CN1657540A - Pegylated derivatives of human parathyroid hormone (1-34), pharmaceutical compositions containing them and uses - Google Patents

Abstract

The invention discloses pegylated derivatives of human parathyroid hormone (1-34), their pharmaceutical composition and application. The PEGylated derivatives of human parathyroid hormone (1-34) of the present invention are PEGylated to the N-terminal, C-terminal or some amino acid side chains of human parathyroid hormone (1-34). A compound with a novel structure obtained after alcohol modification. The pegylated derivatives of human parathyroid hormone (1-34) of the invention can be used for preparing medicines for treating or preventing osteoporosis and related bone metabolism diseases.

Description

PEGylated derivatives of human parathyroid hormone (1-34), pharmaceutical compositions containing them and use

Technical Field

The invention relates to a compound, in particular to a polyethylene glycol derivative of human parathyroid hormone (1-34), and also relates to a pharmaceutical composition containing the same and application thereof.

Background

Parathyroid hormone (PTH) is a polypeptide hormonesecreted by parathyroid chief cells consisting of 84 amino acid residues. The primary physiological role of PTH is to stimulate osteoclasts, releasing calcium and phosphorus from the bone; promoting reabsorption of calcium by renal tubule, and inhibiting phosphorus resorption; increasing the conversion of 25- (OH) D3 to 1, 25- (OH) in the kidney₂D3; promote the absorption of calcium and phosphorus by the intestinal tract. It also acts on osteoblasts and, in addition,PTH can stimulate osteoblast proliferation and promote bone anabolism, and PTH has the functions of inducing bone formation related to insulin-like growth factors-I, -II (IGF-I, -II) and β transforming growth factor (TGF- β).

PTH relieves bone pain in patients with osteoporosis, increases the levels of alkaline phosphatase, osteocalcin and type I collagen carboxy-terminal extension peptides, and increases urinary hydroxyproline. PTH increases the level of bone resorption indicators, but the bone resorption indicators change later and to a lesser extent than bone formation indicators. A multicenter study in 1980 reported that PTH (1-34) increased iliac spine bone mass by 70% in middle aged and elderly osteoporosis patients, and that newly formed bone morphologically was normal and no hypercalcemia or other adverse effects were observed. Hodsman et al treated osteoporosis with PTH (1-38), with an increase in iliac cancellous bone mineralization surface and bone formation rates after 1 month, an increase in osteoclast number and erosion circumference, and an increase in cancellous bone trabecular thickness observed after 6 months. Finkelstein et al, in early menopausal women treated with PTH (1-34), observed a 3.4% increase in lumbar vertebral density and a 3.5% decrease in bone density in the control group, suggesting that PTH prevents primary osteoporosis. In 2000, Hodsman et al treated 29 patients with severe osteoporosis with PTH (1-34) alone or in combination with calcitonin, observed that after 2 years, the bone formation rate and cortical bone thickness of two groups of cancellous bones were significantly increased, the bone turnover rate was increased by 3 times, and the treatment effect of PTH (1-34) alone and in combination with calcitonin was not different.

Studies have demonstrated that the N-terminal 1-34 fragment of PTH has the biological activity of all PTH. PTH-based formulations can be divided into two generations, the first including native PTH, the amino-terminal 1-34 and 1-38 fragments of PTH. The second generation is a tiny PTH fragment. The amino acid sequence of human PTH (1-34) is as follows:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH

the human PTH (1-34) fragment synthesized by American Gift has been approved by the FDA to be marketed at 26.11.2002 under the trade name Forteo and the generic name teriparatide (teriparatide). Is suitable for male and female osteoporosis patients, and has a common dosage of 20 μ g, and can be administered by subcutaneous injection to leg or abdomen every day.

To date, Forteo is the only drug that has been shown to promote osteogenesis, previous osteoporosis treatments are primarily directed to reducing bone loss, and Forteo is an anabolic class of substances that can increase bone mass and significantly reduce the risk of vertebral and non-vertebral fractures. It is expected to sell for over 5 billion dollars annually in the U.S. market.

