WO2004060404A1

WO2004060404A1 - Drug carrier

Info

Publication number: WO2004060404A1
Application number: PCT/JP2004/000004
Authority: WO
Inventors: Tsuyoshi Shimoboji; Teruo Nakamura; Hajime Miyamoto; Rie Shiokawa
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2002-12-27
Filing date: 2004-01-05
Publication date: 2004-07-22

Abstract

A method of regulating the rate of extinction of a hyaluronic acid (HA) derivative, comprising changing the percentage of substituent introduction into the carboxylic acid of glucuronic acid of the HA derivative, or comprising changing the molecular weight of the HA derivative. Further, there is provided a drug conjugate wherein a hyaluronic acid derivative having its extinction rate regulated is used as a carrier so as to prolong or control the half-life period of drug in blood.

Description

Specification

Drug carrier Technical field

The present application relates to a hyaluronic acid (HA) derivative drug carrier that can extend and control the blood half-life of a drug and maintain sufficient drug efficacy, conjugation with a drug using the same, and in vivo The present invention relates to a kinetic control technique for the drug. Background art

In general, conjugates of proteins and water-soluble polymers have been attempted to improve the protein retention and stability in blood. In particular, polyethylene glycol (PEG) is widely used due to its inert property and its effect of preventing protein adsorption, and is in the stage of practical application as a pharmaceutical. However, since PEG is not a biodegradable polymer, there are concerns about problems such as excretion by renal excretion and safety when accumulated.

Hyaluronic acid (HA) is a biomaterial (polysaccharide) isolated from the vitreous of bovine eyes in 1934 by K. Meyer, and has long been known as a major component of fine matrices. HA is a type of darcosamide glycan composed of a disaccharide unit in which D-glucuronic acid and N-acetyltyl glucosamine are linked by a / 3 (1 → 3) glycosidic bond (formula (II)).

HA has no difference in chemical and physical structure, and humans also have a metabolic system. It is the safest biomaterial (Biomateria 1) in terms of immunity and toxicity. In recent years, aspects of bioactive substances such as induction of cell adhesion, proliferation and migration have been reported and attracted attention, and large-scale production of high molecular weight HA by microorganisms has become possible. It has also been put to practical use in the fields of cosmetics and the like.

In the meantime, by conjugating the drug with HA, the drug is targeted to cancer tissue (see International Publication No. WO 92/06714) and liver (see Japanese Patent Application Laid-Open No. 2001-81103). (See Japanese Patent Application Laid-Open No. 2-273176) and prolonged blood retention (see Japanese Patent Application Laid-Open No. 5-85942, International Publication No. 01Z05434, and International Publication No. 01/60412).

HA as a drug conjugate carrier is safer and can be made larger in size than PEG, which has been widely studied so far. Alternatively, different types of drugs can be carried in one molecule. Therefore, compared to PEG, HA has much greater potential as a drug conjugate carrier for designing and developing conjugates with more advanced pharmacokinetic control functions such as targeting and sustained release. It is thought that. HA is one of the most excellent carriers in terms of safety because it is biodegradable and there is no species difference in its chemical structure.

On the other hand, HA has a rapid elimination rate in blood, and its half-life is reported to be 2 minutes when administered intravenously (IV) (J. Intel. Med. Vol. 242, No. 27- 33, 1997). The present inventor has also confirmed that simply conjugating HA to a protein does not extend blood retention and does not lead to improvement in sustained drug efficacy. However, the main factors controlling the pharmacokinetics of HA, which are important in using HA as a drug carrier, are unknown, and increase the kinetics of the HA derivative itself and its retention in blood. There are no reports on the prerequisites required for derivatives to be provided. As described above, the main factors for controlling the pharmacokinetics of HA, which are important for using HA as a drug carrier, are unknown. In addition, there is no report on the kinetics of the modified HA itself or any modification method for increasing the retention in the blood, and there is no known method for avoiding the internal metabolic system of HA and controlling the retention in the blood. Absent.

The inventor of the present invention has made intensive studies to solve the powerful problem, and by changing the rate of introduction of a substituent into the carboxylic acid of the glucuronic acid moiety in hyaluronic acid (HA), It has been found that the rate of HA disappearance can be adjusted. Furthermore, it has been found that the disappearance rate of HA can be adjusted by changing the molecular weight of HA. That is, the present invention relates to a method for controlling the disappearance rate of HA, and a method for producing a conjugate with a substance having a pharmacological action using the HA.

That is, according to one aspect of the present invention, there is provided a method for adjusting the rate of disappearance of hyaluronic acid by changing the rate of introduction of a substituent into the carboxylic acid of the dalcuvic acid moiety in the hyaluronic acid derivative. You. Here, the change in the introduction rate of the substituent may be either increase or decrease, and the disappearance rate may be either decrease or increase. However, the increase in the introduction rate of the substituent may reduce the disappearance rate. It is preferable to reduce the speed.

