JPH0742324B2 - N-acetylcarboxymethyl chitosan derivative and its production method - Google Patents

Description

[0001] TECHNICAL FIELD [0002] The present invention relates to a novel N-acetylcarboxymethylchitosan derivative and a method for producing the same. More specifically, the present invention relates to a technology for in vivo targeting of pharmaceutical compounds, and to a novel N-acetylcarboxymethylchitosan derivative as a polysaccharide polymer carrier useful for increasing the stability of the pharmaceutical compound in the blood, its targeting to organs, and its metabolic potential in the body. The present invention also relates to the novel N-acetylcarboxymethylchitosan derivative in the form of a complex bound to the pharmaceutical compound.

The use of water-soluble polymeric substances as carriers for drugs, i.e., pharmaceutical compounds, has been attempted for a long time, particularly in the field of pharmaceutical formulations, and numerous related technologies have been proposed. These water-soluble polymeric substances for drug carriers are already generally known, and in many cases, cellulose derivatives such as carboxymethyl cellulose, hydroxypropyl cellulose, and hydroxypropylmethyl cellulose are used. However, these prior art technologies are concerned with so-called sustained drug release and do not address drug delivery, i.e., the delivery of the required amount of drug to the tissue where it is needed, at the required time. The current situation is that the use of water-soluble polymers as drug delivery carriers has not yet been fully developed. For example, see the following references 1 and 2. Reference 1 discloses a technology using carboxymethyl chitin as an implantable microparticulate carrier, and reference 2 discloses a technology using carboxylated dextran as a carrier for a drug complex.

1) Watanabe, K. et al., Chem. Pharm. Bull., 38 .506−50
9, (1990) 2) Sezaki Jin, Pharmaceutical Journal, 109 , 611-621, (1989) However, although the technology described in reference 1) focuses on the gelling ability and biodegradability of carboxymethyl chitin and utilizes these properties, it is an implantable type locally in the body, and its function does not go beyond the scope of drug release control, so it cannot be expected to improve the targeting of drugs to cancer tissues or organs. Also, the drug complex described in reference 2) shows the potential for excellent drug delivery, but the functional groups that can be used to modify the molecules of the drug complex to achieve drug delivery are limited to alcoholic hydroxyl groups.

With the gradual development of anticancer drugs and drugs for treating brain diseases, there is an urgent need to perfect targeting techniques for these drugs using water-soluble polymers, but conventional techniques have not provided a satisfactory solution.

In order to perfect a targeting technology for drugs, i.e., pharmaceutical compounds, using a water-soluble polymer carrier, the prerequisites are, first, that the complex between the carrier and drug is stable in the blood within the time it takes for the complex to reach the target organ after intravenous administration; in other words, that the required drug concentration in the blood can be maintained; second, that the carrier is gradually degraded in the body so that it does not remain in the body for a long period of time; and third, that the carrier itself, bound in the form of a complex with the drug, exhibits an organ-targeting tendency; therefore, a carrier must first be provided that simultaneously satisfies all of these conditions.

DISCLOSURE OF THE INVENTION In order to solve the above problems, the present inventors have repeatedly conducted various studies. As a result, it has been found that the low molecular weight carboxymethyl chitosan obtained by enzymatically treating the above carboxymethyl chitin and alkali-treating it is possible to obtain a low molecular weight carboxymethyl chitosan by N-substituted amino groups.
By introducing a peptide chain and an N-acetyl group, the present inventors have now succeeded in synthesizing a novel N-acetylcarboxymethylchitosan derivative and have found that this derivative is surprisingly useful as a carrier that satisfies the above-mentioned requirements and can be used to achieve the above-mentioned objectives. Furthermore, the inventors have found that by selecting an appropriate peptide chain, the introduced N-peptide chain can broaden the range of applicable pharmaceutical compounds to a wide range of carboxylic acid compounds, amino compounds, and alcohol compounds, and also imparts organ-targeting properties to the complex. Based on these findings, the present invention has been completed.

The present invention will be described in detail below.

The novel substance of the present invention is a compound represented by the following general formula (I):
It is an acetylcarboxymethylchitosan derivative, and one of its characteristics is that some of the H's at the 6th and/or 3rd position of the hydroxyl groups of glucosamine, which is a constituent sugar unit of chitosan, are substituted with carboxymethyl groups, and in some sugar units, the H's at the 2nd position of the amino group are substituted with an acetyl group, and in other sugar units, the H's at the 2nd position of the amino group are substituted with a peptide chain or one amino acid.

The novel N-acetylcarboxymethylchitosan derivative according to the first aspect of the present invention is represented by the following formula (I):

In formula (I), R ₁ and R ₂ each represent H or a carboxymethyl group, but cannot both represent H.

In addition, P represents an R ₃ CO group, an R ₄ NH group, or an R ₅ O group.
Here, P may be linked to the N-terminal amino acid of the N-peptide chain that constitutes X, in which case P is an R ₃ CO group, and may also be linked to the C-terminal amino acid of the N-peptide chain that constitutes X, in which case P is an R ₄ NH group or
R ₅ O group. Here, R ₃ COOH is a carboxylic acid compound, R
₄ NH ₂ means an amino compound, and R ₅ OH means an alcohol compound.

In many cases, the R ₃ CO group is specifically a protecting group for an amino group or an acyl group derived from a carboxylic acid-based pharmaceutical compound, for example, an amino-protecting group such as an alkoxycarbonyl group such as a tert-butoxycarbonyl group or an aralkyloxycarbonyl group such as a p-methoxybenzyloxycarbonyl group, or further, the R ₃ COOH corresponding to the R ₃ CO group can be a carboxylic acid-type pharmaceutical compound, for example, methotrexate.

The R ₅ O group can be a lower alkoxy group such as a tert-butyloxy group or an aralkyloxy group such as a benzyloxy group as a protecting group for a carbonyl group.
Alternatively, the R ₅ O group may be an alcohol-type pharmaceutical compound R
It can be a residue obtained by removing H from the alcoholic hydroxyl group of ₅ OH.

The R ₄ NH group may be an amide-type protecting group for protecting a carboxyl group, for example _, a lower alkylimino group such as a methylimino group.
R ₄ NH ₂ can represent a pharmaceutical compound of the amino compound type, such as daunorubicin or triprolidine.

Q represents H or an OH group. In some cases, Q is linked to the N-terminal amino acid of the N-peptide chain that constitutes X, in which case Q is H. Conversely, Q may be linked to the C-terminal amino acid of the N-peptide chain that constitutes H, in which case Q is an OH group.

Furthermore, in formula (I), X denotes a peptide chain containing 1 to 10 identical or different amino acids (however, in this specification, the term "peptide" also includes cases where there is only one amino acid), and this peptide chain not only includes peptide chains composed only of amino acids, but also peptide chains containing compounds other than amino acids as part of the chain. In the latter case, for example, a dibasic acid such as succinic acid, particularly a dibasic carboxylic acid, may be linked to an amino acid at the middle or end of the peptide chain, resulting in the formation of a peptide chain containing the same or different amino acids as a whole.

Furthermore, the N-acetylcarboxymethylchitosan derivatives according to formula (I) include salts thereof, particularly alkali metal salts of the carboxymethyl group of the derivatives, such as sodium salt or potassium salt or ammonium salt.

In the derivative compound of formula (I) according to the first aspect of the present invention, the number of amino acids constituting the peptide chain X is usually 1 to 4, preferably 3 to 4, taking into consideration drug release and antigenicity. Examples of XP in which the peptide chain X in XP is composed of only 1 to 4 amino acids and P is bound to the N-terminal amino acid are as follows: In such cases, the peptide bond in X is -NHCO- from the chitosan side.

P-Phe-Phe-Gly- P-Gly-Phe-Gly-Gly- P-Phe-Gly-Phe-Gly-Phe-Gly- P-Gly-Phe-Gly-Phe-Gly-Phe-P-Gly-Gly-Gly- P-Gly-Gly-Gly- P-Ala-Gly-Gly-Gly-Furthermore, when the peptide chain X in XP contains a dibasic acid and is 1
An example of XP where P is bound to the C-terminal amino acid of a peptide chain containing 1 to 4 amino acids is shown below. In this case, the peptide bond in X is -CONH- from the chitosan side. In the sequence, Suc represents a succinic acid residue.

-Suc-Ala-Ala-Ala-P -Suc-Ala-Ala-Val-Ala-P Further examples of amino acid sequences for peptide chain X that can be used in the present invention include the following:

(i) H-Gly-Gly-Gly-Val-Ala-OH, -Gly-Gly-Gly-Leu-Ala-, -Gly-Gly-Phe-Leu-Gly-, -Gly-Gly-Phe-Tyr-Ala-, -Gly-Gly-Gly-Gly-Gly-, (ii) -Gly-Gly-Gly-Phe-Leu-Gly-, -Gly-Gly-Gly-Gly-Leu-Ala-, -Gly-Gly-Gly-Gly-Gly-Gly-, (iii) -Gly-Gly-Gly-Gly-Phe-Leu-Gly-, -(Gly) ₇ -, -(Gly) ₅ -Leu-Ala-, (iv) -(Gly) ₅ -Phe-Leu-Gly-, -(Gly) ₆ -, (v) -(Gly) ₉ -, (vi) -(Gly) ₁₀ - Examples of parent compounds from which the residue (P-) of the pharmaceutical compound that can be used in the present invention is derived include the following.

Methotrexate, levodopa, bumetanide, furosemide, dinoprost, daunorubicin, doxorubicin,
Mitomycin-C, triprolidine, acriflavine and the like.

Furthermore, the substance of formula (I) according to the first aspect of the present invention is characterized by the degree of carboxymethylation, the molecular weight (gel filtration method), the a/(a+b) value and the molar ratio of P/X.

In the present invention, the degree of carboxymethylation is determined by colloid titration or alkali titration. For colloid titration, the following document 3) is referred to.

3) Okimasu, Satoshi, Journal of Agricultural Chemistry, 32 , 303-308 (1958). The molecular weight of the compound of formula (I) of the present invention is determined by gel filtration using dextran as a standard substance. In the examples below, TSK-gel G4000PW _XL is used as the column, eluent: 0.1M NaCl, flow rate: 0.8 ml/min,
Column temperature: 40°C, detection: measured with a differential refractometer.

The value a/(a+b) is what can be called the degree of peptidation, where a represents the sum of the number of -XP-substituted sugar units _a1 and the number of -XQ-substituted sugar units _a2 , and b represents the number of acetyl-substituted sugar units. Since all sugar units in the substance represented by formula (I) are either -XP-substituted, -XQ-substituted, or acetyl-substituted, a+b represents the total number of sugar units in the molecule.

Note that a/(a+b) can be calculated using the following formula (1).

where A is the content (wt%) of P in formula (I), _Mm is the molecular weight of P, _Ms is the average molecular weight of the sugar unit of N-acetylcarboxymethylchitosan, and C is the value when _a2 = 0 in formula (I).
where C is calculated by the following formula (2):

C = B (100 - A) x ^10-2 (2) Here, B represents the content (% by weight) of X when _a1 = 0 in formula (I), and can be determined, for example, by measuring the peptide content after peptide formation and before introducing P, or after eliminating P from the substance of formula (I). If the amino acid in X has a characteristic absorption, B can be determined directly by absorbance measurement using this. However, if the amino acid in X does not have a characteristic absorption, B can also be determined by the following formula (3):

Here, D represents the content (wt%) of XP when _a2 = 0 in formula (I), and if P has a characteristic absorption, this can be used to determine the content by absorbance measurement. Also, r represents the value determined by the following formula (4).

r=M _PX /M _X (4) where M _PX and M _X represent the molecular weights of XP and X, respectively.

