U.S. patent application number 11/204636 was filed with the patent office on 2006-02-16 for hyaluronidase preparation for ophthalmic administration and enzymatic methods for accelerating clearance of hemorrhagic blood from the vitreous body of the eye.
Invention is credited to Gabriel Arturo Carpio Aragon, Jose Luis Gutierrez Flores, Hampar L. Karageozian, Vicken H. Karageozian, Maria Cristina Kenney, Anthony B. Nesburn.
Application Number | 20060034822 11/204636 |
Document ID | / |
Family ID | 24242789 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060034822 |
Kind Code |
A1 |
Karageozian; Hampar L. ; et
al. |
February 16, 2006 |
Hyaluronidase preparation for ophthalmic administration and
enzymatic methods for accelerating clearance of hemorrhagic blood
from the vitreous body of the eye
Abstract
A thimerosal-free hyaluronidase preparation wherein the
preferred hyaluronidase enzyme is devoid of molecular weight
fractions below 40,000 MW, between 60-70,000 MW and above 100,000
MW. Also disclosed is a method for accelerating the clearance of
hemorrhagic blood from the vitreous humor of the eye, said method
comprising the step of contacting at least one hemorrhage-clearing
enzyme (e.g., a .beta.-glucuronidase, matrix metalloproteinase,
chondroitinase, chondroitin sulfatase or protein kinase) with the
vitreous humor in an amount which is effective to cause accelerated
clearance of blood therefrom.
Inventors: |
Karageozian; Hampar L.; (San
Juan Capistrano, CA) ; Karageozian; Vicken H.; (San
Juan Capistrano, CA) ; Kenney; Maria Cristina;
(Malibu, CA) ; Gutierrez Flores; Jose Luis;
(Tijuana, MX) ; Carpio Aragon; Gabriel Arturo;
(Tijuana, MX) ; Nesburn; Anthony B.; (Malibu,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
24242789 |
Appl. No.: |
11/204636 |
Filed: |
August 15, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10369372 |
Feb 14, 2003 |
6939542 |
|
|
11204636 |
Aug 15, 2005 |
|
|
|
09453012 |
Dec 2, 1999 |
6551590 |
|
|
10369372 |
Feb 14, 2003 |
|
|
|
09139282 |
Aug 24, 1998 |
6039943 |
|
|
09453012 |
Dec 2, 1999 |
|
|
|
08561636 |
Nov 22, 1995 |
5866120 |
|
|
09139282 |
Aug 24, 1998 |
|
|
|
Current U.S.
Class: |
424/94.62 ;
424/94.61; 424/94.63 |
Current CPC
Class: |
A61K 38/47 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/7016
20130101; A61K 38/47 20130101; A61P 27/02 20180101; A61P 27/00
20180101; A61K 31/7016 20130101; Y10S 514/912 20130101 |
Class at
Publication: |
424/094.62 ;
424/094.61; 424/094.63 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61K 38/46 20060101 A61K038/46; A61K 38/48 20060101
A61K038/48 |
Claims
1. A method for accelerating the clearance of hemorrhagic blood
from the vitreous humor of a mammalian eye, said method comprising
the step of: contacting with said vitreous humor an amount of an
enzyme which is active to accelerate the clearance of hemorrhagic
blood from the vitreous humor.
2. The method of claim 1 wherein said enzyme is selected from the
group consisting of: hyaluronidase; keratinase; chondroitinase AC;
chondroitinase B; chondroitinase ABC; chondroitin 4 sulfatase;
chondroitin 6 sulfatase; matrix metalloproteinase-1; matrix
metalloproteinase-2; matrix metalloproteinase-3; matrix
metalloproteinase-9; streptokinase; urokinase; and, combinations
thereof.
3. The method of claim 1 wherein said enzyme is a
.beta.-glucuronidase enzyme.
4. The method of claim 1 wherein said enzyme is a matrix
metalloproteinase enzyme.
5. The method of claim 1 wherein said enzyme is a protein kinase
enzyme.
6. The method of claim 1 wherein said enzyme is a chondroitin
sulfatase enzyme.
7. The method of claim 1 wherein said enzyme is in liquid solution,
and wherein the step of contacting of said enzyme with the vitreous
humor comprises: injecting said liquid solution into the vitreous
humor.
8. The method of claim 1 wherein said enzyme is hyaluronidase.
9. The method of claim 8 wherein said amount of said hyaluronidase
enzyme is 10-300 International Units.
10. The method of claim 1 wherein said enzyme is administered in a
single intravitreal injection.
11. The method of claim 10 herein the single intravitreal injection
has an injectate volume of less than 50 .mu.l.
12. The method of claim 8 wherein said hyaluronidase enzyme is
devoid of molecular weight fractions above 100,000 MW.
13. The method of claim 8 wherein said hyaluronidase is devoid of
hyaluronidase molecular weight fractions below 40,000 MW.
14. The method of claim 8 wherein said hyaluronidase is devoid of
molecular weight fractions between 60,000-70,000 MW.
15. The method of claim 8 wherein said hyaluronidase is devoid of
molecular weight fractions above 100,000 MW below 40,000 MW and
between 60,000-70,000 MW.
16. The method of claim 8 wherein hyaluronidase enzyme is devoid of
gelatinolytic hyaluronidase having molecular weights between
approximately 60,000-100,000 MW.
17. The method of claim 8 wherein said hyaluronidase enzyme is
devoid of caseinolytic hyaluronidase having molecular weight above
approximately 45,000 MW.
