CN116162147B

CN116162147B - Long-acting insulin analogue

Info

Publication number: CN116162147B
Application number: CN202111403061.9A
Authority: CN
Inventors: 高俊萍; 周述靓; 王鹏; 邓岚; 潘文强
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2021-11-24
Filing date: 2021-11-24
Publication date: 2023-10-03
Anticipated expiration: 2041-11-24
Also published as: CN116162147A; WO2023093758A1

Abstract

The invention relates to the field of medicine synthesis, and discloses a long-acting insulin analogue. The long-acting insulin analogue is used for preparing a pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for treating various diseases, including type I diabetes, type II diabetes and gestational diabetes.

Description

Long-acting insulin analogue

Technical Field

The invention relates to a long-acting insulin analogue and application thereof.

Background

Insulin is a protein hormone secreted by islet beta cells in the pancreas by stimulation with endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc., insulin is the only hormone in the body that reduces blood glucose, and promotes glycogen, fat, protein synthesis, exogenous insulin is mainly used for diabetes treatment.

Unlike other recombinant protein medicines, insulin is used as hormone for regulating blood sugar, has very narrow treatment window, has to strictly control blood concentration, is easy to generate hypoglycemia, shock and death when being too high, and is easy to generate hyperglycemia when being too low. Thus, insulin cannot be administered intravenously and can only be slowly released into the blood by subcutaneous injection. In addition, the sugar intake of three meals a day requires the insulin regulation at the time of meal, and the basal blood sugar is regulated by basal insulin, so that the physiological insulin curve is extremely complex, and the design significance of quick-acting insulin and long-acting insulin is also achieved.

Insulin drugs are typically formulated at 100IU/ml, at which concentration insulin typically forms dimers and thus hexamers. After subcutaneous injection of insulin, the hexamer will gradually release dimer-monomer and then enter the blood for onset. Because the insulin action time is 4-6 hours, the insulin has longer action time than the meal insulin, is easy to be hypoglycemia after 2 hours after meal, and has shorter action time than the basal insulin. Protamine and insulin combine to form precipitate, which results in slow dissolution and release after subcutaneous injection of insulin, and the action time is prolonged to tens of hours, typically twice daily. Although the protamine insulin partially solves the problems, the insulin secretion curves still have great differences in physiological states, and the precipitation property of the protamine insulin determines that the protamine insulin is a suspension, so that the accuracy of dosage control is difficult to ensure.

Insulin glargine of Sainofil is similar to the slow release effect of protamine, and by adding 2 basic amino acids arginine at the end of the B chain, the isoelectric point of insulin glargine is changed from 5.4 to 6.7, and after subcutaneous injection, precipitation is formed near the isoelectric point, and slow dissolution is explained and put into blood. However, the change of isoelectric point determines that insulin glargine must be prepared into an acidic preparation to be soluble, and asparagine which is easy to undergo deamidation of A21 is mutated into glycine, which is the name of insulin glargine, the half life of insulin glargine is 12 hours, the action time is 20-24 hours, and the insulin glargine is injected once a day.

Noand Nord developed a new generation of long acting insulin, degu insulin, which was successfully marketed in 2013, removed threonine at position B30, and linked a 16-carbon fatty acid chain to lysine at position B29 via a glutamic acid linker, which can bind to albumin in plasma and prolong half-life, this mechanism being the same as Det insulin. In addition, by optimizing the proportion of the linker to the fatty acid chain and zinc ions, the deluge insulin is in the form of double hexamers in the preparation, phenol diffuses after subcutaneous injection, the deluge insulin undergoes conformational change, and linear polyhexamethylene is rapidly assembled from the double hexamers, generally up to several thousand molecules or more, and the polyhexamethylene slowly releases the hexamer-dimer-monomer into the blood for effect. By both mechanisms, the half-life of insulin deluge reaches 24 hours and the duration of action reaches 42 hours.

Although the long-acting insulin achieves the purpose of once daily administration, it cannot achieve a longer administration time and cannot achieve once weekly administration. It is an object of the present invention to provide a long acting insulin analogue with a longer half-life.

Disclosure of Invention

The invention provides a long-acting insulin analogue and application thereof.

To achieve the above object, the present invention provides a compound of the formula I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, a prodrug based on the compound, or a mixture of any of the above forms.

