CA1322716C - Ophthalmological collagen coverings - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Ophthalmologic collagen coverings derived from animal eyes and a process for preparing such coverings comprising treatment of a fibrous tunic of an animal eye with an alkali metal hydroxide in a saturated solution of a salt of an alkali metal, followed by neutralization, dissolution in an aqueous solution of an organic acid, purification of the resulting solution of collagen by dialysis against a buffer solution while bringing the solution of collagen to a pH of 4.5 to 7.5 and drying the thus-obtained solution of collagen simultaneously with shaping spherical coverings therefrom.

Description

OP~THAL~OLOGICAL COLLAGEN COVE.RINGS

BAC~GROUND OF THE INVE~TION

This invention relates to a form of collagen that is uniquely suited for ophthalmic uses. In more specific embodiments, this invention relates to a process for preparing ophthalmological collagen coverings and to coverings prepared by this process and useful in all kints of ophthalmological ope~ations, as well as in a eonservative trea~ment of t~aumatic and t~ophic inju~ies of the cornea of the eye having different etiology.
10 Ihe present stage of the develoFment of eye surgery features a high quality of suryical treatnent. In this resFect there is a problem L
o developing new meth3ds and agents making it Fx~;sible to re~u oe the r number of po5t-operaticnal ccmçlications, to s~rten the time of ;
hospital stays, and to ensure higher unctional rE~I~lts of operations on the globe o~ the eye.
~ nown in the art are materials produced from collagen and methods of dissolution of collagen derived from the skin and tendons of animals.
~ Thus, a known procoss for dissolution of collagen of an animal s~in (USSR Inventor's Certificate No. 162280! 1964) comprises an alkali-salt treatment of an animal skin with a subsequent neutralization and residence in an acidic medium to give a dispersion of collagen having pH
of not higher than 2.5.
~ nown in the art is a process for preparing a hemostatic material from a dispersion of collagen ~cg. USSR Inventor's Certificate No.
5615~4, 1~77~. Acidification of neutral dispersions of ~ollagen obtained a~ter an inten~ive alkali~salt treatment of animal skin an~
tendons are inte~mixed with thrombin and antibiotics, where after the mixture is frozen and dried in ~acuum.
These processes cannot be employed for the preparation of collagen co~rings to be used in ophthalmology, since the materials ~rod~lced by these procssses have clearly pronounc~d antigenic properties.
It is an objec~ of the presant invention to provide a proces5 for preparing and using ophthalmologic~l collagen coverings. A particular object, ls a covering for use in conn&ction with microsurgical o~er~tions on the globe o~ the eye. ~ further object is an improved ~/
1~ ~3~27 / conservative treatment of traumatic and trvphic injuries of the cornea of the eye having different etiology. Other objects will be apparent from consideration of the following description of the invention.

SUM~5ARY OF T~IE lNVENTIl)N

It has been found tha~ collagen isolated from animal eyes, especially the fibrous tumic of ani~al eyes and preferably the sclera of animal eyes, is specially suited for topical application to the eye, avoiding antigenic charac~ristics associated with collagen derived from, e.g., animal dermis. The collagen also possesses therapeutic properties making it desirable for the treatment of traumatic and trophic injuries of the cornea. The collagen further possesses lubricating properties suggesting the potential for the treatment of dry eye.
The collagen may be provided in a variety of forms adapted for ophthalmic use. Examples include me~branes, gels, and solutions.
Especially preferred for the treatment of traumatic and tTophic injuries are shaped collagen articles (described in more detail below). Collagen applied to the eye in the form of, e.g., drops or ointments is washed ~elatively rapidly from the eye by tear flow. By contrast, collagen applied as a shaped article is maintained in the eye for a longer periods, extending the desirable therapeutic effects of the application.
Even when applied as a shaped article, however, the collagen is bioerodible and will dissolve in the eye.
The preferred process for isolating collagen from animal eyes and for preparing ophthalmological collagen coverings comprises treating the fibrous tunic of an animal eye with an alkali metal hydroxide in a saturated solution of an alkali metal salt, then neutrali~ing the ~esulting tissue to a pH of from 6.0 to 7.0, and dissolving the tissue in an aqueous solution of an organic acid; the thus-prepared solution of colIagen is decontaminated from low-molecular impurities by dialysis against a buffer solution while bringing pH of the solution of collagen to 4.5-7.5 and the obtained solution of collagen is dried simultaneously wi~h shaping therefrom spherical coverings repeating the cur~ature of the front section Df ~he eye.
With the view to improve quality of the resulting covering, as the starting material - fibrDus tunic of an animal eye use is preferably ~ade of ~he sclera of animal eyes.
The neutrali~ation of the tissue should be preferably effec~ed by means of a 2S solution of boric acid. For dissolution of collagen any organic acid can be used~ acetic acid being prefcrable. The ophthalmological collagen coverings prepared by the p~ocess accor~ling to the presen~ invention has ~ spherical sh~pe Tepeating the c~r~ature o~

