JP4344279B2 - Polymeric drug carrier system for drug delivery - Google Patents

Description

  The present invention relates to polymers useful as carriers for drug delivery, in particular block copolymers comprising hydrophilic polymer chain segments and polyamino acid derivative segments, their use, and methods of making the block copolymers.

  Drug delivery or targeting is defined as “selectively delivering a drug to a site requiring drug treatment”, and the target increases the drug concentration at the site requiring drug treatment. On the other hand, side effects can be remarkably reduced by reducing the delivery amount to other sites. In particular, the need for drug targeting has been recognized in anticancer agents and the like, where large side effects have become a serious therapeutic problem. Drug targeting is accomplished by using a carrier that carries the drug to the target and delivering the drug to the site in need of treatment. Among such carriers, polymer micelles are attracting attention because they have many excellent features. Polymer micelles are mainly formed by associating many AB type block copolymers, etc., and have a two-phase structure of inner core (or core) and outer shell (shell). A spherical structure with a submicron diameter.

  Among the many features of the polymeric micelle drug carrier system, what is important for medical applications is its applicability to hydrophobic or water-insoluble drugs. It is high in content and can be sealed without losing the water solubility of the system. The encapsulating method includes a method of chemically binding a drug molecule to the inner core constituting polymer chain and a method of encapsulating the drug in the hydrophobic inner core. Physical encapsulation of a hydrophobic, water-insoluble or poorly water-soluble drug in the inner micelle core is achieved by the interaction between the inner core-constituting polymer chain and the drug. This interaction is considered to be mainly a hydrophobic interaction, but in addition, it is considered that hydrogen bonds and π-π interactions also contribute to encapsulation.

Typical AB type block copolymers that form such polymer micelles include those in which the hydrophilic polymer chain segment is made of poly (ethylene oxide) and the hydrophobic polymer chain segment is made of a hydrophobic polyamino acid ( For example, see Patent Documents 1 and 2). In these, the hydrophobic strength of the polymer chain constituting the inner core of the polymer micelle is recognized as an important factor for drug encapsulation. In particular, in Patent Document 2, the benzyl group of poly (benzylaspartic acid) constituting the hydrophobic segment in Patent Document 1 is partially transesterified with a higher alcohol in the presence of an acid catalyst to hydrophobicize the inner core of the polymer micelle. It enhances the properties, and physically encapsulates poorly water-soluble drugs such as taxol and camptothecin in the inner core of polymer micelles. On the other hand, unlike Patent Document 2, poly (benzylaspartic acid) is used to physically encapsulate topotecan, which has been introduced with an N, N-dimethylaminomethyl group, which is a camptothecin derivative, to enhance water solubility in the core of the polymer micelle. It has also been proposed to use a segment (α-amide type) benzyl group which has been partially hydrolyzed by acid partial hydrolysis to make some carboxyl groups free (see, for example, Patent Document 3).
Japanese Patent No. 2777530 (page 4, formula [III]) JP-A-11-335267 (page 8) JP 2003-342167 A (page 7)

  Patent Documents 2 and 3 described above suggest that the encapsulation characteristics of the drug in the core of the polymer micelle may vary depending on the ester form of the carboxyl group in the polymer side chain and the ratio of the carboxyl group. However, for example, in the partial hydrolysis of the benzyl group of the poly (benzylaspartic acid) segment described in Patent Documents 2 and 3, it is often difficult to achieve a desired ester partial hydrolysis rate. In addition, when the polymer composition obtained by such partial hydrolysis is considered at the molecular level of the polymer, for example, as a mixture of 100% ester hydrolyzed and undecomposed molecules, it is apparently constant. It may also indicate a hydrolysis rate.

  On the other hand, conventionally, in order to generally form an ester on a carboxyl group of a polymer side chain, the carboxyl group of the polymer side chain is left in a highly reactive active state by a carbodiimide coupling agent, A compound having a hydroxyl group was reacted. However, this conventional method includes a process in which two carboxyl groups react with a coupling agent to form an acid anhydride. However, since the polymer is a polymer, all or most of the carboxyl groups in the polymer side chain are acid anhydrides. It is difficult to become an ester through a product. This is because when the esterification reaction proceeds and the number of unreacted carboxyl groups on the polymer decreases, the distance between the two carboxyl groups to be reacted on one polymer molecule increases, and the two react closer to each other. Probability decreases. Moreover, it is difficult for the carboxyl groups in two different polymers to approach and react because the diffusion of polymer chains is significantly slower than that of low molecules.

  However, it has been found here that a polymer having a high esterification rate can be produced by performing a halogen compound substitution reaction using an organic strong base as a catalyst.

  In addition, the polymer having a certain esterification rate obtained in this way efficiently achieves physical encapsulation of a water-insoluble or poorly water-soluble drug in the core of micelles even in the presence of a free carboxyl group. It has also been found that drug-encapsulated micelles that can be maintained stably under physiological conditions can be provided.