Structure I

AA1 in structure I is Asp, or Glu, or Ada, or Apm, or Asu;

AA2 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain SH groups, or is absent;

AA3 in structure I is His, or Tyr, or Phe;

AA4 in structure I is His, or is Tyr, or is Phe;

AA5 in structure I is His, or is Tyr, or is Phe;

AA6 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain SH groups, or is absent;

AA7 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain SH groups, or is absent;

AA8 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain SH groups, or is absent;

AA9 in structure I is Lys, or is Dah, or is Orn, or is Dab, or is Dap, asp [ NH (CH 2) mNH ], or is Glu [ NH (CH 2) mNH ], or is Ada [ NH (CH 2) mNH ], or is Apm [ NH (CH 2) mNH ], or is Asu [ NH (CH 2) mNH ], or is absent;

wherein: m is an integer of 2 to 10

AA10 in structure I is any encodable amino acid other than Cys, or is any non-encodable amino acid that does not contain SH groups, or is absent;

AA11 in structure I is Lys, or is Dah, or is Orn, or is Dab, or is Dap, asp [ NH (CH 2) mNH ], or is Glu [ NH (CH 2) mNH ], or is Ada [ NH (CH 2) mNH ], or is Apm [ NH (CH 2) mNH ], or is Asu [ NH (CH 2) mNH ], or is absent;

wherein: m is an integer of 2 to 10

R1 and R2 in structure I are HO ₂ C(CH ₂ )n1CO-(γGlu)n2-(PEGn3(CH ₂ )n4CO)n5-；

Wherein: n1 is an integer from 10 to 25;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5;

r1 and R2 in structure I cannot be present at the same time;

the invention also provides a composition comprising a compound according to the invention and Zn ²⁺ The complex formed.

The invention also provides a composition comprising a compound according to the invention and Zn ²⁺ Pharmaceutical compositions of the complexes formed and pharmaceutical compositions of the compounds of the invention are provided for use in the preparation of pharmaceutical compositions for the treatment of diseases.

Use of the pharmaceutical composition in the treatment of various diseases including type I diabetes, type II diabetes, gestational diabetes.

Further details of the invention are set forth in the accompanying drawings and the description below, or may be learned by practice of the invention. Unless otherwise indicated, the amounts of the various components, reaction conditions, and the like, are used herein and are to be construed in any sense as "generally", "about". Accordingly, unless explicitly indicated otherwise, the numerical parameters set forth in the following claims are approximations that may vary depending upon the standard deviation employed under the particular circumstances.

Herein, when the chemical structural formula and chemical name of a compound are divergent or ambiguous, the compound is defined exactly by the chemical structural formula. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and stereoisomers, including isomers of double bonds (such as geometric isomers), optical enantiomers or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description herein, whether partial or whole containing such structures, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer), and mixtures of any of these isomers. These racemic isomers and mixtures of stereoisomers may also be resolved further into their constituent enantiomers or stereoisomers by methods known to those skilled in the art using continuous separation techniques or chiral molecule synthesis.

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 optical isomers, may be obtained by asymmetric synthesis or resolution of racemates. Resolution of the racemate can be accomplished in various ways, such as recrystallization with conventional resolution-aiding reagents, or by chromatographic methods. In addition, the compounds of the formula I also contain cis-and/or trans-isomers with double bonds.

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

Detailed Description

The invention discloses a long-acting insulin analogue and application thereof, and a person skilled in the art can properly improve related parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the compounds and methods of preparation described herein, or in appropriate combinations, without departing from the spirit and scope of the invention.

The Chinese names corresponding to the English abbreviations in the invention are shown in the following table:

english abbreviations	Chinese name	English abbreviations	Chinese name
				Fmoc	9-fluorenylmethoxycarbonyl	OtBu	Tert-butoxy radical
tBu	Tert-butyl group	Boc	Boc acid tert-butyl ester
				Trt	Trityl radical	Pbf	(2, 3-dihydro-2, 4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl
Ala	Alanine (Ala)	Leu	Leucine (leucine)
				Arg	Arginine (Arg)	Lys	Lysine
Asn	Asparagine derivatives	Phe	Phenylalanine (Phe)
				Asp	Aspartic acid	Pro	Proline (proline)
Cys	Cysteine (S)	Ser	Serine (serine)
				Gln	Glutamine	Thr	Threonine (Thr)
Glu	Glutamic acid	Trp	Tryptophan
				Gly	Glycine (Gly)	Tyr	Tyrosine
His	Histidine	Val	Valine (valine)
				Ile	Isoleucine (Ile)	Dah	2, 7-diaminoheptanoic acid
Aib	Amino isobutyric acid	Dao	2, 8-diamino octanoic acid
				5-Ava	5-Aminopentanoic acid	Ada	2-aminocaproic acid
Dap	2, 3-diaminopropionic acid	Apm	2-amino heptanoic acid
				Dab	2, 4-diaminobutyric acid	Asu	2-Aminooctanoic acid
Orn	Ornithine

