NOVEL DRUG DELIVERY CONJUGATED MOIETY FOR ORAL ADMINISTRATION OF DRUG UNSUITABLE FOR ORAL ADMINISTRATION AND PREPARATION METHOD THEREOF

Abstract

The present invention provides a novel drug delivery conjugated moiety for oral administration of a drug that is not suitable for oral administration or a pharmaceutically acceptable salt thereof. When the drug delivery conjugated moiety of the present invention or a pharmaceutically acceptable salt thereof is combined with a drug, which is not suitable for oral administration, and is administered orally, it exhibits an excellent absorption rate without decreasing the biological activities of the drug. Moreover, the drug delivery conjugated moiety of the present invention or a pharmaceutically acceptable salt thereof can be easily prepared in a few steps, which is very advantageous in terms of mass production.

Claims

1-21. (canceled)

22. A method for preparing a drug delivery conjugated moiety-low molecular weight heparin (LMWH) complex, comprising: (S1) preparing a compound of Formula 1 and a compound of Formula 2 from lysine; (S2) preparing a compound of Formula 3 by a reaction of the compound of Formula 1 with the compound of Formula 2; (S3) preparing a compound of Formula 4 by a deprotection of amine protecting groups of the compound of Formula 3; (S4) preparing a compound of Formula 5 by a reaction of the compound of Formula 4 with bile acids; (S5) preparing a compound of formula I by connecting a linker to the compound of Formula 5; and (S6) preparing the complex by reductive amination of the compound of Formula I and an end site of LMWH: ##STR00004## ##STR00005## wherein B is a bile acid residue; wherein L is a linker; wherein P1 is a carboxyl protecting group; and wherein P2 is an amine protecting group.

23. The method of claim 22, wherein B is a bile acid residue selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyodeoxycholic acid residues.

24. The method of claim 22, wherein P1 is C.sub.1-C.sub.6 alkyl or benzyl.

25. The method of claim 22, wherein P2 is tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (Moz), or fluorenylmethyloxycarbonyl (FMoc).

26. The method of claim 22, wherein the lysine in (S1) is L-lysine.

27. The method of claim 22, wherein the reaction in (S2) is performed under peptide coupling reaction conditions.

28. The method of claim 22, wherein the reaction in (S4) is performed under peptide coupling reaction conditions.

29. The method of claim 22, wherein the connecting in (S5) is performed under conditions for amidation of an ester.

30. The method of claim 22, wherein the linker is ethylenediamine (EDA).

Description

MODE FOR INVENTION

[0056] Hereinafter, preferred examples will be provided for better understanding of the present invention. However, the following examples are provided only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of Drug Delivery Conjugated Moiety of the Present Invention

[0057] Step 1: Protection of Lysine

[0058] Preparation of Compound of Formula 1

[0059] To a solution of 2,2-dimethoxypropane (70 mL) and conc. hydrochloric acid (18.0 mL) in methanol (110 mL) was added L-lysine (10 g, 54.75 mmol). The reaction mixture was heated to reflux for 3 hours, cooled down to room temperature and stirred overnight. The mixture was concentrated under reduced pressure to afford L-lysine methyl ester dihydrochloride as a white solid (9.88 g, 42.38 mmol).

[0060] Preparation of Compound of Formula 2

[0061] To a solution of L-lysine (20 g, 109.5 mmol) in 160 mL of water and 160 mL of tetrahydrofuran was added sodium carbonate (24 g, 226.4 mmol). After stirring for 15 minute, the reaction mixture was cooled down to 0° C. and di-tert-butyl-dicarbonate (48.93 g, 224.4 mmol) was slowly added. After stirring overnight at room temperature, the reaction mixture was diluted with ethyl acetate (66.7 mL) and 6N hydrochloric acid solution (56.7 mL) was added to adjust pH to 3 or lower. The aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over sodium sulfate and concentrated under reduced pressure to provide the desired product (34.2 g, 98.8 mmol).

[0062] Step 2: Preparation of Lysine Trimer (Formula 3)

[0063] To a solution of L-lysine methyl ester dihydrochloride (1 g, 4.3 mmol) prepared in step 1 in ethyl acetate (20 mL) was slowly added triethylamine (0.9 g, 8.9 mmol). After stirring for 10 minutes at room temperature, di-boc lysine (2.6 g, 7.4 mmol) prepared in step 1 and N-hydroxysuccinimide (0.9 g, 7.4 mmol) were added. To the reaction mixture dicyclohexylcarbodimide (1.5 g, 7.4 mmol) was added at 0° C. After stirring overnight, the reaction mixture was cooled down to 0° C., filtered to remove precipitate. The organic layer was washed with saturated sodium bicarbonate solution, 12% sodium bisulfate solution, saturated sodium bicarbonate solution and brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol/triethylamine=95/5/0.1) to provide the desired product (2.3 g, 2.8 mmol).

