KR100375939B1 - Novel water soluble polymer-cyclosporine conjugated compounds and process for preparing the same - Google Patents

Abstract

The present invention relates to a novel cyclosporin precursor which chemically binds a water-soluble carrier to cyclosporin (CsA) to increase its bioavailability, and more specifically, general formula (I), which can be hydrolyzed to cyclosporin which has physiological activity in vivo again. A water-soluble polymer-cyclosporine conjugate of Cyclosporin is almost sparingly soluble in water and lipophilic. Until now, the use of cyclosporin in vegetable oil was very low in bioavailability and tissue utilization, but an object of the present invention is to increase the bioavailability by providing a new water-soluble cyclosporin precursor.

Wherein R is the same as

-C (= O) -CH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( II )

-C (= O) -OCH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( III )

-C (= O) -OCH ₂ XC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( Ⅳ )

-C (= O) -OCH ₂ XCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( Ⅴ )

X represents O, S or NH,

m is 1 to 6,

n is 10 to 460.

Description

Water-soluble polymer-cyclosporine conjugated compounds and process for preparing the same

The present invention relates to cyclosporin useful as an immunosuppressive agent, and more specifically, to providing a new water-soluble polymer-cyclosporine conjugate by binding cyclosporin to a chemically water-soluble polymer or macromolecular carrier, and also to the water solubility and bioavailability of cyclosporin. It is increased. In other words, the present invention relates to a cyclosporin prodrug which, when hydrolyzed from the carrier, again has physiological activity.

Cyclosporin is a peptide compound formed of an 11 amino acid backbone and is known to have useful pharmacological activity. Cyclosporine, in particular, is mainly used for the treatment of organ transplantation or bone marrow transplantation as an immunosuppressive agent, and also for the treatment of a wide range of autoimmune diseases such as inflammatory diseases and baldness, rheumatoid disease (rheumatic polyarthritis), blood diseases (aplastic anemia, unknown platelets). It is also applied to the treatment of gastrointestinal diseases (ulcerative colitis, chronic diseases), skin diseases (psoriasis, scleroderma), eye diseases (uveitis) and psoriasis, uveitis.

Cyclosporin is very lipophilic and almost insoluble in water. Therefore, to use for treatment, dissolved in olive oil or peanut oil, etc. However, it has been reported that the cyclosporin oil solution has a very low bioavailability and a large difference in the range of tissue utilization (4-25%) (Takada, K. et al, J. Pharmacobio-Dyn ., 11, 80-7, 1988). ). The bioavailability of cyclosporin is known to be affected by food, bile and other interactions (Fahr, A., Pharmacokinetics , 24, 472-95, 1993). Recent studies have shown that for cyclosporine microemulsions, absorption differs in the small and large intestine (duodenum, jejunum, ileum, and descending colon), and absorption is dominant in the small intestine (Drewe, J., et al, J. Clin. Pharmac ., 33, 39-43, 1992).

Cyclosporine should be taken in large doses due to low bioavailability and tissue utilization, and side effects such as renal toxicity, hypertension, hyperkalemia, uric acid hyperemia, hepatotoxicity, anemia, gastrointestinal resistance, tremor and paresthesia often occur. . The biggest side effect is kidney failure. The acute nephrotoxicity of cyclosporin is proportional to the dose of the drug, which is related to blood levels and can be improved by reducing the dose or by stopping the treatment of cyclosporin. However, reversible and irreversible kidney damage has been reported in transplant patients.

Cyclosporine is neutral, poorly soluble in water and hexane, and very well soluble in all other organic solvents. Due to the low solubility of cyclosporin in water, it is necessary to dissolve an adjuvant to dissolve the drug. Cyclosporin (Sandimun), which is currently used in the clinic, is prepared as a solution and used as an injection and oral drug, or a soft capsule filled with a solution. Cyclosporines formulated orally or by injection are in the form of microemulsions.

A liquid cyclosporine microemulsion comprising a surfactant, an oil and an auxiliary surfactant (US Patent No. 4,388,307) is a transesterified product of vegetable oils and natural vegetable oil triglycerides, which are cyclosporine oils, and polyalkylene glycols and auxiliary surfactants. It consists of ethanol which is surfactant. Cremopoell, a nonionic surfactant commonly used in injections made in this form, has been shown to cause side effects of hypersensitivity reactions (Lorence, W., et al, Agents and actions , 12, 64-80, 1982) in some patients. The addition of non-aqueous solvents such as ethanol, propylene glycol and polyethylene glycol 400 to the development of injectables is a consideration for parenteral use. In addition, the use of an organic solvent has problems of hemolysis and local irritation at the injection site.