13227~
.,e front section of the eye and ensuring a full contact wi~h the surface of the cornea of the eye; they also have dimensions ensurin~ a full C08ting of the cornea.
The ophthalmological collagen coverings according to the present invention are appli~d onto the front surface of the cornea and serve as a temporary hydrophilic 5phe~ical "bantage" and kept on the front 5urface of the cornea without ~ny additional fixation while exhibi~ing therapeutiCal properties of a Daturally-occuring copolymer - collagen.
~ he coverings according to the present invention make it possible to reduce the nu~ber of post-operational complications, accelerate h~aling of injured tissues of the eye. In the case of traumatic and trophic injuries of the cornea the collagen coverings according to ~he present invontion enablc normalization of metabolic processes in the injured cornea. The co~orings of this invention are useful in the front radial keratotomy, especially in the case of eppearance of nicroperforations, in keratoplasty, keratoprosthetics, in the treatment of post-operational keratopathy, ontothelial-epithelial dystrophy, erosion of the cornea. ~he use of ophthalmologicsl collagen coverings mnkes ie poss~ble to r~duce the number of post-operational complications, in 98% of cases there has been noted the formation of more soft cicatricos on the cornea after keratotomy; in 98% of cases there has taken place a lesser d~gree of edema of the cornea and the tr~nsplantat~ ~dema in keratoplasty. In trophic injuries of the cornea hsving different etiology positive dynamics has been noticed in 95% of cases as compared to proYiously carried out conservative therapy with the use of pharmaceutical preparations.

DETAILED DESCRIPTION OF THE lNVENTlON

The source of the collagen is mammalian eyes, preferably the eyes of animals, and most prefer~bly agricultural animals, such as pigs. The collagen may be extr~cted from any collagen containing, discrete portion of the eye or fro~ the whole intact eye, but the preferred source of coll~gen is from th~ sclera of the ~ye. Collagen isolated from animal eyes and used in ophthslmological products according to this invention causes ~inimal allergic reaction and less eye infla~mation as compared to coll~en isolated from other sources, such as bovine hide~
The fibrous tunic of an animal eye is thoroughly cleaned from internal coats of the eye, residual-epithelium, conjunctiva, muscles and ~iscellaneous; and seroma is isola~ed. -The isolated stroma is cut into small ~ieces. The cut tissue is~insed with distilled water to completely remove m~rhani,cal ~rei~n mattex and bloc~, then it is transferred into a flask to which is chen added an alka1i metal hydroxide in a saturated solution of a sa1t of an alkali ~ 3227~ ~
metal> for example, with a 10% solution of caustic soda in a saturated solution of sodium sulphate (at a rate of S00 ml of the solution per 10 g of the tissue) for 48 hours st a temperature of 18-25C. The solution is decanted and the tissues are subjected to neutralization to a pH of 6.0-7.0 under sti~ring, e.g. i~ a 2% solution of boric acid and repeatedly changing the solution. The tissue is ~insed with distilled water until a complete removal of the sulphate ion in the rinsing liquid The rinsed tissue is then dissolved i~ a Q.5-l~S solution of an organic acid such as acetic acid in such quantity that the final concentration of p~otein in the solution be equal to 1%. As the organic acid use can be made of citric acid, lactic acid, ascorbic acid and the like. The mass is stirred and allowed to stand in a refrigerator for 1-3 days at a low temperature. Then the mass is homogenized, centrifuged and allowed to stand for one day at a low temperature. The resulting solution is filtered. To carry out neutralization and desolvation, the acetic-acid sslution of collagen is diluted with acetic acid to a concentration of protein of 0.7-0.8% and dialy~ed against a phosphate or citrate buffer at a temperature of 18-20C while bringing pH of the solution of collagen to 4.5-7.5. The resulting collagen solution is centrifuged, poured into matrices repeating the shape of the front section of the eye and air-dried in a dust-free csbinet at a temperature within the range of from 10 to 27C. Ophthalmological collagen coverings are thus obtained which are elastic, transparent, of a spherical shape repeating the curvature of the front section of the eye. The size and shape of coverings ensure a full coating of the cornea of the eye and its full contact with the front surace thereof. The final coverings are sterilized.
Preferred embodiments of the foregoing process steps are:

