JPS6317843B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to fluorescently labeled acidic mucopolysaccharides and methods for their production, and more particularly to fluorescein amidated acidic mucopolysaccharides, their salts, and their production methods. Acidic mucopolysaccharide according to the present invention (hereinafter referred to as
AMPS) includes hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, heparin, heparan sulfate, dermatan sulfate, and salts thereof, and their chemical structures and The characteristics are shown in Table 1.

[Table] 〓No
〓

[Table] High content of acetyl and D〓glucuronic acid, N〓sulfuric acid, O〓sulfuric acid, N〓
Low content of iduronic acid.
The above AMPS exists as a component of various binding tissue substrates in vivo, and each is involved in many physiological functions. It is known that the distribution pattern of AMPS in animal tissues and body fluids changes with maturation or age. this
When the normal metabolism of AMPS is disrupted, a serious clinical disorder called mucopolysaccharidosis occurs, which is characterized by various symptoms. Heparin exists in the spleen, lungs, liver, intestinal mucosa, etc. of mammals, and has an anticoagulant effect.
It is known as an important factor with Because of its strong anticoagulant activity, heparin extracted from animal organs is widely used in clinical medicine. Furthermore, dermatan sulfate and heparan sulfate are also known to have anticoagulant effects, although they are weak. AMPS other than heparin are found in many animal tissues,
connective tissues such as skin, bones, cartilage, and ligaments;
Found in secretions. Together with collagen and elastin fibers, these form intercellular interstitium or matrix, in which connective tissue cells are embedded to construct connective tissue. Hyaluronic acid is found in the umbilical cord, skin, joint synovial fluid,
It is found in ocular vitreous fluid, etc., and has functions such as preventing tissue cell infection. It is also known that it is abundant in malignant tumors. Chondroitin-4 and 6-sulfate play a useful role in smoothing cell metabolism and maintaining tissue due to their strong water retention properties. These are the main components of cartilage, and the weakening of cartilage associated with scurvy, for example, is based on a decrease in the synthetic function of chondroitin-4 or 6-sulfate. Additionally, these are used to treat painful diseases, kidney diseases, and the like. In addition, mucopolysaccharidoses include Hurler syndrome,
Hunter syndrome, Marfan syndrome, etc. are known, and both are caused by the use of dermatan sulfate, heparan sulfate, etc.
Because it is accompanied by abnormal excretion of AMPS, it is considered a disease caused by abnormal AMPS metabolism. Also, Hurler
In cases of this syndrome, heparan sulfate accumulates in the patient's liver and dermatan sulfate accumulates in the spleen.
Abnormal distribution of AMPS may occur. Mucopolysaccharidosis often presents with systemic symptoms, including skin diseases,
These include rheumatoid arthritis, arteriosclerosis, chronic liver disease, and eye disease. As mentioned above, AMPS is an important biological component, both as a physiologically active substance like heparin and as a structural factor of biological tissues involved in various physiological functions, and the normal metabolism and distribution of AMPS in the body are important. It is extremely important for maintaining life activities. Therefore, measurement of the metabolism, excretion, and biodistribution of AMPS and AMPS are necessary for both basic medical research on the relationship between the metabolism and biodistribution of AMPS in humans and diseases, as well as clinical medical responses such as diagnosis and treatment of mucopolysaccharidoses. It is essential to measure the distribution and activity of enzymes involved in biosynthesis and decomposition. An object of the present invention is to provide a fluorescently labeled acidic mucopolysaccharide that is useful in the above-mentioned bioscientific research and medical applications regarding AMPS. That is, according to the fluorescently labeled acidic mucopolysaccharide of the present invention, for example, when a small amount of the acidic mucopolysaccharide is administered into a living body, it can be detected with high sensitivity by fluorescence measurement.
It becomes easy to track behaviors such as movement, accumulation, and excretion. It also becomes easier to measure the location and distribution of AMPS degrading enzymes. Furthermore, we clarified the in vivo behavior of anticoagulants such as heparin, which have already been developed for medicinal use.
Not only can it be useful for pathological research, but it can also be used to develop other drugs that are expected to have potential as coagulation and fibrinolytic drugs.
