Drug containing liver targeting specific ligand and thyroid hormone receptor agonist

Abstract

Provided is a drug containing a liver targeting specific ligand and a thyroid hormone receptor agonist in its structure, which is a new drug structure formed by linking the liver targeting specific ligand with the thyroid hormone receptor agonist through a branched chain, a linker and a linking chain. Thyroid hormone receptors (TRs) are divided into two subtypes, TR-α and TR-β, wherein, TR-β is mainly expressed in liver, and TR-α is mainly expressed in heart, nervous system, etc. In certain embodiments, it is envisaged that the provided drug has the action of liver targeting, can specifically bring the thyroid hormone receptor agonist into liver, without entering heart and other issues, and may thereby avoid side effects caused by the action of the thyroid hormone receptor agonist on other issues, and maintain its therapeutic efficacy in the treatment of lipid metabolism disorders and related complications.

Claims

1. A compound containing a liver targeting specific ligand and a thyroid hormone receptor agonist in the structure, wherein the liver targeting specific ligand X is connected to the thyroid hormone receptor agonist T sequentially through a branched chain L containing a structure for stabilizing steric hindrance, a linker B and a linking chain D, and the compound is represented by Formula (I) of
(X-L).sub.n-B-D-T wherein n is 3, Formula (I) has a structure as shown below: ##STR00144## the compound has one of the structures shown in Kylo-0101 to Kylo-0103: ##STR00145##

2. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, which is an injectable or oral preparation.

4. A method for treating a liver-derived disease wherein the liver-derived disease is non-alcoholic fatty liver or non-alcoholic steatohepatitis comprising administering a pharmaceutically acceptable amount of a compound of claim 1 to a subject in need thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) The present invention provides the following description of the drawings to make the objectives, technical solutions and beneficial effects of the present invention clearer:

(2) FIG. 1 is a high-resolution mass spectrum of Kylo-0101;

(3) FIG. 2 is a high-resolution mass spectrum of Kylo-0102:

(4) FIG. 3 is a high-resolution mass spectrum of Kylo-0103:

(5) FIG. 4 shows detection results of total cholesterol (TC) content in the serum of db/db mice in Example 4;

(6) FIG. 5 shows detection results of low-density lipoprotein (LDL) content in the serum of db/db mice in Example 4;

(7) FIG. 6 shows detection results of triglyceride (TG) content in the serum of db/db mice in Example 4:

(8) FIG. 7 shows detection results of bone mineral density (BMD) in db/db mice in Example 4;

(9) FIG. 8 shows effects of experimental drugs on body weight, hit mass and lean mass of db/db mice in Example 4;

(10) FIG. 9 shows effects of Kylo-0101 at different doses on TC level in the serum of db/db mice in Example 5;

(11) FIG. 10 shows effects of Kylo-0101 at different doses on LDL level in the serum of db/db mice in Example 5;

(12) FIG. 11 shows effects of Kylo-0101 at different doses on TG level in the serum of db/db mice in Example 5;

(13) FIG. 12 shows effects of Kylo-0101 at different doses on body weight of db/db mice in Example 5:

(14) FIG. 13 shows effects of Kylo-0101 at different doses on fat and lean mass in db/db mice in Example 5;

(15) FIG. 14 shows effects of Kylo-0101 at different doses on bone mineral density of db/db mice in Example 5;

(16) FIG. 15 shows effects of Kylo-0101 at different doses on liver weight of db/db mice in Example 5;

(17) FIG. 16 shows histopathological pictures of oil red and HE stained liver slices in Example 5;

(18) FIG. 17 shows average quantitative results of pathological examination of liver tissue by staining in Example 5;

(19) FIG. 18 shows effects of Kylo-0101 at different doses on fT3 concentration in the serum of db/db mice in Example 5;

(20) FIG. 19 shows effects of Kylo-0101 at different doses on TSH in the serum of db/db mice in Example 5.

BEST MODE FOR THE INVENTION

(21) The following examples illustrate some embodiments disclosed in the present invention, but are not limited thereto. In addition, when providing specific embodiments, the inventors anticipated application of some specific embodiments, for example, compounds with specifically same or similar chemical structures for treatment of different liver-derived diseases.

