WO2024222770A1 - Long-acting teduglutide compound - Google Patents

Abstract

A long-acting teduglutide compound. The long-acting teduglutide compound is used for preparing a pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for the treatment and prevention of diseases such as short bowel syndrome, intestinal mucosal injury caused by radiotherapy and chemotherapy, ulcerative enteritis, and chronic enteritis.

Description

A long-acting teduglutide compound

This application claims the priority of the Chinese patent application filed with the China Patent Office on April 28, 2023, with application number 202310480890.X and invention name “A Long-Acting Teduglutide Compound”, and the priority of the Chinese patent application filed with the China Patent Office on May 19, 2023, with application number 202310566678.5 and invention name “A Long-Acting Teduglutide Compound”. The entire contents of the above two applications are incorporated into this application by reference.

Technical Field

The present invention relates to the technical field of biomedicine, and in particular to a long-acting teduglutide compound and uses thereof.

Background Art

Teduglutide is a glucagon-like peptide 2 (GLP-2) compound that reduces gastric emptying and secretion and regulates the growth, proliferation and repair of small intestinal lining cells. Teduglutide increases the number of these cells, which can increase small intestinal absorption and reduce diarrhea.

Short bowel syndrome refers to a series of syndromes caused by the inability of the body to absorb nutrients normally due to severe small intestinal disease or surgical removal of most of the small intestine. Before the approval of teduglutide, there was no drug to treat this disease. Most patients can only rely on full or partial parenteral nutrition (intravenous route) to obtain the nutrients needed by the body. However, parenteral nutrition not only seriously affects the quality of life of patients, but also often causes serious complications, such as infection caused by the intravenous channel, bacterial overgrowth in the small intestine, liver toxicity and biliary tract disease. Clinical trials have shown that teduglutide can reduce the demand for parenteral nutrition in patients with short bowel syndrome.

Since teduglutide has a short half-life in vivo, patients need to be subcutaneously administered every day, and patient compliance is poor. The purpose of the present invention is to provide patients with a long-acting teduglutide compound, with an estimated medication frequency of once a week, to improve patient medication compliance, and to have good social and economic benefits.

Summary of the invention

The invention provides a long-acting teduglutide compound and use thereof.

To achieve the above objectives, the present invention first provides a compound shown in structural formula I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the compound, a drug precursor based on the compound, or any mixture of the above forms.

His-AA1-AA2-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp- Leu-Ile-Gln-Thr-AA3-Ile-Thr-Asp-(Lys)n-AA4

Structural formula Ⅰ

AA1 in structural formula I is Gly, or Aib, or Acpc, or Accb;

AA2 in structural formula I is Asp, or Glu, or Ada;

AA3 in structural formula I is Lys, or Arg, or His;

AA4 in structural formula I is NH2, or OH;

In formula I, n is an integer of 1-10.

The long-acting teduglutide compound of the present invention comprises a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex, a drug precursor based on the compound, or any mixture of the above forms.

The present invention also provides a pharmaceutical composition comprising the compound according to the present invention, and provides use of the pharmaceutical composition of the compound according to the present invention in preparing a medicament for treating a disease.

Furthermore, the pharmaceutical composition is used for the treatment and prevention of diseases such as short bowel syndrome, intestinal mucosal damage caused by radiotherapy and chemotherapy, ulcerative enteritis and chronic enteritis.

More contents involved in the present invention are described in detail below, or some of them can also be experienced in the embodiments of the present invention.

Unless otherwise indicated, the quantities of different components and reaction conditions used herein may be interpreted as "roughly" or "approximately" in any case. Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under respective experimental conditions due to different standard errors.

In this article, when there is a discrepancy or ambiguity between the chemical formula and chemical name of a compound, the chemical formula shall be used to accurately define the compound. The compounds described herein may contain one or more chiral centers and/or double bonds and the like, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optical enantiomers or non-isomeric isomers. Enantiomers. Accordingly, any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structure in part or in its entirety, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereomer) and any mixture of these isomers. These racemic isomers and mixtures of stereoisomers can also be further separated into their constituent enantiomers or stereoisomers by those skilled in the art using continuous separation techniques or chiral molecular synthesis methods.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above cases, single enantiomers or diastereomers, such as optically active isomers, can be obtained by asymmetric synthesis or racemate resolution. The resolution of the racemate can be achieved by different methods, such as conventional recrystallization with a resolving agent, or by chromatographic methods. In addition, the compounds of formula I also include cis and/or trans isomers with double bonds.

