Bimodal molecular weight hyaluronate formulations and methods for using same

BIMODAL MOLECULAR WEIGHT HYALURONATE FORMULATIONS AND METHODS FOR USING SAME

0002Related Application

0003This application is a continuation-in-part of co- pending application Serial No. 621,290 filed November 30, 1990.

0004Background of the Invention This invention relates to compositions and methods useful to protect human or animal eye cell layers and tissue subject to exposure to trauma. More particularly, the invention relates to compositions and methods involving hyaluronate fractions having differing molecular weights for protecting eye cell layers and tissues exposed to trauma, for example, during surgery.

0005When the natural lens of the eye becomes hazy or clouded, surgery is often indicated to remove the impaired lens. The current trend in such surgery is toward breaking the lens into a plurality of particles and then removing the particles, e.g., using the conventional phacoemulsification procedure. The use of a protective agent, in particular a viscoelastic fluid, that adheres to and protects the corneal endothelium during the surgical procedure is highly beneficial. Fluids containing high molecular weight range hyaluronates, that is hyaluronates having weight average molecular weights over 750,000, do not provide entirely acceptable adhesion to and/or protection of the corneal endothelium during certain surgical procedures. An example of such a fluid is that sold by Pharmacia Ophthalmics of Monrovia, California under the trademark Healon.

0006Formulations which include hyaluronic acid having a molecular weight of at least about 750,000, and preferably at least about 1,200,000 are disclosed in Balazs U.S. Patent 4,141,973. Such formulations, which are not described in this patent as including any lower molecular weight hyaluronic acid, are disclosed as being useful in a number of applications, such as in the replacement of the aqueous humor after various intraocular surgical procedures, and as a biological prosthesis in the anterior chamber after cataract surgery.

0007Another formulation which includes high molecular weight hyaluronate (molecular weight of 1x10 to 4.5x10 ) is that disclosed in Balazs U.S. Patent 4,303,672. The cosmetic formulations of this patent also include a low molecular weight hyaluronate fraction (molecular weight of 10,000 to 200,000) and protein in an amount ranging from 50% to 400% the weight of the hyaluronate. Such compositions are taught as having emollient, moisturizing, elasticizing and lubricating properties where applied to the skin. Such protein-containing hyaluronate compositions are not ophthalmically acceptable and would not be useful as protective agents in eye surgery. Bracke et al U.S. Patent 4,517,295 discloses hyaluronic acid having an average molecular weight of about 55,000 which is produced from bacterial sources. Such hyaluronic acid is disclosed as having potentially significant use as an eye drop ingredient and as an ingredient of cosmetic formulations, as well as being useful in post-surgical applications for reducing complications due to fibrotic response and/or adhesion formation. This patent does not disclose the use of such hyaluronic acid as a protective agent in eye surgery. Other formulations incorporate combinations of different materials, such as sodium hyaluronate and chondroitin sulfate. This type of product is described in Australian Patent 555,747. A product for use in providing protection during eye surgery and including sodium hyaluronate and chondroitin sulfate is that sold by Alcon Surgical, Inc. of Forth Worth, Texas under the trademark Viscoat. Sources of chondroitin sulfate often include significant amounts of protein which need to be removed, using relatively complex and expensive separation techniques, before the purified chondroitin sulfate can be used in the eye.

0008There continues to be a need for compositions useful in protecting ocular or eye cell layers and tissues exposed to trauma, in particular trauma involved in ophthalmic surgical procedures. Summary of the Invention

0009New compositions and methods useful to protect human or animal ocular or eye cell layers and tissues subject to exposure to trauma have been discovered. The present ophthalmically acceptable compositions, which include two distinct molecular weight fractions of alkali metal and/or alkaline earth metal hyaluronates, have been found to provide a substantial degree of protection against trauma, such as that trauma which occurs during eye surgery, in particular eye surgery such as the removal of a diseased natural lens. The present compositions, which are based on hyaluronate fractions, are relatively easy to manufacture and to obtain governmental regulatory approval on, in comparison to compositions which include two or more significantly dissimilar adhesion/protective components.

0010In one embodiment, the present ophthalmically acceptable compositions, in particular in the form of solutions, comprise water, a first alkali and/or alkaline earth metal hyaluronate fraction having a molecular weight of at least about 300,000 and a second alkali and/or alkaline earth metal hyaluronate fraction having a molecular weight of less than about 200,000.