AMPS is also a powerful means for elucidating the mechanism of action as a drug. Furthermore, in terms of medical applications for diseases such as mucopolysaccharidosis,
This study is expected to contribute to the elucidation of metabolic mechanisms such as abnormal accumulation and excretion of AMPS, and may contribute to its diagnosis and treatment. The useful effects of the present invention described above will be further clarified by the following description. The fluorescently labeled acidic mucopolysaccharides of the present invention include hyaluronic acid fluoresceinamide, chondroitin-4-sulfate fluoresceinamide, chondroitin-6-sulfate fluoresceinamide, heparin fluoresceinamide, heparan sulfate fluoresceinamide and dermatan sulfate fluoresceinamide, and their A fluorescein amidated acidic mucopolysaccharide selected from the group consisting of salts. That is, in the fluorescently labeled acidic mucopolysaccharide of the present invention, the constituent uronic acid residues of the mucopolysaccharide have the following formula: [In the formula, one of R ₁ and R ₂ represents a hydrogen atom;
The other is the following formula, (In the formula, the bonding position of the residue -HNCO- to the fluorescein residue is the 5th or 6th position. , the rest is the following equation, (In the formula, one of R ₃ and R ₄ represents a hydrogen atom;
The other side represents a carboxyl group). In the following explanation, fluorescein amine is abbreviated as FA, fluorescein amidated acidic mucopolysaccharide is referred to as FA-AMPS, and fluorescein amidated chondroitin-4-sulfate is referred to as FA-AMPS.
FA-A fluorescein amidate such as chondroitin-4-sulfate is represented by the prefix "FA-." Then, the degree of fluorescein amino group substitution is
Abbreviated as FA substitution degree. The method for producing FA-AMPS of the present invention includes AMPS selected from the group consisting of hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, heparin, heparan sulfate, dermatan sulfate, and salts thereof, and fluorescein amine (FA). The method is characterized by comprising a step of reacting them in an acidic to neutral aqueous medium in the presence of a dehydration condensation agent. Here, the acidic or neutral conditions are preferably PH3-7, more preferably PH4.5-5.
is preferred. Each AMPS used in the present invention
The molecular weight of may be within any range (see Table 1). The FA-AMPS of the present invention, as schematically shown by the following formula (), contains uronic acid (D-
It is an amide bond between the carboxyl group of glucuronic acid or L-iduronic acid) and fluoresceinamine (), which is a fluorescent labeling reagent. When AMPS and FA are bonded using a dehydration condensation agent, the uronic acid carboxyl group of AMPS mainly reacts with the amino group of FA to form an amide bond, but some reacts with the phenolic hydroxyl group of FA to form an ester. Form (). This ester bond is chemically and biochemically more unstable than the amide bond, and it is difficult to treat it by alkaline hydrolysis (e.g. 0.1NNaOH,
selectively cleaved by treatment at 30°C for 24 hours). Therefore, in carrying out the production method of the present invention, if the reaction product is subjected to this alkaline hydrolysis, a fluorescent label consisting only of an amidated form of fluorescein, which is chemically and biochemically more stable as described above, can be obtained.
AMPS is manufactured. The amount of FA added to the reaction system is determined by the desired FA−
AMPS (FA substitution degree, AMPS type, molecular weight, etc.)
Although it varies depending on the situation, the amount is generally several to several tens of times the amount to be replaced, and the addition may be done all at once or in portions. The fluorescein amine herein refers to 5-aminofluorescein, 6-aminofluorescein, or a mixture thereof. As the dehydration condensation agent added to the reaction system, so-called water-soluble carbodiimide compounds such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (generally abbreviated as "EDC") and 1
-cyclohexyl-3-(2-morpholinyl-4
-ethyl)carbodiimide metho-p-toluenesulfonate and 1-benzyl-3-(3-dimethylaminopropyl)carbodiimide are most preferred, but general carbodiimide compounds such as dicyclohexylcarbodiimide, di-p-tolylcarbodiimide, Diisopropylcarbodiimide or the like may also be used. In addition, dehydration condensation agents such as trichloroacetonitrile, cyanamides, and carbonic anhydride derivatives can also be used. The dehydration condensation agent may be added all at once or in portions. The amount of dehydration condensation agent added to the reaction system varies depending on the target FA-AMPS and also depends on the amount of FA added. Generally used FA
The amount is equimolar to 10 times the molar amount. As an aqueous medium, it does not have a negative effect on the reaction,
Any solution that can dissolve the starting materials (AMPS and FA) may be used, but preferred is a mixed aqueous solution consisting of an inorganic acid and an organic base with a pH of 3 to 7, and particularly preferred is
It is a mixture of HCl and pyridine with a pH of 4.5 to 5.0. These buffer solutions, such as dimethyl sulfoxide, dimethyl formamide, acetonitrile, etc., can be mixed with water and also in the presence of the dehydration condensation agents mentioned above.