The hyaluronic acid derivative is not particularly limited as long as a substituent of the carboxylic acid of the glucuronic acid moiety is introduced, but is preferably represented by the formula (I):

(Where X is the number of one-one, one N (——, one S—, -N (-R ^ N (-R ₂ ) —, -N (-R _x ) N (-R ₂ ) CO — Or a single bond,

X represents a linear or branched - alkyl group, straight or branched C - alkoxy group, a linear or branched C ₂ _ ₁₂ alkenyl, C ₆ _ ₁₈ Ariru group, C ₃ - ₈ cycloalkyl group, or a heterocyclic Each substituent has at least one protecting group-introduced or free hydroxy group, amino group, hydrazide group, mercapto group, maleimide group, olepoxyl group, aldehyde group, or sulfonic acid group. And also includes a polyaddition product or a polycondensate of a substituent having the above functional group,

And R ₂ are each independently a hydrogen atom, a linear or branched alkyl group, a linear or branched Ci-hydroxyalkyl group, an alkylene oxide group or a polyalkylene oxide group. )

Is a hyaluronic acid derivative having one or more repeating structures in the molecule.

Here, in the formula (I), preferably X ₀ is _N (-Ri) N (_R ₂ ) — or _N (-Ri) N (_R ₂ ) CO—, and preferably X ₂ is an amino A linear or branched alkyl group substituted with a hydrazide group or a hydrazide group, and preferably and R ₂ is a hydrogen atom.

Further, the hyaluronic acid derivative may form a conjugate with a substance having a pharmacological action. The rate of disappearance is not particularly limited, but is preferably a rate of disappearance in blood.

According to another aspect of the present invention, there is provided a conjugate wherein a substance having a pharmacological action is bound to a hyaluronic acid derivative whose disappearance rate has been adjusted or reduced by the above method.

Here, the substance having a pharmacological action is not particularly limited, but is preferably a protein or a peptide.

According to yet another aspect of the present invention, formula (II):

(Where X is _〇_, — N (-R _x ) —, one S—, one N (_R N (—R ₂ ) —, — N (-Ri) N (-R ₂ ) CO — Or a single bond,

X ₂ is linear or branched. ₁₂ alkyl group, straight or branched (^ - ₁₂ alkoxy group, linear or branched C ₂ _ ₁₂ alkenyl group, ₍₆ _ ₁₈ Ariru group, C ₃ - is ₈ cycloalkyl group or a heterocyclic group, Each substituent is a protecting group-introduced or free hydroxyl group, amino group, hydrazide group, mercapto group, maleimide group, hepoxyl group

, An aldehyde group or a sulfonic acid group, and also includes a polyadduct or a polycondensate of a substituent having the above functional group,

And R ₂ are each independently a hydrogen atom, a straight-chain or branched C-alkyl group, a straight-chain or branched- ^ hydroxyalkyl group, an alkylene oxide group or a polyalkylene oxide group. )

The use of the above-mentioned hyaluronic acid derivative in the preparation of a conjugate in which a substance having a pharmacological action is bound to a hyaluronic acid derivative having one or more repeating structures represented by the following formulas in the molecule is provided.

Here, in the formula (II), X ₀ is preferably —N (-R _x ) N (—R ₂ ) — or one N (~ R _X ) N (—R ₂ ) CO—, preferably X ₂ is a straight-chain or branched alkylene group substituted with an amino group or a hydrazide group, and preferably and R ₂ is a hydrogen atom.

According to yet another aspect of the present invention, the following steps: (a) binding a substance having a pharmacological action to two or more hyaluonic acid derivatives having different introduction ratios of the substituents to the carboxylic acid in the darcnic acid moiety,

(b) measuring the disappearance rate of the conjugate prepared in (a),

(c) calculating the introduction rate of the substituent corresponding to the target disappearance rate based on the measurement result,

(d) A method for preparing a conjugate of a hyaluronic acid derivative and a substance having a pharmacological action, comprising: a step of preparing a hyaluronic acid derivative having an introduction rate calculated in (c).

In addition, the method for preparing the conjugate comprises the following steps:

(a) measuring the rate of disappearance of two or more hydranoic acid derivatives having different rates of introduction of substituents into the carboxylic acid of the glucuronic acid moiety,

(b) calculating the introduction ratio of the substituent corresponding to the target disappearance rate based on the measurement result,

(c) a step of binding a substance having a pharmacological action to the hyaluronic acid derivative having the introduction rate calculated in (b),

May be included.

Here, the elimination rate is not particularly limited, but is preferably a blood elimination rate. According to yet another aspect of the invention, the following steps are provided:

(a) Different rates of introduction of substituents to carboxylic acid in Daryrek's carboxylic acid moiety Different in introduction rates of substituents to carboxylic acid in two or more hyal sulfonic acid derivatives or darcylic acid moiety Measuring the disappearance rate of a conjugate in which a substance having a pharmacological action is bound to two or more hyaluronic acid derivatives,

(b) obtaining a correlation between the rate of introduction of the substituent and the rate of disappearance based on the result measured in (a),

(c) Based on the correlation obtained in (b), estimating the disappearance rate from the substituent introduction rate,

The present invention provides a method for predicting the disappearance rate of a hyaluronic acid derivative containing:

Again, the rate of disappearance is not particularly limited, but is preferably the rate of disappearance in blood. According to still another aspect of the present invention, the adjustment of the molecular weight of the hyaluronic acid derivative and the adjustment of the rate of introduction of a substituent into the carboxylic acid of the glucuronic acid portion of the hyaluronic acid derivative are combined to provide the hyaluronic acid derivative. A method is provided for adjusting the rate of disappearance. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the time-dependent change in the concentration of the HA derivative (a0-a3) in a plasma sample collected from a rat to which the HA derivative (a0-a3) was administered.