Explanations

(22) pip refers to piperidine:

(23) DMF refers to N,N-dimethylformamide;

(24) Dde-Lys(Fmoc)-OH refers to N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-N′-fluorenylmethoxycarbonyl-L-Lysine;

(25) HBTU refers to O-benzotriazole-tetramethylurea hexafluorophosphate.

(26) DIPEA (DIEA) refers to N,N-diisopropylethylamine;

(27) Fmoc-Glu-OtBu refers to fluorenylmethoxycarbonyl-L-glutamic acid 1-tert-butyl ester;

(28) TBTU refers to O-benzotriazole-N,N,N′,N′-tetramethylurea tetrafluoroborate;

(29) ACN refers to acetonitrile;

(30) MTBE refers to methyl tert-butyl ether;

(31) refers to a solid phase carrier, such as a resin;

(32) Unless otherwise specified, the ratio of two substances involved in this application refers to volume ratio;

(33) Unless otherwise specified, the content involved in this application refers to volume percentage concentration.

Example 1: Preparation of Drug 1 (Kylo-0101)

(34) (1) Compound 1-1 undergoes the following chemical reaction to produce Compound 1-2:

(35) ##STR00044##

(36) Compound 1-1 (0.31 g, 0.1 mmol) was weighed into a syntheisi tube, added with pip/DMF (2 ml/8 ml), bubbled with nitrogen for 30-40 min, and vacuumed out. Then Compound 1-2 was washed with DMF 3 times, 10 ml each time, to wash away pip and impurities produced by the reaction.

(37) (2) Compound 1-2 undergoes the following chemical reaction to produce Compound 1-3:

(38) ##STR00045##

(39) Dde-Lys(Fmoc)-OH (0.16 g, 0.3 mmol) and HBTU (0.114 g, 0.3 mmol) were weighed into a synthesis tube, added with DMF (10 mL) to dissolve the above solids, added with DIPEA (55 μL) and bubbled with nitrogen for 30-60 min. After removing the reaction liquid, the remaining solid compound 1-3 was washed with DMF 3 times, 10 ml each time, to remove HBTU, DIPEA and impurities produced by the reaction.

(40) (3) Compound 1-3 undergoes the following chemical reaction to produce Compound 1-4:

(41) ##STR00046##

(42) To a synthesis tube containing Compound 1-3, pip/DMF (2 ml/8 ml) was added, bubbled with nitrogen for 20-30 min, and vacuumed out the reaction liquid and impurities generated by the reaction. The remaining solid Compound 1-4 was washed with DMF twice, 10 ml each time, to remove pip and impurities generated by the reaction.

(43) (4) Compound 1-4 undergoes the following chemical reaction to produce Compound 1-5:

(44) ##STR00047##

(45) Fmoc-Glu-OtBu (0.13 g, 0.3 mmol) and HBTU (0.114 g, 0.3 mmol) were weighed into Compound 1-4 (0.1 mmol), added with DMF (10 mL) to dissolve the above solids, added with DIPEA (55 μL) and then bubbled with nitrogen for 15-30 min. After removing the reaction liquid, the remaining solid compound 1-5 was washed with DMF 3 times, 10 ml each time, to remove HBTU, DIPEA and impurities produced by the reaction.

(46) (5) Compound 1-5 undergoes the following chemical reaction to produce Compound 1-6:

(47) ##STR00048##

(48) To Compound 1-5 (0.1 mmol), pip/DMF (2 ml/8 ml) was added, bubbled with nitrogen for 15-30 min, and vacuumed out the reaction liquid and impurities generated by the reaction. The remaining solid Compound 1-6 was washed with DMF 6 times, 10 ml each time, to remove pip and impurities generated by the reaction.

