WO1992000745A1

WO1992000745A1 - Collagen-based viscoelastic solution for visco-surgery

Info

Publication number: WO1992000745A1
Application number: PCT/US1991/004572
Authority: WO
Inventors: Charles D. Kelman; Dale P. Devore
Original assignee: Autogenesis Technologies, Inc.
Priority date: 1990-07-03
Filing date: 1991-06-26
Publication date: 1992-01-23
Also published as: US5631243A; CA2086014A1; JPH05508417A; JP3224815B2

Abstract

The present invention relates to a method of treating a patient with visco-surgery, particularly ocular visco-surgery, utilizing a collagen-based viscoelastic solution having a reversibly high viscosity. The invention also relates to collagen-based viscoelastic solutions which further include mucopolysaccharides or growth factors.

Description

COLLAGEN-BASED VISCOELASTIC SOLUTION FOR VISCO-SURGERY

FIELD OF THE INVENTION

The present invention relates to viscoelastic solu¬ tions of collagen suitable for use in visco-surgery, particu¬ larly ophthalmic visco-surgery. Specifically, the present invention relates to such viscoelastic solutions wherein their viscosity is dramatically reduced upon the addition of solu¬ tions containing cations, such as saline solution, balanced salt solution, etc.

BACKGROUND OF THE INVENTION

Aqueous lubrication solutions have widespread use in ophthalmic surgery as protective agents for the easily damaged intraocular tissues. A major concern in ophthalmic surgery has been the frequent observation of elevated intraocular pressure associated with the surgical use of visco-elastic solutions.

Elevated intraocular pressure can reduce the flow of blood-born oxygen and nutrients to the eye and to the optic nerve. Not surprisingly, such elevated pressure is strongly correlated with loss of vision (glaucoma) . It is, thus, desirable that solutions for ophthalmic surgery be easily removed from eye tissues by irrigation and aspiration. It is further desirable that any residues remaining in the eye show reduced tendency to induce elevated ocular pressure.

A number of solutions have been used or proposed for use as viscoelastic solutions. These include solutions of sodium hyaluronate (Healon™, Phamacia LKB Biotechnology, Inc. and Amvisc™, IOLAB); chondroitin sulfate; mixtures with chon- droitin sulfate with sodium hyaluronate (Viscoat™, Cooper- vision/ALCON) ; methylcellulose; carboxymethylcellulose; polyacrylamide or polymethacrylamide; and collagen derivatives. A number of these solution are reviewed by Arshinoff, Curr. Can. Ophthalmic Prac. 1: 64-74, 1986.

Sodium hyaluronate solutions are the most widely used viscoelastic solutions for ophthalmic surgery. Balazs (U.S. Patent 4,141,973) or Pape (U.S. Patent 4,328,803) describe their manufacture and/or use. These solutions, however, have been found to induce transient elevations in intraocular pressure. They have the further disadvantage of being expen¬ sive and failing to adhere to ocular prostheses and surgical instruments. Chondroitin sulfate solutions are not pseudoplastic

(and not viscoelastic) and consequently are difficult to inject through the narrow gauge needles used in ophthalmic surgery (see MacRae et al., "The Effects of Sodium Hyaluronate, Chon¬ droitin Sulfate and Methyl Cellulose on the Corneal Endothelium and Intraocular Pressure," Amer. J. Qphthal. £5:332-341, 1983). Additionally, these solutions are less effective than sodium hyaluronate solutions in maintaining space between separated ocular tissues. Finally, such solutions are associated with sharp elevations in intraocular pressure. Unmodified collagen is usually unsuitable for oph¬ thalmic solutions due to its low solubility at pH values close to neutral pH. However, Devore et al. (U.S. Patents 4,713,446 and 4,851,513) and Miyata et al. (U.S. Patent 4,748,152) have developed collagen-based viscoelastic solutions wherein the collagen is derivatized at lysyl amino acids to increase its solubility at neutral pH.