FIG. 2 is a graph showing a time-dependent change in the concentration of the HA derivative (b 0 -b 3) in a plasma sample collected from a rat to which the HA derivative was administered.

FIG. 3 is a graph showing a time-dependent change in the concentration of the HA derivative in a plasma sample collected from a rat to which the HA derivative (c 0 -c 3) was administered. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically.

The factors considered by the present inventors to be important for controlling the half-life of hyaluronic acid (HA) in blood are two factors, the carboxylic acid modification rate and the HA molecular weight. By synthesizing an HA derivative with these different elements and conjugating the protein to the HA derivative, the usefulness of HA as a protein conjugate carrier and the parameters for optimizing its effects are clarified. I made it.

The main sites of metabolism of HA are the liver and lymph glands, and its metabolism is mainly mediated through receptor membrane-mediated receptors such as CD44, RHAMM, and HARE (Receptor Mediated) cells. It is due to internal uptake and subsequent degradation by hyaluronidase. Both of these molecules have been reported to use the continuous free carboxylic acid (hexasaccharide) of HA as the main recognition site (Exp. Cell Res., Vol. 228, No. 16). — See page 228, 1996.) The present inventors have paid attention here, and have found that by modifying a carboxylic acid to Δt so as to be hardly recognized by metabolic molecules, the blood retention can be controlled and prolonged. The modification rate of the carboxylic acid is determined by proton NMR.

The molecular weight of the HA derivative of the present invention is also an important factor for its pharmacokinetics. The blood retention of the HA derivative of the present invention also depends on the molecular weight of HA, and the higher the molecular weight of HA, the longer its half-life in blood. Therefore, the half-life in blood of the HA derivative can be controlled by changing the molecular weight of HA and the modification rate of the carboxylic acid of HA. As a method of measuring the weight average molecular weight of hyaluronic acid, various known methods such as a light scattering method and a viscosity method can be used.

In the present invention, the adjustment of the disappearance rate of HA refers to changing the disappearance rate of HA by increasing or decreasing the disappearance rate of HA, and is usually changed so as to have a target disappearance rate. In general, when the rate of introduction of the substituent is increased, the disappearance rate of HA decreases, and conversely, when the rate of introduction of the substituent is decreased, the disappearance rate of HA increases. In addition, the change in the introduction ratio of the substituent is usually changed in the range of 0.001% to 99.999%, preferably in the range of 1% to 99%, More preferably, it is varied between 10% and 90%.

In the present invention, the substituent to be introduced into the carboxylic acid of dalc carboxylic acid in HA is not particularly limited, and any substituent that can be introduced can be used. However, when the introduced functional group is hydrophobic, the solubility of the HA derivative is reduced, so that a hydrophilic functional group is preferable.

The method for introducing the substituent is not particularly limited as long as a person skilled in the art can usually carry out the method.For example, a propyloxyl group is converted into an amide group, an ester group, or a hydrazide group, and the amide group and the hydrazide group are converted. It can be introduced as a substituent on a nitrogen atom or as a substituent on an oxygen atom of an ester group. Specific examples of the substituent include a linear or branched alkyl group, an alkoxy group, an alkenyl group, an aryl group, an alkylene oxide group, a cycloalkyl group, and a heterocyclic group. , For example, one or more selected from functional substituents such as a hydroxyl group, an amino group, a hydrazide group, a mercapto group, a hydroxyl group, and an aldehyde group. May have a substituent. Further, the functional substituent may have a protecting group in some cases.

Here, the alkyl group has 1 to 12 carbon atoms, and preferably has 1 to 12 carbon atoms. ~ 6. The alkoxy group has 1 to 12 carbon atoms, and preferably has 1 to 6 carbon atoms. The alkenyl group has 2 to 12 carbon atoms, and preferably has 2 to 6 carbon atoms. The aryl group has 6 to 18 carbon atoms, and preferably has 6 to 12 carbon atoms. The alkylene oxide group is a group represented by — (CH (—R) CH (—R ′) 〇) _n- H ( _where R and R ′ are a hydrogen atom or —5 alkyl group). It is preferably a polyethylene oxide group or a polypropylene oxide group, and preferably n is an integer of 1 to 20. The cycloalkyl group has 3 to 8 carbon atoms, and preferably has 3 to 6 carbon atoms. The heterocyclic group is, for example, a 5- to 6-membered heterocyclic group having one or more hetero atoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and is unsaturated, saturated, or partially unsaturated. It may be saturated.

The polyaddition product or polycondensate of the substituent having a functional group is not particularly limited, and examples thereof include poly (oligo) C ₆ alkylene glycol, poly (oligo) amino acid, and a combination of diamine and dicarponic acid. .