上記各式中、Ｒは水素原子または式（３）： Thus, according to the present invention, the general formula (1)
X- <H.philic> -block- <Carbo-E> -Z (1)
In the formula, X- <H.philic> is the formula (E):

_{X- (OCH 2 CH 2) n} - (CH 2) s - (E)

Wherein X is a hydrogen atom, C ₁ -C ₆ alkyl, hydroxy-C ₁ -C ₆ alkyl, acetal or ketalized formyl-C ₁ -C ₆ alkyl, amino-C ₁ -C ₆ alkyl or benzyl group N represents an integer of 10 to 10,000, and s represents an integer of 0 to 6)
A hydrophilic polymer block of
<Carbo-E> -Z is
Formula (A-1), Formula (A-2), Formula (B-1), Formula (B-2), Formula (C-1), Formula (C-2), Formula (D-1) or Formula (D-2):

In the above formulas, R is a hydrogen atom or formula (3):

(Wherein R ¹ represents a hydrogen atom or a phenyl group, R ² represents C ₃ -C ₁₇ alkyl, phenyl, p-, m- or o-biphenyl, or 1 or 2-naphthyl, and phenyl and naphthyl The benzene ring is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, fluorine, chlorine, diazo, carbamoyl, hydroxy, carboxy, C ₁ -C ₆ alkoxycarbonyl, amino and mono- or di-C ₁ -C ₆ alkyl. 1 to 4 substituents selected from the group consisting of amino may be substituted, and a represents an integer of 0 to 4)
Represents an amino acid polymer segment forming an ester bond of
The group of formula (3) accounts for at least 40% of the total R, p and q are independently an integer from 1 to 300, but p + q is up to 350;
Z is a hydrogen atom, C ₁ -C ₆ alkyl, phenyl-C ₁ -C ₄ alkyl, C ₁ -C ₄ alkylphenyl, C ₁ -C ₆ alkoxycarbonyl, phenyl-C ₁ -C ₄ alkoxycarbonyl, C _1- C ₆ alkylaminocarbonyl, or phenyl-C ₁ -C ₄ alkylaminocarbonyl group,
A process for producing a block copolymer of
General formula (1 _P )

_{X- <H.philic- Pre> -block- <Carbo} > -Z (1 P)

In the formula, X- <H.philic- _Pre > is a hydrophilic polymer block represented by the above formula (E) (wherein X, n and s are as defined above), and <Carbo> is the above Formula (A-1), Formula (A-2), Formula (B-1), Formula (B-2), Formula (C-1), Formula (C-2), Formula (D-1) or Formula (D-2) (wherein R represents a hydrogen atom, p and q are the same as defined above), and Z is the same as defined for formula (1))
In the presence of a strong organic base in solution.

In which halo represents chlorine, bromine or iodine, and R ¹ , R ² and a are as defined for general formula (1),
There is provided a process for producing a block copolymer represented by the general formula (1), characterized by reacting with a halide of

(Wherein R ¹ represents a hydrogen atom or a phenyl group, R ² represents C ₃ -C ₁₇ alkyl, phenyl, p-, m- or o-biphenyl, or 1 or 2-naphthyl, and phenyl and naphthyl The benzene ring is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, fluorine, chlorine, diazo, carbamoyl, hydroxy, carboxy, C ₁ -C ₆ alkoxycarbonyl, amino and mono- or di-C ₁ -C ₆ alkyl. 1 to 4 substituents selected from the group consisting of amino may be substituted, a represents an integer of 0 to 4, and the group of formula (3) is at least 40% of the total R; preferably at least 50%, accounting for more preferably at least 55% p and q is an integer of 1 to 300, independently of one another, p + q is up to 350, Y ¹ is - H- or _{^{-R a - (CH 2) r}} -R b - ^represents, R a represents OCO, OCONH, NHCO, NHCONH, the COO or CONH, ^{R b} represents NH or O, ^{Y 2} is CO or _{^{-R c - (CH 2) r}} -R d - ^represents, R c represents OCO, OCONH, NHCO, NHCONH, the COO or CONH, ^{R d} represents CO, and r represents an integer of 1 to 6 , And s represents an integer from 0 to 6 is also provided.
[The invention's effect]

  The process for producing a block copolymer of the present invention can effectively perform esterification of a carboxyl group of a polymer side chain, and includes the block copolymer, particularly a block copolymer intended to provide the polymer itself. The carrier for drug loading can efficiently encapsulate a water-insoluble or poorly water-soluble drug, particularly a drug having a structure rich in benzene rings, even if free carboxyl groups still remain. The stability of molecular micelles under physiological conditions can be significantly increased.

