CS276775B6 - A process for preparing (H) radioisotope-labeled fractions of hyaluronic acid or a salt thereof - Google Patents

Abstract

The solution relates to a method of preparing (3H) radioisotope-labeled fractions of hyaluronic acid (HA) or its salts of defined molecular weight. The procedure consists in labeling native HA or its salt with (3H) radioisotope, measuring the radioactivity of the sample taken by the liquid scintillation spectrometry (LSC) method. Subsequently, HA or its salt is allowed to pass through a cation exchanger layer, preferably from the group of carboxymethylcellulose derivatives, and then divided into fractions by the high-performance gel permeation chromatography method, and then measuring the radioactivity of the sample taken of the relevant fraction of HA or its salt by the LSC method. The product has high radiochemical purity and high specific radioactivity.

Description

y 1

CS 276 775 BS o

The present invention relates to a process for the preparation of (H) radiolabelled fractions of hylauronic acid or a salt thereof with a definitive molecular weight and an evaluation of the quality of said bodies.

Hyaluronic acid (IIa) and its derivatives are glycosaminoglycan polysaccharides composed of repeating units of glucuronate and N-acetylglucosamine. The growing interest of HA and its derivatives is due to the expanding use of a variety of preparations containing HA in ophthalmology, rheumatology, dermatology and other fields of medical practice. The possibilities of extending the application of HA and its derivatives also depend on the greater knowledge of ichbiochemistry in animal organisms. For in vivo studies, the radioisotope-labeled HA and the like, respectively, are preferably used. its derivatives. These substances must have a high (= 95%) radiochemical purity and a high specific radioactivity. HA resp. its salts can be obtained from cohesive tissue, from human umbilical cord, from vitreous humor or from bovine trachea, but from Streptococcus zooepidemicus. HA isolated from any source is structurally identical, but can be isolated with varying degrees of purity and degree of polymerization.

Two methods are used to prepare radiolabeled HA and its derivatives: 1. Add radioactive precursors to the given biosynthesizing system / cell, the HA culture producing cells (e.g. (3H) -, (CC) -glucose, N-acetylglucosamine) This method produced (3H) -1 (C4C) -labeled HA with a molecular weight of up to 106 Da / Gallagher 3.T. + col .: Biochem. 148, 187 (1975); Winterbourna D. 3rd + Biochem. 182, 707 (1979); Underhill C.B., Toole B.P .: 3. Cell Biol. 82, 475, (1979)). The advantage of this method is that a high molecular weight HA is obtained and, using specifically labeled precursors, the radioactivity can be introduced into specific sites in the HA molecule.

The disadvantage of this method of preparation of radioactive HA is the laboriousness of isolating the labeled HAz reaction mixture, the need for its purification from the ballast radioisotope-labeled endogenous substances, the relatively low radiochemical extract, often low or unknown non-radioactivity, the instrumental intensity. 2. The chemical method for the preparation of radiolabelled HA was first published in 1982 / Hook M. + al .: Anal. Biochem. 119, 235, (1982), the authors prepared hyaluronic acid by N-deacetylation of native HA with hydrazine followed by N (H) -acetylation with (H) -acetanhydride. The isolation of the high molecular weight fraction of (3H) -hyaluronic acid by the removal of the molecular fractions was done by equilibrium dialysis and gel chromatography. This method produced HA with specific radioactivity (3-4) x 10 cpm / µg of uronic acid.

A disadvantage of this process is that the entire process of preparing and isolating (3H) -hyaluronic acid is both material and time consuming and provides a relatively radioactivity derivative.

Another method for the preparation of high molecular weight HA / o mol. wt. 3 x 10 3 Da / tritiated by Orlando et al. / Orlando P. + al .: 3. Label, Comp. Radiopharm. 22, 951 (1985)). HA was oxidized with sodium periodate and, after purification of the oxidation product by ultrafiltration and equilibrium dialysis, was poorly reduced with (H) -NaBH. After purification of the product3 from * by equilibrium dialysis and ultrafiltration, (H) -hyaluronic acid with 95% purity was obtained % and with specific radioactivity from 5.55 MBq / mg.

