CS276775B6 - Process for preparing (3h)radiolabelled fractions of hyaluronic acid or salts thereof - Google Patents

Abstract

The solution relates to the preparation method ("^ H)" radioisotope-labeled acid fractions of hyaluronic acid (HA) or its salts of defined molecular weight. Approach is that the HA is native or its salt is labeled with (3H) radioisotope, measured the radioactivity of the sample taken by liquid scintillation spectrometry (LSC). Followed by HA or letting the sol pass through the layer a cation exchanger, preferably a group carboxymethylcelyl derivatives, and thereafter is separated into fractions by the high-efficiency method gel permeation chromatography and then the radioactivity is measured sample of the respective HA fraction or salts thereof by LSC. The product has high radiochemical purity and high radioactive.

Description

The invention relates to a process for the preparation of (H) radiolabeled fractions! hyaluronic acid or its salts of defined molecular weight and the evaluation of the quality of these substances.

Hyaluronic acid (Ha) and its derivatives are glycosaminoglycan polysaccharides composed of repeating units of glucuronate and N-acetylglucosamine. The growing interest in the development of HA and its derivatives is due to the expanding application of a number of preparations containing HA in ophthalmology, rheumatology, dermatology and in the Salsh fields of medical practice. The possibilities of expanding the application of HA and its derivatives also depend on Salší's knowledge of their biochemistry in the animal organism. For in vivo studies, radiolabeled HA or HA is preferably used. its derivatives. These substances must have high (= 95%) radiochemical purity and high specific radioactivity. HA resp. its salts can be obtained from the tissue of rooster combs, from the human umbilical cord, from the vitreous or from the trachea of bovine animals, but from the bacterium Streptococcus zooepidemicus. HA isolated from any source is structurally identical, but can be isolated with both varying degrees of purity and degree of polymerization.

For the preparation of radioactively ^- labeled HA and its derivatives are used in two ways:

1. Radioactive precursors are added to a given biosynthetic system (cell, tissue culture) producing HA. (1 H) -, (1 C) -glucose, N-acetylglucosamine]. In this way, ( ³ H) -i (1 C) -labeled HA with a molecular weight of the order of ΙΟθ Da / Gallagher 3.T. + col .: Biochem. 9. 148, 187, (1975); Winterbourne D.

3. + col .: Biochem. □. 182, 707, (1979); Underhill C.B., Toole B.P .: 3. Cell Biol. 82, 475, (1979)).

The advantage of this method is that high molecular weight HA is obtained, and using specifically labeled precursors, radioactivity can be introduced at a specific site in the HA molecule. ·

The disadvantages of this method of preparation of radioactive HA are the laboriousness of isolating labeled HA from the reaction mixture, the need to purify it from ballast radiolabeled endogenous substances, relatively low radiochemical yield, often low or unknown specific radioactivity, instrumentation.

2. A chemical method for the preparation of radiolabeled HA was first published in 1982 / Hook M. + et al .: Anal. Biochem. 119, 236, (1982), when the authors prepared hyaluronic acid 3 by N-deacetylation of native HA with hydrazine and subsequent N (H) -acetylation with (H) 3 *

-acetic anhydride. Isolation of the high molecular weight (H) -hyaluronic acid fraction from the low molecular weight fractions was performed by equilibrium dialysis and gel permeation chromatography. HA with specific radioactivity (3-4) x 10 cpm / μg uronic acid was prepared by this method.

The disadvantage of this process is that the whole process of preparation and isolation of (H) -hyaluronic acid is time-consuming and time-consuming and provides a derivative with a relatively low specific radioactivity.