The compounds of the present invention include, but are not limited to, compounds of formula I and all of their pharmaceutically acceptable forms. The pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures of the above forms.

DETAILED DESCRIPTION

The present invention discloses a long-acting teduglutide compound and its use. Those skilled in the art can refer to the content of this article and appropriately improve the relevant parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. The Chinese names corresponding to the English abbreviations involved in the present invention are shown in the table below:

Table 1

Example 1 Preparation of Compounds

The preparation method is a peptide solid phase synthesis method, including: preparing a peptide resin by a solid phase peptide synthesis method, the peptide resin is then subjected to acid hydrolysis to obtain a crude product, and finally the crude product is purified to obtain a pure product; wherein the step of preparing the peptide resin by the solid phase peptide synthesis method is to sequentially connect the corresponding protected amino acids in the following sequence to the carrier resin by a solid phase coupling synthesis method to prepare the peptide resin:

In the above preparation method, the amount of the Fmoc-protected amino acid is 1.2 to 6 times the total molar number of the resin fed, preferably 2.5 to 3.5 times.

In the above preparation method, the substitution value of the carrier resin is 0.3-1.5 mmol/g resin, and the preferred substitution value is 0.6-1.0 mmol/g resin.

As a preferred embodiment of the present invention, the solid phase coupling synthesis method is: the protected amino acid-resin obtained in the previous step is deprotected from the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time of the Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

The coupling reaction requires the addition of a condensation reagent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

The coupling reaction requires the addition of an activation reagent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of the activation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

As a preferred solution of the present invention, the Fmoc-removing reagent is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, wherein the piperidine content in the mixed solution is 10-30% (V). The amount of the Fmoc-removing reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and the side chain protecting groups and then subjected to oxidative cyclization to obtain a crude product:

Further preferably, the acid hydrolysis agent used in the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA is 80-95%, EDT is 1-10%, and the balance is water.

More preferably, the volume ratio of the mixed solvent is: 89-91% TFA, 4-6% EDT, and the balance is water. Optimally, the volume ratio of the mixed solvent is: 90% TFA, 5% EDT, and the balance is water.

The amount of the acid hydrolysis agent is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.

The acid hydrolysis agent is used for 1 to 6 hours at room temperature, preferably 3 to 4 hours.

The oxidative cyclization uses an oxidant such as iodine, H2O2 or DMSO, preferably iodine. The oxidant is added in a titration manner and the addition is stopped when the oxidation endpoint is reached.

Furthermore, the crude product was purified by high performance liquid chromatography and freeze-dried to obtain a pure product.

1. Synthesis of peptide resin

Take the carrier resin, remove the Fmoc protection and couple the reaction, and sequentially connect the protected amino acids corresponding to the sequence to obtain the peptide resin:

(1) Insertion of the first protected amino acid

Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, slowly add it into the protected amino acid DMF solution with stirring, and stir the reaction at room temperature for 30 minutes to obtain the activated protected amino acid solution for use.

Take 0.01 mol of Rink amide MBHA resin (substitution value is about 0.4 mmol/g), use 20% PIP/DMF solution to deprotect for 25 minutes, wash and filter to obtain the de-Fmoc resin.

The activated first protected amino acid solution is added to the Fmoc-free resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing one protected amino acid is filtered and washed to obtain.

(2) Accession of other protected amino acids

The same method as the first protected amino acid in the main chain is used to sequentially insert the other corresponding protected amino acids in the main chain to obtain a resin containing main chain amino acids.

(3) Finally, go to protect

Deprotection was performed using 20% PIP/DMF solution for 25 minutes, followed by washing, filtration and vacuum drying to obtain the peptide resin free of Fmoc.

2. Preparation of crude product

Take the above peptide resin, add a cleavage reagent with a volume ratio of TFA: water: EDT = 95:5:5 (cleavage reagent 10 mL/gram of resin), stir evenly, and react at room temperature for 3 hours. The reaction mixture is filtered using a sand core funnel, and the filtrate is collected. The resin is washed 3 times with a small amount of TFA, and the filtrate is combined and concentrated under reduced pressure. Anhydrous ether is added to precipitate, and the precipitate is washed 3 times with anhydrous ether. It is dried in vacuo to obtain a crude product.