0011The first hyaluronate fraction preferably has a mole ular weight in the range of about 350,000 to about 800,000, more preferably in the range of about 500,000 to about 750,000. The second hyaluronate fraction preferably has a molecular weight in the range of about 25,000 or about 35,000 to about 150,000 or about 175,000. The second hyaluronate fraction may have a molecular weight in the range of about 30,000 to about 100,000. Preferably, the compositions are essentially non-pyrogenic and protein-free.

0012The ophthalmically acceptable compositions may, and preferably do, include at least one buffer component in an amount effective to control the pH of the composition and/or at least one tonicity adjuster component in an amount effective to control the osmolality of the composition. More preferably, the present compositions include both a buffer component and a tonicity adjuster component. The ophthalmically acceptable compositions of the present invention are preferably sterile.

0013In another embodiment of the present invention, methods to protect human or animal eye cell layers and tissues subject to exposure to trauma are provided. These methods comprise administering an amount effective to protect the ocular or eye cell layers and tissues of an ophthalmically acceptable composition, such as described herein, to the cell layers and tissues prior to exposure to trauma.

0014In a particularly useful embodiment, methods are provided for removing the natural lens from the eye of a human or animal. These lens removal methods comprise introducing a protective amount of an ophthalmically acceptable composition, such as described herein, into the eye of a human or animal. This introduced composition acts to adhere to and/or protect the eye cell layerπ and tissues, in particular the corneal endotheliu-", in proximity to the natural lens in the eye. The natural lens is caused to break into a plurality of particles, e.g., using a conventional surgical procedure which involves a potentially traumatic force associated with a relatively high degree of turbulence. These particles are removed from the eye. The hyaluronate-containing composition is also removed from the eye. In this manner, the defective natural lens, for example, a natural lens which has developed a cataract condition, is effectively removed from the eye without causing undue trauma or adverse effects to the eye cell layers and tissues in proximity to the natural lens which is removed. The presently useful compositions are relatively easy to administer to the eye, provide a very effective and high degree of protection against the potential trauma of the surgical procedure, and are relatively easy to remove, such as by irrigation/aspiration from the eye, after the protection is no longer needed. In particular, the present compositions are maintained in a protective amount on the eye cell layers and tissues in spite of the relative high degree of turbulence associated with causing the natural lens to break into a plurality of particles and/or removing the lens particles from the eye. Without such protection, lens fragments may impact and damage sensitive cell layers and tissues, such as the corneal endothelium. Detailed Description of the Invention Ophthalmically acceptable compositions, in particular in the form of solutions, comprising water, a first alkali and/or alkaline earth metal hyaluronate fraction having a moderate to high molecular weight and a second alkali and/or alkaline earth metal hyaluronate fraction having a relatively low molecular weight, and preferably at least one buffer component and/or at least one tonicity adjuster component, have been found to provide substantial advantages, e.g., in terms of providing adhesion and/or protection to human or animal cell layers and tissues located in the eye which are exposed to trauma, in particular during surgical procedures. The present compositions also have other properties useful in viscoelastic fluids, such as high viscosity at zero shear, elasticity and pseudoplasticity. The molecular weights noted herein are the weight average molecular weights of the fraction or fractions. This weight average molecular weight can be determined or measured by an indirect method, in particular the limiting viscosity method. The weight average molecular weight may be calculated from the limiting viscosity number by the published equation of Laurent et al, Fractionation of Hyaluronic Acid, Biochimica Et Biophysics Acta, 42, pps 476-485 (1960). Alternately, the weight average molecular weight can be determined using size exclusion chromatography techniques.

0015The first hyaluronate fractions useful in the present invention have moderate to high molecular weights, in particular, molecular weights in the range of at least about 300,000, more particularly in the range of about 300,000 to about 1,000,000. The first hyaluronate fractions preferably have molecular weights in the range of about 350,000 to about 800,000, more preferably about 500,000 to about 750,000.

0016The presently useful second hyaluronate fractions have molecular weights of less than about 200,000, preferably in the range of about 25,000 or about 35,000 to about 150,000 or about 175,000. Second hyaluronate fractions having molecular weights in the range of about 30,000 to about 100,000 are very useful. As is conventional and well known in the art, the first and second hyaluronate fractions can be obtained from various sources, such as rooster combs and other connective tissue, and from bacterial sources, in particular bacterial fermentation. The molecular weight fractions can be obtained using conventional separation procedures. Alternately, high molecular weight hyaluronates can be converted, for example, hydrolyzed, to lower molecular weight hyaluronates, which are then recovered and used. The first hyaluronate fraction and second hyaluronate fraction are each preferably independently selected from the group consisting of sodium hyaluronates, potassium hyaluronates, magnesiumhyaluronates, calciumhyaluronates and mixtures thereof. More preferably, each of these fractions involves the same metal or metals. Sodium hyaluronate fractions are particularly useful.