" _Of 5

Another method for the preparation of high molecular weight HA / o mol. mass 3 x 10 Da / tritium labeled by Orlando et al. / Orlando P. + col .: □. Label, Comp. Radiopharm. 22, 961, (1985)). HA was oxidized with sodium periodate and after purification of the oxidation product by ultrafiltration and equilibrium dialysis, it was badly reduced with (H) -NaBH. After purification of the product by equilibrium dialysis and ultrafiltration, (H) -hyaluronic acid with radiochemical purity of 95% and specific radioactivity was obtained. zv5.55 MBq / mg ·

The disadvantage of this method is that the molecular weight of HA is reduced during oxidation and that ultrafiltration is not the most suitable, as biomacromolecules often clog pores in ultrafiltration membranes. ....... ’

The method by which the integrity of the HA molecule is maintained after the introduction of a isotope with high specific radioactivity consists in the reduction of HA oligosaccharides with NaBH 4 / modified

CS 276 775 86 only terminal reducing sugar (oxidation of vicinal hydroxyl groups and reaction with alkyldiamines and Bolton-Hunter reagent (N-succinimidyl-3- (4-hydroxyphenyl) propionate). The resulting hydroxyphenyl derivative of HA is easily radioiodidized with Na I at the reducing end of the chain. By this method, oligosaccharides can be obtained with HA radioactivity up to 1000 times higher (Raja R.3. + Et .: Anal. Biochem. 139, 168, (1984)) than stated by the authors of the methods described above.

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

Hyaluronic acid can be easily converted to (H) -hyaluronic acid by isotope exchange of hydrogens for tritium in aqueous solution under Pd / CaCO ₃ catalysis. ·

The (H) -hyaluronic acid derivative can also be prepared by alkylation of the native biopolymer with (H) -methyl bromide with high specific radioactivity (1.5 TBq / mol), in a liquid ammonia medium at low temperature (-33.5 ° C) without special treatment. starting HA. In this way, a reaction mixture with high specific radioactivity is prepared, from which the high molecular weight derivative of H-hyaluronic acid, resp. its fractions are further isolated by some suitable method and characterized.

The following techniques were used to characterize the molecular weights of HA:

a) viscosimetry (H. Bothner, T. Waaler, O. Wik: Int. 3. Biol. Macrotnol. 1988, 10, 287-291; E. Shimada, G. Matsumura: Bio. Biochem. 78, 513-517, 1975;

b) light scattering (H. Bothner; T. Waaler, 0. Wik: Ing. □. Biol. Macromol. 1988, 10, 287-291; RL Cleland: Arch. Biochem. Biophys. 1977, 180, 57-63; Barret TW, Baxter 3. E .: Physiol. Chen. Phys. 1982, 14, 19-29);

c) sedimentation analysis (Swann D. A .: Biochem. Biophys. Acta 1963, 156, 17-30; T. C. Laurent, M. Ryan; A. Pietruszkiewicz: Biochim. Biophys. Acta I960, 42, 476-485);

d) equilibrium ultracentrifugation (E. Shimada, G. Matsumura: O. Biochem. 78, 513-517, 1975);

e) goal chromatography (N. Motohashi, I. Mori: O. Chromatogr. 1984, 299, 508-512; M. Terbojevich, A. Česaní, M. Palumbo: Carbohydr. Res, 1986, 157, 269-272; Beaty NB, Tew WP, Mello R. Anal. Biochem. 1985, 147, 387-395).

The main disadvantage of viscosimetry and light scattering is that these methods only determine the average molecular weight (viscosity, R _v , or mass, M ^, average) and polymers with different molecular weight distributions can be considered as the result of the same values of M _v , or for identical substances. Sedimentation analysis as well as equilibrium ultracentrifugation provide a broader indication of the shape of the molecular weight distribution of the analyzed polymer, but the operation of these methods is disproportionately high. Goal chromatography, in contrast to its high performance modification (HP GPC), is relatively slow. One analysis usually takes several hours to tens of hours.

The method of high-performance gel permeation chromatography (HP GPC) is the most frequently used in the world today for the characterization of the molecular weight distribution as well as the individual defined average molecular weights of polymers (biopolymers).