In the development of oral preparations, soft capsules filled with microemulsions as the main ingredient have some drawbacks in the absorption process. When the eluted oil component is in contact with oral or gastrointestinal fluids, the composition of the drug often separates into a solid state, which can reduce the bioavailability of the drug to less than 30%. Moreover, during long-term storage, cyclosporin tends to crystallize by evaporation of the ethanol contained in the formulation, and patients must suffer the unpleasant odor of ethoxylated castor oil during storage.

Although there are several other formulation types that have successfully improved the stability of the formulation through minimization of ethanol content, it still has other drawbacks. For example, the use of surfactants of rather complex composition, which is not easy or practically suitable for the development of formulations. For liposomes (WO 90-00389), this is complicated by the entire manufacturing process and the particle size or content of each preparation. Its disadvantage is that it is difficult to control because of the low reproducibility of the ratio.

In addition, formulations using polymeric polysaccharides (German Patent No. 293,499) have the disadvantage that the final volume is too large for administration. Moreover, the above mentioned methods are not yet cyclosporin formulations with a composition with a satisfactory dissolution rate in aqueous solution.

In order to overcome problems such as the toxicity of surfactants and solvents in the development of the formulation, the present inventors can significantly alleviate the above problems by attaching a water-soluble macromolecule to a poorly soluble drug and acting as a carrier, oral or parenteral administration. Intensive research was deemed appropriate for all.

Until now, it has not been considered to attach a water-soluble polymer such as polyethylene glycol to cyclosporine to a drug and to apply it to body delivery.

Polyethyleneglycol is a linear or branched neutral polymer with various molecular weights, and is an organic solvent, methylene chloride and water-soluble material. A molecular weight of 1,000 or less is a viscous colorless liquid, whereas a large molecular weight exists as a waxy white solid. The melting point of the solid is proportional to the molecular weight and approaches the plateau at 67 ° C. In general, molecular weights from hundreds to 20,000 are applied in biology and biotechnology.

Most interesting in the biomedical field is that polyethylene glycol is non-toxic and polyethylene glycol has been approved for internal use by the US Food and Drug Administration (FDA). Polyethylene glycol is widely used in drug synthesis, cosmetics and hygiene products, and one of the most widely studied drug transport technologies is to covalently bind a monomethoxy poly (ethylene glycol) polymer to a protein surface. Harris, MJ, Poly (ethylene Glycol) Chemistry, Biotechnical and Biomedical Applications.

1 is a hydrolysis of the water-soluble polymer-cyclosporin conjugate (KI-306, Cyclosporine 3'-methoxypropyleneglycol 5000-succinyloxymethyl-oxy carbonate ester) of the present invention in a human homogenous solution at 37 ° C. Graph showing pharmacokinetics.

2 is a linear graph showing the pharmacokinetics of hydrolysis of graph KI-306 to cyclosporin in a human homogenate at 37 ° C.

Figure 3 is a linear graph showing the change in blood concentration over time after intravenous injection of a solution of KI-306 dissolved in saline to red.

Figure 4 is a graph showing the change in blood concentration over time after oral administration of physiological saline solution of KI-306 and Sandimun neoral oral solution (Switzer Novartis).

The present invention is made by chemically bonding cyclosporin (CsA) to a water-soluble carrier, and relates to a water-soluble polymer-cyclosporine conjugate of the following general formula ( I ). That is, the present invention is to separate and act on the cyclosporin having a physiological activity when hydrolyzed from the polymer carrier.

In the above formula, R is the same as the following group.

-C (= O) -CH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( II )

-C (= O) -OCH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( III )

-C (= O) -OCH ₂ XC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( Ⅳ )

-C (= O) -OCH ₂ XCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( Ⅴ )

Wherein X means O, S or NH, m represents an integer of 1-6, preferably 1-3, n is 10-460, preferably 10-220, more preferably 10-120 . The structure of the cyclosporine is when R = H in the formula ( I ).

Water-soluble polymers such as polyethylene glycol (PEG) and monomethoxypolyethylene glycol (mPEG) are used to increase the solubility of the drug in water by binding to the water-insoluble drug. Therefore, in the present invention, the poorly soluble drug cyclosporin (CsA) is covalently linked to the water-soluble polymer and was developed to be dissolved in water, the compound of the present invention can be used as an immunosuppressant, anti-inflammatory, antifungal, anti-proliferative .