0017The weight ratio of the first hyaluronate fraction to the second hyaluronate fraction in the presently useful compositions is preferably in the range of about 0.25 to about 4, more preferably about 0.5 to about 2. The concentration of the first hyaluronate fraction in the presently useful compositions is preferably in the range of about 5 mg/ml to about 50 mg/ml, more preferably about 10 mg/ml or about 20 mg/ml to about 40 mg/ml. The present ophthalmically acceptable compositions are preferably essentially free of protein and essentially non-pyrogenic. The present compositions preferably include less than about 0.5% by weight of protein based on the total weight of first and second alkali and/or alkaline earth metal hyaluronates. More preferably, the present compositions have no detectable protein content and no detectable pyrogenicity.

0018The present compositions preferably include a major amount of liquid water, e.g., as a carrier or liquid medium for the hyaluronate fractions. The present ophthalmically acceptable compositions are preferably sterile, in particular prior to being used in the eye.

0019The present compositions preferably include at least one buffer component in an amount effective to control the pH of the composition and/or at least one tonicity adjuster component in an amount effective to control the osmolality of the composition. More preferably, the present compositions include both a buffer component and a tonicity adjuster component. Such components are useful in maintaining the present compositions ophthalmically acceptable so that, for example, the presence of these compositions in the eye do not result in any undue adverse effect or permanent damage to the eye. The use of one or more tonicity adjuster components is particularly important when the composition is to be applied to one or more portions of the cornea. Such tonicity adjuster components act to enhance the compatibility between the composition and the corneal tissue and/or to avoid damage to the corneal tissue. Such buffer components and tonicity adjuster components may be chosen from those which are conventional and well known in the art. Examples of useful buffer components include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, borate buffers and the like and mixtures thereof. Phosphate buffers are particularly useful. Useful tonicity adjuster components include, but are not limited to, salts, particularly sodium chloride, potassium chloride, any other suitable ophthalmically acceptable tonicity adjuster component and mixtures thereof. The amounts of buffer component and osmolality control component employed are preferably sufficient to maintain the pH of the compositions in the range of about 6 to about 8, more preferably about 7 to about 7.5, and the osmolality of the compositions in the range of about 200 to about 400, more preferably about 250 to about 350, mOsmol/kg, respectively.

0020The present compositions may include one or more other components in amounts effective to provide one or more useful properties and/or benefits to the present compositions. The present compositions are useful in a method for protecting human or animal ocular or eye cell layers and tissues which are subject to being exposed to trauma, for example, during surgery. In one embodiment, the present method comprises administering a protective amount of such a composition to the ocular or eye cell layers and tissues which are subject to exposure to trauma prior to exposure to the trauma. In this manner, such compositions preferably act to adhere to, or at least partially coat, the cell layers and tissues and/or provide protection for such cell layers and tissues from an imposed trauma.

0021The present compositions provide particularly good protection in situations where the trauma or potential trauma to which the eye cell layers or tissues is exposed occurs in a dynamic, turbulent environment, such as during a surgical procedure in which a natural lens to be removed is broken into particles, such as by using the conventional phacoemulsification technique.

0022A specific application in which the present compositions find significant usefulness is in a method for removing the natural lens, e.g., which is diseased, from the eye of a human or animal. In this embodiment, a protective amount of a composition in accordance with the present invention is introduced, such as by being injected, into the eye of a human or animal, for example, through an incision made in the eye. Such compositions can be relatively easily introduced into the eye, for example, using conventional injection cannulas or the like. The composition preferably adheres to at least a portion of the eye cell layers and tissues, in particular the corneal endothelium. in proximity to the natural lens to be removed. With the present composition in place, the natural lens is caused to break into a plurality of particles. One particularly useful approach to breaking up the natural lens is to use a conventional lens emulsification procedure, such as the well known phacoemulsification procedure. Unless the portions of the eye in proximity to the natural lens are provided with protection, the dynamic force used to break up the natural lens and/or the turbulence which often occurs during such lens removal procedures may also damage these other portions of the eye. The use of the present compositions has been found to provide adequate protection for these eye portions, in particular the corneal endothelium, against such potentially traumatic force and/or turbulence. The plurality of lens particles is removed, for example, using a conventional irrigation/aspiration procedure, from the eye. The present hyaluronate- containing composition is also removed, along with the lens particles and/or after further irrigation/aspiration, from the eye. Such removal occurs relatively easily and substantially without detrimentally affecting the remaining portions of the eye.

