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WO2004092361A1 - Procede d'isolation et de purification d'hyaluronidase ovine - Google Patents

Procede d'isolation et de purification d'hyaluronidase ovine Download PDF

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Publication number
WO2004092361A1
WO2004092361A1 PCT/US2004/011692 US2004011692W WO2004092361A1 WO 2004092361 A1 WO2004092361 A1 WO 2004092361A1 US 2004011692 W US2004011692 W US 2004011692W WO 2004092361 A1 WO2004092361 A1 WO 2004092361A1
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WIPO (PCT)
Prior art keywords
hyaluronidase
acs
vitreous
treating
dose
Prior art date
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PCT/US2004/011692
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English (en)
Inventor
William S. Craig
John Chesham
Original Assignee
Ista Pharmaceuticals, Inc.
Biozyme Laboratoreis, Ltd
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Publication date
Application filed by Ista Pharmaceuticals, Inc., Biozyme Laboratoreis, Ltd filed Critical Ista Pharmaceuticals, Inc.
Priority to JP2006510093A priority Critical patent/JP2006523461A/ja
Priority to CA002522544A priority patent/CA2522544A1/fr
Priority to AU2004230482A priority patent/AU2004230482A1/en
Priority to EP04759568A priority patent/EP1616004A1/fr
Publication of WO2004092361A1 publication Critical patent/WO2004092361A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the disclosure relates to a process for preparing a mammalian testicular hyaluromdase preparation suitable for pharmaceutical applications.
  • the process is used to purify hyaluromdase from ovine testes and includes the use of viral filtration steps to increase the purity of the final product.
  • the process also provides a method that enhances the purity of hyaluromdase preparations presently available in commerce.
  • the methods are preferably used to purify hyaluromdase from mammalian sources.
  • the methods disclosed can be used to purify recombinant hyaluromdase. Background of the Invention
  • Hyaluromdase is a versatile class of enzymes that are expressed in vertebrates and invertebrates alike.
  • the mammalian hyaluromdase catalyzes the random hydrolysis of 1,4- linkages between 2-acetamido-2-deoxy-b-D-glucose and D-glucose residues in hyaluronate.
  • the hyaluromdase from bovine testes has a reported molecular weight of 65,000 (Lathrop et al. 1990 J Cell Biol 111:2939).
  • bovine testicular hyaluromdase hydrolyzes the endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin sulfuric acids A and C (but not B), primarily to tetrasaccharide residues (Ludovig et al. 1961 J Biol Chem 236:333).
  • Figures 1-11 show flow charts of the described hyaluromdase preparation methodology.
  • Figure 12. Typical chromatograms: A. for Standard; B. for Sample.
  • Ovine hyaluromdase is an enzyme product purified from ovine testes and capable of hydrolyzing mucopolysaccharides of the type of hyaluronic acid.
  • Amino Acid Sequence - a-form (SEQ ID NO: 1)
  • the consensus sites for glycosylation are underlined.
  • the site of cleavage that yields the ⁇ -form of hyaluromdase is assigned by homology with the bovine sequence and is indicated as bold and underlined.
  • the molecular weight based on mobility in 4-20% gradient reduced sodium dodecylsulfate (SDS) polyacrylamide gels is 70-74 kDa for the ⁇ -form and 60-63 for the ⁇ - form (see hyaluromdase content assay below).
  • Identification test The identification of hyaluromdase in samples of the drug substance is based on demonstration of a hyaluromdase enzymatic activity.
  • Annexin II - The annexin ⁇ content of drug substance is determined by electrophoretic content assay, quantitated against an internal standard curve of in-house aimexin IT reference standard. Specification: between 0.29 and 0.57 mg annexin per mg protein.
  • the annexin ⁇ reference standard is purified from drug substance by Protein G affinity chromatography and size-exclusion chromatography. Batches of reference standard are qualified for use by testing for purity (SDS PAGE), identity (western blot), protein concentration and amino acid content (amino acid analysis) and in the electrophoretic content assay described here.
  • Assay Method Assay Method:
  • Annexin II content is assessed with 12-well 4-20% gradient Tris-glycine sodium dodecylsulfate (SDS) polyacrylamide gels.
  • Samples of drug substance are reduced and denatured in 2X Tris-glycine SDS sample preparation buffer containing 2-mercaptoethanol at 500 ⁇ L per 10 mL of buffer at a final concentration of 0.12 mg/mL.
  • a standard curve of annexin II reference standard is prepared at 5 concentrations: 7.42, 14.84, 29.68, 44.52 and 74.2 ⁇ g/mL (Stds 1-5, respectively). These are diluted 1:1 in reducing 2x sample preparation buffer (see above).
  • Vitrase ® finished product is used as a system suitability standard, also prepared at 0.12 mg/mL.
  • One sample lane is used by a sample of broad range reduced molecular weight standards. All samples and standards are loaded at a volume of 10 ⁇ L of sample per well in the following order:
  • the gel is quantitated on a scanning laser densitometer.
  • the densitometer yields a band density histogram that is integrated to give an area under the "peaks" in the histogram.
  • %Annexin U. mean annexin II in sample (in ⁇ g) x 100/total protein loaded (in ⁇ g). The sample meets the requirement if the content is between 0.29 and 0.57 mg annexin per mg protein.
  • results from this assay are unacceptable if the following criteria are not met: 1.
  • the densitometer must pass its internal calibration check 2.
  • the annexin II band must migrate between the 31 and 36.5 kDa molecular weight standards in gel sample lane 12
  • the calculated r 2 value for the standard curve must be > 0.98.
  • the percentage coefficient of variance for triplicate data points must be ⁇ 10%.
  • the mean calculated annexin II for samples must fall within the range of 0.076-0.39 ⁇ g for drug substance.
  • IgG fragment - The IgG heavy chain fragment ("IgG") content of drug substance is quantitated with a high performance liquid chromatography (HPLC) method using an affinity column and IgG reference standard curve. Specification: ⁇ 0.23 mg IgG per mg protein.
  • the IgG reference standard is prepared in a one-step affinity purification by Protein G chromatography. Batches of IgG reference standard are qualified for use by testing for purity (SDS PAGE), identity (Western blot), concentration (colorimetric protein assay) and content (HPLC method). ' The method uses a standard HPLC system with a tunable UV detector set at 280 nm. The column is a 4.6 x 100 mm Poros OH pre-column, attached in sequence to a 4.6 x 100 mm Poros G affinity column. Samples of drug substance are loaded in a mobile phase of 0.05 M sodium phosphate, pH 7.2, supplemented with 0.15 M sodium chloride.
  • Solvent A 0.05 M sodium phosphate, pH 7.2, 0.15 M sodium chloride
  • Solvent B 0.1 M glycine, 5% acetic acid, pH 2.5
  • the IgG content is determined in comparison to an in-house purified ovine testicular IgG reference standard.
  • the IgG standards are prepared at concentrations of 500, 250, 120, 60 and 30 ⁇ g/mL in Solvent A.
  • the IgG standards are loaded onto the column at 100 ⁇ L per injection. Prepare drug substance samples by dissolving 10 mg of drug substance in 3.5 mL of
  • Check samples of Vitrase ® finished product are prepared by dissolving the contents of 2 vials in 0.5 mL (each) of Solvent A. Mix by vortexing briefly and then allow them to stand for not less than 30 minutes to ensure complete dissolution. Combine the vial contents and filter as described for drug substance. Each check sample preparation is analyzed with 2 injections of 100- ⁇ L volume. Check sample preparations can be stored at 2-8°C for up to 3 days.
  • System Equilibration The system is equilibrated with Solvent A for 20 minutes, first at a flow rate of 2 mL/minute for 5 minutes, and then at 4 mL/minute for 15 minutes. Four 100- ⁇ L injections of Solvent A are used to establish that the UV signal is stable. Assay Method:
  • the sample protein concentration is determined with a colorimetric protein assay and used to calculate the IgG content per mg of total protein in the sample.
  • the theoretical plate count for the 25- ⁇ g IgG standard must be > 2300.
  • ' The peak retention time for IgG must be within 4.17 ⁇ 0.5 minutes and non-bound proteins must elute at 0.79 ⁇ 0.5 minutes.
  • the % coefficient of variation for the 3 injections of the 250 ⁇ g/mL (or 25 ⁇ g) IgG standard must be ⁇ 10%.
  • the r 2 value for the standard curve must be > 0.99.
  • the Relative Retention Time of IgG must be within 0.9 - 1.1 minutes.
  • Loss on drying - Tare a glass-stoppered weighing vial that has been dried overnight at 110°C. Place 150 milligrams + 10 milligrams of the drug substance sample in the bottle and accurately weigh the bottle and the contents. Place the bottle in the drying chamber, evacuate the chamber to less than 10 inches of Hg, and dry two (2) hours ⁇ 1 minute at 60°C with the lid removed, but also in the chamber. Release the vacuum, open the chamber door and immediately replace the lid. Weight the dried container and sample after it has cooled to ambient temperature and subtract the weight obtained from the tare weight. Calculate the percent of the original weight that was lost on drying. The loss on drying should not be more than 5% of the original tare weight. Product-related Impurities Quantitation
  • Hyalnronidase content The hyaluromdase content of drug substance samples is quantitated by an electrophoretic method. Hyaluromdase is quantitated against a standard curve of in-house hyaluromdase reference standard with a system suitability sample provided by Vitrase ® finished product. Specification: between 0.10 and 0.23 mg hylauronidase per mg protein.