18. The method of claim 8 wherein said hyaluronidase enzyme is
devoid of hyaluronic acid lysing hyaluronidase having molecular
weights above approximately 100,000 MW.
19. The method of claim 1 wherein said method further comprises:
contacting said enzyme with said vitreous humor in the absence of
thimerosal.
20. The method of claim 8 wherein said hyaluronidase enzyme is
prepared in a solution for injection which is free of thimerosal
and which has the general formulation: TABLE-US-00006 Hyaluronidase
(ACS) 0-8000 I.U.; Lactose, USP 13.3 mg; and Phosphate, USP 5
mmoles;
and, wherein the hyaluronidase enzyme is devoid of molecular weight
fractions below 40,000.
21. The method of claim 20 wherein said solution for injection has
the following specific formulation: TABLE-US-00007 Hyaluronidase
(ACS) 7,200 I.U.; Lactose, USP 13.3 mg; and Phosphate, USP 5
mmoles.
22. The method of claim 20 wherein said solution for injection is
dissolved in balanced salt solution.
23. A hyaluronidase preparation for ophthalmic administration, said
preparation being free of thimerosal and free of hyaluronidase
which is less than 40,000 MW.
24. The preparation of claim 23 wherein said preparation is further
free of hyaluronidase having molecular weights between
60,000-70,000 MW.
25. The preparation of claim 23 wherein said preparation is further
free of hyaluronidase having a molecular weight in excess of
100,000 MW.
26. The preparation of claim 23 wherein said preparation is further
devoid of hyaluronic acid lysing hyaluronidase having molecular
weight above approximately 100,000 MW.
27. The preparation of claim 23 wherein said preparation is further
devoid of caseinolytic hyaluronidase having molecular weight above
approximately 45,000 MW.
28. The preparation of claim 23 wherein said preparation is further
devoid of gelatinolytic hyaluronidase having molecular weight
between approximately 60,000-100,000 MW.
29. The preparation of claim 23 wherein said preparation is a
solution for injection having the following general formulation:
TABLE-US-00008 Hyaluronidase (ACS) 0-8000 I.U.; Lactose, USP
13.3-133.3 mg; and Phosphate, USP 5-200 mmoles;
and, wherein the hyaluronidase enzyme is devoid of molecular weight
fractions below 40,000.
30. The preparation of claim 29 wherein the amount of hyaluronidase
(ACS) in said formulation is 7,200 I.U.
31. The preparation of claim 29 wherein the components of said
formulation are dissolved in balanced salt solution.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/369,372, filed Feb. 14, 2003, which is a
continuation of U.S. patent application Ser. No. 09/453,012, filed
Dec. 2, 1999, now U.S. Pat. No. 6,551,590, which is a continuation
of U.S. patent application Ser. No. 09/139,282, filed Aug. 24,
1998, now U.S. Pat. No. 6,039,943, which is a continuation of U.S.
patent application Ser. No. 08/561,636, filed Nov. 22, 1995, now
U.S. Pat. No. 5,866,120, all of which are hereby expressly
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to enzyme
preparations for therapeutic administration to the eyes of humans
or other mammals, and more particularly to a) a method for
utilizing one or more enzymes to accelerate the rate at which
hemorrhagic blood is cleared from the vitreous body of the
mammalian eye and b) an improved hyaluronidase preparation for
ophthalmic administration.
BACKGROUND OF THE INVENTION
i. Anatomy of the Human Eye
[0003] In human beings, the anatomy of the eye includes a "vitreous
body" which occupies approximately four fifths of the cavity of the
eyeball, behind the lens. The vitreous body is formed of gelatinous
material, known as the vitreous humor. Typically, the vitreous
humor of a normal human eye contains approximately 99% water along
with 1% macromolecules including; collagen, hyaluroriic acid,
soluble glycoproteins, sugars and other low molecular weight
metabolites.
[0004] The retina is essentially a layer of nervous tissue formed
on the inner posterior surface of the eyeball. The retina is
surrounded by a layer of cells known as the choroid layer. The
retina may be divided into a) an optic portion which participates
in the visual mechanism, and b) a non-optic portion which does not
participate in the visual mechanism. The optic portion of the
retina contains the rods and cones, which are the effectual organs
of vision. A number of arteries and veins enter the retina at its
center, and splay outwardly to provide blood circulation to the
retina.
[0005] The posterior portion of the vitreous body is in direct
contact with the retina. Networks of fibrillar strands extend from
the retina and permeate or insert into the vitreous body so as to
attach the vitreous body to the retina.
ii. The Causes, Treatments and Clinical Sequelae of Intravitreal
Hemorrhage
[0006] Diabetic retinopathy, trauma and other ophthalmological
disorders sometimes result in rupture or leakage of retinal blood
vessels with resultant bleeding into the vitreous humor of the eye
(i.e., "intravitreal hemorrhage). Such intravitreal hemorrhage
typically manifests as clouding or opacification of the vitreous
humor.
[0007] Intravitreal hemorrhage is sometimes, but not always,
accompanied by tearing or detachment of the retina. In cases where
the intravitreal hemorrhage is accompanied by a retinal tear or
detachment, it is important that such retinal tear or detachment be
promptly diagnosed and surgically repaired. Failure to promptly
diagnose and repair the retinal tear or detachment may allow
photoreceptor cells of the retina, in the region of the tear or
detachment, to become necrotic. Such necrosis of the photoreceptor
cells of the retina may result in loss of vision. Furthermore,
allowing the retinal detachment to remain unrepaired for such
extended period of time may result in further intravitreal
hemorrhage and/or the formation of fibrous tissue at the site of
the hemorrhage. Such formation of fibrous tissue may result in the
formation of an undesirable fibrous attachment between the vitreous
body and the retina.
