JP2006523461A - Method for isolating and purifying sheep hyaluronidase - Google Patents

Abstract

A method for preparing a high-purity hyaluronidase preparation suitable for pharmaceutical use is provided.
In a preferred embodiment, the process includes the use of a viral filtration step that increases the purity of the final product. The process provides a way to increase the purity of currently marketed hyaluronidase formulations. The method is preferably used to purify hyaluronidase from mammalian sources. In another embodiment, the disclosed method can be used to purify recombinant hyaluronidase.

Description

  This disclosure relates to a method for preparing a mammalian testicular hyaluronidase formulation suitable for pharmaceutical use. In a preferred embodiment, the process is used to purify hyaluronidase from sheep testis and includes using a viral filtration step to increase the purity of the final product. The process also provides a way to increase the purity of currently marketed hyaluronidase formulations. The method is preferably used to purify hyaluronidase from mammalian sources. In another embodiment, the disclosed method can be used to purify recombinant hyaluronidase.

  This application claims the benefit of US Provisional Patent Application No. 60 / 463,516, filed Apr. 15, 2003, and is hereby incorporated by reference in its entirety.

  Hyaluronidase is a versatile enzyme that is equally expressed in vertebrates and invertebrates. Mammalian hyaluronidase catalyzes the random hydrolysis reaction of 1,4-bonds between 2-acetamido-2-deoxy-bD-glucose and D-glucose residues in hyaluronate.

  Hyaluronidase from bovine testis has been reported to have a molecular weight of 65,000 (Lathrop et al. 1990 J Cell Biol 111: 2939). This bovine testicular hyaluronidase hydrolyzes the endo-N-acetylhexosamine bond of hyaluronic acid and chondroitin sulfate A and C (but not B) to produce mainly tetrasaccharide residues (Ludovig et al. 1961 J Biol Chem 236: 333).

Typical purification protocols require the use of standard chromatographic techniques that produce a purified solution having hyaluronidase activity. Tolksdorf et al., 1949 J Lab Clin Med 34:74 and Kass & Seastone, 1944 J Exp Med 79: 319 describe generally accepted assay protocols for measuring the activity of hyaluronidase. These purification protocols were sufficient to provide a relatively crude hyaluronidase formulation.
Lathrop et al. 1990 J Cell Biol 111: 2939 Ludovig et al. 1961 J Biol Chem 236: 333 Tolksdorf et al., 1949 J Lab Clin Med 34:74 Kass & Seastone, 1944 J Exp Med 79: 319

  Provided is a method for preparing a high-purity hyaluronidase preparation suitable for pharmaceutical use.

The present invention provides a hyaluronidase purified from sheep testis and capable of hydrolyzing hyaluronic acid type mucopolysaccharides, which is based on mobility in a 4-20% gradient SDS polyacrylamide gel. Containing hyaluronidase with a molecular weight of 70-74 kDa for α form and 60-63 kDa for β form and a specific activity in the range of 1.2 × 10 ^{4 to} 1.9 × 10 ⁴ USP units / mg protein; And it is a composition which does not contain thimerosal.

  The present invention also provides a method for preparing a purified mammalian hyaluronidase comprising: a) providing a testis source; b) extracting the testis source to obtain a crude testis extract. Preparing a liquid; c) precipitating the crude testis extract to produce a precipitate and suspending the precipitate to produce a crude testis suspension; d) the crude testis suspension; Dialyzing with a food grade dialysate to prepare a crude dialysate; e) subjecting the crude dialysate to ion exchange chromatography to isolate the crude eluate; and f) the crude Lyophilizing the eluate to produce a crude lyophilizate, g) suspending the crude lyophilizate to produce a crude formulation, and h) subjecting the crude formulation to ion exchange chromatography. Isolating the purified eluate; i) dialyzing the purified eluate using a food grade dialysate to prepare a purified dialysate; j) Performing viral filtration of the purified dialysate to prepare purified mammalian hyaluronidase.

  Sheep hyaluronidase is an enzyme product purified from sheep testis and can hydrolyze hyaluronic acid type mucopolysaccharides.

Amino acid sequence-α type (SEQ ID NO: 1)
The consensus site for glycosylation is underlined. The cleavage site that produces the β-type hyaluronidase is homologous to the bovine sequence and is shown in bold and underlined.
Sheep 1 LDFRAPPLIS N TSFLWAWNAPAERCVKIFKLPPDLRLFSVKGSPQKSATG
Sheep 51 QFITLFYADRLGYPHIDEKTGNTVYGGIPQLGNLNKHLEKAKDIAYYI
Sheep 101 P N DSVGLAVIDWENWRPTWARNWKPKDVYRDESVELVLQKNPQLSFPEAS
Sheep 151 KIAKVDFETAGKSFMQETLKLGKLLLRPNHLWGYYLFPDCYNHNYNQPTYN
Sheep 201 GNCSDLEKRRNDDDWLWKESTALFPPSVYLNIKLKSTPKAAFYVRNRVQE
Sheep 251 AIRLSKIASVESPLPVFVYHRPVFTTDGSSTYLSQGDLVNSVGGEIVALGAS
Sheep 301 GIIMWGSL N LSLTMQSCMNLGNYL N TTLNPYII N VTLAAKMCSQVLCHDE
Sheep 351 GVCTRLKWNSSDYLHLNPNMFAIQTGKGGGKYTVPGKVTLEDLQTFSDKFY
Sheep 401 CSCYANINCKKRVDIKNVHSVNVCMAEDICIEGPVKLQPSDDH S SSQNEAS
Sheep 451 TTTVSSISPSTTTTVSPCTPEKQSPECLKVRCLEAIANVTQTGCQGVKW
Sheep 501 KNTSSQSQSSIQNIKNQTTY.

The molecular weight based on mobility in a reduced sodium dodecyl sulfate (SDS) polyacrylamide gel having a molecular weight gradient of 4 to 20% is 70 to 74 kDa for α form and 60 to 63 kDa for β form (the following hyaluronidase content) (See the test results in).