It is also possible to add a polar organic solvent that does not react with AMPS and FA to advantageously carry out the reaction. The appropriate reaction time is usually 30 minutes to 2 hours, but when preparing FA-AMPS with a high degree of FA substitution, the reaction may be repeated at the above reaction time. The reaction is usually carried out around room temperature (15 to 35°C), but may be carried out at lower or higher temperatures if necessary. Its range is 0-80°C. Once FA-AMPS is produced as described above, its purification is performed, for example, as follows. The reaction mixture is dialyzed, and an excess amount (for example, 3 times the volume) of ethanol or acetone is added to the dialysate solution to precipitate all FA-AMPS. The precipitate obtained here was dissolved in 0.1N−NaOH solution.
After keeping at 30°C for 24 hours, neutralize with hydrochloric acid, etc., and add an excess amount (for example, 3 times the volume) of ethanol or acetone. The resulting precipitate is separated, dissolved in water again, dialyzed, and the dialyzed solution is precipitated with ethanol or acetone, or lyophilized to produce FA-AMPS.
is refined. Next, the properties of the FA-AMPS of the present invention are shown below. (1) Fluorescence: Fluorescence of fluorescein amine itself is extremely weak (approximately 1/150 of fluorescein), but by converting AMPS into fluorescein amidation, its fluorescence increases significantly. F.A.
-The fluorescence intensity of AMPS depends on pH and becomes stronger on the alkaline side than around pH8. The maximum excitation wavelength at this time is 490 nm, and the maximum fluorescence wavelength is 510 ~
It is 512.5 nm (see measurement example 1...FA-heparin). (2) Electrophoresis: When each FA-AMPS was run using pH 2.0 and 5.2 buffers, it showed a single fluorescent band that matched the characteristic Alcian blue staining band of each AMPS. . Their electrophoretic mobility is almost the same as that of the original AMPS. (3) Molecular weight: The elution curve of FA-AMPS obtained by gel filtration using a suitable gel filtration agent such as Sepharose 2B or 6B and Sephadex G-100 is as follows:
The elution curves almost match the elution curves of each starting material except for hyaluronic acid (see Measurement Example 2...FA-heparin). In the case of hyaluronic acid, a change in the elution curve was observed, which is presumed to be due to slight lowering of the molecular weight during the manufacturing process of FA-hyaluronic acid (see Measurement Example 3...FA-hyaluronic acid). Hyaluronic acid is susceptible to depolymerization, which is thought to be due to radical reactions, due to a combination of various factors (dissolved oxygen, light, transition metal ions, vitamin C, etc.)
Known as AMPS. Since alkali treatment alone does not cause hyaluronic acid to have a lower molecular weight, it is assumed that the combination of reaction steps including alkali treatment in this production method is responsible for the lower molecular weight of hyaluronic acid. In this way, it was found that FA-AMPS is almost the same as the original AMPS in terms of physical properties, despite the introduction of a fluorescein group into the AMPS molecule. Therefore, FA-AMPS produced by the method of the present invention is expected to behave similarly to natural AMPS in vivo or under biochemical conditions. In fact, as shown in Table 2, FA-heparin obtained by this production method almost completely retains the anticoagulant activity of the raw material heparin, and FA-hyaluronic acid and FA-chondroitin sulfate each have the corresponding It was confirmed that, like the original AMPS, it serves as a substrate for the enzyme hyaluronidase (see Measurement Example 4 "Decomposition of FA-hyaluronic acid by hyaluronidase"). AMPS as a raw material and Examples 1-
Table 2 shows the chemical analysis values and biological activity of FA-AMPS produced in Example 5. These FA−AMPS
The degree of FA substitution is not necessarily large. FA−AMPS
In order to retain their respective biological activities,
As long as the fluorescence is detectable by ordinary measuring means, it is preferable to keep the degree of FA-substitution to an appropriate level. As shown in Table 2, each FA−
The number of FA substituents per molecule of AMPS is less than 1 for heparin and heparan sulfate, which have small molecular weights, clearly indicating that they are a mixture of FA-substituted and unsubstituted molecular species. Furthermore, even if the number of FA substituents per molecule is one or more, such as chondroitin-4 and 6-sulfate and dermatan sulfate, all AMPS molecules are uniformly FA-substituted.