(49) (6) Triiodothyronine undergoes the following chemical reaction to produce Compounds 1-7:

(50) ##STR00049##

(51) 0.5 g of triiodothyronine was weighed and put into an eggplant-shaped bottle, added with 4 ml of dioxane. 1 ml of purified water, 0.3 ml of triethylamine and 232 mg of Boc-anhydride, stirred for 2 h at room temperature protected from light, added with 4 ml of water and 10 ml of dichloromethane, adjusted pH to 4 by dropwise addition of hydrochloric acid, stood still to separate the layers. The water layer was added with 10 ml of dichloromethane to extract again. The combined organic layer was dried over 5 g of anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness with a rotary evaporator. The concentrate was added with 20 ml of petroleum ether, slurried and filtered. The filter cake was washed with 40 ml of petroleum ether and dried under reduced pressure to obtain 630 mg of Compound 1-7 (a white solid).

(52) (7) Compound 1-6 undergoes the following chemical reaction to produce Compound 1-8:

(53) ##STR00050##

(54) Compound 1-7 (0.23 g, 0.3 mmol) and HBTU (0.114 g, 0.3 mmol) were weighted and put into a synthesis tube containing 0.1 mmol of Compound 1-6, added with DMF (10 mL) to dissolve the above solids and then added with DIPEA (55 μL), and bubbled with nitrogen for 10-20 min. After removing the reaction liquid, the remaining compound 1-8 was washed with DMF 3 times, 10 ml each time, to remove HBTU, DIPEA and active esters produced by the reaction.

(55) (8) Compound 1-8 undergoes the following chemical reaction to produce Compound 1-9:

(56) ##STR00051##

(57) A solution of hydrazine hydrate/DMF (0.2 ml/9.8 ml) was added to Compound 1-8 (0.1 mmol), and bubbled with nitrogen for 10 min. The DMF solution of hydrazine hydrate was removed under vacuum, and Compound 1-9 was washed with DMF 6 times, 10 ml each time, to remove hydrazine hydrate and impurities produced by the reaction.

(58) (9) Preparation of compound 1-10:

(59) Step One:

(60) ##STR00052##

(61) 0.21 g of 1,5-glutaric acid monobenzyl ester was weighed and dissolved in 2 ml of DMF, added with 0.36 g of TBTU and 0.4 ml of DIEA, and stirred and reacted for 5 min. 1.09 g of Compound a was dissolved in 10 ml of DMF, and slowly added into the above reaction solution. The reaction solution was stirred overnight at room temperature, evaporated to dryness under reduced pressure, added with 40 ml of dichloromethane and 20 ml of water, stirred for 15 min and layered. The organic layer was dried over 10 g of anhydrous sodium sulfate, and passed through a chromatography column (eluent: dichloromethane:methanol=1%-10%). The target compound b to be collected was identified by thin Layer chromatography (the developing solvent contains dichloromethane and methanol in a volume ratio of 10:1). The eluent containing the pure Compound b was collected and evaporated off the solvent under reduced pressure to obtain 0.85 g of Compound b as a white product.

(62) Step Two:

(63) ##STR00053##

(64) 0.85 g of Compound b was put into a 100 ml single-necked flask, added with 127 mg of palladium-carbon, cammed with a water pump and supplemented with hydrogen, repeating for three times, and then pressed with hydrogen and reacted overnight. On the second day, TLC of the reaction mixture showed no compound b. The reaction mixture was filtered (aided with 12 g of diatomite), and the filtrate was evaporated under reduced pressure to obtain 90.76 g of Compound 1-10.

(65) (10) Compound 1-9 and Compound 1-10 undergo the following chemical reaction to produce Compound 1-11:

(66) ##STR00054##

(67) Compound 1-10 (0.57 g, 0.3 mmol) and HBTU (0.114 g, 0.3 mmol) were weighed and put into a synthesis tube containing Compound 1-9 (0.1 mmol), added with DMF (10 mL) to dissolve the above solids, added with DIPEA (55 μL) and bubbled with nitrogen for 10-20 min. The reaction liquid was removed and the remaining Compound 1-11 was washed with DMF, 10 ml each time.