The water-soluble polymer-cyclosporine conjugates of the present invention in the form esterified at the 3'-OH position of the cyclosporin are esterified by using a compound ( VI ) having a carbonyl group at 3'-OH of the cyclosporin in the presence of a base such as pyridine. the compounds of the resultant compound of Preparation of intermediate (ⅶ), and polyethylene glycol derivatives herein (ⅷ), (ⅸ) or general formula (ⅰ) respectively by reacting iodine, potassium carbonate and a crown ether present purpose the (ⅹ) Can be obtained.

Y (C = O) OCH ₂ Y or YCH ₂ (C = O) O (C = O) CH ₂ Y ( Ⅵ )

CsA-OC (= O) OCH ₂ Y or CsA-OC (= O) CH ₂ Y ( Ⅶ )

OCH _3- (CH ₂ CH ₂ O) nCH ₂ CH ₂ OC (= O) (CH ₂ ) _m C (= O) -XH ( Ⅷ )

OCH _3- (CH ₂ CH ₂ O) nCH ₂ CH ₂ OC (= O) -XH ( Ⅸ )

OCH _3- (CH ₂ CH ₂ O) nCH ₂ CH ₂ -XH ( Ⅹ )

Where Y is a suitable leaving group such as halogen, X is O, S or NH, m is 1-6, preferably 1-3, n is 10-460, preferably 10-220, more Preferably it represents the integer of 10-120.

Compounds belonging to the present invention are intended to treat organ transplant rejection such as kidney, heart, liver, lung, bone marrow, pancreas, cornea and small intestine and to treat skin allograft and rejection of heart xenograft, or to treat and inhibit graft versus host reaction. Can be used as It can also be used to treat and prevent diseases such as lupus, lumatis arthritis, diabetes mellitus, myasthenia gravis, dermatosis, eczema, seborrheic dermatitis, enteritis, pneumonia (asthma, chronic lung disease, emphysema, respiratory syndrome, bronchitis, etc.) and uveitis. Can be. In view of the pharmacological effects of cyclosporine, the compounds of the present invention are also expected to have antifungal and antiproliferative effects and to be effective in hyperproliferative vascular diseases such as fungal infections, relapse stenosis and atherosclerosis. When used for this purpose, the compounds of the present invention can be administered before disease induction, during treatment, after cure, and for simultaneous purposes.

When administered for the treatment or inhibition of such disease states, the compounds of the present invention can be administered orally, parenterally, intranasally, transdermally, intratracheally, locally vaginally, or rectally. Since the compound of the present invention has high water solubility, it is particularly useful as a drug for immunosuppression, anti-inflammatory action, antifungal action and anti-proliferative action.

When the compounds belonging to the present invention are administered as an immunosuppressive agent or an anti-inflammatory agent, they may be co-administered with one or several kinds of immunomodulators. The immunomodulators administered in combination include azathioprine, adrenocortical hormones such as prednisolone and methylprednisolone, cyclophosphamide, rapamycin, tacrolimus, and OKT- 3, ATG, and the like, but are not limited to these. The desired effect can be obtained with a small amount of the drug by co-administration of the drug used for immunosuppression or inflammation treatment with the compound of the present invention.

The compounds belonging to the present invention can be formulated purely or as necessary with pharmaceutical excipients. Pharmaceutical excipients may be solid or liquid.

Solid excipients may include one or more of fragrances, lubricants, solubilizers, suspending agents, fillers, lubricants, tablets, binders, substances which act as disintegration aids in tablets, and may also be used as the main ingredient encapsulant. Works. In powders, the excipient is a fine powder that can be mixed evenly with the refined main ingredient. In tablets, the active component is mixed with the necessary moldable excipients in suitable proportions and compacted in a suitable shape and size. Suitable solid excipients include calcium phosphate, magnesium stearate, talc, sugar, lactose, dextran, starch, gelatin, cellulose, methylcellulose, sodium CMC, povidone, low melt wax, ion exchange resins.