The molar ratio of P/X then determines the degree of P (or P
The molar ratio can be calculated by the following formula (5): a ₁ /(a ₁ +a ₂ ).

The substance represented by formula (I) according to the first aspect of the present invention has a degree of carboxymethylation, molecular weight (gel filtration method), a/(a+b) value and P/X value, each calculated as described above, of 0.5 to 1.
2, 3,000 to 300,000, 0.01 to 1, and 0.1 to 1, respectively.

In this specification, the description of the formula (I) merely indicates that the molecule of the substance represented by formula (I) contains _a1 -XP substituted sugar unit (i.e., glucosamine sugar unit), _a2 -XQ substituted sugar units, and b _-COCH3 substituted sugar units,
The same kind of sugar units are bonded consecutively in the respective numbers, or these three kinds of sugar units are represented by the formula (I).
This does not mean that the sequences are bonded in the order described above.

Furthermore, partial N-acetylcarboxymethyl chitosan derivatives in which some of the N-acetyl groups in the compound of formula (I) according to the first invention are missing are also novel substances and have the usefulness described below.
According to the present invention, the compound of formula (II) In formula (II), R ₁ ,
R ₂ and X have the same meanings as in formula (I), except that P has a more or less expanded meaning than in formula (I) and represents H, an OH group, an R ₃ CO group, an R ₄ NH group, or an R ₅ O group.
P may be linked to the N-terminal amino acid of the peptide chain that constitutes X, in which case P is H or an R ₃ CO group, and P
may be linked to the C-terminal amino acid of the peptide chain that constitutes X, in which case P is an OH group, an R ₄ NH group or an R ₅ O group. a, b ₁ and b ₂ each represent a positive integer.

The substance of formula (II) is specified by the degree of carboxymethylation, molecular weight (gel filtration method), a/(a+b) value, and P/X molar ratio, which are determined by the same methods as those for formula (I). However, in formula (II), b in the a/(a+b) value has a slightly different meaning from b in the substance represented by formula (I), and the number of acetyl-substituted sugar units, b, is ₁ and 2.
The number of sugar units whose amino groups remain free without substitution
b represents the sum of _2. The characteristic values of the substance represented by formula (II) are the same as those of the substance represented by formula (I).

In this specification, the description of the formula (II) above means the formula (II)
This merely indicates that in the molecule of the substance represented by formula (II), there are a -XP-substituted sugar units, b _{-COCH3 -} substituted sugar units, and _b2 sugar units in which the 2-amino group remains free without being substituted. It does not mean that the same types of sugar units are bonded consecutively in the respective numbers, or that these three types of sugar units are bonded in the sequence described in formula (II).

The substance represented by formula (II) is a synthetic intermediate for producing the substance represented by formula (I), and therefore has the same industrial application field as the substance represented by formula (I) and the same main part of its constitution.

For example, in the compound of formula (II) above, when P represents a residue _R3CO group, _R4NH group or _R5O group derived from a pharmaceutical compound, the amino group of the compound of formula (II) can be acetylated with acetic anhydride or other suitable acetylating agent such as acetyl chloride in a suitable solvent, for example, pyridine, to give the compound of formula (I) of the present invention in the form of a complex when bound to a pharmaceutical compound. Also, in the compound of formula (II), when P is a protecting group, after removing the protecting group from the compound of formula (II), or when P is H or
When the compound of formula (II) is an OH group, it _is possible to bind pharmaceutical compounds _R3COOH , _R4NH2 or _R5OH , and then acetylate the remaining amino group in the same manner as above to produce the compound of the present invention of formula (I).

BEST MODE FOR CARRYING OUT THE INVENTION Next, the production of the compound of formula (I) according to the first aspect of the present invention will be described. The outline thereof is as follows, but the present invention is not limited thereto.

Carboxymethylchitin, the raw material for the present invention, can be easily prepared by reacting chitin (β-1,4-poly-N-acetylglucosamine), which is widely found in the exoskeleton of crustaceans such as crabs and shrimp, insects, and fungal cell walls, with monochloroacetic acid in the presence of alkali. By changing the reaction conditions, carboxymethylchitins with different degrees of carboxymethylation can be obtained, and commercially available products can also be used.

Next, the carboxymethylchitin is depolymerized. For example, commercially available carboxymethylchitin is reacted with egg white lysozyme, and the molecular weight of the resulting carboxymethylchitin can be adjusted by controlling the reaction conditions. The reaction conditions vary depending on the degree of carboxymethylation (ds: degree of carboxymethylation per sugar residue) of the carboxymethylchitin used. For example, when ds = 1.0, a depolymerized carboxymethylchitin with a molecular weight reduced to about 1 x 10 ⁵ will have a molecular weight of 1 x 10 ^6.
This can be obtained by allowing egg white lysozyme in an amount approximately 1/400 of that of carboxymethylchitin to act on the chitin at pH 6.0 and 37°C for approximately 2 hours.

The deacetylated carboxymethylchitin is then subjected to alkaline treatment. For example, the deacetylated carboxymethylchitin is dissolved in 1N NaOH and refluxed at 100°C for several hours to several tens of hours, preferably 3 to 8 hours, to remove some of the N-acetyl groups, yielding carboxymethylchitosan with some N-acetyl groups remaining. This is called partial N-acetylcarboxymethylchitosan.

Next, partially N-acetylcarboxymethyl chitosan,
For example, after dissolving the chitosan in a mixed solvent of dimethylformamide and 0.5% aqueous _NaHCO3 solution, the N-hydroxysuccinimide ester (active ester of the protected peptide) of the peptide according to the present invention, in which a protecting group has been introduced into the amino group of the terminal amino acid of the peptide chain (hereinafter simply referred to as the protected peptide; it can be represented by the formula "HO-X-protecting group"), is added to the solution and reacted to bind the protected peptide to the free amino group of the partially N-acetylcarboxymethylchitosan. By changing the amount of the active ester of the protected peptide added, "partial N-acetylcarboxymethylchitosan" with different amounts of the protected peptide bound can be obtained.
An acetylcarboxymethylchitosan-protected peptide conjugate is obtained. If a large excess of the activated ester of the protected peptide is reacted, it is possible to obtain a conjugate containing almost no free amino groups. The activated ester of the protected peptide can be obtained by dissolving the protected peptide in dimethylformamide using a conventional peptide synthesis method, for example, and then adding N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide to the solution and reacting.

As described above, the concentration of the partially N-acetylcarboxymethyl chitosan used in the binding reaction with the active ester of the protected peptide is preferably 0.1 to 5% (by weight), and the molar ratio of the sugar residue of the polysaccharide to the active ester of the protected peptide is preferably 20:1.
The ratio is preferably 1:10 or less. The number of amino acids in the protection peptide width is in the range of 1 to 10.

Furthermore, by using a drug instead of a protecting group in the above procedure and adding an N _- hydroxysuccinimide ester of the peptide of the present invention to which a drug has been introduced [which can be represented by the formula HO-X-P (where P is an R3CO group, _R4NH group, or _R5O group derived from a pharmaceutical compound)], a "partially N-acetylcarboxymethylchitosan-peptide-drug" complex can be obtained.

Furthermore, by removing the protecting group or drug residue (P) from the conjugate obtained as described above, a "partial N-acetylcarboxymethylchitosan-peptide" conjugate can be obtained.

The substance of the present invention represented by formula (II) includes three types of substances obtained as described above: a "partial N-acetylcarboxymethylchitosan-protected peptide" conjugate, a "partial N-acetylcarboxymethylchitosan-peptide-drug" conjugate, and a "partial N-acetylcarboxymethylchitosan-peptide" conjugate.

The compound of the present invention represented by formula (II) can be dissolved in saturated aqueous NaHCO ₃ solution, and then treated with acetic anhydride or other suitable acetylating agent,
For example, the free amino groups remaining in the complex substance of formula (II) can be acetylated by reaction with acetyl chloride to obtain the corresponding compound of the present invention represented by formula (I), provided that the compound obtained here has a structure in which a = _a1 , _a2 in formula (I)
= 0 or a = _a2 , _a1 = 0. For example, "N-
An "acetylcarboxymethyl chitosan-protected peptide" conjugate, an "N-acetylcarboxymethyl chitosan-peptide-drug" conjugate, and an "N-acetylcarboxymethyl chitosan-peptide" conjugate can be obtained, with the former two being substances corresponding to a = _a1 , _a2 = 0, and the latter being a substance corresponding to a = _a2 , _a1 = 0. The former two can usually be converted to the latter by acid treatment. For example, the "N-acetylcarboxymethyl chitosan-protected peptide" conjugate can be treated with a weak acid, e.g., 0.5N
By treating in HCl at 30° C. for 16 hours, an “N-acetylcarboxymethylchitosan-peptide” conjugate can be obtained.

Next, this "N-acetylcarboxymethyl chitosan-peptide" complex is dissolved in, for example, a 1% _NaHCO3 aqueous solution, and then
An "N-acetylcarboxymethylchitosan-peptide-drug" conjugate can be obtained by reacting an N-hydroxysuccinimide ester (active ester) of a carboxylic acid-type pharmaceutical compound, i.e., a drug having a carboxyl group, with the N-terminus of the peptide chain in the conjugate. However, the substance obtained here is not necessarily limited to the substance in which a = _a1 , _a2 = 0 in formula (I), but is actually shown as a substance in which a = _a1 + _a2 , _a2 ≠ 0. That is, if methotrexate (MTX) is selected as the drug here, an "N-acetylcarboxymethylchitosan-peptide N-MTX" conjugate can be obtained, but the MTX content in the conjugate will usually be smaller than the peptide content.

Therefore, according to the third aspect of the present invention, a compound represented by the formula (III) (wherein _R1 and _R2 each represent H or a carboxymethyl group) and having the following properties (1) to (2): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000, is treated with a compound of the following formula: PX-OH (IV) (wherein P represents H, an OH group, an _R3CO group, an _R4NH group, or an _R5
and X represents a peptide chain containing 1 _to 10 identical or different amino acids. _R3COOH represents a carboxylic acid compound, _R4NH2 represents an amino compound, and _R5OH represents an alcohol compound. (wherein _R1 , _R2 , P, and X are as defined above in formula (III), and a, _b1 , and _b2 are positive integers.) The partially N-acetylcarboxymethyl chitosan derivative is represented by the formula (III) and has the following properties (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a + b): 0.01 to 1 (wherein b = _b1 + _b2 ) (4) P/X (molar ratio): 0.1 to 1.