Although this does not limit the present invention by theory, it can be understood as follows. As described above, when encapsulating a drug in the inner core of a polymer micelle, the contribution of hydrophobic interaction, hydrogen bonding or π-π interaction between the polymer chain and the drug has been considered. However, steric factors such as physical properties other than hydrophobicity of the polymer chain in the micelle core (eg, fluidity of the polymer chain) and the arrangement of the hydrophobic group and the shape with the drug molecule may be important. There is. According to the present invention, among the factors contributing to the physical encapsulation of the hydrophobic drug by systematically changing the chemical structure of the polymer chain forming the inner core of the polymer micelle, factors other than the hydrophobic strength A block copolymer structure with more favorable physical encapsulation is provided. Specifically, for example, when camptothecin having a structure rich in benzene rings is encapsulated, a polymer having high encapsulation stability of camptothecin by adjusting the arrangement of benzene rings in a block copolymer having a benzene ring as a hydrophobic group A micelle is obtained. In addition, regarding the configuration of the hydrophobic group of the core polymer chain in the micelle, by using a block copolymer rich in β-amide type aspartic acid residues, the spacing of the benzyl group of the hydrophobic group on the polymer chain can be reduced. It can be understood that camptothecin could be stably encapsulated by making it larger than the mold.
<Specific Description of the Invention>
The alkyl moiety in a group such as C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy used in connection with the description of the invention is alkyl having 1 to 6 carbon atoms and is methyl, ethyl, n-propyl, iso -Propyl, n-butyl, tert-butyl, n-hexyl and the like. Also, a bond or linking group as used herein is understood to bind or link each group or segment or block with the indicated orientation. Examples of the alkyl group up to C ₁₈ that can be taken with respect to the definition of R ² include, in addition to the above, optionally branched octadecane, heptadecane, tetradecane, undecane, nonane and the like. it can.

As used herein, a bond or linking group is understood to be that each group or segment or block is bonded or connected with the indicated orientation. For example, referring to the formula (1 _{A −1} ), when (CH ₂ ) _s —Y ¹ — (Y ^{1 in} CO ₂ represents —R ^a — (CH ₂ ) _r —R ^b —, R ^a is ( It means bonding to CH ₂ ) _s .

The block copolymer represented by the general formula (1 _P ) that can be used to produce the block copolymer represented by the general formula (1) corresponds to X- <H.philic- _Pre >, and optionally has one end. modified poly to correspond to the X prepared (ethylene oxide), while corresponding to <Carbo> -Z, optionally one terminal is modified so as to correspond to Z, the formula (a-1) , Formula (A-2), Formula (B-1), Formula (B-2), Formula (C-1), Formula (C-2), Formula (D-1) or Formula (D-2) poly giving a group represented (alpha amide type - aspartic acid), poly (beta amide type - aspartic acid), poly (alpha amide type and beta-type - aspartic acid), poly (alpha amide type - glutamic acid) or poly ( (γ-amide type-glutamic acid) can be prepared and both can be obtained by subjecting them to a known binding reaction. On the other hand, a polymer corresponding to the hydrophilic polymer chain segment is prepared, and if necessary, an appropriate functional group is introduced into the ω-terminal, and then the terminal functional group is used to make known sequential chain lengths known per se. By performing an elongation reaction, a block copolymer represented by the general formula (1 _P ) into which the desired polyamino acid is introduced can be obtained. For example, by subjecting the corresponding benzyl esterified product of each amino acid obtained by the method described in Patent Literature 1 to alkali or oxidized water decomposition, substantially all benzyl groups are eliminated to form a free carboxylic acid. A block copolymer represented by the general formula (1 _P ) can be provided. As poly (γ amide type-glutamic acid), those produced by Bacillus bacteria may be used.

When the polymer chain of poly (β-benzyl aspartic acid) is alkalized hydrolyzing the benzyl ester bond, usually about 75% of the main chain is β-amidated. For example, R in the formula (1 _{A −1} ) is hydrogen. It becomes a polymer chain in which α-amide type-aspartic acid units and β-amide type-aspartic acid units are randomly mixed and represent atoms. On the other hand, in place of the above alkaline hydrolysis, acid hydrolysis can provide a block copolymer represented by the general formula (1 _P ) consisting only of α-amide-aspartic acid units.

  The block copolymer thus prepared is dissolved in the presence of an organic strong base in a solution of the general formula (2)

In which halo represents chlorine, bromine or iodine, and R ¹ , R ² and a are as defined for general formula (1),
Can be esterified with at least 40% of the total carboxyl groups. Such conditions are not limited, but preferably a dipolar aprotic solvent such as N, N-dimethylformamide, dimethyl sulfoxide or the like (optionally mixed with a halogenated hydrocarbon such as chloroform or methylene chloride). And may be allowed to react at a temperature not higher than the boiling point of the solvent until the desired esterification proceeds.

  In this reaction, it is essential to use a strong organic base as a catalyst. Examples of strong organic bases include 1,8-diazabicyclo [5.4.0] 7-undecene (DBU), 1,5-diazabicyclo [4.3.0] non-5-ene (DBN), 1,5, 7-triazabicyclo [4.4.0] dec-5-ene (TBD), 7-methyl-1,5,7-triazabicyclo [4.4.0] dec-5-ene (MTBD), Examples include 2,2,6,6-tetramethylpiperidine (TMP).

