Sustained release-microsphere formulation comprising semaglutide or pharmaceutically acceptable salt thereof and preparation method therefor

Abstract

The present invention relates to a pharmaceutical composition that contains sustained-release microspheres composed of semaglutide or a pharmacologically acceptable salt thereof, a bioavailability enhancer, and a biodegradable polymer and which is designed to avoid rapid initial drug release (initial burst), contain a high concentration of the drug relative to particle size, have high bioavailability, and minimize patient discomfort and inflammatory reactions when administered to the human body, whereby the composition is useful in the prevention or treatment of diabetes, hypertension, hyperlipidemia, obesity, non-alcoholic steatohepatitis, or degenerative neurological diseases such as Alzheimer's and Parkinson's disease and in the preservation of beta-cell functions.

Description

0.125 to 0.450 g of semaglutide as a drug (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) and 0 to 1.2 g of a bioavailability enhancer (see Table 1) were used, and finally, the biodegradable polymer was weighed using at least one of R203H, RG203H, RG503H, RG653H, RG753H, PDL04A, and PDLG7504A in the range of 0.75 to 2.55 g, so that the batch size was 1.0 to 3.0 g (in Preparation Examples 1 to 32, Preparation Examples 16, 19, and 30 have a batch size of 3.0g, Preparation Examples 1, 13, 14, 15, 29, and 30 have a batch size of 2.0g, and the remainder have a batch size of 1.0g.). Dichloromethane was used as a solvent to prepare the dispersed phase, and Preparation Examples 4 to 29 and Preparation Examples 31 and 32 were homogeneously dissolved using at least one of glacial acetic acid and methanol as a cosolvent. The primary emulsion was prepared by dispersing the aqueous phase in the organic phase using a 40 pm porous emulsion membrane. In Preparation Examples 2 to 32, 2,000 ml of an aqueous solution containing 0.1% of polyvinyl alcohol (viscosity: 4.8-5.8 mPa.s) and 2.0% of Na2HPO4 (disodium phosphate) were used as the continuous phase, and in Preparation Example 1, 2,000 ml of an aqueous solution containing polyvinyl alcohol (viscosity: 4.8- 5.8 mPa.s) and 1.0% of NaCl were used. The continuous phase was connected to an emulsification device equipped with a porous membrane with a diameter of 40 pm, and the prepared dispersed phase was injected into the porous membrane together with the continuous phase. An emulsion having dispersed biodegradable polymer microdroplets containing semaglutide was prepared, and the suspension was placed in a preparation container and stirred at a speed of 200 to 300 rpm. The temperature of the preparation container was maintained at 25 °C, and when the injection of the dispersed phase was completed, the organic solvent was removed while maintaining the suspension temperature at 40°C for 3 hours. Subsequently, the temperature was cooled to 25°C, the microspheres were filtered, treated for removing 5 residual polyvinyl alcohol by using tertiary distilled water, and lyophilized. Preparation Examples 5 and 10 did not include an initial-release inhibitor in the continuous phase. [Table 1] Preparati on Example No. Type of polymer Used amount of polymer (g) Used amount of semaglutid e(g) Type       / used amount       of bioavailability enhancer (g) Solvent Co-solvent Initialrelease inhibitor and concentratio n Preparati on Example 1 a)d) RG653H:R G753H=1:1 1.52 0.150 Poloxamer F127 / 0.33 DCM - l%NaCl Preparati on Example 2a) d) PDL04A 0.78 0.200 Disodium phosphate / 0.02 DCM - 2% Na2HPO4 Preparati on Example 3 b) d) PDLG7504 A 0.76 0.200 Sodium Decanoate / 0.02 , Disodium phosphate / 0.02 DCM - 2% Na2HPO4 Preparati on Example 4 b) c) PDLG7504 A 0.79 0.200 Benzathine      / 0.006 DCM DCM:acetic acid:mcthanoH 16:4:1 2% Na2HPO4 Preparati on Example 5 b) c) PDLG7504 A 0.80 0.200 N / A DCM DCM:acetic acid:methanol 16:4:1 2.5% NaCl Preparati on Example 6 b) c) PDLG7504 A 0.80 0.200 N / A DCM DCM:acetic acid:mcthanoH 16:4:1 2% Na2HPO4 Preparati on Example 7 b) c) PDLG7504 A 0.79 0.200 Benzathine      / 0.012 DCM DCM:acetic acid:methanol 16:4:1 2% Na2HPO4 Preparati on Example 8 b) c) PDLG7504 A 0.78 0.200 Benzathine      / 0.023 DCM DCM:acetic acid:mcthanoH 16:4:1 2% Na2HPO4 Preparati PDLG7504 0.75 0.200 Benzathine / 0.05 DCM DCM:acetic 2% on Example 9 b) c) A acid:methanoH 16:4:1 Na2HPO4 Preparati on Example 10 b) c) RG653H:R G753H=1:1 0.80 0.200 N / A DCM glacial acid acetic 2.5% NaCl Preparati on Example 11 b) c) RG503H:R G653H=1:1 0.86 0.125 Benzathine 0.012 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 12 b) c) RG653H:R G753H=1:1 0.84 0.150 Benzathine 0.012 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 13 b)c) RG653H:R G753H=1:1 1.63 0.350 Benzathine 0.024 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 14 b) c) RG653H:R G753H=1:1 1.58 0.400 Benzathine 0.024 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 15 b) c) RG653H:R G753H=1:1 1.60 0.400 N / A DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 16 b) c) R203H 2.55 0.450 N / A DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 17b)c) RG653H:R G203H=l:2 0.85 0.150 N / A DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 1 8 b) c) RG503H 0.84 0.150 Benzathine 0.012 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 19b)c) RG653H 2.51 0.450 Benzathine 0.036 