JP3208570B2 - Galactosamine-substituted poly-ω-substituted-L-glutamic acid (or aspartic acid) - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a galactosamine-substituted product of poly-ω-methyl (or benzyl) -L-glutamic acid (or aspartic acid), which is useful as a medical polymer material, particularly a missile pharmaceutical carrier.

[0002]

2. Description of the Related Art Glycoproteins in serum generally have a sugar structure of sialic acid-galactose-N-acetylglucosamine at the terminal. In the late 1960's G. Ashwell and A.M. Morell found that this trisaccharide structure was necessary for serum proteins to be stable in blood. When the terminal sialic acid is removed, galactose becomes the new sugar terminal. The glycoprotein from which sialic acid is removed to expose galactose is called asialoglycoprotein. In this condition, asialoglycoprotein cannot be stably present in the bloodstream and rapidly disappears from the bloodstream. It has been found that about 80% or more of the asialoglycoprotein lost is taken up by the liver.

By the way, a specific sugar recognition receptor exists on the membrane surface of hepatocytes, and asialoglycoprotein is taken into cells via this asialoglycoprotein receptor. The present inventors have focused on the asialoglycoprotein receptor on the liver cell membrane and studied repeatedly with the aim of developing a polymer material for a drug carrier used for a missile drug or the like. The present inventors have found that amino acids have excellent properties and completed the present invention.

[0004]

[Means for Solving the Problems] That is, the present invention provides:
General formula

[0005]

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Wherein x represents a degree of polymerization of 60 to 250, n represents 1 or 2, and R represents a methyl group or a benzyl group, respectively. Part or all of the general formula

[0007]

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## STR1 ## wherein n is as defined above, and a ω-galactosamil-L-glutamic acid (or aspartic acid) residue and optionally a compound of the formula

[0009]

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In the formula, galactosamine of poly-ω-alkyl (or benzyl) -L-glutamic acid (aspartic acid) substituted with an L-glutamic acid (or aspartic acid) residue represented by the following formula: Pertaining to substitutions. The polypeptide of the present invention is further described as follows. Structural unit: ω-alkyl (or benzyl) -L-glutamic acid (aspartic acid) residue

[0011]

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(Wherein n and R are the same as above) L-glutamic acid (aspartic acid) residue

[0013]

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(Wherein, n is the same as above.) Ω-galactosamil-L-glutamic acid (or aspartic acid) residue

[0015]

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A galactosamilylated ω- methyl (or benzyl) -L-glutamic acid (or aspartic acid) linear polymer having a structural unit represented by the following formula (A) to (C): A: degree of polymerization 60 to 250 B: molecular weight 8,000 to 71,000 C: ratio of each structural unit ω- methyl (or benzyl) -L-glutamic acid (or aspartic acid) residue 0 to 97 Mol% L-glutamic acid (or aspartic acid) residue 0 to 87 mol% ω-galactosamil-L-glutamic acid (or aspartic acid) residue 3 to 100 mol%

[0017]

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(Wherein n and R are the same as above.
And Z are numbers smaller than 1 and satisfy Y ≧ Z. ) Heavy This method comprises poly -ω- side chain carboxyl group by hydrolyzing the side-chain methyl Et <br/> ester (or benzyl ester) substituent -L- glutamic acid (or aspartic acid) (II) is liberated The compound (III) is obtained (first step), and then galactosamine is introduced into the side chain carboxyl group of the polymer (III) to obtain the target compound (I) of the present invention (second step).

In the hydrolysis in the first step, poly-γ- methyl (or benzyl) -L-glutamic acid or poly-β- meth
It can be easily carried out by treating tyl (or benzyl) -L-aspartic acid with a base in a suitable organic solvent. As the organic solvent, for example, halogenated hydrocarbons (helical solvents) such as chloroform and dichloromethane are suitable, but random coil solvents such as dichloroacetic acid and trifluoroacetic acid can be used.