1. The sclera pieces are treated with an aqueous solution comprising from 1.0 to 3.0 molar alkali metal hydroxide and from 0.8 to 1.5 molar alkali metal sulfate, preferably 2.5 molar sodium hydroxide and 1.4 molar sodium sulfate having a pH
of about 12-14. Sclera is treated with stirring for about one to three days~ preferably for two days. The weight to volume ~atio of sclera to treatment solution is from about 15 to 25 grams per liter, preferably about 20 grams per liter.

2. The scl~ra is neutralized to a pH of about 6 to 7 by draining the hydroxide solution and treating first with distilled water alld then with a dilute aqucous acid solution havin~ a pll of about 3-5, preferably 4 to 4.5. Acids useful for neutralization include boric acid, tartaric acid, citric acid, ccetic acid, l~ctic acid and ascorbic acid. Boric acid is preferred for neutralization, with a concentration of about .02 2~7~
to .04 molar, preferably .030 to .033 molar. Treatment cycles Are typically 15 ~inutes to two houTs, with constant stirring.
The weight to ~olume ratio i~ typically 20 grams (initial starting ~eight) to frD~ about S00 ~illiliters to one liter treatment solution. Two to five acid treat~ents are used to neutralize the sclera; prefer~bly, three treatments are used.
The sclor~ is rinsed with distillcd water following neu~raliza~ion; preferably, three tim~s for about 1 hour e~ch rinse.

3. The sclera is then dissolved in an aqueous organic scid solution tD a concentration of about .5~ to 1.5~ by weigh~
collagen, preferably to a concentration of lS. Acids suitable ~or prepsration of collagen solution are the organic ones listed previously for neutralization of scler~. She collagen is dissolved by incubation in a suitable squeous organic acid, preferably ~retic acid, with a concentrstion of about .1 to 2 molar, preferably 1 oolar, with stirring ~or about two to four days, preferably three days, at a temperature of about 2 to 10 degrees cen~igrade, prefersbly 4 to 6 degrees centigrsde.

4. me oollagen solution i~ conveniently homcgem zed in a blender, centrifuged and filtered. The collagen solution is then dislyzed against sn aqueous buffer, see e.g. U.S. Pharmacopeis XXI, 1985, page 1420, sush as phosphate or citrate, preferably citrate, ha~ing a concentration of sbou1: .002 to 0.2 molar, and a pH of about 7.0 to 8.0, preferably 7.2. Dialysis is continuet with addition of fresh citrate buffer until the pl~ of the collagen is from about 4.5 to 7.5, preferably about 5.0 to 5.5, and the collagen solution has formed a cloudy, ho~ogenous gel. Dialysis m~y be ~ccomplished by various techniques known to the art. Typically, dislysis membranes are used having moleçular weight cut-off limi~s of from about 3,000 to 100,~00, preferably from abnut 10,000 to 100,000. These membranes easily retain the extracted collagen, ~hich has an sverage molecular ~eight of about ~00,000. This molecular weight is typical of tropocollagen, defined herein and in the li~erature as the basic moleculsr subunit of collagen, existing as a rigid rod consistinQ of three polypeptide chains wound together in a triple helix.