It is unlikely that it has been replaced. Even in the biological science field as mentioned above, the fact that FA-AMPS is a mixture of labeled and non-labeled molecular species does not seem to pose any problem in the use of FA-AMPS. If a product consisting only of substituted AMPS species is required, FA-substituted and non-FA-substituted species can be separated by applying hydrophobic affinity chromatography, as described in Example 7, for example. It can be separated into molecular species. The present invention will be explained in more detail by showing examples below. Example 1 200 mg of hyaluronic acid mixed with N-HCl/pyridine (3:1) (adjusted to PH4.75 with 6N-HCl)
Dissolved in 100ml. 300 mg of fluorescein amine was dissolved in 10 ml of a mixture of N-HCl and pyridine (1:1). After mixing both solutions and adjusting the pH to 4.75, water-soluble carbodiimide (1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide)
0.25 ml was added dropwise at room temperature while stirring. of reaction solution
The pH was constantly kept at 4.75 with 6N HCl and stirred for 1 hour. The reaction solution was transferred to a dialysis membrane (Visking tube 30/20) and dialyzed against purified water for 24 hours.
Three times the volume of ethanol was added to the dialysis fluid. The resulting precipitate was collected by centrifugation and washed twice with ethanol to obtain a yellow powder (164 mg). This was dissolved in 40 ml of 0.1N NaOH, kept at 30°C for 24 hours, neutralized with dilute hydrochloric acid, added with 3 times the volume of ethanol, and the resulting precipitate was centrifuged. Wash this precipitate three times with ethanol and then wash it with purified water.
The solution was dissolved in 200 ml and dialyzed against purified water. The pH of the dialysate solution was adjusted to 6.5 with 0.1N-NaOH, and then lyophilized to obtain 140 mg of hyaluronic acid fluoresceinamide (sodium salt). Example 2 Chondroitin 4-sulfate (sodium salt) 500
mg of N-HCl/pyridine (3:1) mixture (PH
4.75) Dissolved in 100ml. fluorescein amine
570mg in 30ml of N-HCl/pyridine (1:1) mixture
dissolved in After mixing both solutions and adjusting the pH to 4.75, 0.484 ml of water-soluble carbondiimide (EDC) was added dropwise while stirring at room temperature. PH of reaction solution is 4.75
After stirring for 1 hour, the reaction solution was transferred to a dialysis membrane, followed by dialysis and ethanol precipitation in the same manner as in Example 1 to obtain a yellow powder (398 mg).
This was dissolved in 100 ml of 0.1N-NaOH, kept at 30°C for 24 hours, neutralized with dilute hydrochloric acid, precipitated with ethanol in the same manner as in Example 1, dialyzed, and freeze-dried to produce chondroitin-4-sulfate fluoresceinamide (sodium salt). ) 362 mg was obtained. Example 3 In the case of chondroitin-4-sulfate (Example 2)
By the same reaction and operation as above, chondroitin-6
- 354 mg of chondroitin-6-sulfate fluoresceinamide (sodium salt) was obtained from 500 mg of sulfuric acid (sodium salt). Example 4 500 mg of heparin (sodium salt) was mixed with N-HCl.
It was dissolved in 100 ml of pyridine (3:1) mixture (PH4.75). 1.15 g of fluorescein amine was dissolved in N-HCl.
It was dissolved in 50 ml of pyridine (1:1) mixture. After mixing both solutions and adjusting the pH to 4.75, 1.33 ml of water-soluble carbodiimide (EDC) was added dropwise while stirring at room temperature. The pH of the reaction solution was maintained at 4.75 and stirred for 1 hour.
