SOLUTION FOR USE IN VISCOSURGERY AND/OR AS A VITREOUS SUBSTITUTE

Abstract: An improved, viscous, transparent, pyrogen-free, isoto­ nic, succinylated atelocollagen solution provides an effective medium for use in viscosurgery and as a replacement for vitreous.

Description: SOLU lQI FOR USE IN VISCOSURGERY AND/OR AS A VITREOUS SUBSllTUl E This invention relates to viscous, transparent, pyrogen-free, isotonic solutions for use in viscosur9ery and/or as a vitreous substihit e. Solutions of fractionatedodiurnhyaIuronate have been used in ophthalmic visosurgery. The surgical uses of these compositions are described in US Patent 4, 141, 973 (Balazs); in Pape, L.G., Balazs, E.A. "The Use of Sodium Hyaluronate (Healon) in Human Interior Segment Surgery" Ophalmology 87:699-705 (1980) and in Healon (sodium hyaluronate) A guide to its Uses in Ophthalmic Surgery, D. Miller and R. Stigmann (John Wily & Sons). Succinylated collagen compositions are known and have been used in contact lenses, US Patent 4, 223, 984 (Miyata et al). in skin or wound dressings, US Patent 4, 294, 241 (Miyata), and in thin membranes suitable for ophthalmic drug delivery, US Patent 4, 164, 559, (Miyata et al). Stabilized collagen gels made from purified tropocollagen have been used as replacements for vitreous in rabbit and monkey eyes. Stenzel, K.H., Dunn, M.W., Rubin A.L. and Miyata, T. "Collagen Gels: Design for a Vitreous Replacement", Science, June 13, 1969 vol. 164 pp. 1282-83. These unmodified collagen gels, however, have proved deficient for they become opaque under pH conditions found within the eye. In accordance with the present invention there is provided a solution of succinylated atelocollagen suitable for use in ophthalmic viscosurgery or as a vitreous replacement medium comprising atelocollagen succinylated to more than about 10% of the total lysine residue and dissolved in a buffered physiologic solution to a concentration of between 0.5% and 5% by weight. According to one embodiment of the invention, an improved viscoelastic medium is provided to facilitate ophthalmic surgery procedures by preventing tissue damage, inter alia, as a result of its ftJnction as a lubricant. The medium also allows more zoom for a surgical ipuJetion while i/lsuring post - aperative tissue separation which precludes adhesion formation. Ophttlairnic surgery performed with the aid of a viscoelastic medium is known in the art as viscosurgey. According to another embodiment of the invention, the succinylated collagen solution (nay be used as an irnproved vitreous replacement suitable for intraocular implantation. The human vitreous humour is a clear gel-like structure located in the posterior part of the eye. In caseS of damaged vitreous, replacement of the damaged vitreous with a clear solution of succinylated collagen may aid in restoring sight. The following non-limiting Examples serve to illustrate the preparation of succinylated collagen for viscosurgery and vitreous replacement in accordance with the invention: EXAMPLE 1 Fresh calf skin was split into three layers, epidermal, corium and flesh. One kilogram of the corium layer was minced using a microcutter and then washed with a 5% NaC1 solution to remove soluble substances from the corium insoluble collagen. After washing with water, the minced corium collagen was treated with pepsin (pepsin:collagen = 0.5:100 in weight) in 20 litres aqueous acidic solution (pH 3) at 200C for 3 days. During the pepsin treatment the mixture was slowly stirred. The corium insoluble collagen was completely solubilized by the pepsin treatment. Such pepsin treatment of collagen is preferred when preparing a biomaterial because the antigenic portion (telopeptides) of the collagen molecule are eliminated by the pepsin treatment. The resulting collagen (atelocollagen) is superior for medical surgical uses to a collagen untreated with pepsin. The solubilized pepsin-treated collagen solution was then filtered successively through 1.0, 0.6 and 0.45 m pore size membranes. The pH of the filtrate was adjusted to 7.5 and the reconstituted collagen fiber (atelocollagen) was collected by centrifuge. The atelocollagen fiber was redissolved in 5 litres of pld 3 aqueou5sololion arid the pH was adjusted to 8 with Z4aOId solution. The alelocollagen which had dissolved in the acidic medium formed a precipitate when the pH was adjusted to 8 and a milky white atelcollagen suspension was obtained. To this su perxsiun, 40 ml of acetone containing 4 g of succinc anhydride was gradually added under mixing at constant pH 8. To maintain pil 8 during addition of succine anhydride, 1 N NaOH was gradually added. The reaction mixture was maintained at pH 8 under rnixing overnight. During the succinylation reaction milky white atelocollagen precipitate was dissolved to give a clear solution. After the reaction, the pH of the mixture was adjusted to 4.5 to form a precipitate of succinylated atelocollagen. The precipitate was collected by centrifuge and washed with water at pH 4.5, five (5) times to remove any unreacted succinc acid. The washed succinylated atelocollagen was freezed-dried for storage. To make the final viscous succinylated atelocollagen solution useful for eye surgery, 10 g of freeze-dried succinylated atelocollagen was dissolved in 1 litre of phosphate buffered saline of pH 7.4 which contained 30 m mol Na2tHPO4 and 200 m mol NaC1. The viscosity of the physiological solution was 1.2 Pa.s and it was pyrogen-free. When the sterile, pyrogen-free succinylated atelocollagen solution thus prepared was implanted into the anterior chamber of a rabbit eye, it showed minimal inflammatory reaction and remained clear, as is required for viscosurgery of the eye. This same viscous solution was used as a vitreous body substitute in a rabbit eye, and remained clear and showed minimal inflammatory reaction, demonstrating its utility as a vitreous substitute. As will be appreciated by those skilled in the art, the viscosity of a succinylated atelocollagen solution can be controlled by adjusting the concentration of the atelocollagen, i.e., while a 1% solution has a viscosity of 1.2 Pa.s a 2% solution has a viscosity of approximately 2 Pa.s, and a 3% solution has a viscosity of approximately 10 P.a.s. Useful buffered physiological solution will range from 0.5% to 5% by weight in concentration of ateJocolIagc!n. 150, instead of steriIizatiun of the succinyloied at elocollagen solution with a 0.45)m millipore filter ethylene oxide gas (goy) can lae utilized. Freeze-dried succinylated atclocollagcn rnay be conveniently treated with EcG and and their absorbed in phosphate buffereo saline after complete rcrnoval of excess EOG. Such an EOG siliz succinylated atelocollagen solution is sterile, pyrogen-free, and useful as a viscosurgery material and as a vitreous body substitute. The degree of succinylation of the E-NH2 group of atelocollagen can be controlled by controlling the amount of succinic anhydride, the pH and the time of reaction. The degree of succinylation of the atelocollagen prepared by the method described above was over 85% and the isoelectric point of the atelocollagen was 4.5. The degree of succinylation can be determined by the TNBS (Trinitrogenen sulfuric acid) method (Kakade, M.L. and Liener, I.E. (1969) Anal. Biochem. 27, 273-280). Antelocollagen which is succinylated to more than 10% of the total lysine residue (E-NH2) is soluble at physiologic conditions (pH 7.0, under isotonic conditions. Therefore, for use in eye visco-surgery, succinylation of more than 10% of the atelocollagen E-NH2 groups is essential in order that the resulting solution is clear. The denaturation temperature (TD) of the succinylated atelocollagen is dependent on the degree of succinylation. Since the TD drops with an increasing degree of succinylation, an upper limit on the desirable degree of succinylation is generally imposed. For example, the TD is 39.50C at 24% succinylation, 39.2 0C at 35% succinylation and 34.00C at 90% succinylation. A higher TD is of course, preferable for eye use. Therefore, while a 10% degree of succinylation of the E-NH2 groups is necessary, a 20-50% succinylation degree is preferable. EXAMPLE 2 Preparation of atelocollagen succinylated to varying degrees was accompli hed as follows: To 300 rnl of 0.5% atelocollagen solution at pH 3.0 was added 11.1 N NaOH to adjust the pH to 9.0. To this atelocollagen at pH 9.0, was added 0.1 ml of 1% succinic anhydride in acetone solution, and the pH of the reaction mixture was maintained at pH 9.0 by adding 0.1 N tJaCH for 3 hours. The mixture was then dialyzed against water to remove succinic acid. The dialyzed succinylated atelocollagen was freeze-dried, and redissolved in physiological saline, pH 7, at an atelocollagen concentration of 2%. The degree of succinylation of this atelocollagen was 11.3% EXAMPLES 3 and 4 The above procedure was repeated using 0.3 ml and 1 ml of the 1% succinic anhydride acetone solution. The resulting atelocollagen samples had degrees of succinylation of 24% and 35% respectively.