[0008] The typical surgical procedure used for repair of retinal
tears or detachment requires that the surgeon be able to look
through the vitreous humor, to visualize the damaged region of the
retina (i.e., "transvitreous viewing of the retina"). When
intravitreal hemorrhage has occurred, the presence of the
hemorrhagic blood within the vitreous can cause the vitreous to
become so cloudy that the surgeon is prevented from visualizing the
retina through the vitreous. Such hemorrhagic clouding of the
vitreous can take 6-12 months or longer to clear sufficiently to
permit trans-vitreal viewing of the retina. However, in view of the
potential complications which may result from delayed diagnosis or
treatment of a retinal tear or detachment, it is generally not
desirable to wait for such natural clearance of the hemorrhagic
blood to occur.
[0009] Furthermore, even when the intravitreal hemorrhage is not
accompanied by retinal tear or detachment, it is often difficult to
verify that retinal tear or detachment has not occurred, because
the hemorrhagic clouding of the vitreous prevents the physician
from performing routine funduscopic examination of the retina.
Moreover, the presence of hemorrhagic blood within the vitreous may
significantly impair the patient's vision through the affected eye,
and will continue to do so until such time as the hemorrhagic blood
has been substantially or fully cleared.
[0010] Thus, the presence of hemorrhagic blood within the vitreous
body causes multiple clinical problems including a) inability to
visually examine and diagnose the site of the hemorrhage and/or any
accompanying tear or detachment of the retina, b) full or partial
impairment of vision in the affected eye and c) impairment or
prevention of the performance of trans-vitreal surgical procedures
of the type typically utilized to repair the site of hemorrhage
and/or to repair any accompanying retinal tear or detachment.
[0011] In cases where intravitreal hemorrhage has resulted in
substantial clouding or opacification of the vitreous, the treating
physician may have the option to perform a procedure known as a
vitrectomy, wherein all (or a portion of) the vitreous body is
removed from the interior of the eye, and replaced with a clear
liquid. The performance of such vitrectomy procedure is intended to
allow the surgeon to visualize the retina sufficiently to proceed
with the necessary retinal examination and/or surgical repair of
the hemorrhage and any accompanying retinal tear or detachment.
However, such vitrectomy procedures are highly skill-intensive, and
are associated with several significant drawbacks, risks and
complications. Among these drawbacks, risks and complications are
the potential that the act of removing the vitreous will cause
further detachment or tearing of the retina and/or that such
removal of the vitreous will cause further hemorrhage from the
already-weakened retinal blood vessels.
iii. Prior Ophthalmic Applications of Hyaluronidase and Other
Enzymes
[0012] In an effort to minimize the potential for causing further
detachment or tearing of the retina during performance of
vitrectomy, it has previously been proposed in U.S. Pat. No.
5,292,509 (Hageman), to inject certain protease-free
glycosaminoglycanase enzymes into the vitreous body, to cause the
vitreous body to become uncoupled or "disinserted" from the retina,
prior to removal of the vitreous body. Such disinsertion or
uncoupling of the vitreous body is purported to minimize the
likelihood that further tearing or detachment of the retina will
occur as the vitreous body is removed. Examples of specific
protease-free glycosaminoglycanase enzymes which may be used to
bring about this vitreal disinsertion purportedly include;
chondroitinase ABC, chondroitinase AC, chondroitinase B,
chondroitin 4-sulfatase, chondroitin 6-sulfatase, hyaluronidase and
.beta.-glucuronidase.
[0013] Although hyaluronidase enzyme has been known to be usable
for various ophthalmic applications, including the vitrectomy
adjunct application described in U.S. Pat. No. 5,292,509 (Hageman),
published studies have indicated that the hyaluronidase enzyme may
itself be toxic to the retina and/or other anatomical structures of
the eye. See The Safety of Intravitreal Hyaluronidase; Gottleib, J.
L.; Antoszyk, A. N., Hatchell, D. L. and Soloupis, P., Invest
Ophthalmol Vis Sci 31:11, 2345-52 (1990).
[0014] The ophthalmic toxicity of some hyaluronidase preparations
has been confirmed by other investigators, who have proposed that
such hyaluronidase preparations be used as a toxic irritant for
causing experimentally-induced neovascularization of the eye, in
animal toxicity models. See, An Experimental Model of Preretinal
Neovascularization in the Rabbit; Antoszyk, A. N.; Gottlieb, J. L.,
Casey, R. C., Hatchell, D. L. and Machemer, R., Invest Ophthalmol
Vis Sci 32:1, 46-51 (1991).
[0015] Unfortunately, it has not been previously known whether the
reported therapeutic activities and toxicities of hyaluronidase are
universally applicable to all hyaluronidase preparations, or
whether such efficacies and/or toxicities are applicable only to
hyaluronidase preparations containing certain excipient materials
or to hyaluronidase enzymes derived from specific sources. This is
an important consideration in view of the fact that the purity and
characterization (e.g., molecular weight distribution) of the
various hyaluronidase preparations used in the prior art may vary,
depending on the source of the hyaluronidase and the solvents
and/or other formulation components with which the hyaluronidase is
combined.
iv. Purity and Characterization of Hyaluronidase Preparations
Previously used for Ophthalmic Administration
[0016] The term "hyaluronidase" is commonly used to describe a
group of endo-(.beta.-glucuronidase enzymes which depolymerize
certain mucopolysaccharides, such as hyaluronic acid. Myer, K. et
al. in The Enzymes Vol. 4, 2d, Ed., pp 447, Academic Press, Inc.,
New York (1960).