(68) (11) Compound 1-11 undergoes the following chemical reaction to produce Compound 1-12:

(69) ##STR00055##

(70) F solution (6 ml, trifluoroacetic acid:triisopropylsilane:water=90:3.5:5.5) was slowly poured into a centrifuge tube containing Compound 1-11 (0.3 g, 0.05 mmol), took out after cutting for 1 hour while controlling temperature at 30-35° C. and rotation speed at 200 r/min, and filtered to remove the solid. The filtrate (5 ml) was slowly poured into MTBE (20 ml), and the suspension was centrifuged with a centrifuge at 3000 r/min. The solid was dispersed again with MTBE (20 ml), and the suspension was centrifuged again. The solid was collected and vacuum dried for 1 hour to obtain 46 mg of a white powder with a yield of 34% calculated based on Compound 1-11.

(71) The resultant white powder was dissolved in 2 ml of ACN/water (0.2 ml/1.8 ml) and loaded on a column using a filler with a trade name of GE Resource 15RPC (50 ml) and a mobile phase being a mixture of water and acetonitrile (acetonitrile content is 10% to 90%) to carry out gradient elution. All products were collected and lyophilized to obtain 12 mg of the pure compound 1-12.

(72) (12) Compound 1-12 undergoes the following chemical reaction to produce the drug 1 (Kylo-0101):

(73) ##STR00056##

(74) A solution of sodium methoxide/methanol (20 mg/ml) was added into the lyophilized compound 1-12 (12 mg, 0.0043 mmol), shook for 20 min while setting shaker temperature at 30-35° C. and rotation speed at 200 r/min, adjusted pH to 7 fay addition of 1 mol/L HCl (5-7 drops), and rotary evaporated to remove the solvent with a water bath at a temperature of 40-45° C. The residue was dissolved in 2 ml of ACN/water (0.2 ml/1.8 ml), and loaded on a column using a filler with a trade name of GE Resource 15RPC (10 ml) and a mobile phase being a mixture of water and acetonitrile (acetonitrile content is 10% to 90%) to carry out purification. All eluted products were collected and lyophilized to obtain 7 mg of the pure drug 1 (Kylo-0101) with a yield of 67.3%. As shown in FIG. 1, the target peak is 1232.35108(2+) when the mass-to-charge ratio is 2 in mass spectrometry of Kylo-0101.

Example 2: Preparation of Drug 2 (Kylo-0102)

(75) Compound 2-1 undergoes the following chemical reaction to produce Drug 2 (Kylo-0102):

(76) ##STR00057##

(77) A solution of sodium methoxide/methanol (20 mg/5 ml) was added into Compound 2-1 (20 mg, 0.0078 mmol), shook for 30 min while setting shaker temperature at 30-35° C. and rotation speed at 200 r/min. adjusted pH to 7 by addition of 1 mol/L HCl (5-7 drops), and rotary evaporated to remove the solvent with a water bath at a temperature of 40-45° C. The residue was dissolved in ACN/water (0.2 ml/1.8 ml), and loaded on a column using a filler with a trade name of GE Resource 15RPC (10 ml) and a mobile phase being a mixture of water and acetonitrile (acetonitrile content is 10% to 90%) to carry out purification. All eluted products were collected and lyophilized to obtain 12 mg of the pure drug 2 (Kylo-0102) with a yield of 71.1%. As shown in FIG. 2, the target product is 2201.55613(1+) when the mass-to-charge ratio is 1, and 1111.75962(2+) when the mass-to-charge ratio is 2 in mass spectrometry of Kylo-0102.

Example 3: Preparation Method of Drug 3 (Kylo-0103)

(78) Compound 3-1 undergoes the following chemical reaction to produce Drug 3 (Kylo-0103):

(79) ##STR00058##

(80) A solution of sodium methoxide/methanol (20 mg/5 ml) was added into Compound 3-1 (20 mg, 0.0076 mmol), shook for 30 min while setting shaker temperature at 30-35° C. and rotation speed at 200 r/min, adjusted pH to 7 by addition of 1 mol/L HCl (5-7 drops), and rotary evaporated to remove the solvent with a water bath at a temperature of 40-45° C. The residue was dissolved in ACN/water (0.2 ml/1.8 ml), and loaded on a column using a filler with a trade name of GE Resource 15RPC (10 ml) and a mobile phase being a mixture of water and acetonitrile (acetonitrile content is 10% to 90%) to carry out purification. All eluted products were collected and lyophilized to obtain 12.5 mg of the pure drug 3 (Kylo-0103) with a yield of 72.7%. As shown in FIG. 3, the target product is 2283.69975(1+) when the mass-to-charge ratio is 1 in mass spectrometry of Kylo-0102.