According to the fourth aspect of the present invention, a compound represented by the formula (II') [In the formula, _R1 and _R2 each represent H or a carboxymethyl group, _P1 represents H or an OH group, or an _R3'CO group, _R4'NH group, or _R5'O group, and X represents a peptide chain containing 1 to 10 identical or different amino acids. _R3'COOH represents a carboxylic acid compound, _R4'NH2 represents an amino compound, and _R5'OH represents an alcohol compound. _a , _b1 , and _b2 represent positive integers. and (4) P/X (molar ratio): 0.1 to 1. acetylating a partially N-acetylcarboxymethylchitosan derivative represented by the formula (I), which has the following characteristic values (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2, (2) molecular weight (gel filtration method): 3,000 to 300,000, (3) a/(a+b): 0.01 to 1 (wherein b = _b1 + _b2 ), and ( ₄ ) P/X (molar ratio): 0.1 to 1, and further removing the _P1 group when the P1 group is other than H or OH, and finally reacting the resulting chitosan with a compound represented by the formula P-H or P-OH (V) (wherein P represents _R3CO , _R4NH , or _R5O ). (wherein _R1 , _R2 , P, and X are as defined above, and Q represents H or an OH group; _a1 and _a2 represent 0 or a positive integer, except when both are 0; and b represents a positive integer.) and has the following characteristic values (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2; (2) molecular weight (gel filtration method): 3,000 to 300,000; (3) a/(a + b): 0.01 to 1 (where a = _a1 + _a2 ); and (4) P/X (molar ratio): 0.1 to 1.

Furthermore, the present inventors have found that the N-
We have conducted research to further improve the properties of acetylcarboxymethyl chitosan derivatives. As a result, we have found that several of the N-acetylcarboxymethyl glucosamine units, which are the constituent sugars of the derivative of formula (I), are 2-substituted with groups containing polyethylene glycol chains (hereinafter sometimes abbreviated as PEG).
A carboxymethylglucosamine unit having a substituent on an amino group, specifically, a carboxymethylglucosamine unit represented by the following formula (A): wherein R ₁ and R ₂ have the same meanings as above, and PEG is a compound represented by the following formula (V
I), (VII), (VIII) or (IX):- -CO- _{CH2CH2 -} CO-O-( _CH2CH2O ) _{n- CH3} (VII) -CO-O-( _CH2CH2O ) _n - _CH3 (VIII) -CO- _{CH2 -} O-( _CH2CH2O ) _n - _CH3 (IX), where n represents the average degree _of polymerization of the polyethylene glycol chain, to give a compound of the following formula (X) _: wherein R ₁ , R ₂ , P, Q, and X have the same meanings as in formula (I), PEG has the same meaning as above, a ₁
and _a2 each represent 0 or a positive integer, but cannot both be 0, and b and c each represent a positive integer. ] and have the following properties (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a+b+c): 0.01 to 1 (where a = _a1 + _a2 ) (4) P/X (molar ratio): 0.1 to 1. We have succeeded in synthesizing a novel N-acetylcarboxymethylchitosan derivative.
It has been found that this novel N-acetylcarboxymethylchitosan derivative represented by formula (X) has enhanced aqueous solution compared with the N-acetylcarboxymethylchitosan derivative of formula (I) itself, and can be used as a drug delivery carrier, and can remain in the blood for a long period of time after administration to the body.

Furthermore, the present inventors have found that a plurality of N-acetylcarboxymethylglucosamine units, which are the constituent sugars of the derivative of formula (I), can be substituted with N-acetylcarboxymethylglucosamine units represented by the following formula (B): (wherein R ₁ represents hydrogen or a carboxymethyl group as defined above) to form a polyol unit represented by the following formula (XI): (wherein _R1 , _R2 , P, Q, and X each have the same meaning as in formula (I), _a1 and _a2 each represent 0 or a positive integer, but cannot both be 0, and b and c each represent a positive integer) and have the following properties (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2, (2) molecular weight (gel filtration method): 3,000 to 300,000, (3) a/(a + b + c): 0.01 to 1 (where a = _a1 + _a2 ), and (4) P/X (molar ratio): 0.1 to 1.
It has been found that the novel N-acetylcarboxymethylchitosan derivative represented by formula (XI) has enhanced water solubility compared to the N-acetylcarboxymethylchitosan derivative of formula (I) itself, can be used as a drug delivery carrier, and can remain in the blood for a long period of time after administration to the body.

Therefore, in the fifth aspect of the present invention, the compound represented by formula (X) or formula (X
I) wherein R ₁ and R ₂ are each H or a carboxymethyl group, P is R ₂ CO group, R ₄ NH group or R ₅ O group, and Q is H or OH.
The group X represents a peptide chain containing 1 to 10 identical or different amino acids. R ₃ COOH represents a carboxylic acid compound. R _4
NH2 represents an amino compound, and _R5OH represents an alcohol compound. _a1 and _a2 represent 0 or a positive integer, except when both are 0. b and c represent positive integers.

In addition, in formula (X), -PEG is represented by the following formula (VI), (VII),
(VIII) or (IX) -CO- _{CH2CH2 -} CO-O-( _CH2CH2O ) _{n- CH3} (VII) -CO-O-( _CH2CH2O ) _n - _CH3 (VIII) -CO- _CH2 -O-( _CH2CH2O ) _{n- CH3} (IX), where n represents the average degree _of polymerization of the polyethylene glycol chain _, and having the following characteristic values (1) to ( ₃ ):
(4) An N-acetylcarboxymethylchitosan derivative having the following characteristics is provided: (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a + b + c): 0.01 to 1 (where a = _a1 + _a2 ) (4) P/X (molar ratio): 0.1 to 1.

The derivative of formula (X) according to the fifth aspect of the present invention is an N-acetylcarboxymethylchitosan derivative in which some of the sugar units in formula (I) are replaced by sugar units of formula (A) below, and the derivative of formula (XI) according to the fifth aspect of the present invention is an N-acetylcarboxymethylchitosan derivative in which some of the sugar units are replaced by polyol units of formula (B) below. Examples 17 to 21 below show examples of compounds of formula (X), and Examples 22 and 23 show examples of compounds of formula (XI).

The sugar derivative of formula (A) is a sugar unit in which the 2-amino group of the carboxymethylglucosamine unit is replaced by PEG, where PEG specifically represents a group of the following formulae (VI) to (IX) containing a polyethylene glycol chain.

-CO- _{CH2CH2 -} CO-O-( _CH2CH2O ) _{n- CH3 (} VII) -CO-O-( _CH2CH2O ) _n - _CH3 (VIII) -CO- _{CH2 -} O-( _CH2CH2O ) _{n- CH3} (IX) The polyethylene glycol chains present in the groups represented by _formulas (VI) to (IX) above can be those of various molecular weights, but a preferred polyethylene glycol chain is one having an average molecular weight of about 5000 because it is generally easy to obtain. Therefore, the preferred average degree of polymerization n of the polyethylene glycol chains in the groups of formulas (VI) to (IX) is about 100 to 120, but the present invention is not limited to this. The polyethylene glycol content in the derivative of formula (X) can be determined by measuring the peak area based on methylene protons in the chain using NMR, and the higher this content, the better, for example, 1% (by weight or more) or more, but there is no particular limitation in the present invention.

On the other hand, the polyol unit of formula (B) in the derivative of formula (XI) according to the fifth aspect of the present invention has a chemical structure characterized in that the bond between the 2- and 3-carbon atoms of the carboxymethylglucosamine unit is cleaved by oxidation, and then converted into a polyalcohol by reduction after the cleavage.

The a/(a+b+c) value in the derivatives of formula (X) and formula (XI) of the fifth aspect of the present invention is
The degree of peptidation is defined corresponding to the value of b), and therefore, the method for determining it can be performed using the above-mentioned formula (1) as is, and in practice, it is calculated in exactly the same manner as in formula (I), applying formulas (2) to (5) mutatis mutandis. The range of this value is also the same as in formula (I). Similarly, the ranges of the degree of carboxymethylation and molecular weight (gel filtration method) are also the same as in formula (I).

The fifth method for producing the substance of formula (X) according to the present invention is to react a compound of formula (II)
The method is carried out by preparing a "partially N-acetylcarboxymethylchitosan-protected peptide" conjugate represented by the formula (II) as a starting material and treating it as follows: First, the free carboxymethylglucosamine unit that is not N-acetylated in the conjugate of formula (II) is reacted with an active PEG derivative to convert the free 2-amino group into the compound represented by formula (VI) to (VI).
The PEG group (IX) is introduced as a substituent, and the remaining free carboxymethylglucosamine units are N-acetylated. Next, the protecting group is removed from the protected peptide portion in the conjugate to deprotect it. A reactive derivative of a drug is reacted with the terminal of the peptide portion to bind the drug (P) to the peptide terminal.

In the above reaction steps, the intermediates and final products after N-acetylation are represented by formula (X).

The above-mentioned active derivative of PEG may be, for example, 2-O-monomethoxypolyethyleneglycol-3,5-dichloro-s-triazine or an active ester of methoxypolyoxyethylenecarboxylic acid (for example, N-succinimide ester).

The fifth method for producing the substance of formula (XI) according to the present invention is to react a compound of formula (X)
Similar to the preparation of the substance (1), a "partially N-acetylcarboxymethylchitosan-protected peptide" conjugate represented by formula (II) is prepared as a starting material, and then the following treatments are carried out. First, the unacetylated free carboxymethylglucosamine units in this starting conjugate are oxidized, for example, by periodic acid, to cleave the ring. The aldehyde produced by cleavage is then converted into a polyol by reduction, for example, with sodium borohydride. Next, the protected peptide in the conjugate is deprotected by removing the protecting group, and a reactive derivative of a drug is reacted with the terminal of the peptide moiety to bind the drug (P) to the peptide terminal. The intermediates and final product after conversion to a polyol in the above reaction steps are represented by formula (XI).

The substances of formula (X) and formula (XI) according to the fifth invention are highly water-soluble, and therefore the fifth invention provides a drug delivery carrier and drug delivery product with improved water solubility.
According to Experimental Example 4 below, for example, the substance of formula (X) has excellent retention in blood, and when an anticancer drug is loaded as a drug, it has improved cancer selectivity.

The derivatives of formula (I) and of formula (X) and formula (XI) according to the present invention have the following advantageous properties:

As will be shown in the experimental examples below, the derivatives of formula (I) and formula (X) of the present invention are stable in the blood after intravenous administration until they reach the organs, i.e., the necessary blood concentration of these substances of the present invention can be maintained, and on the other hand, since they are gradually decomposed enzymatically in the body, there is no concern that the N-acetylcarboxymethylchitosan moiety of these substances will remain in the body for a long period of time.Furthermore, it has been observed that the derivatives of formula (I) of the present invention and the derivatives of formula (X) and formula (XI) of the present invention have an organ-targeting tendency in the body.

Here, the compounds of formula (I), (X) and (XI) of the present invention have a tendency to be directed to the target organ, which means that the compounds have a tendency to be directed to the target organ, compared to when the means of the present invention is not added.
It merely means that the concentration of the compound in a specific target organ tends to increase, and does not mean that the compound is selectively concentrated in only that specific organ.

The present invention will be further illustrated by the following examples.

The first to fourth aspects of the present invention are illustrated by Examples 1 to 16,
The fifth invention is illustrated by Examples 17-23.