Miyata et al. (supra) teach partially succinylated ateleopeptide collagen dissolved in physiologic saline. The viscosities taught therein are modest (highest is 10,000 centipoise for a 3% solution). Miyata et al. have no teachings relating to a collagen solution that is readily irrigated out of eye tissues. Nor are there any disclosures in Miyata et al. of the effects of the solution on intraocular pressure. Devore et al. (supra) teach a method or producing collagen suitable for viscoelastic solutions by acylating the lysyl amino acids of collagen with a combination of monofunc- tional and bifunctional reagents. The result of this acylation treatment is that some collagen lysine amino groups are odi- fied to substitute a carboxylic acid group in place of the basic amino function. The residue of the lysine amino groups are covalently linked to lysine amino groups of the same or an adjacent collagen molecule. The viscoelastic solution is produced by reconstituting this derivatized collagen in a physiologic saline solution. These are no disclosures in the Devore et al. patents of collagen viscoelastic solutions that are readily irrigated out of ocular cavities. Neither is there any disclosure of the effects of the collagen solutions taught therein on intraocular pressure. Desirable properties of viscoelastic solutions include: transparency; stability (i.e. they maintain their useful properties over long term storage); high viscosity; pseudoplasticity; viscoelasticity; thixotropy; and biological compatibility. Pseudoplastic solutions are useful in visco-surgery because their viscosity drops when they are subjected to high shear flow, such as occurs during injection through the high gauge (narrow) needles used in ophthalmic surgery. (Pseudo- plastic fluids are non-Newtonian fluids wherein the kinetic viscosity (centipoise'sec or cps) decreases as the sheer rate increases.) Thus, pseudoplastic surgical solutions are inject- able despite their viscosity.

Viscous solutions are useful due to their tendency to resist flowing out of the ocular cavity into which they have been injected.

Viscoelastic solutions are useful in viscosurgery because they resist having their shape deformed (i.e., they are elastic), thus helping to maintain the shape of body cavities into which the solutions are injected. (The elasticity of the solution results in the Weissenberg Effect, which describes the tendency of viscoelastic solutions to flow at right angles to an applied force. ) During surgery, viscoelastic materials protect cell and tissue surfaces from mechanical trauma; create space by separating two adjacent but not adherent tissue surfaces, or by breaking normal or pathological tissue adhe¬ sions; maintain space, for instance, in the interior chambers of the eye or in the lens sack, allowing for safe surgical manipulations by permitting the insertion of implants without dislocating or touching sensitive tissues; contain hemorrhages; and also act as a "soft instrument" or "surgical tool" to move, manipulate or relocate tissues.

Following injection of a viscoelastic solution, at high shear, through a narrow gauge needle (e.g. into a chamber of the eye) , it is useful for the solution to regain the majority of its viscosity. Solutions having this recovery property are "thixotropic."

A biocompatible substance, as used herein, shall be noninflammatory, nontoxic, nonimmunogenic, pH buffered and shall have osmolarity between about 200 milliosmoles and about 400 milliosmoles, preferably between about 250 milliosmoles and about 350 milliosmoles, most preferably between about 280 milliosmoles and about 330 milliosmoles.

What is needed in the art is a viscoelastic solution that has transparency, stability, high viscosity, pseudoplas- ticity, viscoelasticity, thixotropy, and biological compatibil- ity, but moreover, can be used in ophthalmic surgery with reduced risk of elevated intraocular pressure.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have unexpectedly discovered that soluble collagen, when reconstituted in low ionic strength buffers, shows markedly increased viscosity and continues to have viscoelastic properties. This increased viscosity is maintained as the osmolarity of the solution is increased using nonionic solutes.

By this invention, high viscosity solutions useful for ophthalmic surgery (or any visco-surgery involving delicate tissue in need of protection) can be prepared at lower collagen concentrations. The viscosity of the solution can, subsequent- ly, be dramatically lowered by simply diluting or irrigating with saline solutions. Thus, the viscoelastic solutions of the present invention are easily irrigated out of ocular cavities following ophthalmic surgery. Therefore, their use reduces the risk of elevated intraocular pressure associated with residues of viscoelastic solution remaining in the eye following sur¬ gery.

It has been discovered that viscoelastic solutions according to the present invention reduce the risk of elevated ocular pressure even when they are not washed out of ocular spaces.

The addition of mucopolysaccarides, such as chondroi¬ tin sulfate, and growth factors, such as epidermal growth factor (EGF) or growth hormone (GH), to the viscoelastic solutions used in the present invention is expected to provide further benefits.

DETAILED DESCRIPTION OF THE INVENTION

The viscoelastic collagen solutions of the present invention have osmolarities ranging between about 260 and 340 milliosmoles, about 75% to about 95% of the osmolarity provided by nonionic solutes such as glycerol.