5. The collagen gel is conveniently homogenized in a blender, centrifuged, deaerated, and filtered to remove particulate matter from the gel.

f~, ~ ' fl3227~L~
The collagen of this invention can be used as a vehicle for drug delivery. For example, shaped collagen may be impregnated with an o~hthalmically active drug or collagen solution or gels may be employed ~o produce soluble preparations for ophthalmic use. Selection of the ophthalmically active drug is not critical to this invention, although materials su~h as pilocarpine, d~xamethazone, and gentamycin may be mentioned as being exewpla~y.
Shaped collagen aTticles p~epared as described above have very little cross-linking, and, consequently, dissolve in a ~elatively slow period of time. Dissolution rate of the collagen ~ay be slowed if desired by introducing a minor amount of cross-linking into the collagen structure. This may be accomplished by any of the techniques well-known in the art. Radiation induced cross-linking is particularly convenient. However, the degree of cross-linking, if induced is limited to that which does Dot undermine the bioerodible properties of the articles.
The process according to the present invention makes it possible to prepare ophthalmological collagen coverings the use of which contributes to reducing post-operational complications, accelerates healing of injured tissues of the eye, reduces hospital stays of post-operational treatment of patients. The use of coverings according to the present invention in the case of traumatic and trophic injuries of the cornea exerts a favorable influence on metabolic processes in the cornea which is objectively revealed in diminished photophobia, lachry~ation, edema o the cornea~ better acuity of vision. Thls invention, and properties of the products, will be better understood by reference to the following examples.

EX~PLE 1 To 20 g of purifiad and cut stroma of sclera of an animal eye 1 liter of a 10% solution of caustic soda in a saturated solution of sodium sulphate is added and allowed to stand at a temperature of 18-20C for 48 hours. Then the solution is decanted, the tissue is rinsed with a small amount of distilled water, added wi~h 1 liter of a 2%
solution of boric acid and agitated by means of a magnetic stirrer for 2 hours twice changing the solution of boric acid. Under continuous stirring the tissue is ~horoughly rinsed wi~h distilled water (total Yolu~e of water - 5 li~ers) till a complete removal of the sulphate ion from the rinsing liquid. 250 ml of the resulting wa~er-treated tissue are added with 350 ml of a 0.5M acetic acid, stirred and left for one 40 day at the temperature of 4C. Then the mass is homogenized by means of a microdisintegrator of tissues, centrifuged for 30 minutes at 2,000 r.p.m. and allowed to stand for 3 days at the temperature of 4C. The resulting solution is filtered through a glass filter. The thus ~ ~ 2 ~
obtsined 1% solution of collagen is diluted with a 0.5M solution of acetic acid to the concentration of 0.8%. The dialysis of the 0.8%
solution of collagen is effected against a 0.2M citrate buffer while bringing pH of the solution of collagen to 6.7. The dialysis is continued while lowering the buffer cuncentration from 0.2M to O.OOZM in 3-4 stages using, i~ the last stage, heating to a temperatuIe of 28-30C. The resulting solution of coIlagen is centrifuged for 15 minu~es at 1,000 r.p.m., poured into matrices repeating the shapc of the f~ont section of the eye and air-dried in a dust-protected cabinet for 48 hours at the ~emperature of 15C and relative humidity of 40-50%. The final coverings sre sterilized by gamma-rays in the dose of 2.5MRad and dose rate of 0.5~1Rad/h.
The thus-p~epared coverings are transparent, elastic; they have a spherical shape repeating the curvature of the front section of the eye.
The shape ~nd dimensions ensure a full coating of the cornea of the eye and a full contact with the front surface thereof.

Twenty grams of comminuted sclera, derived from porcine eyes, were treated in 8 one liter flask with 980 milliliters of a solution comprised of 100 grams per liter sodium hydroxide and 200 grams per liter sodium sulfate in distilled water by stirring continuously for 48 hours. The solution was then drained and the sclera rinsed by a 5 minute stirring cycles with 1 liter of distilled water. The sclera was then neutralized by three 1 hour stirring cycles, each in one liter of a solution comprised of 20 grams boric acid per liter in distilled water, followed by two 15-minute s*irring cycles, each in one liter of distilled water. During neutralization, the sclera swells significantly and becomes soft and transparent. The swollen sclera was then dissolved in 530 milliliters of 1 molar acetic acid by stirring at 4 degrees centigrade for 48 hours to form a collagen solution having an approximate concentration of 1%o The resulting collagen solution was homogenized in a blender, centrifuged to remove air bubbles and filtered to remove fine retinal fragments. The collagen solution was then placed in dialysis tubing having a molecular weight cut-off of 10,000-12,000 and dialyzed against 25 liters o~ a citrate buffer solution comprised of 22.5 grams of sodium citrate in 25 liters of distilled water, pH
adjusted to 7.2 with 1 molar hydrochloric acid. As a result of the dialysis, the collagen solution formed a cloudy, white, homogeneous gel and hsd a pH of 5.2. The gel was then homogeni~ed in a blender, centrifu~ed, de~erated and filtered, The resulting solution was cast into molds conforming to the curvature of the eye and allowed to dry at room temperature in a laminar flow hood for 72 hours. The corneal coverings have an average thickness of about .002 inches snd weigh about . 003 grams.