As the base, sodium hydroxide, potassium hydroxide and the like are suitable. These bases are usually added to the reaction solution as an aqueous alcohol solution such as methanol or isopropyl alcohol. The reaction is carried out at around room temperature,
Perform for 200 minutes. The hydrolysis rate can be arbitrarily adjusted by appropriately selecting these reaction conditions, particularly the reaction time. The poly-ω-substituted-L-glutamic acid (or aspartic acid) (II) used as the starting compound in this step has a degree of polymerization of about 60 to 250, but is not limited thereto. Absent. In the examples described below, for example, poly-γ-methyl-L-glutamate (abbreviated as PMLG) has a degree of polymerization of about 100.
~ 200 (molecular weight of about 14,000-29,000) was used.

The second step is the peptidation between the side chain carboxyl group of polyglutamic acid (or polyaspartic acid) (III) and the primary amino group of galactosamine. For this peptidation, a method of activating a carboxyl group or an amino group, a method of conducting in the presence of a condensing agent, and the like can be employed. Among these, as the peptide for activating the carboxyl group, the carboxyl group of the hydrolyzate (III) obtained in the first step was activated in the form of, for example, p-nitrophenyl ester, and the activated compound was separated. Later, this is reacted with galactosamine. This reaction is carried out using dimethylformamide (DMF), tetrahydrofuran (THF), dimethylsulfoxide (DMS).
The reaction is carried out in a solvent such as O) at room temperature or under cooling. The reaction time is several hours to several days. The progress of peptidation can be determined by quantifying p-nitrophenol released with the reaction.

Next, as a method using a condensing agent, for example, N, N'-dicyclohexylcarbodiimide (D
CC), the partial hydrolyzate (III) and galactosamine are coupled in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or the like. The reaction conditions are the same as the above-mentioned peptidation due to activation of the carboxyl group. The target compound (I) thus produced can be purified, for example, by dialysis using a cellulose dialysis membrane.

[0023]

As described above, the target compound of the present invention is expected to have a recognition effect on target living cells, and can be applied to the medical field as a biorecognition polymer. In addition, since the target compound of the present invention is a polyamino acid derivative that is a naturally similar polymer, it is biodegradable.
It is water soluble. Therefore, this compound is suitable as a polymer material for a drug carrier used for a missile drug or the like. Next, hepatocyte affinity of the compound of the present invention is shown by animal experiments using rats.

Experimental Example Using SD female rats (4-5 weeks old), rat hepatocytes were separated according to the so-called Seglen perfusion method in which the intercellular adhesion protein was digested with an enzyme. The prepared hepatocytes were suspended in ice-cooled WE medium at a concentration of 400,000 cells / ml. Thereafter, using a disposable pipette, 1.5 ml of the hepatocyte suspension was inoculated on each polymer-coated petri dish (Note 1) prepared in advance, and cultured at 37 ° C. in a carbon dioxide gas culturing apparatus at a carbon dioxide gas concentration of 5% for a predetermined time. . Then, by counting the number of non-adherent cells,
The adhesion rate was calculated.

The polymer used in this experiment was the target compound of the present application and the poly-γ-methyl-L-
Glutamate (abbreviated as PGA) and a polyvinyl-based polymer (polybenzyllactone amide, abbreviated as PVLA) for which the affinity for hepatocytes is conventionally known as a control
It is. FIG. 1 shows the state of initial adhesion of hepatocytes to each petri dish. Looking at the graph, hepatocytes do not adhere much to the main chain polymer, and there is no physiological activity of the main chain itself. On the other hand, the polymer of the present invention having galactosamine in the side chain showed a high adhesiveness like PVLA.

(Note 1) Preparation of polymer-coated petri dish Each sample was dissolved in Milli-Q water at a concentration of 0.05% (W / V). A polymer-coated petri dish was prepared by injecting 2 ml of the polymer solution into a petri dish, freeze-drying, rinsing three times in Milli-Q water, and air-drying.

Next, FIG. 2 shows the adhesion rates of the compounds of the present invention having different galactosamine substitution ratios to each polymer-coated petri dish. The graph shows that hepatocytes do not adhere well to the main chain polymer and the polymer having a sugar content of 25%, and it is considered that the sugar side chain has no effect at a content of about 25%. In addition, the sugar content is 4
The increase in the hepatocyte adhesion rate was observed for the polymers increased to 0%, 60%, 70%, and 85%, and almost the same adhesion behavior was observed for the samples having sugar residues of 60% or more. Indicated.