In each example, gel filtration was carried out under the following conditions: Column: TSK-gel G4000PW _XL , Eluent:
0.1M NaCl, flow rate: 0.8 ml/min, column temperature: 40°C, sample injection amount: about 75 μg Example 1 5.0 g of commercially available carboxymethyl chitin (carboxymethylation degree: 1.0 per sugar residue) (Funakoshi Pharmaceutical Co., Ltd.) was added to the column.
After dissolving H6.0 in 0.05M acetate buffer (500 ml), egg white lysozyme (12.5 mg) was added and the mixture was allowed to react at 37°C for 2 hours. The reaction mixture was added to ethanol (2), and the precipitate was collected and dried in vacuo to obtain 4.25 g of low molecular weight carboxymethylchitin. This material (3.9 g) was dissolved in 1N NaOH aqueous solution (390
ml), and then heated to reflux at 100°C for 6 hours.
After adjusting the pH to 8, the supernatant obtained by centrifugation was added to methanol (1.9). The precipitate was collected and dried in vacuum to obtain 1.91 g of partially N-acetylcarboxymethyl chitosan (hereinafter sometimes simply referred to as polysaccharide) as an example of the substance of formula (III). The molecular weight of this polysaccharide substance was determined by gel filtration (G400) using dextran as a standard substance.
The yield was approximately 1 x 10 ⁵ on a 0PW _XL column.

The above polysaccharide material (200 mg) was dissolved in 0.5% NaHCO3 _aqueous solution (20 ml
After dissolving the polysaccharide in 1), dimethylformamide (17.5 ml) was added to obtain a homogeneous polysaccharide solution.
He-Gly-OH (94 mg) was dissolved in 1.5 ml of dimethylformamide, and then N-hydroxysuccinimide (23 mg) and N,
N'-dicyclohexylcarbodiimide (37 mg) was added and reacted at 4°C for 24 hours to form an active ester. The entire reaction mixture was added to the polysaccharide solution and reacted at 4°C for 16 hours. The reaction mixture was added to ethanol (160 ml), and the precipitate was collected and dried in vacuo to give 200 mg of partial N-
-Acetylcarboxymethylchitosan-Gly-Phe-Phe
-Boc complex (an example of a derivative of formula (II)) was obtained.

This complex (150 mg) was dissolved in saturated aqueous _NaHCO3 solution (15 ml), and then acetic anhydride (0.6 ml) was added. The N-acetylation reaction was carried out at room temperature for 17 hours. After neutralization, the reaction solution was added to ethanol (80 ml). The precipitate was collected and dried in vacuo to obtain 154 mg of N-acetylcarboxymethylchitosan-G.
The N-Boc peptide content of this complex was measured by the ultraviolet absorption spectrum (258 s) and the gel filtration elution pattern (258 s) of this complex.
The absorbance analysis at 1000 nm revealed that the content was 14.1% (wt %).

This complex (130 mg) was dissolved in 0.5 N HCl (13 ml) and then heated at 30°C.
The reaction mixture was reacted with ethanol (70 ml) for 17 hours for deprotection treatment to remove Boc. After neutralization, the reaction mixture was added to ethanol (70 ml) and the precipitate was collected and dried in vacuo to obtain 121 mg of N-acetylcarboxymethylchitosan-Gly-Phe-Phe-H conjugate (another example of the derivative of formula (I)). The ultraviolet absorption spectrum and gel filtration elution pattern of this conjugate are shown in Figures 3 and 4 of the accompanying drawings, respectively. The peptide content of this conjugate was determined to be 11.3 g by ultraviolet (258 nm) absorbance analysis.
% (by weight).

182 mg of methotrexate (MTX) as a pharmaceutical compound in the carboxylic acid form was dissolved in dimethylformamide (4 ml), and then N,N'-dicyclohexylcarbodiimide (82 mg) was added.
The reaction mixture was added with N-hydroxysuccinimide (46 mg) and pyridine (63 μg), and the mixture was reacted at room temperature for 5 hours to prepare the activated ester of MTX. On the other hand, the above N-acetylcarboxymethylchitosan-Gly-Phe-Phe-H complex (50 mg) was dissolved in 0.5% NaHO.
After dissolving in 10 ml of aqueous _H3 solution, 0.5 ml of the reaction solution containing the activated ester of MTX was added, and the mixture was reacted at 4°C for 15 hours to bind MTX to the N-terminus of the peptide chain of the complex.
The resulting reaction mixture was added to ethanol (40 ml), and the precipitate was collected and dried in vacuo to obtain 53 mg of N-acetylcarboxymethylchitosan-Gly-Phe-Phe-MTX conjugate (another example of the conjugate of formula (I)) as a yellow powder. The ultraviolet-visible absorption spectrum and gel filtration elution pattern of this conjugate are shown in Figures 5 and 6 of the accompanying drawings, respectively.
The MTX content of this complex was determined to be 11.5% (wt%) by ultraviolet (307 nm) absorbance analysis. The P/X molar ratio of this complex, specifically the MTX/peptide molar ratio, was calculated to be 0.93 according to the aforementioned formula (5).

In addition, for this complex, a/(a+
b) The value was estimated to be 0.09 according to the formula (1) above.

Example 2: Partially N-acetylcarboxymethyl chitosan (100 mg) obtained in Example 1 was dissolved in 10 ml of 1% aqueous _NaHCO3 solution, and then 6 ml of dimethylformamide was added to obtain a homogeneous polysaccharide solution.
(141 mg) was dissolved in 4 ml of dimethylformamide, and then N-
Hydroxysuccinimide (46 mg) and N,N'-dicyclohexylcarbodiimide (74 mg) were added and reacted at room temperature for 3 hours to form an activated ester. The entire reaction mixture containing this activated ester was added to the above polysaccharide solution, and 10 ml of dimethylformamide was added, followed by reaction at 4°C for 23 hours. 5 ml of water was added to the reaction mixture, followed by centrifugation. The resulting supernatant was added to ethanol (150 ml). The precipitate was collected and dried in vacuo to yield 120 mg of a partially N-acetylcarboxymethylchitosan-Gly-Gly-Gly-pMZ complex (an example of a derivative of formula (II)).

This complex (100 mg) was N-acetylated with acetic anhydride in accordance with Example 1 to obtain an N-acetylcarboxymethylchitosan-Gly-Gly-Gly-pMZ complex (97 mg).
The ultraviolet absorption spectrum and gel filtration elution pattern of this complex are shown in Figures 7 and 8, respectively. The pMZ-peptide content of this complex was determined by ultraviolet (272 nm) absorbance analysis to be 20.
It was 1% (wt%).

This complex (89 mg) was treated with acid in accordance with Example 1 to remove the protecting group pMZ, yielding 78 mg of N-acetylcarboxymethylchitosan-Gly-Gly-Gly-H complex. The ultraviolet absorption spectrum of this complex is shown in Figure 9. The peptide content (B) of this complex was 11.3 g according to formula (3).
It was calculated as %.

This complex (50 mg) was dissolved in 5 ml of 1% _NaHCO3 aqueous solution, and then 1 ml of the MTX active ester solution prepared in the same manner as in Example 1 was added to the solution, followed by a reaction at 4°C for 21 hours. The reaction solution was added to 25 ml of ethanol, and the precipitate was collected and dried in vacuo to obtain 52 mg of N-acetylcarboxymethylchitosan-
The Gly-Gly-Gly-MTX complex was obtained as a yellow powder. The ultraviolet-visible absorption spectrum and gel filtration elution pattern of this complex are shown in Figures 10 and 11, respectively. The MTX content of this complex was determined to be 21.4% (wt%) by ultraviolet (307 nm) absorbance analysis. The P/X molar ratio of this complex was also determined to be 1.4% (wt%).
Specifically, the MTX/peptide (molar ratio) is (5)
For this complex, the a/(a+b) value in formula (I) is calculated to be 1.0 according to the formula (1),
The calculated value was 0.2.

Example 3 In the same manner as in Example 1, 5.0 g of carboxymethylchitin (carboxymethylation degree: 1.0 per sugar residue) was treated with egg white lysozyme to obtain low molecular weight carboxymethylchitin (4.28 g). 4.1 g of this low molecular weight carboxymethylchitin was treated with alkali to obtain a carboxymethylchitin having a molecular weight of about 1.
10× ⁵ partially N-acetylcarboxymethyl chitosan (2.22 g) was obtained.

This substance (200 mg) was treated with N-Boc in the same manner as in Example 1.
The activated ester of N-acetylcarboxymethyl chitosan-Gly-Gly-Phe-Gly-Gly-OH (90 mg) was reacted to give a partially N-acetylcarboxymethyl chitosan-Gly-Gly-Phe-Gly-Boc complex (210 mg),
200 mg of this product was N-acetylated to give 190 mg of N-acetylcarboxymethylchitosan-Gly-Gly-Phe-Gly-Boc conjugate, the N-Boc-peptide content of which was determined to be 9.8% (wt %) by ultraviolet (258 nm) absorbance analysis.

In the same manner as in Example 1, this conjugate (160 mg) was treated with acid for deprotection to obtain an N-acetylcarboxymethylchitosan-Gly-Gly-Phe-Gly-H conjugate (144 mg, peptide content: 7.6%).

This 120 mg was treated with the active ester of MTX (55 mg),
N-acetylcarboxymethyl chitosan-Gly-Gly-Ph
An e-Gly-MTX conjugate (127 mg, MTX content: 9.5%) was obtained.
The MTX/peptide (molar ratio) of the complex and the
a/(a+b) is 1. from equations (5) and (1), respectively.
The calculated values were 0 and 0.07.

Example 4 The partially N-acetylcarboxymethyl chitosan (200 mg) obtained in Example 3 was treated with N-Boc in the same manner as in Example 1.
The chitosan was reacted with the active ester of N-Gly-Phe-Gly-Phe-OH (105 mg) to give a partially N-acetylcarboxymethylchitosan-Phe-Gly-Phe-Gly-Boc conjugate (199 mg).

This 188 mg was N-acetylated to obtain an N-acetylcarboxymethylchitosan-Phe-Gly-Phe-Gly-Boc conjugate (191 mg). The N-Boc-peptide content of this conjugate was 4.9% (wt%) based on ultraviolet (258 nm) absorbance analysis. In the same manner as in Example 1, this conjugate (160 mg) was deprotected by acid treatment to obtain an N-acetylcarboxymethylchitosan-Phe-Gly-Phe-Gly-H conjugate (150 mg, peptide content: 4.0%), and 50 mg of this was then deprotected.
The activated ester of MTX (23 mg) was reacted with N-acetylcarboxymethylchitosan-Phe-Gly-Phe-Gly-
An MTX complex (46 mg, MTX content: 4.5%) was obtained.
MTX/peptide (molar ratio) and a/(a+) in formula (I)
b) The values are 1.1 and 0.03 from equations (5) and (1), respectively.
It was calculated that:

Example 5 The partially N-acetylcarboxymethyl chitosan (100 mg) obtained in Example 3 was treated with N-Boc in the same manner as in Example 1.
The chitosan was reacted with the active ester of N-Phe-Gly-Phe-Gly-OH (54 mg) to give a partially N-acetylcarboxymethylchitosan-Gly-Phe-Gly-Phe-Boc conjugate (109 mg).

90 mg of this was N-acetylated to give N-acetylcarboxymethylchitosan-Gly-Phe-Gly-Phe-Boc complex (8
The N-Boc-peptide content of this complex was 12.7% (wt%) based on ultraviolet (258 nm) absorbance analysis.
was deprotected by acid treatment to give N-acetylcarboxymethylchitosan-Gly-Phe-Gly-Phe-H conjugate (63 mg,
The peptide content was 10.4%).