  • the ovine hyaluromdase standard is purified from drug substance by Protein G affinity chromatography, followed by size-exclusion chromatography and Concanavalin A affinity chromatography. Batches of reference standard are qualified for use by testing for purity (SDS PAGE), identity (western blot), protein concentration and amino acid content (amino acid analysis) and in the electrophoretic content assay. Under reducing conditions, ovine testicular hyaluromdase migrates as a pair of discreet bands that differ in apparent mass by about 7 kDa. Both forms retain full enzymatic activity. Assay Method:
  • Hyaluromdase content is assessed with 12-well 4-20% gradient Tris-glycine SDS polyacrylamide gels.
  • Samples of drug substance are reduced and denatured in 2X Tris- glycine SDS sample preparation buffer containing 2-mercaptoethanol at 500 ⁇ L per 10 mL of buffer, a final concentration of 0.1 mg/mL.
  • a standard curve of annexin ⁇ reference standard is prepared at 5 concentrations: 0.092, 0.0506, 0.0276, 0.0138 and 0.0092 ⁇ g/mL (Stds 1-5, respectively). These are diluted 1:1 in reducing 2x sample preparation buffer (see above).
  • a designated lot of Vitrase ® finished product is used as a system suitability standard, also prepared at 0.1 mg/mL.
  • One sample lane is used by a sample of broad range reduced molecular weight standards. All samples and standards are loaded at a volume of 10 ⁇ L of sample per well.
  • Std 1 thru Std 5 standard curve of in-house annexin H " reference standard
  • MWS broad range molecular weight standard
  • Gels are electrophoresed at a constant voltage of 120 V for 120 +/- 5 minutes, or until the bromphenol blue tracking dye line reaches the bottom of the gel.
  • the gel is carefully removed from its plastic cassette and placed in 50 mL of colloidal Coomassie blue stain solution. Gels are left to stain 15-17 hours with constant mixing provided by a gel rocker platform.
  • the gel Upon completion of the stain, the gel is destained over a period of not less than 7 hours in deionized water on the gel rocker platform, using multiple changes of water during the process.
  • the gel is quantitated densitometrically on a scanning laser densitometer. Calculations: Plot the band density total peak area against the theoretical mass values for each standard in the standard curve, to yield an r 2 value and equation for the line.
  • the mean hyaluromdase mass is expressed as a percentage of the total protein by:
  • %Hyaluronidase 100 x [mean sample hyaluromdase ( ⁇ g)] / total protein ( ⁇ g) loaded.
  • Results from this assay are unacceptable if the following criteria are not met:
  • the densitometer must pass its internal calibration check.
  • the hyaluromdase bands must migrate between the phosphorylase b (97.4 kDa) and glutamic dehydrogenase (55.4 kDa) molecular weight standards in gel sample lane 1.
  • Each standard curve must be comprised of at least 5 points.
  • the mean hyaluromdase content of drug substance samples must fall between 0.092 and 0.32 ⁇ g.
  • Total Protein - The total protein assay is a colorimetric method based on the binding of Coomassie Brilliant Blue G-250 to proteins in solution. Proteins are quantitated relative to a standard curve of bovine serum albumin. Specification: between 0.55 and 0.83 mg protein per mg drug substance. Solutions:
  • the acidic dye concentrate, bovine serum albumin standard and 0.9% saline solutions are obtained from commercial sources.
  • the dye concentrate is diluted 5:1 in deionized water and filtered through Whatman #1 filter paper prior to use. This 5x-diluted dye solution is stable for up to 14 days when stored at 2-8°C.
  • the bovine serum albumin stock solution is 2.0 mg/mL. Dilutions are prepared in
  • 0.9% saline to final concentrations of 0.9, 0.7, 0.5, 0.3 and 0.1 mg/mL in volumes of 500 ⁇ L. These standards are prepared fresh daily. A blank consisting solely of 0.9% saline is included. All samples and standards are pipetted in triplicate into 13x100 mm glass tubes at 100 ⁇ L per tube. The assay is initiated by pipetting 5.0 mL of dilute dye reagent into each tube at timed intervals of 20 seconds. After each addition, the tube is covered with a plastic cap and vortexed briefly to mix. The assays are then incubated at room temperature for 10 minutes.
  • the saline blank is used to blank the spectrophotometer at 595 nm - the tubes are read directly in the spectrophotometer cuvette holder. After 10 minutes incubation, the samples are read in the same order as initiated, at the same 20-second intervals.
  • Protein concentrations are interpolated from the regression line of standard curve absorbance at 595 nm (A 595 ) versus theoretical standard concentration.
  • the assay reporting range is from 0.35 - 0.9 mg/mL - drug substance samples outside this range must be re- assayed with fresh dilutions to bring them into the standard curve range.
  • the drug substance contains proteins (in particular, the IgG fragment) that do not respond in the same way as BSA to the Coomassie Blue dye, a 1.2 correction factor is applied to all concentrations obtained by this assay.
  • the sample meets requirement if the protein content is between 0.55 and 0.83 mg protein per mg drug substance.
  • %CV coefficient of variance
  • Results from this assay are unacceptable if the following criteria are not met:
  • the spectrophotometer calibration must be current.
  • the baseline reading on the blank must be stable at 0.00 ⁇ 0.01 AU for at least 20 seconds.
  • the r 2 value for the standard curve must be > 0.98.
  • Potency is measured with a hyaluromdase activity assay in which enzyme is incubated with hyaluronic acid substrate for a fixed time period and the non-degraded substrate detected by the turbidity formed when reacted with an acidic albumin solution.
  • Turbidity is measured spectrophotometrically at 600 nm.
  • Hyaluromdase activity is quantitated relative to a USP hyaluromdase standard curve run simultaneously. Specific activity is calculated using the values from the activity and total protein assays. Potency specification: between 7.32 x 10 3 and 1.37 x 10 4 USP Units per mg drug. Specific activity specification: between 1.2 x 10 4 and 1.9 x 10 4 USP Units per mg protein.
  • the sodium hyaluronate substrate solution is prepared at 0.5 mg/mL in 0.3 M sodium phosphate at pH 5.30-5.35. All USP hyaluromdase standard, drug substance and check sample solutions are prepared in a diluent buffer of 20 mM sodium phosphate, pH
  • the acidic albumin solution is 24 mM sodium acetate, pH 3.75 ⁇ 0.05, with 0.1% bovine serum albumin.
  • the USP hyaluromdase standard solution is prepared to yield a nominal activity of 15 USP U/mL.
  • Samples of drug substance are initially diluted to approximately 1 mg/mL in 0.9% saline in a 10-n ⁇ L volume. This initial saline dilution is further diluted, based upon the manufacturer's release activity value for the particular drug substance lot to be assayed, to yield a final concentration of about 9 USP U/mL. This secondary dilution is made in the 20 mM sodium phosphate buffer.
  • Check samples are made from lots of Vitrase ® finished product in the following manner.
  • a single vial of finished product is dissolved in 5.4 mL of 0.9% saline, delivered from a 10-mL syringe. This solution is filtered through a 5- ⁇ m filter needle and returned to its original vial. Dilute 125 ⁇ L of this solution to a final volume of 25 mL of the 20 mM sodium phosphate buffer to yield the check sample.
  • All assays consist of a 10-point standard curve, a check sample, and the drug substance test samples.
  • the standard curve contains USP hyaluromdase standard at concentrations of 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5 and 15.0 USP U/mL in a final volume of 1 mL of the 20 mM sodium phosphate buffer in 16x125 mm glass test tubes.
  • drug substance samples are of 1-mL volumes, pipetted into the test tubes.
  • Assays are initiated by uncapping the tube, pipetting 1 mL of the 0.5 mg/mL hyaluronate solution into the tube, recapping it and immediately vortexing before returning it to the water bath. Care should be taken to vortex at a relatively low speed to avoid foaming the solution. Assays are initiated every 30 seconds until all have received the substrate solution. The incubation time at 37-38°C is 45 minutes. At t - 45 minutes, the assays are quenched by the addition of 10 mL of acidic albumin solution: uncap the tube, add the acidic albumin, recap it, and mix by gently inverting.
  • the tube is first re-mixed by gentle inversion, and then the sample decanted into a disposable 4.5-mL polystyrene cuvette. Samples are read every 30 seconds, maintaining the temporal lockstep of the process.
  • the spectrophotometer is initially blanked with a cuvette full of deionized water.
  • the standard curve is plotted as a third-order polynomial function of USP U/mL versus the observed absorbance at 600 nm (A 60 o).
  • Check sample and drug substance sample activity values are then interpolated from this standard curve from their A 600 values.
  • analysts may find it most convenient to set up spreadsheet activity calculation tables whereby entering the raw A 600 data, the polynomial coefficient values from the standard curve equation, the mass value (drug substance samples only) and the sample dilution factors for drug substance and check samples yields calculated activity values for these samples from pre-entered equations in fixed cells of the spreadsheet.
  • the sample meets the potency requirement if the activity content is between 7.32 x 10 3 and 1.37 x 10 4 USP Units per mg drug substance.
  • Results from this assay are unacceptable if the following criteria are not met:
  • the correlation coefficient (r 2 value) of the USP standard curve must be > 0.999. 3.
  • the check sample activity value must fall within +/- 6% of the historically established value for that lot of finished product. 4.
  • the % relative error between replicate assays (e.g. check samples) must be ⁇ 6%.
  • Bacterial endotoxins - less than 60 eu per milligram drug substance.
  • the disclosure relates to a process for preparing a hyaluronidase preparation suitable for pharmaceutical applications, hi a preferred embodiment, the process includes the use of viral filtration steps to increase the safety level of the final product.
  • the process provides a method that enhances the purity of hyaluronidase preparations presently available in commerce.
  • the methods are preferably used to purify hyaluronidase from mammalian sources.
  • the methods disclosed can be used to purify recombinant hyaluronidase.