Such solutions have been found to have higher viscos¬ ities or the same viscosity at lower concentrations of soluble collagen then comparable collagen solutions dissolved in physiologic saline solutions. Collagen suitable for use in the present invention can be prepared by reacting collagen (at its lysyl amino acids) with an acylating agent, e.g., glutaric anhydride, succinic anhydride, heptafluorobutyric anhydride, methacrylic anhydride, phthalic anhydride, methyl glutaric anhydride, dimethyl glutaric anhydride, benzene sulfonic acid, B-styrene sulfonyl chloride, hydroquinone sulfonic acid, polyvinyl sulfonic acid, etc. For instance:

(I) P-(CH2)₄NH₂ + 0 H

where P is the collagen backbone.

The above reaction reverses the ionic charge at physiologic pH of the reacted lysyl amino acids (plus to minus). The reaction also increases the solubility at physiologic pH of the collagen product.

Such collagen can be reconstituted (following inter¬ mediate processing steps) in sodium phosphate buffer to give a kinetic viscosity of 30,000 cps (at 1 sec"¹) and dialyzed against distilled water. After 24 to 72 hours of dialysis, the viscosity is dramatically increased (e.g. 200,000 cps). The viscosity of the solution can be reduced to about 60,000 cps by dilution with distilled water. The osmolarity of such a solution of collagen can be increased to the physiologic range (205 to 405 milliosmoles) by addition of a nonionic solute, such as glycerol, without substantial loss of viscosity.

However, the addition of salt to increase the ionic strength to at least 0.0022u produces a drop in viscosity of as much as 88% (with dilution effects accounting for none of the viscosity drop). Preferably, the addition of salt to increase the ionic to 0.0043u produces a drop in viscosity of at least 50 fold. The dialysis step is not essential to the invention. Compar¬ able high, reversible viscosities are seen with collagen solutions that are reconstituted simply by dissolving precipi¬ tated or lyophilized collagen in low saline buffer.

It is known in the prior art that dialysis of colla- gen against water results in long strands of end-to-end associ¬ ate collagen molecules (see Bruns et al., U.S. Patent 4,581,030). Such end-to-end association results in increased effective molecular weight and some associated increase in viscosity. While not wanting to be restricted to theory, this previously observed phenomenon does not adequately explain the dramatic viscosity effects described herein. The dramatic increase in viscosity seen in the solutions of the present invention (e.g., 30,000 cps increased to 200,000 cps) suggest more complex, multiple interactions such as seen for hyaluro- nate solutions (see Balasz, E., Sodium Hyaluronate and Visco- surgery, Healon, Miller, D. and Stegman, R. , eds. John Wiley & Sons, New York, 1983).

The viscoelastic collagen solutions of the present invention are prepared by reconstitution of collagen in a low saline physiologic buffer. "Reconstitution," as used herein, means any method of obtaining collagen dissolved in a desired buffer. This may be done by a number of methods including:

1) Dialysis to exchange the buffer dissolving collagen for the desired buffer. 2) Redissolving precipitated collagen in the desired buffer.

3) Dilution of a collagen solution with a buffer concentrate to obtain the desired buffer composition. As used herein, "low saline" buffer shall mean buffer having ionic solutes that yield no more than about O.OOlu of the ionic strength, preferably no more than about O.OOOδu. A "low saline physiologic" buffer shall meet the above saline requirement and have nonionic solutes for a total osmolarity between about 200 and about 400 milliosmoles, preferably between about 250 and about 350 milliosmoles, most preferably between about 280 and about 330 milliosmoles.

Appropriate nonionic solutes include glycerol, sor- bitol, xylitol, threitol, mannitol, arabitol, ribitol, adon- itol, erythritol, dulcitol, altritol, iditol, various heptoses, hexoses and pentoses, polyglycols, etc.

Since mucopolysaccarides such as chondriotin sulfates (e.g., chondroitin sulfate A, B or C) adhere to endothelial cells, it is anticipated that the addition of mucopolysacca¬ rides to the viscoelastic collagen solutions will also increase the adherence of the solution. This increase in adherence should enhance the protection of endothelial tissues (such as the tissues lining the ocular cavities). While the source of the affinity of such mucopolysaccarides for endothelial cells is not completely understood, it may, in part, be due to the receptors for mucopolysaccarides which are present on the endothelium (Lutjen-Drecoll, E., ARVQ Annual Meeting Abstracts, April 29-May 4, 1990, Assoc. for Res. in Vision and Opthamo- logy, publisher, Vol. 31(4), p. 184). Other mucopolysaccarides which should be useful adherence promoting additives to the viscoelastic collagen solutions are salts of hyaluronic acid, dermatan sulfate (also known as chondroitin sulfate B), keratan sulfate, heparan sulfate, etc. A useful range of collagen to mucopolysaccaride weight ratios is anticipated to be between about 100:1 and about 10:1, more preferably between about 40:1 and about 20:1.