Regarding the charge of the HA derivative into which the substituent has been introduced, if the introduced substituent is cationic, the total charge shifts to the positive side, leading to a reduction in the retention in blood. In order to reduce the disappearance rate of the compound, the modification charge is preferably nonionic or anionic.

In the present invention, the HA derivative disappearance rate is a rate at which HA is eliminated at invivo or invitro. Since the adjustment method of the present invention is particularly effective in invivo, it is preferable to measure the disappearance rate at invivo, in particular, blood retention (blood disappearance rate) as an index.

The measurement of the disappearance rate is not particularly limited, and can be measured by a method known to those skilled in the art. For example, when measuring the disappearance rate of HA using the retention of HA in the blood as an index, the change over time in the blood concentration is measured, and the kinetic analysis is performed to quantify the clearance from the blood. This is defined as the disappearance rate. When the elimination rate is measured in vitro, for example, hyaluronidase is added, and the hyaluronan derivative is applied over time by gel permeation chromatography (ge 1 permeation on chroma to og raphy, GPC). May be measured.

HA used in the present invention is not particularly limited as long as it has an HA skeleton. For example, modified HA in which a part of HA is modified, or a salt of HA and modified HA (sodium salt, potassium salt, magnesium salt, calcium salt) Salt, aluminum salt, etc.). The HA used in the present invention may be HA obtained by any method, such as HA extracted from animal fiber, HA obtained by fermentation, or HA obtained by chemical synthesis. Not limited.

The substance having a pharmacological action is not particularly limited, and low molecular compounds, proteins, peptides, and the like can be used.

Examples of low molecular compounds include, for example, anti-cancer agents (eg, alkylating agents, chemotherapy drugs, alkaloids, etc.), immunosuppressants, anti-inflammatory drugs (steroid drugs, non-steroidal anti-inflammatory drugs, etc.), anti-rheumatic drugs Agents, anti-J (J-lactam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfa drugs, etc.).

Examples of proteins and peptides include, for example, erythropoietin (EP ニュ), granule oral site colony stimulating factor (G-CSF), interferon-one, β ゝ r, (INF-hi, β, r), thrombopoietin (TPO), Serial Neutrokinetic Factor (CNTF), Tumeric Necrosis Factor-1 (TNF), Tumeric Necrosis Factor-1 Binding Protein (TNFbp), Interleukin-10 (IL-10), Similar to FMS Tyrosine kinase (F1t-3), Growth hormone (GH), Insulin, Insulin-like growth factor_1 (IGF-1), Platelet-derived growth factor (PDGF), Interleukin-11 receptor antagonist (IL — 1 ra), Brain-derived neurotrophic factor (BDNF), keratinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte Growth differentiation factor (MGDF), osteoprotegerin (OPG), lebutin, parathyroid Examples include glandular hormone (PTH), basic fibroblast growth factor (b-FGF), bone morphogenetic protein (BMP), glucagon-like peptide-11 (GLP-1), antibodies, and Diabody. The proteins and peptides referred to herein are, respectively, one or more substitutions, deletions, or modifications of one or more amino acids or covalently modified modifications of the constituent amino acids. including.

As a method for preparing the HA derivative, a known carboxylic acid modification method can be used. For example, the carboxylic acid of HA is condensed with ethylenediamine (EDA) or adipic dihydrazide (ADH) with 1-ethyl-3- (3-dimethylaminopropyl) carposimide (EDC) to have an amino group or hydrazide group. Synthesize HA (HA-AM, HA-HZ). At this time, AM and HZ are preferably treated with, for example, succinic anhydride, etc., and converted to carboxylic acid to make the total charge anion. When preparing a conjugate of the HA derivative of the present invention and a substance having a pharmacological action, the order of conjugate of the substance having a pharmacological action with regulation of the rate of HA elimination is not limited. For example, a substance having a pharmacological action may be conjugated to the HA derivative whose disappearance rate has been adjusted, or the rate of HA disappearance may be adjusted after conjugating the pharmacological substance.

Furthermore, the method for preparing the conjugate comprising the HA derivative with a controlled blood elimination rate obtained in this way and a protein having a medicinal effect is based on the method used for conjugates of known polymers and proteins. Can be used. For example, the above-mentioned amino- or hydrazide-modified HA (HA-AM, HA-AZ) is synthesized, and a part of this is synthesized with N-succinimidyl 3- [2-pyridyldithio] propionate (N-Succinimidyl 3- [ 2-pyr i dy 1 dithio] roionate, SPDP) to introduce a mercapto group and prepare HA-SA. At this time, surplus AM and HZ are treated with, for example, anhydrous succinic acid, etc., and — converted into a group having a carboxyl group such as NHCOCH ₂ CH ₂ C〇OH or NHNHCOC H ₂ CH ₂ COOH, and the total charge It is more preferable to use anion. On the other hand, maleimide group, pinyl sulfo Introduce a functional group that specifically reacts with thiols such as thiol groups For example, a conjugate may be prepared by introducing a maleimide group into an amino group of a protein with maleimidobutylyloxysulfosuccin ether, and reacting this with HA-SH.