  Typical examples of the halide represented by the general formula (2) include, but are not limited to, benzyl bromide, n-butyl bromide, n-butyl iodide, lauryl bromide, (2-bromomethyl ) Naphthalene, (2-bromoethyl) benzene, 1-bromo-3-phenylpropane, 4-phenylbenzyl chloride, 4-bromobiphenyl, bromodiphenylmethane, and 2-phenylbenzyl bromide.

  Specifically, in the block copolymer of the general formula (1) thus obtained, <Carbo-E> -Z is represented by the formula (A-1), the formula (A-2), the formula (B-1), the formula ( B-1), formula (C-1), formula (C-1), formula (D-1) and formula (D-2):

  [In the above formulas, R is a hydrogen atom or formula (3):

(Wherein R ¹ , R ² and a are as defined above for general formula (1)), the group of formula (3) is at least 40% of total R, preferably Occupy at least 50, more preferably at least 55%, most preferably at least 60%, p and q are each independently an integer from 1 to 300, but p + q is up to 350, and Z is of the general formula ( 1) is selected from the group consisting of polyamino acid blocks represented by the general formula (1 _P having <Carbo> -Z corresponding to <Carbo-E> -Z. ) Starting material).

More specifically, X- <H.philic> in the general formula (1) is represented by the formula (E):

X— (OCH ₂ CH ₂ ) _n — (CH ₂ ) _s − (E)

Wherein X is as defined above for general formula (1), n represents an integer from 10 to 10,000, and s represents an integer from 0 to 6 The above block copolymer is obtained (using a starting material of the general formula (1 _P ) having X- <H.philic- _Pre > corresponding to X- <H.philic>).

More specifically, the above formula (A-1), formula (A-2), formula (B-1), formula (B-1), formula (C-1), formula (C-1), equations (D-1) and formula (D-2), a combination of the formula (E), formula _{(1 a-1)} below having a linking group represented by the ^{Y 1} or ^{Y 2,} (1 _A-2 ), (1 _B-1 ), (1 _B-2 ), (1 _C-1 ), (1 _C-2 ), (1 _D-1 ) and (1 _D-2 ) A block copolymer represented by the following formula selected from the group is obtained by the above method.

In the above formulas, X, Z, R, Y ¹ , Y ² , n, p, q, and s are as described above.

  Block copolymers that can be conveniently obtained in this manner are useful as drug-carrying carriers.

  Such drugs include camptothecin and its derivatives, vincristine, vinplastin, vindesine, taxol and its derivatives such as taxolate, etoposide, mitomycin C, bleomycin, peplomycin, mesoorexate, 6-mercaptopurine and indomethacin Mention may be made of drugs selected from the group consisting of water-soluble anti-inflammatory agents, tetracycline antibiotics, peptide antibiotics including vancomycin, and quinolone antibacterial agents.

  Therefore, according to the present invention, there is preferably provided a composition comprising a drug-encapsulating polymer micelle comprising the block copolymer and the drug, wherein the drug is encapsulated or encapsulated in the inner core of the polymer micelle. Is done. As a drug, camptothecin and a derivative thereof containing a benzene ring (for example, an ethyl group at the 19-position of camptothecin replaced with methyl, 2-hydroxyethyl, 2-bromoethyl group, or 9 of camptothecin can be used conveniently. One in which hydrogen atoms at any of the 10th, 11th, 12th positions are substituted with amino, hydroxy or nitro, and the hydrogen atoms at the 10th and 12th positions are substituted together by methylenedioxy, In addition, those described in Patent Document 3), taxol, and derivatives thereof such as taxolate can be mentioned.

  According to the present invention, specifically, as shown in the specific examples described later, a drug-encapsulating polymer micelle that can be stably maintained under physiological conditions and has an increased drug encapsulation rate can be provided.

  A method for encapsulating the drug in the polymer micelle formed by the block copolymer is a method known per se, such as (i) a dialysis method, (ii) an emulsion method, (iii) an evaporator method, specifically described later. Can be used.

Further, the block copolymers represented by the formulas (1 _A-1 ) and (1 _A-2 ) can be particularly preferably used for encapsulating the above-mentioned drugs, and are also novel substances. Therefore, according to the present invention, block copolymers represented by the formulas (1 _A-1 ) and (1 _A-2 ) themselves are also provided.

  The action and effect of the drug-encapsulating polymer micelle thus provided will be further described with reference to camptothecin. Camptothecin usually has a lactone ring at a position called the E ring, and exists in both a lactone form and an open form in which it is hydrolyzed, and the two forms are in a pH-dependent equilibrium relationship. At pH 7.4, the majority exists in an open form. Of these two forms, only the lactone form has anticancer activity, and the other open form has no anticancer activity. According to the present invention, water-insoluble camptothecin is encapsulated in polymer micelles so that water-insoluble camptothecin is water-soluble, that is, can be easily injected into blood. Can be increased. In addition, the anticancer effect can also be enhanced by suppressing the hydrolysis of camptothecin in the blood after administration into the body and keeping the lactone body, which is an active substance that brings about its anticancer action, stably.

