JPH0784481B2 - Carboxymethyl mannoglycan and its derivatives - Google Patents

Description

[Detailed Description of the Invention] [Background of the Invention] (Field of Industrial Application) The present invention relates to a novel carboxymethyl mannoglucan and its derivatives and salts. More specifically, the present invention relates to carboxymethyl mannoglucan and its derivatives and salts as carriers useful for delaying the disappearance of pharmaceuticals from the blood and increasing the targeting of the pharmaceuticals to cancer tissues.

(Prior Art) The use of water-soluble polymers as drug carriers has been attempted in the past, particularly in the field of pharmaceutical formulations, and many related technologies have been proposed. In many cases, cellulose derivatives such as carboxymethyl cellulose, hydroxypropyl cellulose, and hydroxypropylmethyl cellulose have been used, and the physicochemical properties of these substances themselves have been utilized to disperse and sustain the release of drugs. However, in these examples, the drug is integrated with the cellulose derivative as a carrier by pharmaceutical mixing, but is not chemically bound to the carrier.

In the so-called organ-directed technology, which delivers drugs to the required tissues at the required time in the required amount, when using water-soluble polymers as drug carriers, it is necessary to chemically bind the drug to the carrier, rather than simply mixing it. One such attempt is to bind mitomycin C to dextran (Jin Sezaki, Pharmaceutical Journal, 109 , 611-621 (1999)).
89)), binding of mitomycin C to mannan (Article from the 49th Annual Meeting of the Japanese Cancer Association (1990), p. 425, presentation number 2155), and binding of bleomycin to mannan (Article from the 49th Annual Meeting of the Japanese Cancer Association (1990), p. 425, presentation number 2154). However, the reality is that attempts to synthesize new water-soluble polysaccharide polymers and chemically bind drugs to them for drug delivery have not yet been fully developed.

Summary of the Invention

In view of the above, the present inventors focused on a polysaccharide polymer known as mannoglucan and attempted to carboxymethylate it. The obtained substance was a novel water-soluble polysaccharide polymer, which they found to be useful as a carrier for drug delivery techniques in which drugs are chemically bound to the polymer, particularly for techniques that reduce the rate at which drugs disappear from the blood and increase their migration to cancer tissue, and thus completed the present invention.

That is, an object of the present invention is to provide a water-soluble polysaccharide polymer that can hold a drug via a chemical bond and enable appropriate drug delivery.

Furthermore, the present invention provides a method for delaying the elimination of a pharmaceutical agent from the blood,
Another object of the present invention is to provide a water-soluble polysaccharide polymer that is useful for increasing the targeting of the pharmaceutical to cancer tissues.

The carboxymethyl mannoglucan and salts thereof according to the first aspect of the present invention are composed of tetrasaccharide units represented by the following general formula (I):

(Wherein, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 , ^R11 and R
¹² may be the same or different and each represents a hydrogen atom or CH ₂ COOH.) The carboxymethyl mannoglycan derivative and salts thereof according to the second aspect of the present invention are represented by the following general formula (III):
and is composed of tetrasaccharide units represented by the formula:

(In the formula, R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² ,
^R23 and ^R24 are each a hydrogen atom, _CH2COOH , _CH2CONR ^*1 R ^*2
(where NR ^*1 R ^*2 represents a residue obtained by removing one hydrogen atom from the amino group of a pharmaceutical compound having an amino group represented by the general formula HNR ^*1 R ^*2 ), _CH2COOR ^*3 (where OR ^*3 represents a residue obtained by removing a hydrogen atom from the alcoholic hydroxyl group of a pharmaceutical compound having an alcoholic hydroxyl group represented by the general formula HOR ^*3 ), or _CH2COO.1 /2[P
t(NH ₃ ) ₂ ] (where Pt represents divalent platinum).

However, at least one of R ¹³ to ^{R 24} in the molecule is CH ₂ CONR
^*1 R ^*2 CH ₂ COOR ^*3 or CH ₂ COO·1/2 [Pt(NH ₃ )
₂ )]. Furthermore, the oxidized carboxymethyl mannoglucan and its derivatives and salts according to the third aspect of the present invention are composed of units represented by the following general formula (IV) and/or units represented by the following general formula (V):

[Wherein, ^R25 , ^R26 , ^R27 , ^R28 , ^R29 and ^R30 may be the same or different and each represent a hydrogen atom or _CH2COOH , ^W1 and ^W2 each represent =0 or =N-R ^*4 (wherein R represents a residue obtained by removing two hydrogen atoms from the amino group of a pharmaceutical compound having an amino group represented by the general formula _H2N -R ^*4 ), ^A1 and ^A2 may be the same or different and each represent the following formula (VI), formula (VII), formula (VIII) or formula (IX), ^A3 and ^A4 may be the same or different and each represent the following formula (VII), formula (VIII) or formula (IX), provided that when the molecule consists only of the general formula (IV), not all of ^A1 and ^A2 in the molecule represent formula (VI).

(where X ⁱ¹ , X ⁱ² , X ⁱ³ , X ⁱ⁴ , X ⁱ⁵ , X ⁱ⁶ , X ⁱ⁷ , X ⁱ⁸ and X ⁱ⁹ are
may be the same or different, and each may be a hydrogen atom or a CH
₂ COOH, and ^Wi1 , ^Wi2 , ^Wi3 , ^Wi4 , ^Wi5 and ^Wi6 may be the same or different and each represent =0 or =N-R ^*4 (where N-
R ^*4 represents a residue obtained by removing two hydrogen atoms from the amino group of a pharmaceutical compound having an amino group represented by the general formula _H2N -R ^*4 ), provided that the formula (VI), the formula (VII), the formula (VIII) and the formula (I
X), the subscript i of X ⁱ¹ to X ⁱ⁹ and W ⁱ¹ to W ⁱ⁶
represents an integer of 1 to 4, and each of ^A1 , ^A2 , ^A3 and ^A4 will generally be referred to as ^Ai .) Furthermore, the carboxymethyl ring-opened mannoglycan and its derivatives and salts thereof according to the fourth aspect of the present invention are composed of units represented by the following general formula (X) and/or units represented by the following general formula (XI):

[wherein R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ and R ³⁸ may be the same or different and each represents a hydrogen atom, CH ₂ COOH, C
H ₂ CONR ^*1 R ^*2 , CH ₂ COOR ^*3 (where NR ^*1 R ^*2
and OR ^*3 has the same meaning as defined in claim 5) or
_CH2COO.1 /2[Pt( _NH3 ) ₂ ] (wherein Pt represents divalent platinum), ^B1 and ^B2 may be the same or different and represent the following formula (XII), (XIII), (XIV) or (XV), respectively, ^B3 and ^B4 may be the same or different and represent the following formula (XIII), (XIV) or (XV), respectively, provided that when a molecule consists only of the general formula (X), not all of ^B1 and ^B2 in the molecule represent formula (XII).

(Here, Y ^j1 , Y ^j2 , Y ^j3 , Y ^j4 , Y ^j5 , Y ^j6 , Y ^j7 , Y ^j8 , Y ^j9 ,
Y ^j10 , Y ^j11 , Y ^j12 , Y ^j13 , Y ^j14 and Y ^j15 may be the same or different and each represents a hydrogen atom, CH ₂ COOH, CH ₂ CO
NR ^{*1R *2} or _CH2COOR ^*3 (wherein NR ^{*1R *2} and OR ^*3 have the same meanings as defined in claim 5), or
CH ₂ COO·½[Pt(NH ₃ ) ₂ ] (wherein Pt represents divalent platinum), provided that the formula (XII), the formula (XIII), the formula (XIV) and the formula (X
In each of the above (V), the subscript j of Y ^j1 to Y ^j15 represents an integer of 1 to 4, and each of B ¹ , B ² , B ³ and B ⁴ will generally be referred to as B ^j ). BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the time course of plasma concentration in Walker 256 tumor-bearing rats when administered at 18.0 μg/kg.