Identification test The identification of hyaluronidase in a sample of drug substance is based on demonstrating the enzymatic activity of hyaluronidase.

<Activity ID test>
Hyaluronidase enzyme activity is demonstrated by non-quantitative variation of activity assay (see “Titer” below). A solution of approximately 0.2 mg / mL drug substance is prepared in 20 mM sodium phosphate dilution buffer at pH 6.90. In a 16 × 125 mm glass test tube, 1 mL of the drug substance solution is reacted with 1 mL of 0.5 sodium hyaluronate substrate solution and then incubated at 37 ° C. for 10 minutes. A blank solution consisting of 1 mL of 20 mM sodium phosphate buffer and 1 mL of substrate solution is prepared simultaneously. After 10 minutes, the absorbance of the blank solution and drug substance solution is read with a spectrophotometer at 600 nm. The value obtained by subtracting the absorbance of the sample solution from the absorbance of the blank solution should be greater than 0.4 if hyaluronidase activity is present in the drug substance. This indicates that the existing hyaluronidase originates from testicular hyaluronidase. This is because, of the six known mammalian hyaluronidases, only this enzyme has significant enzymatic activity at pH higher than 5.0.

Microbial limit test E. coli, S. aureus, P. aeruginosa or Salmonella are not present. Total microbial contamination is less than 10 ³ per gram.

  pH: 3 g solution in 1 mL of deionized water with a pH of 5.2-7.2.

[Impurity test]
<Process-related impurities>
<Annexin II> The annexin II content of a drug substance is measured by an electrophoretic content assay, and is quantified against an internal standard curve of an annexin II reference standard in a company. Specification: 0.29-0.57 mg Annexin II per mg protein. The annexin II reference standard was purified from drug substance by protein G affinity chromatography and size exclusion chromatography. Each batch of reference standard is used by testing for purity (SDS PAGE), identification (Western blotting), protein concentration and amino acid content (amino acid analysis) and testing with an electrophoretic content assay as described herein. Qualification is given.

Test method:
Annexin II content is assessed on 12-well 4-20% gradient sodium tris-glycine dodecyl sulfate (SDS) polyacrylamide gels. The drug substance sample is reduced and denatured at a final concentration of 0.12 mg / mL with a buffer solution containing 500 μL of 2-mercaptoethanol per 10 mL of 2X Tris-Glycine SDS sample preparation buffer. Annexin II reference standard standard curves were generated at five concentrations (referred to as Std1-5, respectively) of 7.42, 14.84, 29.68, 44.52 and 74.2 μg / mL. These solutions are diluted in a 1: 1 ratio when reducing 2x sample preparation buffer (see above). The designated lot of Vitrase® finished product is used as the system suitability standard and is also prepared at 0.12 mg / mL. One sample lane is used for samples with a wide range of reduced molecular weight standards. Samples and standards are all loaded with a sample volume of 10 μL per well in the following order.

TS = Drug substance test sample SSS = System suitability standard lot Std1-Std5 = Standard curve of in-house Annexin II reference standard MWS = A wide range of molecular weight standards.

  The gel is electrophoresed at a constant voltage of 120V for 120 ± 5 minutes or until the bromphenol blue tracking dye dye line reaches the bottom of the gel. When the experiment is complete, the gel is carefully removed from its plastic cassette and then placed in 50 mL of colloidal Coomassie blue dye solution. The gel continues to stain for 13-17 hours with constant agitation on a gel rocker platform.

  When staining is complete, the gel is depigmented on a gel rocker platform in deionized water for at least 7 hours using multiple changes of water during the process.

  Following depigmentation, the gel is quantitatively analyzed with a scanning laser densitometer. The densitometer creates a band density histogram and integrates the histogram to obtain the area under the “peak” in the histogram.

Calculation:
1. The peak area data points of the band concentration for each Annexin II concentration of the standard curve are plotted against their theoretical concentration in Microsoft Excel with correlation coefficient (r ² ) values and curve equations.
2. Annexin II band mass (in μg) in all drug substance and system suitability samples is calculated from the area of the quantified band peak and interpolated from the standard curve.
3.3 Calculate the average mass of Annexin II for each set of samples (drug substance or system suitability sample).
4). The average mass of Annexin II for each sample is expressed as a percentage of total protein using the following formula:

Percentage of Annexin II = Average value of Annexin II in sample (in μg) × 100 / total protein loaded (in μg)
If the annexin II content is between 0.29 and 0.57 mg / mg protein, the sample meets the requirements.

System suitability criteria:
Results obtained from this test 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 in the gel sample lane 12 between a molecular weight standard of 31 kDa and 36.5 kDa.
3. The average value of system suitability samples tested three times must be within 15% of the historically required value.

Acceptance criteria:
Data obtained from this test is unacceptable if the following criteria are not met:
1. The calculated r ² value for the standard curve should be ≧ 0.98.
2. Percentage coefficient of variance for 3 data points (SSS and TS samples) should be ≦ 10%.
3. The calculated average value of Annexin II for the sample should be in the range of 0.076-0.39 μg for the drug substance.
Must be in the μg range.

    <IgG Fragment> The IgG heavy chain fragment (“IgG”) content of the drug substance is quantified by a high performance liquid chromatography (HPLC) method using an affinity column and an IgG reference standard curve. Specification: ≦ 0.23 mg IgG / mg protein.

  The IgG reference standard is prepared in a one-step affinity purification method by protein G chromatography. Each batch of IgG reference standard is qualified for use by testing for purity (SDS-PAGE), identification (Western blotting), concentration (colorimetric protein assay) and content (HPLC method).

  This method utilizes a standard HPLC system with a tunable UV detector set at 280 nm. The column is a 4.6 × 100 mm Poros-OH pre-column connected in series to a 4.6 × 100 mm Poros-G affinity column. The drug substance sample is loaded onto a mobile phase of 0.05 M sodium phosphate pH 7.2 supplemented with 0.15 M sodium chloride. All IgG is retained on the column and removed with elution buffer of 0.1 M glycine, 5% acetic acid pH 2.5. All chromatography was performed at ambient temperature. The elution gradient has a cycle time of 10 minutes and takes the following form:

Solvent A = 0.05M sodium phosphate, pH 7.2, 0.15M sodium chloride Solvent B = 0.1M glycine, 5% acetic acid, pH 2,5.