[0064] Step 3: Deprotection of Amine Groups (Preparation of Compound of Formula 4)

[0065] A mixture of methanol (198 mL) and acetyl chloride (15.6 g, 0.2 mol) was stirred for 1 hour at 0° C. To the reaction mixture was slowly added lysine trimer (7.8 g, 9.55 mmol) prepared in step 2. The reaction mixture was allowed to warm up to room temperature slowly and then stirred overnight. The reaction mixture was concentrated under reduced pressure to afford the desired product Formula 4 (5.04 g, 8.97 mmol).

[0066] Step 4: Binding of Bile Acids (Preparation of Compound of Formula 5)

[0067] To a solution of amine-deprotected compound (5.04 g, 8.97 mmol) obtained in step 3 in methanol (25 mL) and dimethylformamide (151 mL) was added slowly N-methylmorpholine (10.87 g, 107 mmol) over 0.5 hours. After stirring for 1 hour, the reaction mixture was cooled down to 5° C. To the reaction mixture was slowly added N-succinimidyl deoxycholic ester (19.75 g, 40.3 mmol) dissolved in 92 mL of dimethylformamide. The reaction mixture was allowed to warm up to room temperature and stirred overnight. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (dichloromethane/methanol=9/1) to provide the desired product (10.08 g, 5.26 mmol).

[0068] Step 5: Connection of Linker (Preparation of Drug Delivery Conjugated Moiety of Formula I)

[0069] To a solution of Formula 5 (5 g, 2.61 mmol) obtained in step 4 in ethanol (40 mL) was slowly added ethylenediamine (21.3 g, 0.35 mol) at 5° C. or lower. The reaction mixture was allowed to warm up to room temperature and stirred for 3 days. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (chloroform/methanol/ammonia solution=8/2/0.25) to afford the desired product Formula I (2.54 g, 1.31 mmol).

Example 2: Preparation of Drug Delivery Conjugated Moiety-Heparin Complex of the Present Invention

[0070] To a solution of enoxaparin (50 mg, 11 μM) in a mixture of H.sub.2O/DMF ( 1/7 mL) was added drug delivery conjugated moiety prepared in Example 1 (130 mg, 0.067 mmol). After stirring at 60° C. for 24 hours, sodium cyanoborohydride (7.0 mg, 0.11 mmol) was added. After stirring for 4 hours, the reaction mixture was diluted with ethanol and the precipitate was filtered to provide the desired product as an off-white powder.

Experimental Example 1: Measurement of PK Parameters

[0071] The following experiment was performed to determine the pharmacokinetic (PK) behavior of heparin administered orally using the drug delivery conjugated moiety of the present invention.

[0072] Prior to drug administration, experimental animals (rats) were fasted for more than 4 hours to empty the stomach. Then, after the dorsal skin was fixed, forced oral administration was performed using a sonde and a syringe for oral administration, and the animals were fed again about 4 hours after the administration. The drug delivery conjugated moiety-heparin complex prepared in Example 2 was administered orally, and blood samples were collected over 0.25, 0.5, 1, 2, 3, 4, 6, 8 hours. Blood samples (450 μL) were collected in a tube containing sodium citrate (50 μL) and centrifuged at 4500×g for 20 minutes to isolate plasma, which was then stored in a deep freezer at −70° C. The concentration of the drug delivery conjugated moiety-heparin complex in the plasma was analyzed using the COATEST HEPARIN FXa assay (Chromogenix). The results are shown in the following table 1:

TABLE-US-00001 TABLE 1 Dose E.sub.max.sup.a) T.sub.max.sup.b) AUC.sup.c) t.sub.1/2.sup.d) CL.sup.e) F.sup.(f) Substances (mg/kg) (IU/ml) (h) (IU .Math. h/ml) (h) (ml/h/kg) (%) Intravenous administration LMWH 2 1.61 ± 0.02 0.2 ± 0.0  1.8 ± 0.1 1.2 ± 0.1 0.7 ± 0.0 — Oral administration Example 2 5 0.46 ± 0.04 1.0 ± 0.0 1.99 ± 0.4 2.3 ± 0.6 2.4 ± 0.5 44.1 ± 9.0 .sup.a)Maximum effective concentration; .sup.b)Time to reach maximum effective concentration; .sup.c)Area under the concentration-time curve from 0 to 8 h; .sup.d)Half-life of drug; .sup.e)Clearance; and .sup.f)Absolute bioavailability.

