HK1018401A - Enzymatic method and compositions for treating intravitreal hemorrhagic blood - Google Patents

Description

Enzymatic method and composition for treating intravitreal hemorrhage

Technical Field

The present invention relates generally to enzyme preparations for use in therapeutic administration to the eye of a human or other mammal, and in particular to a) methods of using one or more enzymes to accelerate the clearance of blood from vitreous hemorrhage of the eye of a mammal and b) an improved hyaluronidase for ophthalmic administration.

Background

i. Anatomy of the human eye

In humans, the anatomy of the eye includes the "vitreous body" located behind the lens, accounting for almost four fifths of the cavity of the eye. The vitreous body is formed of a gel-like substance, so-called molten glass. In general, the vitreous humor of a normal human eye contains about 99% water and 1% of macromolecular substances including: collagen, hyaluronic acid, soluble glycoproteins, sugars and other low molecular weight metabolites.

The retina is basically a layer of nerve tissue formed inside the posterior surface of the eyeball. The retina is surrounded by a layer of cells called the choroid. The retina can be divided into a) an optical portion that participates in the visual mechanism and b) a non-optical portion that does not participate in the visual mechanism. The optical portion of the retina contains rods and cones, which are visual effectors. Many arteries and veins enter the retina at its center and are spread outward to provide blood circulation to the retina.

The posterior portion of the vitreous body is in direct contact with the retina. The network of fibril bundles extends from the retina and distributes or penetrates into the vitreous body so that it meets the retina.

Etiology, treatment and clinical sequelae of intravitreal hemorrhage

Diabetic retinopathy, trauma and other ophthalmic diseases sometimes result in retinal vessel destruction or leakage with resultant bleeding into the vitreous humor of the eye (i.e., "intravitreal hemorrhage"). This intravitreal hemorrhage is typically manifested as a cloudy or opaque vitreous humor.

Intravitreal hemorrhage is sometimes, but not always, accompanied by tears or separation of the retina. In the case of intravitreal hemorrhage with retinal tears or separations, it is important that such retinal tears or separations be diagnosed and surgically repaired in a timely manner. Failure to diagnose and repair a retinal tear or separation in a timely manner can lead to necrosis of the retinal photoreceptor cells in the area of the tear or separation. This necrosis of retinal photoreceptor cells can lead to vision loss. Furthermore, retinal detachment for a period of time in an unrepaired state can lead to further intravitreal hemorrhage and/or the formation of fibrous tissue at the site of the hemorrhage. This fibrous tissue formation can lead to poor adhesion between the vitreous and the retina.

The general surgical procedure used to repair retinal tears or separations requires the surgeon to look through the vitreous to visualize the damaged area of the retina (i.e., "transvitrectomy"). When intravitreal hemorrhage occurs, the presence of the bleeding blood within the vitreous can cloude the vitreous to the point of preventing the surgeon from viewing the retina through the vitreous. This vitreal hemorrhagic turbidity may take 6-12 months or more to clear enough for transvitreous examination of the retina. However, waiting for the natural clearance of such bleeding blood to occur is generally undesirable, as delayed diagnosis or treatment of a retinal tear or separation can create potential complications.

Moreover, even in the absence of intravitreal hemorrhage associated with retinal tears or separations, it is often difficult to confirm that retinal tears or separations have not occurred because hemorrhagic opacification prevents the ability of the physician to perform routine ophthalmoscopy of the retina. Moreover, the presence of hemorrhage within the vitreous can significantly impair the vision of the affected eye of the patient, and the damage caused by such hemorrhage can persist until the bleeding blood is substantially or completely removed. .

Thus, the presence of bleeding blood within the vitreous body can cause a variety of clinical problems, including a) the inability to visually inspect and diagnose the bleeding site and/or any corresponding tears or separations of the retina, b) total or partial impairment of vision in the affected eye and c) adverse and obstructive performance of the type of vitrectomy surgery typically employed to repair the bleeding site and/or to repair any accompanying retinal tears or separations.

In a case where intravitreal hemorrhage has led to substantial opacity or opaqueness of the retina, the treating physician may choose to perform a so-called vitrectomy, in which all (or part) of the vitreous is removed from the inside of the eye and replaced with clear fluid. Such vitrectomy procedures are performed in order for the physician to view the retina sufficiently for the necessary retinal examination and/or surgical repair of the hemorrhage and any accompanying retinal tears or separations. However, this vitrectomy procedure is highly technically intensive and is associated with several significant drawbacks, risks and complications. These disadvantages, risks and complications are so latent that the removal of the vitreous may cause further retinal detachment or tears and/or cause further bleeding of the otherwise fragile retinal blood vessels.