[0017] Hyaluronidase causes hydrolysis of the endo-N-acetyl
hexosaminic bonds of hyaluronic acid and of the chondroitin sulfate
acids A and C, primarily to tetrasaccharide residues.
[0018] Significant evidence indicates that hyaluronidase enzymes
derived from different sources differ in enzyme molecular weight
distribution and in specific enzymatic activities. Such variability
in molecular weight distribution and specific enzymatic activity
are noteworthy considerations in view of the fact that
hyaluronidase enzymes may be isolated from a variety of sources,
including bovine testes, ovine testes, certain bacteria such as
streptomyces and certain invertebrate animals such as leaches.
[0019] The Wydase.RTM. hyaluronidase preparation is reported to
have been previously administered to the eyes of mammals for
various clinical and experimental applications, including the
treatment of glaucoma and the promotion of liqudifacation of the
vitreous body during vitrectomy procedures wherein the vitreous
body is removed from the eye.
[0020] Although some hyaluronidase preparations have been reported
to exhibit desirable therapeutic effects when injected into or
administered topically to the eye, the potential toxicities of
hyaluronidase and/or the thimerosal preservative are cause for
concern regarding the safety of routine clinical administration of
such, preparations by intraocular injection.
[0021] Accordingly, there exists a need in the art for the
formulation and development of a new hyaluronidase preparation
which may be administered to the eye at dosage levels which are
sufficient to bring about optimal therapeutic effects, but which do
not cause ocular toxicity.
[0022] Additionally, in view of the above-discussed problems
associated with the slowness of natural clearance of hemorrhagic
blood from the vitreous body, there exists a need in the art for
the elucidation and development of new methods and procedures for
accelerating the clearance of hemorrhagic blood from the vitreous
body of the eye so as to permit trans-vitreal viewing of the
posterior aspect of the eye, including the retina, without the need
for removal of the vitreous body (i.e., total or partial
vitrectomy).
SUMMARY OF THE INVENTION
[0023] The present invention provides a method for accelerating the
clearance of hemorrhagic blood from the vitreous humor of a
mammalian eye, wherein the method comprises the step of contacting,
with the vitreous humor, an amount of an enzyme which is active to
accelerate the clearance of hemorrhagic blood from the vitreous
humor. Specific enzymes which may be administered to bring about
the hemorrhage clearing effect of the present invention include
.beta.-glucuronidases such as hyaluronidase, keratinase,
chondroitinase AC, chondroitinase B and chondroitinase ABC;
chondroitin sulfatases such as chondroitin 4 sulfatase and
chondroitin 6 sulfatase; matrix metalloproteinases such as matrix
metalloproteinase 1, matrix metalloproteinase 2, matrix
metalloproteinase 3 and matrix metaloproteinase 9; and
protein-kinases such as streptokinase and urokinase.
[0024] Further in accordance with the present invention, there is
provided an improved, thimerosal free, hyaluronidase preparation
which is suitable for administration onto or within the eye as a
therapeutic agent for the treatment of various disorders including,
but not limited to, the acceleration of the clearance of
hemorrhagic blood from the vitreous humor in accordance with the
hemorrhage-clearing methodology of the present invention. This
hyaluronidase preparation of the present invention comprises a
preferred hyaluronidase enzyme which is substantially devoid of
hyaluronidase molecules having molecular weights in excess of
100,000 MW, between 60,000-70,000 MW and/or below 40,000 MW. The
preferred hyaluronidase of the present invention may be obtained
from ovine testes, and may be combined in aqueous solution with
quantities of lactose and phosphate, to provide a thimerosal-free
aqueous hyaluronidase solution for intraocular injection.
[0025] Further objects and advantages of the present invention will
become apparent to those skilled in the area upon reading and
understanding of the following detailed description, the
accompanying figures, and the examples set forth therein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows an electrophoresis gel having lanes 1-6, said
lanes indicating 1) molecular weight standards from 31,000 MW
through 200,000 MW 2) ovine hyaluronidase (ACS), 3) bovine
hyaluronidase type VI-S, 4) ovine hyaluronidase type V, 5) bovine
hyaluronidase type IV-S and 6) bovine hyaluronidase type I-S.
[0027] FIG. 2 is a table which summarizes the zymographically
determined hyaluronic acid lysing, gelatinolytic and caseinolytic
activities of the hyaluronidase ACS of the present invention, in
comparison to bovine hyaluronidases of types VI-S, IV-S and I-S and
ovine hyaluronidase of type V.
[0028] FIG. 3 is a table which summarizes the toxic effects of
single dose intravitreal injections of BSS, BSS+thimerosal,
hyaluronidase (ACS) and hyaluronidase Wydase.RTM. in rabbits, in
accordance with Example I herebelow.
[0029] FIG. 4 is a table which summarizes the efficacy of
single-dose intravitreal hyaluronidase (ACS) in rabbits, in
accordance with Example II herebelow.
[0030] FIG. 5 is a table which summarizes the safety and efficacy
of multiple doses of intravitreal hyaluronidase (ACS) in rabbits,
in accordance with Example III herebelow.