Example 4: Animal Experiment 1

(81) Materials: 30 genetically obese model mice (db/db mice) (6 weeks old, male, SPF level, provided by Nanjing University-Nanjing Institute of Biomedicine with a production license NO. SCXK (SU) 2015-0001, an animal certificate NO. 201820469 and a use license NO. SCXK(SU)2018-0027) were selected as the administration group. The mice needed to adapt to the environment before the experiment, and healthy mice were selected as test animals, and reared in IVC cages at a density of 5 animals/cage with the litter being changed twice a week. Requirements on Laboratory animal room: room temperature 22 to 24° C., relative humidity 40 to 70%, automatic lighting, 12 h alternating light and dark (lights were tinned on at 08:00, and turned off at 20:00), the standard of laboratory animal room meets the national standard of the People's Republic of China GB14925-2010.

(82) Experimental Drugs: see Table 1.

(83) TABLE-US-00001 TABLE 1 Experimental drug Lot number Source Kylo-0101 20180607 Kylonova (Xiamen) Kylo-0102 20180622 Biopharma Co., Ltd. Kylo-0103 20180626 Kylo-0100 20180612 Storage precautions: frozen storage (−20° C.)
Remarks: Kylo-0100 is T3 as a positive control drug, the experimental drugs were dissolved in saline.

(84) Preparation of anesthetic of xylazine combined with ketamine: the concentrations of xylazine and ketamine in the mixed solution were 10 mg/ml and 0.5 mg/ml respectively.

(85) The grouping and dosing schedule are shown in Table 2.

(86) TABLE-US-00002 TABLE 2 Mouse Experimental Administration type Number drug manner Dose Control 6 Saline Subcutaneous 150 μL/mouse group injection Adminis- 6 Kylo-0100 The injection 13.5 μg/kg tration 6 Kylo-0101 volume is 48.4 μg/kg group 6 Kylo-0102 150 μL/ 43.3 μg/kg 6 Kylo-0103 mouse/day 43.3 μg/kg

(87) The body weight was weighed twice a week, and the administration was carried out continuously for 21 days. After the last administration, the mice were fasted overnight for 15-16 hours, sacrificed by CO.sub.2 anesthesia. The blood was collected by cardiac puncture, left to stand at room temperature for 2 hours, and centrifuged on a low temperature centrifuge at 3000 RPM for 10 minutes to collect serum, which was then stored in a −80° C. refrigerator for later use. The contents of total cholesterol (TC), low-density lipoprotein (LDL) and triglyceride (TG) in the serum were measured on a blood biochemical analyzer.

(88) On the 18th day of the administration, the mice were intraperitoneally injected with the anesthetic (xylazine combined with ketamine, 10 ml/Kg, IP), and the bone mineral density and body fat ratio of the mice were measured on a bone density meter.

(89) The experimental data in FIG. 4 shows that the serum TC levels of the mice in the Kylo-0100 to Kylo-0103 administration groups were significantly lower than that of the saline control group, and the reduction rates reached 34.68%, 52.25%, 37.61% and 44.37%, respectively.

(90) The experimental data in FIG. 5 shows that the serum LDL levels of the mice in the Kylo-0100 to Kylo-0103 administration groups were significantly lower than that of the saline control group, and the reduction rates reached 40.68%, 54.24%, 22.03% and 15.25%, respectively.

(91) The experimental data in FIG. 6 shows that the serum TG levels of the mice in the Kylo-0100 to Kylo-0103 administration groups were significantly lower than that of the saline control group, and the reduction rates reached 42.55%, 62.42%, 44.88% and 43.94%, respectively.

