JP2003327693A - HYDROPHILIC/HYDROPHOBIC MODIFIED POLY(gamma-GLUTAMIC ACID) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a hydrophilic/hydrophobic modified poly(γ-glutamic acid) having a novel function. <P>SOLUTION: The modified poly(γ-glutamic acid) is prepared by introducing a graft of a partially hydrophilic group and a partially hydrophobic group into poly(γ-glutamic acid) through side chain carboxyl groups. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to poly (γ-glutamic acid) modified with a parent-hydrophobic group.

[0002]

2. Description of the Related Art Poly (γ-glutamic acid) is produced very efficiently by fermentation of a microorganism of the genus Bacillus (for example, 50 g per liter of culture solution), and a carboxyl group derived from an α-carboxyl group has a side chain. Which is a linear water-soluble biodegradable polymer. Taking advantage of such characteristics, poly (γ-glutamic acid) serves as a drug carrier for supporting an anticancer agent through a chemical bond or encapsulating a drug in a gel matrix through gelation by chemical crosslinking. Uses have been proposed (Japanese Patent Laid-Open No. Hei 6)
-92870, JP-A-6-256220).

The above chemically crosslinked poly (γ-glutamic acid) (hereinafter sometimes abbreviated as γ-PGA)
Can be said to be a derivative of γ-PGA which has opened up a new application by being crosslinked by a bifunctional group via its side chain carboxyl group.

[0004]

DETAILED DESCRIPTION OF THE INVENTION γ-PGA is a polymeric material useful as such or as a derivative thereof as described above. However, if possible, γ-PG with additional functions
It would also be desirable to provide an A derivative. The functionalization of γ-PGA has been studied from such a viewpoint.
Γ-PGA by individually introducing a certain hydrophilic group and hydrophobic group via a part of the side chain carboxyl groups of
We have found that some of them form polymeric micelles that are associated molecular aggregates in aqueous media. Such polymer micelles can also be used as a new drug carrier capable of encapsulating a drug or the like in micelles. Even if γ-PGA alone cannot form a polymeric micelle, a suitable drug is physically or chemically bound, for example, through a hydrophobic group or a remaining carboxyl group. In some cases, polymeric micelles containing the drug can be formed. Thus, the present inventors succeeded in imparting a new function to γ-PGA.

Therefore, according to the present invention, a parent-hydrophobic modified γ-PGA having a new function is provided. Specifically, the present invention provides a compound of general formula (I)

[0006]

[Chemical 2]

Wherein A is a group-(CH ₂ CH ₂ O) _n --R ¹ , R ¹ represents C ₁ -C ₆ alkyl or C ₁ -C ₅ alkylcarbonyl, and n is 1, preferably 10
Represents any integer of 200, B is a group --CHR ² R ³ , R ² represents C ₃ -C ₂₁ alkyl, and R ³ represents hydrogen or C ₁ -C ₆ alkyloxycarbonyl. ,
X and Y independently represent -O- or -NH-,
p, q and r independently represent an integer of 1 or more, and p +
The total number of q + r represents any integer of 20 to 2500, and each repeating unit may partially form a block, but it is essentially random and is represented by a modified poly (γ- Glutamic acid) or a salt thereof.

[0008]

DETAILED DESCRIPTION OF THE INVENTION Alkyl or alkyl moiety of alkylcarbonyl or alkyloxycarbonyl defining each group in the general formula (I) may be straight-chain or branched in common. Therefore, C ₁
Specific examples of -C ₆ or C ₁ -C ₅ alkyl include methyl, ethyl, n-propyl, isopropyl and n.
-Butyl, isobutyl, s-butyl, t-butyl, n-
Pentyl, isopentyl, t-pentyl, C
Examples of _6- alkyl include n-hexyl and isohexyl.