Liquid excipients are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, high pressure compositions. Liquid excipients, such as water or organic solvents, mixtures of water and organic solvents, pharmaceutically suitable oils or fats, are used to dissolve or disperse the active ingredient. Liquid excipients may contain other pharmaceutically usable additives, such as buffers, preservatives, sweeteners, flavorings, emollients, colorants, viscosity modifiers, stabilizers, osmotic pressure regulators and the like. Suitable excipients used for oral or parenteral administration include water (partially containing additives such as sodium CMC solution, a cellulose derivative), alcohols (polyhydric alcohols such as monohydric or glycols), lecithin, oils (e.g. For example, coconut oil and arachis oil).

Excipients in parenteral administration are used as sterile liquid compositions. Liquid excipients for high pressure compositions in aerosol form may include halogenated hydrocarbons or other pharmaceutically usable sprays. Pharmaceutically usable liquid compositions such as sterile solutions or suspensions can be used, for example, for intramuscular injection, intraperitoneal injection or subcutaneous injection, and can also be administered intravenously. Of course, the compounds belonging to the present invention may be prepared in liquid or solid composition and administered orally.

The compounds belonging to the present invention may be prepared in the form of general suppositories and administered intrarectally. For nasal administration, bronchial inhalation administration, or gas injection, the compounds belonging to the present invention must be formulated in aqueous or partial aqueous solutions which can be used in the form of aerosols. The compounds belonging to the present invention can be administered to the skin using a transdermal patch containing an excipient which is inert with the active ingredient and non-toxic to the skin and allows the transport of drugs that enter the blood vessels through the skin and are systemically absorbed. At this time, the transport vehicle may be in the form of cream, ointment, pasta, gel, occlusive devices. Creams and ointments are oil-in-water or semi-solid emulsions or viscous liquids in water-in-oil form. Pasta is made from hygroscopic powder dispersed in petroleum or hydrophilic petroleum containing the active ingredient. Occlusive devices, such as semipermeable membranes surrounding an active ingredient or a reservoir containing an active ingredient and an excipient or a matrix containing the active ingredient, are used to deliver the active ingredient into the bloodstream.

In addition, the compounds belonging to the present invention can be used in solutions, creams and lotions prepared using pharmaceutically usable adjuvants containing 0.1-5% suitably 2% of active ingredients that can be directly applied to the fungal infection. have.

The formulation required may vary with a particular composition depending on the route of administration or the extent of the symptoms and the particular purpose of the treatment. In particular, unit dosage forms, such as tablets or capsules of pharmaceutical compositions, may be divided and used again in unit dosages containing suitable doses of the active ingredient. For example, they are packaged and used in compressed powders, vials, ampoules, prefilled injectables or in small bags filled with liquids. The unit dosage form contains a suitable number of compositions depending on the form of tablet, capsule or package.

The following examples are described in more detail by representative examples of the present invention, but the present invention is not limited in any way by these examples.

Example 1 Synthesis of Cyclosporine Chloroacetyl

Dissolve 1.07 g (0.9 mmol) of cyclosporin and 3.69 g (21.6 mmol) of chloroacetyl anhydride in 15 mL of anhydrous tetrahydrofuran (THF). 5 ml of pyridine was added thereto, followed by reaction for 6 hours while maintaining 45 ° C in a water bath. After the reaction, the solvent was dried under reduced pressure, and extracted with ether and water to the residue. It was washed several times with water and then dried over magnesium sulfate. The solvent was dried again under reduced pressure and then recrystallized from ether / petroleum ether to give the title 818 mg (72% yield). IR (KBr, cm ⁻¹ ) 1760 (ester); ¹ H NMR (300 MHz, CDCl ₃ ) δ 4.0 (2H, s, ClCH ₂ COO); EI-MS: 1265/1263 (M ⁺ ), 1169 (M ⁺ -ClCH ₂ COOH)

Example 2 Synthesis of Cyclosporine Chloromethyl Carbonate

1.0 g (0.832 mmol) of cyclosporine is dissolved in 50 ml of anhydrous methylene chloride or tetrahydrofuran (THF). 2 ml of pyridine was added thereto, and the reaction solution was cooled to 5 ° C. with an ice bath. 2.0 mL (20.8 mmol) of chloromethylchloroformate was slowly added to the above reaction solution and reacted at room temperature for 20 hours. 50 ml of ether was added to the reaction, the resulting inorganics were filtered off and the filtrate was dried under reduced pressure. Recrystallization from ether / petroleum ether (5: 1) gave the title 970 mg (yield 90%). IR (KBr, cm ⁻¹ ) 1760 (ester); ¹ H NMR (500 MHz, CDCl ₃ ) δ 5.70 (2H, dd, J = 58.98 and 6.33 Hz, ClCH ₂ OCOO); ¹³ C NMR (125 MHz, CDCl ₃ ) δ 173.7, 173.4, 173.1, 172.8, 171.6, 171.2, 170.9, 170.8, 170.0, 169.9, 167.6, 153.5 (C = O, 12); MS (FAB) m / z 1294 (M ⁺ ).

