key parts that play a role in the prevention and treatment of liver damage.

[0003] After research by the inventors, it was found t...

Examples

Embodiment 1

[0023] The synthesis of embodiment 1 peptide

[0024] By solid-phase peptide synthesis method, 413A automatic peptide synthesizer (purchased from Perkin Elmer) was used to synthesize 8 peptides as shown in the following sequences: GQTYTSG (abbreviated as 7P), GQTYTS (abbreviated as P1), QTYTSG (abbreviated as P2), GQTYT (abbreviated as P3), QTYTS (abbreviated as P4), QTYT (abbreviated as P5), TYTS (abbreviated as P6), TYT (abbreviated as P7). The amino acid residues in these peptides are all L-form amino acids. The synthesized peptides were purified by reverse phase HPLC with a gradient of 37-42% acetonitrile / 0.9% TFA. It was then concentrated and freeze-dried. From this, peptides 7P, P1, P2, P3, P4, P5, P6, and P7 were synthesized, respectively. The 8 synthetic peptides were confirmed to have a purity of ≥90% by HPLC, and were used in Example 3 to test their curative effects.

Embodiment 2

[0025] The preparation of embodiment 2 peptide conjugates

[0026] The 7P synthesized in Example 1 was cross-linked with bovine serum albumin (BSA) by the glutaraldehyde method to form a conjugate. The specific conjugation process is as follows: take 1mg of 7P synthesized in Example 1 and dissolve it in 0.5ml PBS (pH7.4, 0.02mol / L); take 4.5mg BSA and dissolve it in 4.5ml PBS (pH7.4, 0.02mol / L); / L). Mix the above 7P and BSA solutions, then slowly add 0.1% glutaraldehyde 1ml, and let the cross-linking reaction proceed for 12 hours at room temperature in the dark. Then glycine solution (1 mol / L) was slowly added to terminate the reaction, followed by dialysis with PBS (pH7.4, 0.02 mol / L) overnight and freeze-dried to obtain the peptide conjugate.

Embodiment 37

[0027] Example 37P and its truncated peptide preventive effect on rat galactosamine liver injury model

[0028] 1. Experimental method

[0029]Drug dosage and grouping: The experimental animals were randomly divided into 11 groups, namely, the blank control group, the model group, the positive drug (Ganlixin injection) group (the dosage was calculated as 13.5 mg / kg.day by diammonium glycyrrhizinate), 7P group (87μg / kg·day), 7 truncated peptide groups (i.e. P1, P2, P3, P4, P5, P6, P7 groups, the doses were 87μg / kg·day), each group 10 SPF grade SD rats (body weight 180g ~ 220g, male and female half and half).

[0030] Administration process: 7 days before the rats were injected with galactosamine, the positive drug group was injected intraperitoneally with Ganlixin injection every day; 14 days before the rats were injected with galactosamine, the 7P group and the P1-P7 groups were subcutaneously injected every other day The corresponding peptide was administered, and at the sa...

technical field

[0001] The present invention relates to a truncated peptide of hepatitis C virus immunogenic peptide 7P and a method for preventing or treating liver injury using it. The invention also relates to a pharmaceutical composition containing the peptide, a preparation method and a pharmaceutical application. Background technique

[0002] The peptide whose sequence is GQTYTSG (abbreviated as 7P herein) is an immunogenic peptide originally designed according to hepatitis C virus. Peptide 7P and its derivatives. In addition, in PCT application WO / 2007 / 137456, the inventors also found that 7P and its derivatives can also be used for the prevention and treatment of liver damage, especially for the prevention or treatment of immune liver damage and hepatotoxic chemical substances. of liver damage. However, there is no indication of the key parts that play a role in the prevention and treatment of liver damage.

[0003] After research by the inventors, it was found t...

Examples

Embodiment 1

[0023] The synthesis of embodiment 1 peptide

[0024] By solid-phase peptide synthesis method, 413A automatic peptide synthesizer (purchased from Perkin Elmer) was used to synthesize 8 peptides as shown in the following sequences: GQTYTSG (abbreviated as 7P), GQTYTS (abbreviated as P1), QTYTSG (abbreviated as P2), GQTYT (abbreviated as P3), QTYTS (abbreviated as P4), QTYT (abbreviated as P5), TYTS (abbreviated as P6), TYT (abbreviated as P7). The amino acid residues in these peptides are all L-form amino acids. The synthesized peptides were purified by reverse phase HPLC with a gradient of 37-42% acetonitrile / 0.9% TFA. It was then concentrated and freeze-dried. From this, peptides 7P, P1, P2, P3, P4, P5, P6, and P7 were synthesized, respectively. The 8 synthetic peptides were confirmed to have a purity of ≥90% by HPLC, and were used in Example 3 to test their curative effects.

Embodiment 2

[0025] The preparation of embodiment 2 peptide conjugates

[0026] The 7P synthesized in Example 1 was cross-linked with bovine serum albumin (BSA) by the glutaraldehyde method to form a conjugate. The specific conjugation process is as follows: take 1mg of 7P synthesized in Example 1 and dissolve it in 0.5ml PBS (pH7.4, 0.02mol / L); take 4.5mg BSA and dissolve it in 4.5ml PBS (pH7.4, 0.02mol / L); / L). Mix the above 7P and BSA solutions, then slowly add 0.1% glutaraldehyde 1ml, and let the cross-linking reaction proceed for 12 hours at room temperature in the dark. Then glycine solution (1 mol / L) was slowly added to terminate the reaction, followed by dialysis with PBS (pH7.4, 0.02 mol / L) overnight and freeze-dried to obtain the peptide conjugate.

Embodiment 37

[0027] Example 37P and its truncated peptide preventive effect on rat galactosamine liver injury model

[0028] 1. Experimental method

[0029]Drug dosage and grouping: The experimental animals were randomly divided into 11 groups, namely, the blank control group, the model group, the positive drug (Ganlixin injection) group (the dosage was calculated as 13.5 mg / kg.day by diammonium glycyrrhizinate), 7P group (87μg / kg·day), 7 truncated peptide groups (i.e. P1, P2, P3, P4, P5, P6, P7 groups, the doses were 87μg / kg·day), each group 10 SPF grade SD rats (body weight 180g ~ 220g, male and female half and half).

[0030] Administration process: 7 days before the rats were injected with galactosamine, the positive drug group was injected intraperitoneally with Ganlixin injection every day; 14 days before the rats were injected with galactosamine, the 7P group and the P1-P7 groups were subcutaneously injected every other day The corresponding peptide was administered, and at the sa...

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