When R ² in the group --CHR ² R ³ related to B in the general formula (I) is C ₃ -C ₂₁ alkyl, among the above-mentioned alkyl, C ₃ (having 3 carbon atoms) ) In addition to the above, C ₂₁ is a long-chain aliphatic hydrocarbon group which may be branched.

Of the alkyls thus defined, R ¹
Is preferably an alkyl group having up to 3 carbon atoms because the A moiety in the general formula (I) is intended to form a hydrophilic domain. Of course, in part A,-(CH ₂ CH ₂
If the number of n in O) _n -R ¹ is large, the carbon number of the alkyl of R ¹ may be 6.

The number of n is not limited as long as such a part A functions as a hydrophilic domain in the molecule represented by the general formula (I), but it is generally 1 or more, preferably n is 10 to 10. Any integer of 200, more preferably 40 to
It is an integer of 120. As will be described later, the -X-A part is usually a polyethylene glycol derivative having an amino group or a hydroxyl group at one end, and those groups and γ-
It is preferable that each X is —NH— or —O— because it can be introduced by reacting PGA with a side chain carboxyl.

On the other hand, since part B is intended to form a hydrophobic domain, R ² and R ³ are selected so that the group —CHR ² R ³ as a whole exhibits hydrophobicity. Further, in order to maintain the biodegradable property of γ-PGA, it is preferable that the B part mainly contains the residue of the compound of biological origin. Thus, the group —CHR ² R ³ is
It is preferably a hydrophobic amino acid residue in which the carboxyl group is protected together with -NH- or -O- of Y linked to the GA main chain, or a residue of a higher aliphatic alcohol.

Without being limited thereto, typical residues when Y is --NH-- can include, for example, those derived from carboxylic acid esters of valine, leucine or isoleucine, while Typical residues when Y is --O-- include saturated alcohols in natural fats and oils, such as C ₂₂ docosanol, C ₁₉ 16-methyloctadecanol, C ₁₈ stearyl alcohol, C _18. It can be the residue of ₁₆ cetyl alcohols, C ₁₂ lauryl alcohol, C ₁₀ decanol, C ₈ octanol.

The total number of p, q and r is -Y-B and -X, respectively, using γ-PGA as a raw material as described later.
When -A is grafted, it varies depending on the molecular weight of the raw material γ-PGA. Bacillus s
γ-PGA obtained by fermentation of ubtilis has been obtained with a molecular weight of several hundred thousand.
Γ-PGA having a molecular weight of about 300,000 has been obtained from strain 0-1 and is also commercially available. Modified γ-PG of the present invention
A is a commercially available product as it is, or by culturing a strain of Bacillus or Subtilis, and by mechanically or chemically hydrolyzing a high molecular weight γ-PGA, the molecular weight is lowered. Can be obtained as a starting material. Therefore, in general, the total number of p, q and r can be any integer from 20 to 3000, preferably 180 to 2400. Further, in the modified γ-PGA of the present invention, in order to achieve the intended function, the side chain carboxyl group, —COYB
And p, q, and r are independently integers of 1 or more, because it is essential that the repeating units each having —COXA and —COXA are present together.

The modified γ-PGA of the present invention has a content of about 300,000.
The amount of γ-PGA is used as a raw material, and -Y-B is used as L-leucine.
Methyl ester and -X-A is converted to H₂N
(CH ₂CH₂O)_nCH₃[Poly (ethylene oxide) content
In the case of forming with a weight of 5000], q / (p +
When the ratio of q + r) becomes a constant value of 0.25 or more, water
To form polymeric micelles, which are molecular aggregates associated with each other
be able to. On the other hand, the above ratio is well below 0.25.
In other cases, the modified γ-PGA becomes water soluble,
These alone do not form polymeric micelles in water.

The ability of such modified γ-PGA to form polymeric micelles varies depending on the molecular weights of the units having the A and B parts and the types of residues constituting them. In addition, when the modified γ-PGA can form a polymer micelle, n of the A part is
The particle size of the polymeric micelle can be adjusted by varying the number.

