CS275626B6 - A method for preparing fractions of hyaluronic acid or salts thereof - Google Patents

Abstract

It solves the method of preparing fractions of hyaluronic acid (HA) or its salts with a defined molecular weight. The procedure consists in that a solution of HA or its salts with a concentration of preferably 0.01 to 1.00 ¾ wt. is treated with ultrasound at a frequency of 16 to 450 kHz, for a period of 1 to 90 min. Before and/or after the ultrasound treatment, the solution of the substance is preferably allowed to pass through a layer of an ion exchanger, preferably from the group of carboxymethylcellulose derivatives. Characterization and distribution of molecular weights is performed by the HP GPC method. The product is not contaminated with foreign matter. The effect of ultrasound is homogeneous and immediate. The procedure allows for use on a preparative and analytical scale.

Description

The invention relates to a process for the preparation of fractions of hyaluronic acid or its salts of defined molecular weight.

Hyaluronic acid (HA) resp. its salts of formula I

wherein R is hydrogen, sodium, potassium or another cation or different cations, the polysaccharide is a glycosaminoglycan with repeating glucuronate and N-acetylglucosamines (Balazs E.A. U.S. Patent 4141973, 1979). These substances are endogenous polymeric products present in the connective tissues of organisms and microorganisms. HA resp. its salts can be obtained from the tissue of the cockscombs, from the human umbilical cord, from the boxwood or from the trachea of bovine animals, or from the bacteria Streptococcus zooepidemicus. Hyaluronic acid or its salts isolated from any source are structurally identical, but may be isolated with varying degrees of purity as well as degrees of polymerization.

One of the decisive factors in the successful use of isolated hyaluronic acid in human medicine is, in addition to its low toxicity and tolerability of the human body (no immune, inflammatory or allergic side effects have been observed), the significant molecular weight of the biopolymer. In eye surgery, rheumatology, etc. samples with a molecular weight of the order of 5 x 10 6 daltons are required (Holmberg AS, Philipson BT: Ophthalmology 91, 53-59, 1904; Pape LC, Balazs EA: Ophthalmology 06, 109, 1979; Miller 0, Stegmann R .: Ophthalmology 86, 109, 1979; Heaton (sodium hyaluronate) Technical Information and Clinical Experience, Pharmacia, Sweden 1905). On the contrary, for applications in cosmetics, dermatology, or in some veterinary indications, biopolymers with a molecular weight of the order of 10 daltons or less are required (Van Brunt J .: Biotechnology 4, 780-782, 1986).

The safe use of HA and its salts in clinical and veterinary medicine, as well as in other human applications, requires the use of defined fractions and thus the control of molecular weight resp. control of molecular weight distribution. For characterization of the mol. The high-performance gel permeation chromatography (HP GPC) method is the most frequently used method in the world today for determining the average molecular weights of polymers.

The HP GPC method is not an absolute physico-chemical method and therefore the experimental equipment needs to be calibrated with defined fractions of hyaluronic acid or hyaluronic acid. its salt. Isolation of HA fractions or its salts from biological material is of course possible, but the fractions obtained are contaminated to varying degrees and practically unusable in practice. One possible solution is to select a defined animal tissue as the source of the starting HA or its salt, where a reproducible biotechnological process yields a biopolymer of defined purity, from which suitable fractions are then obtained by depolymerization.

Currently, as one of the most progressive sources of HA is the tissue of rooster combs. Of these, HA is isolated by a process according to patents and copyright certificates (Balazs EA: US Patent 414 1973, 1979; Czech Copyright Certificate No. 267 079) of defined biological purity. Butyrate process the resulting hyaluronic acid has a very high molecular weight of the order of 1 to 5 x 10 ^s daltons (NB Beaty, Tew WP, Mello RJ Ana 1yt.Biochem. 147, 38 7-395, 1985 Dark WA: Am Lab./Fa irfi eld Comn./ 20 / 8A /, 22, 19B8; Bothner H., Waaler T., Wik 0 .: Int. J.8 io 1.Macromo1. 10, 287-291, 1908). '

CS 275626 0 6

In principle, two methods have been published on an analytical scale for the preparation of HA fractions or its salts from this biopolymer: physicochemical and biological (enzymatic).

Enzymatic procedure (Beaty NB, Tew WP, Mello R.3 .: Ana 1yt.0 i echem. 14 7, 307-395, 1985; Turner R. I., Lin P., Cowman MK: Arch.Biochem.Biopriys. 265/2 /, 404-495, 1908; Iwata S .: Folia OphthaImo 1.3pn. from the enzyme used-hya 1uroni vase.

Physicochemical procedures circumvent the need for separation-purification procedures of the resulting biopolymer from the enzyme used in the biological process in such a way that the physical πο-chemical agent does not increase the complexity of the resulting sample. Various physicochemical agents have been used to depolymerize HA and its salts:

1. Effect of elevated temperature in an autoclave (Beaty NB, Tew WP, Meloo R.3 .: Analyt. Biochem. 147, 307-395, 1985; Oother M., Waaler T., Wik 0 .: Int.3.0iol.Macromo1 10, 207 291, 1900). ·

2. Irradiation with UV light or electron flux (Balazs EA, Laurent TC, Howe AF, Varga L .: Radiat.Res. 11, 149, 1950; Oalazs EA, Davies 3.V., Philips GO, Young M.0 .: Radiat.Res. 31, 243, 1967).