0023An intraocular lens can then be implanted in the eye, e.g., using a conventional technique, to replace the removed natural lens. After this implantation, the incision in the eye is closed, such as by suturing. The use of the present compositions, as described above, allows for effective removal of the natural lens from the eye without substantially damaging the eye cell layers and tissues in proximity to the natural lens.

0024The following non-limiting examples illustrate certain^' aspects of the present invention.

0025EXAMPLES 1 TO 3 A composition (Composition 1) was prepared by mixing two sodium hyaluronate fractions with a standard phosphate buffered saline solution. This composition included 20 mg/ml of a^' sodium hyaluronate fraction having a weight average molecular weight of about 700,000, and 20 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of about 50,000.

0026Composition 2 (comparative) was a phosphate buffered saline solution containing 30 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of about 500,000, and 40 mg/ml of chondroitin sulfate having a weight average molecular weight of about 50,000. Composition 2 is commercially available from Alcon Surgical, Inc., Forth Worth, Texas, and is sold under the trademark Viscoat. Composition 3 (comparative) was a phosphate buffered saline solution containing 10 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 3 to 4 million. Composition 3 is commercially available from Pharmacia Ophthalmics, Monrovia, California, and is sold under the trademark Healon.

0027Each of these compositions was tested as follows. Healthy New Zealand white rabbits were selected for surgery. The surgical procedure performed, using conventional techniques, on the eye of the selected rabbit involved anterior capsulotomy, phacoemulsification of the eye's natural lens (using a standard phacoemulsification unit), irrigation/aspiration and intraocular lens implantation.

0028Each of the compositions was loaded into a standard BD Hypack syringe. A 25g cannula was attached to each syringe for the injections. Each rabbit received one (1) fluid sample for each eye prior to the procedure. This fluid was replenished as necessary as the surgical procedure progressed. The surgeon's observations relating to these compositions included the following: Composition 1 was difficult to inject through a 25g cannula. It maintained the anterior chamber well and adhered strongly to the corneal endothelium cells during the phacoemulsification procedure, but was difficult to remove during irrigation/aspiration due to its relatively high viscosity and stickiness.

0029Composition 2 was somewhat easier to inject than Composition 1 and it maintained the chamber approximately the same as Composition 1. Composition 2 coated the corneal endothelium well and was somewhat difficult to remove by irrigation/aspiration.

0030Composition 3 was very easy to inject. However, it did not appear to maintain the anterior chamber as well as Compositions 1 and

00312 during the anterior capsulotomy procedure. Furthermore, Composition 3 flushed completely out of the eye upon initiation of phacoemulsification and did not appear to coat either the endothelial cells or the intraocular lens well. Composition 3 was the most readily removed during irrigation/aspiration.

0032These results indicate that Composition 1 provided the desired protection during the phacoemulsification procedure. Such protection was as good as or better than the protection achieved using the other compositions tested. Also, the relatively high viscosity and stickiness of Composition 1 can be reduced by reducing the concentrations of one or both of the sodium hyaluronate components and/or by adjusting the ratio of the different molecular weight sodium hyaluronate components and/or by adjusting the molecular weight of one or both of the sodium hyaluronate components.

0033EXAMPLES 4 TO 8 A composition (Composition 4) was prepared by mixing two sodium hyaluronate fractions with a standard saline solution. This composition included 30 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 700,000, and 5 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 120,000. The viscosity of Composition 4 was 75,000 centipoises at a shear rate of 1 sec^" . A composition (Composition 5) was prepared by mixing two sodium hyaluronate fractions with a standard saline solution. This composition included 25 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 700,000, and 10 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 120,000. The viscosity of Composition 5 was 45,000 centipoises at a shear rate of l sec-1.

0034A composition (Composition 6) was prepared by mixing two sodium hyaluronate fractions with a standard saline solution. This composition included 20 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 700,000, and 30 mg/ml of a sodium hyaluronate fraction having a weight average molecular weight of 120,000. The viscosity of Composition 6 was 20,000 centipoises at a shear rate of 1 sec^" .

0035Composition 7 (comparative) was similar to Composition 2. The viscosity of Composition 7 was 45,000 centipoises at a shear rate of 1 sec^" .

0036Composition 8 (comparative) was similar to Composition 3. The viscosity of Composition 8 was 45,000 centipoises at a shear rate of 1 sec .