Standard, sample and reference sample preparation:
The content of IgG is determined by comparison with a purified sheep testis IgG reference standard in the company. The IgG standards are prepared in solvent A at concentrations of 500, 250, 120, 60 and 30 μg / mL. This IgG standard is loaded onto the column at 100 μL per injection.

  Prepare a sample of drug substance by dissolving 10 mg of drug substance in 3.5 mL of solvent A in a 5 mL volumetric flask. The samples are mixed briefly and then incubated at 2-8 ° C for at least 12 hours to completely dissolve. Additional solvent A is added to bring the drug substance sample to a final volume of 5.0 mL and mixed. The sample is filtered through a 0.45 μm syringe filter attached to a 3 mL syringe. All drug substance samples are analyzed with 100 μL of infusate. The IgG content of a given drug substance is weighed 3 times separately and analyzed with n = 2 injections each. Drug substance samples may be stored at 2-8 ° C. for up to 3 days.

  A Vitrase® finished product reference sample is prepared by dissolving the contents of two vials in 0.5 mL of solvent A each. Stir briefly to mix, then let stand for at least 30 minutes to completely dissolve. The contents of the vial are mixed and then filtered as described for the drug substance. Each reference sample formulation is analyzed by injecting two 100 μL volumes. The reference sample formulation can be stored at 2-8 ° C. for up to 3 days.

System equilibration:
The system is equilibrated with solvent A for 20 minutes. That is, equilibrate at a flow rate of 2 mL / min for the first 5 minutes and then at a flow rate of 4 mL / min for 15 minutes. Inject 100 μL of solvent A 4 times to ensure stabilization of the UV signal.

Test method:
Once a stable baseline is established, a standard curve of five IgG standard concentrations is generated with one injection per standard concentration. Subsequently, all drug substance samples are injected and then the reference sample is injected. Use the last pair of injections of the 250 μg / mL (or 25 μg) IgG standard to verify that the retention time and signal amplitude did not change during the chromatographic test.

Calculation:
To determine the concentration of IgG in a sample, first, linear regression analysis of the peak area of the standard curve versus the injected theoretical mass was performed for each standard, and the correlation coefficient (r ² ) value, slope, and y-intercept were calculated. Ask.
Injected IgG mass (μg) = (peak area of sample−section) / gradient IgG concentration (μg / mL) = injected IgG mass (μg) / injection volume (mL).

The protein concentration of the sample is measured with a colorimetric protein assay, and the measured value is used to calculate the IgG content per mg of total protein in the sample.
IgG concentration (μg / mL) / protein concentration (μg / mL) = (μg _IgG / μg _protein ) = IgG content / total sample protein (μg)
If the content is IgG of ≦ 0.23 mg per mg of protein, the requirement is satisfied.

The relative error (%) is calculated by the following formula (using the absolute value of the subtraction).
RE (%) = 100 × [injection 1 (μg) −injection 2 (μg)] / 2
The relative retention time of IgG is calculated by the following formula.
RRT = RT _sample / RT ₂₅₀ μg _{/ mL (or 25} μg _{) standard} .

System suitability:
The results obtained from this assay are unacceptable unless the following chromatographic conditions are met.
The theoretical plate count for a 1.25 μg IgG standard should be ≧ 2300.
2. Peak retention times for IgG must be within 4.17 ± 0.5 minutes and unbound protein must elute at 0.79 ± 0.5 minutes.
3. The baseline shift when switching from load to elution buffer should not exceed 0.005 AU during blank injection.
4. The coefficient of variation (%) for 3 injections of IgG standard of 250 μg / mL (or 25 μg) should be ≦ 10%.

Acceptance criteria:
Data obtained from this test is not acceptable unless it meets the following criteria:
1. The r ² value for the standard curve must be ≧ 0.99.
2. All sample concentrations must fall within the IgG standard curve.
Each injection volume of the 3.25 μg standard should fall within the range of 22.5 to 27.5 μg (± 10%).
4). For any two injections of the same sample, the relative error (%) must be ≦ 5%.
5. The relative retention time of IgG must be within 0.9-1.1 minutes.

    <Weight loss during drying> Weigh a taring bottle with a glass stopper dried at 110 ° C. overnight. A 150 mg ± 10 mg sample of drug substance is placed in the bottle, and then the bottle and its contents are accurately weighed. Place the bottle in a drying chamber, depressurize the drying chamber below 254 mm (10 inches) Hg, remove the lid and leave in the drying chamber and dry at 60 ° C. for 2 hours ± 1 minute. Release the vacuum, open the drying chamber door, and immediately return the lid. The dried container and sample are weighed after cooling to ambient temperature, and then the weight determined by tare measurement is subtracted. Calculate the percentage of the original weight lost by drying. Weight loss due to drying should not exceed 5% of the original tare weight.

《Impurities related to products》
<Quantitative>
<Hyaluronidase content> The hyaluronidase content of a drug substance sample is quantified by electrophoresis. Hyaluronidase is quantified against a standard curve of an in-house hyaluronidase reference standard with a system suitability sample provided by the Vitrase® finished product. Specification: 0.10-0.23 mg hyaluronidase / mg protein.

  The sheep hyaluronidase standard is purified from drug substance by protein G affinity chromatography followed by size exclusion chromatography and concanavalin A affinity chromatography. Batches of reference standards are quantified by testing for purity (SDS-PAGE), identification (Western blotting), protein concentration and amino acid content (amino acid analysis), and with an electrophoretic content assay. Under reducing conditions, sheep testicular hyaluronidase migrates as a pair of distinct bands that differ in apparent mass by approximately 7 kDa. Both forms retain the overall enzyme activity.

