Note: Descriptions are shown in the official language in which they were submitted.
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.
-5
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.
EXAMPLE 2
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.
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~' 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.