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 20 b) C) RG753H 0.84 0.150 Benzathine 0.012 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example 21 b)c) RG653H:R G203H=l:2 0.84 0.150 Benzathine 0.012 / DCM glacial acid acetic 2% Na2HPO4 Preparati on Example RG653H:R 203H-3:l 0.84 0.150 Benzathine 0.012 / DCM glacial acid acetic 2% Na2HPO4 22 b> C) Preparati on Example 23 b) c) RG653H:R 203H=l:l 0.84 0.150 Benzathine      / 0.012 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 24 b) C) RG653H:R 203H=l:3 0.84 0.150 Benzathine      / 0.012 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 25 b) c) RG653H:R 203H=3:l 0.80 0.200 N / A DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 26 b) c) RG653H:R 203H=l:l 0.80 0.200 N / A DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 27 b) c) RG653H:R 203H=l:3 0.80 0.200 N / A DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 28 b) C) RG653H:R 203H=l:l 0.80 0.200 N / A DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 29 b) C) RG653H:R 203H=l:l 0.85 0.150 N / A DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 30 b) c) RG653H:R G753H=1:1 2.55 0.450 N / A DCM 2% Na2HPO4 Preparati on Example 31 b) c) RG653H:R G753H=1:1 1.69 0.300 Benzathine      / 0.006 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 32 b) c) RG653H:R G753H=1:1 1.68 0.300 Benzathine      / 0.024 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 52 a) d) 653H:753H =1:1 0.4 0.150 N / A DCM 2.5% NaCl Preparati on Example 53 a) d) 653H:753H =1:1 0.4 0.150 HPbCD / 0.15 DCM 2.5% NaCl Preparati on Example 54 a) d) 653H:753H =1:1 0.4 0.150 Sucrose / 0.04 DCM 2.5% NaCl Preparati 653H:753H 0.42 0.150 Benzathine      / DCM glacial acetic - on Example 55 b) c) =1:1 0.006 acid Preparati on Example 56 b) c) 653H:753H =1:1 0.42 0.150 Benzathine      / 0.006 and 2% Na2HPO4 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 57 b) c) 653H:753H =1:1 0.85 0.150 2% K2HPO4 DCM glacial acetic acid 2% K2HPO4 Preparati on Example 58 b) c) 653H:753H =1:1 0.43 0.150 2% NaH2PO4 DCM glacial acetic acid 2% NaH2PO4 Preparati on Example 59b)C) 653H:753H =1:1 0.43 0.150 0.5% Na2HPO4 DCM glacial acetic acid 0.5% Na2HPO4 Preparati on Example 60 b) c) 653H:753H =1:1 0.43 0.150 l%Na2HPO4 DCM glacial acetic acid 1.0% Na2HPO4 Preparati on Example 61 b) c) 653H:753H =1:1 0.43 0.150 3% Na2HPO4 DCM glacial acetic acid 3.0% Na2HPO4 Preparati on Example 62 b) c) 653H:753H =1:1 0.43 0.150 4% Na2HPO4 DCM glacial acetic acid 4.0% Na2HPO4 Preparati on Example 63 b) c) 653H:753H =1:1 0.43 0.075 2% Na2HPO4 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 64 b) c) 653H:753H =1:1 0.85 0.15 2% Na2HPO4 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 65 b) c) 653H:753H =1:1 0.85 0.15 2% Na2HPO4 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 66 b> C) 653H:753H =1:1 0.85 0.15 DCM glacial acetic acid Preparati on Example 67 b) c) 653H:753H =1:1 46.1 8.25 Benzathine / 0.66 and 2% NaH2PO4 DCM glacial acetic acid 2% Na2HPO4 Preparati on Example 653H:753H =1:1 3.4 0.6 2% Na2CO3 DCM glacial acetic acid 2% Na2CO3 68 b) c) Preparati on Example 69 b) c) 653H:753H =1:1 0.85 0.15 2% NaHCO3 DCM glacial acetic acid 2% NaHCO3 Preparati on Example 70 b) C) 653H:753H =1:1 0.85 0.15 2% (NH4)2SO4 DCM glacial acetic acid 2% (NH4)2SO4 Preparati on Example 71 b)c) 653H:753H =1:1 0.85 0.15 2% (NH4)2HPO4 DCM glacial acetic acid 2% (NH4)2HPO4 a) Semaglutide sodium salt, b) Semaglutide free base, c) O / W method, d) W / O / W method Preparation Examples 1 to 4 show the preparation of microsphere depending on different bioavailability enhancers by using poloxamer Fl27, disodium phosphate, sodium decanoate and disodium phosphate, and benzathine as bioavailability enhancers, respectively. Preparation Examples 4 to 9 show the preparation of microsphere depending on the content of benzathine, Preparation Examples 11 to 15 show the preparation of microsphere depending on the content of semaglutide, and Preparation Examples 16 to 29 show the preparation of microspheres depending on the polymer. Preparation Examples 33 to 37 (O / W method): Preparation of biodegradable polymer microsphere containing dexamethasone acetate or a pharmaceutically acceptable salt thereof For the preparation of dexamethasone microsphere, the dispersed phase includes at least one of Purasorb PDLG 7502A (i.v 0.16-0.24 dl / g; manufacturer: Purac, Netherlands), RG 752H (i.v 0.14-0.22 dl / g; manufacturer: Evonik, Germany) and RG 753H (i.v 0.32-0.44 dl / g; manufacturer: Evonik, Germany), PDL04A, PDL02, PDLG 7504A (manufacturer: Corbion, Netherlands), which are biocompatible polymers, and 0.200 to 0.400 g of dexamethasone acetate (manufacturer: Pfizer, USA) was mixed with dichloromethane (manufacturer: J.T Baker, USA), dimethyl sulfoxide (manufacturer: J.T Baker, USA), and benzyl alcohol (manufacturer: Junsei, Japan) to prepare a microsphere. In Preparation Examples 33 to 37, 2,000 ml of an aqueous solution containing 0.5% polyvinyl alcohol (viscosity: 4.8 to 5.8 mPa.s) and 2.5% of NaCl were used as the continuous phase. 5            [Table 2] Preparation Example No. Type of polymer Used amount of polymer (g) Used amount of Dexamethasone (g) Solvent Co-solvent Preparation Example 33 7502A:PDL04A=l:4 0.80 0.200 DCM benzyl alcohol Preparation Example 34 7502A:RG752H:RG75 3H -44:9:20 0.73 0.268 DCM benzyl alcohol Preparation Example 35 7502A:7504A=l:l 0.60 0.400 DCM benzyl alcohol Preparation Example 36 PDL02 0.60 0.400 DCM benzyl alcohol Preparation Example 37 PDL04A 0.60 0.400 DCM benzyl alcohol Preparation Examples 33 to 37 show the preparation of microspheres depending on the type of polymer and the used amount of dexamethasone acetate. 