  • mammalian beta-glucuronidase enzymes are prepared preferably from mammalian testes.
  • mammalian sources include ovine, bovine, porcine, and equine.
  • any mammalian source can be used with the described methods.
  • a taxonomic hierarchy that includes Order, Family, Subfamily, and Genus is found in Wilson, D. E., and D. M. Reeder (eds.) 1993 Mammal Species of the World, Smithsonian Institution Press, 1206 p. (Available from Smithsonian Institution Press), which is hereby incorporated by reference. Extraction of hyaluronidase
  • a preliminary step of the purification of hyaluronidase is to isolate testes from a preferred mammalian source.
  • the testes can be processed immediately or preferably are frozen for later use. When frozen, the testes should be thawed at 2-8°C for 40-44 hours.
  • the testes are then minced and filtered to extract the hyaluronidase.
  • the isolated material is then precipitated for a first time, preferably at 15% saturated ammonium sulphate.
  • the temperature of the buffers is preferably maintained at 2-8°C.
  • the pH of the precipitation step is preferably maintained at a pH of 3.60 ⁇ 0.1.
  • the precipitate is filtered, preferably for less than 5 hours.
  • Figures 3 and 4 illustrate the second step in the preparation of hyaluronidase from mammalian testes.
  • the product of the purification Step 1 (15/85) purification is thawed and ammonium sulphate is added to 35% saturation.
  • the solution is stirred and filtered.
  • the pH of the filtrate is adjusted to 4.10 + 0.20 and a sample is typically taken for quality control analysis.
  • the solution is then brought to an 85% saturated concentration of ammonium sulphate and stirred.
  • the solution is then filtered and the precipitate (35/85 precipitate) is collected and stored.
  • Figures 5 through 8 illustrate the third step of the purification protocol.
  • the 35/85 precipitate is thawed and resuspended for further processing.
  • the solution is dialyzed against 20mM potassium phosphate solution.
  • the dialyzed solution is then filtered and concentrated.
  • the concentrate is stored at 2-8°C overnight. Samples are typically taken as indicated in Figure 5 for quality control purposes.
  • the concentrate is then subjected to DEAE sephadex fractionation. As described in Figure 6, fractions are collected and assayed for hyaluromdase activity. Selected fractions are combined and then precipitated with 85% saturated ammonium sulphate. This solution is stirred and then filtered.
  • the precipitate (0/85) is collected, weighed, filtered, and clarified.
  • the solution is precipitated with PEG6000 to a concentration of 20%. This suspension is centrifuged and the precipitate is collected. The precipitate is then resuspended and filtered. Quality control testing is typically performed. The product is dried and stored for further processing.
  • the fourth step of the procedure is illustrated in Figure 9 through 11.
  • the product is dissolved for CM sephadex column fractionation. The sample is applied to the column and fractions are collected. Active fractions are pooled and precipitated with ammonium sulphate to 85%. The precipitate (0/85) is collected and weighed. The sample is dialyzed against a 20 mM potassium phosphate. As illustrated in Figure 10, the dialysate is then filtered and the pH is adjusted.
  • Hyaluronidase activity was measured using a turbidity assay. This assay is used to determine the activity of hyaluronidase in the final product, the drug substance [active pharmaceutical ingredient (API)], and in-process intermediates during manufacture of the API or final product.
  • Hyaluronidase activity is determined using a modification of the turbidity assay described in USP 26 for Hyaluronidase for Injection.
  • the dissolved hyaluronidase enzyme is allowed to react with the substrate, hyaluronic acid, for a set period of time followed by inactivation of the enzyme and precipitation of non-degraded hyaluronic acid by an acidic albumin solution.
  • the degree of resultant turbidity is measured by absorbance determination at 600 nm using a UV- Visual Spectrophotometer.
  • Enzyme activity is inversely proportional to the turbidity of the solution. Quantitation is based on comparison to turbidity data from a primary USP bovine hyaluronidase standard of known enzyme activity, run under the same conditions.
  • the hyaluronidase preparation has 7.32 x 10 3 - 1.37 x 10 4 USP Units/mg.
  • the hyaluronidase preparation has a
  • Total protein Total protein of the hyaluronidase preparation is measured by generally accepted protein concentration assays. These assays are used to determine the protein concentration of in-process intermediates, active pharmaceutical ingredient (API), and final product. This assay is based on the method of Bradford for protein quantitation. It is a dye-binding assay in which a proportional color change of the dye occurs in response to various concentrations of protein. The absorbance maximum for an acidic solution of the dye, Coomassie Brilliant Blue G-250, shifts from 465 nm to 595 nm when binding to protein occurs. The Coomassie blue dye binds to primarily amine containing amino acid residues, especially arginine. The color yield for an individual protein may depend on its amino acid composition. Total protein of tl e hyaluronidase preparation is 0.55 - 0.83 mg/mg protein.
  • S Sppeecciifificc activity of the preparation ranges from 1.2 x 10 4 -1.9 x 10 4 USP Units per mg protein.
  • the hyaluromdase preparation typically has a water content of ⁇ 12% by Karl
  • the Karl Fischer Coulometric assay is used to determine the amount of water present within in- process intermediates, active pharmaceutical ingredient (API), and final product using the Karl Fischer Coulometric method as delineated in the U.S. Pharmacopoeia 25 (United States Pharmacopoeia), which is hereby incorporated by reference in its entirety.
  • the Karl Fischer Coulometric assay is based on the titration of iodine against water and sulfur dioxide in the presence of a base and an alcohol. When all the water is used up excess iodine is generated and detected at the double platinum electrode.
  • the Karl Fischer coulometer calculates and prints the percent (%) water of the injected sample based on weight.
  • the water content of the hyaluronidase preparation typically is ⁇ 5% loss on drying.
  • the hyaluronidase preparations disclosed possess a limited concentration of bacterial endotoxins.
  • the Limulus amoebocyte lysate (LAL) test is used to determine the concentration of bacterial endotoxins in a given sample.
  • the LAL test is based on the observation that bacterial endotoxins react with a lysate derived from circulating cells associated with the blood clotting mechanism of the horseshoe crab, Limulus polyphemus.
  • Bacterial endotoxins are present in the hyaluronidase preparation at ⁇ 60 endotoxin units per mg.
  • Microbial limits are determined using the assay outlined in USP 61. This test is designed to demonstrate that the viable aerobic microorganisms present in the product are free of E. coli, S. aureus, P. aeruginosa or Salmonella. Total microbial contamination less than 10 3 organisms per gram.
  • the hyaluronidase preparations for use. in the disclosed method have a prescribed concentration of annexin 13.
  • a preferred embodiment of the purified solution typically contains four major protein components (alpha hyaluronidase, beta hyaluronidase, annexin IL and an IgG fragment), as well as lactose and other buffer components.
  • SDS-PAGE separates proteins based on their apparent molecular weight.
  • This method describes the procedure for running SDS-PAGE gels, scanning gels by densitometry, and quantitating annexin II by ImageQuant ® densitometry software.
  • Annexin II content determined by SDS-PAGE analysis from 0.29 - 0.57 mg annexin per mg protein.
  • the hyaluronidase preparations for use with the disclosed methods contain a particular level of immunoglobulin.
  • the amount of immunoglobulin (IgG) heavy chain fragment, referred to as "IgG,” in drug substance [active pharmaceutical ingredient - (API)], in-process intermediates and final product was measured by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • a preferred embodiment is a formulation containing several proteins, lactose, and phosphate buffer.
  • the formulation contains hyaluronidase (the active ingredient) and two major impurities, annexin ⁇ and IgG. h order to characterize the drug product, set product specifications and provide process controls, the amounts of various components need to be determined.
  • the HPLC procedure used for quantitation of IgG is discussed below.
  • the procedure uses an affinity column in which separation is achieved based on the binding affinity of the protein of interest to a ligand attached to the stationary phase. This procedure utilizes protein G as the ligand.
  • the IgG is bound to the stationary phase using a buffer at neutral pH, and then is eluted in a step-wise manner with a buffer of low pH. The critical factor in the elution step is low pH. Salt is present to reduce nonspecific binding.
  • IgG content determined by HPLC analysis is approximately ⁇ 0.23 mg IgG per mg protein.
  • Hyaluronidase content is determined by SDS-PAGE analysis To quantitate hyaluronidase protein content by running sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) for final product, various process intermediates, and active pharmaceutical ingredient (API).
  • a preferred embodiment typically contains four (4) major protein components [alpha hyaluronidase, beta hyaluronidase, annexin ⁇ , and an immunoglobulin (IgG) fragment], as well as lactose and other buffer components.
  • SDS-PAGE separates proteins based on their apparent molecular weight.
  • This method describes the procedure for running SDS-PAGE gels, scanning gels by densitometry, and quantitating hyaluronidase by ImageQuant ® densitometry software.
  • the hyaluronidase preparations contain 0.1-0.23 mg hyaluronidase per mg protein.
  • EDTA content is 23 - 33 ⁇ g per mg.
  • the disclosed hyaluronidase preparations are highly purified preparations typically containing 0.10-0.23 mg hyaluronidase per mg protein determined by SDS-PAGE analysis.
  • the preparation typically has a specific activity ranging from 1.2 x 10 4 - 1.9 x 10 4 USP
  • the preparation will comprise bacterial endotoxins ⁇ 60 endotoxin units per mg drug substance.
  • the composition will have an absence of E. coli, S. aureus, P. aeruginosa or Salmonella, and a total microbial contamination of less than 10 organisms per gram protein.