FIG. 2 is a graph showing the time course of plasma concentration in Walker 256 tumor-bearing rats when administered 1 mg/kg.

FIG. 3 shows the ultraviolet-visible absorption spectrum of the carboxymethyl mannoglucan-daunorubicin complex formed by a Schiff base type bond obtained in Example 2.

FIG. 4 shows the ultraviolet-visible absorption spectrum of the carboxymethyl mannoglucan-daunorubicin complex via an amide bond obtained in Example 3.

FIG. 5 shows the ultraviolet-visible absorption spectrum of the carboxymethyl mannoglycan-mitomycin C complex via an amide group obtained in Example 15.

FIG. 6 shows the ultraviolet-visible absorption spectrum of the carboxymethyl ring-opened mannoglycan-mitomycin C complex via an amide group obtained in Example 18.

FIG. 7 shows a gel filtration chromatogram of the carboxymethyl ring-opened mannoglycan-mitomycin C complex via an amide group obtained in Example 18.

FIG. 8 shows the ultraviolet-visible absorption spectrum of the oxidized carboxymethyl mannoglucan-daunorubicin complex obtained in Example 23 via Schiff base type coupling.

FIG. 9 shows the ultraviolet-visible absorption spectrum of the oxidized carboxymethyl mannoglucan-daunorubicin complex obtained in Example 24 via Schiff base type coupling.

FIG. 10 shows the ultraviolet-visible absorption spectrum of the oxidized carboxymethyl mannoglucan-daunorubicin complex obtained in Example 25 via Schiff base type coupling.

FIG. 11 shows the gel filtration elution pattern of the cis-diammineplatinum (II) complex obtained in Example 26 via a coordinate bond.

[Specific Description of the Invention]

The carboxymethyl mannoglucan according to the first aspect of the present invention is composed of tetrasaccharide units represented by the general formula (I). Here, "composed of tetrasaccharide units" means that the carboxymethyl mannoglucan according to the present invention is a polymer compound having a structure in which the units are repeated.

Here, the tetrasaccharide unit represented by general formula (I) has a basic skeleton represented by the following formula (II).
Therefore, the carboxymethyl mannoglycan of the present invention can be rephrased as one having a polysaccharide skeleton composed of tetrasaccharide units represented by the following formula (II).

This polysaccharide polymer has already been reported by one of the present inventors (Inoue K. et al., Carbohydrate Re
s. 114 , 245−256, (1983)).

The basic polysaccharide is D-manno-D-
It is a glucan, and the backbone of this polysaccharide is β(1
→ 4) D bond cellulose that constitutes the main chain
- α- in positions 3 and 6 of every other glucose residue
The D-mannosyl group is bonded with an α(1→3) bond and an α
It has a double-branched structure with a (1→6) bond.

The structure can be represented by the following formula in addition to the formula (II).

The carboxymethyl mannoglycan of the present invention is not limited in terms of its molecular weight as long as it is composed of tetrasaccharide units represented by the general formula (I). However, it is preferably 1×10 ⁴ to 2×10 ⁶ .
More preferably, it is about 1×10 ⁶ .

The carboxymethyl mannoglucan of the present invention can be said to have a structure in which the hydrogen atoms of the hydroxyl groups in the basic skeleton are substituted with carboxymethyl groups, and the ratio of the introduction of these substituents can be expressed by the substitution degree, which is defined as the number of substituents per sugar residue. This can be expressed as:

The upper limit of the degree of substitution is 3 when all hydroxyl groups are substituted, but in the present invention, the degree of substitution is preferably 0.01 or more. In the present invention, it is necessary that at least one carboxymethyl group is present in the molecule, and in this sense, compounds with a degree of substitution of 0 are excluded. It goes without saying that, as long as the structure of each tetrasaccharide unit is within the scope of the above general formula (I), the positions at which the substituents are introduced in adjacent tetrasaccharide units may be the same or different.

The carboxymethyl mannoglycan according to the present invention can exist as a salt thereof. Suitable examples of the salt include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt, and calcium salt, and amino acid salts such as arginine salt and lysine salt.

The carboxymethyl mannoglycan derivative according to the second aspect of the present invention is derived from the carboxymethyl mannoglycan of the general formula (I). That is, the mannoglycan derivative composed of units represented by the general formula (III) has a structure in which a pharmaceutical compound is supported on some or all of the carboxymethyl groups in the general formula (I) via an acid amide bond, an ester bond, or a coordinate bond.

Examples of pharmaceutical compounds that can be introduced include the following. First, pharmaceutical compounds that can be introduced via an acid amide bond include pharmaceutical compounds having an amino group represented by the general formula HNR ^*1 R ^*2 , and specific examples include daunorubicin, doxorubicin, mitomycin C, and bleomycin. Furthermore, pharmaceutical compounds that can be introduced via an ester bond include pharmaceutical compounds having an alcoholic hydroxyl group represented by the general formula HOR ^*3 ,
Examples include cyclocytidine, vincristine,
Examples include vinblastine, adrenaline, etc. Furthermore, examples of compounds that can be introduced via coordinate bonds include platinum complexes such as cisplatin.

The molecular weight of the carboxymethyl mannoglycan derivative according to the second aspect of the present invention is not limited, but is preferably 1×10 ⁴ to 2×10 ⁶ , more preferably about 1×10 ⁵ .
The upper limit of the degree of substitution is always less than 3, and the lower limit exceeds 0. The preferred degree of substitution is about 1 to 2.

The carboxymethyl mannoglycan derivative according to the second aspect of the present invention can also exist as a salt of the carboxymethyl group. Suitable examples of the salt include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt, and calcium salt, and amino acid salts such as arginine salt and lysine salt.

Furthermore, the oxidized carboxymethyl mannoglucan and its derivatives according to the third aspect of the present invention are composed of units represented by the general formula (IV) and/or units represented by the general formula (V). In this specification, the term "derivative" is used only to refer to a compound to which a pharmaceutical compound has been introduced via a chemical bond. Therefore, the oxidized carboxymethyl mannoglucan according to the third aspect can be said to have a structure having an aldehyde group at the cleavage terminal, obtained by cleaving some or all of the mannose residues in the tetrasaccharide units constituting the carboxymethyl mannoglucan represented by the general formula (I) and then cleaving some or all of the glucose residues that are not branched from the mannose residues in the main chain. Furthermore, the oxidized carboxymethyl mannoglucan derivative according to the third aspect can be said to have a pharmaceutical compound introduced to the terminal aldehyde via a Schiff base bond.

In the general formula (IV), ^A1 and ^A2 represent the formula (VI), formula (VII), formula (VIII) or formula (IX).
The case where the molecule consists only of the general formula (IV) and A ¹ and A ² are all formula (VI) is excluded.
The units represented by formula (III) or formula (IX) are represented by formula (V
I) can be said to be a compound in which the bond between the 2nd and 3rd positions of the mannose residue is cleaved, the bond between the 3rd and 4th positions is cleaved, or the bond between the 2nd and 3rd positions and the bond between the 3rd and 4th positions are cleaved.

In addition, in the general formula (V), ^A3 and ^A4 are each independently selected from the group consisting of the groups represented by the formula (V
I), formula (VII), (VIII) or formula (IX).