10          Preparation Examples 38 to 51 The co-administered formulation of semaglutide microsphere and dexamethasone acetate microsphere are shown as Preparation Examples 38 to 51 (see Table 3). [Table 3] Semaglutide microsphere Dexamethasone microsphere API     ratio     of semaglutide: dexamethasone Ratio of semaglutide microsphere: dexamethasone microsphere Semaglutide content when used in combination Preparation Example 38 Preparation Example 12 Preparation Example 35 200 : 1 533 : 1 12.9 Preparation Example 39 Preparation Example 13 Preparation Example 35 200 : 1 457 : 1 16.4 Preparation Example 40 Preparation Example 14 Preparation Example 35 200 : 1 400 : 1 17.1 Preparation Example 41 Preparation Example 15 Preparation Example 35 200 : 1 400 : 1 13.0 Preparation Example 42 Preparation Example 28 Preparation Example 36 200 : 1 400 : 1 Preparation Example 43 Preparation Example 29 Preparation Example 36 200 : 1 533:1 Preparation Example 44 Preparation Example 16 Preparation Example 37 22 : 1 59 : 1 10.4 Preparation Example 45 Preparation Example 21 Preparation Example 33 22 : 1 30 : 1 12.7 Preparation Example 46 Preparation Example 30 Preparation Example 34 67 : 1 40 : 1 13.1 Preparation Example 47 Preparation Example 31 Preparation Example 34 67 : 1 40 : 1 13.0 Preparation Example 48 Preparation Example 32 Preparation Example 34 67 : 1 40 : 1 14.0 Preparation Example 49 Preparation Example 15 Preparation Example 34 67 : 1 30 : 1 15.7 Preparation Example 50 Preparation Example 22   : Preparation Example 24 = 1:1 Preparation Example 36 200 : 1 533 : 1 Preparation Example 51 Preparation Example 25   : Preparation Example 27 = 1:1 Preparation Example 36 200 : 1 400 : 1 Experimental Example 1: Measurement of the encapsulated amount of semaglutide in the microsphere In order to measure the amount of encapsulated semaglutide in the microsphere 5 prepared in Preparation Examples 1 to 32 and the amount of encapsulated dexamethasone in the microsphere prepared in Preparation Examples 33 to 37, 10 mg of microspheres were completely dissolved in DMSO, and then diluted with a mobile phase. 20 pL of the diluted solution was injected into HPLC and measured at a detection wavelength of 214 nm. The column used in this experiment was ZORBAX 10   300SB-C18, 5pm, 4.6x150 mm, and the mobile phase was a mixture of 45% of 0.1% TFA acetonitrile and 55% of 0.1% TFA aqueous solution in isocratic mode. The measured amount of encapsulated drug is shown in Table 4 below. [Table 4] Formulation Target loading amount (% w / w) Drug content (% w / w) Preparation Example 1 7.5 6.67 Preparation Example 2 20 14.50 Preparation Example 3 20 17.90 Preparation Example 4 20 18.20 Preparation Example 5 20.0 18.0 Preparation Example 6 20.0 20.1 Preparation Example 7 20.0 17.1 Preparation Example 8 20.0 17.7 Preparation Example 9 20.0 18.0 Preparation Example 10 20.0 19.7 Preparation Example 11 12.5 11.6 Preparation Example 12 15.0 12.9 Preparation Example 13 17.5 16.5 Preparation Example 14 20.0 17.1 Preparation Example 15 20.0 16.0 Preparation Example 16 15.0 10.5 Preparation Example 17 15.0 13.1 Preparation Example 18 15.0 13.1 Preparation Example 19 15.0 13.6 Preparation Example 20 15.0 13.1 Preparation Example 21 15.0 13.1 Preparation Example 30 15.0 13.3 Preparation Example 31 15.0 13.3 Preparation Example 32 15.0 14.2 Preparation Example 33 20.0 22.1 Preparation Example 34 26.8 25.2 Preparation Example 35 40.0 37.1 Preparation Example 36 40.0 43.9 Preparation Example 37 40.0 40.1 Preparation Example 52 7.5 6.89 Preparation Example 53 7.5 7.53 Preparation Example 54 7.5 7.01 Preparation Example 55 15.0 14.6 Preparation Example 56 15.0 13.4 Preparation Example 57 15.0 12.4 Preparation Example 58 15.0 12.9 Preparation Example 59 15.0 12.0 Preparation Example 60 15.0 12.1 Preparation Example 61 15.0 12.3 Preparation Example 62 15.0 11.3 Preparation Example 63 15.0 11.7 Preparation Example 64 15.0 12.6 Preparation Example 65 15.0 12.8 Preparation Example 66 15.0 13.8 Preparation Example 67 15.0 13.5 Preparation Example 68 15.0 12.0 Preparation Example 69 15.0 11.8 Preparation Example 70 15.0 13.6 Preparation Example 71 15.0 12.5 Experimental Example 2: In-vivo pharmacokinetic test using rats Experimental Example 2-1: Change in blood concentration of semaglutide depending on bioavailability enhancer and release controlling agent 5           In order to evaluate the drug release profile in the body of the semaglutide sustained-release microsphere according to the present disclosure, the blood concentration of semaglutide was measured after administration to rats. The microspheres were measured so as to the amount of semaglutide be 1.2 mg (4.0 mg / kg) or 3.6 mg (12.0 mg / kg), dispersed in 0.5 mL suspension, and then injected 10 into SD rats (Sprague-Dawley Rat, 300 g). 