Hereinafter, the present invention will be described more specifically with specific examples, but the present invention is not intended to be limited to these specific embodiments.
Example 1: Hydrolysis of benzyl ester (1) -1 alkaline hydrolysis Polyethylene glycol of polyethylene glycol-poly (β-benzyl L-aspartate) block copolymer (hereinafter abbreviated as PEG-PBLA) has a molecular weight of 5,000. In this case, 3.00 g of β-benzyl L-aspartate having a polymerization degree of 27 was taken, and a 1.5N aqueous solution of sodium hydroxide of 1.5N was added in an amount of 1.5 times molar equivalent to the β-benzyl L-aspartate unit. Stir for minutes. When the solution became transparent in about 15 minutes, 6N hydrochloric acid was added in an amount of 10-fold molar equivalent to the β-benzyl L-aspartate unit. The mixture was then dialyzed against 0.1N hydrochloric acid and then distilled water. Finally, it was freeze-dried to obtain a polyethylene glycol-poly (aspartic acid) block copolymer (PEG-P (Asp)). It has been confirmed in the art that about 75% of the backbone of the poly (aspartic acid) portion of polyethylene glycol-poly (aspartic acid) is β-amidated by this alkaline hydrolysis.
(1) -2 Acid hydrolysis Polyethylene glycol of a polyethylene glycol-poly (β-benzyl L-aspartate) block copolymer (hereinafter abbreviated as PEG-PBLA) having a molecular weight of 5,000 and β-benzyl L-aspartate 0.50 g having a polymerization degree of 27 was taken and dissolved in 5 mL of trifluoroacetic acid. To this, 10-fold molar equivalents of trifluorometasulfonic acid, thioanisole, and m-cresol were added to β-benzyl L-aspartate unit, and the mixture was stirred at 0 ° C. for 4 hours. The reaction solution was dropped into 10 times its volume of diethyl ether, and the reprecipitated polymer was collected and dialyzed against water. Finally, it was freeze-dried to obtain a polyethylene glycol-poly (aspartic acid) block copolymer (PEG-P (Asp)). It was confirmed by 1H-NMR measurement that the structure of the amide of the polyaspartic acid moiety of PEG-P (Asp) obtained by this acid hydrolysis was almost 100% (95% or more) α-amide.

In the following examples, unless otherwise noted, polyethylene glycol-poly (aspartic acid) obtained by alkaline hydrolysis is used for ester formation reaction and camptothecin encapsulation.
Example 2. Ester formation reaction 200.0 mg of polyethylene glycol-poly (aspartic acid) block copolymer (PEG-P (Asp)) having a polyethylene glycol molecular weight of 5,000 and an aspartic acid polymerization degree of 27 is taken, and N, N,- Dissolved in 2.7 mL of dimethylformamide. To this solution, 116.9 mg of benzyl bromide and 103.9 mg of 1,8-diazabicyclo [5,4,0] 7-undecene (DBU) were added and reacted at 50 ° C. overnight. The reaction solution was added dropwise to about 10 times the volume of diethyl ether, and the reprecipitated polymer was recovered by filtration. The recovered polymer was dissolved in a small amount of dimethyl sulfoxide, 10 times the amount of 6N HCl was added to the aspartic acid residue, dialyzed against water, and lyophilized. Yield 144.0 mg. Various esterifications shown in Table 1 were performed in the same procedure.

As shown in Table 1, various hydrophobic groups could be introduced into the block copolymers having various chain lengths by ester formation. In particular, a very high ester formation rate was obtained for the aspartic acid residue on the block copolymer side, as 75% ester formation rate was achieved in the case of the benzyl group.
Example 3 Comparison of encapsulation efficiency of camptothecin into polymer micelles by the following three polymer micelle formation methods (1) Dialysis method Dissolve 5.0 mg of block copolymer and 0.5 mg of camptothecin in 1.0 mL of dimethylform sulfoxide and add it to distilled water. Dialyzed overnight. The solution remaining in the dialysis membrane was filtered with a filter paper to obtain a micelle solution.
(2) Emulsion method 5.0 mg of block copolymer and 0.5 mg of camptothecin were dissolved in 0.5 mL of methylene chloride and added to 2.0 mL of distilled water with stirring. After the methylene chloride was evaporated, the aqueous solution was filtered with a filter paper to obtain a micelle solution.
(3) Evaporation method 5.0 mg of block copolymer and 0.5 mg of camptothecin were dissolved in 0.5 mL of chloroform and placed in a glass container. While stirring, dry nitrogen gas was sprayed. After evaporating chloroform, 3.0 mL of distilled water was added and ultrasonic irradiation was performed for 2 minutes (model VC100 manufactured by Sonics & Materials). This aqueous solution was filtered with a filter paper to obtain a micelle solution.