Said disadvantages of the known processes are eliminated by the process for the preparation of ( ³ H) radiolabeled fractions of hyaluronic acid or its salt nevý defined molecular weight according to the invention. In essence, the native hyaluronic acid or its salt is radiolabeled with (H) and the radioactivity of the sample is measured by liquid scintillation spectrometry (LSS). The labeled HA is preferably made by treating an aqueous solution of hyaluronic acid or a salt thereof with tritium gas in the presence of a Pd / CaCO 3 catalyst, removing the solvent and labile radioactivity. Another suitable method of labeling HA is that it is alkylated with (H) -methyl bromide in liquid ammonia, which is removed after neutralization. ⁽³ H) labeled with a radioisotope hyaluronic acid or its salt is not

CS 275 775 B6 can be passed through a cation exchanger layer, preferably from the group of carboxymethylcellulose derivatives. Subsequently, the HA or its salt is divided into fractions and their modular mass is determined by high performance gel permeation chromatography (HP GPC). HP GPC Isolation and Fraction Identification! (H) -HA as well as salts are made using chromatographic columns packed with hydroxyethyl methacrylate. A 0.1 M aqueous solution of sodium nitrate, NaNO 2, is used as the mobile phase. Finally, the radioactivity of the sample taken of the relevant fraction of HA or its salt is measured by liquid scintillation spectrometry.

The advantages of the proposed method are, in particular, that without prior modification of the starting HA, which often resulted in a decrease in molecular weight, the labeling of HA with tritium with a molar radioactivity several times higher than with the hitherto used methods is achieved. Furthermore, the isolation of (H) -hyaluronic acid or its salt from the reaction mixture by means of GPC, which is simple and undemanding, is more efficient than hitherto used (dialysis or ultrafiltration); high-efficiency GPC modification is also significantly faster. In addition to the high specific radioactivity, (H) -hyaluronic acid or its salt with high radiochemical purity, while the combination of GPC and LSS methods can also easily characterize their molecular distribution and polymolecularity and determine the specific radioactivity of the gray fractions.

The following examples illustrate the proposed process without, however, limiting the scope of the invention in any way.

Example 1

Weigh 10 mg of catalyst (10% PdO / CaCO 3) into a 1.0 mL reaction flask and pipette 0.5 mL of aqueous hyaluronic acid (10 mg). The reaction flask is then connected! to the titration apparatus. The contents are cooled with liquid nitrogen and the titration apparatus is evacuated. Using a Toepler pump, 200 GBq of unsupported tritium is introduced into the reaction flask into a reservoir. The reaction mixture is stirred electromagnetically at a temperature of 295 K. After 100 min. the reaction is terminated, the contents of the reaction flask are frozen and the residual tritium is transferred to a reservoir. Freeze sublimation in a closed system removes solvent and labile radioactivity. The residue is dissolved in 2 ml of redistilled water and the catalyst is separated by centrifugation. Residues of labile moistened tritium are removed by repeated freeze sublimation in a closed system. A sample of the reaction mixture was injected into the HP GPC system and on columns packed with hydroxyethyl methacrylate (Separon HEMA S-1000 and Separon HEMA S-300) connected in series, the reaction mixture was fractionated! of different molecular weight. Mobile stage creature! 0.1 M aqueous solution of nitrate, sodium, NaNO3. To isolated 3,. ^J

An aliquot of the liquid scintillator is added to the (H) -hyaluronic acid fractions and their radioactivity is measured by liquid scintillation spectrometry on a Packard 300 CD.