This 50 mg was treated with the active ester of MTX (23 mg),
N-acetylcarboxymethylchitosan-Gly-Phe-Gl
A y-Phe-MTX complex (49 mg, MTX content: 9.4%) was obtained. The MTX/peptide (molar ratio) of this complex and the a/
(a + b) is 0.94 and 0.94 from equations (5) and (1), respectively.
The calculated value was 07.

Example 6 The partially N-acetylcarboxymethyl chitosan (200 mg) obtained in Example 3 was treated with N-Boc in the same manner as in Example 1.
The activated ester of chitosan-Gly-Gly-Gly-OH (180 mg) was reacted with chitosan to give a partially N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-Boc conjugate (218 mg), and 150 mg of this was N-acetylated to give an N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-Boc conjugate (143 mg). In the same manner as in Example 1, this conjugate (130 mg) was treated with acid to give an N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-H conjugate (12
0 mg) was obtained.

This 50 mg was treated with the active ester of MTX (46 mg),
N-acetylcarboxymethyl chitosan-Gly-Gly-Gl
A y-Ala-MTX complex (55 mg, MTX content: 21.5%) was obtained.
The ultraviolet-visible absorption spectrum and gel filtration elution pattern of this complex are shown in Figures 12 and 13, respectively.

Example 7 Partially N-acetylcarboxymethyl chitosan (45 mg) with a molecular weight of about 1 × 10 ⁵ , prepared in accordance with Example 1 using carboxymethyl chitin (carboxymethylation degree: 0.7 per sugar residue), was dissolved in 0.1 M borate buffer (pH 8.0) (2
On the other hand, N-succinyl-Ala-Ala-Ala-p-nitroanilide (45 mg) was dissolved in dimethylformamide (2 ml), and then N-hydroxysuccinimide (12 mg) and N,N'-dicyclohexylcarbodiimide (41 mg) were added. The mixture was then incubated at room temperature for 1 hour, and then at 4°C.
The reaction mixture was reacted with HCl for 18 hours to prepare an activated ester of the peptide bound to a nitroanilide moiety. The solvent was removed under reduced pressure, and the residue was washed with isopropanol and then dissolved in dimethylformamide (0.8 ml). This was added to the polysaccharide solution and reacted at room temperature for 40 hours. Ethanol was added to the reaction mixture, and the resulting precipitate was collected and dried in vacuo to yield 46 mg of a partially N-acetylcarboxymethylchitosan-Suc-Ala-Ala-Ala-p-nitroanilide conjugate.

Furthermore, this complex (26 mg) was taken and dissolved in 3 ml of saturated NaHCO ₃ solution.
After dissolving in acetic anhydride (0.14 ml), add acetic anhydride (0.14 ml) and keep at 4°C for 24 hours.
The reaction mixture was reacted for 1 hour to form N-acetylated chitosan. The reaction mixture was dialyzed against water and then added to ethanol. The precipitate was dried under vacuum to obtain 25 mg of N-acetylcarboxymethylchitosan-Suc-Ala-Ala-Ala-p-nitroanilide conjugate. The ultraviolet absorption spectrum and gel filtration elution pattern of this conjugate are shown in Figures 14 and 15, respectively. The -Suc-Ala-Ala-Ala-p-nitroanilide content of this conjugate was determined to be 3.9% (wt%) based on absorbance analysis at 315 nm.

Example 8: In a manner similar to that of Example 7, partially N-acetylcarboxymethyl chitosan (45 mg) was converted to N-succinyl-Ala-Ala.
The chitosan was reacted with the active ester of Suc-Ala-Ala-Val-Ala-p-nitroanilide (55 mg) to give a partially N-acetylcarboxymethylchitosan-Suc-Ala-Ala-Val-Ala-p-nitroanilide complex (44 mg).

Furthermore, using this 30 mg, N
Acetylation was performed to obtain N-acetylcarboxymethylchitosan-Suc-Ala-Ala-Val-Ala-p-nitroanilide complex (22 mg).
The content of α-Val-Ala-p-nitroanilide was determined to be 4.5% (wt %) from the absorbance at 315 nm.

Example 9: Partially N-acetylcarboxymethyl chitosan (50 mg) obtained in Example 1 was dissolved in 0.25% aqueous _NaHCO3 solution (7 ml), and then dimethylformamide (6 ml) was added to obtain a homogeneous polysaccharide solution. Separately, phenylalanine N-Boc-Phe-OH (265 mg) protected with the Boc protecting group was dissolved in 3 ml of dimethylformamide, and then N-hydroxysuccinimide (115 mg) and N,N'-dicyclohexylcarbodiimide (190 mg) were added and reacted at 4°C for 20 hours to obtain an activated ester. 1 ml of the reaction solution containing this activated ester was added to the above polysaccharide solution and reacted at room temperature for 20 hours. The reaction solution was added to ethanol (35 ml), and the resulting precipitate was collected and analyzed.
After vacuum drying, 51 mg of a partially N-acetylcarboxymethylchitosan-Phe-Boc complex was obtained.
The c-phenylalanine (Phe-Boc) content is
m) was found to be 14.4% (wt %) by absorbance measurement.

Example 10 The depolymerized carboxymethyl chitin (4.
The reaction solution was adjusted to pH 8.5 by alkali treatment of 1 g of thiamin mononitrate.
The mixture was added to 4.4 times the amount of methanol, and separated into a precipitate and a supernatant. Ethanol (300 ml) was added to the supernatant, and the precipitate was collected and dried in vacuum to obtain a product with a molecular weight of approximately 2x.
¹⁰⁴ partially N-acetylcarboxymethyl chitosan (0.54 g) was obtained.

This substance (100 mg) was reacted with the protected peptide N-Boc-Phe-Gly-Phe-Gly-OH (217 mg) in the same manner as in Example 1, and then N-acetylated with acetic anhydride to give N-acetylcarboxymethylchitosan-Gly-Phe-Gly-Phe
This conjugate (80 mg) was deprotected by acid treatment in the same manner as in Example 1 to obtain an N-acetylcarboxymethylchitosan-Gly-Phe-Gly-Phe-H conjugate (46 mg, peptide content: 24%).

35 mg of this complex plus methotrexate (MTX) (69 mg)
The activated ester of the above compound was reacted in the same manner as in Example 1 to obtain N-acetylcarboxymethylchitosan-Gly-Phe-Gly-Phe
A MTX complex (35 mg, MTX content: 19%) was obtained. The MTX/peptide (molar ratio) of this complex and the a/(a+) ratio in formula (I) were
b) is calculated from the above formula (5) and formula (1), respectively.
The calculated values were 0.92 and 0.19.

Example 11 Partially N-acetylcarboxymethyl chitosan (50 mg) having a molecular weight of about 2 x 10 ⁴ as described in Example 10 was dissolved in water (1.25 ml).
l)-dimethyl sulfoxide (1.25 ml)-dimethylformamide (8 ml), and then N-Succinyl-Gly-Phe-Gly-Lys(ε-N-Bo
c) Partially N-acetylcarboxymethyl chitosan-Suc-Gly was prepared by the reaction of the active ester of -O-tBu (169 mg).
Then, N-acetylation and deprotection by acid treatment were performed to obtain N-acetylcarboxymethylchitosan-Suc-Gly.
The resulting 47 mg of the -Phe-Gly-Lys-H conjugate was obtained.

This complex (35 mg) was combined with methotrexate (MTX) (23 mg
The activated ester of chitosan (Suc-Gly-Phe-Gly-Lys-MTX) was reacted with chitosan to give an N-acetylcarboxymethylchitosan-Suc-Gly-Phe-Gly-Lys-MTX conjugate (29 mg, MTX content: 1.5%). The UV-visible absorption spectrum and gel filtration elution pattern of this conjugate are shown in Figures 20 and 21, respectively.

Example 12: Degraded carboxymethylchitin (4.37 g) was obtained by reacting 25 mg of egg white lysozyme with 5.0 g of carboxymethylchitin (carboxymethylation degree: 0.7 per sugar residue) in the same manner as in Example 1. 3.7 g of this product was partially deacetylated by alkali treatment to obtain partially N-acetylcarboxymethylchitosan (2.58 g) with a molecular weight of approximately 1 × ¹⁰ (dextran standard).

This partially N-acetylcarboxymethyl chitosan (30
0 mg) was added to N-Boc-Phe-Gly in the same manner as in Example 1.
The active ester (N-hydroxysuccinimide ester) of chitosan-Gly-OH (141 mg) was reacted to give a partial N-acetylcarboxymethylchitosan-Gly-Phe-Phe-Boc conjugate (340 mg). 300 mg of this was N-acetylated with acetic anhydride to give N-acetylcarboxymethylchitosan-Gly
The N-Phe-Phe-Boc complex (298 mg, N-Boc-peptide content: 12.0%) was obtained.

This complex (100 mg) was deprotected (removal of the Boc group) by acid treatment in the same manner as in Example 1 to give N-acetylcarboxymethylchitosan-Gly-Phe-Phe-H complex (8
9 mg) was obtained.

This complex (75 mg) was dissolved in 1% NaHCO3 _aqueous solution (7.5 ml), and the activated ester of methotrexate (MTX) (75 mg) was added. The reaction was carried out at 4°C for 24 hours to bind MTX to the N-terminus of the peptide in the complex. The reaction solution was diluted with ethanol (40 ml).
l) and collect the precipitate, dry it in vacuum,
N-acetylcarboxymethylchitosan-Gly-Phe-Ph
The e-MTX complex (75 mg, MTX content: 10.1% by weight) was obtained as a yellow powder. The ultraviolet-visible absorption spectrum and gel filtration elution pattern of this complex are shown in Figures 22 and 23, respectively.

Example 13 Carboxymethyl chitin (carboxymethylation degree: 0.7)
2.0 g of the chitosan was dissolved in 200 ml of 0.05 M acetate buffer at pH 6.0, and egg white lysozyme (2.5 mg) was added. The mixture was allowed to react at 37°C for 1 hour and 30 minutes to depolymerize the chitosan. This was then treated with 1N NaOH in the same manner as in Example 1 to give 1.72 g of partially N-acetylcarboxymethylchitosan with a molecular weight of approximately 3 x ¹⁰ (dextran standard).
This substance (150 mg) was dissolved in 0.5% NaHCO ₃ aqueous solution (15 ml
l) and N-Boc-Ala
The active ester of chitosan-Gly-Gly-Gly-OH (135 mg) was reacted to give partially N-acetylcarboxymethyl chitosan-Gly.
The γ-Gly-Gly-Ala-Boc conjugate (150 mg) was obtained.
100 mg of the chitosan was N-acetylated with acetic anhydride to obtain an N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-Boc complex (154 mg).
The Boc group was deprotected to give N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-H conjugate (106 mg). This conjugate (95 mg) was dissolved in 1% _NaHCO3 aqueous solution (4.8 ml).
and reacted with the activated ester of MTX (86 mg).
N-acetylcarboxymethyl chitosan-Gly-Gly-Gl
A y-Ala-MTX complex (88 mg, MTX content: 13.7%) was obtained.