Example 3 (KI-301) of Cyclosporine-Monomethoxypolyethylene Glycol Binder

synthesis

Cyclosporine chloroacetyl 140.7 mg (1.1 equiv), monomethoxy polyethylene-succinate 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg (1.0 Equivalent weight) and then suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 482 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of a reactant having an average molecular weight of 6314 and a starting material of 5,054. The mass difference (1,260) was consistent with the cyclosporine chloroacetyl group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 4.20 (2H, OCOCH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 174.18, 173.79, 173.30, 173.07, 172.31, 171.83, 171.74, 171.59, 171.20, 171.16, 171.13, 170.16, 168.09, 167.84 (C = O, 14); MS (MALDI / TOF) m / z 6314 (ave.M. Wt.)

Example 4 Synthesis of Cyclosporine-Monomethoxy Polyethylene Glycol Binder (KI-306)

Method 1

100 mg (0.077 mmol) of cyclosporine chloromethyl carbonate was dissolved in 10 ml of acetonitrile, and 385 mg (0.077 mmol) of monomethoxypolyethylene-succinate 5000 and 50 mg (0.154 mmol) of cesium carbonate were added thereto for 24 hours. Reflux for a while. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 350 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of reactants having an average molecular weight of 6,377 and starting materials of 5,117. The mass difference (1,260) was consistent with the cyclosporine chloromethyl carbonate group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.77 (2H, q, J = 5.67 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.59, 173.27, 172.91, 172.71, 171.87, 171.46, 171.17, 170.87, 170.76, 170.57, 169.98, 169.81, 167.58, 154.07 (C = O, 14); MS (MALDI / TOF) m / z 6377 (Average molecular weight)

HPLC analysis conditions:

1.column: water C8, 3μ, 4.6 × 150 mm

2. Mobile Phase: Acetonitrile: Water (60:40)

3. Flow rate: 1.0 ml / min

4. Column temperature: 65 ℃

5. Detector: UV at 215 ㎚

6. Retention time: 17.85 min

Method 2

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxypolyethylene-succinate 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg ( 1.0 equivalent) was added all at once, suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 480 mg of the title product.

Method 3

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxypolyethylene-succinate 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), potassium carbonate 20.7 mg (1.5 equiv), crown ether 26.4 mg ( 1.0 equivalent) was added all at once, suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 465 mg of the title product.

Example 5 (KI-309) of Cyclosporin-Monomethoxypolyethylene Glycol Binder

synthesis

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxy polyethylene-propionic acid 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg (1.0 equiv) was added all at once, suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 465 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of reactants having an average molecular weight of 6,610 and starting material of 5,350. The mass difference (1,260) was consistent with the cyclosporine chloromethyl carbonate group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.69 (2H, q, J = 5.65 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.60, 173.24, 173.02, 171.76, 171.50, 171.20, 171.12, 170.31, 170.15, 170.02, 169.80, 167.90, 154.41 (C = O, 13); MS (MALDI / TOF) m / z 6610 (ave.M. Wt.)

Example 6 (KI-311) of cyclosporin-monomethoxypolyethylene glycol conjugate

synthesis

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), amino-monomethoxy polyethylene 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg (1.0 Equivalent weight) and then suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 481 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of reactants having an average molecular weight of 6,420 and starting materials of 5,160. The mass difference (1,260) was consistent with the cyclosporine chloromethyl carbonate group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.75 (2H, q, J = 5.67 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 174.25, 173.73, 173.20, 173.13, 171.78, 171.66, 171.24, 171.19, 171.10, 170.27, 168.71 (C = O, 12); MS (MALDI / TOF) m / z 6420 (ave.M. Wt.)

Example 7 Synthesis of Cyclosporine-Monomethoxypolyethylene Glycol Binder (KI-312)

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxy polyethylene-glutanate 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg (1.0 equiv) was added all at once, suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 473 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of reactants having an average molecular weight of 6,388 and starting materials of 5,128. The mass difference (1,260) was consistent with the cyclosporine chloromethyl carbonate group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.75 (2H, q, J = 5.67 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.59, 173.27, 172.91, 172.71, 172.62, 171.47, 171.18, 171.10, 170.87, 170.76, 169.98, 169.81, 167.58, 154.10 (C = O, 14); MS (MALDI / TOF) m / z 6388 (ave.M. Wt.)