From high molecular weight fractions! The high radioactivity (H) -HA is re-injected into the HP GPC system for an aliquot time and the molecular characteristics of t (100 .mu.l) ( ^d H) -hyaluronic acid are determined. From the obtained values, the following characteristics were calculated:

- 3 5

- average molecular weight Mp (H) -HA = 2.28 x 10 Da

- polymolecularity of sample D and 1.98 q

- specific radioactivity of pure (H) -HA = 3.3 MBq / mg

Example 2

To an aqueous solution of hyaluronic acid (5 mg in 0.5 ml of redistilled water) was added 0.6 ml of ethanol and 9 ml of ether. The resulting white suspension solution was evaporated in a vacuum evaporator and the residue was dried to constant weight. The reaction apparatus with a reflux condenser is rinsed with dried ammonia. 3 ml of dry ammonia are distilled off to the residue in a 10 ml reaction flask. The reaction mixture was stirred for 10 minutes under boiling ammonia, and 2 mg of sodium was added to the reaction mixture. The resulting blue solution decolorizes in one minute. Adds

CS 276 775 B5 o

with 0.5 ml of toluene in which 5 GBq of (H) -methyl bromide are dissolved. The reaction mixture is refluxed for 1 hour with electromagnetic stirring. After neutralization (4 mg HN.C1), the liquid ammonia is allowed to evaporate. The residue is dissolved in redistilled water and evaporated again. After dissolving the residue, pure (H) -hyaluronic acid is isolated from the mixture by gel permeation chromatography on a Superose 6 column (soldered agarose) so that the mixture is fractionated! according to the molecular weights at which the radioactivity is measured by method 3

LSS. High molecular weight fractions of pure (H) -HA with high specific radioactivity are pooled and carefully analyzed! goal permeation chromatography on a FPLC Pharmacia instrument and liquid scintillation spectrometry on a Packard 300 CD instrument.

From the measured values, the characteristics were calculated as follows: * · 3 5

- average molecular weight M (H) -HA = 3.92 x 10 DA

- polymolecularity of sample D = 1.55

- specific radioactivity (H) -HA and 37.2 MBq / mg.

Example 3

From the reaction mixture; prepared according to Example 2, an aliquot of the high molecular weight (H) -hyaluronic acid derivative was injected into the HP GPC system described in Example 1 and fractionated into fractions of 0.65 ml of effluent, in which the radioactivity was measured by the LSC method described in Example 1. From the fraction with the highest radioactivity, characterization into the HP GPC was obtained! diotribution of its molecular weight 3 * (H) -hyaluronic acid derivative:

= 9.91 x 10 ⁴ Da; polymolecularity 0 = 1.37; specific radiothema, inject the sample again. The following values were found: Average molecular weight M activity 7.6 MBq / mg.

Similarly, any fraction of a high molecular weight 3 / (H) -hyaluronic acid derivative can be characterized by a given method.

Example 4 Pure (¾) -hyaluronic acid prepared according to Example 2 was subjected to enzymatic degradation by the enzyme hyaluronidase EC 3.2.1.35 (Sevac, USOL Prague) at 37 ° C. The amount of enzyme added was 48 TRU / 1 mg (H) -HA. At time intervals (0.2, 6, 10 and 24 h), 100 [mu] l of the reaction mixture was taken, boiled for 10 minutes to inactivate the enzyme and centrifuged at 15,000 rpm. HP GPC analysis as well as measurement of the radioactivity distribution of the individual samples was performed as in Example 1. From the radioactivity distribution after 24 hours. digestion showed an elution volume of 14.4 ml, corresponding to an M value of 30,000 Da and a radioactivity of 19,693 dpm. Fri 48 hours digestion, the elution volume was 15 ml and the radioactivity was 21,183 dpm. The resulting peak of radioactive (H) -HA was not altered by further treatment with the enzyme. The resulting low molecular weight (H) -HA fraction is characterized by low polydispersity, therefore its radioactivity has increased. The degradation kinetics of (H) -HA are identical to the degradation kinetics of unlabeled hyaluronic acid.