Hyaluronic acid is available from Sigma Chemical Co., St. Louis, MO (from human umbilical cord, bovine vitreous and rooster comb) and from Calbiochem Biochemicals, San Francisco, CA (from human umbilical cord). Chondroitin sulfate A is available from Sigma Chemical Co. (from bovine trachea) and Calbiochem (from bovine trachea). Chondroitin sulfate B is available from Sigma Chemical Co. (from bovine mucosa, porcine skin) and Calbiochem (from porcine mucosa) . Chondroitin sulfate C is available from Sigma Chemical Co. (from shark cartilage) and Calbiochem (from shark cartilage) . Keratan sulfate is available from Sigma Chemical Co. (from bovine cornea) as is heparan sulfate (from bovine intestinal mucosa and bovine kidney). A number of these mucopolysaccarides are also available from Seikagaku Kogoyo Co., Ltd., Tokyo, Japan. Growth factors (such as EGF, transforming growth factors (TGFs) GH, interleukins, fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), insulin-like growth factors (IGFs) , etc. ) may be added to the collagen viscoelastic solution. These growth factors are anticipated to enhance the viability of the tissues manipulated and exposed during the course of visco-surgery. The amount of growth factor added to the viscoelastic solution of the present invention will vary with the specific growth factor, but will generally be in the range of 10-1,000 ng/ml. The range for EGF is anticipated to be between about 1 ng/ml and about 100,000 ng/ml; for FGFs between about 10 ng/ml and about 500 ng/ml; and for PDGFs and IGFs between about 10 ng/ml and 10,000 ng/ml.

Growth factors have been found to enhance the rate of wound healing in many tissues, including corneal tissues (see Dijke et al.. Bio/Technology, 7:793-798, 1989). Accordingly, it is anticipated that growth factors will enhance the rate of recovery of corneal tissue from the trauma of visσosurgery. For example, EGF has been shown to increase corneal reepitheli- alization and to stimulate corneal cell-proliferation and migration. In addition, preliminary data suggests that EGF enhances corneal reinnervation and stimulates into corneal endothelial cell-proliferation.

EGF is available from Amgen, Thousand Oaks, CA and Sigma Chemical Co. ; fibroblast growth factors are available from Amgen Biochemicals and R&D Systems, Oxford, England; transforming growth factors are available from R&D Systems; and PGFs are available from R&D Systems and Collaborative Research, Inc., Medford, MA. Interleukins are available from R&D Systems and Collaborative Research, Inc.; IGFs are available from R&D Systems; and GH is available from Cambridge Medical Technolo¬ gies Corp., Billerica, MA and Boehringer Mannheim Corp., Indianapolis, IN.

The pH of the viscoelastic collagen solution is preferably stabilized generally by between about 5 mM and about 50 mM of buffer. Buffers that are usable with the invention are any that are biologically compatible. These include phosphate buffers, carbonate buffers, and phosphate-bicar¬ bonate.

The saline solutions that may be used to reduce the viscosity of the viscoelastic solutions of the present inven¬ tion include any of the numerous such solutions that are used in medicine or biology research for non-disruptive manipulation of tissues and cells. These include numerous phosphate buf¬ fered saline solutions (e.g., 0.84 percent by weight NaCl, 0.054 by weight KC1, 0.028 percent by weight Na₂HP0 , 0.004 percent by weight NaH₂PO_ή and, optionally, 0.017 percent by weight CaCl₂), Hank's balanced salt solutions (GIBCO Labs., Grand Island, New York) and Balanced Salt Solutions available from several ophthalmic pharmaceutical companies, including Alcon Laboratories (Ft. Worth, TX), Pharmafair (Hauppauge, NY), Akorn, Inc. (Abita Springs, LA), and Allergan Pharmaceuticals (Irvine, CA) . In general, these saline solutions will have os- molarities ranging from about 280 to about 340 milliosmoles and sodium salts in excess of potassium salts (e.g., 20.5 to 1 ratio such as for the above-described phosphate buffered saline solution) .