Example 8 Synthesis of Cyclosporine-Monomethoxypolyethylene Glycol Binder (KI-313)

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxy polyethylene-malonate 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg ( 1.0 equivalent) was added all at once, suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 478 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of reactants having an average molecular weight of 6,404 and starting materials of 5,144. The mass difference (1,260) was consistent with the cyclosporine chloromethyl carbonate group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.73 (2H, q, J = 5.61 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.59, 173.27, 172.91, 172.71, 172.62, 171.47, 171.18, 171.10, 170.87, 170.76, 169.98, 169.81, 167.58, 154.10 (C = O, 14); MS (MALDI / TOF) m / z 6404 (ave.M. Wt.)

Example 9 Synthesis of Cyclosporine-Monomethoxypolyethylene Glycol Binder (KI-315)

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxy polyethylene-diglycolic acid 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 mg (1.0 equiv) was added all at once, suspended in 50 ml of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 485 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. MS (MALDI / TOF) showed monomethoxypolyethylene-succinate 5000 of reactants having an average molecular weight of 6,303 and starting materials of 5,043. The mass difference (1,260) was consistent with the cyclosporine chloromethyl carbonate group. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.74 (2H, q, J = 5.69 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.95, 173.63, 173.25, 173.0, 171.74, 171.50, 171.22, 171.12, 170.37, 170.14, 169.88, 168.53, 167.86, 154.39 (C = O, 14); MS (MALDI / TOF) m / z 6303 (ave.M. Wt.)

Example 10 Synthesis of Cyclosporine-Monomethoxypolyethylene Glycol Binder (KI-316)

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxy polyethylene-methylsuccinate 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 MG (1.0 equiv) was added all at once, suspended in 50 mL of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 478 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.84 (2H, q, J = 5.58 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.59, 173.27, 172.91, 172.71, 172.62, 171.47, 171.18, 171.10, 170.87, 170.76, 169.98, 169.81, 167.58, 154.10 (C = O, 14).

Example 11 Synthesis of Cyclosporine-Monomethoxypolyethylene Glycol Binder (KI-317)

Cyclosporine chloromethyl carbonate 142.5 mg (1.1 equiv), monomethoxy polyethylene-thiodiglycolic acid 5000 500 mg (0.1 mmol), iodine chloride 33 mg (2.2 equiv), cesium carbonate 50 mg (1.5 equiv), crown ether 26.4 MG (1.0 equiv) was added all at once, suspended in 50 mL of benzene (or THF, toluene) and refluxed for 24 hours. The inorganics were filtered off and the filtrate was dried under reduced pressure. The residue was recrystallized in methylene chloride / ether (1: 5) to give 475 mg of the title product. Unreacted monomethoxypolyethylene glycol was removed by preparative HPLC. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.70 (2H, q, J = 5.77 Hz, OCO ₂ CH ₂ OCO); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.59, 173.27, 172.91, 172.71, 172.62, 171.47, 171.18, 171.10, 170.87, 170.76, 169.98, 169.81, 167.58, 154.10 (C = O, 14).

Example 12 Hydrolysis Test of Cyclosporine-Monomethoxypolyethylene Glycol Binder

In order to prove that the conjugated compound synthesized by the above method was degraded in the body to produce cyclosporin, an enzyme hydrolysis test was performed on the homogenized liver at 37 ° C. Add 3.0 ml of 0.1 M phosphate buffer (pH 7.4) to 3.0 g humans, homogenize in an ice bath and centrifuge for 10 minutes. The supernatant is transferred to another tube. The test solution was prepared by dissolving 51.6 mg (10 mg cyclosporine equivalent / mL) of the conjugated compound in 1.0 mL of 0.1 M phosphate buffer (pH 7.4).

90 μl of supernatant is placed in each eppen rope tube and maintained at 37 ° C. Then, 10 μl of test solution, which was warmed up to 30 ° C., is added. Stir the reaction mixture in each tube for 5 seconds and add 100 μl of acetonitrile (0, 1, 3, 5, 7, 10, 15, 30, 45, 60, 90, 120 minutes apart) and add the reaction for 1 minute. Stir. Centrifuge the tubes at 13,000 rpm for 10 minutes and refrigerate. The theoretical final concentration of the conjugate compound in the tube is 129 μg / ml (250 μg / ml with cyclosporine).