Test method:
Hyaluronidase content is assessed on a 12-well 4-20% gradient Tris-Glycine SDS polyacrylamide gel. The drug substance sample was reduced and denatured at a final concentration of 0.1 mg / mL with a buffer solution containing 500 μL of 2-mercaptoethanol per 10 mL of 2X Tris-Glycine SDS sample preparation buffer. Annexin II reference standard standard curves are generated at five concentrations (referred to as Std1-5, respectively) of 0.092, 0.0506, 0.0276, 0.0138 and 0.0092 μg / mL. These solutions were diluted at a ratio of 1: 1 when reduced with 2 × sample preparation buffer (see above). A designated lot of Vitrase® finished product is used as the system suitability standard and is also prepared at 0.1 mg / mL. One sample lane is used for samples with a wide range of reduced molecular weight standards. All samples and standards are loaded with a sample volume of 10 μL per well.

TS = Drug substance test sample SSS = System suitability standard lot Std1-Std5 = Standard curve of in-house Annexin II reference standard MWS = A wide range of molecular weight standards.

  Each gel is electrophoresed at a constant voltage of 120 V for 120 ± 5 minutes or until the bromphenol blue tracking dye reaches the bottom of the gel. When the experiment is complete, the gel is carefully removed from its plastic cassette and then placed in 50 mL of colloidal Coomassie Blue dye solution. The gel continues to stain for 15-17 hours with constant stirring on the gel rocker platform.

  When staining is complete, the gel is destained on a gel rocker platform in deionized water for at least 7 hours using multiple changes of water during the process.

  Following destaining, the gel is quantitatively analyzed with a scanning laser densitometer.

Calculation:
Plot the total peak area of the band concentration against the theoretical mass value of each standard in the standard curve to obtain the r ² value and the formula for that line.

  The total total mass of the hyaluronidase band in each sample is calculated using the peak area of the sample band concentration and the formula from the standard curve.

  The average mass of each set of hyaluronidases consisting of three hyaluronidase samples is calculated.

The average mass of hyaluronidase is expressed as a percentage of total protein according to the following formula:
Hyaluronidase (%) = 100 × [average amount of sample hyaluronidase (μg)] / total protein loaded (μg)
If the content is between 0.10 and 0.23 mg hyaluronidase / mg protein, the sample meets the requirements.

System suitability criteria:
Results obtained from this test are unacceptable if the following criteria are not met:
1. The densitometer must pass its internal calibration test.
2. Each band of hyaluronidase must migrate between the molecular weight standards of phosphorylase b (97.4 kDa) and glutamate dehydrogenase (55.4 kDa) in gel sample lane 1.
3. The average value of the system suitability samples tested three times must be within 2 standard deviations of the historically determined value.

Acceptance criteria:
Data obtained from this test is unacceptable if the following criteria are not met:
1. Each standard curve must consist of at least 5 points.
2. The calculated value of r ² for each standard curve must be ≧ 0.98.
3. The coefficient of variation (%) for each of the three data point sets must not exceed 15%.
4). The average value of the hyaluronidase content of the drug substance sample should fall between 0.092 and 0.32 μg.

    <Total protein> The total protein assay is a colorimetric method based on the binding of Coomassie brilliant blue G-250 to protein in solution. Protein is quantified relative to a standard curve of bovine serum albumin. Specification: 0.55-0.83 mg protein / mg drug substance.

solution:
Acid dye concentrate, standard bovine serum albumin and 0.9% saline are obtained from commercial sources. The dye concentrate is diluted 5: 1 with deionized water and filtered through Whatman- # 1 filter paper before use. This 5-fold diluted dye solution is stable for up to 14 days when stored at 2-8 ° C.

  Approximately 10 mg (± 0.5 mg) drug substance sample is weighed and diluted to approximately 1 mg / mL with 0.9% saline. Such drug substance formulations are allowed to stand for at least 12 hours to ensure that they are completely dissolved before use. The sample is then mixed with gentle stirring and then filtered through a 0.2 μm cellulose acetate membrane. The reconstituted drug substance sample solution may be used for up to 7 days when stored at 2-8 ° C.

Test method:
The stock solution of bovine serum albumin is a 2.0 mg / mL solution. Prepare each dilution with a final concentration of 0.9, 0.7, 0.5, 0.3 and 0.1 mg / mL and a volume of 500 μL in 0.9% saline. The standard is prepared fresh daily. A blank solution consisting only of 0.9% saline is also included. Pipette 100 μL each of all samples and standards into three 13 × 100 mm glass test tubes.

  The assay is started by pipetting 5.0 mL of diluted dye reagent into each tube at 20 second intervals. After each addition, the test tube is capped with a plastic cap and stirred briefly to mix. The assay is incubated for 10 minutes at room temperature. A blank solution of saline is used to perform a blank measurement (calibration) of the spectrophotometer at 595 nm, and these tubes are read directly in the cuvette holder of the spectrophotometer. After incubating for 10 minutes, the samples are read in the same order as when the test was started, ie at the same 20 second interval.

Calculation:
Protein concentration is determined by interpolation from a regression line of absorbance at 595 nm (A ₅₉₅ ) standard curve versus theoretical standard concentration. Since the range reported by this assay is 0.35-0.9 mg / mL, drug substance samples that exceed this range must be re-assayed with a new diluent that is within the standard curve range. .

  Since the drug substance contains proteins (especially IgG fragments) that do not respond to Coomassie blue dye in the same way as BSA, a correction factor of 1.2 is applied to all concentrations obtained in this assay. Apply.

Drug substance sample concentrations are reported in units of mg protein / mg drug substance. This is calculated from the mg / mL concentration according to the following formula:
mg protein / mg drug substance = (PC × 10 mL) / SW,
Where PC is the protein concentration in mg / mL and SW is the weight of the original drug substance sample in mg.

  If the protein content is 0.55-0.83 mg protein / mg drug substance, the sample meets the requirements.

  In addition, the coefficient of variation (% CV or% RSD) is calculated by testing each drug substance in triplicate.

System suitability:
The results obtained from this test are not acceptable unless the following criteria are met:
1. The calibration of the spectrophotometer must be the latest calibration.
2. The baseline reading with a blank must be stable at 0.00 ± 0.01 AU for at least 20 seconds.