In this conjugate, in order to keep the biological activity of the conjugate effective, the length of the spacer between the protein and the backbone of the HA derivative may be adjusted or a site-specific conjugate may be used. it can.

As a specific method for adjusting the disappearance rate of HA, there is a method of measuring the disappearance time of a plurality of HAs having different substituent introduction rates, and selecting an HA having a desired disappearance time from the measured values. The disappearance times of multiple HAs with different ratios were measured, and the results were used to derive a correlation between the substituent introduction rate and the disappearance time of the HA, and based on the correlation, the substituents that resulted in the desired disappearance time And a method of calculating the introduction rate of the compound.

The correlation between the rate of introduction of the substituent and the rate of disappearance can be determined using a known method such as the least square method. The least squares method is a method of determining a parameter of a model such that a sum of squares of a difference between a measurement result and a value obtained from a model function is minimized. The least squares solution can be obtained by solving a system of linear equations where the model function is linear with respect to the parameters. If the model function is nonlinear linear with respect to the parameters, it is necessary to determine the parameters by iterative improvement, and it is possible to obtain a least squares solution by the steepest descent method, Marquardt method, Gauss-Newton method, etc. Become. The production of the HA derivative in which the substituent is introduced at a desired ratio can be carried out by a method known to those skilled in the art. For example, when ethylenediamine (EDA) or adipic acid dihydrazide (ADH) is condensed with HA carboxylic acid and modified with EDC, the amount of EDC added to HA and the amount of substituent introduced based on the concentration of hyaluronic acid in the reaction solution Can be adjusted.

Further, since the molecular weight of HA also affects the rate of disappearance of HA, the rate of disappearance may be adjusted by changing the introduction ratio of the substituent and the molecular weight of HA. Example Hereinafter, preferred embodiments of the present invention will be described in more detail, but the present invention is not limited to these embodiments.

The NMR measurement was performed using a nuclear magnetic resonance apparatus JNM-ECA500 (manufactured by JEOL Ltd.) using heavy water as a solvent. The rate of introduction of the substituent was determined from the integral ratio of the peak specific to the introduced substituent and the peak derived from hyaluronic acid.

Gel permeation mouth chromatography (GPC) analysis was performed using the HP LC system (Waters ™ 600S Controller, Waters ™ 616 Pump, Waters ™ 717puls Autos ampler, Waters ™ 474 Scanning Fluorescence Detector Aliance) under the following measurement conditions. .

GPC column: TSKg e 1 G6000 PW _XL

Mobile phase: phosphate buffered saline (PBS, pH 7.4)

Elution method: Isocratic

Flow rate: 0.5mL / min

Sample volume used: 40 L

Detection wavelength: fluorescence (excitation 490 nm, fluorescence 5, 18 nm)

Analysis software: M i 11 e n i um 32 V e r. 3.21

Further, the term “unit” means a repetitive structure containing a disaccharide of D-dalcuronic acid and N-acetyldarcosamine as a skeleton contained in a hyaluronic acid (HA) derivative.

Example 1 Synthesis of Hyaluronic Acid Derivative

[Example 1-1-1] Synthesis of hyaluronic acid—HZ (MW 25KDa)

The synthesis of hyaluronic acid derivatives (HA-HZ) into which hydrazide groups (HZ) with different modification rates were introduced was performed by the following procedure.

Dissolve hyaluronic acid (HA) having a molecular weight of 2.5 × 10 ⁴ daltons (manufactured by Denki Kagaku Kogyo Co., Ltd.) at a concentration of 1.0% in distilled water, and adjust the pH to 4.7 to 4.8 with 5N: ^. Was. In the three lots, 1-ethyl-3- (3-dimethylaminopropyl) power Ruposimide (EDC) and adipic dihydrazide (ADH) were converted into HA: EDC: ADH = 1: 0.1: 40 (a0), 1 : 1:40 (a 2) and 1: 5: 40

(a 3) Add each to a molar ratio and adjust the pH to 4.7-4.8 with 5N hydrochloric acid. The reaction was carried out at room temperature for 2 hours while maintaining. Dialysis against large excess of 10 OmM sodium chloride solution, 25% ethanol solution and distilled water in order (Spectrapore 7, molecular weight cutoff)

(MWCO): 12k-14k tareton), and dried to form hyaluronic acid (HA-HZ) into which the title hydrazide group (HZ) was introduced.

When the HZ introduction rate in the obtained HA-HZ was determined by the proton NMR method as the ADH introduction rate, 3% (aO), 42% (a2), and 59% of the carboxylic acid of HA were obtained.

(a 3) was converted to HZ (comparison of N-acetyl group from HA (2.1 ppm) and methylene group X4 from adipic hydrazide (1.7 ppm, 2.4 ppm) [ Example 1-1-1 2] Synthesis of hyaluronic acid-HZ (MW19 OKDa) Except that HA (manufactured by Denki Kagaku Kogyo Co., Ltd.) having a molecular weight of 1.9 × 10 ⁵ daltons was dissolved in distilled water at a concentration of 0.5%. An HA derivative into which the title HZ was introduced (HA-HZ) was obtained in the same manner as in Example 1-1-1 above, and the introduction rate of HZ in the obtained HA-HZ was determined by proton NMR. As a result, 6% (b0), 49% (b2), and 71% (b3) of the carboxylic acid of HA were converted to HZ.