  • Other protein components of the preparation include an annexin content of about
  • a method for accelerating clearance of hemorrhagic blood from the vitreous of the eye comprising the step of contacting, with the vitreous humor, a quantity of hyaluronidase at a dose which is sufficient to accelerate the clearance of hemorrhagic blood from the vitreous without causing damage to the retina or other tissues of the eye.
  • the hyaluronidase is selected to have a molecular weight distribution which allows the hyaluronidase to be administered intravitreally at doses above 1 IU, and preferably above 15 IU, and advantageously above 75 IU, in the absence of thimerosal, without causing toxic damage to the retina or other tissues of the eye.
  • This hemorrhage-clearing method 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.
  • the preferred route of administration of these hemorrhage-clearing enzymes is by intraocular injection directly into the vitreous body.
  • hemorrhage-clearing enzyme(s) 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.
  • the preferred injectable solution may contain a hyaluronidase which has a molecular weight distribution which allows it to be administered intravitreally at doses above 1 IU, and preferably above 15 IU, and advantageously above 75 IU, without causing toxic damage to the eye, along with inactive ingredients which cause the solution to be substantially isotonic, and of a pH which is suitable for injection into the eye.
  • This preferred hyaluronidase preparation is preferably devoid of thimerosal.
  • Such solution for injection may be initially lyophilized to a dry state and, thereafter, may be reconstituted prior to use.
  • hyaluronidase ACS
  • ACS hyaluronidase
  • Such hyaluronidase may be derived from ovine testicles and is available commercially from Biozyme Laboratories Limited, San Diego, California, which source may be a starting material for the disclosed process for isolating and purifying ovine hyaluronidase.
  • 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.
  • hyaluronidase 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, without causing significant toxicity to the eye or associated anatomical structures.
  • 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. 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.
  • the fundus photography was performed by restraining the animals and visualizing the optic nerve, retinal arcades and fundas with a KOWA® RC-3 Fundus Camera loaded with Kodak Gold 200 ASA film.
  • 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.
  • 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.
  • the 13 male rabbits that had received the fundus photography and fluorescein angiography at pre-dose 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.
  • 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.
  • 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.
  • one randomly selected rabbit was subjected to the same intracardiac glutaraldehyde fixation procedure described hereabove for the previously randomly selected animal.
  • 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 Table 1. hi summary, the eyes of the BSS-treated control group were free of toxicity at 2 and 7 days post dose.
  • the Group No. 3 animals treated with BSS+thimerosal exhibited severe treatment-related toxic effects, at days 2 and 7 post dose.
  • the Group No. 4 animals treated with Wydase® at the 1 IU. dose were free of toxicity at days 2 and 7, however, the eyes of the animals in Group Nos. 5-8 treated with Wydase® at dosages ranging from 15 I.U.-150 LU. exhibited generally dose-related toxic effects at days 2 and 7 post dose.
  • Wydase® formulation do cause toxic effects in the eyes of rabbits at the dosages tested, however, the hyaluronidase (ACS) caused no toxic effects in these animals at the dosages tested.
  • ACS hyaluronidase
  • Table 1 The results of the examinations conducted on day 7 are summarized in Table 1. As shown, in Table 1, 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 LU. hi contrast, no toxic effects were observed in the eyes of animals treated with the hyaluronidase (ACS) at doses between 1 and 150 LU.
  • Table 1 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 LU.
  • ACS hyaluronidase
  • EXAMPLE 2 Safety and Efficacy of the Hyaluronidase (ACS) Injected Intravitreally in Rabbit Eyes
  • 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.
  • the animals were randomly divided into four (4) treatment groups of three (3) animals each.
  • 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. Following the initial examination of the animals eyes, 100 ⁇ l or 10 ⁇ l of blood was injected intravitreally into each eye of each animal.
  • ACS hyaluronidase
  • the observed hemorrhage-clearing efficacy of the hyaluronidase (ACS) is summarized in Table 2.
  • 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.
  • the retinas of the rights eyes of all animals in Treatment Groups B-D appeared normal and free of treatment- related toxicity.
  • ACS hyaluronidase
  • 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.
  • 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).
  • ACS hyaluronidase
  • Procedures used to evaluate the safety of the test articles were completed at various intervals throughout the study, and included indirect ophthalmoscopy, fundus photography, fluorescein angiography, electroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, and autorefraction.
  • a concurrent placebo control group was included in the study so that adverse events peculiarly related to hyaluronidase (ACS) could be distinguished from those attributable to the vehicle (BSS)/injection procedure. Only visually impaired eyes were treated, moreover, since the test articles were injected proximate to the retina and any untoward responses of a serious nature could have been sight threatening. Patients were assigned to treatment using a computer generated randomization scheme beginning with the number 601 for the first phase of the study, and 701 for the second. Neither the patients nor investigators were aware of whether it was the BSS vehicle or hyaluronidase (ACS)ZBSS solution that was being injected intravitreally. Following establishment of a baseline for each patient, the subjects were injected with either the enzyme or the placebo control.
  • ACS hyaluronidase
  • Retinal/cortical responses as measured by electroretinography/visual evoked potential, deteriorated over time in one patient treated with BSS and two who were given 50 IU. of hyaluronidase (ACS)/BSS.
  • ACS hyaluronidase
  • alterations in elecfroretinographic patterns were always bilateral and did not occur in either the treated or untreated eyes of the patients assigned to high dose (100 IU.) hyaluronidase (ACS)/BSS, nor did fluorescein angiographic test results indicate that retinal ischemia was present in any eye irrespective of treatment.
  • the indirect ophthahnoscopic exams revealed liquefaction and the establishment of posterior vitreal detachment (PVD) in the eyes of the test subjects.
  • the vitreous was characterized as exhibiting a high degree of motility and/or liquefaction soon after injecting the test articles, which was expected for the hyaluronidase (ACS)-containing preparations.
  • Example 2 Given the results from Example 2 where injection of hyaluronidase (ACS) into the vitreous of rabbits at various doses up to 150 LU. did not result in any significant histopathologic changes in an earlier preclinical study, it was expected that doses below 150 LU. would be well-tolerated in humans. Consistent with this expectation, the intravitreal administration of hyaluronidase (ACS)/BSS into visually impaired eyes in the current trial elicited few symptoms, all of which were believed attributable to the injection procedure itself as they occurred with comparable frequency in each of the study groups, and treatment-related adverse sequelae were relatively mild and of short duration. Furthermore, treatment of human eyes with hyaluronidase (ACS) was observed to increase the incidence of observed posterior vitreal detachment. The observed increase in
  • PVD in patients injected intravitreally with hyaluronidase shows that the methods described herein are effective in inducing liquefaction and detachment of the vitreal humor.
  • the hyaluronidase (ACS) administered in this experiment was prepared by the formulation, described hereabove and shown in Table 9.
  • the hemorrhagic vitreous became sufficiently clear to permit trans- vitreal 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.
  • Hyaluronidase is capable of being administered intravitreally at doses of or in excess of 1 LU. without causing toxic damage to the eye and thus is useable to effect prompt liquefaction of the vitreous body and concomitantly the disconnection or detachment of the vitreous body from the retina and other tissues (e.g., epiretinal membranes, macula).
  • ACS Hyaluronidase
  • the physical pulling forces of the vitreous on the retina and other tissues are minimized and the rate of natural turnover of fluids within the vitreous is accelerated.
  • hyaluronidase is particularly suitable for the treatment of many disorders (e.g., proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates and cystoid macular edema) which benefit from liquefaction/disconnection of the vitreous and/or accelerated clearance of toxins or other deleterious substances (e.g., angiogenic factors, edema fluids, etc.) from the posterior chamber of the eye and/or from tissues adjacent the posterior chamber (e.g., the retina or macula).
  • disorders e.g., proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates and cystoid macular edema
  • toxins or other deleterious substances e.g., angiogenic factors, edema fluids,
  • liquefaction of the vitreous is also believed to remove the matrix, in the form of the polymerized vitreous, necessary to support neovascularization.
  • the present method is useful in preventing or reducing the incidence of retinal neovascularization.
  • many ophthalmic disorders have as a causative component, a destabilization of the blood-retina membrane. This destabilization permits various components (e.g., serum components, lipids, proteins) of the choriocapillaries to enter the vitreal chamber and damage the retinal surface. This destabilization is also a precursor to vascular infiltration of the vitreal chamber, known as neovascularization.
  • embodiments of the present method are directed toward the prevention and treatment of various disorders of the mammalian eye which result from damage or pathology to the vascularization of the eye or which result in damage to the blood-retinal barrier.
  • diseases include but are not limited to proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates, and cystoid macular edema, and others in which the clinical symptoms of these disorders respond to the hyaluronidase (ACS) treatment.
  • ACS hyaluronidase
  • PDR Proliferative Diabetic Retinopathy
  • IRMA intraretinal microvascular abnormalities
  • vascular endothelial growth factor from a hypoxic retina in areas of capillary nonperfusion is thought to result in the development of extraretinal neovascularization.
  • neovascularization and its associated fibrous components may spontaneously involute or be complicated by vitreous hemorrhage or traction retinal detachment.
  • Neovascularization may be easily seen on fluorescein angiogram by the profuse leakage of dye from these new vessels since they lack the tight endothelial junctions of the retinal vasculature. Impaired axoplasmic flow in areas of retinal hypoxia result in cotton wool spots.
  • Proliferative diabetic retinopathy requires careful screening of diabetics for early identification and treatment since PDR remains largely asymptomatic in the early stages.
  • Proliferative diabetic retinopathy can be classified into three subgroups: (1) nonproliferative retinopathy; (2) preproliferative retinopathy; (3) proliferative retinopathy. Each classification has certain morphological characteristics.