3. Acid or alkaline hydrolysis (Cleland R.L .: Oiopolymers 23, 647, 1904; Cleland R.L .: Arch. 3iochem. Oiophys. 100, 57-60, 1977).

4. Effects of free radicals (Wong SF, Halliwell 0., Richmond R., Skowroneck WR: 3. Inorg.Oiochem. 14, 127, 1901; Lál M .: 3.Radioana1.Nucl.Chem. 92, 105, 1905) .

However, the successful use of the above procedures is considerably limited due to the fact of inhomogeneity of kinetically controlled reactions (such as heat transfer through the sample during thermal degradation; or diffusion of radicals in the depolymerization effects of radicals), which is particularly pronounced on a semi-preparative and preparative scale.

The disadvantages of all previously proposed and used processes are eliminated by the process for the preparation of fractions of HA or its salts with a defined molecular weight according to the invention, the essence of which is to apply a solution of hyaluronic acid or its salt, preferably at a concentration of 0.01 to 1.00. acts with ultrasound at a frequency of 16 to 450 kHz, for 1 to 90 minutes. Before and / or after the sonication, the solution of the substance is purified at least once by passing it through a layer of ion exchanger, preferably from the group of carboxymethylcellulose derivatives, preferably before the sonication.

The advantage of using ultrasound is that the biopolymer sample is not contaminated with another chemical or biochemical agent. The effect of high-energy electromagnetic radiation is not limited by effective kinetic factors, the action of ultrasound is homogeneous and immediate, which allows its use in both analytical and preparative scale. The ultrasonic depolymerization reaction of hyaluronic acid or its salt is energetically, instrumentally and materially inexpensive, clean and simple.

The following examples illustrate the proposed process without limiting in any way the scope of protection to which the present invention is intended to apply '.

Example 1

An ultrasonic probe with a frequency of 20 kHz and an energy of 100 W was immersed in 1 ml of 0.05 wt. By sonication, the molecular weight of sodium hyaluronate decreased from the original M = 1.26 x 10 ° Dana 6.67 x 10 °. The molecular weights of the biopolymers were determined by high performance gel permeation chromatography (HP GPC) on two columns packed with hydroxy sorbent.

CS 275626 B 6 ethyl methacrylate (Separon HEMA-S 1000 and Separon HEMA-S 300), connected in series. The mobile phase consisted of a 0.1 M aqueous solution of sodium nitrate NaNO 3 and a refractometric detector was used for detection.

Example 2

0o 1 ml 0.05 t wt. A solution of the magnesium sodium salt of hyaluronic acid in 0.1 M aqueous NaNO 3, pre-purified on a separation minicolumn with a carboxymethylcellulose layer, was immersed in an ultrasonic probe with a frequency of 40 kHz and an energy of 150 W for 75 min. The molecular weight of sodium hyaluronate decreased from the original M = 1.13 6 5 P x 10 O and 2.16 x 10 0a by sonication. The molecular weights of sodium hyaluronate were determined by the above HP GPC method.

Example 3

10 ml of a 0.5% strength by weight solution of sodium hyaluronic acid in 0.1 M aqueous NaNO 3, pre-purified on a separation minicolumn with a carboxymethylcellulose layer, were sonicated at a frequency of 150 kHz and an energy of 400 W for 24 min. The molecular weight of sodium hyaluronate was reduced from 2.05 x 10 6 to 5.66 x 10 6 Da by sonication of the sealed sample. The molecular weights of the biopolymers were determined by the indicated HP GPC method.

Example 4

An ultrasonic probe with a frequency of 20 kHz and an energy of 150 W for 15 minutes was immersed in 1 ml of a 1.0 .mu.l solution of hyaluronic acid in 0.1 M aqueous NaNO3, pre-purified on a separation minicolumn with a layer of carboxymethylcellulose. Ultrasound reduced the mol. weight of hyaluronic acid from Mp = 1.49 x 10 ^ Da to 3.32 x 10 $ 0a.

The molecular weights of HA were measured by the indicated HP GPC method.

in

The ultrasound equipment used has a power output of about 150 W.

in the range of 40 to 400 W, preferably

Claims (3)

PATENT CLAIMS A process for the preparation of fractions of hyaluronic acid or its salts of defined molecular weight, characterized in that the solution of hyaluronic acid or its salts is treated with ultrasound at a frequency of! 16 to 450 kHz, for 1 to 90 min. 2. The preparation method according to item 1, characterized in that before and / or after the sonication, the solution of the substance is passed at least once through an ion exchange layer, preferably from the group of carboxymethylcellulose derivatives. 3. The process according to item 1, characterized in that a solution of hyaluronic acid or its salt in a concentration of 0.01 to 1.00 wt.