[Example 1-1-1-3] Synthesis of hyaluronic acid mono-HZ (MW58 OKDa) In addition to dissolving HA (manufactured by Denki Kagaku Kogyo Co., Ltd.) with a molecular weight of 5.8 × 10 ⁵ reton at a concentration of 0.25% in distilled water In the same manner as in Example 1-1-1 described above, an HA derivative (HA-HZ) into which the title HZ was introduced was obtained. The HZ incorporation rate of the obtained HA-HZ was determined by proton NMR, and 8% (c 0), 56% (c 2), and 73% (c 3) of the carboxylic acid in HA were converted to HZ. It had been.

[Example 1-2] Synthesis of fluorescently labeled hyaluronic acid mono-HZ derivative

Example 11 Nine types of HA-HZ prepared in Example 11 were dissolved in distilled water, and the final concentration was adjusted to 1 mgZmL by adding an equal amount of 10 OmM sodium carbonate (pH 9.0). Fluorescein-1-isothiocyanate (Fluo rescein-4-isothiocyanate, FITC) dissolved in 1/10 volume of dimethyl sulfoxide (DMSO) of HA-HZ solution was added to FITC / HA. Unit = 3.0 (a 0, b 0, c 0), 0.5 (a 2, b 2.c 2), 0.3 (a 3, b 3, c 3) The mixture was added at a charge ratio of (mo 1 / mo 1) and reacted at room temperature for 1 hour. 25 mL of the reaction solution was poured into 10 PD-10s previously equilibrated with 5 OmM sodium carbonate (pH 9.0) to remove unreacted FITC. SA / HZ (TNBS) = 250 (a 0, b 0, c 0), 80 (a 2, b 2.c 2), 40 (a 3, b 3, c 3) (mo 1 / At a charging ratio of mo 1), succinic anhydride (SA) dissolved in 3.5 mL of DMSO was added and reacted similarly. The reaction mixture was purified by dialysis against a large excess of distilled water, and the resulting aqueous solution was freeze-dried.

Each of the obtained samples was dissolved to a predetermined concentration, and the FITC concentration was quantified from the absorbance at 494 nm of the sample solution diluted to 0.25 mg ZmL, and the concentration of each unit was calculated according to the following formula. In addition, conversion to molar fraction and calculation of the weight fraction derived from HA in the derivative were performed.

For each sample, quantification of amino groups with trinitrobenzenesulfonic acid (TNBS) was performed. The quantification was performed according to the method (TNBS method) described on page 37, “Society Publishing Center, Biochemistry Experimental Method, 12 Chemical Modification of Proteins, First Edition”, page 37. However, the TNBS solution was adjusted to 0.5 M, and the absorbance at 500 nm was measured to quantify the hydrazide group.

Unmodified (unmodified) HA unit: X nmo1 / mL

Units with succinic anhydride introduced into the remaining HZ groups: y nmo 1 ZmL

-Formula 1: (379.3 X x) + (635.57 X y) + (924.88 X (FITC cone.)) = 250 mgFormula 2: x / (y + (FITC cone.)) = (100-HZ (¾ )) HZ (%)

The results obtained are summarized in Table 1.

Table 1. F ITC-labeled HA derivatives for the calculation of pharmacokinetic parameters Residual HA-FITC HA-SUC unmodified HA introduced

HZ group (%) HZ group (%) Unit Unit Unit HA

HA-HZ NMR TNBS (mol%) (mol%) (mol%) (weight%) a0 3-1.06 1.94 97.00 99.75 b0 6-1.35 4.65 94.00 98.24 c0 8-1.48 6.52 92.00 97.15 a2 42-1.36 40.64 58.00 79.30 b2 49 1 1.50 47.50 51.00 76.57 c2 56-1.77 54.23 44.00 74.14 a3 59-1.22 57.78 41.00 72.56 b3 71-1.25 69.75 29.00 68.55 c3 73-1.09 71.91 27.00 67.80

-: Below the limit of quantification Example 2: Evaluation of blood retention

Rat plasma sample administered with HA derivative

The nine HA derivatives of Example 1 were administered once intravenously at a dose of 10 mg / kg to rats intravenously, before administration, and after administration at 0.25, 1, 2, 4, 6, 8, 10, 12 and After 24 hours, the mixture was centrifuged at 0 L (heparin treatment) to obtain plasma. This plasma sample was stored at 120 ° C until measurement.

Measuring method

The standard sample for calibration curve and the sample for measurement were analyzed by GPC.

Preparation of sample for calibration curve

Dilute each fluorescently labeled HA derivative using PBS (pH 7.4) to prepare standard solutions of 1, 5, 10, 50, 100, 500 gZmL and 0 / gZmL (control PBS (pH 7.4)). An equal volume of normal rat plasma was added to this standard solution to prepare a sample for a calibration curve.

Preparation of sample for measurement

An equal volume of PBS (pH 7.4) was added to the HA derivative-administered rat plasma sample to prepare a measurement sample.