  • Features of nonproliferative retinopathy include capillary microangiopathy (microvascular obstructions and permeability changes, nonperfusion of capillaries, retinal capillary microaneurysms, basement membrane thickening, and internal microvascular abnormalities (IRMA)); intraretinal hemorrhages; exudates; and macular changes.
  • Preproliferative retinopathy is indicated by any or all of the changes described for nonproliferative retinopathy and the following: significant venous beading, cotton-wool exudates, extensive IRMA and extensive retinal ischemia.
  • Proliferative retinopathy is indicated by extraretinal neovascularization and fibrous tissue proliferation, vitreous alterations and hemorrhage, macular disease, and retinal detachment.
  • fibrovascular tissue is an especially important complication of PDR since it often will lead to retinal damage mediated by the vitreous.
  • the fibrovascular tissue may form preretinal membranes that create dense adhesions with the posterior hyaloid membrane. These adhesions are responsible for transmitting the forces of vitreous traction to the retina, which may result in retinal detachments.
  • the vitreous base is normally firmly attached to the adjacent retina and to the outer circumference of the optic nerve head, known as the ring of Martegiani.
  • the attachment of the vitreous to the retina in all other sites between the ring of Martegiani and the vitreous base is much less firm.
  • Neovascularization from the retina leads to the formation of vascular strands extending into the vitreous from the nerve head or elsewhere in the fundus. Contraction of these strands may cause partial or complete retinal detachment.
  • Retinal detachment at the macula is a major complication of PDR.
  • Most retinal detachments resulting from PDR begin as fractional detachments without holes, but they may become rhegmatogenous by the formation of retinal holes at some later point in the disease.
  • the fractional detachments are caused by abnormal vitreoretinal adhesions or vitreal traction with subsequent shrinkage of the fibrous bands and elevation of the retina.
  • the present method contemplates freatment of PDR in the preproliferative and proliferative states using hyaluronidase (ACS) intravitreal injections. Without being limited to a particular mechanism, it is believed that the effect of intravitreal hyaluronidase (ACS) injection is to promote the clearance of the liquid phase of the vitreous. The rate of transfer of intravitreally injected tritiated water from the mid vitreous to the choroid was significantly increased after depolymerization of vitreous hyaluronic acid by injected hyaluronidase (ACS).
  • ACS hyaluronidase
  • the present method capitalizes upon this observation to liquefy the vitreous, for example, in order to promote the clearance of various growth inducing factors and other serum products leaked into the vitreous due to the presence of PDR. It is further contemplated that the hyaluronidase (ACS) treatment of the present method may be performed alone or in combination with other treatments of PDR.
  • ACS hyaluronidase
  • hyaluronidase ACS
  • NPDR non-proliferative diabetic retinopathy
  • PVD induced posterior vitreous detachment
  • a diabetic patient manifesting preproliferative diabetic retinopathy is treated for this complication of diabetes mellitus through the intravitreal injection of hyaluronidase (ACS).
  • ACS hyaluronidase
  • the purpose of this treatment is to reduce or prevent the development of proliferative diabetic retinopathy manifested by extraretinal neovascularization and fibrous tissue proliferation, vitreous alterations and hemorrhage, macular disease, and retinal detachment.
  • PDR proliferative diabetic retinopathy
  • hyaluronidase (ACS) treatment is commenced. This stage is defined as the presence of venous beading in 2 or more quadrants, LRMA in one or more quadrants, and/or microaneurysm and dot hemorrhages in all quadrants. Once these indicia are present, hyaluronidase (ACS) method of treatment is initiated.
  • ACS hyaluronidase
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, electroretinography and A-scan measurements.
  • hyaluronidase hyaluronidase
  • ACS hyaluronidase
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60).
  • the patient is monitored for vitreous liquefaction.
  • the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression.
  • the extent of PDR presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, LRMA, and retinal ischemia.
  • a diabetic patient manifesting proliferative diabetic retinopathy is treated by the intravitreal injection of hyaluronidase (ACS).
  • ACS hyaluronidase
  • the purpose of this treatment is to reduce the extent of proliferative diabetic retinopathy, to prevent further manifestations of the disease after removal of any extraretinal neovascularized tissue, and to reduce the likelihood of retinal detachment.
  • a patient presenting proliferative diabetic retinopathy is to receive the hyaluronidase (ACS) method of treatment in combination with surgical treatment of the neovascularized tissue.
  • ACS hyaluronidase
  • the proliferation usually begins with the formation of new vessels with very little fibrous tissue component. They arise from primitive mesenchymal elements that differentiate into vascular endothelial cells. The newly formed vascular channels then undergo fibrous metaplasia; that is, the angioblastic buds are transformed into fibrous tissue.
  • the new vessels leak fluorescein, so the presence of proliferation is especially noticeable during angiography.
  • the new vessels and fibrous tissue break through the internal limiting membrane and arborize at the interface between the internal limiting membrane and the posterior hyaloid membrane.
  • the fibrovascular tissue may form preretinal membranes that create dense adhesions with the posterior hyaloid membrane. These adhesions are extremely important because they are responsible for transmitting the forces of vitreous traction to the retina during the later stage of vitreous shrinkage.
  • the proliferative stage of PDR is defined as the presence of three or more of the following characteristics: new vessels, new vessels on or within one disc diameter of the optic nerve, severe new vessels (as defined by one-third disc area neovascularization at the optic nerve or one-half disc area neovascularization at the optic nerve or one-half disc area neovascularization elsewhere), and preretinal or vitreous hemorrhage.
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected visual acuity) symptomatology, fundus photography, fluorescein angiography, electroretinography and A-scan measurements.
  • hyaluronidase hyaluronidase
  • ACS hyaluronidase
  • Panretinal photocoagulation may be used to treat patients presenting PDR in conjunction with the hyaluronidase (ACS) method of treatment.
  • Panretinal photocoagulation is a form of laser photocoagulation.
  • lasers such as the argon green (614 nm), argon blue-green (488 and 514 nm), krypton red (647 nm), tunable dye, diode and xenon arc lasers, are used for retinal surgery. Laser energy is absorbed predominantly by tissues containing pigment (melanin, xanthophyll, or hemoglobin) producing thermal effects on adjacent structures.
  • Krypton red lasers are the preferred method of treatment, as they are better able to penetrate nuclear sclerotic cataracts and vitreous hemorrhage than the argon lasers, which require more energy to produce equal levels of penetration.
  • the parameters used during laser retinal surgery may be modified depending on the goal of the photocoagulation.
  • the laser has a coagulative effect on small vessels.
  • Focal laser photocoagulation is used in diabetes to stop leakage of microaneurysms.
  • the laser spot is place directly over the microaneurysm to achieve a slight whitening and closure of the aneurysm.
  • the laser may reduce microvascular leakage.
  • Panretinal photocoagulation is thought to be effective by destroying tissue, reducing the amount of ischemic tissue in the eye.
  • Confluent laser spots may be used over a neovascular membrane to obliterate the abnormal vessels.
  • the present method does not require a particular order of treatment.
  • the patient is first treated with hyaluronidase (ACS) and then laser treatment, hi another embodiment the patient is first undergoes laser treatment followed by the hyaluronidase (ACS) treatment.
  • ACS hyaluronidase
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60).
  • the patient is monitored for vitreous liquefaction.
  • the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression.
  • the extent of PDR presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, IRMA, retinal ischemia, neovascularization, and vitreal hemorrhage.
  • Evidence of new neopolymerization or incomplete depolymerization of the vitreous would warrant a repeat treatment of the patient as described above.
  • Age-related macular degeneration consists of a gradual, often bilateral decrease of vision. It is the most common cause of legal blindness in adults. It is probably caused by aging and vascular disease in the choriocapillaries or the afferent retinal vessels. There are basically two morphologic types of AMD: “dry” and “wet”.
  • the underlying abnormality of AMD is the development of involutional changes at the level of Bruch's membrane and the retinal pigment epithelium (RPE).
  • RPE retinal pigment epithelium
  • the hallmark lesion of such changes is the druse.
  • drusen the plural form of druse
  • Drusen may be categorized as hard, soft or basal laminar drusen.
  • the present method is directed both to the treatment and prevention of wet and dry forms of AMD.
  • the condition is thought to affect the choriocapillaries.
  • the choriocapillaries are a component of the choroid which serves to vascularize the globe.
  • the choriocapillaries consists of a rich capillary network that supply most of the nutrition for the pigment epithelium and outer layers of the retina. Damage to the choriocapillaries is thought to result ultimately in neovascular complications, a cause of macular degeneration.
  • the dry form nondisciform macular degeneration results from a partial or total obliteration of the underlying choriocapillaries.
  • degeneration of the retinal pigment epithelium and hole formation may be observed.
  • subpigment epithelial deposits of material such as calcium chelates or proteinaceous material and others may be observed.
  • secondary retinal changes generally occur gradually, resulting in the gradual loss of visual acuity. Nevertheless, in some percentage of patients, a severe loss of vision results.
  • the present method contemplates utility in treating dry AMD and preventing macular degeneration tlirough liquefaction of the vitreous. It is contemplated that the liquefaction of the vitreous would result in an increase in the rate of clearance from the retina of deposited material that later results in macular degeneration.
  • Neovascularization also is thought to occur as an adaptation of retinal vascularization to inadequate oxygenation as a result of vesicular damage. Neovascularization may also cause several other disorders such as detachment of the pigment epithelium and sensory retina. Typically the disease usually begins after 60 years of age, manifesting in both sexes equally and in patients presenting the disease, bilaterally.
  • age-related macular degeneration is the development of defects in Bruch's membranes of the globe through which new vessels grow.
  • This epithelial neovascularization may result in the production of exudative deposits in and under the retina.