The dosage form for the pharmaceutical preparation using the compound of the present invention includes, for example, nanosphere formation. An example of a method for preparing nanospheres is as follows.
Dissolve lipiodol, isobutyl cyanoacrylate and drug in ethanol. On the other hand, the nonionic surfactant and the compound of the present invention are dissolved in water, and the above ethanol solution is gradually poured into this aqueous solution with vigorous stirring. After concentrating the reaction solution and freeze-drying,
A nanosphere containing or adhering the compound of the present invention and a drug is obtained.

[0029]

EXAMPLES Next, the target compound of the present invention and the method for producing the same will be further described with reference to examples. Example 1 (1) Hydrolysis of side chain methyl ester of PMLG 11.57 g of PMLG was dissolved in 100 ml of chloroform to prepare an 8% solution, and 35.8 ml of 2N-sodium hydroxide and methanol were added to the solution while stirring. A mixed solution of 71.5 ml and 71.5 ml of isopropyl alcohol (volume ratio 1: 2: 2) was added dropwise over 15 minutes, and then stirring was continued at room temperature to carry out a hydrolysis reaction of the side chain methyl ester. At that time, the reaction was carried out while changing the stirring time. Thereafter, the reaction was terminated by neutralization with glacial acetic acid, and the reaction solution was added to 500 ml of diethyl ether with stirring to precipitate the product. Then, the precipitate was filtered and washed several times with diethyl ether. A small amount of distilled water was added to the gel to form a gel. The gel was placed in a dialysis tube, dialyzed at room temperature for 2 days, and then the side-chain hydrolyzed polymer was freeze-dried. Prepared. The dialysate was replaced as appropriate.

¹ H—N of the obtained side chain hydrolyzed polymer
FIG. 3 shows the MR spectrum. In the figures, the spectra (a), (b) and (c) are obtained by increasing the reaction time in order from the top, and the lower spectrum (c) is obtained by reacting at room temperature for 3 days. Looking at the results, the peak of the side chain methyl ester decreases in order from the top, indicating that the side chain hydrolysis reaction proceeds according to the reaction time.

(2) Activation of Side Chain Carboxyl Group 0.8 g (5.9 × 10 ⁻³ m) of side chain partially hydrolyzed polymer
ol, a value calculated from an apparent molecular weight per unit in monomer units) and p-nitrophenol 0.5
5 g (4.0 × 10 ⁻³ mol) was added to 20 ml of DMF, and 0.82 g (4.0 × 10 ⁻³ mo) of DCC was added to the solution.
1) was added and the reaction was carried out by stirring at 0 ° C. for 30 minutes and then at room temperature for 2 days. Thereafter, the sample was left in a refrigerator for 2 hours, and the precipitate was thoroughly washed with DMF, water and hot ethanol in that order, and then dried to prepare a sample.
(This method is based on the modification of a polymer having a side chain ester hydrolysis rate of 28.6%. In other reactions, the charged amounts of p-nitrophenol and DCC are also determined by the molar amount of carboxyl groups in the polymer side chain. The amount was 1.5 to 2 times the number.)

FIG. 4 shows the UV spectrum of the compound obtained. In the figure, a peak of p-nitrophenol was observed at 310 nm, and it was confirmed that p-nitrophenol was introduced into the polymer side chain. The side chain activation rate (p-nitrophenol introduction rate) in this reaction was determined by UV
It was identified by measuring the spectrum. As a method, 0.
The reaction product was dissolved in methanol at a concentration of 2 g / l, and 0.1 N potassium hydroxide was added to the solution to add 10 g.
After stirring vigorously for a minute, the p-
A method for measuring the absorption of nitrophenol was used.

(3) Coupling with galactosamine (Active ester method) 0.22 g (1.04 × 10 ⁻³ mol) of galactosamine hydrochloride was added to 10 m of DMF.
dissolved in 0.15 ml of triethylamine (1.05 ml).
After adding 4 × 10 ⁻³ mol), PGA-ONp0.3 was added.
0 g (1.84 × 10 ⁻³ mol, calculated from the apparent molecular weight per unit) was added, and the mixture was reacted at room temperature for 2 days. Thereafter, the solution including the precipitate was dialyzed (for 2 days), and a sample was prepared by freeze-drying. (The amount of charge given here is that of a sample prepared by performing side chain activation using a polymer having a side chain hydrolysis rate of 28.6% as described above. About twice as much sugar and triethylamine were used depending on the rate.)