Calculation of plasma HA derivative concentration. The peak area was calculated using analysis software Millenium. The concentration of the HA derivative in plasma was calculated from the calibration curve obtained from the peak area of each standard sample.

Changes in the blood concentration transition of the HA derivative are shown in FIGS.

The pharmacokinetics of HA derivatives in blood concentration data were determined using WinNonlin Ver 2.1 (Belgium Science) and MULTI (RUNGE) (K. Yamaoka, et al., J Pharmacobiodyn 6; 595-606, 1983). The target parameters were calculated overnight. WinNonlin performs a model-independent analysis using the data of the last three measurement points of each individual, and calculates the half-life (t 1/2), mean blood residence time (MRT), and total clearance (C 1) did. (MULT KRUNGE), as an analysis taking into account nonlinearity, based on Equation 3, for each HA derivative with the same molecular weight, the maximum elimination rate (Vmax) and the steady state distribution volume (Vds s) are common variables, and the Michaelis constant For (Km), three simultaneous differential equations were set as unique variables for each HA derivative, and Km, Vmax, and Vdss were calculated by fitting analysis.

dC / dt =-(Vmax I (Km I c (t)))% c (t) I Vdss

Pharmacokinetic parameters of F ITC labeled HA derivatives

(Calculated by inNonlin)

HZ introduction rate CI MET tl72

(%: NME) (mL / hrkg) (h) (h) a0 3 29.6 ± 1.8 1.36 Sat 0.08 0.871 ± 0.068 a2 42 22.3 ± 10 2.16 0.48 1.43 ± 0.30 a3 59 12.4 ± 0.7 3.87 Sat 0.63 2.40 ± 0.55 b0 6 16.9 ± 3.3 2.02 0.22 1.30 ± 0.20 b2 49 12.5 ± 2.7 2.74 0.27 1.41 ± 0.15 b3 71 4.58 ± 0.42 10.1 + 1.1 7.01 ± 0.78 cO 8 14.6 ± 1.7 2.19 0.16 1.42 ± 0.15 c2 56 4.97 ± 0.58 5.72 0.72 3.17 ± 0.73 c3 73 2.52 ± 0.41 16.3 + 3.7 11.2 ± 2.9 Pharmacokinetic parameters of FITC-labeled HA derivatives

(Calculated by MULTI (RUNGE))

HZ introduction rate Vmax Vdss Em

(% NME) (mgmnv (mLkg) (mg / mL)

aO 3 127.2 51.7 146.9 a2 42 127.2 51.7 290.3

a3 59 127.2 51.7 523.9

cO 8 44.9 38.0 48.6 c2 56 44.9 38.0 396.7

c3 73 44.9 38.0 '916.5

Table 2 shows the results of model-independent kinetic analysis. Looking at the total clearance (C 1) value, a 0 is 2.4 times a 3, b 0 «b 3 3.7 times, and so on. 0 is. It was 5.8 times that of 3, indicating that the higher the introduction rate of HZ, the slower the HA derivative disappears from the blood. Comparing HA derivatives with similar HZ introduction rates, the HA derivative with a higher molecular weight has a smaller C1 value, and the higher the molecular weight of HA, the greater the degree of improvement in blood retention. Indicated. The same tendency was observed for MRT and t12 shown in Table 2.

Table 3 shows the results of the kinetic analysis of the HA derivative taking into account the nonlinear region. MULTI (RUNGE) for aO-a3, cO-c3, assuming that Vds s and Vmax are equal for HA derivatives with the same molecular weight and that the affinity for the receptor involved in HA clearance changes with the HZ introduction rate. The Vds s value, Vmax, and Km were calculated by fitting analysis (Table 3). The Vmax of a0-a3 was 2.8 times the Vmax of c0_c3, indicating that the HA derivative having a smaller molecular weight disappeared from the blood earlier. For HA derivatives with the same molecular weight, the introduction rate of HZ is high, such as the Km value of a3 is 3.6 times the Km value of a0 and the Km value of c3 is 18.9 times the Km value of c0. It was shown that Km became larger. That is, it was suggested by the fitting analysis that the higher the introduction rate of HZ, the lower the affinity with the receptor and the slower the disappearance of the HA derivative from the blood. As shown above, the results of both model-independent analysis and kinetic analysis taking into account nonlinearity indicate that the half-life in blood is prolonged as the carboxylic acid modification rate of the HA derivative and the molecular weight of the HA derivative increase. Rukoto has been shown.

By using the hyaluronic acid derivative exemplified in the above example, it is possible to avoid the in vivo metabolic system of the HA derivative, which is important in using the HA derivative as a drug carrier, and to prolong the maximum blood retention. Is possible. Industrial potential

The hyaluronic acid derivative of the present invention can control the rate of metabolism of the HA derivative in the body, and can control and prolong the blood retention property. To provide a biodegradable and safe pharmacokinetic control technology that enables adjustment of the blood exposure pattern that is optimal for the subject.

Claims

The scope of the claims

1. A method of adjusting the rate of disappearance of hyaluronic acid by changing the rate of introduction of a substituent into the carboxylic acid in the darkuvic acid moiety in the hyaluronic acid derivative.