  • the neovascularization may also lead to hemorrhage into the vitreous, which may lead to degeneration of the retina's rods and cones, and cystoid macular edema (discussed below).
  • a macular hole may form which results in irreversible visual loss.
  • neovascular complications of AMD account for the overwhelming majority of cases of severe visual loss.
  • Risk factors include increasing age, soft drusen, nongeographic atrophy, family history, hyperopia, and retinal pigment epithelial detachments.
  • Symptoms of choroidal neovascularization in AMD include metamorphopsia, paracentral scotomas or diminished central vision.
  • Ophthalmoscopic findings include subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane. Fluorescein angiography is often an effective method of diagnosis.
  • choroidal neovascular membranes as delineated by fluorescein angiography include elevated blocked fluorescence, flat blocked fluorescence, blood, and disciform scar.
  • neovascular AMD suggests that classic choroidal neovascularization is the lesion component most strongly associated with rapid visual deterioration. Accordingly, treatment of AMD must encompass all neovascular and fibrovascular components of the lesion. At present, treatment is only indicated when classic neovascularization has boundaries that are well demarcated, and photocoagulation has been shown to be beneficial. hi eyes with extrafoveal choroidal neovascularization (>-200 microns from the foveal center), argon laser photocoagulation diminished the incidence of severe visual loss,
  • Recurrent neovascularization developed in one-half of laser-treated eyes, usually in the first year after treatment. Recurrent neovascularization was invariably associated with the development of severe visual loss.
  • krypton laser photocoagulation diminished the incidence of severe visual loss from 45% to 31% at 1 year, although the difference between untreated and treated groups was less marked at 5 years.
  • Laser treatment remains an essential therapeutic method for the treatment of AMD, however, the present method would augment this approach by reducing the reoccurrence of neovascularization.
  • ACS hyaluronidase
  • increased ophthalmic surveillance is performed to detect the presence of AMD.
  • This increased surveillance should include periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane.
  • a regime of hyaluronidase (ACS) treatment is commenced coupled with or without other treatments such as photocoagulation.
  • ACS hyaluronidase
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, electroretinography and A-scan measurements.
  • ACS hyaluronidase
  • ACS hyaluronidase
  • Laser photocoagulation treatment of the hyaluronidase (ACS) injected eyes may be required.
  • the laser treatment protocol described in Examples 8 and 9 should be followed when treating AMD. hi an alternative embodiment, photocoagulation treatment occurs before the enzyme treatment of the present method.
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). Because of the possibility of reoccurrence, the patient should return for periodic examinations on a monthly basis thereafter. On each examination day the patient is monitored for vitreous liquefaction. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression. Finally, the extent of AMD presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane.
  • ACS hyaluronidase
  • laser treatments may be required if indicia of reoccurring neovascularization are observed.
  • the following Example demonstrates the efficacy of the present method, even without the use of photocoagulation. Improvement in Symptoms of Macular Degeneration Following Intravitreal
  • ACS hyaluronidase
  • the patient was repeatedly examined post-dose and the vision in his left (untreated) eye remained unchanged, while the vision in his right (treated) eye was observed to improve as follows:
  • amblyopia is derived from Greek and means dull vision (amblys— dull, ops—eye). Poor vision is caused by abnormal development in visual areas of the brain, which is in turn caused by abnormal visual stimulation during early visual development.
  • the pathology associated with amblyopia is not specific to the eye, rather, it is located in the visual areas of the brain including the lateral geniculate nucleus and the striate cortex.
  • This abnormal development is caused by three mechanisms: (1) blurred retinal image called pattern distortion; (2) cortical suppression, or (3) both cortical suppression plus pattern distortion.
  • the present method is primarily concerned with pattern distortions caused by media opacity. More specifically, the present method addresses issues of vitreous opacity.
  • Amblyopic vision is usually defined as a difference of at least two Snellen lines of visual acuity.
  • Critical to the treatment of amblyopia is early detection and early intervention.
  • the strategy for treating amblyopia caused by vitreous opacity is to provide a clear retinal image by altering the opacity of the vitreous so that clear vision results.
  • a patient manifesting amblyopia resulting from vitreal opacity was treated with an intravitreal injection of hyaluronidase (ACS).
  • ACS hyaluronidase
  • ACS hyaluronidase
  • the patient was examined repeatedly post-dose and the vision in her left (untreated) eye remained unchanged while the vision in her right (treated) eye was observed to improve as follows:
  • Retinitis pigmentosa is the name given to a group of heritable disorders of progressive retinal degeneration characterized by bilateral nyctalopia constricted visual fields and abnormality of the electroretinogram. Early symptoms include difficulty with dark adaptation and midperipheral visual field loss. As the disease progresses, visual field loss advances, typically leaving a small central field of vision until eventually even central vision is affected. Central acuity may also be affected earlier in the course of disease either by cystoid macular edema, macular atrophy, or development of a posterior subcapsular cataract.
  • RP represents a varied group of diseases whose common thread is the abnormal production of at least one protein in photoreceptor outer segments critical to light transduction.
  • RP retinitis pigmentosa
  • the uncorrected vision in his left eye was 20:400 and with correction was also 20:400.
  • a single intravitreal injection of 100 LU. of the hyaluronidase (ACS) was administered to the left eye of the patient. The other eye remained untreated.
  • the patient was examined repeatedly following the dose of hyaluronidase (ACS) and the vision in the patient's right (unfreated) eye remained unchanged, while the vision in the patient's left (treated) eye was observed to improve as follows:
  • ACS hyaluronidase
  • HM* denotes "hand movement”
  • cf** denotes "finger counting”
  • EXAMPLE 13 Hyaluronidase Treatment of Macular Holes A rupture or bursting open of the macula is known as a macular hole, rnterestingly, this condition usually occurs in women in their sixth through eighth decades, or after trauma such as lightening injury, solar injury, scleral buckling, or in staphylomatous eyes. Symptoms include metamorphopsia and diminished visual acuity.
  • Macular hole formation is thought to result from tangential traction across the retinal surface induced by the posterior cortical vitreous with involvement of fluid movement within a posterior vitreous synergetic cavity.
  • the posterior vitreous syneresis cavity is present in the vast majority of patients presenting macular holes. It is thought that as the posterior vitreal gel retreats from the retinal surface, the resulting gap between the two surfaces creates an area wherein movement of the vitreous humor may negatively interact with the retinal surface.
  • the tangential movement of the vitreous humor within the space of the posterior vitreous syneresis cavity is thought to promote tears of the retinal membrane, resulting in the creation of macular holes.
  • the present method contemplates the use of hyaluronidase (ACS) to depolymerize the vitreous so as to eliminate the conditions which result in macular hole formation.
  • ACS hyaluronidase
  • the posterior vifreous syneresis cavity is eliminated as a result of hyaluronidase (ACS)-mediated reorganization of the vitreous.
  • ACS hyaluronidase
  • a patient presenting the early signs of macular hole formation is freated with an intravitreal injection of hyaluronidase (ACS).
  • ACS hyaluronidase
  • the patient to be freated presents the various signs of premacular hole formation. These include loss of the foveal depression associated with a yellow foveal spot or ring.
  • the fovea has begun to thin in the region of hole formation and the lesion may obtain a reddish appearance. Fluorescein angiography at this stage may appear normal or show faint hyperfluorescence.
  • the appearance of an eccentric full thickness dehiscence denotes an advanced early stage of the disease. Upon observance of these symptoms hyaluronidase (ACS) treatment is commenced.
  • ACS hyaluronidase
  • the hyaluronidase (ACS) treatment of the present method is commenced when the formation of a macular hole is diagnosed.
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, electroretinography and A- scan measurements.
  • hyaluronidase hyaluronidase
  • both eyes are affected, they may be treated separately.
  • the eye to be treated is injected with 50 ⁇ l of 50 LU. of the hyaluronidase (ACS) ophthalmic solution described above intravitreally to promote the depolymerization of vitreous hyaluronic acid, resulting in the liquefaction of the vitreous.
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). On each examination day the patient is monitored for vitreous liquefaction. Fluorescein angiography, considered a particularly effect method of monitoring the course of the treatment, is also performed. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression.
  • Macular exudates are material that penetrates the blood-retina barrier and seeps through the macula into the vitreal chamber. There are two kinds, soft exudates and hard exudates. The soft exudates are actually not exudates but clusters of ganglion cell axons in the nerve fiber layer that have undergone a bulbous dilation at a site of ischemic damage or infarction. Hard exudates are commonly exuded as a result of microvascular changes in background retinopathy. Hard exudates appear yellow and waxy are often deposited in a circular fashion about the macula.
  • lipid and proteinaceous material derived from the exudation of serum components from leaking vessels or from the lipid products of degenerating neural elements within the retina. Adsorption of hard exudates is primarily mediated by macrophagic resorption, however, the rate of this process may be slow since exudation often occurs in the outer plexiform layer within the avascular zone of the retina.
  • the present method is particularly useful in reducing the extent of exudative accumulation resulting from the destabilization of the retinal membrane since hyaluronidase (ACS) depolymerization of the vitreous promotes an increased turn-over rate of the aqueous components of the vitreous.
  • ACS hyaluronidase
  • a patient presenting macular exudates is treated with hyaluronidase (ACS) injection method of treatment.
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, electroretinography and A-scan measurements.
  • an intravitreal injection of hyaluronidase included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, electroretinography and A-scan measurements.
  • ACS hyaluronidase
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60).
  • the patient is monitored for vitreous liquefaction. Fluorescein angiography, considered a particularly effect method of monitoring the course of the treatment, is also performed. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression.