Early applications of hyaluronidase and other enzymes

In an effort to minimize the potential for further retinal detachment or tears during the performance of a vitrectomy, it has previously been proposed in U.S. patent No. 5,292,509(Hageman) to inject certain protease-free glycosaminoglycanases into the vitreous to separate (uncouple) or "free" (disinsert) the vitreous from the retina and then remove the vitreous. This vitreous detachment or separation is intended to minimize the likelihood of further retinal tears or separations that may occur when the vitreous is removed. Examples of specific protease-free glycosaminoglycanases intended to be useful for producing such vitreous freedom include; chondroitinase ABC, chondroitinase AC, chondroitinase B, chondroitin 4-sulfatase, chondroitin 6-sulfatase, hyalinase and beta-glucuronidase.

Although it is known that the hyalinase enzyme can be used for various ophthalmic applications, including the vitrectomy adjunct application described in U.S. patent 5,292,509(Hageman), published studies have shown that the hyalinase enzyme itself can be toxic to the retina and/or other anatomical structures of the eye. Referring to the description of the preferred embodiment,safety of hyaluronidase in vitreous(ii) a Gottleib, j.l.; antosszyk, a.n., Hatchell, d.l., and Soloupis, p., Invest Ophthalmol Vis Sci 31: 11, 2345-52(1990).

The ophthalmic toxicity of some hyaluronidase preparations has been demonstrated by other investigators who have suggested using such hyaluronidase preparations as a toxic in animal modelsThe stimulant induces experimental induced neovascularization of the eye. Referring to the description of the preferred embodiment,experimental animal models of rabbit pre-retinal neovascularization;Antoszyk，A.N.，Gottleib，J.L.，Casey，R.C.，Hatchel，D.L.andMachemer，R.，Invest Ophthalmol Vis Sci 32：1，46-51(1991)。

unfortunately, it has not previously been known whether the reported therapeutic activity and toxicity of the hyaluronidase is generally applicable to all hyaluronidase formulations, or whether the efficacy and/or toxicity is applicable only to hyaluronidase formulations containing certain excipient materials or to hyaluronidase from a particular source. This is an important consideration because the various hyaluronidase formulations used in the foregoing work may vary in purity and characteristics (e.g., molecular weight distribution) depending on the source of the hyaluronidase and the solvent and/or other formulation ingredients to which the hyaluronidase is bound.

Purity and characterization of early stage hyaluronic acid esterase preparations for ophthalmic administration

The term "hyaluronidase" is commonly used to describe a group of endo-beta glucuronidases that cleave certain mucopolysaccharides, such as hyaluronic acid. Myer, k. et al, enzymes; volume four, page 447, science publishing company, new york (one nine six zero years).

The hyaluronidase causes hydrolysis of endo-N-acetylhexosamine linkages of hyaluronic acid and chondroitin sulfates A and C, mainly to tetrasaccharide residues.

There is clear evidence that hyaluronidase from different sources differs in both the molecular weight distribution and the specific enzymatic activity of the enzyme. This difference in molecular weight distribution and specific enzyme activity is considerable in view of the fact that the hyaluronidase can be isolated from a variety of sources including bovine testes, ovine testes, certain bacteria such as streptomyces and certain non-vertebrates such as leeches.

It has been reported that Wydase formulations have previously been administered to mammalian eyes for a variety of clinical and experimental applications, including the treatment of glaucoma and the promotion of vitreous liquefaction during a vitrectomy procedure to remove the vitreous from the eye.

Although some hyaluronidase formulations have been reported to exhibit satisfactory therapeutic efficacy when injected or topically applied to the eye, the potential toxicity of hyaluronidase and/or thimerosal preservatives remains a cause of concern with respect to the safety of conventional clinical administration of intraocular injection formulations.

Accordingly, there exists a need in the art to combine and develop new formulations of hyaluronidase that can be administered to the eye at dosage levels sufficient to produce the most desirable therapeutic effect without causing toxicity to the eye.