[0031] FIG. 6 is a table which summaries the hemorrhage-clearing
efficacy of single dose hyaluronidase ACS in human patients having
diabetic retinopathy, in accordance with Example IV herebelow.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] The following detailed description and the accompanying
examples are provided for purposes of describing and explaining
certain preferred embodiments of the invention only, and are not
intended to limit the scope of the invention in any way.
i. Enzymatic Method for Accelerating Clearance of Hemorrhagic Blood
from the Vitreous of the Eye
[0033] In accordance with the invention, applicant has determined
that certain types of enzymes, when contacted with the vitreous
humor following hemorrhage thereinto, will accelerate the rate at
which the hemorrhagic blood is cleared from the vitreous humor.
[0034] In this regard, Applicant has devised a method for
accelerating clearance of hemorrhagic blood from the vitreous of
the eye, said method generally comprising the step of contacting,
with the vitreous humor, at least one enzyme in an amount which is
active to accelerate the clearance of hemorrhagic blood from the
vitreous humor. This hemorrhage-clearing method of the present
invention may be performed without any vitrectomy or other surgical
manipulation or removal of the vitreous humor, thereby avoiding the
potential risks and complications associated with such vitrectomy
procedures.
[0035] Specific .beta.-glucuronidase enzymes which exhibit this
hemorrhage-clearing effect include:
[0036] Hyaluronidase
[0037] Keratinase
[0038] Chondroitinase AC
[0039] Chondroitinase B
[0040] Chondroitinase ABC
[0041] Chondroitin 4 sulfitase; and
[0042] Chondroitin 6 sulfatase
[0043] Specific metalloproteinase enzymes which exhibit this
hemorrhage-clearing effect include:
[0044] Matrix Metalloproteinase-1
[0045] Matrix Metalloproteinase-2
[0046] Matrix Metalloproteinase-3
[0047] Matrix Metalloproteinase-9
[0048] Specific protein-kinase enzymes which exhibit this
hemorrhage-clearing effect include:
[0049] Streptokinase
[0050] Urokinase
[0051] The preferred route of administration of these
hemorrhage-clearing enzymes is by intraocular injection, whereby an
injectable solution containing one or more of the above-listed
hemorrhage clearing enzymes is injected, through a needle, directly
into the vitreous body located within the posterior chamber of the
eye. Alternatively, however, the hemorrhage-clearing enzyme(s), of
the present invention may be administered by any other suitable
route of administration (e.g., topically) which results in
sufficient distribution of the enzyme(s) to the vitreous body to
cause the desired hemorrhage-clearing effect.
[0052] The preferred injectable solution of the hemorrhage-clearing
enzyme(s) may contain, in addition to the hemorrhage-clearing
enzyme(s), certain inactive ingredients which cause the solution to
be substantially isotonic, and of a pH which is suitable for
injection into the eye. Such solution for injection may be
initially lyophilized to a dry state and, thereafter, may be
reconstituted prior to use.
ii. A Preferred Hyaluronidase Preparation for Ophthalmic
Administration
[0053] A general formulation for an injectable thimerosal-free,
hyaluronidase preparation, of the present invention is shown in
Table I as follows: TABLE-US-00001 TABLE I General Information
Ingredient Quantity Hyaluronidase (ACS) 0-8000 International units
Lactose, USP 13.3 mg-133.0 mg Phosphate, USP 5-200 mmoles
[0054] These formulation ingredients are initially dissolved in
sterile water, sterile filtered and subsequently lyophilized to a
dry composition. The lyophilized composition is packaged for
subsequent reconstitution prior to use, in balanced salt solution.
Such balanced salt solution typically contains: 0.64% sodium
chloride, 0.075% potassium chloride, 0.048% calcium chloride
dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% sodium
acetate trihydrate, 0.17% sodium citrate dihydrate, sodium
hydride/hydrochloric acid to adjust the pH, and water for injection
is 100%.
[0055] The term "hyaluronidase ACS" as used herein describes a
preferred hyaluronidase which is devoid of hyaluronidase molecular
weight fractions above 100,000, between 60,000-70,000 and below
40,000. Such hyaluronidase may be derived from ovine testicles and
is available commercially from Calbiochem Biochemicals, P.O. Box
12087, La Jolla, Calif. 92039-2087. Applicants have determined that
this specific molecular weight distribution of the hyaluronidase
ACS results in less ophthalmic toxicity than other hyaluronidase
preparations, while exhibiting desirable therapeutic efficacy in a
number of ophthalmic applications.
[0056] FIG. 1 shows an electrophoresis gel (10% SDS-PAGE) which
demonstrates the molecular weight distribution of the preferred
hyaluronidase ACS in comparison to the molecular weight
distributions of bovine type VI-S, IV-S and I-S and ovine typw V
hyalduronidases obtained from Sigma Chemical Company, P.O. Box
14508, St. Louis, Mo. 63178. Standardized amounts (i.e., equivalent
units of hyaluronidase activity) of each enzyme was loaded into
each lane (lanes 2-6) of the electrophoresis gel shown in FIG. 1.