Acceptance criteria:
Data obtained from this test is unacceptable if the following criteria are not met:
1. The r ² value for the standard curve must be ≧ 0.98.
2. All samples must fall within the reporting range of this assay, from 0.35 to 0.9 mg / mL.
3. The% CV value of triplicate test values for all drug substance samples should be ≦ 10%.

<Titration test>
The enzyme is incubated with the substrate hyaluronic acid for a period of time, and then the titer is measured by a hyaluronidase activity assay that detects the undegraded substrate by the turbidity of the turbidity generated when reacted with an acid albumin solution. Turbidity is measured at 600 nm with a spectrophotometer. Hyaluronidase activity is simultaneously quantified relative to the USP hyaluronidase standard curve. Specific activity is calculated using values from the above activity and total protein assays. Potency specification: 7.32 × 10 ^{3 to} 1.37 × 10 ⁴ USP units / mg drug. Specific activity specifications: 1.2 × 10 ^{4 to} 1.9 × 10 ⁴ USP units / mg protein.

solution:
A solution of 0.5 mg / mL substrate sodium hyaluronate is prepared with a 0.3 M sodium phosphate solution having a pH of 5.30-5.35. USP hyaluronidase standard, drug substance and reference sample solutions are all 20 mM sodium phosphate pH 6.90 ± 0.05 supplemented with 0.45% sodium chloride and 0.01% bovine serum albumin. Prepare with dilution buffer. The acidic albumin solution is a 24 mM sodium acetate solution containing 0.1% bovine serum albumin and having a pH of 3.75 ± 0.05.

  USP hyaluronidase standard solution is prepared to give a nominal activity of 15 USP-U / mL. For the latest lot of USP hyaluronidase, this means that 45.3 mg of standard is dissolved in 20 mM sodium phosphate buffer in a final volume of 20 mL.

  The drug substance sample is first diluted to about 1 mg / mL with 10 mL volume of 0.9% saline. This initial saline dilution is further diluted to give a final concentration of about 9 USP-U / mL based on the manufacturer's published activity value for the specific drug substance lot to be assayed. To do. This secondary dilution is performed with 20 mM sodium phosphate buffer.

  The reference sample is prepared from a plurality of lots of Vitrase (registered trademark) finished product by the following method. One 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 μm filter needle and returned to its original vial. 125 mL of this solution is diluted with 20 mM sodium phosphate buffer to a final volume of 25 mL to obtain a reference sample.

Test method:
All assays consist of a 10-point standard curve, a reference sample, and a drug substance test sample. The standard curve is 1.5, 3.0, 4.5, 6.0, 7.5, 9.0 in a final volume of 1 mL of 20 mM sodium phosphate buffer placed in a 16 × 125 mm glass test tube. USP hyaluronidase standards are included at concentrations of 10.5, 12.0, 13.5 and 15.0 USP-U / mL. The reference sample consists of 1 mL of secondary diluent collected by pipette into a glass test tube. Similarly, the drug substance sample has a volume of 1 mL and is pipetted into a test tube. Typically, drug substance samples are assayed with n = 2 assay / formulation multiple times for multiple formulations, taking into account the natural variation of the assay procedure. All standards, reference samples and drug substance samples are mixed by briefly stirring, then capped with a plastic lid and then pre-incubated in a circulating bath at 37-38 ° C. for 5 minutes.

  Remove the lid of the test tube, pipet 1 mL of 0.5 mg / mL hyaluronate solution into the test tube, plug the test tube again, stir immediately and place the test tube in the hot water. Return to the bath. Care must be taken to stir the solution at a relatively low speed to avoid foaming. The assay starts 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 assay is cooled by adding 10 mL of acidic albumin solution. That is, remove the stopper of the test tube, add acid albumin, stopper again, and mix slowly by inversion. These assays were cooled exactly in the same order as they were started, then allowed to stand at room temperature for 20 minutes, then at the t = 65 minute time point (for each start time point) on a spectrophotometer. Read at 600 nm. To read at 600 nm, the tube is first slowly inverted and remixed, then the sample is decanted into a disposable polystyrene 4.5 mL cuvette. Samples are read every 30 seconds to continue the temporal lockstep of the process. The spectrophotometer first performs a blank measurement with a cuvette filled with deionized water.

  Note: Throughout this test, the relative timing of each of these events is important. The occurrence of turbidity is non-linear with time, so improper timing results in incorrect or inaccurate activity from mistimed samples.

Calculation:
The standard curve is plotted as a third order polynomial function of the absorbance measurement at USP-U / mL vs. 600 nm (A ₆₀₀ ). The activity values of the reference sample and drug substance sample are then determined by interpolating from the standard curve based on the A ₆₀₀ values of the samples. For simplicity and reproducibility purposes, the analysts set up a spreadsheet activity calculation table, whereby raw A ₆₀₀ data, multinomial coefficient values from standard curve equations, mass values (drug substance It is most convenient to obtain the calculated activity values for these samples from the formulas pre-filled in each fixed cell of the spreadsheet, including the sample dilution factor for the drug substance and reference sample only) You will notice.

If the activity value is in the range of 7.32 × 10 ^{3 to} 1.37 × 10 ⁴ USP units / mg drug substance, the sample meets the titer requirement.

System suitability criteria Results from this test are not acceptable unless the following criteria are met:
1. The calibration of the spectrophotometer must be the latest calibration.
2. The correlation coefficient (r ² value) of the USP standard curve must be ≧ 0.999.
3. The activity value of the reference sample must fall within ± 6% of the historically established value for the lot of finished product.
4). The relative error (%) between repeated assays (eg, reference samples) should be ≦ 6%.

Acceptance criteria Data obtained from this test is not acceptable unless the following criteria are met:
1. The measured concentration in each sample assay must fall within the range of 3.0-13.5 USP units / mL.
2. The relative error (%) between repeated tests should be ≦ 6%.