(92) The experimental data in FIG. 7 shows that the bone mineral density (BMD) of the mice in the Kylo-0100 administration group was significantly lower than that of the saline control group; and the bone mineral density of the mice in the Kylo-0101 administration group was basically no change compared to the saline control group.

(93) The experimental data in FIG. 8 shows that Kylo-0100 has a significant effect on the body weight of mice, and Kylo-0101 has no obvious effect on the body weight of mice, indicating that Kylo-0101 mainly acts in the liver and does not produce adverse effects on mice as a whole.

Example 5: Animal Experiment 2

(94) Animals and feeding: 36 male 6-week-old genetically obese mouse models (db/db mice) and 5 wild type littermates (provided by Nanjing University-Nanjing Institute of Biomedicine with a production license NO. SCXK (SU) 2015-0001, an animal certificate NO. 201826897 and a use license NO. SCXK(SU)2018-0027) were selected as the administration group.

(95) The mice needed to adapt to the environment before the experiment, and healthy mice were selected as test animals, and reared in IVC cages at a density of 5 animals/cage with the litter being changed twice a week. Requirements on Laboratory animal room: room temperature 22 to 24° C., relative humidity 40 to 70%, automatic lighting, 12 h alternating light and dark (lights were turned on at 08:00, and turned off at 20:00), the standard of laboratory animal room meets the national standard of the People's Republic of China GB14925-2010.

(96) The experimental drugs are shown in Table 3.

(97) TABLE-US-00003 TABLE 3 experimental Lot No. drug number Source 1 Kylo-0101 20180828 Kylonova (Xiamen) 2 Kylo-0100 20180612 Biopharma Co.,Ltd.

(98) Preparation of anesthetic of xylazine combined with ketamine: the concentrations of xylazine and ketamine in the mixed solution were 10 mg/ml and 0.5 mg/ml respectively.

(99) The grouping and dosing schedule are shown in Table 4.

(100) TABLE-US-00004 TABLE 4 Dose of experi- Adminis- mental tration Group drug volume Intervention Group Number No. (μg/kg) (ml/kg) method Wild type 5 Group 1 saline 5 Subcutaneous control (G1) injection once group a day Model 6 Group 2 saline 5 control (G2) group Kylo-0100 6 Group 3 65 5 Adminis- (G3) tration group Kylo-0101 6 Group 4  1 5 Adminis- (G4) tration 6 Group 5  3 5 group (G5) 6 Group 6 10 5 (G6) 6 Group 7 30 5 (G7)
Remarks: the administrated drug was dissolved in saline.

(101) Experimental design: Before starting the experiments, the mice were weighed and randomly grouped according to body weight. The mice were weighed every Monday and Thursday. On the 18th day of administration, the mice were anesthetized by intraperitoneal injection of the anesthetic (xylazine combined with ketamine, 10 ml/kg, IP), and the bone mineral density and body hit ratio of the mice were measured. The administration was carried out continuously for 21 days.

(102) After the last administration, the mice were fasted overnight for 15-16 hours, sacrificed by CO.sub.2 anesthesia. The whole blood was collected by cardiac puncture, and left to stand at room temperature for 2 hours to collect serum. The serum is divided into three parts. One part (100 μl) was used for detecting blood glucose, triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL) and high-density lipoprotein (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and phosphatase (AP) levels in the serum. The remaining two parts were divided equally for detecting contents of triiodothyronine (bonded and free T3), thyroxine (bonded and free T4), and TSH (thyroid stimulating hormone).

(103) The weights of the liver and the heart were weighted. The liver is divided into three parts. One part is frozen in liquid nitrogen, one part is fixed with 10% neutral formaldehyde, and the other part is frozen and embedded in OCT (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol).