The reaction solution was transferred to a dialysis membrane, followed by dialysis and ethanol precipitation in the same manner as in Example 1 to obtain a yellow powder (413 mg). This was dissolved in 100ml of 0.1N-NaOH, kept at 30℃ for 24 hours, and then neutralized with dilute hydrochloric acid.
Ethanol precipitation, dialysis and lyophilization were carried out in the same manner as in Example 1 to obtain 352 mg of heparin fluoresceinamide (sodium salt). Example 5 500 mg of heparan sulfate (sodium salt) was prepared by the same reaction and operation as in the case of heparin (Example 4).
370 mg of heparan sulfate fluoresceinamide (sodium salt) was obtained. Example 6 500 mg of dermatan sulfate (sodium salt) was added to N-
HCl/pyridine (3:1) mixture (PH4.75) 100ml
dissolved in Fluorescein amine 570mg N-
It was dissolved in 30 ml of HCl/pyridine (1:1) mixture.
After mixing both solutions and adjusting the pH to 4.75, 0.484 ml of water-soluble carbodiimide (EDC) was added dropwise while stirring at room temperature. After stirring the reaction solution for 1 hour while keeping the pH of the reaction solution at 4.75, the reaction solution was transferred to a dialysis membrane, and the following steps were taken.
Dialysis and ethanol precipitation were performed in the same manner as in Example 1. A yellow powder (402 mg) was obtained. This is 0.1N−
Dermatan sulfate fluoresceinamide (sodium salt) 370 was dissolved in 100 ml of NaOH, kept at 30°C for 24 hours, neutralized with dilute hydrochloric acid, precipitated with ethanol in the same manner as in Example 1, dialyzed, and freeze-dried.
I got mg. Example 7 100 mg of heparin fluoresceinamide (sodium salt) produced in Example 4 was added to 10 ml of 3M ammonium sulfate/0.05M phosphate buffer (PH6.6).
This was applied to a hydrophobic affinity chromatography agent column (Octyl Sepharose CL-4B, 1.5 x 25.5 cm) equilibrated with the same buffer, and eluted with 180 ml of the same buffer. Next, it was eluted with 200 ml of 1M ammonium sulfate/0.05M phosphate buffer (PH 6.8), and finally with 140 ml of 0.05M phosphate buffer (PH 7.0). FIG. 1 shows an elution curve prepared by measuring the uronic acid content (solid line) and relative fluorescence intensity (wavelength 512 nm) (dotted line) for each eluate fraction. Fractions with an eluate volume of 215 to 255 ml were collected, adjusted to pH 9 with 0.1 N NaOH solution, dialyzed against purified water for 72 hours, and then lyophilized to produce heparin fluoresceinamide (sodium salt), which consists only of fluoresceinamide molecular species. Obtained 29 mg.

[Table] Measurement Example 1 Excitation and Fluorescence Spectrum of FA Heparin When the FA-heparin produced in Example 4 was measured, the results shown in FIG. 2 were obtained. The solid line shows the fluorescence spectrum, and the dashed line shows the excitation spectrum.
Maximum fluorescence wavelength is 512nm, maximum excitation wavelength is 490nm
It was hot. A fluorescence spectrophotometer was used for measurement, and the fluorescence intensity was
It is expressed as a relative value with the fluorescence intensity of 10 ^-6 M quinine sulfate (0.1M sulfuric acid solution) as 10. The sample is dissolved in 0.05M borate buffer (PH10.0). In the case of gel chromatography, the same borate buffer is diluted 6 times and used. Measurement Example 2 Gel filtration chromatography of FA-heparin The FA-heparin produced in Example 4 and raw material heparin were subjected to gel filtration chromatography.
The column used was a column packed with Sephadex G-100 (size 1.5 x 80 cm), and the eluent was
A 0.9% NaCl solution was used. The bond is shown in Figure 3. In the figure, the solid line is the elution curve of FA-heparin according to the uronic acid content, and the dotted line is the elution curve according to the relative fluorescence intensity. Moreover, the broken line is an elution curve depending on the uronic acid content of the raw material heparin. Measurement Example 3 Gel filtration chromatography of FA-hyaluronic acid Gel filtration chromatography of the FA-hyaluronic acid produced in Example 1 and the raw material hyaluronic acid was performed. Using a column (size 1.5 x 80 cm) packed with Sepharose 2B, 0.9% NaCl was used as the eluent.