0.5 mL of blood was collected at preplanned times, and the blood concentration of semaglutide was measured using HPLC. The measured AUC and Cmax are shown in Table 5 below. [Table 5] Category Dose (mg / head) Dose (mg / kg) AUC total (ng*day / mL ) / (mg / mL) Cmax (ng / mL) Dose-normalized AUC total (ng*day / m L) / (mg / kg) Dose-normalized Cmax (ng / mL) / (m g / kg) AUC (%)o- 24hrs Preparation Example 01 3.6 12 14217 2426.7 1184.8 202.2 9.2 Preparation Example 02 1.2 4 2564 405.3 641.0 101.3 8.3 Preparation Example 03 3.6 12 11490.0 1139.7 957.5 95.0 6.6 Preparation Example 04 3.6 12 21564 1940.0 1815 161.7 8.9 15           As can be seen in Table 5, the microsphere containing semaglutide prepared in Preparation Examples 1 to 4 of the present disclosure, shows a high AUC and less than 10 % of the cumulative AUC ratio of from administration time to 24 hours after adminstration, which can represent the initial releasing profile at the time of administration. Experimental Example 2-2. Improvement of semaglutide bioavailability by the content of benzathine In order to evaluate the drug release profile in the body of the semaglutide sustained-release microsphere according to the present disclosure, the bioavailability (%) after administration to rats was measured. Specifically, the content of benzathine relative to the total weight of the batch was set at 0.6 (w / w)%, 1.2 (w / w)%, 2.3 (w / w)%, and 5 (w / w)%, respectively. For a semaglutide sustained-release microsphere to which benzathine was added, and a semaglutide sustained-release microsphere to which benzathin was not added, 1.2 mg (4.0 mg / kg) or 3.6 mg (12.0 mg / kg) of semaglutide contained in the sustained-release microsphere were dispersed in 0.5 mL suspension, and then injected into SD rats (Sprague-Dawley Rat, 300 g). After that, in order to compare the improvement in bioavailability in rats, the pharmacokinetic parameters were analyzed. As a result, it was confirmed that Preparation Examples 4, 7, 8, and 9 showed an increase in AUC of about twice or more as compared to Preparation Example 6. It was confirmed that overall, the AUC was similar, and the bioavailability was higher than 40% or more. [Table 6] Category Bioavailability enhancer Used amount of bioavailability enhancer (g) Weight      ratio      of bioavailability enhancer / semaglutide (w / w(%)) AUCnorm. (ng*day / ml) / (mg / kg) Preparation Example 4 Benzathine 0.006 3 1815.0 Preparation Example 6 None N / A - 959.4 Preparation Example 7 Benzathine 0.012 6 1861.0 Preparation Example 8 Benzathine 0.023 11.5 1721.0 Preparation Example 9 Benzathine 0.050 25 2026.0 Experimental Example 2-3. Improvement of AUC by semaglutide content In order to evaluate the in vivo drug releasing profile of semaglutide sustained-release microsphere according to the present disclosure, the AUC was measured after 5 administration to rats. In the experiment, the semaglutide sustained-release microspheres containing 0.125, 0.150, 0.350, and 0.400 g of semaglutide, respectively, and 1.2 g of benzathine as a bioavailability enhancer, and semaglutide sustained-release microspheres containing 0.400 g of semaglutide and no bioavailability enhancer were injected into rats. For 10 injection of the semaglutide microspheres, the sustained-release microspheres containing 3.6 mg (12.0 mg / kg) as semaglutide was dispersed in 0.5 mL suspension and then injected into SD rats (Sprague-Dawley Rat, 300 g). [Table 7] Category Bioavailabili ty enhancer Used amount of semaglutide (g) Content        of semaglutide (w / w)% AUCnorm. (ng *day / ml) / (mg / kg) AUCnorm. 0-28d (ng*day / mL) / (mg / kg) Preparation Example 12 benzathine 0.150 15.0 1379.6 1016.0 Preparation Example 14 benzathine 0.400 20.0 3572.8 3296.6 It was confirmed that AUC increased as the drug content increased. In 15 particular, when the microspheres of Preparation Example 12 were administered to an experimental animal, the cumulative drug release rate within 28 days after administration was 62.28% of the total drug release amount according to the results of Fig. 1 showing the change in blood drug concentration and the pharmacokinetic parameters. These confirm that the ratio of the drug release amount within the drug administration period relative to the total drug release amount was 80% or less, showing a preferable drug release profde. Experimental Example 2-4. Initial release depending on whether initialrelease inhibitor is used or not Table 8 below shows the initial release depending on whether or not an initialrelease inhibitor is included in the continuous phase. [Table 8] Initial-release inhibitor Initial release Preparation Example 10 N / A 9.1 Preparation Example 15 Disodium phosphate 1.5 Preparation Example 5 N / A 40.8 Preparation Example 6 Disodium phosphate 1.9 As a result of the experiment, it was confirmed that the initial release of the drug in the microsphere containing a high content of semaglutide was significantly reduced becuase the initial-release inhibitor was contained in the continuous phase during the preparation of microspheres. Experimental Example 3-1. Comparison of microsphere properties by the addition of disodium phosphate and the use of similar salts This experiment was carried out using the salts like disodium phosphate as the initial-release inhibitor to compare their characteristics during the preparation of microparticles. A control formulation without addition of the initial release inhibitor in Table 1 (Preparation Example 55) and formulations prepared by adding Na2HPO4, K2HPO4, and NaH2PO4 as initial-release inhibitor were shown as Preparation Examples 56 to 58. Table 9 shows the main characteristics and initial release of Preparation Examples 55 to 58. [Table 