Thus, in view of the above-discussed problems associated with the slow removal of blood from vitreous humor natural hemorrhage, there exists a need in the art to develop new methods and procedures for accelerating the removal of blood from the vitreous humor of an eye such that posterior segments of the eye, including the retina, can be subjected to vitrectomy procedures without the need for removal of the vitreous humor (i.e., total or partial vitrectomy).

Summary of The Invention

The present invention provides a method for accelerating the clearance of blood from a vitreous hemorrhage of a mammalian eye, wherein the method comprises contacting said vitreous with an amount of an enzyme active in accelerating the clearance of blood from said vitreous hemorrhage. Specific enzymes may be applied to produce the bleeding clearance effects of the present invention and include beta-glucuronidases such as hyaluronidase, keratinase, chondroitinase AC, chondroitinase B and chondroitinase ABC; chondroitin sulfatase such as chondroitin 4 sulfatase and chondroitin 6 sulfatase; matrix metalloproteinases such as matrix metalloproteinase 1 and matrix metalloproteinase 2, matrix metalloproteinase 3 and matrix metalloproteinase 9; and protein kinases such as streptokinase and urokinase.

Further in accordance with the present invention, there is provided an improved, thimerosal-free, hyaluronidase formulation suitable for ocular or intraocular administration of therapeutic agents for a variety of conditions including, but not limited to, accelerated clearance of blood from vitreous hemorrhage in accordance with the inventive methodology of hemorrhage clearance. Such a hyaluronidase preparation of the present invention comprises a preferred hyaluronidase enzyme that is substantially free of hyaluronidase molecules having a molecular weight in excess of 100,000MW, between 60,000 MW and 70,000MW and/or below 40,000 MW. The preferred hyaluronidase of the present invention is available from sheep testis and can be combined with large amounts of lactose and phosphate in aqueous solution to provide an aqueous hyaluronidase solution free of thimerosal for intraocular injection.

Further objects and advantages of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description, the accompanying drawings and the embodiments illustrated therein.

Brief Description of Drawings

FIG. 1 shows an electrophoresis gel containing lanes 1-6, which represent 1) molecular weight standards 31,000MW to 200,000MW, 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.

FIG. 2 is a table summarizing the hyaluronic acid hydrolysis, gelatin hydrolysis and casein hydrolysis activities of the hyaluronidase ACS of the present invention as determined by enzymatic mapping compared to bovine hyaluronidase types VI-S, IV-S and I-S and ovine hyaluronidase type V.

FIG. 3 is a table summarizing the toxic effects of single dose intravitreal injections of BSS, BSS + thimerosal, hyaluronidase (ACS) and hyaluronidase Wydase in rabbits, according to example 1 below.

FIG. 4 is a table summarizing the efficacy of single dose intravitreal hyaluronidase (ACS) in rabbits, according to example II below.

FIG. 5 is a table summarizing the safety and efficacy of multiple doses of intravitreal hyaluronidase (ACS) in rabbits, according to example III below.

FIG. 6 is a graph summarizing the efficacy of single dose hyalase ACS hemorrhaging in patients with diabetic retinopathy according to example IV below.

Detailed description of the preferred embodiments

The detailed description and the corresponding examples below are provided for the purpose of describing and explaining certain preferred embodiments of the present invention only and are not intended to limit the scope of the present invention in any way.

i. Enzymatic method for accelerating the removal of blood from the vitreous humor of the eye

In accordance with the present invention, applicants have determined that certain types of enzymes, when contacted with the vitreous following bleeding, accelerate the rate of blood removal from the vitreous hemorrhage.

To this end, applicants have invented a method for accelerating the clearance of blood from vitreous bleedings of the eye, said method generally comprising the step of contacting the vitreous with at least one enzyme in an amount effective to accelerate the clearance of blood from vitreous bleedings. The methods of hemorrhage clearance of the present invention may be performed without any vitrectomy or other surgical procedure or removal of the vitreous humor, thereby avoiding the potential risks and complications associated with such vitrectomy procedures.

Specific beta-glucuronidases that exhibit this hemorrhagic clearance include:

hysteryl esterase

Keratinase

Chondroitinase AC

Chondroitinase B

Chondroitinase ABC

Chondroitin-4 sulfatase; and

chondroitin 6 sulfatase

Specific metalloproteases exhibiting such hemorrhage clearance include:

matrix Metalloproteinase-1

Matrix Metalloproteinase-2

Matrix Metalloproteinase-3

Matrix Metalloproteinase-9

Specific protein kinases that exhibit this hemorrhagic clearance include:

streptokinase (Tak. RTM.)