PTH (1-34) is a peptide drug, is easily degraded by gastric acid, is quickly reduced to amino acid by protease and aminopeptidase in human plasma, has short half-life and needs injection administration. The PTH (1-34) preparations available at present are expensive and require long-term injection administration. Therefore, the modification of PTH(1-34) prolongs the action time of PTH in vivo, and has better application prospect in developing long-acting, economical and convenient medicaments and preparations.

Researches show that certain protein drugs can keep better biological activity after being modified by polyethylene glycol (PEG) and can obviously prolong the half-life period in organisms. Polyethylene glycol modified adenosine deaminase was approved by FDA to market in 1991 and polyethylene glycol modified interferon was approved by FDA to market in 2001. Peptide drugs have many similarities with protein drugs in structure and properties, and the research on the pegylation modification of peptide drugs is later than that of protein drugs, and some progress has been made. No report on the pegylation modification of PTH (1-34) is available.

Disclosure of Invention

An object of the present invention is to provide a pegylated derivative of human PTH (1-34) having a structure in which cysteine is introduced into N-terminal or C-terminal of human PTH (1-34), maleimide-based polyethylene glycol, vinyl polyethylene glycol or iodoacetyl polyethylene glycol is coupled thereto, or amino or carboxyl groups introduced into carboxyl or amino groups are coupled to amino, C-terminal carboxyl or side chain of N-terminal, C-terminal or side chain of human PTH (1-34).

The polyethylene glycol derivative of human PTH (1-34) of the invention is a compound with a structure shown in a formula (I), and comprises a compound modified by PEG-MAL, PEG-VS or PEG-IODO after cysteine is introduced into the N-terminal of the human PTH (1-34):

PEG-M-Cys-PTH(1-34) (I)

or

Or

PEG represents: RO (CH)₂CH₂O)n-CH₂CH₂R is H or CH₃N is 5-1000; cys is cysteine, covalently linked to the M group by a side chain thioether atom.

The polyethylene glycol derivative of human PTH (1-34) of the present invention is also a compound having the structure of formula (II), including compounds modified with PEG-MAL, PEG-VS or PEG-IODO after introduction of cysteine at the C-terminus of human PTH (1-34):

PTH(1-34)-Cys-M-PEG (II)

PEG, Cys and M are as defined for formula (I).

The polyethylene glycol derivative of human PTH (1-34) of the present invention is also a compound having the structure of formula (III), comprising a PEG covalent compound of amino groups including N-terminal amino group and lysine side chain amino group at any site of human PTH (1-34).

[PEG-X-(CH₂)mCO-NH]z-PTH(1-34) (III)

PEG is defined by the same formula (I); x ═ O, NH or NHCO; m is 0-6; and z is 1-4.

The polyethylene glycol derivative of human PTH (1-34) of the present invention is also a compound having the structure of formula (IV), including compounds in which the carboxyl group at any site of human PTH (1-34), including the C-terminal carboxyl group, the carboxyl groups of the side chains of aspartic acid and glutamic acid, is covalently modified with PEG.

PTH(1-34)-[CO-Y-PEG]z (IV)

PEG is defined by the same formula (I); y is O or NH, and z is 1-5.

Another object of the present invention is to provide a process for the preparation of pegylated derivatives of human PTH (1-34).