  • Cystoid macular edema is a common ocular abnormality resulting form a diverse group of etiologies. Most the causes of this condition stem from a disturbance of the blood- retinal barrier of the perifoveal capillaries and the optic nerve head that result in fluid leakage which accumulates in microcysts of the outer plexiform layer. This region is a relatively thin and under vascularized area of the retina. Clinically, a cystoid macular edema produces a honey-comb appearance when examined with fluorescein angiography. As the edema progresses, the outer plexiform layer may rupture, producing a lamellar hole.
  • the hole may be confined to the inner layer of the retina or it may eventually progress to a complete macular hole.
  • the present method contemplates the treatment of cystoid macular edema and the prevention of macular hole formation through the hyaluronidase (ACS)-mediated depolymerization of the vitreous.
  • ACS hyaluronidase
  • Hyaluronidase has been used therapeutically for many years now. Its proven and diverse effects, which occur mainly in intercellular connective tissue, are primarily attributable to the breakdown of hyaluronic acid in the tissue. The therapeutically useful consequences of this action, i.e. reduced viscosity of intercellular cement and increased membrane and vascular permeability, are due to its "spreading effect". The permeability- enhancing effect of hyaluronidase after administration of fluids and/or radiopaque media is of therapeutic significance. Hyaluronidase - The Spreading Effect:
  • Hyaluronidase accelerates and enhances the absorbtion of injected drugs (antibiotics, cytostatic agents, local anesthesia, chemotherapeutic agents, antivirals, etc.) by the tissue, even when large volumes of the medications are administered in solution, suspension or emulsion form.
  • Hyaluronidase has been successfully used in orthopedics, surgery, ophthalmology, internal medicine, oncology, gynecology, dermatology, etc. for many years.
  • Ophthalmology is now an important and well documented area of indication for hyaluronidase (Fair C. ⁇ t al. 1997 Wien Med Wschr 147:1-8).
  • Hyaluronic acid is often applied during ophthalmic surgery (e.g., cataract surgery), for example, to keep the anterior chamber of the eye intact or to protect the corneal endothelium during lens implantation. This results in an increase in intraocular pressure. Measurements have shown that introduction of hyaluronidase in the anterior chamber of the eye can effectively decrease the intraocular pressure postoperatively.
  • Hyaluronidase was also found to be effective in reducing the intraocular pressure in patients who underwent frabeculectomy for treatment of wide-angle glaucoma (doses of 300 IU were administered as a subconjunctival injection). The authors concluded that hyaluronidase reduced the number of complications and improved the prognosis of trabeculectomy. Hyaluronidase can also be helpful in retro- and peribulbar anesthesia for cataract surgery when used in combination with local anesthetics such as lidocaine and bupivacaine (with or without adrenaline). The effects of hyaluronidase in local anesthesia of the eye were reported back in 1949.
  • hyaluronidase was found to be highly effective when added to lidocaine and noradrenaline in retrobulbar anesthesia in senile cataract operations. i one clinical study, the addition of 75 IU of hyaluronidase to 0.75%> bupivacaine and 2% mepivacaine lead to improved motor blockade, analgesia and hypotonus of the eyeball.
  • peribulbar anesthesia investigators recommended that a mixture of 2% lidocaine, 0.5% bupivacaine, and 1550 IU hyaluronidase be warmed to body temperature to eliminate symptoms of pain in the target areas, hi a separate study, two concentrations of hyaluronidase were used as a supplement to peribulbar anesthesia. Three groups received either 0, 50 or 150 IU of the hyaluronidase supplement, and the onset of effect, analgesia and akinesia were assessed. No statistically significant differences between the 50 IU and 150 IU hyaluronidase groups could be detected. The authors concluded that the addition of hyaluronidase did not lead to any complications.
  • Hyaluronidase makes is possible for lidocaine and bupivacaine to spread more rapidly within the peribulbar space.
  • the injection pressure of local anesthesia administered prior to cataract operations was investigated in 50 patients in a double-blind study.
  • the study concluded that significant (sufficient) akinesia of the exfraocular muscles can be achieved by administration of 1% etidocaine, 0.5%o bupivacaine, and 50 IU hyaluronidase.
  • Hyaluronidase is useful for treatment of glaucoma or to alleviate intraocular pressure.
  • hyaluronidase When hyaluronidase was combined with a local anesthetic, e.g., procaine or lidocaine, the onset of effect was quicker, the analgesic region was enlarged, and postoperative pain was significantly reduced.
  • a local anesthetic e.g., procaine or lidocaine
  • Orthopedics, diseases of the supportive and locomotive apparatus For many years now, hyaluronidase has been successfully used for treatment of various diseases of the supportive and locomotive apparatus, e.g., acute conditions of the synovial sheath, surrounding connective tissue and varied inflammations in these areas (paratendinitis crepitans, humeroscapular periarthritis, humeral epicondyhtis, tibial condylitis, radial styloiditis, etc.).
  • Good treatment results can usually be achieved (especially in combination with exercise or physiotherapy) if hyaluronidase therapy is started as early as possible, even if the affected limb cannot be immobilized.
  • Hyaluronidase was successfully used in combination with mephenesin for treatment of arthrosis, primarily in patients with gonarthrosis. The success rate in chronic cases was particularly impressive.
  • hyaluronidase When used as a supplement to chemotherapy of malignant tumors, hyaluronidase can dissolve hyaluronic acid-containing areas around tumor cells and tumor cell conglomerates, thereby enabling a higher concentration of the cytostatic agent to take effect in the desired target area. Moreover, hyaluronidase may induce a related enhancement of immunological defensive processes, e.g., by creating direct contact between immunocompetent cells ("natural killer cells") and antigens on the tumor surface.
  • natural killer cells immunocompetent cells
  • malignant diseases hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ENT region, adenocarcinomas of the lung and colon, and carcinomas of the bladder
  • malignant diseases hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ENT region, adenocarcinomas of the lung and colon, and carcinomas of the bladder
  • hyaluronidase was usually found to increase the response rate to cytostatic agents if high doses of the enzyme are admimstered prior to adminisfration of the cytostatic agent.
  • Hyaluronidase supplements can improve the patient's response to chemotherapy when used in therapy-resistant patients with malignant hematological diseases.
  • hyaluronidase has proven to be particularly useful when administered as a supplement to cytostatic agents like doxorubicin and adriamycin.
  • the enzyme improves the penetration of doxorubicin in the cells and increases the activity of adriamycin in breast cancer.
  • Hyaluronidase led to a decrease in adhesion-related multicellular drug resistance in carcinomas of the breast. This mechanism of action is based on the reduction of cell- contact-dependent inhibition of growth and on the sensitization of cells for the cytostatic agents.
  • Hyaluronidase increases the effects of cytostatic agents used for freatment of such malignant diseases as hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ear, nose and throat region, adenocarcinomas of the lung and colon, and carcinomas of the bladder.
  • Hyaluronidase can improve the subjective well-being and the quality of life of tumor patients.
  • hyaluronidase is useful in certain dermatological diseases, such as, for example, progressive scleroderma, which is a systemic disorder of the entire vascular connective tissue system, with its most important characteristic being the displacement of collagen fractions.
  • progressive scleroderma which is a systemic disorder of the entire vascular connective tissue system, with its most important characteristic being the displacement of collagen fractions.
  • histomorphological skin changes that occur in scleroderma begin with a dermal edema rich in acidic mucopolysaccharides (hyaluronic acid, chondroitin sulphate). Histopathological and chemical tests have shown that part of the ground substance occurs as cement in the collagen fibers. It would therefore appear that acidic mucopolysaccharides, soluble collagen, and polymeric collagen are responsible for the sclerosis.
  • hyaluronidase for treatment of acute myocardial infarction was first described in 1959. Studies have shown that the administration of hyaluronidase in the acute stage, i.e., in the early stage of fresh myocardial infarction (2 to 4 hours after the onset of infarction) can reduce the size of the necrotic area in the heart.
  • Hyaluronidase was found to have a favorable effect on concomitantly administered thrombolytic agents such as streptokinase. This appears to be attributable to the ability of hyaluronidase to entrap oxygen radicals.
  • Statisticians performed meta-analyses in which they studied the role of hyaluronidase and other cardiovascular drugs with a potential for reducing the size of myocardial infarction.
  • hyaluronidase in addition to conventional agents for treatment of acute myocardial infarction (nitrates, beta receptor blockers, calcium antagonists).
  • Miscellaneous indications Another indication is for treatment of submucosal fibrosis.
  • Experience with 150 patients over a 10-year period has shown that the combination of hyaluronidase and dexamethasone is able to reduce symptoms over a long period of time in most cases.
  • hyaluronidase for prevention of postoperative adhesions following surgery has also been reported.
  • hi cerebral abscess hyaluronidase was alternatively combined with dexamethasone or antibiotics, primarily to eliminate edema in high-risk patients.
  • Hyaluronidase was found to improve the absorption of locally administered drugs and to reduce the risk of progression of skin necrosis in patients treated with intravenously administered Vinca alkaloids.
  • Hyaluronidase is one of the few antidotes that can be used as an antidote for Vinca alkaloids or epipodophyllotoxins such as etoposide.
  • Gynecology is another area of application for hyaluronidase.
  • hyaluronidase When injected in the perineal region prior to the expulsive stage of labor, hyaluronidase was found to soften the consistency of the birth canal of first-time mothers, which often eliminated the need for episiotomy.
  • Hyaluronidase is also useful for facilitation of partial and complete aspiration of viscous joint effusions and pleural effusions, i.e., it liquefies the effusions.
  • the enzyme is also used for treatment of edema of various origins and for treatment of arthritic joint changes.
  • Hyaluronidase is a treatment for corneaplasty, corneal scars, opacification, and haze, and cornea in need of delamination.