9 Preparation Example No. Preparation Example 55 Preparation Example 56 Preparation Example 57 Preparation Example 58 Initial-release inhibitor N / A 2% Na2HPO4 2% K2HPO4 2% NaH2PO4 Initial release (%) 4.6 0.4 0.9 17.0 pH = 3 = 7 = 7 = 5 Referring to Preparation Examples 55 to 58 of Tables 1 and 9, when Na2HPO4 or K2HPO4 was added as an initial-release inhibitor, the initial release decreased to less than 1% as compared to the control formulation. However, when NaH2PO4 was added to the continuous phase as an initial-release inhibitor, the initial release was 17%, which appeared to be very higher than that of the control formulation. These confirmed that even the salts similar to disodium phosphate did not show the effect of suppressing initial release. Preparation Example 55 had about pH 3, Preparation Examples 56 and 57 had about pH 7, Preparation Example 58 had about pH 5. Although it does not show linear data, the reason of high initial release in Preparation Examples 55 and 58 may be considered as a pH factor. When the cross-sectional image was observed with SEM, a large number of internal pores were distributed in the formulation with acidic pH, and conversely, the internal pores were reduced in the formuation with neutral or alkaline pH. The result was considered to explain the reason of suppressed initial release. Experimental Example 3-2. Comparison of microsphere characteristics by the type of initial-release inhibitor added This experiment was carried out to compare the characteristics due to the type of an initial-release inhibitor when preparing the microsphere using Na2COs, NaHCOs, (NH4)2SO4, and (NH4)2HPO4 as an initial-release inhibitor. A control formulation without addition of the initial-release inhibitor was shown in Table 1 (Preparation Example 66) and the formulations prepared by adding Na2COs, NaHCOs, (NH4)2SO4, and (NH4)2HPO4 as an initial-release inhibitor, respectively, were shown as Preparation Examples 68 to 71. Table 10 below, shows the main characteristics and the initial release of Preparation Examples 66 and 68 to 71. [Table 10] Preparation Example No. Preparation Example 66 Preparation Example 68 Preparation Example 69 Preparation Example 70 Preparation Example 71 Initial-release N / A 2(w / v)% 2(w / v)% 2(w / v)% 2(w / v)% inhibitor Na2CO3 NaHCO3 (NH4)2SO4 (NH4)2HPO4 Initial release (%) 8.2 1.4 1.0 8.1 2.5 pH = 3 = 12 = 8 = 5 = 7 Referring to Preparation Examples 66, 68 to 71 of Tables 1 and 10, the initial release appeared to be slightly higher in Preparation Examples 66 and 70. Preparation Examples 66 and 70 having high initial release showed acidic pH values of about 3 and about 5, respectively, and Preparation Examples 68, 69 and 71 with neutral and basic pH values exhibited less than 3% of an initial release. Based on these results, when the continuous phase exhibited an acidic pH by addition of an initial-release inhibitor to a continuous phase, the initial release of semaglutide appeared to be significantly high. However, when using an initial-release inhibitor that causes basic pH to appear at pH 7 or higher (neutral or basic), the initial release of semaglutide was clearly suppressed. Here, by considering the initial release of Preparation Examples 68, 69, and 71, the initial release was suppressed more when it was basic than when it was neutral, but the suppression of initial release did not increase linearly depending on the degree of basicity. Experimental Example 3-3. Comparison of microsphere characteristics by pH adjustment after addition of disodium phosphate When the pH was intentionally adjusted after adding 2(w / v)% of disodium phosphate as an initial-release inhibitor, the test result of the change in initial release confirmed that the effect of suppressing the initial release on the microsphere was maintained even if the pH was changed after adding the initial-release inhibitor. Specifically, the control formulation (Preparation Example 66) without addition of the initial-release inhibitor in Table 1 and the formulation that 2 (w / v)% of disodium phosphate was added as an initial-release inhibitor but pH was not intentionally adjusted was shown as Preparation Example 63, the formulation in which the pH was adjusted to pH 2 (acidic) using HC1 was shown as Preparation Example 64, and the formulation in which the pH was adjusted to pH 12 using NaOH was shown as Preparation Example 65. Table 11 below shows the main characteristics and initial release of Preparation Examples 63 to 66. [Table 11] Preparation Exampfe No. Preparation Exampie 63 Preparation Exampie 64 Preparation Example 65 Preparation Example 66 tnitiaf-refease inhibitor 2(w / v)% Na2HPO4 2(w / v)% Na2HPO4 2(w / v)% Na2HPO4 N / A initiaf refease (%) 0.86 0.7 0.8 8.2 pH = 8 = 2 = 12 = 3 Remarks Adjusting pH after adding Na2HPO4 (using HO) Adjusting pH after adding Na2HPO4 (using NaOH) As could be seen in Table 11, when the microspheres were prepared using a continuous phase containing 2 (w / v)% of Na2HPO4 as an initial-release inhibitor, the effect of suppressing the initial release of semaglutide was maintained, even if the pH of the continuous phase was intentionally adjusted to acidic at a later time. Experimental Example 4. Comparison of microsphere characteristics 5 depending on the used amount of disodium phosphate This experiment was carried out to compare the characteristics of microspheres depending