The PEG-OH structure used by the invention is as follows: RO (CH)₂CH₂O)n-CH₂CH₂-OH, R ═ H orCH₃And n is 5-1000. PEG-OH with an average molecular weight of several hundred to several tens of thousands is commercially available as a commercial reagent, PEG-NH₂Can be purchased or obtained by the following reaction:

adding PEG-NH₂Respectively reacting with maleic anhydride, vinyl thionyl chloride and iodoacetic anhydride to obtain PEG-MAL, PEG-VS and PEG-IODO. PEG-MAL was obtained by the following reaction:

PTH (1-34) can be synthesized by solid-phase or liquid-phase peptide synthesis, in which a Cys is easily introduced at the N-or C-terminus. Dissolving the peptide chain containing Cys in water, adjusting pH with sodium bicarbonate 7-8, adding 3 times equivalent of PEG-MAL or PEG-VS and PEG-IODO, stirring at room temperature for reaction, and purifying with reversed phase high performance liquid chromatography to obtain PEG modified PTH (1-34) derivative.

The reaction of PEG-MAL to modify Cys-PTH (1-34) is as follows:

PEG-NH₂reacting with succinic anhydride to obtain PEG-NHCOCH₂CH₂COOH, then the compound can be used as a carboxyl component, and can be coupled to the N-end of the polypeptide on a solid phase, and finally the product is obtained by trifluoroacetic acid cleavage and reversed phase high performance liquid chromatography purification. Introducing amino group, carboxyl group or preparing PEGylated modified amino acid at one end of PEG, such as Fmoc-Lys (NH-COCH)₂-PEG) -OH and Fmoc-Asp (CO-NH-PEG) -OH, and then the coupling is carried out in a peptide sequence by a liquid phase or solid phase method to realize the polypeptideN-terminal amino group, C-terminal carboxyl group, Lys side chain amino group, AModification of side chain carboxyl of sp or Glu.

Wang resin, Rink resin, DCC, HOBT, HBTU, NMM, TMBS, TFA and Fmoc-amino acids according to the invention are commercially available.

According to the invention, among the pegylated derivatives of human PTH (1-34), the following compounds are preferred:

Cys(mPEG₅₀₀₀-MAL)-PTH(1-34)

PTH(1-34)-Cys(mPEG₅₀₀₀-MAL)-NH₂

it is also an object of the present invention to provide pharmaceutical compositions containing pegylated derivatives of human PTH (1-34) and the use of pharmaceutical compositions containing them. The composition comprises a pegylated derivative of human PTH (1-34) and a pharmaceutically acceptable excipient.

The inventor of the invention observes that the C-terminal of the human PTH (1-34) still has better biological activity after being modified by pegylation through activity evaluation. The pharmaceutical compositions containing them can be used for the treatment or prevention of osteoporosis and related diseases. The polyethylene glycol derivative of human PTH (1-34) can be combined with one or more excipients to make into dosage forms suitable for human, such as injection with mannitol as excipient, and administered subcutaneously for treating osteoporosis and related diseases.

The human PTH (1-34) polyethylene glycol derivative has the characteristics of novel chemical structure, better biological activity, long half-life period in organisms and the like, and has good application prospect.

In the present invention, all amino acids are in the L-form.

The meanings of the English abbreviations used in the present invention are as follows:

PTH-parathyroid hormone, PEG-polyethylene glycol, PEG-MAL-maleimido polyethylene glycol, PEG-VS-vinylsulfonyl polyethylene glycol, PEG-IODO-iodoacetamido polyethylene glycol, Arg-arginine, Asn-asparagine, Asp-aspartic acid, Cys-cysteine, Gly-glycine, Gln-glutamine, Glu-glutamic acid, His-histidine, Ile-isoleucine, Leu-leucine, Lys-lysine, Met-methionine, Ser-serine, Trp-tryptophan, Val-valine, Boc-tert-butoxycarbonyl, cAMP-cyclic adenosine monophosphate, DCC-dicyclohexylcarbodiimide, DMF-dimethylformamide, EDT-dithioglycol, Fmoc-fluorenylmethyloxycarbonyl, HOBT-1-hydroxybenzotriazole, MALDI-TOF-MS-matrix assisted laser desorption ionization time-of-flight mass spectrometry, Mtr-2, 3, 5-trimethyl-p-methoxy-benzenesulfonyl, RP-HPLC-reversed phase high performance liquid chromatography, TFA-trifluoroacetic acid, TLC-thin layer chromatography, TMBS-trimethylbromosilane, Ts-Cl-p-toluenesulfonyl chloride, Trt-trityl.