<Specific activity> The specific activity is calculated as the quotient of the activity and the total protein test result. If the specific activity is between 1.2 × 10 ^{4 and} 1.9 × 10 ⁴ USP units / mg protein, the sample meets the requirements.

    <Bacterial endotoxin> Less than 60 eu per 1 mg of drug substance.

[Method for preparing hyaluronidase]
This disclosure relates to a process for preparing a hyaluronidase formulation suitable for pharmaceutical use. In a preferred embodiment, the process includes using a virus filtration step to increase the safety level of the final product. This process provides a way to increase the purity of currently marketed hyaluronidase formulations. The method is preferably used to purify hyaluronidase from mammalian sources. In another embodiment, the disclosed method can be used to purify recombinant hyaluronidase.

Organization source
Purified formulations of mammalian β-glucuronidase enzyme are preferably prepared from mammalian testis. Examples of preferred mammalian sources are sheep, cows, pigs and horses. However, any mammalian source can be used in the methods described herein. There are currently 4,629 types of currently known mammalian species. A hierarchy of classifications including eyes, families, subfamilies and genera can be found in Wilson, DE and DMReeder (ed.) 1993 “Mammal Species of the World” Smithsonian Insitution Press, 1206 p. (Available from Smithsonian Insitution Press) . This document is incorporated herein by reference.

[Extraction of hyaluronidase]
Typically, the preparatory step for purifying hyaluronidase is the step of isolating testis from a preferred mammalian source. The testis can be processed immediately or preferably frozen for later use. When frozen, the testis must be thawed at 2-8 ° C. for 40-44 hours. The testis is then minced and filtered to extract hyaluronidase. The isolated material is then first precipitated, preferably with a 15% saturated ammonium sulfate solution. The temperature of this buffer is preferably maintained at 2-8 ° C. The pH of this precipitation step is preferably maintained at a pH of 3.60 ± 0.1. The precipitate is preferably filtered in less than 5 hours. After filtration, the material is assayed for hyaluronidase activity and protein concentration, and total units are calculated. The isolate is then subjected to a second salt cut or precipitation operation. Add ammonium sulfate to a level of saturation of about 85% while maintaining the pH of the solution at 3.60 ± 0.1. These processes are described more fully in FIGS.

  Figures 3 and 4 detail the second step in preparing hyaluronidase from mammalian testis. Thaw the product from the purification of purification step 1 (15/85) and then add ammonium sulfate to 35% saturation. The solution is stirred and then filtered. The pH of the filtrate is adjusted to 4.10 ± 0.20 and then a sample is typically taken for quality control analysis. The solution is then placed in 85% saturated ammonium sulfate and stirred. The solution is then filtered and the precipitate (35/85 precipitate) collected and stored.

  Figures 5-8 detail the third step of the purification protocol. The 35/85 precipitate is thawed and resuspended for further processing. The solution is dialyzed against 20 mM potassium phosphate solution. The dialysis solution is then filtered and concentrated. Store the concentrate at 2-8 ° C. overnight. Samples are typically taken for quality control as shown in FIG. The concentrate is then subjected to DEAE sephadex fractionation. Fractions are collected and assayed for hyaluronidase activity as described in FIG. Selected fractions are combined and precipitated with 85% saturated ammonium sulfate. The solution is stirred and then filtered. The precipitate (0/85) is collected, weighed, filtered and then clarified. The solution is precipitated with PEG 6000 to a concentration of 20%. The suspension is centrifuged to collect the precipitate. The precipitate is then resuspended and filtered. Quality control tests are typically performed. The product is dried and stored for subsequent processing.

  The fourth step of the procedure is described in detail in FIGS. Dissolve the product for fractionation on a CM Sephadex column. The sample is injected into the column and the fractions are collected. The active fractions are pooled and precipitated by adding ammonium sulfate to 85% saturation. Collect the precipitate (0/85) and weigh. The sample is dialyzed against 20 mM potassium phosphate. As detailed in FIG. 10, the dialysate is then filtered and its pH adjusted.

  As depicted in FIG. 11, the product is filtered to remove possible viral contamination, lyophilized, and tested.

[Hyaluronidase activity]
Hyaluronidase activity was measured using a turbidity assay. This assay is used to determine the activity of the final product, drug substance (active pharmaceutical ingredient (API)), and intermediate hyaluronidase in the course of manufacturing the API or final product. Hyaluronidase activity is determined using a modification of the turbidity assay described in USP 26 for injectable hyaluronidase. Briefly, the dissolved hyaluronidase enzyme is reacted with the substrate hyaluronic acid for a defined time, then the enzyme is inactivated, and the undegraded hyaluronic acid is then precipitated with an acidic albumin solution. The turbidity of the suspension that has occurred is measured by determining the absorbance at 600 nm using an ultraviolet-visible light spectrophotometer. The activity of the enzyme is inversely proportional to the turbidity of this solution. Quantification is based on comparison of turbidity data obtained from experiments under the same conditions from a primary USP bovine hyaluronidase standard with known enzymatic activity. The hyaluronidase formulation has an activity of 7.32 × 10 ^{3 to} 1.37 × 10 ⁴ USP units / mg. Typically, hyaluronidase formulations have a pH of 5.2 to 7.2.

[Total protein]
The total protein of the hyaluronidase preparation is measured with a widely accepted protein concentration assay. These assays are used to determine manufacturing process intermediates, active pharmaceutical ingredients (APIs) and final product protein concentrations. This assay is based on Bradford protein quantification. This method is a dye binding assay in which the color of the dye changes in proportion to various concentrations of protein. The maximum absorbance of the acidic solution of the dye Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when the dye binds to the protein. Coomassie blue dye naturally binds to amines containing amino acid residues, particularly arginine residues. The color development for an individual protein depends on its amino acid composition. The total protein of the hyaluronidase formulation is 0.55-0.83 mg / mg protein. The specific activity of this formulation is in the range of 1.2 × 10 ^{4 to} 1.9 × 10 ⁴ USP units / mg protein.