Collagen suitable for use in the present invention is soluble at physiologic Ph. Usually, this means a collagen that has been reacted with an acylating agent having two or more carboxylic acids or carboxylic acid derivatives. The degree of reaction being effective to substantially solubilize collagen at physiolic pH. For Type I collagen an effective degree of acylation is thought to be in excess of 35% of collagen lysyl amino acids acylated. Type IV collagen, when isolated from the placenta, is soluble a physiologic pH without further process¬ ing. Type II and Type III collagen are not as abundant as Type I and Type IV and are, for this reason alone, not anticipated to have as much utility in the practice of the present inven¬ tion as Types I and IV. However, these collagens are antici- pated to be solubilized by substantially the same degree of acylation as is effective for Type I collagen.

It is anticipated that collagen from unusual sources or tissues not traditionally used as a source of commercial collagen may have somewhat different solubility properties. The degree of acylation required to prepare such collagen for use in viscoelastic solutions with the viscosity properties of the present invention can be determined by routine experimenta¬ tion. Such viscoelastic solutions are within the scope of the present invention. A preferred acylating reaction for the solubilization of collagen is taught in U.S. Patents 4,851,513 and 4,713,446. Also preferred is acylation using glutaric anhydride (reaction pH between about 7.0 and about 9.0). Another source of colla¬ gen suitable for use in the present invention is disclosed in U.S. Patent Application No. 157,638, filed February 18, 1988, wherein a method is taught for solubilizing telopeptide-con- taining collagen from comminuted animal tissue using acylating reagents. (All patents, patent publications and literature citations found herein are incorporated by reference into this disclosure.)

The acylating reagents useful for preparing the collagen used in the present invention include C₃ to C₈ organic compounds having two or more carboxylic acid-related functional groups, at least one of which is an amino-reactive form (e.g., acid halide, acid anhydride, ester, alkyl cyanate). Usually, the collagen will be prepared from a non- human crude collagen source. The method for obtaining the collagen from the crude collagen source, e.g., tendon, hide, etc. , is normally not critical, and some flexibility may be used in the selection of the particular tissue and the method applied thereto. Applicants prefer to extract collagen from a connective tissue, such as bovine hide. If the collagen is to be used for ophthalmic applications, it is preferred that it be obtained solely from the corium layer of the bovine hide, otherwise known as "split" hides. Split hides are commercially available from the Andre Manufacturing Co., Newark, New Jersey.

The collagen may be solubilized by any of the stan¬ dard extraction methods, e.g., acid or salt extraction, enzyme- digestion, or a combination of these. The acylation-dependent solubilization of U.S. Patent Application No. 157,638 (supra) may also be used. Preferably, dehaired and cleaned hide is solubilized with a proteolytic enzyme (pepsin, for example) and solubilized collagen is precipitated at pH 7, after inactiva- tion and removal of the enzyme, by addition of NaCl to about 2.5M. Pepsin-treated collagen precipitates leaving behind in solution (to be discarded) the digested nonhelical terminal peptides of the collagen molecule and other non-collagenous contaminates, e.g., saccharides, mucopolysaccharides, etc. Inactivated enzymes are removed by filtration and centrifuga- tion at 4^CC. The pepsin-treated collagen is then further purified by repeated redissolution in acidic water (pH 2-4) and reprecipitation by salt treatment, e.g., by the addition of 0.8 M sodium chloride solution at pH 3.

It is preferred that the chemicals used during collagen processing, particularly the final stages of process¬ ing, be free of inflammatory contaminants (e.g., pyrogens). It is preferred that the kinetic viscosity of the viscoelastic solutions of the present invention be at least about 1,000 cps, but less than about 500,000 cps. It is particularly preferred that the viscosity be between about 45,000 cps and about 80,000 cps. It is preferred that upon addition of at least equal volumes of irrigating solution, such as Balanced Salt Solution, the kinetic viscosity should drop at least 50%. It is particularly preferred that this drop in viscosity be at least 75%.

The invention is described below with specific working examples which are intended to illustrate the invention without limiting the scope thereof.