(Condensing agent method) 0.45 g of PGA in an aqueous solution
And 1.5 times, 1 time, 0.
Galactosamine (Gal-NH ₂ ) of 75 times, 0.5 times, and 0.25 times mol was continuously dissolved, and a solution was prepared with 0.1N hydrochloric acid to adjust the pH to 4.7. An aqueous solution of pH 4.7 in which 1.5 times mol of EDC of galactosamine used in this solution was dissolved was added dropwise at 0 ° C. over 8 hours. Subsequently, after reacting at room temperature for 24 hours, the sample was dialyzed for 2 days, and lyophilized to prepare a sample.
Compounds obtained by these reactions (PGA-Gal) ¹
FIG. 5 shows the measurement results of the H-NMR spectrum. As is clear from the figure, a saccharide peak was observed at around 4 ppm in each sample, confirming that the saccharide was introduced into the polymer side chain.

A galactosamine-substituted product obtained by adding 1.5-fold, 1-fold, 0.75-fold and 0.5-fold galactosamine to the side chain carboxyl group and coupling the same by the above condensing agent method. ¹ H-NMR of (PGA-Gal)
The spectra are shown in FIGS. 6, 7, 8 and 9. As is clear from the figure, the substitution ratio of galactosamine was 85 mol % [PGA, corresponding to the amount of galactosamine used.
-Gal (85)], 70 mol % [PGA-Gal (7
0)], 60 mol % [PGA-Gal (60)] and 40 mol % [PGA-Gal (40)].

[Brief description of the drawings]

FIG. 1 shows a compound of the present invention (PGA-Gal: substitution ratio 7)
5) is a diagram showing the affinity of a starting compound (PGA) and a control (PVLA) for rat hepatocytes.

FIG. 2 shows the compounds of the present invention having different sugar contents.
FIG. 4 is a diagram showing the difference in hepatocyte adhesion rate for each culture time.

FIG. 3 is a ¹ H-NMR spectrum showing the progress of hydrolysis of a side chain methyl ester of PMLG. (C) in the figure
Was measured for the product after reacting at room temperature for 3 days.

FIG. 4 shows a UV spectrum of the compound obtained in Example 1 (2).

FIG. 5 shows a ¹ H-NMR spectrum of a compound obtained by an active ester method of Example 1 (3).

FIG. 6 shows a ¹ H-NMR spectrum of PGA-Gal (85).

FIG. 7 shows a ¹ H-NMR spectrum of PGA-Gal (70).

FIG. 8 shows a ¹ H-NMR spectrum of PGA-Gal (60).

FIG. 9 shows a ¹ H-NMR spectrum of PGA-Gal (40).

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C08G 69/00-69/50 A61K 47/48 C07K 9/00 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A compound of the general formula (Where x is a degree of polymerization of 60 to 250, and n is 1
R represents a methyl group or a benzyl group, respectively. ), A part or all of the constituent peptides are represented by the general formula: (Wherein, n represents the above-mentioned meaning)
Poly-ω- methyl (or benzyl) substituted with a galactosamil-L-glutamic acid (or aspartic acid) residue
-A galactosamine-substituted product of L-glutamic acid (or aspartic acid). 2. An ω- methyl (or benzyl) -L-glutamic acid (or aspartic acid) residue. (Wherein, n represents 1 or 2, R represents a methyl group or a benzyl group, respectively), an L-glutamic acid (or aspartic acid) residue (Wherein n represents the same meaning as above) and a residue of ω-galactosamil-L-glutamic acid (or aspartic acid) (Wherein, n represents the above-mentioned meaning) is a galactosamilated ω- methyl (or benzyl) -L-glutamic acid (or aspartic acid) linear polymer having a constitutional unit, and satisfies the following AC: Polymer A: degree of polymerization 60 to 250 B: molecular weight 8,000 to 71,000 C: ratio of each structural unit ω- methyl (or benzyl) -L-glutamic acid (or aspartic acid) residue 0 to 97 mol% L -Glutamic acid (or aspartic acid) residue 0 to 87 mol% ω-galactosamil-L-glutamic acid (or aspartic acid) residue 3 to 100 mol%