on the amount of disodium phosphate added as an initial-release inhibitor. A control formulation without addition of Na2HPO4 in Table 1 (Preparation Example 55) and formulations containing 0.5(w / v)%, l(w / v)%, 2(w / v)%, 3(w / v)%, and 10   4(w / v)% of Na2HPO4 were shown as Preparation Examples 59, 60, 56, 61 and 62, respectively. Table 12 below shows the main characteristics and initial release of Preparation Examples 55, 56 and 59 to 62. [Table 12] Preparation Example No. Preparation Example 55 Preparation Example 59 Preparation Example 60 Preparation Example 56 Preparation Example 61 Preparation Example 62 initial-release N / A 0.5(w / v)% l(w / v)% 2(w / v)% 3(w / v)% 4(w / v)% inhibitor Na2HPO4 Na2HPO4 Na2HPO4 Na2HPO4 Na2HPO4 initial release (%) 4.6 0.64 0.56 0.4 0.77 0.86 Referring to Preparation Examples 55, 56, and 59 to 62 in Tables 1 and 12, the 15 control formulation had 4.6 % of the initial release, whereas the initial release of the formulation containing 0.5 (w / v)% of Na2HPO4 decreased to 0.64%. When the Na2HPO4 concentration was 1 (w / v)% or 2(w / v)%, the initial release rates were confirmed to be lowered to 0.56% and 0.4%, respectively. However, when 3 (w / v)% or 4 (w / v)% of Na2HPO4was added, the initial release tended to increase to 0.77% and 20   0.86%, respectively, as compared to when 2% of Na2HPO4 was added. When Na2HPO4 was used at 4 (w / v)% or more, the polymer agglomeration phenomenon occured largely in the preparation of the microsphere. It was suggested that the tendency for initial release to increase as the Na2HPO4 content increased by starting from 2 (w / v)% was due to the polymer agglomeration phenomenon described above. Experimental Example 5. Method for measuring residual amount of sodium and phosphorus 5           In order to measure the content of sodium and phosphorus in the microsphere used in Examples and Comparative Examples, 6 mL of nitric acid aqueous solution containing 300mg of the microsphere mixed with ultrapure water at a ratio of 1:1 was mixed with 3 mL of hydrogen peroxide, and then heated at 100°C or more. Acid was added until the gas generated during the dissolution process changed from yellow to 10 white. The specimen obtained thereby was weighed, dissolved in ultrapure water, and then injected into an inductively coupled plasma emission spectrometer (ICP-OES) (Thermo Scientific Co., iCAP 6300 Duo, UK), and measured at a detection wavelength of 598.5 nm. The results are shown in the following Experimental Examples 5-1 and 52. 15 Experimental Example 5-1. Comparison of the residual amount of sodium and phosphorus in microspheres depending on the amount of disodium phosphate added The residual amounts of sodium and phosphorus in the microsphere according 20 to Preparation Examples 59 to 61 and 63 was measured and shown in Table 13 below. [Table 13] Preparation Example No. Preparation Example 59 Preparation Example 60 Preparation Example 63 Preparation Example 61 C.P Excipients 0.5(w / v)% l(w / v)% 2(w / v)% 3(w / v)% Na2HPO4 Na2HPO4 Na2HPO4 Na2HPO4 Residual sodium (mg / kg) 21 67 103 166 Residual phosphorus 6 14 32 55 (mg / kg) Referring to Table 13, it was confirmed that as the added amount of NaiHPOi increased, the residual amounts of sodium and phosphorus increased significantly. The residual content of the initial-release inhibitor in the microspheres was 10 ppm to 200 ppm based on sodium, and 5 ppm to 100 ppm based on phosphorus. 5 Experimental Example 5-2. Comparison of the residual amount of sodium and phosphorus in microspheres depending on changes in biodegradable polymer and addition of benzathine The residual amounts of Na and P in Preparation Examples 16, 63, and 67 were 10 measured and shown in Table 14. [Table 14] Preparation Example No. Preparation Example 16 Preparation Example 67 Preparation Example 63 C.P Excipients 2(w / v)% Na2HPO4 2(w / v)% Na2HPO4 2(w / v)% Na2HPO4 Additive N / A 1.2% benzathine N / A Polymer 203H 653H:753H 653H:753H Residual sodium (mg / kg) 397 154 103 Residual phosphorus (mg / kg) 80 16 32 Referring to Table 14, it was confirmed that depending on the preparation conditions of the microspheres containing semaglutide, and the initial-release inhibitor contained in the continuous phase, the contents of residual sodium and residual 15 phosphorus were different, and the residual content of the initial-release inhibitor in the microspheres was 50 ppm to 500 ppm based on sodium, and 10 ppm to 100 ppm based on phosphorus. Experimental Example 6. Comparison of SEM results of microspheres depending on whether or not disodium phosphate is used in the continuous phase FIG. 3A shows the microspheres prepared using only 0.1 (w / v)% of PVA in the continuous phase, and FIG. 3B shows the microspheres prepared using 0.1 (w / v)% of 5 PVA and 2 (w / v)% of Na2HPO4 in the continuous phase. Referring to FIGs. 3A and 3B, it was confirmed that the microparticle prepared using only 0.1% of PVA in the continuous phase had many pores present in the cross section of the microsphere. It was confirmed that the microspheres prepared using 0.1 (w / v)% of PVA and 2 (w / v)% of Na2HPO4 in the continuous phase, most pores 10 disappeared or that even if pores existed, the number and size of pores decreased significantly. This may be suggested to exhibit the effect of reducing the channels being capable of affecting the initial release of the drug.