[moisture]
Hyaluronidase formulations typically have a water content of ≦ 12% by Karl Fischer analysis. Using this assay, in-process intermediates, active pharmaceutical ingredients (APIs) and final products using Karl Fischer coulometric methods as detailed in USPharmacopia-25. Determine the amount of moisture present. In addition, the whole pharmacopoeia is used here. The Karl Fischer coulometric method is based on the titration of iodine against water and sulfur dioxide in the presence of base and alcohol. When all the water is consumed, excess iodine is generated and detected by the double platinum electrode. The Karl Fischer coulometer calculates and prints the weight percent of moisture in the injected sample. Alternatively, the moisture content of the hyaluronidase formulation is ≦ 5% weight loss upon drying.

[Bacterial endotoxin concentration and microbial limits]
The hyaluronidase formulations disclosed herein have a limiting concentration of bacterial endotoxin. A Limulus amoeba-like cell lysate (LAL) gelation test is used to determine the concentration of bacterial endotoxin in a given sample. The LAL test is based on the observation that bacterial endotoxins react with circulating cell-derived lysates associated with the blood coagulation mechanism of horseshoe crab: Limulus polyphemus. Bacterial endotoxin is present in the hyaluronidase formulation at a concentration of ≦ 60 endotoxin units / mg.

Microbial limits are determined using the assay described in USP61. This test is designed to demonstrate that viable aerobic microorganisms present in the product are free of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa or Salmonella. Total microbial contamination is less than 10 ³ microorganisms / g.

[Quantification of Annexin II and IgG in the final product]
The hyaluronidase formulations used in the methods disclosed herein contain a defined concentration of Annexin II. Annexin II in the final product, active pharmaceutical ingredient (API) and in-process lyophilized intermediate (HYO5A) is quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preferred embodiments of the purified solution typically contain four major protein components (α hyaluronidase, β hyaluronidase, annexin II and 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 an SDS-PAGE gel, scanning the gel with densitometry, and quantifying Annexin II with ImageQuant® densitometry software. Annexin II content determined by SDS-PAGE analysis is 0.29 to 0.57 mg annexin / mg protein.

  The hyaluronidase formulations used in the methods disclosed herein contain a specific level of immunoglobulin. The amount of drug substance (active pharmaceutical ingredient (API)), in-process intermediate, and immunoglobulin (IgG) heavy chain fragment called “IgG” in the final product was measured by high performance liquid chromatography (HPLC). A preferred embodiment is a formulation containing several proteins, lactose and phosphate buffer. The formulation contains hyaluronidase (active ingredient) and two main impurities, Annexin II and IgG. In order to characterize drug products, set product specifications, and perform process control, it is necessary to determine the amounts of various components.

  The HPLC procedure used to quantify IgG is discussed below. This procedure uses an affinity column that performs separation based on the binding affinity of the protein of interest for the ligand bound to the stationary phase. This procedure utilizes protein G as a ligand. While being separated by HPLC, IgG is bound to the stationary phase using a neutral pH buffer and then eluted stepwise with a low pH buffer. An important factor in this elution step is the low pH. In the presence of salt, non-specific binding is reduced. The IgG content determined by HPLC analysis is approximately ≦ 0.23 mg IgG / mg protein.

[Hyaluronidase content is determined by SDS-PAGE analysis]
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is run to quantify the hyaluronidase protein content in the final product, various process intermediates and active pharmaceutical ingredients (API). Preferred embodiments typically contain four major protein components (alpha hyaluronidase, beta hyaluronidase, annexin II and immunoglobulin (IgG) fragments) as well as lactose and other buffer components. SDS-PAGE separates proteins based on their apparent molecular weight. This method describes a procedure for running an SDS-PAGE gel, scanning the gel with densitometry, and quantifying hyaluronidase with ImageQuant® densitometry software. Typically, hyaluronidase formulations contain 0.1-0.23 mg hyaluronidase per mg protein. The content of EDTA is 23 to 33 μg / mg.

[Preferred hyaluronidase preparation]
The hyaluronidase formulations disclosed herein are highly purified formulations that typically contain 0.10 to 0.23 mg of hyaluronidase per mg of protein as measured by SDS-PAGE analysis. This formulation typically has a specific activity in the range of 1.2 × 10 ^{4 to} 1.9 × 10 ⁴ USP units / mg protein and a total protein concentration of 0.55 to 0.83 mg / mg protein. is there. Once administered, it is 7.32 × 10 ^{3 to} 1.37 × 10 ⁴ USP units / mg as measured by turbidity assay, pH is 5.2 to 7.2, and water by Karl-Fischer analysis is ≦ 12% or loss by drying is ≦ 5%. This formulation contains ≦ 60 toxin units of bacterial endotoxin per mg of drug substance. This composition does not contain E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, or Salmonella and total microbial contamination is less than 10 ³ microorganisms per gram protein. Other protein components of this formulation include an annexin content of about 0.29 to 0.57 mg annexin / mg protein as measured by SDS-PAGE analysis, and approximately ≦ 0. There is an IgG content of 23 mg IgG.

[Toxirosal, hyaluronidase (ACS) and hyaluronidase (Wydase®) toxicity to rabbit eyes]
One type of enzyme accelerates the rate at which bleeding blood is removed from the vitreous humor when contacted with the vitreous humor after bleeding in the vitreous humor.

  In this regard, a method is provided for accelerating the rate at which bleeding blood is removed from the vitreous of the eye, which generally eliminates damage from the vitreous without causing damage to the retina and other tissues of the eye. Contacting a quantity of hyaluronidase with the vitreous humor at a dosage sufficient to accelerate clearance of bleeding blood. Preferably, the hyaluronidase is in excess of 1 IU, preferably in excess of 15 IU, and predominantly greater than 75 IU, without thimerosal and without causing toxic damage to the retina and other tissues of the eyeball. The dosage is selected to have a molecular weight distribution that allows administration in the vitreous. This bleeding removal method can also be performed without vitrectomy or other surgical operations or removal of vitreal fluid, thus avoiding possible risks and complications associated with such vitrectomy.