Urokinase

The preferred route of administration of these hemorrhage-clearing enzymes is by intraocular injection. Whereby an injection solution containing one or more of the hemorrhage-clearing enzymes listed above is injected directly through the needle into the vitreous cavity located in the posterior chamber of the eye. However, the hemorrhage-clearing enzyme of the invention may additionally be administered by any other suitable route of administration (e.g., topical administration) sufficient to distribute the enzyme to the vitreous to cause the desired hemorrhage-clearing effect.

In addition to the hemorrhage-clearing enzyme, the hemorrhage-clearing enzyme preferably contains an inactive ingredient that renders the solution substantially isotonic and has a pH suitable for injection into the eye. Such injectable solutions may be initially lyophilized to a dry state and then allowed to dry prior to use.

Preferred hyaluronidase formulations for ophthalmic administration

The general formulation of injectable thimerosal hyalase of the invention is given in table I below:

TABLE I

General formulation

Composition (I)	Number of
		Hyaluronidase (ACS) lactose, USP phosphate, USP	0-8000 International units 13.3 mg-133.0 mg 5-200mM

These formulation components are initially dissolved in sterile water, sterile filtered and then lyophilized to a dry formulation. The lyophilized formulation is packaged for subsequent reconstitution in a balanced salt solution prior to use. Such balanced salt solutions typically contain: 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, supplementing water to 100%.

The term "hyaluronidase (ACS)" as used herein refers to a preferred hyaluronidase which is free of molecular weight components of the hyaluronidase which are between greater than 100,000, 60,000 and 70,000 and less than 40,000. This hyaluronidase was obtained from sheep testis and was purchased from Calbiochemicals, P.O.Box 12087, La Jolla, Ca 92039-. Applicants have determined that this particular molecular weight distribution of hyaluronidase ACS results in less ophthalmic toxicity than other hyaluronidase formulations, while exhibiting desirable therapeutic effects in many ophthalmic applications.

FIG. 1 shows an electrophoretic gel (10% -SDS-PAGE) which verifies the molecular weight distribution of the preferred hyaluronidase ACS in comparison to bovine type VI-S, IV-S and I-S and ovine type V hyaluronidase purchased from Sigma chemical Company, P.O.Box 14508, St.Loius, Missouti 63178. The normalized amount of each enzyme (i.e., the equivalent unit of hyaluronidase) was added to each lane of the gel shown in FIG. 1 (lane) (lanes 2-6). Lane 1 of the electrophoretic gel shown in FIG. 1 contains molecular weight markers of 200,000MW, 116,000MW, 97,400MW, 66,000MW, 45,000MW, and 31,000MW, respectively. Lanes 2-6 of the electrophoresis gel shown in FIG. 1 contain the respective tested hyaluronidase preparations as follows:

the object in the lane

2 Hyaluronidase ACS

3 bovine hyaluronidase form VI-S

4 ovine hyaluronidase type V

5-Boehringer-esterase type IV-S

6-bovine transparentin esterase type I-S

Lane 2 shows the molecular weight distribution of hyaluronidase ACS, including molecular weight components 97,000, 50,000 (approx.) and 45,000 (approx.), but completely free of molecular weight components between above 100,000, 60,000 and 70,000 and below 40,000.

Electrophoresis gel lanes 3, 4, 5 and 6 in FIG. 1 show that all bovine testicular hyaluronidase types VI-S, IV-S and I-S and ovine testicular hyaluronidase type V tested differ from the hyaluronidase ACS of the present invention in that they include molecular weight components between 60,000 and 70,000MW and below 40,000 MW. Furthermore, three (3) (i.e., types VI-S, IV-S and I-S) of the four (4) bovine testicular hyaluronidase tested included a hyaluronidase molecular weight component of greater than 100,000 MW.

In addition, zymograms were made to compare the relative hydrolytic activity of the above-described hyaluronidase types ACS, VI-S, V, IV-S and I-S bovine hyaluronidase on hyaluronic acid, gelatin and casein in standardized amounts (i.e., equivalent units of hyaluronidase activity). With respect to FIG. 2, the specific methods used for each of these zymograms are as follows:

transparent esterase activity zymogram

Gelatin-1 mg/ml gelatin; 10% polyacrylamide; overnight buffer 50mM tris hci, 5mM calcium chloride, 0.05% Triton-100 ph 7.5; coomassie blue staining; decolorizing with 10% acetic acid/50% methanol.