Claims: CLAIIAS 1. A solution of succinglated atclocollegen suitable for use in ophtilallnic viscr,surgery or as a vitreous rel)lacerrlent medium comprising atelocollagen succinylated to more Lhan about 10% of the total lysine residue and dissolved in a buffered physiologic solution to a concentration of between 0.5% and 5% by weight. 2. The solution of claim 1 wherein the degree of succinylation of the atelocollagen is between about 20% to 50%. 3. A viscoelastic solution which remains transparent when subjected to an intraocular environment, said solution comprising succinylated atelocollagen dissolved in an aqueous buffer physiologic solution to a concentration of between about 0.5% and 5% by weight. 4. A succinylated atelocollagen wherein 10% or more of the total lysine residues are succinylated. 5. A physiologically acceptable solution of the succinylated atelocollagen according to claim 1 in an aqueous medium. 6. A solution in accordance with claim 5 wherein the concentration of succinylated atelocollagen is such that the viscosity of the solution is suitable for ophthalmic viscosurgery or asavitreous humor replacement medium. 7. A solution in accordance with either of claims 5 and 6 which is substantially transparent.

Inventors: DUNN, MICHAEL W., MIYATA, TERUO
Applicants: KOKEN CO. LTD.
Application year: 1985
Main classification: A61L27
IPC codes: C08H1, A61F2, A61L27