This invention relates to methods for the controlled molecular weight reduction of polymers using specific amounts of high-energy irradiation, such as gamma irradiation. The methods of this invention permit the direct reduction of the molecular weight of such polymers to preselected lower molecular weight levels with low polydispersity. Polymers which are particularly suitable for use in the present invention include the polyanionic polysaccharides, and more particularly hyaluronic acid (xe2x80x9cHAxe2x80x9d) and carboxymethyl cellulose (xe2x80x9cCMCxe2x80x9d).
Hyaluronic acid is a linear long-chain polysaccharide comprising repeating D-glucuronate and N-acetylglucosamine disaccharide units. Hyaluronic acid is not species specific, and it may be obtained, for example, either by extraction from animal tissues, such as rooster combs and umbilical cords (Klein, J., and Meyer, F. A., 1983, Biochem. and Biophys. Acta, 755(3), 400-411), or by the removal of hyaluronic acid capsular material form bacterial species, e.g. Streptococcus (Van Brunt, J., 1986, Biotechnology, 4, 780-782). Hyaluronic acid from such sources exists as a mixture of different molecular weight species, and the overall molecular weight of the product is expressed as a weight average molecular weight.
Hyaluronic acid has a variety of therapeutic applications, such as its use in ophthalmic surgery and for post-operative adhesion prevention, as well as having potential uses in a number of other areas. The key to many of the uses of hyaluronic acid lies in its hydrodynamic properties (Van Brunt, J., 1986, Biotechnology, 4, 780-782), which produces highly viscous solutions at low concentrations. The viscosity of a hyaluronic acid solution is primarily dependent on the molecular weight, and the concentration of hyaluronic acid in solution.
The viscosity properties may be altered by the dilution of the polymer, but in many applications this approach is not acceptable because a specific concentration of the polymer is required for particular applications. Proposed strategies to produce hyaluronic acid having a variety of preselected molecular weights include the selection of bacterial mutants capable of producing the desired hyaluronic acid molecular weight range, or the alteration of the physiological conditions during growth of a bacterium to enhance the production of a particular molecular weight range. However, neither of these techniques provides both the range and diversity of molecular weight species required for specific applications due to the inherent limitations of these methods.
Alternatively, the reduction of high molecular weight hyaluronic acid to moieties of lower molecular weight may be achieved by enzymatic, chemical or physical means. Enzymatic methods of degrading hyaluronic acid are known, (Hamai, A., et al, 1989, Agric. Biol. Chem. , 53 (8), 2163-2168), but are relatively uncontrollable and tend to broaden the hyaluronic acid molecular weight distribution and therefore increase the polydispersity of the material. This makes the material particularly unsuitable for certain applications where highly defined molecular weight ranges are required. Chemical methods (Harris, M. J., et al, 1972, JACS, 94, 7570-7572), suffer similar problems and, moreover, may result in residual concentrations of the reacting chemicals remaining in a therapeutic product. Fractionation of hyaluronic acid into defined molecular weight species is feasible (Armand, G., and Reyes, M., 1983, Biochem. and Biophys. Res. Comm., 112(1), 168-175), but this is a complex operation which is not easily controllable in large scale manufacturing operations.
U.S. Pat. No. 5,491,227 describes a method for the molecular weight reduction of polysaccharides, such as hyaluronic acid, carboxymethyl cellulose and guar gum, by subjecting the polymers to pressure homogenization in a single pass through the device at pressures ranging from 10 Mpa to 25 Mpa. The hyaluronic acid product obtained as a result of the pressure homogenization has an average molecular weight of from about 1.2 to about 1.5xc3x97106D, based upon an initial molecular weight of 1xc3x97107D to 2xc3x97107D. One potential shortcoming of this method is that it requires the polymer, in this case hyaluronic acid, to be processed in the solution phase. This requires the additional processing step of solidification and drying of the polymer so if, as is typically the case, a powdered product is desired. In addition, some polymers may not be amenable to being readily solubilized, and since this method requires a rather precise measurement of apparent viscosities, this can present processing problems.
The use of irradiation, such as gamma irradiation, to sterilize polymers and non-polymeric materials is known. It is also known that certain types of irradiation can be used to initiate polymerization reactions in certain polymers, and for the addition or substitution of certain pendant groups onto a polymer chain.
European Patent Application No. 269937 describes a process for the controlled preparation of low molecular weight glucosaminoglycans by treating the corresponding high molecular weight glucosaminoglycans with rectilinear gamma radiation at doses within the range of 2.5 to 20 Mrad. The glucosaminoglycans which can be used in this process include heparin, chondroitin sulphate, keratan sulphate and hyaluronic acid, with heparin being the preferred glucosaminoglycan. The gamma radiation is supplied in successive irradiation stages, followed by intervening cooling steps to prevent alteration of the molecular structure of the heparin. The reference is directed to low molecular weight heparin molecules that are reduced to fragments which are claimed to still possess biological activity.
It will be appreciated that it would be desirable to develop an improved process for the molecular weight reduction of high molecular weight polymers, such as hyaluronic acid and carboxymethyl cellulose, to preselected lower values without the necessity of additional purification or other processing steps required in conventional approaches.
The present invention features a method for the reduction in the molecular weight of high molecular weight polymers to substantially lower, preselected levels. The method of this invention comprises subjecting the high molecular weight polymer to a dose of irradiation sufficient to achieve the desired degree of molecular weight reduction. The present method is applied to the polymer in the solid phase, thereby eliminating the necessity of preparing a polymer solution. The use of polymer solutions requires a solubility step which is difficult to perform for some polymers because of their limited solubility and/or the high viscosity of the solutions. The present method also avoids the expense and trouble of solidifying the polymer after the proper molecular weight has been achieved. Using the method of this invention, the molecular weight of polymers can be reduced directly in a controlled and reproducible manner, without the use of chemicals or enzymes that must be subsequently neutralized or removed.
The type and dosage of the irradiation that can be employed in the practice of this invention will vary depending on the type of polymer treated, the degree of molecular weight reduction desired, and the form of the polymer, i.e. whether the polymer is in the form of a salt. The preferred type of irradiation is gamma irradiation, although other types of irradiation, including heat sources, such as microwave irradiation, can also be used. Typically, the dosage of irradiation used will vary from about 1 kGy to about 120 kGy.
In one embodiment, the polymer which can be treated according to the method of this invention is a polysaccharide polymer, preferably hyaluronic acid with an initial molecular weight in the range of from about 3,000 kdaltons to about 1,000 kdaltons. In another embodiment, the polysaccharide polymer is carboxymethyl cellulose with an initial molecular weight in the ranger of from about 300 kdaltons to about 100 kdaltons.
The process of this invention does not require the use of additional processing steps in order to avoid structural changes to the polymer, or to remove impurities which may have been introduced into the polymer preparation as a direct result of the polymer processing.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder, including published patent applications, and issued or granted patents, are incorporated by reference herein. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.