In addition, the compounds of formula (VI), formula (VII), formula (VIII) and formula (IX)
In each of the above, the subscript i of X ⁱ¹ to X ⁱ⁹ and W ⁱ¹ to W ⁱ⁶ is 1.
^A1 , ^A2 , ^A3 and ^A4 are generally referred to as ^Ai . This means that, for example, when a unit represented by formula (VII) is bonded to each of ^A1 and ^A2 branched from the same D-glucose, ^Xi5 , i.e., ^X15 and ^X25 , are independent and different from each other, and this also falls within the scope of the present invention.
In addition, in the units represented by the general formula (IV) and/or the general formula (V) adjacent to each other in the molecule, R ²⁵ to R ³⁰ and A ¹
to ^A4 , and X ⁱ¹ to X ⁱ⁹ and W ⁱ¹ to W ⁱ⁶ may be different.

the abundance ratio of general formula (IV) and general formula (V) in the molecule;
Furthermore, the compounds of formula (VI), formula (VII), formula (VIII) and formula (I)
The abundance ratio of X) is not particularly limited, but may be determined taking into consideration the type of drug to be introduced and the degree of hydrophilicity.

Pharmaceutical compounds that can be introduced via a Schiff base bond include pharmaceutical compounds having an amino group represented by the general formula _H2N -R ^*4 , and specific examples thereof include daunorubicin, doxorubicin, and bleomycin.

Furthermore, the carboxymethyl ring-opened mannoglycan and its derivatives according to the fourth aspect of the present invention are composed of units represented by the general formula (X) and/or units represented by the general formula (XI). The carboxymethyl ring-opened mannoglycan according to the fourth aspect has a structure in which some or all of the mannose units in the tetrasaccharide units constituting the mannoglycan are first ring-opened, and then some or all of the glucose units constituting the main chain that are not branched from the mannose are ring-opened, and some or all of the hydrogen atoms of the hydroxymethyl groups formed at the open ring ends are substituted with carboxymethyl groups. The carboxymethyl ring-opened mannoglycan derivative according to the fourth aspect also has a structure in which a pharmaceutical compound is supported on the carboxymethyl groups via an acid amide bond, an ester bond, or a coordinate bond.

In the general formula (X), ^B1 and ^B2 are each a group represented by the formula (XII),
It represents formula (XIII), formula (XIV) or formula (XV). Here, the case where the molecule consists only of the general formula (X) and ^B1 and ^B2 are both formula (XII) is excluded. Formula (XII
The units represented by formula (I), formula (XIV) or formula (XV) are those in which the bond between the 2nd and 3rd positions of the mannose residue represented by formula (XII) is cleaved, the bond between the 3rd and 4th positions is cleaved, or the bonds between the 2nd and 3rd positions and the 3rd and 4th positions are cleaved.
It can be said that the bond between the positions is cleaved.

In addition, in the general formula (X), ^B3 and ^B4 are each independently selected from the group represented by the formula (XI).
The formula (II), (XIV) or (XV) is used. Here, the case where ^B3 and ^B4 represent formula (XII) is excluded. This is because, when mannoglucan is ring-opened by oxidation, the branched sugar mannose undergoes oxidative cleavage preferentially over the unbranched D-glucose of the D-glucose that constitutes the main chain.

In addition, the compounds of formula (XII), formula (XIII), formula (XIV) and formula (XV)
In each of Y ^j1 to Y ^j15 , the subscript j represents an integer of 1 to 4, and each of B ¹ , B ² , B ³ and B ⁴ will generally be referred to as B ^j . This means that, for example, when B ¹ and B ² branched from the same D-glucose have the formula (XIII)
When the unit represented by is bonded, Y ^j5 , i.e., Y ¹⁵
and ^Y25 are independent of each other, and the case where they are different from each other is also included in the scope of the present invention. In units represented by general formula (X) and/or general formula (XI) that are adjacent to each other in a molecule, ^R31 to ^R38 , ^B1 to ^B4 , and ^Yj1 to ^Yj15 may be different.

the abundance ratio of general formula (X) and general formula (XI) in the molecule;
Furthermore, the compounds of formula (XII), formula (XIII), formula (XIV) and formula (X
The ratio of V) is not particularly limited, and may be determined taking into consideration the type of drug to be introduced and the degree of hydrophilicity.

The carboxymethyl ring-opened mannoglycan according to the fourth embodiment is preferable in that it retains a carboxymethyl group to which a pharmaceutical compound can be introduced, and has high water solubility in proportion to the carboxymethyl mannoglycan according to the first embodiment of the present invention.

Pharmaceutical compounds that can be introduced into some or all of the carboxymethyl groups of the carboxymethyl ring-opened mannoglycan of this fourth embodiment via an acid amide bond, an ester bond or a coordinate bond include pharmaceutical compounds that can be introduced into the mannoglycan derivative of the second embodiment described above.

The oxidized carboxymethyl mannoglucan and its derivatives of the third embodiment and the carboxymethyl ring-opened mannoglucan and its derivatives of the fourth embodiment are not limited in molecular weight as long as they are composed of units as defined above, but preferably have a molecular weight of 1 x 10 ⁴ to 2 x 10 ⁶ .

The upper limit of the ratio of carboxymethyl group introduction, i.e., the degree of substitution, is always less than 3, and the lower limit is greater than 0. In the case of oxidized carboxymethyl mannoglycan and its derivatives, the preferred degree of substitution is 0.4-1.

Both oxidized carboxymethyl mannoglycans and derivatives thereof and carboxymethyl ring-opened mannoglycans and derivatives thereof can exist as salts of their carboxymethyl groups. Suitable examples of the salts include those exemplified for the carboxymethyl mannoglycans of the first and second embodiments.

Preparation of Compound and Uses The carboxymethyl mannoglucan according to the first aspect of the present invention can be obtained by substituting the hydrogen atoms of the hydroxyl groups of a mannoglucan composed of tetrasaccharide units represented by formula (II) with carboxymethyl groups. Specifically, it can be obtained by reacting a mannoglucan composed of tetrasaccharide units represented by formula (II) with a halogenated acetic acid or its salt in the presence of an alkali. For example, the raw material is dissolved in water, sodium hydroxide is added, and monochloroacetic acid is added while cooling. The mixture is stirred at room temperature for about 20 hours, the pH is adjusted to about 8-9 with acetic acid, and the mixture is poured into methanol. The precipitate is collected, washed with methanol and acetone, and dried. The degree of substitution of the carboxymethyl group can be adjusted by changing the amount of alkali and monochloroacetic acid or its salt added.

The mannoglucan composed of the tetrasaccharide unit represented by the formula (II) can be obtained from the bacteria belonging to the genus Microellobosporia, for example, Actinomonas aeruginosa.
It can be obtained by preparing a product separated and purified from the culture filtrate of the yeast Microellobosporia grisea (JP-B 1-5240
(See Publication No. 2).

The carboxymethyl mannoglycan according to the first aspect of the present invention has a low rate of disappearance from the blood and is cancer tissue-specific (see the Experimental Examples below for details). Meanwhile, the carboxymethyl mannoglycan according to the present invention contains numerous hydroxyl and carboxyl groups, allowing drugs to be bound to the carboxymethyl mannoglycan by utilizing these functional groups. Therefore, the carboxymethyl mannoglycan according to the first aspect of the present invention is a useful carrier for drug delivery techniques that involve chemically binding drugs, particularly techniques that reduce the rate of drug disappearance from the blood and enhance drug delivery to cancer tissues.

The introduction of a drug into the carboxymethyl mannoglycan of the present invention can be carried out by selecting an appropriate method depending on the properties of the drug. For example, in the case of a drug having an amino group (e.g., daunorubicin, doxorubicin, etc.), the carboxymethyl mannoglycan of the present invention can be oxidized in advance with periodic acid or the like to cleave the mannose moiety and form an aldehyde group, to which the drug can be bound as a Schiff base. Similarly, in the case of a drug having an amino group, it is possible to form an amide bond with the carboxyl group, or, after activating the hydroxyl group with cyanogen bromide, to form an isourea bond with the drug having an amino group. The above-mentioned drug having an amino group may be one that itself has an amino group, or one to which an amino group has been newly added for the purpose of binding. Furthermore, by selecting and adding an appropriate spacer having an amino group, it may be possible to obtain a drug having an amino group.