  • Hyaluronidase can be used as an alternative or adjunct to conventional mechanical vitrectomy.
  • Hyaluronidase is also useful for the induction of retinal detachments.
  • Hyaluronidase is indicated as an adjuvant to increase the absorption and dispersion of other injected drugs; for hypodermoclysis; and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. Summary: The proven and diverse activity of hyaluronidase occurs mainly in the intercellular connective tissue. This action is clearly attributable to the breakdown of hyaluronic acid in the tissue. The therapeutically useful consequences of this action
  • the hyaluronidase-related enhancement of diffusion and increase in permeability that occurs after administration of liquids and/or radiopaque media is of therapeutic significance.
  • the substance is able to accelerate and increase the absorption of drugs (antibiotics, cytostatics, local anesthetics, etc.) by the tissue, even when large volumes of the medication are injected in solution, suspension or emulsion form. If it is not possible to administer a certain drug infravenously in cases where a rapid onset of effect is necessary, a "pre-injection" of hyaluronidase can accelerate (by 200 to 300%) the absorption of subcutaneous or intramuscular doses of the drug in the bloodstream, which is of particular significance for internal medicine.
  • the efficacy of hyaluronidase in treatment of disorders of the supportive and locomotive system can be attributed to the so-called "softening effect" of the enzyme.
  • the "antiphlogistic" effect of hyaluronidase makes it possible to control acute symptoms involving the synovial sheaths and the surrounding connective tissue (peritendinitis crepitans, humeroscapular periarthritis, humeral epicondyhtis, tibial condylitis, radial styloiditis, etc.)
  • Joint stiffness (e.g., due to supracondylar fracture) can also be treated successfully.
  • hyaluronidase can also be used for treatment of posttraumatic hematomas or edemas of any origin, and for liquefaction of joint and pleural effusions in orthopedics.
  • Hyaluronidase is indicated to be useful as an anti-edema and anti-inflammatory agent in the prevention of transplant rejection. It has been shown in pre-clinical experiments to lend itself to this role, because it breaks down hyaluronan in damaged tissues. Hyaluronan, a glucosaminoglycan with unique water-binding capacity, draws water into some transplanted organs causing edema. This in turn impairs organ function which may lead to the transplanted organ failure and being rejected. In addition to attracting water, hyaluronan attracts certain cells of the immune system and therefore may be instrumental in initiating inflammatory reactions. Studies have confirmed that hyaluronidase treatment can be used to reduce edema and inflammation after organ transplantation.
  • pre-injection When hyaluronidase is administered prior to administration of a local anesthetic such as procaine ("pre-injection"), the onset of effect of the anesthetic is quicker, the anesthetic region is larger, and the pain after completion of the procedure is significantly lower.
  • a local anesthetic such as procaine
  • hyaluronidase and local anesthetics are now widely used, particularly in ophthalmology and especially in cataract surgery.
  • the preoperative administration of hyaluronidase with certain local anesthetics (procaine, lidocaine, bupivacaine, etc.) for retro- and peribulbar anesthesia is useful in various ophthalmologic operations.
  • the enzyme accelerates the onset of effect of the anesthetic agent and causes reliable blockade of the eye muscles which, in turn, creates excellent conditions for surgery.
  • vasopressors such as adrenaline
  • hyaluronidase increases the duration of anesthesia in the treated area and prevents the rapid diminishment of local anesthesia.
  • Hyaluronidase is also effective for treatment of postoperatively increased internal eye pressure due to the administration of viscoelastic substances such as sodium hyaluronate during ophthalmic surgery.
  • Hyaluronidase is also widely used in dermatology, i.e., in selected skin disorders involving the connective tissue system and characterized by degeneration of it (scleroderma, keloid formation, psoriasis, chronic varicose ulcer, etc.).
  • Hyaluronidase is also useful in gynecology, i.e., for prevention of episiotomy.
  • the usefulness of hyaluronidase has been validated for treatment of myocardial infarction.
  • hyaluronidase is helpful as a supplement to chemotherapy in patients with cancer (myeloma, Hodgkin's disease, non- Hodgkin's lymphoma, breast cancer [also with concomitant cerebral metastasis], cerebral lymphomas, gliomas, squamous cell carcinomas in the ear, nose and throat region, and carcinomas of the bladder).
  • the enzyme not only increases the patient's response to the cytostatic agents, but also drastically improves the patient's overall subjective feeling of well-being and the remission rate.
  • hyaluronidase is a therapeutically versatile enzyme that promises to be a therapeutically useful agent in new areas of medical practice now and in the future.
  • Hyaluronidase for Injection In this Example for use of hyaluronidase as a spreading agent, hyaluronidase for injection dehydrated in the solid state under high vacuum with the inactive ingredients listed below, is supplied as a sterile, nonpreserved, white, odorless, amorphous solid. The product is to be reconstituted with Sodium Chloride Injection, USP, before use.
  • Each vial of 6200 USP units contains 5 mg lactose, 1.92 mg potassium phosphate dibasic, and 1.22 mg potassium phosphate monobasic.
  • the USP/NF hyaluronidase unit is equivalent to the turbidity-reducing (TR) unit and equal to 0.81 International Units (IU).
  • the reconstituted solution is clear and colorless, with an approximate pH of 6.7 and osmolality of 290 to 310 mOsm.
  • Hyaluronidase for injection is to be reconstituted in a vial to a concenfration of 1000 Units/mL of Sodium Chloride Injection, USP by adding 6.2 mL of solution to the vial.
  • the reconstituted solution Prior to administration, the reconstituted solution should be further diluted to the desired concenfration, commonly 150 Units/mL, see table below. The resulting solution should be used immediately after preparation.
  • a ImL syringe and a 5-micron filter needle are supplied in a hyaluronidase for injection kit. Following reconstitution of hyaluronidase for injection, as described above, apply the 5-micron filter needle to the ImL syringe. Draw the desired amount of hyaluronidase for injection into the syringe, and dilute according to the table below. Remove the filter needle and apply a needle appropriate for the intended injection.

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Abstract

L'invention concerne un procédé servant à préparer une préparation hyaluronidase destinée à des applications pharmaceutiques. Dans un mode de réalisation préféré, ce procédé consiste à effectuer une filtration virale afin d'augmenter la pureté du produit final. L'invention concerne également un procédé permettant d'augmenter la pureté des préparations hyaluronidase actuellement disponibles dans le commerce. Les procédés selon l'invention sont utilisés de préférence pour purifier l'hyaluronidase d'origine mammifère. Dans un autre mode de réalisation, lesdits procédés peuvent être utilisés pour purifier l'hyaluronidase recombinée.
PCT/US2004/011692 2003-04-15 2004-04-15 Procede d'isolation et de purification d'hyaluronidase ovine WO2004092361A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2471867C2 (ru) * 2007-06-19 2013-01-10 Тамара П. Уваркина Гиалуронидаза и способ ее применения
US9562223B2 (en) 2003-03-05 2017-02-07 Halozyme, Inc. Methods for reducing intraocular pressure by administering a soluble hyaluronidase glycoprotein (sHASEGP)
US9682149B2 (en) 2012-11-05 2017-06-20 Bmi Korea Co., Ltd Stabilizer for hyaluronidase and liquid formulation comprising hyaluronidase
US10179172B2 (en) 2013-09-11 2019-01-15 Eagle Biologics, Inc. Liquid pharmaceutical formulations for injection comprising yellow 5 or orange G and uses thereof
EP4017524A4 (fr) * 2019-10-15 2022-09-21 Standard of Care Corporation Compositions comprenant de l'hyaluronidase et/ou de la collagénase et/ou de la 4-méthylumbelliférone (4-mu) et procédés de traitement l'utilisant
US11471479B2 (en) 2014-10-01 2022-10-18 Eagle Biologics, Inc. Polysaccharide and nucleic acid formulations containing viscosity-lowering agents

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US9682149B2 (en) 2012-11-05 2017-06-20 Bmi Korea Co., Ltd Stabilizer for hyaluronidase and liquid formulation comprising hyaluronidase
US10821183B2 (en) 2013-09-11 2020-11-03 Eagle Biologics, Inc. Liquid protein formulations containing 4-(3-butyl-1-imidazolio)-1-butane sulfonate (BIM)
US10646571B2 (en) 2013-09-11 2020-05-12 Eagle Biologics, Inc. Liquid protein formulations containing cimetidine
US10179172B2 (en) 2013-09-11 2019-01-15 Eagle Biologics, Inc. Liquid pharmaceutical formulations for injection comprising yellow 5 or orange G and uses thereof
US10821184B2 (en) 2013-09-11 2020-11-03 Eagle Biologics, Inc. Liquid protein formulations containing thiamine pyrophosphate (TPP)
US10849977B2 (en) 2013-09-11 2020-12-01 Eagle Biologics, Inc. Liquid Protein Formulations Containing Thiamine
US11819550B2 (en) 2013-09-11 2023-11-21 Eagle Biologics, Inc. Liquid protein formulations containing cyclic adenosine monophosphate (cAMP) or adenosine triphosphate (ATP)
US11986526B2 (en) 2013-09-11 2024-05-21 Eagle Biologics, Inc. Liquid protein formulations containing 4-ethyl-4-methylmorpholinium methylcarbonate (EMMC)
US11471479B2 (en) 2014-10-01 2022-10-18 Eagle Biologics, Inc. Polysaccharide and nucleic acid formulations containing viscosity-lowering agents
EP4017524A4 (fr) * 2019-10-15 2022-09-21 Standard of Care Corporation Compositions comprenant de l'hyaluronidase et/ou de la collagénase et/ou de la 4-méthylumbelliférone (4-mu) et procédés de traitement l'utilisant

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