Example 1 preparation of Compounds

The preparation method comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, acidolysis is carried out on the peptide resin to obtain a crude product, and finally, the crude product is purified to obtain a pure product; wherein the step of preparing peptide resin by solid phase polypeptide synthesis method comprises the steps of sequentially accessing corresponding protected amino acid or fragment in polypeptide sequence on carrier resin by solid phase coupling synthesis method, and preparing peptide resin:

in the preparation method, the dosage of the Fmoc-protected amino acid is 1.2 to 6 times of the total mole number of the resin; preferably 2.5 to 3.5 times.

In the preparation method, the substitution value of the carrier resin is 0.2-0.6 mmol/g resin, and the preferred substitution value is 0.2-0.4 mmol/g resin.

As a preferred scheme of the invention, the solid phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is subjected to Fmoc protecting group removal and then is subjected to coupling reaction with the next protected amino acid. The deprotection time for 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 needs to add a condensation reagent, wherein the condensation reagent is selected from DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 2- (7-aza-1H-benzotriazol-1-yl) -1, 3-tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar amount of the condensing agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total molar amount of the amino groups in the amino resin.

The coupling reaction needs to add an activating reagent, and the activating reagent is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, and is preferably 1-hydroxybenzotriazole. The amount of the activating agent to be used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total mole number of the amino groups in the amino resin.

As a preferred scheme of the invention, the Fmoc protection removing reagent is PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution, and the mixed solution contains 10-30% (V) of piperidine. The Fmoc-removing protective agent is used in an amount of 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acidolysis and simultaneously the resin and side chain protecting group are removed to obtain a crude product:

further preferably, the acidolysis agent used in acidolysis 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 as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

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

The dosage of the acidolysis agent is 4-15 mL of acidolysis agent required by each gram of peptide resin; preferably, 7 to 10mL of acidolysis agent is required per gram of peptide resin.

The time for cleavage with acidolysis agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

Further, purifying the crude product by high performance liquid chromatography, and lyophilizing to obtain pure product.

1. Synthesis of peptide resins

The Rink Amide MBHA resin is used as carrier resin, and is coupled with the protected amino acid corresponding to the polypeptide sequence in sequence through Fmoc protection removal and coupling reaction, so as to prepare the peptide resin.

(1) Cys side chain protecting group

Chain A: the side chain protecting groups of the 6 th and 11 th Cys are Trt, and the side chain protecting group of the 7 th Cys is Acm.

Chain B: the side chain protecting group of the 7 th Cys is Acm, and the side chain protecting group of the 19 th Cys is Mtt.

(2) Access to the 1 st protected amino acid of the B chain

Taking 0.03mol of the 1 st protected amino acid of the B chain sequence and 0.03mol of HOBt, and dissolving the protected amino acid and the HOBt with a proper amount of DMF; and (3) adding 0.03mol of DIC into the protected amino acid DMF solution slowly under stirring, and stirring and reacting for 30 minutes in a room temperature environment to obtain an activated protected amino acid solution for later use.

0.01mol of Rink amide MBHA resin (substitution value about 0.3 mmol/g) was taken and deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed resin.

And adding the activated 1 st protected amino acid solution into Fmoc-removed resin, performing coupling reaction for 60-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.

(3) Access to B-chain protected amino acids

And sequentially accessing the protective amino acids corresponding to the B chain sequence by adopting the same method for accessing the 1 st protective amino acid of the B chain to obtain the protective peptide resin of the B chain.

(4) Access to the 1 st protected amino acid of the A chain

Removing the Mtt protecting group of the Cys side chain at the 19 th position of the B chain by adopting 50% HFIP/DCM solution, repeating for 5 times, each time for 35 minutes, filtering and washing to obtain the resin with the Mtt protecting group removed for later use.

Taking 0.3mol of 2,2' -dithiodipyridine, dissolving the obtained product by using a proper amount of DMF, adding the obtained product into the resin with the Mtt protection removed, stirring the obtained product for reaction for 3 hours, and filtering and washing the obtained product to obtain SH activated resin for later use.