A specific example of a carboxymethyl mannoglycan according to the first aspect of the present invention, in which a pharmaceutical compound is introduced, is the carboxymethyl mannoglycan derivative according to the second aspect of the present invention. The derivative in which a pharmaceutical compound is introduced via an acid amide bond or an ester bond is a carboxymethyl mannoglycan or a salt thereof composed of a unit represented by the general formula (I) and a compound represented by the general formula HNR ^*1 R ^*2 or the general formula H
OR ^*3 under conditions for forming an acid amide bond or an ester bond.
For example, a derivative into which daunorubicin has been introduced can be obtained by reacting carboxymethyl mannoglucan with daunorubicin hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a condensing agent in borate buffer (pH 8), followed by precipitation with ethanol. Also, a derivative into which a pharmaceutical compound has been introduced via a coordinate bond can be obtained by reacting a platinum complex, cis-dinitrate diammineplatinum(II), with carboxymethyl mannoglucan in an aqueous solution, followed by dialysis and precipitation with ethanol.

The oxidized carboxymethyl mannoglucan according to the third aspect of the present invention can be obtained by oxidizing and ring-opening a carboxymethyl mannoglucan composed of units represented by general formula (I). First, an oxidizing agent (e.g., periodic acid or a salt thereof) is added to the carboxymethyl mannoglucan under ice-cooling, and the mixture is allowed to react gently at room temperature or below. The oxidized carboxymethyl mannoglucan can be obtained as a precipitate by, for example, dialyzing the reaction solution against water, adding sodium acetate as a precipitation aid, and dropping the mixture into ethanol. Here, by varying the amount of periodic acid or its salt added, it is possible to aldehyde the branched mannose residues and the main chain glucose residues to various degrees, thereby obtaining a carrier to which a desired amount of a pharmaceutical compound can be bound. In addition, the degree of aldehyde formation can also be controlled by the reaction time and temperature. Regarding aldehyde formation, see Inoue K. et al., Carbohydrate Reactions, Vol. 1, No. 1, pp. 111-114, 2003.
See s. 123, 305-314 (1983).

The oxidized carboxymethyl mannoglucan thus obtained can be reacted with a pharmaceutical compound represented by the general formula _H2NR ^*4 under Schiff base bond-forming conditions to obtain a derivative carrying the pharmaceutical compound. For example, a derivative carrying daunorubicin as the pharmaceutical compound can be obtained by reacting it with daunorubicin hydrochloride in a borate buffer (pH 8)-ethanol mixed solution.

The carboxymethyl ring-opened mannoglucan according to the fourth aspect of the present invention can be obtained by oxidizing mannoglucan to open the ring, reducing the aldehyde groups formed by the ring-opening to hydroxymethyl groups, and then introducing carboxymethyl groups to some or all of the hydroxymethyl groups. For example, an oxidizing agent (e.g., sodium periodate) is added to an aqueous solution of mannoglucan, and the mixture is allowed to react gently at room temperature or below room temperature in the dark, and then dialyzed against water. Sodium borohydride is then added to the dialyzed solution to react, and the pH of the reaction solution is adjusted to 5, then 7.
The polyalcohol form of the ring-opened mannoglucan can be obtained by adjusting the pH to 8, dialyzing it against water, and concentrating it. This polyalcohol form is dissolved in an aqueous sodium hydroxide solution, and monochloroacetic acid is added to react at room temperature. The pH of the reaction solution is then adjusted to 8, and the mixture is poured into ethanol to obtain carboxymethyl ring-opened mannoglucan.

Furthermore, a pharmaceutical compound can be introduced into the carboxymethyl ring-opened mannoglycan thus obtained in the same manner as in the case of the carboxymethyl mannoglycan derivative of the second embodiment described above.

[Example]

Reference Example The mannoglucan used as a raw material in the following examples was produced as follows using Microerobosporia grisea (Osaka Fermentation Research Institute Deposit No. IFO12518) as the producing microorganism.

GC medium (glucose 2%, peptone 0.5%, corn steep liquor 0.5%, yeast extract 0.3%, sodium chloride
0.5%, calcium carbonate 0.3%, agar 1.5%; pH 7.0) 100ml
The strain was inoculated into a Sakaguchi flask containing 100 ml of ethanol by slant inoculation.
After 5 days of shaking culture at 28°C, 2 ml of the culture was inoculated into a Sakaguchi flask containing 100 ml of GC medium and similarly cultured for 3 days. 200 ml of this culture was inoculated into a 30 L jar fermenter containing 20 L of production medium (3% glucose, 2% corn steep liquor; pH 7.2), and the culture was incubated at 28°C for 92 hours with aeration and agitation (10 L/min, 250 rpm).
The medium was then heated at 80°C for 20 minutes, cooled to room temperature, and filtered. The filtrate was passed through a Diaion PA306 (Cl ^- type) column, and the effluent was treated with 0.5 L of 10% cetylpyridinium and 1.0 L of ethanol.
The resulting precipitate was collected, washed with water, and then dissolved in 2% acetic acid (2.0 L). Ethanol (6.0 L) was added, and the resulting precipitate was collected. This precipitate was washed with ethanol and then dissolved in 0.02% aqueous sodium acetate (3.0 L). The supernatant was centrifuged and reprecipitated with ethanol. The collected precipitate was washed sequentially with 75% ethanol, ethanol, and acetone, and then vacuum-dried over phosphorus pentoxide at 50°C for 8 hours to yield 36 g of the desired mannoglucan. The molecular weight of the resulting mannoglucan (gel filtration method/standard: dextran, column: G5000PW) was approximately 1 x ¹⁰⁶ .

Example 1 The mannoglucan (500 mg) obtained in Preparation Example 1 was added to water (20 ml)
To this solution, monochloroacetic acid (1.5 g) and sodium hydroxide (1.05 g) were added under cooling to obtain a clear solution. Monochloroacetic acid (1.5 g) was added under cooling, and after dissolution, the mixture was stirred at room temperature for 20 hours to allow the reaction to proceed. After the pH of the reaction solution was adjusted to 8 by adding acetic acid, the mixture was poured into methanol (80 ml) and the resulting white precipitate was collected by filtration. The precipitate was washed successively with methanol and acetone and then dried in vacuo to obtain carboxymethyl mannoglucan.
This substance was named CM-1, and the degree of substitution (DS) per sugar residue was measured by the following method, and was found to be 0.08.

Measurement of the Degree of Substitution The degree of substitution (DS) was determined by back titration of the free acid form as follows. First, the carboxymethyl mannoglucan obtained as described above was shaken with 70% nitric acid/methanol (1:10 V/V) for 3 hours at room temperature, washed with 80% methanol and methanol using Melti Red as an indicator, and dried to obtain a sample. Next, this sample was dissolved in a specified excess amount of 0.1N sodium hydroxide solution and back titrated with 0.1N hydrochloric acid using phenolphthalein as an indicator. If the amount of sample collected is S (mg), the specified excess amount of 0.1N sodium hydroxide is A (ml), and the back titration amount of 0.1N hydrochloric acid is B (ml), then:
DS was calculated by the following formula (I).

DS = 16.2 (A - B) / [S - 5.8 (A - B)] Examples 2 to 4 The same procedures as in Example 1 were carried out except that the amounts of sodium hydroxide and monochloroacetic acid used in Example 1 were changed as shown in Table 1.

The yields, degrees of substitution and names of the materials obtained in Examples 1 to 4 are shown in Table 1.