Detailed Description

The following examples represent illustrative embodiments of the present invention, but the present invention is not limited by these examples. In the examples, Wang resin as a solid phase synthesis carrier was ACT, Rink resin, DCC, HOBT, Fmoc-protected amino acids were Shanghai Gill biochemical products, TFA was ACROS, TMBS was Fluka, and mPEG-OH having an average molecular weight of 5000 was Sigma.

Example 1 Cys (mPEG)₅₀₀₀Synthesis of-MAL) -PTH (1-34)

Weighing mPEG₅₀₀₀-OH 50.0g (10mmol) was placed in a 250ml reaction flask and 50ml CH was added₂Cl₂After the solid had dissolved, 7.5ml Et was added₃N (50mmmol) and 9.5g Ts-Cl (50mmol), the reaction was stirred at room temperature. After TLC monitoring reaction completion, the solvent was removed by rotary evaporation and solid precipitated by addition of 100ml of anhydrous ether to yield 33.5g mPEG₅₀₀₀OTs, yield 67%.

30.0g mPEG₅₀₀₀OTs (6mmol) were dissolved in 30ml of DMF, and 3.33g (18mmol) of potassium phthalimide salt was added to react at 120 ℃ for 4 hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in 50ml of anhydrous ethanol, 4.0ml of hydrazine hydrate was added, and the reaction was refluxed for 4 hours. The solvent was removed by rotary evaporation and the residue was dissolved in CH₂Cl₂Precipitating with anhydrous diethyl ether to obtain solid, and recrystallizing with anhydrous ethanol-diethyl ether to obtain 22.5g mPEG₅₀₀₀-NH₂The yield thereof was found to be 75%.

5.0g mPEG₂₀₀₀-NH₂Dissolved in 10ml dioxane, added with 2.0g maleic anhydride, and stirred at 80 ℃ for reaction for 30 min. Evaporating the solvent under reduced pressure, adding 50ml of anhydrous ether, cooling and precipitating to obtain a solidThe solid was collected by filtration and dried to give 4.9 g. The resulting solid was dissolved in 15ml of acetic anhydride, 5.0g of sodium acetate was added, and the reaction was stirred at 100 ℃ for 45 min. Evaporating under reduced pressure to remove solvent, dissolving the residue with dichloromethane, filtering to remove insoluble substances, adding appropriate amount of active carbon into the filtrate, standing for 30min, filtering to remove active carbon, concentrating the filtrate to dryness, adding anhydrous ether, precipitating to obtain solid, filtering, and drying to obtain light yellow solid 2.5g mPEG₅₀₀₀MAL, yield 50%.

250mg Wang resin (0.05mmol) as solid phase carrier, Fmoc-Cys (Trt), Fmoc-Arg (mtr) -OH, Fmoc-His (Trt), Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Trp-OH, Fmoc-Phe-OH as raw material, DCC-HOBT as condensing agent, H-Cys (Trt) -Ser (tBu) -Val-Ser (Val) -Glu (OtBu), (OtBu) -Ile-Gln-Met-Trt-His-Gly-Lys (Asn-Gly-Lys (Fmoc-Gly-Trp-OH) as standard Fmoc solid phase polypeptide synthesis method based on the amino acid sequence of PTH (1-34) ) -His-Leu-Asn-Ser (tBu) -Met-Glu (OtBu) -Arg (mtr) -Val-Glu (OtBu) -Trp-Leu-Arg (mtr) -Lys (Boc) -Leu-Gln-Asp (OtBu) -Val-His-Asn-Phe-Wang resin. EDT (0.5mL) -m-cresol (0.1mL) -TMBS (1.0mL) -TFA (7.5mL) was used as a lysate and reacted at 0 ℃ for 90 minutes, the resin was filtered off, the filtrate was rotary evaporated to remove TFA, and anhydrous ether was added to precipitate a white solid, which was collected by filtration, dissolved in water and freeze-dried to give 70mg of a white dry powder.