A disadvantage of this method is that in the oxidation the molecular weight of HA is reduced and that the ultrafiltration of HA is the most suitable, since biomacromolecules often clog pores in the ultra-filtration membranes. '

The method by which the integrity of the HA molecule is maintained after introduction of the high radioactivity isotope consists of reducing the HA oligosaccharides with NaBH 4 / modifying

CS 275 775 BS 2 only suggests the end-productive sugar, oxidation of vicinal hydroxyl groups and reaction with alkyldiamines and Bolton-Hunter reagent (N-succinimidyl-3- (4-hydroxyphenyl) propionate). I on a reducing constructor. By this method, oligosaccharides can be obtained by a radioactive radioactivity of up to 1000 times higher (Raja R.3. + Kol. Anal. Biochem. 139, 158, (1984)) as reported by the authors.

The disadvantage of the method is that it is not suitable for polymer but only for oligomer HA.

Hyaluronic acid can easily be converted to (H) -hyaluronic acid by isotonic exchange of hydrogen to tritium in aqueous Pd / CaCO 3 catalysis.

The (3H) -hyaluronic acid derivative can also be prepared by alkylating a native biopolymer with (H) -methyl bromide with a high specific radioactivity (1.5 TBq / mole) in low-temperature liquid ammonia (-33.5 ° C) without special In this way, a high specific radioactivity reaction mixture is prepared from which the H-hyaluronic acid derivative or the H-hyaluronic acid derivative is formed. its fractions further isolate by some suitable method and characterize! *

The following techniques have been used to assess HA molecular weights: a) viscosimetry (H. Bothner, T. Waaler, O. Wik: Int. Biol. Macromol. 1988, 10, 287-291; E. Shimada, G. Matsumura : O. Biochem., 78, 513-517, 1975. (b) light scattering (H. Bothner; T. Waaler, 0. Wik: 0 Biol. Macromol. 1988, 10, 287--291; RL Cleland: Arch. Biochem Biophys 1977, 180, 57-53 Barret TW, Baxter E, Physiol Chen, 1982, 14, 19-29; c) sedimentation analysis (Swann O. Biochem. Biophys. Acta 1958, 155, 17-30; TC Renting, M. Ryan; A. Pietruszkiewicz: Biochim. Biophys. Acta 1950, 42, 476-485) ; d) equilibrium ultracentrifugation (E. Shimada, G. Matsumura: O. Biochem. 78, 513-517, 1975); e) Goal Chromatography (N. Motohashi, I. Sea: O. Chromatogr. 1984, 299, 508-512; M. Terbojevich, A. Cesani, M. Palumbo: Carbohydr. Res, 1985, 157, 259-272; Beaty NB,

Tew W. P., Mello R., Anal. Biochem. 1985, 147, 387-395). A major disadvantage of viscosimetry and light scattering is that only the average molecular weight (viscosity, Rv, respectively, mass, M i, diameter) is determined by the three methods and apolymers with different molecular weight distributions can be considered as the same Mv, or R w for the same substances. Sedimentation analysis as well as equilibrium ultracentrifugation provide a broader expression of the molecular weight distribution of the polymer being analyzed, but the operation of these methods is cost-intensive. Gel chromatography, unlike its high-performance modification (HP GPC), is relatively slow. As a rule, one analysis takes several hours to tens of hours.

Highly efficient gel permeation chromatography (HP GPC) is the most frequently used method in the world today to characterize the molecular weight distribution as well as the individual molecular weight diameters of the polymers (biopolymers).

The aforementioned disadvantages of the known processes are eliminated by the preparation of (H) a radioisotope-labeled fraction of hyaluronic acid or a salt thereof with a defined molecular weight of the invention. The essence of this is that native hyaluronic acid or its salt is labeled with (3H) radioisotope and the radioactivity of the collected sample is measured by liquid scintillation spectrometry (LSS). Preferably, the labeled HA is gaseous triturated with an aqueous solution of hyaluronic acid or its salt in the presence of a Pd / CaCO 3 catalyst, the solvent and the labile radioactivity are removed. Another suitable way of labeling HA is to alkylate it with (H) -methyl bromide in a liquid ammonia which is removed after neutralization. (3H) radioisotope-labeled hyaluronic acid or a salt thereof is not passed through a cation exchange layer, preferably a carboxymethylcellulose derivative, Subsequent to HA or a salt thereof being separated into fractions and determined by their high performance gel method permeation chromatography (HP GPC). HP GPC isolation and identification of the (3H) -HA as well as salt fractions are done by chromatographic colony supplementation with hydroxyethyl methacrylate. A 0.1 M aqueous sodium bisulfate solution, NaNOg, is used as the mobile phase. Finally, the radioactivity of the sample of the appropriate fraction of HA or its salt is measured by liquid scintillation spectrometry. In particular, the advantages of the present invention are that without prior modification of the starting HA, which often results in a reduction in molecular weight, HA tritiation with a molar radioactivity is achieved several times higher than previously used. Further isolating (H) -hyaluronic acid or its salt from the reaction mixture by the auxiliary GPC, which is simple and undemanding, is more effective than currently used (dialysis or ultrafiltration); the highly efficient GPC modification is also substantially faster. In addition to the high specificity of radioactivity, the (H) -hyaluronic acid (s) is prepared according to the invention. its solubility with high radiochemical purity with combination of GPC and LSS methods can furthermore be characterized by their molecular distribution and polymolecularity and the specific radioactivity of the various fractions. The following examples illustrate the proposed process, but do not limit the scope of the invention in any way. Example 1