Example 5 CM-4 (substitution degree:
500 mg of 0.55) was added to 20 ml of water and 3.5 g of sodium hydroxide to obtain a clear solution.
After dissolving under cooling, the mixture was reacted at room temperature for 20 hours. After adjusting the pH of the reaction mixture to 8 with acetic acid, the mixture was poured into 100 ml of methanol. The precipitate formed was collected, washed with methanol, and dried in vacuo to obtain CM-5 (531 mg).
The degree of substitution was 0.81.

Example 6: 10 ml of water and 1.75 g of sodium hydroxide were added to CM-5 (250 mg) obtained in Example 5 to obtain a clear solution. 2.5 g of monochloroacetic acid was added to this solution under cooling and dissolved, and then the mixture was stirred at room temperature.
The reaction was carried out for 21 hours. After adjusting the pH of the reaction mixture to 8 with acetic acid, the mixture was poured into 60 ml of methanol. The precipitate formed was collected, washed with methanol, and dried in vacuo to give CM-6 (261 mg).
The degree of substitution of CM-6 was 1.0.

Experimental Example 1 (1) Samples and specimens CM-1 and CM-4 obtained in Examples 1 and 4 were used as samples. The following preparatory steps were carried out to prepare specimens for animal experiments from each sample. First, each sample was dissolved in water, and 0.5 M sodium periodate was added in an amount equivalent to 0.1 mole of periodate ions per mole of sugar residue in the sample. The reaction was carried out at room temperature for 25 hours, and then the sample was dialyzed against water at 4°C. Sodium acetate was then added to the resulting solution, and the resulting solution was diluted with 0.5 M sodium periodate in an amount equivalent to 0.1 mole of periodate ions per mole of sugar residue in the sample. The reaction was carried out at room temperature for 25 hours, and the sample was then dialyzed against water at 4°C.
The resulting precipitate was washed with ethanol and acetone and then dried. Finally, the resulting powder was reacted with tritium-labeled sodium borohydride in 2.5 mM aqueous sodium carbonate at room temperature for 20 hours, adjusted to pH 5 with acetic acid under cooling, dialyzed against water, and the resulting solution was freeze-dried to obtain samples 1 and 2, respectively.

(2) Experimental Methods A. Maintenance of Tumor Cells Walker 256 cells were cultured in Wistar/S rats (6-9 weeks old, female).
3-5 x ¹⁰⁶ cells were administered intraperitoneally to each mouse, and the mice were passaged every 7 days.

S-180 cells were administered intraperitoneally to ICR mice (4-6 weeks old, male) at 2-5 x 10 ⁶ cells and passaged every 7 days.

B. Preparation of tumor-bearing animals: 1.0 x 10 ⁷ tumor cells were transplanted subcutaneously into the inguinal region of Wistar/S rats (6 weeks old, female), and six days later, Walker 256 tumor-bearing rats were used for the experiment.

ICR mice (4 weeks old, male) were injected with 1.5x tumor cells subcutaneously in the groin.
10 ⁶ cells were transplanted, and 10 days later, the mice were used for the experiment as S-180 tumor-bearing mice.

C. Biodistribution experiment (Walker 256 tumor-bearing rats) The experiment was conducted in rats with a 18.0 μg/kg dose administered for up to 6 hours and a 10 mg/kg dose administered for 24 hours.
Two methods were performed: 1) administering the test substance to tumor-bearing rats through the jugular vein under light ether anesthesia, and 2) collecting blood samples after a set time period to examine the plasma concentration of the test substance. The rats were sacrificed by exsanguination 6 hours after the 18.0 μg/kg administration and 24 hours after the 10 mg/kg administration, and the tumor and plasma concentrations of the test substance were examined.

(S-180 tumor-bearing mice) The sample was administered to the tail vein of tumor-bearing mice (18.0 μg/kg).
After 4 hours, the mice were sacrificed by exsanguination, and the intratumoral and plasma concentrations of the test substance were examined.

The tumor and plasma concentrations were determined by burning the tissue and plasma, respectively, using a combustion device and measuring the radioactivity by liquid scintillation spectrometry.

(3) Results The results are shown in Figures 1 and 2, and Tables 2 and 3.

Figures 1 and 2 are graphs showing the time course of plasma concentrations in Walker 256 tumor-bearing rats when administered 18.0 μg/kg and 10 mg/kg, respectively. In the figures, the lines marked with a triangle and the lines marked with a circle represent the results for sample 1 and sample 2, respectively.

1 and 2, it is apparent that the substance of the present invention is not rapidly eliminated from the blood, and the elimination rate is slow.

Tables 2 and 3 show the intratumor concentrations, plasma concentrations, and tumor tissue Kp values (abbreviated simply as Kp values in the tables) at the indicated time points for Sample 1 and Sample 2, respectively. The Kp values were calculated using the following formula:

Kp value = analyte concentration per gram of tissue/analyte concentration per ml of plasma. Tables 2 and 3 demonstrate that the substance of the present invention has specificity for cancer tissues.

Experimental Example 2 (Synthesis of carboxymethyl mannoglucan-daunorubicin conjugate via Schiff base type bond) 200 mg of CM-4 obtained in Example 4 was dissolved in 40 ml of water. To this was added 21 mg of sodium periodate (equivalent to 0.1 mole per mole of sugar residue) dissolved in a small amount of water while stirring under ice cooling. After reacting at room temperature for 25 hours,
The mixture was dialyzed against water, and 200 mg of sodium acetate was added to the resulting solution, which was then added dropwise to 350 ml of ethanol. The precipitate was collected and dried to obtain 192 mg of aldehyde-carboxymethyl mannoglucan. 20 mg of this was dissolved in 4 ml of 0.1 M borate buffer (pH 8.0). A solution of 16.9 mg of daunorubicin hydrochloride dissolved in 4 ml of ethanol and 400 μl of 0.1 M borate buffer (pH 8.0) was added to the resulting solution, and the mixture was allowed to react overnight at room temperature. 12 ml of ethanol was then added to the reaction solution, and the precipitate was collected and dried to obtain a Schiff base-linked carboxymethyl mannoglucan-daunorubicin complex.
The complex was water-soluble, and the daunorubicin content was 10.5% (by weight). Figure 3 shows the ultraviolet-visible absorption spectrum (concentration: 200 μg/ml, solvent: water).
Shows.

Experimental Example 3 (Synthesis of carboxymethyl mannoglucan-daunorubicin conjugate via amide bond) 20 mg of CM-4 obtained in Experimental Example 4 was dissolved in 6 ml of 0.1 M borate buffer (pH 8.0).
Dissolve 5.6 mg of ethanol in 4 ml of 0.1 M borate buffer (pH 8.0) in 1 ml of ethanol.
ml of solution was added, and then 1-ethyl-3-(3-
A solution of 60 mg of (dimethylaminopropyl)carbodiimide hydrochloride dissolved in 1 ml of water was added, and the mixture was allowed to react overnight at room temperature. 24 ml of ethanol was then added to the reaction mixture, and the precipitate was collected and dried to give a carboxymethyl mannoglucan-daunorubicin complex bound to the carboxyl group via an amide bond.
The complex was water-soluble, and the daunorubicin content was 5.3% (by weight). Figure 4 shows the ultraviolet-visible absorption spectrum (concentration: 500 μg/ml, solvent: water).
Shows.

Example 7 Mannoglucan (3.00 g) was carboxymethylated according to the method of Example 4 to give 3.25 g of CM-4 (degree of substitution: 0.53).
This CM-4 (2.00 g) was further carboxymethylated in the same manner as in Example 5 to give 2.22 g of CM-5 (degree of substitution: 0.79). This CM-5 (1.00 g) was further carboxymethylated in the same manner as in Example 6 to give 1.08 g of CM-
6 (degree of substitution: 1.0) was obtained.