Dissolving Cys-PTH (1-34) purified by RP-HPLC in water, adjusting pH to 7-8 with sodium bicarbonate, and adding 3 equivalents of mPEG₅₀₀₀MAL, reaction at room temperature, monitoring the progress of the reaction by RP-HPLC and isolation of the product.

Cys(mPEG₅₀₀₀MALDI-TOF-MS analysis of-MAL) -PTH (1-34) shows a series of peaks near 9474, and the difference between the molecular weights of two adjacent peaks is about 44, which has the typical structural characteristics of polyethylene glycol. The analysis of the amino acid composition ratio by acid hydrolysis (6N aqueous hydrochloric acid, 110 ℃, 22 hours) corresponds to the theoretical values: asp, 3.82 (1); ser, 2.64 (3); glu, 5.19 (5); gly, 1.00 (1); val, 2.50 (3); met, 1.52 (2); ile, 0.90 (1); leu, 4.71 (5); phe, 0.87 (1); lys, 2.84 (3); arg, 1.89 (2); his, 2.57 (3).

EXAMPLE 2 PTH (1-34) -Cys (mPEG)₅₀₀₀-MAL)-NH₂Synthesis of (2)

500mg of Rink resin (0.05mmol) as a carrier for solid phase synthesis, Fmoc-Cys (Trt), Fmoc-Arg (mtr) -OH, Fmoc-His (Trt), Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Trp-OH, Fmoc-Phe-OH as a raw material, DCC-HOBT as a condensing agent, H-Ser (tBu), (tBu) -Val-Ser (tBu) -Glu (OtBu) (Ile-Gln-Leu-Met-His-Gly-Leu-Gly-Boc-Gly-Lys (t) -Gly-Lys-) -Gly-Leu-OH as a raw material, and Fmoc-Lys- (Leu) -OH as a raw His-Leu-Asn-Ser (tBu) -Met-Glu (OtBu) -Arg (mtr) -Val-Glu (OtBu) -Trp-Leu-Arg (mtr) -Lys (Boc) -Leu-Gln-Asp (OtBu) -Val-His-Asn-Phe-Cys (Trt) -Rink resin. EDT (0.5mL) -m-cresol (0.1mL) -TMBS (1.0mL) -TFA (7.5mL) was used as a lysate, reacted at 0 ℃ for 90 minutes, the resin was filtered off, the filtrate was rotary evaporated to remove TFA, and anhydrous ether was added to precipitate a white solid, which was collected by filtration, dissolved in water, and lyophilized to obtain 107mg of white dry powder.

RP-HPLC purified PTH (1-34) -Cys-NH₂Dissolving in water, adjusting pH to 7-8 with sodium bicarbonate, adding 3 equivalent of mPEG₅₀₀₀MAL, reaction at room temperature, monitoring the progress of the reaction by RP-HPLC and isolation of the product.

PTH(1-34)-Cys(mPEG₅₀₀₀-MAL)-NH₂Through MALDI-TOF-MS analysis, a series of peaks are arranged around 9484, the difference of the molecular weight of two adjacent peaks is about 44, and the peaks have the typical structural characteristics of polyethylene glycol. The analysis of the amino acid composition ratio by acid hydrolysis (6N aqueous hydrochloric acid, 110 ℃, 22 hours) corresponds to the theoretical values: asp, 3.73 (1); ser, 2.57 (3); glu, 4.96 (5); gly, 1.00 (1); val, 2.65 (3); met, 1.53 (2); ile, 1.00 (1); leu, 4.58 (5); phe, 0.71 (1); lys, 2.78 (3); arg, 1.92 (2); his, 2.87 (3).