The modified γ-PGA capable of forming polymer micelles as described above is generally a hydrophobic drug (a hydrophobic drug is
It is not limited as long as it is known to be encapsulated in a known polymer micelle for the purpose of water solubilization, or includes a wide range of drugs also called poorly water-soluble drugs). By coexisting in a solvent, a polymeric micelle encapsulating the drug can be formed. In such a system, even if the original modified γ-PGA itself could not form polymeric micelles, the drug and -Y-B may not necessarily be formed.
It is presumed that the polymer micelles can be formed by the interaction with the hydrophobic domain by the unit having a.
In addition, the polymer micelle thus formed can expose the carboxyl group on the surface of the micelle, and therefore it can be further modified by utilizing the group. Such modifications are
It could be due to residues that exhibit biological tropism, such as the antibody or functional residues thereof.

Furthermore, even if the modified γ-PGA cannot form a polymeric micelle by itself, instead of the interaction between the hydrophobic drug and the —Y—B unit as described above, a carboxyl group is used. It would also be possible to form a polymeric micelle encapsulating the drug in an aqueous medium by, for example, attaching a hydrophobic (or optionally water-soluble) alkaline drug via the unit having

Therefore, the modified γ-PGA of the present invention is
Even if it cannot necessarily form a polymer micelle by itself, it is useful as, for example, a drug carrier. However, it is preferable that the modified γ-PGA alone can form a polymeric micelle because it can form a more stable polymeric micelle even when the drug as described above is included. Whether or not the modified γ-PGA alone is capable of forming polymeric micelles in an aqueous medium will be understood by those skilled in the art.
It can be confirmed very easily by conducting a small experiment with reference to the examples capable of forming polymer micelles shown in the above description or Examples described later. The aqueous medium forming the above-mentioned polymer micelles may include water itself, a buffered aqueous solution, or a water-miscible organic solvent within a range that does not adversely affect the stability of the polymer micelles. Good aqueous solutions are included.

The modified γ-PGA of the present invention can also be a salt formed via a carboxyl group, preferably a partial salt or a complete salt of an alkali metal (eg, sodium, potassium, lithium).

The modified γ-PG of the present invention as described above
A is conveniently γ as shown in the reaction scheme below.
It can be produced by sequentially introducing -Y-B moiety and -X-A moiety into -PGA. Reaction scheme:

[0022]

[Chemical 3]

(A, B, X, Y, p, q and r in each formula are as defined above.) The modified γ-P represented by the general formula (I) thus produced.
Each of the three repeating units in the GA may be present in a random manner, although it may optionally form a few blocks.

[0024]

The present invention will be described in more detail below with reference to specific examples, but the present invention is not limited to these examples. Examples: (1) Hydrophobization of γ-PGA with L-leucine methyl ester 1.0 g of γ-PGA (Mn = 3.0 × 10 ⁶ , Meiji Seika Co., Ltd.) was added to a 0.3M NaHCO ₃ aqueous solution. It was dissolved in 50 ml. This solution and 150 ml of chloroform were mixed and stirred. A predetermined amount of L-leucine methyl ester (Leu-Met) was dissolved in this solution. Cool the solution with ice, then WS
C (water-soluble carbodiimide, Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred for 1 hour under ice temperature. Then, the mixture was stirred at room temperature for 12 hours. After the reaction, chloroform was distilled off, the remaining water was removed, and the precipitated polymer was dissolved in acetone. This was reprecipitated in water, and the obtained product was dried under reduced pressure.