0037Each of these compositions was tested as follows. Healthy New Zealand white rabbits were selected for surgery. The surgical procedure performed, using conventional techniques, on the eye of the selected rabbit involved anterior capsulorhexis, phacoemulsification of the eye's natural lens (using a standard phacoemulsification unit), irrigation/aspiration and intraocular lens implantation. Each of the compositions was loaded into a 3 cc BD syringe. A 27 g cannula was attached to each syringe for the injections. Each rabbit received one (1) fluid sample for each eye prior to the procedure. This fluid was replenished as necessary as the surgical procedure progressed. The surgeon's observations relating to these compositions included the following:

0038Composition 4 injected through a 27 g cannula with some difficulty. It maintained the anterior chamber well during capsulorhexis, and adhered well to the corneal endothelium during the phacoemulsification procedure. This material was removed fairly easily during irrigation/aspiration.

0039Composition 5 injected through a 27 g cannula with some difficulty. It did not maintain the anterior chamber during capsulorhexis as well as Composition 4. Composition 5 adhered well to the corneal endothelium during the phacoemulsification procedure and was removed easily during irrigation/aspiration.

0040Composition 6 was difficult to inject through a 27 g cannula. It maintained the anterior chamber during capsulorhexis approximately the same as Composition 4. Composition 6 adhered well to the corneal endothelium during the phacoemulsification procedure, and was easily removed during irrigation/aspiration.

0041Composition 7 was somewhat easier to inject than Compositions 4 and 5, and was much easier to inject than Composition 6. However, Composition 7 did not maintain the anterior chamber during capsulorhexis as well as either Composition 4 or Composition 6. It maintained the anterior chamber approximately the same as Composition 5, and adhered about as well to the corneal endothelium during the phacoemulsification procedure as did either Composition 5 or Composition 6. Composition 7 was removed easily during irrigation/aspiration.

0042Composition 8 was very easy to inject. However, it did not maintain the anterior chamber during capsulorhexis as well as any of the other compositions. Composition 8 completely flushed out of the eye upon initiation of phaco¬ emulsification and did not appear to coat the corneal endothelium well. Composition 8 was the most readily removed by irrigation/aspiration.

0043These results indicate that Compositions 4, 5 and 6, in accordance with the present invention, provided superior protection during capsulorhexis and the phacoemulsification procedure relative to Composition 8 which includes a high molecular weight sodium hyaluronate fraction. In addition, the present compositions, that is Compositions 4, 5 and 6, provide substantially as good or better protection than did Composition 7, which includes both a sodium hyaluronate fraction and a chondroitin sulfate fraction. Of course, the present compositions have advantages, for example, in ease of manufacture, relative to Composition 7 which includes two significantly different materials, sodium hyaluronate and chondroitin sulfate.

0044When comparing Compositions 1, 4, 5 and 6 (in accordance with the present invention) with Compositions 3 and 8, the present compositions provide results which are generally superior with respect to compositions containing a single high molecular weight sodium hyaluronate fraction. These results also demonstrate that compositions including a first hyaluronate fraction having a weight average molecular weight in the range of about 500,000 to about 750,000 and a second hyaluronate fraction having a weight average molecular weight in the range of about 25,000 or about 35,000 to about 150,000 or about 175,000 provide substantially comparable results. For example, changing the weight average molecular weight of the second hyaluronate fraction from 50,000 (Composition 1) to 120,000 (Compositions 4, 5 and 6) did not result in any substantial change in the beneficial properties achieved using the present compositions. The first hyaluronate fraction tested, that is having a weight average molecular weight of 700,000 is representative of hyaluronates having weight average molecular weights in the range of about 500,000 to about 750,000, That is, no substantial difference in composition performance would occur by changing the weight average molecular weight of the first hyaluronate fraction within the range about 500,000 and about 750,000.

0045In other words, the data presented in these Examples is representative of compositions including a first hyaluronate fraction having high molecular weight average molecular weight in the range of about 500,000 to about 750,000 and a second hyaluronate fraction having a weight average molecular weight in the range of about 25,000 or about 35,000 to about 150,000 to about 175,000. These Examples make clear that the present compositions provide as good or better performance than the current commercially available materials, as set forth in Compositions 2, 3, 7 and 8. In particular, the present compositions provide improved performance relative to compositions which include only a single high molecular weight hyaluronate fraction, that is Compositions 4 and 8. This result is clearly unexpected and provides substantial benefits, for example, in terms of improved performance while being relatively easy to manufacture and to control the quality of, and to obtain governmental regulatory approval on. While this invention has been described with respect to various specific examples and embodiments, it is to be understood that the invention is not limited thereto and that it can be variously practiced within the scope of the following claims.