Example 3 evaluation of Activity of PEGylated derivative of PTH (1-34)

PTH binds to its receptor and stimulates the cell's signaling system, increasing the level of the second messenger cAMP, and the bioactivity of PTH can be determined by measuring the level of intracellular cAMP after the action of PTH on UMR-106 cells.

The determination result shows that the bioactivity of the synthesized control product human PTH (1-34) is 13849 IU/mg, the bioactivity of the N-terminal modified product Cys (mPEG5000-MAL) -PTH (1-34) is 120IU/mg, and the bioactivity of the C-terminal modified product PTH (1-34) -Cys (mPEG5000-MAL) -NH₂Has biological activity of 4879 IU/mg.

The results show that the C-terminal of human PTH (1-34) can maintain better bioactivity after being modified by pegylation.

Claims (9)

1. A PEGylated derivative of human parathyroid hormone (1-34), characterized in that cysteine is introduced at the N-terminal or C-terminal of human parathyroid hormone (1-34), coupled with There are maleimide-based polyethylene glycol, vinyl polyethylene glycol or iodoacetyl polyethylene glycol, or N-terminal amino, C-terminal carboxyl of human parathyroid hormone (1-34) And the amino group or carboxyl group of certain amino acid side chains, coupled with polyethylene glycol introducing amino group or carboxyl group. 2. The pegylated derivative of human parathyroid hormone (1-34) according to claim 1, characterized in that it is a compound having the structure of formula (I), that is, the N- Compounds modified with PEG-MAL, PEG-VS or PEG-IODO after introducing cysteine at the end:                                                      

or

or

PEG means: RO(CH ₂ CH ₂ O)n-CH ₂ CH ₂ , R=H or CH ₃ , n=5-1000; Cys is cysteine, covalent with M group through side chain thioether atom connected. 3. The pegylated derivative of human parathyroid hormone (1-34) according to claim 1, characterized in that it is a compound having the structure of formula (II), that is, the C- Compounds modified with PEG-MAL, PEG-VS or PEG-IODO after introducing cysteine at the end: PTH(1-34)-Cys-M-PEG (II) The definitions of PEG, Cys, and M are the same as in claim 2. 4. The pegylated derivative of human parathyroid hormone (1-34) according to claim 1, characterized in that it is a compound with the structure of formula (III), including any position in PTH (1-34) A compound in which the amino group of the point is covalently modified by PEG, wherein the amino group includes an N-terminal amino group and a lysine side chain amino group: [PEG-X-(CH ₂ )mCO-NH]z-PTH(1-34) (III) PEG is as defined in claim 2; X=O, NH or NHCO; m=0-6; z=1-4. 5. The pegylated derivative of human parathyroid hormone (1-34) according to claim 1, characterized in that it is a compound with the structure of formula (IV), including any position in PTH (1-34) A compound in which the carboxyl group of the dot is covalently modified by PEG, wherein the carboxyl group includes the side chain carboxyl group of the C-terminal carboxyl group, aspartic acid and glutamic acid: PTH(1-34)-[CO-X-PEG]z (IV) PEG is as defined in claim 2; X=O, NH or NHCO; m=0-6; z=1-5. 6. The pegylated derivative of human parathyroid hormone (1-34) according to claim 2, characterized in that it has the following structure: Cys( _mPEG5000 -MAL)-PTH(1-34). 7. The pegylated derivative of human parathyroid hormone (1-34) according to claim 3, characterized in that it has the following structure: PTH(1-34)-Cys( _mPEG5000 -MAL) _-NH2 . 8. A pharmaceutical composition, characterized by containing the pegylated derivative of human parathyroid hormone (1-34) as claimed in claim 1 and pharmaceutical excipients. 9. The pegylated derivative of human parathyroid hormone (1-34) according to claim 1 or its use as a component of a pharmaceutical composition in the preparation of medicines for treating or preventing osteoporosis and related diseases .