The above synthetics, including KI-306, were added to the let blood and reacted at 37 ° C. to see hydrolysis over time. The blood was put into each reaction tube and the experiment was performed after equilibrating to 37 ° C. in advance. After adding a synthetic material to react with each reaction time, the reaction was stopped with acetonitrile solution. The acetonitrile solution layer was obtained by centrifugation and used for HPLC analysis. In addition, the stability test of each material at pH 1.0 and pH 7.4 was performed. 0.1 M hydrochloric acid solution pH 1.0 and phosphate buffer solution pH 7.4 Each reaction tube was previously equilibrated at 37 ° C, and then reacted with the addition of each substance. 150 μl of a predetermined amount was taken according to the predetermined time, and the reaction was stopped by mixing with the same amount of acetonitrile solution and analyzed by HPLC.

20 μl of each sample solution was analyzed by HPLC. HPLC analysis used a water reversed phase column CN, 5μ (4.6 x 250 mm). The mobile phase was used in concentration gradient method from water: acetonitrile (35: 65) to water: acetonitrile (20:80). The flow rate was 1.0 ml / min and the eluate was observed at 214 nm, 65 ° C.

As shown in Figure 1, the conjugate compound of the present invention is degraded in liver homogenate to produce the active substance cyclosporin. It can be seen in Figure 2 that the conjugated compound is proportionally degraded by the first order kinetics. Therefore, it can be seen that the conjugate compound of the present invention is converted back to cyclosporine by enzymatic action of liver homogeneity. And the hydrolysis half life of the conjugate compound is approximately 2.2 minutes at 37 ° C. This is most ideal as a cyclosporine precursor. Nevertheless, the conversion of non-enzymatic cyclosporine from pH 7.4 phosphate buffer solution showed a half-life of 21 hours at 37 ° C.

The half-life in the lett blood of other compounds including KI-306 is shown in Table 1. The half life is shown in Table 2 through stability tests at pH 1.0 and pH 7.4 of the compounds.

Hydrolysis of Cyclosporin-Prodrug Compounds in Rat Blood Compound number Half-life (minutes) KI-306 9.9 KI-309 65.0 KI-312 14.2 KI-313 3.4 KI-315 2.1 KI-316 9.5 KI-317 1.6

PH-dependent Hydrolysis of Cyclosporine-Prodrug Compounds Compound number Half-life (hours) pH 1.0 pH 7.4 KI-306 18.2 26.4 KI-309 32.0 16.6 KI-312 10.0 28.0 KI-313 20.0 5.5 KI-315 5.5 2.8 KI-316 43.4 - KI-317 10.4 4.6

Example 13 Pharmacokinetic Experiment

The KI-306 of the present invention was compared with pharmacokinetics by the single oral administration of Sandimun neoral solution currently used in clinical practice. Sprague-Dawley rats with a body weight range of 220 ± 30 g were used for the experiment, and the animals were fasted one day before the experiment and water was freely taken. Comparative drug Sandimun Neoral (Switzerland Novartis) and KI-306 of the present invention were dissolved in physiological saline immediately before administration, and orally administered to the respective doses of 7 mg / kg of cyclosporin. Oral sonde was used for oral administration, and samples were collected from the rat tail vein using an intravenous cannula to collect about 200-250 μl of blood at intervals of 10 μl of heparin at a predetermined time interval. 300 µl of acetonitrile was added to 100 µl of the collected blood, mixed for about 1 minute, and then centrifuged. Only the supernatant was analyzed by HPLC.

As a result, the KI-306 of the present invention was hydrolyzed after absorption and detected as cyclosporin, but the precursor KI-306 was not detected. Also, as shown in FIG. 3, the disappearance half-life of KI-306 in the intravenous injection of red was 2.5 minutes, which is almost identical to the half-life of 2.2 minutes in the homogenized human liver.

The pharmacokinetic parameter lektimes for both compounds showed values obtained using the WinNonlin program. According to the results shown in Table 3, the KI-306 of the present invention is about 65% higher than the AUC of blood concentration in the Sandimun neoral solution, indicating that the bioavailability of KI-306 is higher as shown in FIG. appear.