Example 14 The partially N-acetylcarboxymethyl chitosan (150 mg) obtained in Example 12 was treated with N-Boc in the same manner as in Example 1.
Partially N-acetylcarboxymethyl chitosan was synthesized by the action of the active ester of -Gly-Gly-Gly-OH (109 mg).
A Gly-Gly-Gly-Boc complex (167 mg) was obtained.
was N-acetylated with acetic anhydride to give N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Boc conjugate (153mM
Further, 130 mg of the chitosan was treated with acid to remove the Boc group, and N-acetylcarboxymethylchitosan-Gly
The resulting 110 mg of MT-Gly-Gly-H complex was added to 100 mg of this complex.
The active ester of X (90 mg) was reacted to give an N-acetylcarboxymethylchitosan-Gly-Gly-Gly-MTX conjugate (111 mg, MTX content: 17.4 wt %).

Example 15 The partially N-acetylcarboxymethyl chitosan (150 mg) obtained in Example 12 was treated with N-Boc in the same manner as in Example 1.
The active ester of chitosan-Gly-Gly-Gly-Ala-Boc (135 mg) was reacted with chitosan to give a partially N-acetylcarboxymethyl chitosan-Gly-Gly-Gly-Ala-Boc conjugate (162 mg). 150 mg of this chitosan was N-acetylated with acetic anhydride to give N-acetylcarboxymethyl chitosan-Gly-Gly-Gly-Ala-Boc conjugate (162 mg).
Acetylcarboxymethyl chitosan-Gly-Gly-Gly-A
Further, 130 mg of this compound was treated with acid to remove the Boc group, to give N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-H complex (114 mg).
100 mg of this was reacted with the active ester of MTX (90 mg) to give N-acetylcarboxymethylchitosan-
Gly-Gly-Gly-Ala-MTX complex (111 mg, MTX content: 17.4
% by weight was obtained.

Example 16 The partially N-acetylcarboxymethyl chitosan (200 mg) obtained in Example 12 was treated with N-Boc in the same manner as in Example 1.
The partially N-acetylcarboxymethylchitosan-Gly-Gly-Phe-Gly-Gly-OH (87 mg) was reacted with the active ester to give a partially N-acetylcarboxymethylchitosan-Gly-Gly-Phe-Gly-Boc conjugate (206 mg).
100 mg of this was N-acetylated with acetic anhydride to give N-acetylcarboxymethylchitosan-Gly-Gly-Phe-Gly-
A Boc complex (92 mg, N-Boc-peptide content: 7.4%) was obtained. 80 mg of this compound was further treated with acid to remove the Boc group.
N-acetylcarboxymethyl chitosan-Gly-Gly-Ph
The e-Gly-H complex (71 mg) was obtained. 50 mg of this complex was mixed with MTX (45 mg).
g) was reacted with the active ester to give an N-acetylcarboxymethylchitosan-Gly-Gly-Phe-Gly-MTX conjugate (50 mg, MTX content: 7.0%).

Example 17 50 mg of the partially N-acetylcarboxymethylchitosan-Gly-Phe-Phe-Boc complex (carboxymethylation degree: 0.7, N-Boc-peptide content: 13.2%) obtained in Example 12 was added to 0.
Dissolved in 1% _NaHCO3 aqueous solution (2.5 ml) and 2-O-monomethoxypolyethylene glycol-3,5-dichloro-s-
Triazine (molecular weight 5000, manufactured by Sigma) (hereinafter abbreviated as PEG ₁ ) (80 mg) was added and the mixture was reacted at 0° C. for 4 hours.
The reaction mixture containing the PEG ₁ -Gly-Phe-Phe-Boc conjugate was diluted with water (2.
5ml), sodium bicarbonate (500mg), acetic anhydride (200
The reaction mixture was dialyzed against water, and the dialyzed solution was concentrated to 3 ml. Acetone (90 ml) was added to the mixture, and the precipitate was collected.
The product was washed with methylene chloride and dried in vacuo to obtain an N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-Boc conjugate (72 mg, PEG content: 37%).

This complex (60 mg) was dissolved in 0.5 N HCl (3.5 ml) and
The reaction mixture was reacted at 4°C for 16 hours to remove the Boc group, and the reaction mixture was added to a mixed solvent (60 ml) of ethanol and ether (1:2). The precipitate was collected, washed with methylene chloride, and dried in vacuo to give N-acetylcarboxymethylchitosan-PEG ₁ -
A Gly-Phe-Phe-H conjugate (44 mg, PEG content: 26%) was obtained. The gel filtration elution pattern of this conjugate is shown in FIG.
N-acetylcarboxymethyl chitosan-PEG ₁ -Gly-P
He-Phe-H complex (35 mg) was dissolved in 1% NaHCO3 aqueous solution (1.75 _mg ).
ml), and the activated ester of MTX (16 mg) was added.
The reaction mixture was incubated at 5°C for 18 hours. The reaction mixture was dialyzed against water, and the dialyzed solution was concentrated to 3 ml. Acetone (60 ml) was added to the resulting precipitate, which was collected, washed with methylene chloride, and vacuum dried to obtain an N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-MTX conjugate (29 mg, PEG content: 21%, MTX content: 7.9%) as a yellow powder, as an example of a derivative of formula (X). The UV-visible absorption spectrum and gel filtration elution pattern of this conjugate are shown in Figures 25 and 26, respectively.

Example 18 50 mg of the partially N-acetylcarboxymethylchitosan-Gly-Phe-Phe-Boc complex (carboxymethylation degree: 0.7, N-Boc-peptide content: 13.2%) obtained in Example 12 was added to 0.
The resulting solution was dissolved in 2.5 ml of 1% NaHCO ₃ aqueous solution, and 12 mg of PEG ₁ used in Example 17 was added.
-Acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe
The 4-Phe-Boc complex (49 mg, PEG content: 8.0%) was obtained.
The Boc group was deprotected using 0 mg of chitosan, and the N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-H conjugate (3
The N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-H complex (21 mg) was dissolved in 1% NaHCl.
The chitosan was dissolved in an aqueous solution of _O3 (1.1 ml), and the activated ester of MTX (9.5 mg) was added and reacted for 18 hours at 4° C. The reaction solution was added to ethanol (11 ml), and the precipitate was collected, washed with methylene chloride, and dried in vacuo to obtain an N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-MTX conjugate (17 mg, PEG content: 6.1%, MTX content: 8.5%).

Example 19 Partially N-acetylcarboxymethyl chitosan-Gly-Phe-Phe-Boc conjugate synthesized according to Example 12 (carboxymethylation degree: 0.7, N-Boc-peptide content: 14.8%)
50 mg of the above was dissolved in 0.1% NaHCO ₃ aqueous solution (5 ml), and PEG ₁ (2
00 mg) was added, and N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-B was prepared in the same manner as in Example 17.
The Boc group was removed using 90 mg of this product to give N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-H conjugate (6
N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-H complex (50 mg, PEG content: 45%) was obtained.
g) was reacted with the activated ester of MTX in the same manner as in Example 17 to give an N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-MTX complex (42 mg, PEG content: 43
%, MTX content: 6.3%).

Example 20 Partially N-acetylcarboxymethyl chitosan-Gly-Gly-Phe-Gly-Boc complex synthesized according to Example 16 (carboxymethylation degree: 0.7, N-Boc-peptide content:
25 mg of PEG-100 (7.9%) was dissolved in 1.25 ml of 0.1% _NaHCO3 aqueous solution, and 50 mg of PEG- ₁ was added. The mixture was stirred in the same manner as in Example 17.
N-acetylcarboxymethyl chitosan-PEG ₁ -Gly-G
The Boc group was removed using 30 mg of this compound to give N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Gly-Phe-Gly-Boc complex (42 mg, PEG content: 42%).
The N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Gly-Phe-Gly-H conjugate (15 mg) was reacted with the activated ester of MTX in the same manner as in Example 17 to give an N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Gly-Phe-Gly-MTX conjugate (11 mg).
g, PEG content: 27%, MTX content: 6.1%).

Example 21 Partially N-acetylcarboxymethyl chitosan-Gly-Phe-Phe-Boc conjugate synthesized according to Example 12 (carboxymethylation degree: 0.7, N-Boc-peptide content: 14.8%)
100 mg of the above was dissolved in ₁₀ ml of 0.5% NaHCO3 aqueous solution, and 8.5 ml of dimethylformamide was added to make a homogeneous solution. To this solution, methoxypolyoxyethylene carboxylic acid (molecular weight: 5000, manufactured by Sigma) (hereinafter abbreviated as PEG _CO ) was added.
The reaction mixture was _dialyzed against water, and the dialyzed solution (27 ml) was diluted with sodium bicarbonate (3.6%).
g), acetic anhydride (2.1 ml), and reacted at 4°C for 20 hours to obtain partially N-acetylcarboxymethyl chitosan-PEG
_{The CO} -Gly-Phe-Phe-Boc conjugate was N-acetylated.
The reaction mixture was dialyzed against water, and the dialyzed solution was concentrated to 6 ml.
Acetone (120 ml) was added, and the precipitate was collected, washed with methylene chloride, and dried under vacuum to obtain N-acetylcarboxymethylchitosan-PEG _CO -Gly-Phe-Phe-Boc conjugate (109 mg, PEG content: 9.0%). 50 mg of this was used to remove the Boc group by acid treatment to obtain N-acetylcarboxymethylchitosan-PEG _CO -Gly-Phe-Phe-
The resulting complex (35 mg, PEG content: 4.7%) was N-acetylcarboxymethylchitosan-PEG- _CO -Gly-Phe-Phe-
Using 25 mg of the H complex, MT
The activated ester of X was reacted to form an N-acetylcarboxymethylchitosan-PEG _CO -Gly-Phe-Phe-MTX complex (2
5 mg, PEG content: 4.5%, MTX content: 9.1%) was obtained.

Example 22 Partially N-acetylcarboxymethylchitosan (300 mg) prepared in the same manner as in Example 1 was reacted with the active ester of N-Boc-Gly-Gly-Gly-OH (289 mg) in the same manner as in Example 1 to give a partially N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Boc conjugate (334 mg).

Dissolve 125 mg of this in water (12 ml), add 3.3% sodium metaperiodate aqueous solution (5 ml), and let stand at room temperature for 4 hours in the dark.
The reaction mixture was dialyzed against water, and the dialyzed solution was concentrated to about 12 ml.
The reaction mixture was adjusted to pH 4.5.
The temperature was lowered to 0.5°C, then raised to 8.0°C, and the mixture was added to ethanol (60 ml). The precipitate was collected and dried in vacuo to give an N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Boc conjugate (77 mg) in which some of the pyranose rings had been opened to form polyols.

This conjugate (60 mg) was treated with acid in the same manner as in Example 1 to remove the Boc group, yielding a polyol N-acetylcarboxymethylchitosan-Gly-Gly-Gly-H conjugate (46 mg).
This complex (35 mg) was reacted with the active ester of MTX (46 mg) to give a polyol N-acetylcarboxymethylchitosan-Gly-Gly-Gly-MTX complex (42 mg).
The UV-visible absorption spectrum and gel filtration elution pattern of this complex are shown in Figure 27 and Figure 28, respectively.
As shown in Figure 28.