[0073] The oral administration of the complex of Example 2 to rats showed an excellent bioavailability of about 40% or higher, compared to the intravenous administration of low molecular weight heparin (LMWH).

Experimental Example 2: Measurement of FXa

[0074] The term “heparin activity” refers to the anticoagulant ability of heparin. The COATEST HEPARIN FXa assay kit from Chromogenix was used to determine the anticoagulant activity of heparin that was administered orally using the drug delivery conjugated moiety of the present invention.

[0075] Stock solutions were prepared from 10 IU/ml of standard solution by accurately preparing test substances and then diluted to 0.1 IU/ml with buffer working solution (Tris 0.5 mol/L, pH=8.4, 10 ml, Chromogenix) to prepare standard solutions for each dose as shown in the following table 2 and used for the analysis.

TABLE-US-00002 TABLE 2 Heparin Heparin Buffer Standard IU/ml dilution working Normal solution Plasma 0.1 IU/ml solution Plasma Antithrombin Standard 0.1 100 μl 700 μl 100 μl 100 μl solution A Standard 0.3 300 μl 500 μl 100 μl 100 μl solution B Standard 0.5 500 μl 300 μl 100 μl 100 μl solution C Standard 0.7 700 μl 100 μl 100 μl 100 μl solution D

[0076] Each 200 μL of pretreated samples was placed in a cuvette and preheated at 37° C. for 3 to 4 minutes. Each 100 μl of FXa (Bovine Factor Xa 71 nkat., Chromogenix) was added thereto and left at 37° C. for about 30 seconds. Then, each 200 μl of S-2222 (Chromogenic substrate (Bz-Ile-Glu-(g-OR)-Gly-Arg-pNA.HCl), Chromogenix) was added thereto and left at 37° C. for 3 minutes. Subsequently, each 300 μl of 20% acetic acid was added thereto, and then the absorbance was measured at 405 nm. At this time, the measurement should be performed within 4 hours.

[0077] A linear regression equation was obtained using calibration curve of the standard solutions and converted to obtain the titers of the samples. The results of the heparin activities (FXa) are shown in the following table 3. The complex of Example 2 administered orally showed almost the same activities as the low molecular weight heparin (LMWH) administered intravenously.

TABLE-US-00003 TABLE 3 Anti-factor Xa activity LMWH 101 IU/mg Example 2 101.1 IU/mg

Experimental Example 3: Measurement of DVT Activities

[0078] The following experiment was performed to determine the effects of heparin administered orally using the drug delivery conjugated moiety of the present invention on deep vein thrombosis (DVT).

[0079] Low molecular weight heparin (enoxaparin) was administered to one group by subcutaneous injection, and the formulated complex of Example 2 was administered orally to other groups. The animals were anesthetized by abdominal injection of ketamine (45 mg/kg) and xylazine (5 mg/kg) and subjected to surgery to open the abdominal cavities of the rats, and the superior vena cava and inferior vena cava were isolated. The distal end of the vein along about 3 cm was weakly tied off, and the remaining veins were strongly tied off. 60 minutes after the drug treatment, 1 mL/kg of human pooled plasma was administered intravenously to the ends of the tails at 37° C., and the veins were tied off after 15 seconds to prevent blood flow. The veins were isolated 120 minutes after the drug treatment and stored in a Petri dish with 3.8% sodium citrate. Then, thrombus was isolated, and the amount of plasma generated was measured.

[0080] The comparison of the effects of the bile acid tetramer-biologically active agent complex on DVT disease models showed that when the complex of Example 2 was administered at a dose of 5 mg/kg, about 25% thrombus was generated, compared to the group to which the drug was not treated, which was almost the same results as the group to which LMWH was injected subcutaneously.

INDUSTRIAL APPLICABILITY

[0081] When the drug delivery conjugated moiety of the present invention is combined with a drug, which is not suitable for oral administration, and is administered orally, it exhibits an excellent absorption rate while maintaining the biological activities of the drug. Moreover, the drug delivery conjugated moiety of the present invention can be easily prepared in a few steps, which facilitates mass production. Therefore, the drug delivery conjugated moiety of the present invention can be very effectively used for the oral administration of a drug that is not suitable for oral administration.