(104) Tissue analysis and pathological examination: the liver tissue was homogenized with isopropanol at a mass-to-volume ratio of 1:9, left to stand overnight, and centrifuged to collect the supernatant. The contents of TC and TG in the supernatant were detected by blood biochemistry. The liver tissue immersed in 10% neutral formaldehyde is routinely embedded in paraffin, sectioned (3 μm) and stained with hematoxylin-eosin (HE), and then dehydrated and sealed to observe the liver lesions and perform whole slide imaging. The liver tissue immersed in OCT was frozen and sectioned (5 μm), stained with oil red, stained nucleus with hematoxylin, dehydrated and sealed to observe the lipid deposition of liver tissue and take pictures. The liver frit accumulations of the administration group and the model control group were compared.

(105) Statistical analysis: The quantitative indexes were expressed by Mean±sem, and the differences between the model control group and the rest of the groups were compared by T test (TTest2.3). All the statistical analyses were completed in Excel table.

(106) The experimental data in FIG. 9 shows that the TC levels in the serum of the mice in the respective Kylo-0101 dose groups were reduced, and there was a significant dose-effect relationship. At a dose of 30 μg/kg, the TC level in the serum was reduced by to 48%.

(107) The experimental data in FIG. 10 shows that the LDL levels in the serum of the mice in the respective Kylo-0101 dose groups were reduced, and there was a significant dose-effect relationship. At a dose of 30 μg/kg, the LDL level in the serum was reduced by up to 58%.

(108) The experimental data in FIG. 11 shows that the TG levels in the serum of the mice in the respective Kylo-0101 dose groups were reduced, and there was a significant dose-effect relationship. At a dose of 30 μg/kg, the TG level in the serum was reduced by up to 41.8%.

(109) The experimental data in FIG. 12 shows that the body weights of the mice in the respective Kylo-0101 dose groups were decreased, but not significantly.

(110) The experimental data in FIG. 13 shows that each dose of Kylo-0101 did not significantly reduce the fat and lean mass in the body.

(111) The experimental data in FIG. 14 shows that the bone mineral densities of the mice in the respective Kylo-0101 dose groups were basically unchanged compared with the model control group.

(112) The experimental data in FIG. 15 shows that there was an obvious dose-effect relationship between the increase in the dose of Kylo-0101 and the decrease in the liver weight.

(113) Oil red staining is mainly the specific staining of intracellular lipid droplets. The stained lipid droplets are shown as the dark spots in FIG. 16. The histopathological examination results of oil red staining show that, compared with the model control group, the four dose groups of Kylo-0101 showed significantly reduced positive staining degree of lipid droplets, significantly reduced number of lipid droplets, and a significant dose-effect response. Central fatty degeneration in liver lobules will form fatty vacuoles of varying sizes in the liver cells, of which small vacuoles are the main ones, and form light spots as shown in FIG. 16 after HE staining. In FIG. 16, the histopathological examination results of HE stained sections show that the four dose groups of Kylo-0101 showed hepatocyte steatosis degrees significantly lower than that of the model control group, significant reduced hepatocyte vacuoles caused by steatosis in the liver sections, and a significant dose-effect response.

(114) The average quantitative results of the staining of liver pathological examination in FIG. 17 show that HE staining and oil red staining had a higher consistency, and when the dose of Kylo-0101 was 30 μg/kg, the fat content of the liver dropped to be almost close to that of normal (wild type) mice. It is generally considered that the normal average quantization value is less than 1 as normal.

(115) The detection results in FIG. 18 show that at each dose of Kylo-0101, the fT3 concentration in the serum of mice was increased slightly, but there was no significant difference.

(116) The detection results in FIG. 19 show that each dose of Kylo-0101 had no effect on the TSH concentration in the serum of mice.

Example 6: Effect of the Targeting Specific Ligand X on the Binding Rate of the Drag with ASGPR, Heart Rate and Bone Mineral Density

(117) TABLE-US-00005 TABLE 5 No. X L B Kylo- 0101 0 Kylo- 0105 Kylo- 0106 Kylo- 0107 0 Binding Heart Bone No. D T rate with ASGPR rate mineral density Kylo- 0101 6 1 1 Kylo- 0105 4 4 4 Kylo- 0106 5 3 3 Kylo- 0107 3 6 6 Remarks: The numbers of 6 to 1 indicate that the binding rate of the drug formed by the combination with the asialoglycoprotein receptor ASGPR is from high to low; and the effect on cardiotoxicity and bone mineral density is from high to low.