EXAMPLE 1

One liter of sterile filtered, purified, atelopeptide collagen (1.5 mg/ml to 4.0 mg/ml) in 0.1M acetic acid is placed in a pyrogen-burnt glass beaker and stirred with a magnetic stir bar. A sterile solution of ION NaOH is added to bring the pH to about 9.0. Sterile glutaric anhydride (18% by weight of the collagen weight) is added to the solution as a powder. Through the course of the acylation procedure the pH is main¬ tained at about 9.0 by incrementally adding sterile IN NaOH. After approximately 2 minutes, succinic dichloride is added (7% by weight of the collagen weight) via a syringe outfitted with an 0.2um Gelman syringe filter (Gel an Sciences Inc., Ann Arbor, Michigan). After 30 minutes, another portion of glutar¬ ic anhydride (18% by weight of collagen weight) is added. After an additional 60 minutes, the acylation is stopped by addition of 6N HC1 until the pH is 4.3. Under these condi¬ tions, the acylated collagen precipitates.

After 60 minutes, the collagen precipitate is col¬ lected by centrifugation. The precipitate is washed three times by resuspension in pyrogen-free water and centrifugation. The collagen is then resuspended in approximately 80ml of 0.005M sodium phosphate buffer the osmolarity adjusted to 305±25 milliosmoles with glycerol (pH 7.4+0.4). After an overnight incubation, the collagen is completely dissolved. The solution is then diluted by small increments until the viscosity (measured from aliquots using a Brookfield Viscome- ter, Brookfield Instruments, Stoughton, MA) is between about 45,000 cps and about 80,000 cps at a shear rate of 1 sec"¹. The osmolarity and pH of the sampled alloquots are also moni¬ tored. The osmolarity is monitored using a Precision Micro Osmometer (Precision Systems, Natick, MA) and adjusted by dilution with glycerol-free sodium phosphate buffer (0.005M) or by addition of glycerol. The pH is monitored with a pH elec¬ trode (Orion Research, Cambridge, MA) and adjusted with NaOH or HC1 solutions as appropriate.

The concentration of collagen is then measured based on hydroxproline content determined using the method of Woes- sner (In The Methodology of Connective Tissue Research, D. Hall, ed., Joynson-Bruwers, Ltd., Oxford, page 227). Uronic acid content, an indicator of glycosaminoglycan presence, is measured using the standard carbazole method (Bitter & Muir, Anal. Biochem. , 4_, page 330, 1962). As a further quality control measure, a UV scan is performed on each lot of col¬ lagen-based viscoelastic solution.

Once the viscosity, osmolarity and pH are adjusted to the specification values (see Table I), the solution is fil¬ tered through a sterile 5.0um filter.

Table I Viscoelastic Collagen Solution Specifications

Characteristic Acceptable Preferred

Osmolarity 305 ± 100 m Osmol 305 ± 25 m Osmol pH 7.4 0.6 7.4 ± 0.4

Color Colorless

Viscosity 1,000-500,000 cps^a 45,000-80,000 cps

Endotoxin < 500 Eu/ml^ < 5 Eu/ l

Collagen

Concentration < 10% (w/v) < 2.0% (w/v)

Melt Temperature 5-100°C 30-40°C

Sterility Sterile Sterile

Ionic Strength < 0.001 u < 0.0008 u

^aAt a shear rate of 1 sec^" Eu •= endotoxin units.

EXAMPLE 2

Lot A and comparative Lot A' collagen solutions were acylated as outlined in EXAMPLE 1, but were redissolved after the washing step as follows:

Lot A: - Collagen was redissolved in 0.1N sodium phosphate, pH 7.69, with glycerol added to adjust the osmolarity to 326 milliosmoles. - Viscosity was 69,200 cps (at 1 sec"¹) at a collagen concentration of 1.13% (w/v).

Lot A' : - Collagen was redissolved in IN sodium phos¬ phate buffer, pH 7.0, with the final osmo- larity adjusted by dilution with sterile water to 268 milliosmoles. - Viscosity was 43,800 cps (at 1 sec"¹) at a collagen concentration of 3.9% (w/v).