Lane 1 of the electrophoresis gel shown in FIG. 1 contains
molecular weight markers at 200,000 MW, 116,000 MW, 97,400 MW,
66,000 MW, 45,000 MW, and 31,000 MW, respectively. Lanes 2-6 of the
electrophoresis gel shown in FIG. 1 contain the respective
hyaluronidase preparations tested, as follows: TABLE-US-00002 LANE
WHAT IS IN THE LANE 2 Hyaluronidase ACS 3 Bovine Hyaluronidase type
VI-S 4 Ovine Hyaluronidase type V 5 Bovine Hyaluronidase type IV-S
6 Bovine Hyaluronidase type IS
[0057] Lane 2 shows that the molecular weight distribution of the
hyaluronidase ACS includes molecular weight fractions of 97,400,
50,000 (approx.) and 45,000 (approx.), but is clearly devoid of
molecular weight fractions above 100,000, between 60,000-70,000 and
below 40,000.
[0058] Lanes 3, 4, 5 and 6 of the electrophoresis gel of FIG. 1
show that all of the bovine testicular hyaluronidases of types
VI-S, IV-S and I-S and ovine testicular hyaluronidase of type V
tested differ from the hyaluronidase ACS of the present invention
in that they include molecular weight fractions between
60,000-70,000 MW and below 40,000 MW. Also, three (3) of the four
(4) bovine testicular hyaluronidases tested (i.e., types. VI-S,
IV-S and I-S) included hyaluronidase molecular weight fractions
which were in excess of 100,000 MW.
[0059] Additionally, zymograms were preformed to compare the
relative lytic activities of standardized amounts (i.e., equivalent
units of hyaluronidase activity) of the above-described
hyaluronidase ACS, type VI-S, V, IV-S and I-S bovine hyaluronidases
upon hyaluronic acid, gelatine and casein. With respect to FIG. 2,
the specific methods by which each of these zymograms was performed
are as follows:
Zymogram for Gelatinolytic Activity
[0060] GELATIN-1 mg/ml gelatin; 10% polyacrylamide; overnight
buffer=50 mM Tris HCl, 5mM CaCl.sub.2, 0.05% Triton X-100 pH 7.5;
stain Coomassie blue; destain 10% acetic acid/50% methanol.
Zymogram for Caseinolytic Activity
[0061] CASEIN-4 mg/ml; 15% polyacrylamide; overnight buffer=50 mM
Tris/HCl, 5 mM CaCl.sub.2 and 0.05% Triton X-100 pH 7.5; stain
Coomassie blue; destain 10% acetic acid/50% methanol.
Zymogram for hyaluronic Acid Lysing Activity
[0062] HYALURONIC ACID 2 mg/ml, 10% polyacrylamide; overnight
buffer=phosphate buffered saline, pH 7.4; stain 0.5% alcian blue in
3% acetic acid; destain 10% acetic acid/50% methanol.
[0063] The results of these hyaluronic acid, gelatin and casein
zymograms are summarized in the table of FIG. 2. Notably, the
preferred hyaluronidase ACS of the present invention is devoid of
hyaluronic acid lysing molecular weight fractions above
approximately 100,000 MW while each of the bovine testicular
hyaluronidases tested (i.e., types VI-S, IV-S and I-5) contained
hyaluronic acid lysing molecular weight fractions above 100,000
MW.
[0064] Similarly, the hyaluronidase ACS of the present invention
was devoid of gelatinolytic molecular weight fractions between
approximately 60,000-100,000 MW, while each of the bovine
testicular hyaluronidases tested included gelatinolytic molecular
weight fractions between approximately 60,000-100,000 MW.
[0065] Also, the hyaluronidase ACS of the present invention was
devoid of caseinolytic molecular weight fractions above
approximately 45,000 MW while each of the bovine testicular
hyaluronidases (i.e., types VI-S, IV-S and I-S) and ovine
testicular hyaluronidase (type V) tested did contain caseinolytic
molecular weight fractions above approximately 45,000 MW.
[0066] The specific molecular weight distribution and specific
enzyme activity profile of the preferred hyaluronidase (ACS) of the
present invention, and/or the exclusion of thimerosal from its
formulation, provides a hyaluronidase preparation which is
non-toxic to the eye when administered at dosage levels at which
other hyaluronidase preparations would cause toxic effects.
[0067] For use in the examples set forth herebelow, the preferred
hyaluronidase ACS was prepared in a thimerosal free formulation by
the method and general formula described herebelow and shown in
Table I. More specifically, the hyaluronidase used in the following
examples prepared in accordance with the specific formulation shown
in Table II herebelow. TABLE-US-00003 TABLE II Specific Formulation
Ingredient Quantity Hyaluronidase (ACS) 7,200 I.U. Lactose USP 13.3
mg Phosphate USP 5 mmoles
[0068] As described in the following examples, the specific
preferred formulation as hyaluronidase ACS set forth in Table II
(above) may be injected directly into the posterior chamber of the
eye at dosage levels which bring about desirable therapeutic
affects, including but not necessarily limited to the intravitreal
hemorrhage clearing effect of the present invention, without
causing significant toxicity to the eye or associated anatomical
structures.
EXAMPLE I
Ophthalmic Toxicities of Thimerosal, Hyaluronidase (ACS) and
Hyaluronidase (Wydase.RTM.) in Rabbits
[0069] Fifty-Two (52) healthy rabbits of the New Zealand Cross
variety (26 male, 26 female) weighing 1.5 kg to 2.5 kg, were
individually marked for identification and were housed individually
in suspended cages. The animals received a commercially available
pelleted rabbit feed on a daily basis, with tap water available ad
libitum.
[0070] The animals were divided into thirteen groups of 4 animals
each (2 male, 2 female). Two animals in each group (1 male, 1
female) were selected for pretreatment fundus photography and
fluorescein angiography.