(118) The drags Kylo-0101 and Kylo-0105 to Kylo-0107 in Table 5 are only different in structure of X. The experimental data in the table shows that, in the case that the structures of L, B. D and T are the same respectively, the change in the structure of X would impart on the binding rate of the drag with ASGPR, heart rate and bone mineral density, wherein the drag Kylo-0101 has the best effect, it has a high binding rate to ASGPR and the least impact on heart rate and bone mineral density. This indicates that, in the composition prepared by the present invention, although the liver targeting specific ligand X is used to bind with ASGPR, it also has a certain impacted the whole therapeutic efficacy of the drug.

Example 7: Influence of the Branched Chain L Containing a Structure for Stabilizing Steric Hindrance on Drug Stability

(119) TABLE-US-00006 TABLE 6 No. X L B Kylo-0101 0 Kylo-0108 Kylo-0109 Kylo-0110 0 Kylo-0115 No. D T Drug stability Kylo-0101 High Kylo-0108 Meddle Kylo-0109 Meddle Kylo-0110 00 01 High Kylo-0115 02 03 High

(120) The drugs Kylo-0101, Kylo-0108 to Kylo-0110, and Kylo-0115 in Table 6 at only different in structure of L. The experimental data in the table shows that, in the case that the tinctures of X, B, D and T are the same reflectively, the change in the structure of L would impact on the stability of the drug. When the L structures of Kylo-0101, Kylo-0110 and Kylo-0115 were selected, highly stable drugs could be obtained.

Example 8: Effect of the Linker B on the Binding Rate of the Drag with ASGPR, Heart Rate and Bone Mineral Density

(121) TABLE-US-00007 TABLE 7 No. X L B Kylo- 0111 04 05 06 Kylo- 0112 07 08 09 Kylo- 0113 0 Kylo- 0101 Kylo- 0116 Binding Bone rate with Heart mineral No. D T ASGPR rate density Kylo- 0111 0 3 5 5 Kylo- 0112 6 1 1 Kylo- 0113 4 3 3 Kylo- 0101 6 1 1 Kylo- 0116 5 2 1 Remarks: The numbers of 6 to 1 indicate that the binding rate of the drug formed by the combination with the asialoglycoprotein receptor ASGPR is from high to low; and the effect of cardiotoxicity and bone mineral density is from high to low.

(122) The drugs Kylo-0101 and Kylo-0111 to Kylo-0113 in Table 7 are only different in fracture of B. The experimental data in the table shows that, in the case that the structures of X, L, D and T are the same respectively, the change in the structure of B would impact on the binding rate of the drug with ASGPR, heart rate and bone mineral density. When the B structures in Kylo-0101 and Kylo-0112 were selected, a good receptor binding rate and small side effects to heart and bone mineral density could be obtained.

Example 9: Effect of the Linking Chain D on the Hypolipidemic Ability of the Drug

(123) TABLE-US-00008 TABLE 8 No. X L B Kylo-0101 0 Kylo-0102 Kylo-0114 No. D T TC LDL TG Kylo-0101 6 6 6 Kylo-0102 0 4 3 5 Kylo-0114 3 3 3 Remarks: The numbers under TC, LDL and TG represent the strength of the composition's ability to reduce TC, LDL and TG, respectively. The numbers of 6 to 1 indicate that the ability is from strong to weak.

(124) The drugs Kylo-0101, Kylo-0102 and Kylo-0114 in Table 8 are only different in the structure of D. D alone as a linking structure does not have the hypolipidemic ability. However, the experimental data in the table indicates that, in the case that the structures of X, L, B, and T are the same respectively, the change m the structure of D would impact on the effects of lowing TC, TO and LDL of the drugs. Under the experimental conditions of the present invention, the D in the structure of the drag Kylo-0101 could achieve the best TC, TO and LDL lowering effect.