10 mg of catalyst (10% PdO / CaCO 3) was weighed into a 1.0 mL reaction flask and 0.5 mL of an aqueous hyaluronic acid solution (10 mg) was pipetted. The reaction flask was then connected to a titration apparatus. The contents are cooled with liquid nitrogen and the titration apparatus is evacuated. Carrier 200 G3q tritium is fed to the reaction flask with a Toepler pump. The reaction mixture is stirred electromagnetically at 295 K. After 100 min. the reaction is stopped, the contents of the reaction flask are frozen and the residual tritium is transferred to the reservoir. Freeze-sublimation in a sealed system removes solvent and labile radioactivity. The residue is dissolved in 2 ml of redistilled water and the catalyst is separated by centrifugation. The debris-bound tritium residues are removed by repeated freeze-drying in a closed system. A sample of the reaction mixture was injected into the HP GPCsystem, and on the columns packed with hydroxyethyl methacrylate (Separon HEMA S-1000 and SeparonHEMA S-300), the reaction mixture was separated into a fraction of different molecular weights. The mobile phase is 0.1 M aqueous sodium nitrate solution, NaNO. An isolated amount of liquid scintillator is added to the isolated 3 ' -frame of Hyaluronic Acid and their radioactivity is measured by the liquid scintillation spectrometry method on a Packard 300 CD apparatus. From high radioactivity (H) -HA high molecular weight fractions, an aliquot (100 µl) of β 3 is carefully injected into an HP GPC system and the molecular characteristics of (1 H) -hyalonic acid are determined. The following characteristics were calculated from the obtained values: - 3 5 - average molecular weight Mp (H) -HA and 2.28 x 10 Da - polymolecularity of sample D and 1.98 - specific radioactivity of pure (H) -HA = 3.3 MBq Example 2 To an aqueous solution of hyaluronic acid (5 mg in 0.5 roll of redistilled water) was added 0.6 mL of ethanol and 9 mL of ether. The resulting white suspension solution is evaporated in a vacuum evaporator and the residue is dried to constant weight. Rinse the reaction apparatus with dried ammonia. 3 ml of dry ammonia were distilled off in a 10 ml reaction flask. The reaction mixture is stirred for 10 minutes at the boiling point of ammonia and 2 mg of sodium are added to the reaction mixture. The resulting blue solution was decolorized per minute. Adds

Claims (3)

PATENT CLAIMS 3 _Λ Process for the preparation of (H) radiolabeled fractions of hyaluronic acid or its salts with a defined molecular weight, characterized in that the native hyaluronic acid or its salt is labeled with (H) radioisotope, the radioactivity of the sampled sample is measured by liquid scintillation spectrometry hyaluronic acid or a salt thereof is passed through a cation exchange layer preferably from the group of carboxymethylcellulose derivatives, then the hyaluronic acid or its salt is fractionated by high performance gel permeation chromatography and then the radioactivity of a sample of the relevant hyaluronic acid fraction or by liquid scintillation spectrometry. CS 276 775 B6 2. The preparation method according to item 1, characterized in that the native hyaluronic acid or its salt is radiolabeled (¾) by treating the aqueous solution of hyaluronic acid or its salt with tritium gas _{in the presence of a Pd / CaCO 3 catalyst, removing} solvent and labile radioactivity. 3. The method of preparation according to item 1, characterized in that; to the native hyaluronic acid or a salt thereof labeled ⁽³ H) and a radioisotope that is alkylated with a ⁽³ H) -metylbromidom in a liquid ammonia which is removed after neutralization.