Example 8 CM-6 (500 mg) obtained in Example 7 was dissolved in 2-propanol (3
The precipitate was suspended in 200 ml of water (40 ml), and the entire volume of a solution obtained by dissolving 1 g of sodium hydroxide in 3 ml of water was added dropwise to the suspension. Monochloroacetic acid (1 g) was then added and the mixture was stirred at room temperature for 2 hours. The precipitate was collected and reacted again at room temperature for 20 hours using 2-propanol (40 ml), sodium hydroxide (1 g), water (2 ml), and monochloroacetic acid (1 g). The precipitate was collected and dissolved in water (40 ml), and then poured into methanol (240 ml). The resulting precipitate was collected, washed with methanol, and vacuum dried to obtain 620 mg of CM-
7 (degree of substitution: 2.1) was obtained.

Example 9 Mannoglycan (4.00 g) was dissolved in 0.1 N hydrochloric acid (160 ml), acidolyzed at 80°C for 5 hours, and then neutralized with 5 N sodium hydroxide. This solution was poured into ethanol (500 ml), and the resulting precipitate was collected, washed with ethanol, and then dissolved in water (250 ml). This solution was then washed with Dowex 50W-X2 (H ⁺ ).
and Dowex 1-X2 (Cl ^- ) columns (1.5 x 20 cm each), and the flow-through was concentrated to approximately 150 ml.
The resulting precipitate was collected, washed with ethanol, and dried in vacuo to give 3.48 g of low molecular weight mannoglycan.

This 3.30 g was dissolved in 1 M sodium chloride (330 ml), methanol (330 ml) was added, and the mixture was centrifuged. Methanol (110 ml) was added to the resulting supernatant, and the resulting precipitate was collected. This precipitate was dissolved in water (50 ml) and ethanol (20
The resulting precipitate was collected, washed with ethanol, and dried in vacuo to obtain 1.92 g of low molecular weight mannoglycan (MG1
5) was obtained. The molecular weight of MG15 (gel filtration method/standard substance: dextran, column: G4000PW _XL ) was approximately 1.5 x 10 ⁵ .

Example 10 Mannoglucan (7.00 g) was dissolved in 0.1 N hydrochloric acid (280 ml).
After acid decomposition at 80°C for 7.5 hours, the solution was neutralized with 5N sodium hydroxide. The solution was poured into ethanol (900 ml).
The precipitate was collected, washed with ethanol, and then added to water (450 ml
This solution was dissolved in Dowex 50W-X2 (H ⁺ ) and Dowe
The mixture was passed through both columns (2 x 20 cm each) of x1 and x2 ( ^Cl- ), and the flow-through was concentrated to approximately 250 ml, then poured into ethanol (850 ml). The resulting precipitate was collected, washed with ethanol, and dried in vacuo to obtain 6.02 g of low molecular weight mannoglycan.

3.98 g of this was dissolved in 1 M sodium chloride (400 ml), to which methanol (533 ml) was added, and the resulting precipitate was collected by centrifugation. This precipitate was dissolved in water (100 ml) and then poured into ethanol (400 ml). The resulting precipitate was collected, washed with ethanol, and vacuum dried to obtain 2.00 g of low molecular weight mannoglycan (MG10). Methanol (267 ml) was added to the supernatant from the above centrifugation, and the resulting precipitate was collected. This precipitate was dissolved in water (60 ml) and ethanol (24
The resulting precipitate was collected, washed with ethanol, and vacuum dried to obtain 1.26 g of low molecular weight mannoglycan (MG
4) was obtained. The molecular weights of MG10 and MG4 (gel filtration method/standard material: dextran, column: G4000PW _XL ) were approximately 1x
10 ⁵ and approximately 4 x 10 ⁴ .

Example 11 MG15 (1.50 g) obtained in Example 9 was carboxymethylated in the same manner as in Example 4 to obtain 1.80 g of MG15-CM-4 (degree of substitution: 0.52). 1.40 g of this was further carboxymethylated in the same manner as in Example 5 to obtain 1.54 g of MG15-CM-5.
This MG15-CM-5 (1.00 g) was further carboxymethylated in accordance with the method of Example 6 to give 1.08 g of MG15-CM
-6 (degree of substitution: 1.0) was obtained.

Example 12 MG10 (1.80 g) obtained in Example 10 was carboxymethylated in the same manner as in Example 4 to obtain 2.23 g of MG10-CM-4. 2.00 g of this was further carboxymethylated in the same manner as in Example 5 to obtain 2.25 g of MG10-CM-5.
MG10-CM-5 (1.0 g) was further carboxymethylated according to the method of Example 6 to give 1.07 g of MG10-CM-6 (degree of substitution: 1.
0).

Example 13 MG4 (500 mg) obtained in Example 10 was carboxymethylated in the same manner as in Example 4 to obtain 594 mg of MG4-CM-4 (degree of substitution: 0.54).

Example 14 Mannoglucan (1.50 g) was dissolved in water (150 ml), and then
An 8.5% aqueous solution of sodium periodate (70 ml) was added, and the mixture was reacted at room temperature for 64 hours in the dark. Ethylene glycol (1.7 g) was added to the reaction mixture, and the mixture was allowed to stand at room temperature for 2 hours.
The mixture was dialyzed against water, and sodium borohydride (0.75 g) was added to the resulting solution and allowed to react overnight at room temperature. The pH of the resulting solution was adjusted to 5 with acetic acid, and then adjusted to 7 with 2N sodium hydroxide. The resulting solution was then dialyzed against water. Approximately 10 ml of the resulting solution was then dialyzed against water.
After concentrating to 1.29 g, the mixture was poured into a mixture of ethanol (0 ml) and acetone (80 ml). The precipitate formed was collected, washed with acetone, and dried in vacuo to obtain 1.29 g of mannoglycan polyalcohol (MG-PA).

Water (1 ml) and sodium hydroxide (2.0 g) were added to this MG-PA (500 mg) under cooling to obtain a clear solution. Monochloroacetic acid (2.9 g) was added to this solution and the reaction was allowed to proceed at room temperature for 18 hours. The pH of the reaction mixture was adjusted to 8 with acetic acid, and the mixture was poured into ethanol (200 ml) and the resulting precipitate was collected. The precipitate was dissolved in water (5 ml) and poured into methanol (125 ml). The resulting precipitate was collected, washed with methanol, and then eluted.
After drying in vacuo, 459 mg of the carboxymethylated product was obtained.
400 mg of this was suspended in 2-propanol (40 ml), and the entire volume of a solution obtained by dissolving 0.8 g of sodium hydroxide in 1.6 ml of water was added dropwise to the suspension. Then, monochloroacetic acid (0.8 g) was added.
The mixture was stirred at room temperature for 20 hours. The precipitate was collected, dissolved in water (8 ml), and then poured into methanol (200 ml). The resulting precipitate was collected, washed with methanol, and vacuum dried to obtain 631 mg of carboxymethylated mannoglycan polyalcohol (MG-PA-CM). The degree of substitution (DS) of MG-PA-CM was measured as in Example 1 and found to be 9 per tetrasaccharide. The DS per tetrasaccharide was calculated using the following formula:

DS = 59.4 (A - B) / [S - 5.8 (A - B)] Example 15 50 mg of CM-5 obtained in Example 7 and 50 mg of 1-ethyl-3-
3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC) was dissolved in water (10 ml) and then cooled on ice. 4 mg of mitomycin C (MMC) was dissolved in 0.8 ml of water-ethanol (1:
The entire amount of this solution was added to the above ice-cold solution, and the reaction was carried out for 1 hour under ice-cooling while maintaining the pH of the reaction solution at 5-6 with 0.2N hydrochloric acid.
After adjusting the pH to 7.6 with 0.2N sodium hydroxide, the solution was poured into ethanol (50 ml). The resulting precipitate was collected, washed with 95% ethanol, and vacuum dried to obtain a complex (52 mg) of MMC bound to CM-5. The MMC content of this complex was determined by absorbance analysis at 365 nm to be 7.6% (wt%). The UV-visible absorption spectrum of this complex (concentration: 9
6 μg/ml, solvent: water-ethanol (7:3, v/v)) is shown in FIG.