EXAMPLE 3

Two more lots (B and C) were prepared by acylating atelopeptide collagen with glutaric anhydride alone. These lots were redissolved, following three water wash steps (com¬ parable to those outlined in EXAMPLE 1) , in IN sodium phosphate buffer. These lots were then dialyzed against deionized water for two days. Lot B was adjusted to a protein concentration of 1.55% (w/v), a pH of 6.8, and a osmolarity (adjusted with glycerol) of 275 milliosmoles. The kinetic viscosity was 228,000 cps (at 1 sec"¹). FIGURE 1 shows the kinetic viscosity profile (log

(shear rate) vs. log (poise'sec)) for Lot C before (open sym¬ bols) and after dialysis (closed symbols). The collagen concentration was 1.4% (w/v) and the pH was 6.8. Notice the dramatic increase in viscosity obtained after dialysis. EXAMPLE 4

Adult female. New Zealand white rabbits (5-6 lbs.) were used for an ocular vitreous replacement study. One day prior to the study, by slit lamp evaluation both eyes of each animal were examined using fluorescein dye to determine the condition of corneal epithelium. The corneal epithelium was evaluated to determine if any corneal abrasion, ulceration or irregularities were present. On the day of the study, each animal was again evaluated prior to the injection of the test material. Fluorescein dye was not used in this pre-injection evaluation. Animals exhibiting pre-existing corneal, lens or conjunctival injury, irritation or irregularity were not used in this study.

General anesthesia was achieved by intramuscular injection of Rompun (10 mg/kg body weight) and Ketamine HCl (50 mg/kg body weight). The pupil was dilated with 0.05 ml of 1% Cyclopentolate HCl and 2.5% Phenylephrine HCl administered topically. One drop of Proparacaine 0.5% was administered topically prior to injection of the vitreous replacement solution. All injected solutions were sterile and administered undiluted. The effects on intraocular pressure of Lot A and Lot A' (Example 2), Viscoat™ and Healon™ were compared. Viscoat™ was purchased from Alcon-Cooper/Cilco. Healon™ was purchased from Pharmacia (Uppsula, Sweden). All viscoelastic solutions were refrigerated at 6°C until the time of use.

One rabbit eye was prepared for surgery and a 25- gauge butterfly infusion needle was inserted at the 10 o'clock position, 0.2 mm anterior to the limbus. To prevent collapse of the anterior chamber, the outflow tubing remained capped until the injection of the viscoelastic solution was initiated. A 1 cσ syringe with a 27 gauge needle was filled with the viscoelastic solution to be injected into the eye to replace the vitreous and inserted at the 2 o'clock position, 0.2 mm anterior to the limbus. The beveled edges of the needles were positioned away from the corneal endothelium. The anterior chamber of the eye was filled with 0.15 cc of the viscoelastic solution. After replacement of the aqueous humor with the test material, the outflow and the inflow needles were removed. In the other eye, no viscoelastic solution was injected into the anterior chamber. This eye served as a baseline for intraocu¬ lar pressure measurements. The intraocular pressure was moni¬ tored with a pneumotonometer. Eyes were evaluated prior to injection and at 0, 1, 3, 5, 24 and 48 hours post-injection. The results obtained, using three rabbits for each measurement, are shown in TABLES II and III.

The data of TABLES II and III demonstrate that collagen viscoelastic solutions according to the present invention, LOT A, are equal to or superior to prior art solu- tions LOT A' (collagen solution according to U.S. Patents 4,713,446 and 4,851,513), Healon™ (sodium hyaluronate), or Viscoat™ (a mixture of sodium hyaluronate and chondroitin sulfate) when left in the anterior chamber of the eye. Visco¬ elastic solutions according to the present invention are easier to irrigate out of the intraocular cavities. They are antici¬ pated to be substantially removed from the eye following ocular surgery and, thus, result in still fewer incidents of elevated intraocular pressure relative to prior art solutions.

In view of the present specification and appended claims, various additions, modifications, and omissions will be obvious to those skilled in the art and are within the inven¬ tion as claimed below.

Claims

1. A viscoelastic composition for use in viscosur- ge:ry comprising collagen and a mucopolysaccaride in a low saline physiologic diluent.

2. The viscoelastic composition according to claim 1 wherein the weight ratio of said collagen to said mucopoly- saccaride is between about 100:1 and about 20:1.

3. The viscoelastic solution according to claim 2 where said mucopolysaccaride is selected from chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, salts of hyaluronic acid, keratan sulfate, heparan sulfate and mixtures thereof.

4. A viscoelastic composition according to claim 3 wherein the low saline physiologic diluent comprises nonionic solutes, said diluent having an ionic strength of less than about 0.001 u and an osmolarity between about 200 m Osmol and about 400 m Osmol.