The solution was eluted. The results are shown in Figure 4. In the figure, the solid line is the elution curve of FA-hyaluronic acid according to the uronic acid content, and the dotted line is the elution curve according to the relative fluorescence intensity. Moreover, the broken line is an elution curve depending on the uronic acid content of the raw material hyaluronic acid. Vo indicates void volume. Measurement Example 4 Decomposition of FA-hyaluronic acid by hyaluronidase The FA-hyaluronic acid produced in Example 1 was
℃ and treated with hyaluronidase for 17 hours. Gel filtration chromatography was performed on FA-hyaluronic acid before hyaluronidase treatment and on FA-hyaluronic acid after hyaluronidase treatment as described above. Elution was performed using a 0.9% NaCl solution using a column (size 1.5 x 80 cm) packed with Sepharose 6B. The obtained elution curve is shown in FIG. (――●
-, ---●---) are before hyaluronidase treatment.
For FA-hyaluronic acid, (-○-, ---○---) indicates the elution curve of FA-hyaluronic acid after treatment.
The solid line is based on uronic acid content, and the dotted line is based on relative fluorescence intensity.

[Brief explanation of the drawing]

FIG. 1 shows the elution curve of FA-heparin on an Octyl Sepharose CL-4B column, where the solid line is a curve based on uronic acid content and the dotted line is a curve based on relative fluorescence intensity (Example 7). Figure 2 shows the F.A.
- It is a diagram showing the fluorescence spectrum (solid line) and excitation spectrum (broken line) of heparin (Measurement Example 1). Figure 3 shows the elution curves of FA-heparin and raw material heparin by gel filtration chromatography; the curves for FA-heparin according to uronic acid content (solid line), the curves for FA-heparin according to relative fluorescence intensity (dotted line), and the curves for FA-heparin according to relative fluorescence intensity (dotted line), respectively. This is a curve (broken line) of raw material heparin depending on the uronic acid content (Measurement Example 2). Figure 4 shows
Showing the elution curves of FA-hyaluronic acid and raw material hyaluronic acid by gel filtration chromatography, FA-hyaluronic acid (solid line) according to uronic acid content, FA-hyaluronic acid (dotted line) according to relative fluorescence intensity, and according to uronic acid content, respectively. This is a curve (dashed line) of raw material hyaluronic acid (Measurement Example 3). Figure 5 shows the elution curve of FA-hyaluronic acid before hyaluronidase treatment using a Sepharose 6B column (-●-, ---●---) and the elution curve of hyaluronan after hyaluronidase treatment (-○- ,--
○---). The solid line is a curve based on uronic acid content, and the dotted line is a curve based on relative fluorescence intensity (Measurement Example 4).

Claims (1)

[Claims] 1. The constituent uronic acid residues of the mucopolysaccharide are represented by the following formula: [In the formula, one of R ₁ and R ₂ represents a hydrogen atom;
The other is the following formula, (In the formula, the bonding position of the residue -HNCO- to the fluorescein residue is the 5th or 6th position. , the rest is the following equation, (In the formula, one of R ₃ and R ₄ represents a hydrogen atom;
Hyaluronic acid fluoresceinamide, chondroitin, characterized in that it consists of a unit represented by
A fluorescently labeled acidic mucopolysaccharide selected from the group consisting of 4-fluoresceinamide sulfate, chondroitin-6-fluoresceinamide sulfate, heparin fluoresceinamide, heparan fluoresceinamide sulfate and dermatan fluoresceinamide sulfate, and salts thereof. 2 Hyaluronic acid, chondroitin-4-sulfate,
An acidic mucopolysaccharide selected from the group consisting of chondroitin-6-sulfate, heparin, heparan sulfate, dermatan sulfate, and salts thereof and fluorescein amine are mixed in an acidic to neutral aqueous medium in the presence of a dehydration condensing agent. 1. A method for producing a fluorescently labeled acidic mucopolysaccharide, the method comprising a reaction.