Example 16: In the same manner as in Example 15, CM-6 (50 m
g) and 4 mg of MMC were reacted with 50 mg of EDC, and MMC
A complex (50 mg) with a β-glucan content of 7.3% (wt %) was obtained.

Example 17: In the same manner as in Example 15, CM-7 (50 ml) obtained in Example 8 was
g) was reacted with 10 mg of MMC using 150 mg of EDC, and MM
A complex (57 mg) with a C content of 14% (wt %) was obtained.

Example 18: The MG-PA-CM obtained in Example 14 was prepared in the same manner as in Example 15.
(30 mg) and 10 mg of MMC were reacted with 150 mg of EDC to obtain a complex (31 mg) with an MMC content of 24% (wt%).
2 μg/ml, solvent: water-ethanol (7:3, v/v)) and the gel filtration chromatograms are shown in FIGS. 6 and 7, respectively.

Example 19: MG-15-CM- obtained in Example 11 was synthesized in the same manner as in Example 15.
4 (50 mg) was reacted with 4 mg of MMC using 50 mg of EDC to give a complex (53 mg) with an MMC content of 7.2% (wt %).

Example 20: MG-15-CM- obtained in Example 11 was synthesized in the same manner as in Example 15.
50 mg of 6 was reacted with 10 mg of MMC using 150 mg of EDC to give a complex (48 mg) with an MMC content of 15% (wt %).

Example 21: MG-10-CM- obtained in Example 12 was synthesized in the same manner as in Example 15.
6 (50 mg) was reacted with 10 mg of MMC using 150 mg of EDC to give a complex (55 mg) with an MMC content of 17% (wt %).

Example 22: MG4-CM-4 obtained in Example 13 was obtained in the same manner as in Example 15.
(50 mg) was reacted with 4 mg of MMC using 50 mg of EDC to obtain a complex (46 mg) with an MMC content of 7.4% (wt %).

Example 23 CM-4 (degree of substitution: 0.53,
1.25 g) was dissolved in water (300 ml). This solution was diluted with 3.32 g of sodium periodate (3 molar equivalents per mole of sugar residue).
The solution was mixed with water (200 ml).
After reacting for 1 day, 1 g of ethylene glycol was added and reacted for 4 hours. After dialysis against water and concentrating the internal solution,
An ethanol-acetone mixture (approximately 1:1) was added, and sodium acetate-saturated methanol (15 ml) was added dropwise with stirring. The precipitate was collected to obtain 1.11 g of aldehyde-carboxymethyl mannoglycan. 800 mg of this was dissolved in 0.1 M borate buffer (pH = 8.0, 250 ml), mixed with 160 ml of an ethanol solution containing 130 mg of daunorubicin hydrochloride, and incubated at room temperature for 16 hours.
After adding 3M sodium chloride solution (8 ml), the mixture was filtered and mixed with ethanol to collect the precipitate.
677 mg of a carboxymethyl mannoglucan-daunorubicin complex was obtained. This complex was water-soluble and had a daunorubicin content of 10% (wt%). The ultraviolet-visible absorption spectrum of this complex (concentration: 330 μg/ml, solvent:
Water) as shown in FIG.

Example 24: CM-6 (800 mg) obtained in Example 7 was dissolved in water (200 ml). This solution was mixed with an aqueous solution of 2.12 g of sodium periodate (3 molar equivalents per mole of sugar residue) dissolved in water (30 ml). After reacting at room temperature for one day, 620 mg of ethylene glycol was added and reacted for four hours. After dialysis against water and concentrating the internal solution, an ethanol-acetone mixture (approximately 1:1) was added, and sodium acetate saturated methanol (10 ml) was added.
The precipitate was collected to give 643 mg of aldehyde-carboxymethyl mannoglucan. 600 mg of this precipitate was added to 175 ml of 0.1 M borate buffer (pH 8.0).
The resulting solution was mixed with 110 ml of an ethanol solution containing 150 mg of daunorubicin hydrochloride and stirred at room temperature for 16 hours. After adding 4.5 ml of 3 M sodium chloride solution, the mixture was filtered and mixed with ethanol to form a precipitate, which was collected to give 610 mg of a carboxymethyl mannoglucan-daunorubicin complex.
The complex is water-soluble and contains 13% daunorubicin.
The ultraviolet-visible spectrum of this complex (concentration: 200 μ/ml, solvent: water) is shown in FIG.

Example 25 MG10-CM-6 (700 mg) obtained in Example 12 was dissolved in water (175 ml)
This solution was dissolved in 1.8 ml of sodium periodate.
6 g (3 molar equivalents per mole of sugar residue) was mixed with an aqueous solution of 56 g ...
0 mg of ethylene glycol was added and the mixture was allowed to react for 4 hours.
After dialysis against water and concentrating the internal solution, an ethanol-acetone mixture (approximately 1:1) was added, and sodium acetate saturated methanol (8 ml) was added dropwise with stirring. The precipitate was collected and purified to obtain aldehyde-carboxymethyl mannoglucan.
571 mg of this product was dissolved in 0.1 M borate buffer (pH = 8.
The solution was dissolved in 180 ml of daunorubicin hydrochloride (143 mg) and mixed with 112 ml of ethanol solution containing 143 mg of daunorubicin hydrochloride, and stirred at room temperature for 16 hours. After adding 3 ml of 3 M sodium chloride solution, the mixture was filtered.
The precipitate formed after mixing with ethanol was collected to obtain 610 mg of a carboxymethyl mannoglucan-daunorubicin complex. This complex was water-soluble, with a daunorubicin content of 12% (by weight). The UV-visible spectrum of this complex (concentration: 200 μg/ml, solvent: water) is shown in Figure 10.

Example 26 Platinum complex cis-dinitrate diammineplatinum(II) was prepared by known methods (e.g., Inorg. Chem., Vol. 16, p. 1525 (1977),
It was synthesized by B. Lippert et al.

MG-PA-CM (230 mg) obtained in Example 14 was dissolved in water (7 ml), and cis-dinitratodiammineplatinum(II) was added to the solution.
A solution of 24.71 mg of platinum complex (24.71 mg) in 7 ml of water was added, and the mixture was stirred for 24 hours at room temperature in the dark. After confirming by gel filtration chromatography that no unreacted starting platinum complex remained, the reaction mixture was dialyzed against water. The internal solution was dialyzed against 1N Na
After adjusting the pH to 6.5 with OH, the mixture was concentrated to about 10 ml, and then ethanol (80 ml) was added. The precipitate was collected and dried in vacuo to give cis-diammineplatinum(II) complex (218 mg, platinum content (by atomic absorption spectrometry): 5.90%).
The gel filtration elution pattern of this complex (detection: 280
The ultraviolet absorption at 100 nm is shown in FIG.

Example 27: Using CM-6 (14.6 mg) obtained in Example 7 and cis-dinitratediammineplatinum(II) (2.12 mg), a cis-diammineplatinum(II) complex (12.6 mg, platinum content: 7.04%) was obtained in the same manner as in Example 26.

Example 28: Using CM-7 (211 mg) obtained in Example 8 and cis-dinitratediammineplatinum(II) (24.7 mg), a cis-diammineplatinum(II) complex (205 mg, platinum content: 6.60%) was obtained in the same manner as in Example 6.