5. The viscoelastic solution according to claim 4 wherein said nonionic solute is selected from the group con- sisting of glycerol, sorbitol, xylitol, threitol, annitol, arabitol, ribitol, adonitol, erythritol, dulcitol, altritol, iditol, heptose, hexoses, pentoses and mixtures thereof.

6. The viscoelastic solution of claim 5 wherein said nonionic solute is glycerol.

7. The viscoelastic composition according to claim 1 wherein the kinetic viscosity is between about 1,000 cps and 800,000 cps.

8. The viscoelastic composition according to claim 7 wherein the kinetic viscosity is between about 45,000 cps and about 80,000 cps.

9. The viscoelastic composition according to claim 12 wherein the kinetic viscosity drops at least 88% upon the addition of salt to increase the ionic strength to at least 0.0022u.

10. The viscoelastic composition according to claim 1 further comprising between about 10 ng/ml to about 10,000 ng/ml of a growth factor.

11. The viscoelastic composition according to claim 10 wherein the growth factor is selected from the group con- sisting of growth hormone, interleukins, epidermal growth factors, fibroblast growth factors, insulin-like growth factors and mixtures thereof.

12. A viscoelastic composition for use in visco- surgery comprising collagen and a growth factor dissolved in a low saline physiologic diluent, said growth factor having a concentration between about 10 ng/ml to about 10,000 ng/ml.

13. A method of inserting an intraocular lens into the posterior or anterior chamber of a human or animal eye comprising: introducing into said anterior or posterior chamber an effective amount of a viscoelastic solution compris- ing collagen and a low saline physiologic diluent, said amount sufficient to reduce the trauma which normally results from inserting an intraocular lens into said chamber in the absence of a viscoelastic solution, and inserting said lens into said chamber.

14. The method of claim 13 further comprising a physiologic saline irrigation step following said insertion, said irrigation sufficient to remove substantially all of said viscoelastic solution.

15. A process of preparing a viscoelastic solution useful in visco-surgery comprising reconstituting collagen in a low saline physiologic diluent.

16. An aqueous buffer solution useful for medical applications comprising a combination of a buffer and a nonio- nic solute, said solution having an ionic strength of less than O.OOlu, and an osmolarity between about 200 m osmol and about 400 m osmol.

17. The solution of claim 16, wherein said nonionic solute is selected from the group consisting of glycerol, sorbitol, xylitol, threitol, mannitol, arabitol, ribitol, adonitol, erythritol, dulcitol, altritol, iditol, heptose, hexoses, pentoses and mixtures thereof.

18. The solution of claim 17, wherein such nonionic solute is glycerol.

19. The solution of claim 16, wherein said buffer is biologically compatible.

20. The solution of claim 16, wherein said buffer solution is at a physiological pH.

21. A method of treating a patient with visco- surgery comprising using a viscoelastic composition of collagen in a low saline physiologic buffer to separate and protect tissues being treated.

22. The method of claim 21 wherein the low saline physiologic diluent comprises nonionic solutes, said diluent having an ionic strength of less than about 0.001 u and an osmolarity between about 200 m Osmol and about 400 m Osmol.

23. The method of claim 22 wherein said nonionic solute is selected from the group consisting of glycerol, sorbitol, xylitol, threitol, mannitol, arabitol, ribitol, adonitol, erythritol, dulcitol, altritol, iditol, heptose, hexoses, pentoses and mixtures thereof.

24. The method of claim 23 wherein said nonionic solute is glycerol.

25. The method of claim 21 wherein the kinetic viscosity of said viscoelastic composition is between about 1,000 cps and 800,000 cps.

26. The method of claim 25 wherein the kinetic viscosity of said viscoelastic composition is between about 45,000 cps and 85,000 cps.

27. The method of claim 21 wherein the kinetic viscosity of said viscoelastic composition drops at least 88% upon the addition of salt to increase the ionic strength to at least 0.0022u.

28. The method of claim 21 wherein said viscoelastic composition further comprises a mucopolysaccaride, and wherein the weight ratio of said collagen to said mucopolysaccaride is between about 100:1 and about 20:1.

29. The method of claim 28 wherein said mucopolysaccaride is chondroitin sulfate.

30. The method of claim 21 wherein said viscoelastic composition further comprises between about lOng/ml and about 10,000 ng/ml of a growth factor.