2. A method for reducing the in vivo disappearance rate of a hyaluronic acid derivative, which comprises introducing a substituent into the carboxylic acid of the glucuronic acid moiety in hyaluronic acid.

3. A method of reducing the rate of disappearance of the hyaluronic acid derivative by increasing the rate of introduction of a substituent into the carboxylic acid in the darkuvic acid moiety in the hyaluronic acid derivative.

4. The hyaluronic acid derivative has the formula (I):

(Where, X is one O—, one N (-R _x ) one, one S—, one N (-Ri) N (— R ₂ ) one, — N (— RN (— R ₂ ) CO — Or a single bond,

Represents a linear or branched alkyl group, straight or branched C _ ₁₂ alkoxy group, linear or branched C ₂ _ ₁₂ alkenyl, C ₆ _ ₁₈ Ariru group, C ₃ - ₈ cycloalkyl group or a heterocyclic Wherein each substituent has at least one protecting group-introduced or free hydroxy group, amino group, hydrazide group, mercapto group, maleimide group, propyloxyl group, aldehyde group, or sulfonic acid group. And also includes a polyaddition product or a polycondensation product of the substituent having the functional group,

And R ₂ are each independently a hydrogen atom, a straight-chain or branched C- ₁₂ alkyl group, a straight-chain or branched hydroxyalkyl group, an alkylene oxide group or Is a polyalkylene oxide group. )

The method according to claim 1, wherein the molecule has one or more repeating structures represented by the following formulas.

5. The method according to any one of claims 1 to 4, wherein the derivative of here ^! Reonic acid forms a conjugate with a substance having a pharmacological action.

6. The method according to any one of claims 1 to 5, wherein the elimination rate is a blood elimination rate.

7. A conjugate wherein a substance having a pharmacological action is bound to a hyaluronate derivative whose elimination rate has been adjusted or reduced by the method according to any one of claims 1 to 6.

8. The conjugate according to claim 7, wherein the substance having a pharmacological action is a protein or a peptide.

9. Formula (II):

(Where X. is — O—, — N (— R one, one S—, one N (-R _x ) N (— R ₂ ) one, -N (— RN (— R ₂ ) CO— Or a single bond,

X ₂ represents a linear or branched C i ₂ alkyl group, straight or branched ^ - alkoxy group, a linear or branched C ₂ - ₁₂ alkenyl group, C ₆ - ₁₈ Ariru group, C ₃ - ₈ cycloalkyl Group or heterocyclic group, and each substituent is a protecting group-introduced or free hydroxy group, amino group, hydrazide group, mercapto group, maleimide group, olepoxyl group , An aldehyde group or a sulfonic acid group, and also includes a polyadduct or a polycondensate of a substituent having the above functional group,

And R ₂ are each independently a hydrogen atom, a linear or branched Ci-alkyl group, a linear or branched trihydroxyalkyl group, an alkylene oxide group or a polyalkylene oxide group . )

Use of a hyaluronic acid derivative having one or more repeating structures represented by the following formulas in a molecule in which a substance having a pharmacological action is bonded to the hyaluronic acid derivative.

10. A method for preparing a conjugate of a hyaluronic acid derivative and a substance having a pharmacological action, comprising the following steps:

(a) binding a substance having a pharmacological action to two or more hyaluonic acid derivatives having different introduction ratios of the substituents to the carboxylic acid in the darcnic acid moiety,

(b) measuring the disappearance rate of the conjugate prepared in (a),

(d) a step of producing a hyaluronic acid derivative having the introduction ratio calculated in (c).

11. A method for preparing a conjugate of a hyaluronic acid derivative and a substance having a pharmacological action, comprising the following steps:

(a) a step of measuring the rate of disappearance of two or more hyaluronic acid derivatives having different introduction ratios of substituents to the carboxylic acid of the darcynic acid moiety;

(c) a step of binding a substance having a pharmacological action to the hyaluronic acid derivative having the introduction rate calculated in (b).

12. The method according to claim 10 or 11, wherein the elimination rate is a blood elimination rate.

13. A method for predicting the rate of disappearance of a hyaluronic acid derivative comprising the following steps:

(a) The introduction ratio of substituents to carboxylic acids in the darcylic acid moiety is different Two or more hyaluronic acid derivatives, or the introduction rates of substituents to the carboxylic acid in the darcylic acid moiety are different Measuring the disappearance rate of a conjugate in which a substance having a pharmacological action is bound to two or more hyaluronic acid derivatives.

(b) obtaining a correlation between the rate of introduction of the substituent and the rate of disappearance based on the results measured in (a),

(c) A process of estimating the elimination rate from the substituent introduction rate based on the correlation obtained in (b).

14. The method according to claim 13, wherein the elimination rate is a blood elimination rate.

1 5. A method for adjusting the disappearance rate of a hyaluronic acid derivative by combining the adjustment of the molecular weight of the hyaluronic acid derivative with the adjustment of the rate of introduction of a substituent into the carboxylic acid in the glucuronic acid portion of the hyaluronic acid derivative.