[0025]

[Table 1]

(2) Polyethylene glycol (PE
Grafting of G) Leucine-modified PGA (Leu-PGA) obtained in (1) above.
Is dissolved in dimethylformamide (DMF),

[0027]

[Chemical 4]

Hydrochloride (hereinafter PEG-NH ₂ ) and butanol (HOBt) were added. The solution is ice-cooled, WSC
Was added and stirred for 1 hour. Then, the mixture was stirred at room temperature for 12 hours. After the reaction, the solution was dialyzed against water for 3 days, whereby the solution became white and cloudy particles were formed. This was purified by centrifugation (10,000 rpm, 10 minutes x 3),
A fine particle dispersion was obtained. The usage ratios of the raw materials and the reactants are shown in Table 2.

[0029]

[Table 2]

In Table 2, GAunit indicates the proportion of γ-glutamic acid units present.

Immediately after dialysis, both samples were slightly turbid, but in Example 2-2, the turbidity became strong due to aggregation around the 3rd day, and precipitation started to occur. On the other hand, in Example 2-1, the state immediately after the synthesis was maintained for one week or more. For comparison, leucine-modified PGA was dissolved in DMF and dialyzed against water, which initially became cloudy, but as with the sample of Example 2-2, in 2-3 days, cloudiness became strong and precipitation started to occur. From this, the dispersion stability of particles was improved by introducing PEG as a graft chain.

The particles of Example 2-1 were analyzed by dynamic light scattering. As a result, the average particle diameter was 241 nm and the standard deviation was 50 nm.
The CV was 21%. Further, it was confirmed from the observation result by the electron microscope that fine particles were formed. Further, the obtained fine particles were dispersed in distilled water and the conductivity titration was performed. As a result, it was found that 2.65 per 10 mg of nanospheres was obtained.
It was revealed that x10 ^-6 (mol) of carboxyl groups were present.

Continued front page    (72) Inventor Kenichiro Hiwatari             8-10-10 Higashiokyu, Arakawa-ku, Tokyo Asahi Denka               Oku Second House No. 201 F-term (reference) 4C076 AA16 EE13 EE59 FF16 FF43                 4J001 DA01 EA36 GA20 GE02 JA20

Claims (5)

[Claims] 1. A compound represented by the general formula (I): (In the formula, A is a group — (CH ₂ CH ₂ O) _n —R ¹ and R ¹
Represents C ₁ -C ₆ alkyl or C ₁ -C ₅ alkylcarbonyl, and n represents an integer from 1 to 200, B represents a group —CHR ² R ³ , and R ² represents C ₃ Represents —C ₂₁ alkyl, and R ³ represents hydrogen or C ₁ -C ₆ alkyloxycarbonyl, X and Y independently represent —O— or —NH—, and p, q and r are Independently represents an integer of 1 or more, p +
The total number of q + r represents any integer of 20 to 2500, and each repeating unit may partially form a block, but it is essentially random and is represented by a modified poly (γ- Glutamic acid) or a salt thereof. Wherein -X-A is _{-NH- (CH 2 CH 2 O)} n -
The modified poly (γ-glutamic acid) or a salt thereof according to claim ₁ , wherein R ^1a represents R ^1a and R ^1a represents C ₁ -C ₃ alkyl. 3. —Y—B is —NHCHR ^2a R ^3a (wherein R ^2a is C ₃ -C ₅ alkyl, and R ^3a is C ₁
-C is a ₃ alkyloxycarbonyl. ) Or -O
The modified poly (γ-glutamic acid) or a salt thereof according to claim 1 or 2, which represents CH ₂ R ^2b (wherein R ^2b is C ₈ -C ₂₀ alkyl). Wherein -X-A is _{-NH- (CH 2 CH 2 O)} n -
CH ₃ and n is an integer of 10 to 120, and —Y—B is —NHCHCHR ^2a R ^3a , and R ^2a is isopropyl, 2-butylpropyl or 1-methylpropyl. The modified poly (γ-glutamic acid) or a salt thereof according to claim 1, wherein R ^3a represents methoxycarbonyl. 5. The modified poly (γ-glutamic acid) or its alkyl metal salt according to claim 1, which is capable of forming a polymeric micelle in an aqueous medium.