Example 23 Partially N-acetylcarboxymethyl chitosan (250 mg) prepared in the same manner as in Example 1 was reacted with the active ester of N-Boc-Gly-Phe-Phe-Gly-OH (527 mg) in the same manner as in Example 1 to obtain a partially N-acetylcarboxymethyl chitosan-Gly-Phe-Phe-Gly-Boc complex (3
This complex (200 mg) was oxidized with periodate and then reduced in the same manner as in Example 22 to give polyol N-acetylcarboxymethylchitosan-Gly.
The N-Phe-Phe-Gly-Boc conjugate (113 mg, N-Boc-peptide content: 28%) was obtained.

This conjugate (60 mg) was treated with acid in the same manner as in Example 1 to remove the Boc group, yielding a polyol N-acetylcarboxymethylchitosan-Gly-Phe-Phe-Gly-H conjugate (36 mg, peptide content: 24%).
(55 mg) of activated ester was reacted to give polyol N
-Acetylcarboxymethylchitosan-Gly-Phe-Phe
A 1-Gly-MTX complex (22 mg, MTX content: 19%) was obtained. The MTX/peptide (molar ratio) of this complex and the a/ in formula (XI)
(a+b+c) was calculated to be 0.93 and 0.19 using the above-mentioned formulas (5) and (1), respectively.

The properties of the derivatives of formula (I), formula (X) and formula (XI) of the present invention were tested in Experimental Examples 1 to 4 described below.

Experimental Example 1 [Samples and Methods] The N-acetylcarboxymethylchitosan-Gly-Phe-Phe-MTX complex obtained in Example 1 and the N-acetylcarboxymethylchitosan-Gly-Gly-Gl complex obtained in Example 2 were used.
The y-MTX complex was dissolved in saline and administered at 400 μg/ml
Two types of sample solutions were prepared. Blood samples were collected from several mice and centrifuged to obtain plasma (190 μl), to which each of the sample solutions (10 μl) was added, and the mixture was allowed to react at 37°C. The reaction mixture was sampled over time, deproteinized, and then subjected to gel filtration (column: TSK-gel G4000PW _XL , eluent: 0.1 M NaCl, flow rate: 0.8 m
l/min, column temperature: 40°C, detection: ultraviolet absorption at 307 nm)
The percentage of MTX remaining in plasma was determined.

[Results]

The results are shown in Figure 16. Figure 16 is a graph showing the time course of the percentage of MTX remaining in plasma in the form of a complex represented by formula (I).
Acetylcarboxymethyl chitosan-Gly-Phe-Phe-M
The line marked with squares indicates the results for the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-MTX complex.

From FIG. 16, it can be seen that both of the above two complexes are hardly decomposed in blood and are stable.

Experimental Example 2 [Samples and Methods] The N-acetylcarboxymethylchitosan-Gly-Phe-Phe-MTX complex (1 mg) obtained in Example 1 and the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-MTX complex (1 mg) obtained in Example 2 were mixed.
The 1 mg of 1-Gly-Gly-MTX complex was reacted in the presence or absence of egg white lysozyme (10 μg) in 0.1 M acetate buffer (pH 6.0, 1 ml) at 37° C. to obtain 1, 3, 6, and 23
After 20 minutes, the reaction solution was analyzed by the same gel filtration method as described in Experimental Example 1, and the extension of the elution time (in gel filtration) resulting from the degradation of N-acetylcarboxymethylchitosan to lower molecular weight complexes was determined.

[Results]

The results are shown in Figure 17. Figure 17 is a graph showing the relationship between reaction time and the elution time at which a peak appears in the reaction solution at that reaction time. In the figure, the lines marked with circles and circles represent N-acetylcarboxymethylchitosan-Gly-Phe-
The results for the Phe-MTX complex in the absence and presence of lysozyme are shown, and the square and square lines show the results for the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-MTX complex in the absence and presence of lysozyme, respectively.

As shown in Figure 17, the N of the above complex is formed by the action of lysozyme.
The acetylcarboxymethylchitosan moiety is decomposed, resulting in the degradation of all the complexes into smaller molecules, so it is expected that the complexes will not remain in the body for a long period of time.

Experimental Example 3 [Sample] The N-acetylcarboxymethylchitosan-Gly-Gly-Gly-MTX complex of Example 2, in which the MTX moiety was ^3H -labeled, was used as the test sample compound. Separately, a partial N-acetylcarboxymethylchitosan labeled with ^3H , obtained during the process of Example 1, was prepared as a control sample.

[Method]

Sarcoma180 was subcutaneously implanted into male ICR mice.
The tumor-bearing mice were used for the experiment. The sample was dissolved in physiological saline, and 20 mg/kg of the sample was administered via the tail vein to three mice in each group.
The femoral artery and vein were cut and blood samples were collected 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, and 24 hours after administration. The blood was centrifuged, and the radioactivity of the serum and cancer tissue was measured by the combustion method to determine the concentration of the test compound in the serum and cancer tissue.

[Results]

The results are shown in Figures 18 and 19. Figure 18 is a graph showing the time course of serum concentration of the test compound, and Figure 19 is a graph showing the time course of concentration of the test compound in cancer tissue. In both figures, the square mark line shows the results for the control sample, and the ● mark shows the results for the control sample.
The dashed lines represent the results for the specimen samples listed above.

As shown in FIG. 18, the above test sample contains MTX via a peptide chain.
The complex is a complex of chitosan bound to a polysaccharide (N-acetylcarboxymethylchitosan). Therefore, although such a complex would be expected to be rapidly eliminated from the blood, the blood retention of the complexed test sample compound is actually increased to the same extent or even more than that of the simple polysaccharide, i.e., N-acetylcarboxymethylchitosan, before binding. Furthermore, Figure 19 shows that the test sample, compared to the simple polysaccharide, acquires a new property of easily accumulating in cancer tissues due to the inclusion of a peptide chain. Therefore, by selecting an appropriate peptide chain (-X-) in the substance of the present invention of formula (I), it is possible to endow the substance of the present invention of formula (I) with organ-targeting properties.

Experimental Example 4 In this experimental example, the following experiment was carried out using several examples of the substance of the present invention represented by formula (X) as samples.

That is, the conjugates as the final products of Examples 18, 17, and 19 in which the MTX moiety is ^3H -labeled (each conjugate has a polyethylene glycol (PEG) content of 6%)
%, 21%, and 43%) were prepared as labeling compounds for Samples 1, 2, and 3. Separately, a conjugate lacking PEG but with the MTX moiety labeled with ^3H was prepared as a control labeling compound. In all of the conjugates of Samples 1, 2, and 3, as well as the control conjugate, the peptide chain bound to the 2-amino group of the carboxymethylglucosamine unit of the conjugate was Gly-Phe-Phe, and the PEG in Samples 1, 2, and 3 was _PEG1 (molecular weight 5000) as described in Example 17.

(Test method) Walker 256 cancer cells were implanted subcutaneously in the inguinal region of female Wistar rats, and the tumor-bearing rats were used for the experiment 6 days after implantation. The sample administration solution was prepared by appropriately diluting the ^3H -labeled complex with a physiological saline solution containing the corresponding non-radioactive complex, and the sample was administered into the jugular vein of three rats per group at a dose of 10 mg/kg of the complex.

Under ether anesthesia, the blood was collected from the jugular vein over time (30 minutes, 1 hour, 2
Blood samples were taken at 1, 4, and 6 hours after administration, and plasma was separated. 24 hours after administration, blood samples were taken under ether anesthesia, and the animals were sacrificed by exsanguination. Tissues and organs were collected, and radioactivity was measured after weighing in whole or in part.

The radioactivity in the collected tissues was measured by drying the samples collected in a combustible cone, combusting them in an automatic sample combustion device (ASC-113, ALOKA), adding a scintillator (AQUASOL-2, NEN), and measuring it in a liquid scintillation counter (LSC-3600, ALOKA). Correction was performed using the external standard source method.

(Test Results) The test results are shown in Figures 29 and 30 of the attached drawings and in Table 1 below.
Figure 29 is a graph showing the time course of plasma concentration for each sample up to 24 hours after administration, Figure 30 is a graph showing the tissue concentration distribution in vivo for each sample versus the dose, and Table 1 is a table showing the AUC values calculated for each sample based on Figure 29 and the tumor concentration for each sample versus the dose.

As shown in Figure 29, the greater the amount of PEG introduced, the better the blood retention of the complex. This is shown by the AUC values in Table 1. Sample 1 (PEG introduction amount 6%) was approximately 1.3 times longer than the control, and Sample 2 (PEG introduction amount 21%) and Sample 3 (PEG introduction amount 21%) were approximately 1.3 times longer than the control.
It can be seen that the amount introduced (43%) has increased by approximately three times.

As shown in Figure 30, no significant difference was observed in organs other than tumor tissue due to the amount of PEG introduced.
In tumor tissue, the tumor concentration steadily increased with the amount of PEG introduced, as shown by the tumor concentration values in Table 1. That is, when PEG was introduced into the N-acetylcarboxymethyl chitosan derivative, it began to recognize cancer tissue, thereby reducing the required dose of anticancer drugs and, as a result, reducing undesirable effects on other organs.

BRIEF DESCRIPTION OF THE DRAWINGS With reference to the accompanying drawings, Figure 1: Ultraviolet absorption spectrum of the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-Boc complex obtained in Example 1 of the present invention (concentration: 1900 μg/ml, solvent: 30% EtO
H).

Figure 2: Gel filtration elution pattern of the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-Boc complex obtained in Example 1 of the present invention (detection: ultraviolet absorption at 258 nm)
Shows.

Figure 3: Ultraviolet absorption spectrum of the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-H complex obtained in Example 1 of the present invention (concentration: 1900 μg/ml, solvent: 30% EtO
H).

Figure 4: Gel filtration elution pattern of the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-H complex obtained in Example 1 of the present invention (detection: ultraviolet absorption at 258 nm)
Shows.

Figure 5: N-acetylcarboxymethylchitosan-Gly-Phe-Ph obtained as the final product in Example 1 of the present invention
UV-visible absorption spectrum of e-MTX complex (concentration: 100
μg/ml, solution: 0.1% NaHCO ₃ ).

Figure 6: N-acetylcarboxymethylchitosan-Gly-Phe-Ph obtained as the final product in Example 1 of the present invention
1 shows the gel filtration elution pattern of the e-MTX complex (detection: ultraviolet absorption at 307 nm).

FIG. 7 shows the ultraviolet absorption spectrum (concentration: 500 μg/ml, solvent: water) of the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-pMZ complex obtained in Example 2 of the present invention.

Figure 8: Gel filtration elution pattern of the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-pMZ complex obtained in Example 2 of the present invention (detection: ultraviolet absorption at 272 nm)
Shows.

FIG. 9 shows the ultraviolet absorption spectrum (concentration: 1000 μg/ml, solvent: water) of the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-H complex obtained in Example 2 of the present invention.

Figure 10: N-acetylcarboxymethylchitosan-Gly-Gly-Gl obtained as the final product in Example 2 of the present invention
UV-visible absorption spectrum of y-MTX complex (concentration: 48
μg/ml, solvent: 0.1% NaHCO ₃ ).

Figure 11: N-acetylcarboxymethylchitosan-Gly-Gly-Gl obtained as the final product in Example 2 of the present invention
1 shows the gel filtration elution pattern of the y-MTX complex (detection: ultraviolet absorption at 307 nm).