  The amount of camptethecin in the polymer micelle solution obtained by the above three methods was determined from the value obtained by subtracting the light absorbance at 600 nm from the light absorbance at 370 nm (19,900 was used as the molar extinction coefficient). Table 2 shows the yield of camptothecin. In both the dialysis method and the emulsion method, the yield of camptothecin was low even with various block copolymers, up to 36%. On the other hand, a high value of 60% was obtained by the evaporation method. In addition, “benzyl-25” described in the column of the block copolymer, for example, means that about 25% of aspartic acid units are in the form of benzyl ester. The same applies hereinafter.

Example 4 Optimization of encapsulation of camptothecin by evaporation method In Example 3, since a high camptothecin yield was obtained by the evaporation method, encapsulation efficiency and encapsulation stability were measured by focusing on the evaporation method.

  7.5 mg of the block copolymer and 0.75 mg of camptothecin were dissolved in 0.75 mL of chloroform or a mixed solvent of 0.75 mL of chloroform and 0.5 mL of acetonitrile and put into a glass container. While stirring, dry nitrogen gas was blown. After evaporating chloroform, 3.0 mL of distilled water was added, and ultrasonic irradiation was performed for 2 minutes (model VC100 manufactured by Sonics & Materials). This aqueous solution was centrifuged (3,000 rpm, 10 minutes), and after removing the settled aggregate, only the supernatant was passed through a disk-type filtration device (Puradisc 25NYL, manufactured by Whatman) to obtain a polymer micelle solution. The amount of camptethecin in the polymer micelle solution was determined from the value obtained by subtracting the absorbance at 600 nm from the absorbance at 365 nm in DMSO: distilled water = 9: 1 (volume ratio). (20,860 was used as the molar extinction coefficient) The stability of camptothecin encapsulation was evaluated by gel filtration chromatography.

  That is, the polymer micelle solution was injected into a high-performance liquid chromatograph equipped with a column of Tosoh TSKgel G3000PWXL, and the size of the polymer micelle peak flowing out from 3.3 mL to 4.0 mL detected by absorption at 351 nm was determined. Evaluation was based on the value divided by the concentration (peak area / camptothecin concentration). The larger this value, the higher the encapsulation stability.

  As shown in Table 3, the values of camptothecin yield and peak area / camptothecin concentration differed greatly depending on the block copolymer used. Among them, benzyl-61, benzyl-69, and (2-methyl) naphthyl-64 had high encapsulation stability (peak area / camptothecin concentration was large). In these polymers, the camptothecin yield was also as good as 37% or more. Furthermore, comparing the results of a mixture of only organic solvent chloroform with Run11 and Run12 (chloroform + acetonitrile), Run12 with acetonitrile gives higher values for both camptothecin encapsulation efficiency and encapsulation stability. It was. Therefore, the subsequent encapsulation was performed with a mixed solvent of chloroform and acetonitrile.

  As shown in Table 3, the values of camptothecin yield and peak area / camptothecin concentration differed greatly depending on the block copolymer used. Among them, when benzyl-61, benzyl-69, and (2-methyl) naphthyl-64 were used, the encapsulation stability was high (peak area / camptothecin concentration was large). In these polymers, the camptothecin yield was also as good as 37% or more. Furthermore, comparing the results of a mixture of only organic solvent chloroform with Run11 and Run12 (chloroform + acetonitrile), Run12 with acetonitrile gives higher values for both camptothecin encapsulation efficiency and encapsulation stability. It was. Therefore, the subsequent encapsulation was performed with a mixed solvent of chloroform and acetonitrile.

  Next, an experiment was conducted to confirm that the high encapsulation stability obtained with benzyl-61, benzyl-69, (2-methyl) naphthyl-64 is the effect of β-amide contained in the polyaspartic acid residue. . The conditions of the organic solvent were the same as in Table 3 Run12. The results are shown in Table 4.

  Table 4 compares the encapsulation behavior of camptothecin with a block copolymer in which 66% of benzyl ester is introduced into a block copolymer in which 95% or more of all polyaspartic acid residues are occupied by α-amide using trifluoroacetic acid. In all cases where the weight ratio of camptothecin / block copolymer at the time of encapsulation was changed to 5%, 10%, 20% and 40%, the value indicating the encapsulation stability (peak area / camptothecin concentration) was approximately 75 for β-amide. % Of the block copolymer (A) containing 1%. Since the introduction rate of benzyl ester in this block copolymer (A) is 69%, the aspartic acid residue of β-amide is always benzyl esterified. (The proportion of β-amide aspartic acid residues that are benzyl esterified is in the range of 44% to 69% of all aspartic acid residues), whereas in the block copolymer (A), 95% or more is α-amide. . From the above, it can be seen that the benzyl esterified aspartic acid residue, which is a β-amide, greatly contributes to the stabilization of camptothecin encapsulation.