Continued from the front page (72) Inventor: Toshiro Yano 3-1-8-410 Akebono, Kashiwa City, Chiba Prefecture

Claims (15)

[Claims] [Claim 1] Carboxymethyl mannoglucan and its salts, which are composed of tetrasaccharide units represented by the following general formula (I): (Wherein, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 , ^R11 and R
¹² may be the same or different and each represents a hydrogen atom or _CH2COOH . 2. The carboxymethyl mannoglycan according to claim 1, having a molecular weight of 10,000 to 2,000,000. 3. The method according to claim 1, wherein the degree of substitution, defined as the number of carboxymethyl groups per sugar residue, is 0.01 to 3.0.
Or carboxymethyl mannoglycan according to 2. 4. A method for producing carboxymethyl mannoglucan according to any one of claims 1 to 3, which comprises reacting a mannoglucan composed of tetrasaccharide units represented by the following formula (II) with a halogenated acetic acid. 5. A carboxymethyl mannoglucan derivative and its salt, which is composed of a tetrasaccharide unit represented by the following general formula (III): (In the formula, R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² ,
^R23 and ^R24 are each a hydrogen atom, _CH2COOH , _CH2CONR ^*1 R ^*2
(where NR ^*1 R ^*2 represents a residue obtained by removing one hydrogen atom from the amino group of a pharmaceutical compound having an amino group represented by the general formula HNR ^*1 R ^*2 ), _CH2COOR ^*3 (where OR ^*3 represents a residue obtained by removing a hydrogen atom from the alcoholic hydroxyl group of a pharmaceutical compound having an alcoholic hydroxyl group represented by the general formula HOR ^*3 ), or _CH2COO.1 /2[Pt
(NH ₃ ) ₂ )] (wherein Pt represents divalent platinum), provided that at least one of R ¹³ to ^{R 24} in the molecule is CH ₂ CONR
^*1 R ^*2 CH ₂ COOR ^*3 or CH ₂ COO·1/2 [Pt(NH ₃ )
₂ ) 6. The carboxymethyl mannoglycan derivative and its salt according to claim 5, having a molecular weight of 10,000 to 2,000,000. 7. The degree of substitution, defined as the number of carboxymethyl groups per sugar residue, of claim 5 is 0.01 to 3.0.
6. A carboxymethyl mannoglycan derivative and a salt thereof according to claim 6. 8. The compound according to claim 1 or a salt thereof,
_A method for producing the carboxymethyl mannoglycan derivative or a salt thereof according to any one of claims 5 to 7, which comprises reacting ^{*1R *2} , HOR ^*3 or Pt(NH3) ₂ ( _NO3 ) ₂ . 9. An oxidized carboxymethyl mannoglucan, a derivative thereof, and a salt thereof, which is composed of a unit represented by the following general formula (IV) and/or a unit represented by the following general formula (V): [Wherein, ^R25 , ^R26 , ^R27 , ^R28 , ^R29 and ^R30 may be the same or different and each represent a hydrogen atom or _CH2COOH , ^W1 and ^W2 each represent =0 or =N-R ^*4 (wherein R represents a residue obtained by removing two hydrogen atoms from the amino group of a pharmaceutical compound having an amino group represented by the general formula _H2N -R ^*4 ), ^A1 and ^A2 may be the same or different and each represent the following formula (VI), formula (VII), formula (VIII) or formula (IX), ^A3 and ^A4 may be the same or different and each represent the following formula (VI), formula (VII), formula (VIII) or formula (IX), provided that when the molecule consists only of the general formula (IV), not all of ^A1 and ^A2 in the molecule represent formula (VI). (where X ⁱ¹ , X ⁱ² , X ⁱ³ , X ⁱ⁴ , X ⁱ⁵ , X ⁱ⁶ , X ⁱ⁷ , X ⁱ⁸ and X ⁱ⁹ are
may be the same or different, and each may be a hydrogen atom or a CH
₂ COOH, and ^Wi1 , ^Wi2 , ^Wi3 , ^Wi4 , ^Wi5 and ^Wi6 may be the same or different and each represent =0 or =N-R ^*4 (where N-
R ^*4 represents a residue obtained by removing two hydrogen atoms from the amino group of a pharmaceutical compound having an amino group represented by the general formula _H2N -R ^*4 ), provided that the formula (VI), the formula (VII), the formula (VIII) and the formula (I
X), the subscript i of X ⁱ¹ to X ⁱ⁹ and W ⁱ¹ to W ⁱ⁶
represents an integer of 1 to 4, and each of A ¹ , A ² , A ³ and A ⁴ will generally be referred to as ^Ai .) 10. The molecular weight of claim 9 is 10,000 to 2,000,000.
The oxidized carboxymethyl mannoglycan and its derivatives and salts thereof described above. 11. The oxidized carboxymethyl mannoglycan and its derivatives and salts thereof according to claim 9 or 10, wherein the degree of substitution, defined as the number of carboxymethyl groups per sugar residue, is 0.01 to 3.0. [Claim 12] A method for producing oxidized carboxymethyl mannoglucan and its derivatives and salts thereof according to any one of claims 9 to 11, which comprises reacting a mannoglucan composed of tetrasaccharide units represented by formula (II) defined in claim 4 with a halogenated acetic acid, and subsequently reacting it with periodic acid or a salt thereof. 13. A carboxymethyl ring-opened mannoglycan, a derivative thereof, and a salt thereof, which is composed of a unit represented by the following general formula (X) and/or a unit represented by the following general formula (XI): [wherein R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ and R ³⁸ may be the same or different and each represents a hydrogen atom, CH ₂ COOH, C
H ₂ CONR ^*1 R ^*2 , CH ₂ COOR ^*3 (where NR ^*1 R ^*2
and OR ^*3 has the same meaning as defined in claim 5) or
_CH2COO.1 /2[Pt( _NH3 ) ₂ ] (wherein Pt represents divalent platinum), ^B1 and ^B2 may be the same or different and represent the following formula (XII), (XIII), (XIV) or (XV), respectively, ^B3 and ^B4 may be the same or different and represent the following formula (XIII), (XIV) or (XV), respectively, provided that when a molecule consists only of the general formula (X), not all of ^B1 and ^B2 in the molecule represent formula (XII). (Here, Y ^j1 , Y ^j2 , Y ^j3 , Y ^j4 , Y ^j5 , Y ^j6 , Y ^j7 , Y ^j8 , Y ^j9 ,
Y ^j10 , Y ^j11 , Y ^j12 , Y ^j13 , Y ^j14 and Y ^j15 may be the same or different and each represents a hydrogen atom, CH ₂ COOH, CH ₂ CO
NR ^{*1R *2} or _CH2COOR ^*3 (wherein NR ^{*1R *2} and OR ^*3 have the same meanings as defined in claim 5), or
CH ₂ COO·½[Pt(NH ₃ ) ₂ ] (wherein Pt represents divalent platinum), provided that the formula (XII), the formula (XIII), the formula (XIV) and the formula (X
In each of the formulas V), the subscript j of Y ^j1 to Y ^j15 represents an integer of 1 to 4, and each of B ¹ , B ² , B ³ and B ⁴ will generally be referred to as B ^j ). Claim 14: The molecular weight of claim 13 is 10,000 to 2,000,000.
The carboxymethyl ring-opened mannoglycan and its derivatives and salts thereof described above. [Claim 15] A method for producing carboxymethyl ring-opened mannoglycan and its derivatives and salts thereof according to claim 13 or 14, which comprises reacting a mannoglycan composed of tetrasaccharide units represented by formula (II) defined in claim 4 with periodic acid or a salt thereof, subsequently reacting it with sodium borohydride, and further reacting it with a halogenated acetic acid.