3. Preparation of pure product

Take the above crude product, dissolve it with 10% acetic acid solution, filter it with a 0.45 μm mixed microporous filter membrane, and purify it for later use;

The purification was carried out by high performance liquid chromatography, the chromatographic filler for purification was 10 μm reverse phase C18, the mobile phase system was 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of the 30mm*250mm chromatographic column was 20mL/min, the gradient system was used for elution, and the cyclic injection purification was carried out to take the crude solution Load the sample into the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the purified intermediate concentrate;

The purified intermediate concentrate is filtered through a 0.45 μm filter membrane for standby use, and the salt is exchanged by high performance liquid chromatography, the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic filler for purification is 10 μm reverse phase C18, and the flow rate of the 30 mm*250 mm chromatographic column is 20 mL/min (the corresponding flow rate can be adjusted according to the chromatographic columns of different specifications); the gradient elution and the circular loading method are adopted, the sample is loaded into the chromatographic column, the mobile phase elution is started, the spectrum is collected, the change of the absorbance is observed, the main peak of the salt exchange is collected and the purity is detected by the analytical liquid phase, the main peak solution of the salt exchange is combined, and it is concentrated under reduced pressure to obtain a pure acetic acid aqueous solution, and freeze-dried to obtain a pure product.

The following compounds were synthesized using the above method:

Table 2

Example 2 Activity determination

1. Determination method

GLP-2R, under the stimulation of its specific agonist, can activate the intracellular adenylate cyclase pathway, increase the cAMP level, and ultimately lead to the production and release of insulin. By stimulating the cell line stably transfected with GLP-1R with the test substance, the intracellular cAMP level of the cell increases rapidly, and the relative light unit (RLU) after each dose of cell stimulation is measured by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity determination method is currently a common GLP-2 receptor agonist activity detection method at home and abroad.

The CHO-K1 cell line stably expressing GLP-2R was used to stimulate the stably transfected cells with different concentrations of agonists. The EC50 value of the agonist was calculated by measuring the relative light units after the cells were stimulated at each dose.

2. Measurement results

The results of the measurements are shown in the following table:

Table 3

Example 3 Determination of preliminary pharmacokinetic properties

The experimental animals were cynomolgus monkeys, and the drug was administered subcutaneously at a dose of 1.5 mg/kg. Blood was collected venously before drug administration (0h) and 1h, 2h, 3h, 4h, 8h, 12h, 18h, 24h, 48h, 96h, 144h, and 168h after administration. The plasma samples were separated by centrifugation, and the blood drug concentration of the compound in the plasma samples was determined by liquid chromatography-mass spectrometry. The half-life of the compound after subcutaneous (SC) administration is shown in the table below:

Table 4

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (6)

1. A long-acting teduglutide compound having structural formula I:
   His-AA1-AA2-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-As
   n-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-AA3-Ile-Thr-Asp-(Lys)n-AA4
   Structural formula Ⅰ

   AA1 in structural formula I is Gly, or Aib, or Acpc, or Accb;

   AA2 in structural formula I is Asp, or Glu, or Ada;

   AA3 in structural formula I is Lys, or Arg, or His;

   AA4 in structural formula I is NH2, or OH;

   In formula I, n is an integer of 1-10.
2. The long-acting teduglutide compound according to claim 1 comprises a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex, a prodrug based on the compound, or any mixture of the above forms.
3. The long-acting teduglutide compound according to claim 1 or claim 2, wherein the long-acting teduglutide compound is used to prepare a pharmaceutical composition for treating a disease.
4. The long-acting teduglutide compound according to claim 3, wherein the pharmaceutical composition is used for the treatment and prevention of short bowel syndrome, intestinal mucosal damage caused by radiotherapy and chemotherapy, ulcerative enteritis and chronic enteritis.
5. A pharmaceutical composition, characterized in that it comprises the long-acting teduglutide compound according to any one of claims 1 to 4.
6. Use of the long-acting teduglutide compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for preventing and/or treating a disease;

   The disease includes at least one of short bowel syndrome, intestinal mucosal damage caused by radiotherapy and chemotherapy, ulcerative enteritis, and chronic enteritis.