Next, the camptothecin yield was evaluated using a benzyl ester-introduced block copolymer having relatively good camptothecin yield and encapsulation stability, and changing the ratio of drug to block copolymer from 5% to 40%. The results are shown in Table 5. All block copolymers use 5.0 mg. As the solvent, a mixed solvent of CHCl ₃ 0.5 mL and CH ₃ CN 0.33 mL was used. As the content of benzyl ester increased, the yield of camptothecin increased markedly, benzyl-69 yielded very high values, and the amount of camptothecin charged was 95% or more in the range of 5-20% of the block copolymer. . Also, a high yield of 84% was obtained even when the amount charged was 40%. In the case of this run 12, the ratio of camptothecin / block copolymer in the obtained polymer micelle reaches 34%, assuming that 100% of the block copolymer is recovered. Thus, the camptothecin-encapsulated polymer micelle obtained had a good water solubility while encapsulating a very large amount of camptothecin of 34% or more. This is a very beneficial result.

Example 5. In vitro drug release test All of benzyl-44, benzyl-57, benzyl-69, benzyl-75 having a molecular weight of polyethylene glycol of 5,000 and a polymerization degree of aspartic acid of 27 (the above three block copolymers are shown in Table 4). 5) PEG-PBLA block copolymers were used, and drug release experiments were conducted using camptothecin encapsulated at a camptothecin / block copolymer ratio of 40% among the conditions shown in Table 4. A camptothecin-encapsulated polymer micelle solution (1.0 mL) is placed in a dialysis tube (Spectrapor 4), and dialyzed against 100 mL of phosphate physiological buffer at pH 7.4. The liquid outside the tube was sampled in a timely manner, and the amount of camptothecin in the liquid was determined from the value obtained by subtracting the light absorbance at 600 nm from the light absorbance at 365 nm in DMSO: distilled water = 9: 1 (volume ratio). (20,860 was used as the molar extinction coefficient). The amount of camptothecin in the sample solution was defined as the amount of drug released from the polymer micelle. The result is shown in FIG.

With PEG-PBLA, the drug release is very fast, and more than 80% of the drug is released within 12 hours at the concentration of 7 and 70 μg / mL in terms of capsulated camptothecin. Even at the highest 400 μg / mL, over 80% of the drug is released within 24 hours. Release at benzyl-44 is slower than at PEG-PBLA, and is slower at benzyl-57. Characteristic of benzyl-57 is that the drug release rate depends on the initial concentration of polymeric micelles. Release is significantly slower at a high concentration of 553 μg / mL compared to 7 μg / mL at the initial concentration of camptothecin. However, the release rate of benzyl-75 is almost constant regardless of the initial concentration, and the release amount is about 50% in 24 hours. Such a slow release rate is considered to be a suitable property for a polymer micelle carrier system that is transported to cancer tissue while circulating in the blood for a long time in the body.
Example 6 Retention effect of camptothecin active form As described above, camptothecin has anticancer activity in the lactone form, and the other open form has no anticancer activity. The lactone form and the open form have a parallel relationship depending on the pH, and most of the lactone form and the open form exist in the open form at pH 7.4. As shown in FIG. 2, camptothecin alone rapidly changes to an open body in both pH 7.4 phosphate buffer and pH 7.4 phosphate buffer containing 50% fetal calf serum. In contrast, camptothecin encapsulated in polymer micelles composed of benzyl-69 has an active lactone form in pH 7.4 phosphate buffer, pH 7.4 phosphate buffer containing 50% fetal calf serum. Both are held for a long time. From the above, it was found that the lactone compound exhibiting anticancer activity can be greatly suppressed from being converted into an inactive open compound by encapsulating the polymer micelle.
Example 7. Measurement of blood circulation in mice A dose equivalent to 50 μg of camptothecin was administered to the tail vein of a ddy male mouse having a body weight of about 25 g, and blood was collected after 4 hours, from which serum was prepared. The serum was acidified by adding phosphoric acid at pH 1.6, and camptothecin was extracted with chloroform: methanol = 4: 1 (volume), and the amount was analyzed using a TSK-gel ODS-80Ts column. Measured with

  As shown in Table 6, when camptothecin is administered as it is (in order to administer water-insoluble camptothecin as a homogeneous solution, it is made into an emulsion with lipid. This is (polyethylene glycol 400: propylene glycol: Tween80 = 40: 58: 2 , volume ratio) was dissolved at a CPT concentration of 0.26 mg / mL and prepared by applying ultrasound. After 4 hours, only 0.099% of the dose circulated in the blood. Only. On the other hand, in the case where the polymer micelle is encapsulated in benzyl-57 (polyethylene glycol having a molecular weight of 5,000 and an aspartic acid having a degree of polymerization of 27), the amount of encapsulated charge is 5, 10, or 20%. However, an overwhelmingly large amount of 3.1% to 5.0% circulated in the blood. This was enormously increased in blood circulation by encapsulating in polymer micelles. This is considered to have sufficient properties for targeting to solid cancer.