Taking 0.03mol of 1 st protected amino acid (Fmoc-Cys-Asn (Trt) -OtBu) of the A chain, dissolving with a proper amount of DMF, adding into the SH activated resin, adding 2ml of DIPEA, stirring for reaction for 3 hours, filtering and washing, and completing the access of the first amino acid of the A chain.

(5) Access to other protected amino acids of the A chain

And (3) sequentially accessing the protective amino acid corresponding to the A chain sequence by adopting the same method for accessing the 1 st protective amino acid of the B chain, and washing and drying to obtain the peptide resin.

2. Preparation of crude product

Adding a cracking reagent (10 mL/g resin) with a volume ratio of TFA: tis: water=95:5:5 into the peptide resin, uniformly stirring, stirring at room temperature, reacting for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous diethyl ether for precipitation, washing the precipitate with anhydrous diethyl ether for 3 times, and pumping to obtain white-like powder.

Dissolving the obtained white powder with 20% DMSO aqueous solution, regulating pH to 7.5 with ammonia water, stirring for reaction for 10 hours, adding glacial acetic acid to acetic acid 20%, dropwise adding iodine/ethanol saturated solution under stirring until complete cyclization, and concentrating under reduced pressure at 35-40 ℃ to obtain crude concentrated solution.

3. Preparation of pure product

Taking the crude product concentrated solution, filtering with a 0.45 mu m mixed microporous filter membrane, and purifying for later use;

purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is reverse phase C18 with the size of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column with the size of 30mm or 250mm is 20mL/min, eluting by a gradient system, circularly sampling and purifying, sampling the crude product solution into the chromatographic column, starting mobile phase eluting, collecting main peaks, evaporating acetonitrile, and obtaining purified intermediate concentrated solution;

filtering the purified intermediate concentrate with 0.45 μm filter membrane for use, changing salt by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic column flow rate of purification column is 20mL/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications) with reversed phase C18 of 10 μm and 30mm x 250 mm; adopting a gradient elution and cyclic loading method, loading in a chromatographic column, starting mobile phase elution, collecting a spectrum, observing the change of absorbance, collecting a salt-exchange main peak, analyzing the liquid phase to detect the purity, combining the salt-exchange main peak solutions, concentrating under reduced pressure to obtain a pure acetic acid aqueous solution, and freeze-drying to obtain the pure peptide.

The following lipopeptides were synthesized using the above procedure:

example 2 determination of biological Activity

1. Measurement method

The cell lines stably transfected with insulin receptor are stimulated by the test substance, so that the cell insulin phosphorylating receptor level is rapidly increased, and the biological activity of each analogue is evaluated by measuring the phosphorylating receptor after each dose of stimulated cells.

The HEK293-IRA cell strain which stably expresses insulin receptor is adopted, the agonist with different concentrations is used for stimulating stable transfer cells, and the EC of each analogue is calculated by measuring the phosphorylated receptor after each dose of stimulated cells ₅₀ Values.

2. Measurement results

The measurement results are shown in the following table:

compounds of formula (I)	Biological Activity [ EC ₅₀ (nmol)】
		Compound 1	40.7
Compound 2	36.2
		Compound 3	35.9
Compound 4	36.1
		Compound 5	42.5
Compound 6	36.9

EXAMPLE 3 determination of Primary drug substitution Properties

1. Test method

Using male SD rats, plasma samples were isolated by centrifugation using subcutaneous doses of 1mg/kg, taken from the orbital veins of the rats before (0 h), and 1h, 2h, 3h, 4h, 8h, 24h, 48h, 96h, 144h after administration.

Plasma concentrations of the corresponding compounds in SD rat plasma samples were determined by liquid chromatography-mass spectrometry, and the compound SD rat Subcutaneous (SC) administration half-life was calculated.

2. Test results

The test results are shown in the following table:

compounds of formula (I)	t _1/2 (h)
		Compound 1	24.3
Compound 2	21.8
		Compound 3	25.9
Compound 4	28.4
		Compound 5	25.5
Compound 6	27.6

Claims

1. A long acting insulin analogue selected from one of the following structures:

；

the method comprises the steps of carrying out a first treatment on the surface of the Or (b)

。

2. A pharmaceutically acceptable salt of the long acting insulin analogue of claim 1.

3. A pharmaceutical composition comprising the long acting insulin analogue of claim 1.

4. Use of a long acting insulin analogue according to claim 1 for the preparation of a medicament for the treatment of type I diabetes, type II diabetes, gestational diabetes.