Claims

[CLAIMS]1. A pharmaceutical composition for preventing or treating diabetes, type 2 diabetes, preservation of beta-cell function, obesity, non-alcoholic steatohepatitis, or neurodegenerative disease, comprising a sustained-release microsphere consisting of semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer and a biodegradable polymer,wherein the semaglutide or a pharmaceutically acceptable salt thereof is contained in an amount of 8 wt% or more as semaglutide based on the total weight of the microsphere, and the bioavailability enhancer is contained in an amount of 2.5 wt% to 250 wt% based on the weight of semaglutide.

2. A pharmaceutical composition for preventing or treating diabetes, type 2 diabetes, preservation of beta-cell function, obesity, non-alcoholic steatohepatitis, or neurodegenerative disease, comprising a sustained-release microsphere consisting of semaglutide or a pharmaceutically acceptable salt thereof, an initial-release inhibitor and a biodegradable polymer,wherein the semaglutide or a pharmaceutically acceptable salt thereof is contained in an amount of 8 wt% or more as semaglutide based on the total weight of the microspheres, and the initial-release inhibitor is contained at 5 ppm to 2,000 ppm.

3. The pharmaceutical composition according to claim 1 or 2, wherein the sustained-release microsphere releases less than 20% of semaglutide or a pharmaceutically acceptable salt thereof within 24 hours when administered in vivo.

4. The pharmaceutical composition according to claim 1 or 2, wherein the sustained-release microsphere releases less than 15% of semaglutide or apharmaceutically acceptable salt thereof within 24 hours when administered in vivo.

5. The pharmaceutical composition according to claim 1 or 2, wherein the sustained-release microsphere releases less than 10% of semaglutide or a pharmaceutically acceptable salt thereof within 24 hours when administered in vivo.

6. The pharmaceutical composition according to claim 1 or 2, wherein the biodegradable polymer is at least one selected from the group consisting of: a polymer selected from the group consisting of polylactide (PLA), polyglycolide (PGA), polylactide-co-glycolide (PLGA), which is a copolymer of lactide and glycolide, polydioxanone, polycaprolactone (PCL), polylactide-co-glycolide-co-caprolactone (PLGC), polylactide-co-hydroxymethyl glycolide (PLGMGA), polyalkylcarbonate, polytrimethylenecarbonate (PTMC), polylactide-co-trimethylenecarbonate (PLTMC), polyhydroxybutyric acid (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyorthoester, polyanhydride, polyanhydride-co-imide, polypropylene fumarate, pseudo polyaminoacid, polyalkyl cyanoacrylate, polyphosphazene, polyphosphoester, polysaccharide, and poly(butylene succinate lactide) (PBSLA); copolymers or simple mixtures of two or more thereof; a copolymer of the above polymer with polyethylene glycol (PEG); and a polymer-sugar conjugate in which the polymer or the copolymer is conjugated to sugar.

7. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable salt of the semaglutide is sodium salt, acetate, benzoate, hydroxynaphthoate, napadisilate, or pamoate of semaglutide.

8. The pharmaceutical composition according to claim 1, wherein the bioavailability enhancer is at least one selected from the group consisting of sodiumdecanoate, disodium phosphate, choline, meglumine, basic aluminum carbonate, dihydroxyaluminum sodium carbonate, ammonium phosphate, histidine, HEPES, HEPPS, spermine, spermidine, putrescine, methylene blue, proline, sugar, glycerol, surfactant, arginine, glycine, guanidine hydrochloride, urea, sodium chloride, potassium chloride, triethylamine, ethanolamine, triethanolamine, ethylenediamine, poloxamer, benzathine, procaine, lidocaine, bupivacaine, ropivacaine, oxytetracycline, sunitinib, rhizolutin, benzofuran, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium trisilicate, zinc carbonate, zinc hydroxide, zinc phosphate, aluminum hydroxide, aluminum phosphate, dihydroxyaluminum aminoacetate, calcium phosphate, calcium hydroxide, magaldrate, and benzofuran derivatives.

9. The pharmaceutical composition according to claim 1, further comprising 5ppm to 2,000ppm of an initial-release inhibitor.

10. The pharmaceutical composition according to claim 2 or 9, wherein the initialrelease inhibitor is at least one substance selected from alkali metal, alkaline earth metal or ammonium phosphate salt, phosphide salt, carbonate salt, chromate salt, dichromate salt, oxide, oxalate salt, silicate salt, sulfate salt, sulfide salt, sulfite salt, tartrate salt, tetraborate salt, thiosulfate salt, arsenate salt, arsenite salt, citrate salt, ferricyanide salt and nitride salt.

11. The pharmaceutical composition according to claim 1 or 2, wherein thesustained-release microsphere has an average particle size of 5 pm to 100 pm.

12. The pharmaceutical composition according to claim 1 or 2, wherein theweight of the sustained-release microspheres is 20 to 1,000 mg.

13. The pharmaceutical composition according to claim 1 or 2, wherein the biodegradable polymer has an intrinsic viscosity of 0.16 dL / g to 1.7 dL / g.

14. The pharmaceutical composition according to claim 1 or 2, further comprising one or more release controlling agents selected from the group consisting of butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecylic acid, behenic acid, arachidic acid, isocrotonic acid, oleic acid, elaidic acid, sorbic acid, linoleic acid, arachidonic acid, benzoic acid, hydroxynaphthoic acid, naphadicylic acid, naphthalene sulfonic acid and pamoic acid.

15. A method for preparing a microsphere consisting of semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer and a biodegradable polymer, the method comprising the steps of:(a) dissolving semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and one or more biodegradable polymers in an organic solvent to prepare a solution (dispersed phase) containing semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and a polymer;(b) adding the solution containing semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and a polymer prepared in step (a) to an aqueous solution phase (continuous phase) containing a surfactant to prepare an emulsion;(c) extracting the organic solvent from the dispersed phase in the emulsion state prepared in step (b) into a continuous phase and evaporating the organic solvent to form a microsphere; and(d) recovering the microsphere from the continuous phase of step (c).

16. The method according to claim 15, wherein the bioavailability enhancer is at least one selected from the group consisting of: poloxamer, benzathine, procaine, lidocaine, bupivacaine, ropivacaine, oxytetracycline, sunitinib, rhizolutin and benzofuran.