  According to the present invention, the provided block copolymer production method can support a carboxylic acid ester having a desired content, and the block copolymer thus obtained has a unique useful property as a polymeric drug carrier for drug delivery. Show the features. Therefore, the present invention can be preferably used in the pharmaceutical manufacturing industry.

FIG. 4 shows in vitro drug release behavior from drug-encapsulating polymeric micelles according to or in accordance with the present invention. In the figure, the X mark is 7 μg / mL, the white circle is 70 μg / mL, and the black circle is 500 μg / mL (benzyl-44), 553 μg / mL (benzyl-57), 512 μg / mL ( According to data on initial drug concentrations of benzyl-75) and 400 μg / mL (PEG-PBLA). It is a figure which shows the retention effect of the active body using the drug (camptothecin) inclusion polymeric micelle according to this invention or a comparison. In the figure, black circles indicate camptothecin encapsulated in polymer micelles in PBS, black squares indicate camptothecin encapsulated in polymer micelles in 50% fetal bovine serum, and white circles indicate camptothecin alone in PBS. The open squares are from the data for camptothecin alone in 50% fetal bovine serum.

Claims (2)

General formula (1)

X- <H.philic> -block- <Carbo-E> -Z (1)

In the formula, X- <H.philic> is the formula (E):

_{X- (OCH 2 CH 2) n} - (CH 2) s - (E)

Wherein X is a hydrogen atom, C ₁ -C ₆ alkyl, hydroxy-C ₁ -C ₆ alkyl, acetal or ketalized formyl-C ₁ -C ₆ alkyl, amino-C ₁ -C ₆ alkyl or benzyl group N represents an integer of 10 to 10,000, and s represents an integer of 0 to 6)
A hydrophilic polymer block of
<Carbo-E> -Z is
Formula (A-1), Formula (A-2), Formula (B-1), Formula (B-2), Formula (C-1), Formula (C-2), Formula (D-1) or Formula (D-2):

In the above formulas, R is a hydrogen atom or formula (3):

(Wherein R ¹ represents a hydrogen atom or a phenyl group, R ² represents C ₃ -C ₁₇ alkyl, phenyl, p-, m- or o-biphenyl, or 1 or 2-naphthyl, and phenyl and naphthyl The benzene ring is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, fluorine, chlorine, diazo, carbamoyl, hydroxy, carboxy, C ₁ -C ₆ alkoxycarbonyl, amino and mono- or di-C ₁ -C ₆ alkyl. 1 to 4 substituents selected from the group consisting of amino may be substituted, and a represents an integer of 0 to 4)
Represents an amino acid polymer segment forming an ester bond of
The group of formula (3) accounts for at least 40% of the total R, p and q are independently an integer from 1 to 300, but p + q is up to 350;
Z is a hydrogen atom, C ₁ -C ₆ alkyl, phenyl-C ₁ -C ₄ alkyl, C ₁ -C ₄ alkylphenyl, C ₁ -C ₆ alkoxycarbonyl, phenyl-C ₁ -C ₄ alkoxycarbonyl, C _1- C ₆ alkylaminocarbonyl, or phenyl-C ₁ -C ₄ alkylaminocarbonyl group,
A process for producing a block copolymer of
General formula (1 _P )

_{X- <H.philic- Pre> -block- <Carbo} > -Z (1 P)

In the formula, X- <H.philic- _Pre > is a hydrophilic polymer block represented by the above formula (E) (wherein X, n and s are as defined above), and <Carbo> is the above Formula (A-1), Formula (A-2), Formula (B-1), Formula (B-2), Formula (C-1), Formula (C-2), Formula (D-1) or Formula (D-2) (wherein R represents a hydrogen atom, p and q are the same as defined above), and Z is the same as defined for formula (1))
In the presence of a strong organic base in solution.

In which halo represents chlorine, bromine or iodine, and R ¹ , R ² and a are as defined for general formula (1),
A process for producing a block copolymer represented by the general formula (1), characterized by reacting with a halide of The block copolymer of the general formula (1) has the formula (1 _A-1 ), (1 _A-2 ), (1 _B-1 ),
(1 _B-2 ), (1 _C-1 ), (1 _C-2 ), (1 _D-1 ) or (1 _D-2 ):

(In the above formulas, R, p, q, Z and X are the formulas (A-1), (A-2), (B-1), (B-1) and (C-1). , Formula (C-1), formula (D-1) and formula (D-2) are as defined, n and s are as defined for formula (E), Y ¹ is —NH— or —R ^a — (CH ₂ ) _r —R ^b —, where R ^a represents OCO, OCONH, NHCO, NHCONH, COO or CONH, R ^b represents NH or O, and Y ² represents CO 2. or _{^{-R c - (CH 2) r}} -R d - ^represents, R c is OCO, OCONH, NHCO, NHCONH, represents COO or CONH, ^{R d} represents CO, and r is an integer from 1 to 6 Represent )
In represented by The method of claim 1, wherein.

Images