~' 132~7~

CO~lPARATIVE EXA~IPLE A

Dialysis is 8 critical step in ~he production of collagen material suitable for csstiTIg. When cast from acidic, predialysis solutionsS the resulting collagen coverings are typically ~alformed wi~h numerous inhomogeneities and defects. Because of differences in the vapor pressures of water and acetic acid, and in solutions employing other acids, acid concentra~ion can increase upon drying causing denaturation of the collagen. As an example, collagen derived as described in Example 1 but only dialyzed agains~ 10 liters of buffer, has a pl~ of 3.5 and will not cast clear, homogeneous films, free of imperfections.

EX~IPLE 3 The si~nificance of the use of the collagen covering disclosed herein following ophthalmic surgery or corneal trauma has been demonstrated in experiments in rabbits. Rabbits were subjected to standard radial keratotomy procedures in both eyes, and one eye was covered with a collagen covering i~mediately following surgery.
Examination at various times following surgery showed a marked decrease in traumatic inflammation and edema at all time following surgery for the eyes treated with the coverings. :[t was also observed ~hat incisions without the covering widened as they healed, filling with a wedge of epithelium while those treated with collagen covering were held together and "bridged" by the covering. ~ibroblast proliferation at the site was two to three times greater than thle controls indicating higher le~els of collagen synthesis. The coverings dissolved within two to six hours.
It has also been found that if collagen other than that deriYed from the eye, such as bovine dermal collagen, is used to cover ophthalmic wounds, inflammation and edema are more severe than for porcine eye collagen. Further, collagen from the eyes of pigs elicits somewhat less inflammatory response than from the eyes of cows.
The sdvantages of this invention will be apparent ~o those skilled in the art. Greatly impro~ed9 Yirtually non-allergic products are economically made available for the healing arts.
It will be understood that this invention is not limited to the specific examples which have been offered as ~articular embodiments, and that modifications can be made withou~ departing from the spirit thereof.

Claims (13)

1. A shaped article conforming to the curvature of the front section of the eye comprising bioerodible collagen isolated from animal eyes.

2. The article of claim 1, wherein the collagen is isolated from the fibrous tunic of an animal eye.

3. The article of claim 1, wherein the collagen is isolated from sclera of animal eyes.

4. The article of claim 1, wherein the collagen is isolated from pig eyes.

5. The article of claim 4, wherein the collagen is isolated from the sclera of pig eyes.

6. The article of claim 1, further comprising an ophthalmically active drug.

7. An ophthalmic composition comprising bioerodible collagen iso-lated from animal eyes and an ophthalmically active drug.

8. A method for isolating collagen from animal eyes which com-prises:
a) treating the fibrous tunic of an animal eye with an alkali metal hydroxide in a saturated solution of an alkali metal salt;
b) neutralizing the resulting collagen tissue to a pH of 6.0 to 7.0;
c) dissolving the collagen tissue in an aqueous solution of an organic acid; and d) dialyzing the resulting collagen solution against a buffer solution to raise the pH of the collagen to 4.5 to 7.5.

9. The collagen isolated by the method of claim 8.

10. A process for preparing ophthalmological. collagen coverings comprising treatment of a fibrous tunic of an animal eye with an alkali metal hydroxide in a saturated solution of an alkali metal salt, neutral-ization of the resulting tissue to a pH of 6.0 to 7.0, dissolution in an aqueous solution of an organic acid, purification of the thus-obtained solution of collagen from low-molecular impurities by dialysis against a buffer solution while bringing pH of the solution of collagen to 4.5 to 7.5 and drying of the obtained solution of collagen simultaneously with shaping spherical coverings therefrom repeating the curvature of the front section of the eye.

11. A process as claimed in claim 10, wherein the fibrous tunic of an animal eye is the sclera of the eye.

12. A process as claimed in claim 10, wherein neutralization of the tissue is effected by means of a 2% solution of boric acid.

13. A process as claimed in claim 10, wherein as the organic acid for dissolution of collagen acetic acid is used.