Casein hydrolysis Activity zymogram

Casein-4 mg/ml; 15% polyacrylamide; overnight buffer 50mM Tris/HCI, 5mM calcium chloride and 0.05% Triton X-100 pH 7.5; coomassie blue staining; decolorizing with 10% acetic acid/50% methanol.

Transparent esterase acid hydrolysis activity zymogram

2 mg/ml of hyaluronic acid, 10% polyacrylamide; overnight buffer (phosphate buffered saline, ph 7.4); staining 0.5% alcian blue in 3% acetic acid; 10% acetic acid/methanol decolorization.

The results of these hyaluronic acid, gelatin and casein zymograms are summarized in the table of figure 2. Notably, the preferred hyaluronidase ACS of the present invention has no hyaluronan hydrolyzing molecular weight component above about 100,000MW, whereas each bovine testicular hyaluronidase tested (i.e., VI-S, IV-S, and I-S forms) contains a hyaluronan hydrolyzing molecular weight component above 100,000 MW.

Similarly, the hyaluronidase ACS of the present invention has no gelatin hydrolysis molecular weight component of between about 60,000 and 100,000MW, whereas each bovine testicular hyaluronidase tested contained a gelatin hydrolysis molecular weight component of between about 60,000 and 70,000 MW.

Furthermore, the hyaluronidase of the present invention has no casein hydrolyzing molecular weight component above about 45,00MW, whereas each of the bovine testicular hyaluronidase (i.e., types VI-S, IV-S and I-S) and ovine testicular hyaluronidase (type V) tested contains casein hydrolyzing molecular weight component above about 45,000 MW.

The particular molecular weight distribution and particular enzymatic activity profile of the preferred hyaluronidase (ACS) of the present invention (and/or the exclusion of thimerosal from its formulation) provides a hyaluronidase formulation which is non-toxic to the eye and which produces toxic effects in other hyaluronidase formulations when administered at the same dosage.

For use in the examples provided below, the preferred hyaluronidase ACS was prepared in a thimerosal-free formulation by the methods and general formulations described below and given in Table 1. In particular, the transparent esterase used in the examples described below and prepared according to the specific formulation is given in table II below.

TABLE II

Special preparation

Composition (I)	Number of
		Hyaluronidase (ACS) lactose USP phosphate USP	7,200 I.U.13.3 mg 5mM

As described in the examples below, particularly preferred formulations of hyaluronidase ACS as given in Table II (supra) can be injected directly into the posterior chamber of the eye at dosage levels that produce a desirable effect, including but not necessarily limited to the hemorrhage removing effect of the present invention, without causing significant toxicity to the eye or related anatomical structures.

Example 1

Thimerosal, hyaluronidase (ACS) and hyaluronidase (Wydase)^)

Ophthalmic toxicity to rabbits

Fifty-two (52) healthy New Zealand hybrid rabbits (26 male, 26 female) weighing 1.5 kg to 2.5 kg, were individually labeled for identification and individually housed in suspension cages. The animals eat commercial granular rabbit feed every day, and are connected with a water supply pipe at will.

The animals were divided into thirteen groups of four animals per group (2 male, 2 female). Two animals (1 male, 1 female) from each group were selected for pre-treatment fundus photography and fluorescence angiography.

Animals were fixed and the optic nerve, retinal segment tissue and fundus were photographed with a KOWA RC-3 fundus camera equipped with Kodak Gold 200 ASA film.

Fluorescence angiography included intravenous injection of 1.5 ml of 2% fluorescein solution through the ear margin. About 30 seconds after the injection of fluorescein, the optic nerve, retinal blood vessels and fundus were photographed.

On the following day, each animal was anesthetized by intravenous administration of a formulation of 34 mg/kg ketamine hydrochloride and 5 mg/kg xylazine. The eyelids were retracted with the lid speculum and the eyes were disinfected with providone iodine wash.