Claims (3)

5 ml of toluene in which 5 GBq of (H) -methyl bromide are dissolved. The reaction mixture was refluxed for 1 hour under electromagnetic stirring. Liquid ammonia is allowed to evaporate by neutralization (4 mg HN3 Cl). The residue is dissolved! in redistilled water and evaporate again. Dissolution of the residue is pure (3H) -hyaluronic acid isolated from the precipitate by gel-permeation chromatography on a Superose 6 column (meshed agarose) so that the mixture is fractionated! according to the molecular weights in which the radioactivity is measured by the 3 LSS method. High-molecular-weight high (H) -HA high fractions of high purity radioactivity were pooled and analyzed by goal permeation chromatography on a FPLC Pharmacia instrument and quadrant scintillation spoktrometry on a Packard 300 CD. 2 measured values were calculated as follows: * • 3 5 - average molecular weight M (H) -HA = 3.92 x 10 DA - sample polymolecularity D = 1.55 3 - specific radioactivity (H) -HA and 37.2 MBq / mg. Example 3 From the reaction mixture; prepared according to Example 2, an aliquot of the high molecular weight (3H) -hyaluronic acid derivative is injected into the HP GPC system shown in Example 1 and fractionated into fractions of 0.65 ml of effluent in which the LSC radioactivity is measured. In Example 1, 2 fractions with the highest radioactivity are carefully injected into the HP GPC system for characterization! distribution of its molecular weight. The following (3H) -hyaluronic acid derivative values were found: Mean molecular weight Mp = 9.91 x 10 4 Da; polymolecularity D = 1.37; specific radioactivity 7.6 MBq / mg. Similarly, a high-molecular-weight 3? (H) -hyaluronic acid derivative. Example 4 pure (3H) -hyaluronic acid prepared according to Example 2 was subjected to enzymatic degradation by enzyme-hyaluronidase EC 3.2.1.35 (Sevac, USOL Prague) at 37 ° C. The amount of enzyme added was 48 TRU / 1 mg (H) -HA. At time intervals (0.2, 6, 10 and 24 h), 100 µl of reaction mixture was collected, 10 was allowed to boil for enzyme inactivation and centrifuged at 15,000 rpm. HP GPC analysis, as well as the measurement of the radioactivity distribution of individual samples, was performed as in Example 1. 2 radioactivity distributions after 24 hours of digestion showed that the elution volume was 14.4 ml, corresponding to a M = 30,000 Da and radioactivity of 19,693 dpm . After 48 hours of digestion, the elution volume was 15 ml and 21,183 dpm. The resulting radioactive (3H) -HA peak was not altered by the enzyme with the enzyme. The resulting low molecular weight (H) -HA fraction is characterized by low polydispersity, 3 therefore its radioactivity increased. Said kinetics of degradation of (H) -HA is identical to that of unlabeled hyaluronic acid. PATENT CLAIMS A process for the preparation of (3H) radiolabelled fractions of hyaluronic acid or a defined molecular weight thereof, characterized in that the native hyaluronic acid or its salt is labeled with (H) a radioisotope, the radioactivity of the sample is measured by liquid scintillation spectrometry, the radioisotope labeled acid hyaluronic acid, or a salt thereof, is passed through a cation exchange layer preferably from the carboxymethylcellulose derivative group, the hyaluronic acid or a salt thereof is then separated into fractions by the high performance gel permeation chromatography method, then the radioactivity of the respective fraction of hyaluronic acid is measured. new or its salt by liquid scintillation spectrometry. 4 3. 5 EN 276 775 B6 2. A process according to claim 1, wherein the native hyaluronic acid or a salt thereof is labeled with (¾) riodioisotope by transferring a tritium gas to the aqueous solution of hyaluronic acid or a salt thereof in the presence of a Pd / CaCO3 catalyst. solvent and labile radioactivity are lost. A process according to claim 1, wherein the native hyaluronic acid or a salt thereof is labeled with (3H) a radioisotope such that it is alkylated with (3H) -methyl bromide in the liquid ammonia medium to be neutralized! will be removed.