Pharmacokinetic Comparison of KI-306 and Neoral PK Parameter KI-306 Neoral Average (μg h / ml) Correlation Coefficient (%) Average (μg h / ml) Correlation Coefficient (%) AUC 32.79 19.85 21.40 10.03 C _max 1.77 4.40 1.08 3.36 T _max 1.43 11.35 2.55 16.08

The water-soluble polymer-cyclosporin conjugate of the present invention has a half-life of half-hydrolysis in the hydrolysis test by enzymes of liver, and after 10 hours has been converted to more than 90% cyclosporine, orally administered to a rat and collected and analyzed for blood. In this regard, the water-soluble polymer-cyclosporin conjugate was not detected but was detected as a hydrolyzed cyclosporin, so that hydrolysis is easily achieved in vivo. In the pharmacokinetic experiment, the area under the blood curve was 32.79 in water-soluble polymer-cyclosporine conjugate of the present invention, which was about 65% higher than that of 21.4 of Neonal, a conventional product.

That is, the water-soluble polymer-cyclosporin conjugate of the present invention has a high bioavailability and tissue utilization rate and thus obtains the same effect even when a small amount is administered than conventional products.

In addition, a small amount of administration can reduce side effects such as nephrotoxicity, hypertension, hyperkalemia that can be caused by cyclosporin.

Claims (7)

Water-soluble polymer-cyclosporine conjugate of the general formula ( I ) In the above formula, R is the same as the following group. -C (= O) -CH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( II ) -C (= O) -OCH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( III ) -C (= O) -OCH ₂ XC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( Ⅳ ) -C (= O) -OCH ₂ XCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ ( Ⅴ ) X represents O, S or NH, m is 1 to 6, n is 10 to 460. The conjugate of claim 1 wherein m = 1-3. The conjugate of claim 1 wherein n = 10-220. 4. The conjugate of claim 3, wherein n = 10-120. The substituent R according to any one of claims 1 to 4, wherein the substituent R of general formula ( I ) is -C (= O) -CH ₂ OC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ OC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ SC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ NHC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ OC (= O) -CH ₂ OCH ₂ C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= 0) -OCH ₂ OC (= 0) -CH ₂ SCH ₂ C (= 0) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , —C (═O) —OCH ₂ OC (═O) —CH (CH ₃ ) CH ₂ C (═O) —OCH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n —OCH ₃ , -C (= O) -OCH ₂ OC (= O) -CH ₂ C (CH ₃ ) ₂ CH ₂ C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ OC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ SC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ NHC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , —C (═O) —OCH ₂ OCH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n —OCH ₃ , -C (= 0) -OCH ₂ SCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ or -C (= 0) -OCH ₂ NHCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ . Cyclosporine (CsA) is reacted with the formula (Ⅵ) with a group the formula (Ⅶ) manufacturing, and in the manufacture of formula (Ⅶ) polyethylene glycol derivative formula (Ⅷ), formula (Ⅸ) or by reacting with a general formula (ⅹ) process for producing a conjugate of formula (ⅰ). Y (C = O) OCH ₂ Y or YCH ₂ (C = O) O (C = O) CH ₂ Y ( Ⅵ ) CsA-OC (= O) OCH ₂ Y or CsA-OC (= O) CH ₂ Y ( Ⅶ ) OCH _3- (CH ₂ CH ₂ O) nCH ₂ CH ₂ OC (= O) (CH ₂ ) _m C (= O) -XH ( Ⅷ ) OCH _3- (CH ₂ CH ₂ O) nCH ₂ CH ₂ OC (= O) -XH ( Ⅸ ) OCH _3- (CH ₂ CH ₂ O) nCH ₂ CH ₂ -XH ( Ⅹ ) In the above formula R is —C (═O) —CH ₂ XC (═O) — (CH ₂ ) _m C (═O) —OCH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n —OCH ₃ , -C (= O) -OCH ₂ XC (= O)-(CH ₂ ) _m C (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , -C (= O) -OCH ₂ XC (= O) -OCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ or -C (= 0) -OCH ₂ OXCH ₂ CH _2- (OCH ₂ CH ₂ ) _n -OCH ₃ , and Y represents a leaving group, X represents O, S or NH, m is 1 to 6, n is 10 to 460. A pharmaceutical composition for immunosuppression, anti-inflammatory, anti-fungal or anti-proliferative containing the general formula ( I ) water-soluble polymer-cyclosporin conjugate of claim 1 as an active ingredient.