The formulations were administered by injection with saline solution (BSS), BSS + thimerosal, hyaloesterase (Wydase) using a 1 ml tuberculin syringe, No. 30, with a 0.5 inch needle^) Or experimental therapeutics balanced with hyaluronidase (ACS). The hyaluronidase (ACS) solution used in this example was free of thimerosal and comprised the particularly preferred hyaluronidase ACS formulation given in table II above.

The experimental treatments administered to each group of animals were as follows:

group number therapeutic agent

1 BSS

2 BSS +0.0075 mg Mercury ethyl thiosalicylate

3 BSS +0.025 mg Mercury ethyl thiosalicylate

4 Hydressase (Wydase)1 International Unit

5 Hydressase (Wydase)15 International Unit

6 Hydressase (Wydase)30 International Unit

7 Hydressase (Wydase)50 International Unit

8 Hydressase (Wydase)150 International Unit

9 Hydressase (ACS)1 International Unit

10 Hydressase (ACS)15 International Unit

11 Hydressase (ACS)30 International Unit

12 Hydressase (ACS)50 International Unit

13 Hydressase (ACS)150 International Unit

On the day following injection (first day), 26 animals were observed for fundus photography and fluorescence imaging using the same method used for background dose examination.

The next day after injection, 13 male rabbits that had received fundus and fluorescence imaging and 13 female rabbits that had not been selected for imaging at the background dose were euthanized with pentobarbital sodium. The eyes were then surgically removed and placed in a 2.5% glutaraldehyde fixation solution with 0.1 molar phosphate buffered saline at ph 7.37.

Alternatively, a randomly selected rabbit was euthanized by injection of pentobarbital and then intracardiac glutaraldehyde solution into the left ventricle to determine the effect of the fixation method on the enucleated intra-ocular tissue examination.

On day seven, 13 female rabbits, which had previously been photographed and imaged, were observed in the same manner as described above.

The remaining 26 animals were euthanized 7 days after dosing as described above. The eyeball was fixed in the same manner as the fixation on the next day. One rabbit was still randomly selected to receive the same intracardiac glutaraldehyde fixation method as received by the previously randomly selected animal.

The treated animals of this example were visually and microscopically examined for evidence of treatment-related toxicity. Figure 3 presents a table giving an overview of histological evidence of toxicity or non-toxicity for each treatment group. In summary, the eyes of the BSS-treated control group were not toxic at 2 and 7 days after dosing.

The eyeballs of the second group of animals treated with BSS + thimerosal (0.0075 mg) were non-toxic the next day, but presented evidence of breakdown of the retinal blood barrier on day 7.

Group 3 animals treated with BSS + thimerosal (0.025 mg) exhibited severe treatment-related toxic effects on days 2 and 7 post-dose.

In 1 International unit Wydase^Group 4 animals treated were not toxic on days 2 and 7, however, at doses ranging from 15 international units to 150 international units of Wydase^The eyeballs of treated animals in groups 5-8 generally exhibited overall dose-related toxic effects on days 2 and 7.

There was no evidence of toxicity at days 2 and 7 after dosing in animals from the 9 th to 13 th treatment groups treated with hyaluronidase (ACS) at a dose ranging from 1 international unit to 150 international units.

Thus, it was concluded that thimerosal and Wydase containing thimerosal^The formulations did cause toxicity in rabbit eyes at the tested doses, however, in these animals at the tested dosesThe hyaluronidase (ACS) does not produce toxic effects.

The results of the examination performed on day 7 are summarized in fig. 3. As shown in figure 3, a clear toxic effect was observed on day 7 in the eyes of BSS plus thimerosal (0.0075 mg) and hyaluronidase (Wydase) treated at all doses between 1 international unit and 150 international units. In contrast, no toxic effects were observed in eyes of animals treated with hyaluronidase (ACS) at doses between 1 and 50 international units.

Example II

Safety and efficacy of intravitreal injected hyaluronidase (ACS) in rabbit eyes

In this example, 12 healthy New Zealand hybrid rabbits were labeled for identification and housed in suspension cages. Daily, these animals were fed commercial pellet feed and were connected to drinking water pipes ad libitum.

The animals were randomly divided into four (4) treatment groups of three (3) animals per group.

Initially, the eye of each animal was examined with 1-2 drops of 10% tropicamide mydriasis followed by visual inspection, indirect ophthalmoscopy with a 20 diopter lens, and slit-lens examination of the anterior portion of the eye.

After the initial examination of the animal's eyes, 100 microliters or 10 microliters of blood was injected intravitreally into each eye of each animal.