[0071] The fundus photography was performed by restraining the
animals and visualizing the optic nerve, retinal arcades and fundus
with a KOWA.RTM. RC-3 Fundus Camera loaded with Kodak Gold 200 ASA
film.
[0072] The fluorescein angiography involved a 1.5 ml injection of
2% sterile fluorescein solution via the marginal ear vein.
Approximately 30 seconds post-injection the fluorescein was
visualized upon localization of the optic nerve, retinal vessels
and fundas.
[0073] The following day, each animal was anesthetized by
intravenous administration of a combination of 34 mg/kg of ketamine
hydrochloride and 5 mg/kg xylazine. The eyelids were retracted
using a lid speculum, and the eyes were disinfected with an
iodine-providone wash.
[0074] Experimental treatments of either balanced salt solution
(BSS), BSS+thimerosal, hyaluronidase (Wydase.RTM.) or hyaluronidase
(ACS) were administered by injection using a Icc tuberculin syringe
with a 30 gauge, 0.5 inch needle attached thereto. The
hyaluronidase (ACS) solution utilized in this example was free of
thimerosal and constituted the specific preferred hyaluronidase ACS
formulation set forth in Table II hereabove.
[0075] The experimental treatments administered to each animal
group were as follows: TABLE-US-00004 Group # Treatment 1 BSS 2 BSS
+ 0.0075 mg Thimerosal 3 BSS + 0.025 mg Thimerosal 4 Hyaluronidase
(Wydase) 1 IU 5 Hyaluronidase (Wydase) 15 IU 6 Hyaluronidase
(Wydase) 30 IU 7 Hyaluronidase (Wydase) 50 IU 8 Hyaluronidase
(Wydase) 150 IU 9 Hyaluronidase (ACS) 1 IU 10 Hyaluronidase (ACS)
15 IU 11 Hyaluronidase (ACS) 30 IU 12 Hyaluronidase (ACS) 50 IU 13
Hyaluronidase (ACS) 150 IU
[0076] The day following the injections (Day 1), the 26 animals
which were subjected to the fundas photography and fluorescein
angiography were observed using the same methods as for the predose
examination.
[0077] On Day 2 following the injections, the 13 male rabbits that
had received the fundus photography and fluorescein angiography at
predose and Day 1, as well as the 13. female rabbits that were not
selected for photography were euthanized with a sodium
pentobarbital based drug. The eyes were then surgically removed and
placed in a fixture solution of 2.5% glutaraldehyde with 0.1 M
phosphate buffered saline at pH 7.37.
[0078] Alternatively, one randomly selected rabbit was euthanized
by pentobarbital injection but then fixed by intracardiac injection
of the of the glutaraldehyde solution into the left ventricle to
determine the effect of the fixation procedure on the histology
findings within the enucleated eyes.
[0079] On Day 7, the 13 female rabbits that had been previously
photographed and angiography performed were subjected to the same
observations following the, methods previously described.
[0080] The remaining 26 animals were euthanized as described above
7 days after dosing. The eyes were fixed in the same manner as
those which had been fixed on day 2. Also, one randomly selected
rabbit was subjected to the same intracardiac glutaraldehyde
fixation procedure described hereabove for the previously randomly
selected animal.
[0081] The eyes of the animals treated in this example were
examined grossly and microscopically for evidence of
treatment-related toxicities. A table setting forth a summary of
the histological evidence of toxicity or non-toxicity in each
treatment group, is set forth in FIG. 3.
[0082] In summary, the eyes of the BSS-treated control group were
free of toxicity at 2 and 7 days post dose.
[0083] The eyes of the Group No. 2 animals treated with
BSS+thimerosal (0.0075mg) were free of toxicity at day 2, but
exhibited evidence that there was a breakdown of the blood-retinal
barrier at day 7.
[0084] The Group No. 3 animals treated with BSS+thimerosal (0.025
mg) exhibited severe treatment-related toxic effects, at days 2 and
7 post dose.
[0085] The Group No. 4 animals treated with Wydase.RTM. at the 1
I.U. dose were free of toxicity at days 2 and 7, however, the eyes
of the animals in Group Nos. 5-8 treated with Wydase.RTM. at
dosages ranging from 15 I.U. -150 I.U. exhibited generally
dose-related toxic effects at days 2 and 7 post dose.
[0086] The eyes of animals in treatment Groups Nos. 9-13 treated
with hyaluronidase (ACS) at dosages ranging from 1 I.U. through 150
I.U., were free of evidence of toxic effects at days 2 and 7 post
dose.
[0087] Accordingly, it is concluded that thimerosal and the
thimerosal-containing Wydase.RTM. formulation do cause toxic
effects in the eyes of rabbits at the dosages tested, however,
hyaluronidase (ACS) caused no toxic effects in these animals at the
dosages tested.
[0088] The results of the examinations conducted on day 7 are
summarized in FIG. 3. As shown, in FIG. 3, significant toxic
effects were observed on day 7 in the eyes of rabbits treated with
BSS plus thimerosal (0.0075 mg.) and hyaluronidase (Wydase) at all
doses between 1 I.U.-150 I.U. In contrast, no toxic effects were
observed in the eyes of animals treated with hyaluronidase (ACS) at
doses between 1-50 I.U.
EXAMPLE II
Safety and Efficacy of Hyaluronidase (ACS) Injected Intravitreally
in Rabbit Eyes
[0089] In this example, 12 healthy rabbits of the New Zealand Cross
variety were marked for identification and individually housed in
suspended cages. The animals received commercially pelleted rabbit
feed on a daily basis and tap water was available ad libitum.