17. A method for preparing a microsphere consisting of semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer and a biodegradable polymer, the method comprising the steps of:(a’) dissolving semaglutide or a pharmaceutically acceptable salt thereof and a bioavailability enhancer in an aqueous solution to prepare a primary aqueous solution phase, dissolving one or more biodegradable polymers in an organic solvent to prepare an oil phase, and then mixing the primary aqueous solution and the oil phase to prepare a W / O emulsion (primary emulsion) containing semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and a polymer;(b’) adding the primary emulsion containing semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and a polymer prepared in step (a’) to an aqueous solution phase (continuous phase) containing a surfactant to prepare a W / O / W emulsion (secondary emulsion);(c’) extracting the organic solvent from the oil phase in the secondary emulsion state prepared in step (b’) into a continuous phase and evaporating the organic solvent to form a microsphere; and(d’) recovering the microsphere from the continuous phase of step (c’).

18. The method according to claim 17, wherein the bioavailability enhancer is at least one selected from the group consisting of: sodium decanoate, disodium phosphate, choline, meglumine, basic aluminum carbonate, dihydroxyaluminum sodium carbonate, ammonium phosphate, histidine, HEPES, HEPPS, spermine, spermidine, putrescine,methylene blue, proline, sugar, glycerol, surfactant, arginine, glycine, guanidine hydrochloride, urea, sodium chloride, potassium chloride, triethyleneamine, ethanolamine, triethanolamine, and ethylenediamine.

19. A method for preparing a microsphere consisting of semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer and a biodegradable polymer, the method comprising the steps of:(a”) dissolving semaglutide or a pharmaceutically acceptable salt thereof and one or more biodegradable polymers in an organic solvent, and suspending a bioavailability enhancer in this solution to prepare a suspension (dispersed phase) containing semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and a polymer;(b”) adding the suspension containing semaglutide or a pharmaceutically acceptable salt thereof, a bioavailability enhancer, and a polymer prepared in step (a”) to an aqueous solution phase (continuous phase) containing a surfactant to prepare an emulsion;(c”) extracting the organic solvent from the dispersed phase in the emulsion state prepared in step (b”) into a continuous phase and evaporating the organic solvent to form a microsphere; and(d”) recovering the microsphere from the continuous phase of step (c”).

20. The method according to claim 19, wherein the bioavailability enhancer is at least one selected from the group consisting of: magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium trisilicate, zinc carbonate, zinc hydroxide, zinc phosphate, aluminum hydroxide, aluminum phosphate, dihydroxyaluminum aminoacetate, calcium phosphate, calcium hydroxide, magaldrate, and benzofuran derivatives.

21. The method according to any one of claims 15 to 20, wherein the biodegradable polymer is at least one selected from the group consisting of: a polymer selected from the group consisting of polylactide (PLA), polyglycolide (PGA), polylactide-co-glycolide (PLGA), which is a copolymer of lactide and glycolide, polydioxanone, polycaprolactone (PCL), polylactide-co-glycolide-co-caprolactone (PLGC), polylactide-co-hydroxymethyl glycolide (PLGMGA), polyalkylcarbonate, polytrimethylenecarbonate (PTMC), polylactide-co-trimethylenecarbonate (PLTMC), polyhydroxybutyric acid (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyorthoester, polyanhydride, polyanhydride-co-imide, polypropylene fumarate, pseudo polyaminoacid, polyalkyl cyanoacrylate, polyphosphazene, polyphosphoester, polysaccharide, and poly (butylene succinate lactide) (PBSLA); copolymers or simple mixtures of two or more thereof; a copolymer of the above polymer with polyethylene glycol (PEG); and a polymer-sugar conjugate in which the polymer or the copolymer is conjugated to sugar.

22. The method according to any one of claims 15 to 20, further comprising one or more release controlling agents selected from the group consisting of butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecylic acid, arachidic acid, isocrotonic acid, oleic acid, elaidic acid, sorbic acid, linoleic acid, arachidonic acid, hydroxynaphthoic acid, naphadicylic acid, and pamoic acid.

23. The method according to any one of claims 15 to 20, wherein the organic solvent in step (a) or (a') or (a”) is at least one selected from the group consisting of dichloromethane, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile,dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol and benzyl alcohol.

24. The method according to any one of claims 15 to 20, wherein the surfactant in step (b) or (b') or (b”) is polyvinyl alcohol.

25. The method according to any one of claims 15 to 20, wherein the continuous phase in step (b) or (b’) or (b”) is water, or a mixed solvent of water and one or more selected from the group consisting of methyl alcohol, ethyl alcohol, propyl alcohol and ethyl acetate.

26. The method according to any one of claims 15 to 20, wherein the continuous phase in step (b) or (b’) or (b”) comprises an initial-release inhibitor.

27. The method according to claim 26, wherein the initial release inhibitor is contained in an amount of 0.05 w / v% to 20 w / v% based on the total volume of the continuous phase.

28. The method according to claim 26, wherein the initial release inhibitor is at least one selected from the group consisting of disodium phosphate, dipotassium phosphate, and diammonium phosphate.

29. The method according to any one of claims 15 to 20, wherein the continuous phase in step (b) or (b’) or (b”) has a pH of 7 or more.

30. The method according to any one of claims 15 to 20, wherein the release rate of semaglutide or a pharmaceutically acceptable salt thereof within 24 hours is less than15%, when the prepared sustained-release microsphere containing semaglutide or a pharmaceutically acceptable salt thereof and a bioavailability enhancer is administered in vivo.5   31. The method according to any one of claims 15 to 20, wherein the release rate ofsemaglutide or a pharmaceutically acceptable salt thereof within 24 hours is less than 10%, when the prepared sustained-release microsphere containing semaglutide or a pharmaceutically acceptable salt thereof and a bioavailability enhancer is administered in vivo.10

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