On day 2, each treatment group received a single intravitreal injection of BSS or hyaluronidase (ACS) into the right eye according to the following treatment schedule:

group number processing

Left eye and right eye

A No BSS (30 microliter) X1

B30. mu.l X1 without 25 International units of hyaluronidase (ACS)

C No 50 International Unit Hyaluronidase (ACS) 30. mu.l X1

D No 75 International Unit Hyaluronidase (ACS) 30. mu.l X1

The hyaluronidase (ACS) used in this example is the preferred formulation described above and is given in table II.

On days 3, 5, 7, 14 and 21, the eyes of each animal were again slit-specularly examined to assess the cornea, anterior chamber and iris. In addition, the eyes of each animal were dilated with a 10% tropicamide solution and the retinas were examined with an indirect ophthalmoscope with a 20 diopter lens.

The observed efficacy of hemorrhagic clearance of hyaluronidase ACS is summarized in figure 4. Generally, the left eye (untreated) of each animal in each treatment group contained turbid vitreous and some blood clots due to the amount of blood injected therein. The right eye of the BSS-treated (control) animals of group a also contained turbid vitreous and some blood clots, while the right eye of all the hyaluronidase-treated animals in treatment groups B-D contained clear vitreous and was transparent to retinal transvitreous examination. Moreover, the right retina of all animals in the B-D treated group appeared normal and had no treatment-related toxicity.

The results of this experiment show that intravitreally administering a single dose of hyaluronidase (ACS) in the 25-75 international units is effective in increasing the rate of blood clearance from the eyes of treated animals, and further that administration of such a single dose of hyaluronidase (ACS) in this experiment does not cause visible toxic effects in the treated rabbit eyes of this experiment.

Observations after each dose were consistent and summarized in figure 5. In general, the left eye (untreated) of each animal in each treatment group contained turbid vitreous humor and some blood clots due to the amount of blood infused therein. The right eye of the BSS-treated (control) animals of group a also contained turbid vitreous and some blood clots, while the right eye of all animals in treatment groups B-E (i.e. animals treated with hyaluronidase (ACS)) contained clear vitreous through which transvitreous examination of the retina could be performed. Moreover, the retinas of the right eyes of all animals in treatment groups B-D appeared normal and had no treatment-related toxicity, even after multiple doses of hyaluronidase ACS treatment.

The results of this experiment show that intravitreal administration of hyaluronidase (ACS) is effective at increasing the rate of blood clearance from rabbit eyes at 4-fold of a single 25-75 international unit dose and further show that this dose of hyaluronidase (ACS) and that this dose of hyaluronidase ACS does not cause visible toxic effects in treated rabbit eyes even if four (4) consecutive doses of hyaluronidase ACS are administered at 2 week intervals.

Example III

In humans, the efficacy of the enzyme hyaluronate in clearance of bleeding

In this experiment, six (6) patients (5 women, 1 man) with intravitreal hemorrhage were treated with a single intravitreal injection of hyaluronidase (ACS) at a dose of 50-200 International units.

The hyaluronidase (ACS) administered in this experiment was prepared by the preferred formulation described above and given in table II.

All patients treated in this experiment had a history of diabetic retinopathy and were found to have intravitreal bleeding at various stages. The amount of blood present in the vitreous in each patient is sufficient to preclude inspection of the retina with standard fundoscopic procedures.

Each patient received a single hyaluronidase (ACS) intravitreal injection. Four (4) patients received a 50 international unit dose, one (1) patient received a 70 international unit dose, and one patient received a 200 international unit dose.

The observations of this experiment are summarized in fig. 6.

In the six (6) treated patients in this experiment, the hemorrhaged vitreous became clear enough for the transvitreous examination of the retina within 6-16 days of the single injection of hyaluronidase (ACS). Such clearance of the vitreous was subjectively determined to occur significantly faster than clearance occurring in patients not treated with hyaluronidase.

The foregoing detailed description, examples, and drawings describe the invention with respect to certain presently preferred embodiments. Those skilled in the art will recognize that various deviations can be made from the above-described embodiments and formulations without departing from the spirit and scope of the invention. Accordingly, it is intended that all such reasonable deviations be included within the scope of the following claims.