[0090] The animals were randomly divided into four (4) treatment
groups of three (3) animals each.
[0091] Initially, the eyes of each animal were examined by dilation
with 1-2 drops of 10% Tropicanide followed by gross examination,
indirect ophthalmoscopy using a 20 diopter lens, and slit lamp
examination of the anterior anatomy of the eye.
[0092] Following the initial examination of the animals eyes, 100
.mu.l or 10 .mu.l of blood was injected intravitreally into each
eye of each animal.
[0093] On day 2, the animals of each treatment group received a
single intravitreal injection of either BSS or Hyaluronidase (ACS)
into the right eye, in accordance with the following treatment
schedule: TABLE-US-00005 Treatment Group # Left Eye Right Eye A
None BSS (30 .mu.l) .times. 1 B None 25 I.U. Hyaluronidase (ACS) in
30 .mu.l .times. 1 C None 50 I.U. Hyaluronidase (ACS) in 30 .mu.l
.times. 1 D None 75 I.U. Hyaluronidase (ACS) in 30 .mu.l .times.
1
[0094] The hyaluronidase (ACS) preparation used in this experiment
was the preferred formulation described hereabove and shown in
Table II.
[0095] On days 3, 5, 7, 14 and 21 the eyes of each animal were
again examined by slit-lamp to evaluate the cornea, anterior
chamber and iris. In addition, the eyes of each animal were dilated
with 10% tropicamide solution and the retina was examined by
indirect ophthalmoscopy with a 20 dioptor lens.
[0096] The observed hemorrhage-clearing efficacy of hyaluronidase
ACS is summarized in FIG. 4. In general, the left eye (untreated)
of each animal in each treatment group contained hazy vitreous and
some blood clots, due to the quantity of blood which had been
injected therein. The right eyes of the BSS treated (control)
animals of Group A also contained hazy vitreous and some blood
clots, while the right eyes of all hyaluronidase-treated animals in
Treatment Groups B-D contained vitreous which was clear and through
which transvitreal visualization of the retina was possible.
Furthermore, the retinas of the rights eyes of all animals in
Treatment Groups B-D appeared normal and free of treatment-related
toxicity.
[0097] The results of this experiment indicate that intravitreally
administered hyaluronidase (ACS) was effective at single doses of
25-75 I.U., to accelerate the rate at which blood was cleared from
the eyes of the treated animals and further that such single doses
of hyaluronidase (ACS) administered in this experiment did not
cause observable toxic effects in the eyes of the rabbits treated
in this experiment.
[0098] The observations following each dose were consistent and are
summarized in FIG. 5. In general, the left eye (untreated) of each
animal in each treatment group, contained hazy vitreous humor and
some blood clots, due to the quantity of blood which had been
injected therein. The right eyes of the BSS treated (control)
animals of Group A also contained hazy vitreous and some blood
clots, while the right eyes of all animals in treatment Groups B-E
(i.e., the animals treated with hyaluronidase (ACS)) contained
clear vitreous through which transvitreal visualization of the
retina was possible. Furthermore, the retinas of the right eyes of
all animals in treatment Groups B-D appeared to be normal and free
of treatment-related toxicity, even after multiple doses of the
hyaluronidase ACS.
[0099] The results of this experiment indicate that intravitreally
administered hyaluronidase (ACS) was effective, at single doses of
25-75 I.U. x 4, to accelerate the rate at which blood was cleared
from the eyes of rabbits and further that such dosages of
hyaluronidase (ACS), and that such doses of hyaluronidase ACS did
not cause observable toxic effects in the eyes of the treated
rabbits, even after four (4) consecutive doses of hyaluronidase ACS
administered at 2 week intervals.
EXAMPLE III
Hemorrhage Clearing Efficacy of Hyaluronidase (ACS) in Humans
[0100] In this experiment, six (6) human patients (5 female, 1
male) who presented with intravitreal hemorrhage were treated with
single intravitreal injections of Hyaluronidase (ACS) at dosages of
50-200 I.U.
[0101] The Hyaluronidase (ACS) administered in this experiment was
prepared by the preferred formulation described hereabove and shown
in Table II.
[0102] All of the patients treated in this experiment had a history
of diabetic retinopathy, and were found to have intravitreal
hemorrhages of varying duration. In each patient, the amount of
blood present in the vitreous was sufficient to prevent viewing of
the retina by standard funduscopic means.
[0103] Each patient received a single intravitreal injection of
hyaluronidase (ACS). Four (4) patients received a dose of 50 I.U.,
one (1) patient received a dose of 70 I.U., and one patient
received a dose of 200 I.U.
[0104] The observed results of this experiment are summarized in
FIG. 6.
[0105] In the six (6) patients treated in this example, the
hemorrhagic vitreous became sufficiently clear to permit
transvitreal viewing of the retina within 6-16 days of the single
intravitreal injection of the hyaluronidase (ACS). Such clearing of
the vitreous was subjectively determined to have occurred
significantly faster than that which would have been expected to
occur in these patients without hyaluronidase treatment.
[0106] The foregoing detailed description, examples, and
accompanying figures have described the present invention with
reference to certain presently preferred embodiments thereof. It
will be appreciated by those skilled in the art that various
deviations may be made from the specific embodiments and
formulations described hereabove, without departing from the
intended spirit and scope of the present invention. Accordingly, it
is intended that all such reasonable deviations be included within
the scope of the following claims.
* * * * *