FIG. 12 shows the ultraviolet-visible absorption spectrum of the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-MTX complex obtained in Example 6 of the present invention (concentration: 50 μg/ml, solvent: 0.1% NaHCO ₃ ).

FIG. 13 shows the gel filtration elution pattern (detection: ultraviolet absorption at 307 nm) of the N-acetylcarboxymethylchitosan-Gly-Gly-Gly-Ala-MTX complex obtained in Example 6 of the present invention.

Figure 14: N-acetylcarboxymethylchitosan-Suc-Ala-Al obtained as the final product in Example 7 of the present invention
1 shows the ultraviolet absorption spectrum of the a-Ala-p-nitroanilide complex (concentration: 500 μg/ml, solvent: water).

Figure 15: N-acetylcarboxymethylchitosan-Suc-Ala-Al obtained as the final product in Example 7 of the present invention
1 shows the gel filtration elution pattern of a-Ala-p-nitroanilide (detection: ultraviolet absorption at 315 nm).

FIG. 16: A graph showing the time-dependent change (in vitro) in the percentage of remaining pharmaceutical compound methotrexate (MTX) in plasma when present as a complex with chitosan, as measured in Experimental Example 1 according to the present invention.

FIG. 17: As measured in Experimental Example 2 of the present invention, in the reaction of decomposing the complex of the present invention with an acetate buffer solution,
1 shows a graph showing the relationship between reaction time and the elution time at which a peak appears in the reaction solution at that reaction time.

FIG. 18: A graph showing the time-dependent changes (in vivo) in serum concentration of a sample compound, measured in Experimental Example 3 according to the present invention.

FIG. 19: A graph showing the time course of the concentration of the sample compound in cancer tissues, as measured in Experimental Example 3 according to the present invention.

Figure 20: N-acetylcarboxymethylchitosan-Suc-Gly-Phe-Gly-Lys-MTX obtained in Example 11 of the present invention
UV-visible absorption spectrum of the complex (concentration: 490 μg/m
l, solvent: 0.1% NaHCO ₃ ).

Figure 21: N-acetylcarboxymethylchitosan-Suc-Gly-Phe-Gly-Lys-MTX obtained in Example 11 of the present invention
The gel filtration elution pattern of the complex (detection: ultraviolet absorption at 307 nm) is shown.

Figure 22: UV-visible absorption spectrum (concentration: 100 μg/ml, solvent: 0.1% NaHCO ₃ ) of the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-MTX complex (carboxymethylation degree: 0.7) obtained in Example 12 of the present invention.

FIG. 23 shows the gel filtration elution pattern (detection: ultraviolet absorption at 307 nm) of the N-acetylcarboxymethylchitosan-Gly-Phe-Phe-MTX complex (carboxymethylation degree: 0.7) obtained in Example 12 of the present invention.

FIG. 24: Gel filtration elution pattern (detection: ultraviolet absorption at 258 nm) of the N-acetylcarboxymethylchitosan-PEG ₁ -Gly-Phe-Phe-H conjugate produced in Example 17 of the present invention.

Figure 25: N-acetylcarboxymethylchitosan-PEG ₁ -Gly-P obtained as the final product in Example 17 of the present invention
1 shows the ultraviolet-visible absorption spectrum of the he-Phe-MTX complex (concentration: 100 μg/ml, solvent: 0.1% NaHCO ₃ ).

Figure 26: N-acetylcarboxymethylchitosan-PEG ₁ -Gly-P obtained as the final product in Example 17 of the present invention
Gel filtration elution pattern of he-Phe-MTX complex (detection: 307
The figure shows the ultraviolet absorption at 100 nm.

Figure 27: Polyol N- obtained in Example 22 of the present invention
Acetylcarboxymethyl chitosan-Gly-Gly-Gly-M
UV-visible absorption spectrum of TX complex (concentration: 40 μg/m
l, solvent: 0.1% NaHCO ₃ ).

Figure 28: Polyol N- obtained in Example 22 of the present invention
Acetylcarboxymethyl chitosan-Gly-Gly-Gly-M
The gel filtration elution pattern of the TX complex (detection: ultraviolet absorption at 307 nm) is shown.

FIG. 29: A graph showing the time course of plasma concentrations of each sample up to 24 hours after administration, as measured in Experimental Example 4 of the present invention.

FIG. 30: A graph showing the distribution of tissue concentrations in the body relative to the dose for each sample.

The novel N-acetylcarboxymethylchitosan derivatives of the present invention are useful as polysaccharide polymer carriers for pharmaceutical compounds, aiming at in vivo targeting of pharmaceutical compounds, and are effective in enhancing the stability of pharmaceutical compounds in the blood, organ targeting, and metabolic susceptibility of pharmaceutical compounds in the body. Furthermore, the novel N-acetylcarboxymethylchitosan derivatives conjugated with pharmaceutical compounds according to the present invention have organ targeting and other advantageous properties, making them useful as pharmaceuticals.

Claims (12)

[Claims] [Claim 1] Formula (I) wherein R ₁ and R ₂ are each H or a carboxymethyl group, P is R ₃ CO group, R ₄ NH group or R ₅ O group, and Q is H or OH.
X represents a peptide chain containing 1 to 10 identical or different amino acids; _R3COOH represents a carboxylic acid compound;
_R4NH2 represents an amino compound, _{and R5OH} represents an alcohol compound. _a1 and _a2 represent 0 or a positive integer, except when both are 0. b represents a positive integer. ] and has the following characteristic values (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a + b): 0.01 to 1 (wherein a = _a1 + _a2 ) (4) P/X (molar ratio): 0.1 to 1. 2. The N-acetylcarboxymethylchitosan derivative according to claim 1, wherein X in XP is a peptide chain consisting of 1 to 4 identical or different amino acids, and P is bound to the N-terminal amino acid. 3. The N-acetylcarboxymethylchitosan derivative according to claim 2, wherein XP is any one of the following: P-Phe-Phe-Gly- P-Gly-Phe-Gly-Gly- P-Phe-Gly-Phe-Gly-Phe-Gly- P-Gly-Phe-Gly-Phe-Gly-Phe-P-Gly-Gly-Gly- P-Ala-Gly-Gly-Gly- [Claim 4] The N-acetylcarboxymethylchitosan derivative according to claim 1, wherein X in XP is a peptide chain containing a dibasic acid and consisting of 1 to 4 identical or different amino acids, and P is bound to the C-terminal amino acid. 5. The N-acetylcarboxymethylchitosan derivative according to claim 4, wherein XP is any one of the following: -Suc-Ala-Ala-Ala-P -Suc-Ala-Ala-Val-Ala-P (Suc represents a succinic acid residue). 6. The N-acetylcarboxymethylchitosan derivative according to any one of claims 1 to 5, wherein the R ₃ CO group and the R ₅ O group are protecting groups. 7. The N-acetylcarboxymethylchitosan derivative according to claim 6, wherein the R ₃ CO group is a tert-butoxycarbonyl group or a p-methoxybenzyloxycarbonyl group, and the R ₅ O group is a tert-butyloxy group. 8. The N-acetylcarboxymethylchitosan derivative according to any one of claims 1 to 5, wherein R ₃ COOH is methotrexate. [Claim 9] Formula (II) wherein R ₁ and R ₂ are each H or a carboxymethyl group, and P is H, an OH group, an R ₃ CO group, an R ₄ NH group, or an R ₅ O group.
X represents a peptide chain containing 1 to 10 identical or different amino acids; _R3COOH represents a carboxylic acid compound;
_R4NH2 represents an amino compound, and _R5OH represents an alcohol compound. _a , _b1 , and _b2 represent 0 or a positive integer. ] and has the following characteristic values (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a + b): 0.01 to 1 (wherein b = _b1 + _b2 ) (4) P/X (molar ratio): 0.1 to 1. [Claim 10] Formula (III) (wherein _R1 and _R2 each represent H or a carboxymethyl group) and having the following properties (1) to (2): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000, is treated with a compound of the following formula: PX-OH (IV) (wherein P represents H, an OH group, an _R3CO group, an _R4NH group, or an _R5
and X represents a peptide chain containing 1 _to 10 identical or different amino acids. _R3COOH represents a carboxylic acid compound, _R4NH2 represents an amino compound, and _R5OH represents an alcohol compound. (wherein _R1 , _R2 , P, and X have the same meanings as defined above in formula (III), and a, _b1 , and _b2 are positive integers.) and have the following characteristic values (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a + b): 0.01 to 1 (wherein b = _b1 + _b2 ) (4) P/X (molar ratio): 0.1 to 1. [Claim 11] Formula (II') [wherein _R1 and _R2 each represent H or a carboxymethyl group, _P1 represents H or an OH group, or an _R3'CO group, an _R4'NH group, or an _R5'O group, and X represents a peptide chain containing 1 to 10 identical or different amino acids. _R3'COOH represents a carboxylic acid compound, _R4'NH2 represents an amino compound, and _R5'OH represents an alcohol compound. _a , _b1 , and _b2 represent positive integers.] and has the following characteristic values (1) to (4):
A partially N-acetylcarboxymethyl chitosan derivative having (1) a carboxymethylation degree of 0.5-1.2, (2) a molecular weight (gel filtration method) of 3,000-300,000, (3) a/(a+b): 0.01-1 (wherein b = _b1 + _b2 ), and (4) a molar ratio of P/X of 0.1-1 is acetylated, and when the _P1 group is other than H or OH, the _P1 group is further eliminated, and finally, the chitosan is reacted with a compound represented by the following formula: P-H or P-OH (V) (wherein P represents _R3CO , _R4NH , or _R5O ). (wherein _R1 , _R2 , P, and X are as defined above, and Q represents H or an OH group; _a1 and _a2 represent 0 or a positive integer, except when both are 0; and b represents a positive integer.) and has the following characteristic values (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2; (2) molecular weight (gel filtration method): 3,000 to 300,000; (3) a/(a+b): 0.01 to 1 (where a = _a1 + _a2 ); and (4) P/X (molar ratio): 0.1 to 1. Claim 12: Formula (X) or Formula (XI) wherein R ₁ and R ₂ are each H or a carboxymethyl group, P is R ₃ CO group, R ₄ NH group or R ₅ O group, and Q is H or OH.
X represents a peptide chain containing 1 to 10 identical or different amino acids; _R3COOH represents a carboxylic acid compound;
_R4NH2 represents an amino compound, _{and R5OH} represents an alcohol compound. _a1 and _a2 represent 0 or a positive integer, except when both are 0. b and c represent positive integers. In the formula, -PEG represents a group represented by the following formula (VI), (VII), or (VIII):
or (IX) -CO- _{CH2CH2 -} CO-O-( _CH2CH2O ) _{n- CH3} (VII) -CO-O-( _CH2CH2O ) _n - _CH3 (VIII) -CO- _{CH2 -} O-( _CH2CH2O ) _{n- CH3} ( _IX ), where n represents the average degree _of polymerization of the polyethylene glycol chain, and having the following properties (1) to (4): (1) degree of carboxymethylation: 0.5 to 1.2 (2) molecular weight (gel filtration method): 3,000 to 300,000 (3) a/(a+b+c): 0.01 to 1 (where a = _a1 + _a2 ) (4) P/X (molar ratio): 0.1 to 1.