Claims (31)

1. A method of accelerating the clearance of blood from a vitreous hemorrhage of a mammalian eye, the method comprising the steps of:

contacting said vitreous humor with an amount of an enzyme active to accelerate blood removal from the vitreous humor.

2. The method of claim 1, wherein the enzyme is selected from the group consisting of:

a hyaluronidase;

a 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;

a streptokinase;

urokinase; and

combinations thereof.

3. The method of claim 1, wherein the enzyme is a β -glucuronidase.

4. The method of claim 1, wherein the enzyme is a matrix metalloproteinase.

5. The method of claim 1, wherein the enzyme is a protein kinase.

6. The method of claim 1, wherein said enzyme is chondroitin sulfatase.

7. The method of claim 1, wherein the enzyme is in a fluid solution, and wherein the step of contacting the enzyme with the molten glass comprises:

injecting the fluid solution into the molten glass.

8. The method of claim 1, wherein the enzyme is a hyaluronidase.

9. The method of claim 8, wherein the amount of the hyaluronidase is 10-300 international units.

10. The method of claim 1, wherein the enzyme is administered in a single intravitreal injection.

11. The method of claim 10, wherein the single intravitreal injection volume is less than 50 microliters.

12. The method of claim 8, wherein the hyaluronidase has no molecular weight component above 100,000 MW.

13. The method of claim 8, wherein the hyaluronidase has no less than 40,000MW hyaluronidase molecular weight component.

14. The method of claim 8, wherein said hyaluronidase has no molecular weight component between 60,000 and 70,000 MW.

15. The method of claim 8, wherein said hyaluronidase has no molecular weight component between 100,000MW above, 40,000MW below and 60,000 MW 70,000 MW.

16. The method of claim 8, wherein the hyaluronidase is free of gelatin hydrolyzing hyaluronidase with a molecular weight between about 60,000 and 100,000 MW.

17. The method of claim 8, wherein the hyaluronidase is free of caseinolytic hyaluronidase that has a molecular weight of about 45,000MW or greater.

18. The method of claim 8, wherein the hyaluronidase is free of hyaluronidase which is a hyaluronic acid hydrolysis hyaluronidase with a molecular weight above about 100,000 MW.

19. The method of claim 1, wherein the method further comprises:

the enzyme is contacted with the glass bath in the absence of thimerosal.

20. The method of claim 8, wherein the hyaluronidase is prepared as a solution of thimerosal free from ethylmercurial for injection and which contains the general formulation:

hyaluronidase (ACS) … … 0-8000 international units;

lactose, USP … … 13.3.3 mg; and

phosphate, USP … … 5 mM;

and wherein the hyaluronidase has no molecular weight fraction of 40,000 or less.

21. The method of claim 20, wherein the injectable solution comprises the following specific formulation:

hyaluronidase (ACS) … … 7,200, 200 international units;

lactose, USP … … 13.3.3 mg, and

phosphate, USP … … 5 mM.

22. The method of claim 20, wherein the injectable solution is dissolved in a balanced salt solution.

23. A hyaluronidase preparation for ophthalmic administration, said preparation being free of thimerosal and having no hyaluronidase below 40,000 MW.

24. The formulation of claim 23, wherein the formulation further does not contain a hyaluronidase with a molecular weight between 60,000 and 70,000 MW.

25. The formulation of claim 23, wherein the formulation further does not contain a hyaluronidase with a molecular weight in excess of 100,000 MW.

26. The formulation of claim 23, wherein the formulation is further free of a hyaluronic acid hydrolyzing hyaluronic acid enzyme having a molecular weight above about 100,000 MW.

27. The formulation of claim 23, wherein the formulation further does not contain a caseinolytic hyaluronidase that has a molecular weight of about 45,000MW or greater.

28. The formulation of claim 23, wherein the formulation further comprises a gelatin hydrolyzing hyaluronidase with a molecular weight between about 60,000 and 70,000.

29. The formulation of claim 23, wherein the formulation is an injectable solution comprising the following general formulation:

hyaluronidase (ACS) … … 0-8000 international units;

lactose, USP … … 13.3.3-133.3 mg; and

phosphate, USP … … 5-200 mM;

and wherein the hyaluronidase has no components with a molecular weight below 40,000.

30. The formulation of claim 29, wherein the amount of hyaluronidase (ACS) in the formulation is 7,200 international units.

31. The formulation of claim 29, wherein the ingredients of the formulation are dissolved in a balanced salt solution.