  The preferred route of administration of these bleeding removal enzymes is the route of intraocular injection directly into the vitreous. Instead, however, the bleeding-removing enzyme (s) may be any other suitable route of administration (eg, a local route of administration) that distributes the enzyme well in the vitreous to exert the desired bleeding-removing effect. ) Can also be administered.

  Preferred injectable solutions are those with an inactive ingredient that makes the solution substantially isotonic and the pH is suitable for injection into the eye, at doses exceeding 1 IU without causing toxic damage to the eye. It may contain hyaluronidase having a molecular weight distribution that allows administration into the vitreous, preferably at doses greater than 15 IU, and advantageously at doses greater than 75 IU. This preferred hyaluronidase formulation is preferably devoid of thimerosal. Such injection solutions may be first lyophilized to a dry state and then reconstituted prior to use.

  US Pat. Nos. 6,610,292 and 6,551,590, which are hereby incorporated by reference in their entirety, the term “hyaluronidase (ACS)” is used herein. Fractions containing no thimerosal and having a molecular weight greater than 100,000 as determined by electrophoresis gel (4-20% gradient SDS-PAGE), fractions between 50,000 and 60,000 and 20 It means a hyaluronidase solution for intravitreal injection that does not contain a fraction of less than 1,000. Such hyaluronidases can be extracted from sheep testis and are commercially available from Biozyme Laboratories Limited, San Diego, California, USA, the source of which is for the process disclosed herein for isolating and purifying sheep hyaluronidase. Starting material. This particular molecular weight distribution of hyaluronidase (ACS) is less toxic to the eye than other hyaluronidase formulations and exhibits desirable therapeutic efficacy in many ophthalmic applications.

  As described in the Examples below, hyaluronidase (ACS) is desirable including but not necessarily limited to the removal of intravitreal hemorrhage without causing significant toxicity to the eyeball or related anatomical structures. It can be injected directly into the posterior chamber of the eye at dosage levels that span the therapeutic effect.

  52 healthy New Zealand Cross rabbits (male 26, female 26) weighing 1.2 kg to 2.5 kg were individually housed in a hanging basket with an identifying mark. These animals were given a commercial pelletized rabbit diet daily and tap water without restriction.

  These animals were divided into 13 groups of 4 animals (2 males and 2 females). Two animals in each group (one male and one female) were selected for use in fundus photography and fluorescein angiography before treatment.

  Fundus photography was performed by restraining the animals and then visualizing the optic nerve, retinal arc and fundus with a KOWA® RC-3 Fundus Camera loaded with Kodak Gold 200 ASA film.

  Fluorescein angiography was performed by injecting 1.5 ml of a 2% sterile fluorescein solution via the marginal ear vein. Approximately 30 seconds after injection, fluorescein was visualized to locate the optic nerve, retinal blood vessels and fundus.

  The next day, each animal was anesthetized by intravenous injection of a mixture of 34 mg / kg ketamine hydrochloride and 5 mg / kg xylazine. The eyelids of the animals were retracted using an ophthalmoscope and the eyeballs were disinfected with iodopovidone detergent.

  Any buffered saline solution (BSS), BSS + thimerosal, hyaluronidase (Wydase®) or hyaluronidase (ACS) experimental treatment, 1 cc with a 30 gauge 12.7 mm (0.5 inch) needle attached The injection was made using a tuberculin syringe. The hyaluronidase (ACS) solution utilized in this example did not contain thimerosal and had the specific formulation described in [Table 5].

  The experimental treatments administered to each animal group were as shown in [Table 6].

  On the day after injection (Day 1), 26 animals with fundus and fluorescein angiography were observed using the same method as in the pre-dose experiment.

  On the second day after injection, 13 male animals that had undergone fundus photography and fluorescein angiography on the first day before administration, as well as 13 female animals that were not selected for fundus photography, Euthanized with a drug based on sodium pentobarbital. The eyeball was then surgically removed and placed in a fixative solution obtained by dissolving 2.5% glutaraldehyde in 0.1 M phosphate buffered saline at pH 7.37.

  Separately, a randomly selected rabbit was euthanized with an injection of pentobarbitol, then fixed with a glutaraldehyde solution injected intracardiac into the left ventricle, and the histology in the removed eyeball. The effect of the fixing operation on the findings was confirmed.

  On the seventh day, the same observations were made according to the method described above for 13 female rabbits that had been previously subjected to fundus imaging and angiography imaging.

  The remaining 26 animals were euthanized as described above on the seventh day after dosing. The eyeball was fixed in the same manner as the eyeball fixed on day 2. One randomly selected rabbit was also subjected to the same intracardiac glutaraldehyde fixation procedure as described above for the previously randomly selected animals.

  The eyeballs of animals treated in this example were examined visually and microscopically to find evidence of toxicity associated with the treatment. A table outlining the histological evidence of toxicity or non-toxicity for each treatment group is shown in [Table 1].

  In summary, the control group eyes treated with BBS were not toxic on days 2 and 7 after administration.

  The eyeballs of group 2 animals treated with BBS + thimerosal (0.0075 mg) were not toxic on day 2 but showed evidence that the blood-retinal barrier was destroyed on day 7.

  Group 3 animals treated with BBS + thimerosal (0.0025 mg) showed severe treatment-related toxic effects on days 2 and 7 after dosing.

  1I. U. Group 4 animals treated with different doses of Wydase® were not toxic on days 2 and 7, but 15I. U. ~ 150I. U. Eyes of Group 5-8 animals treated with Wydase® at doses in the range of 2 to 7 days after administration generally showed toxic effects related to administration.

  1I. U. -150I. U. The eyeballs of group 9-13 animals treated with a range of doses of hyaluronidase (ACS) had no evidence of toxic effects on days 2 and 7 after dosing.

  Thus, thimerosal and thimerosal-containing Wydase® formulations cause toxic effects on the rabbit eye at the doses tested, whereas hyaluronidase (ACS) does not have any toxic effects on these animals at the doses tested. It is concluded that it will not happen.

  The results of the inspection conducted on the seventh day are summarized in [Table 1]. As shown in Table 1, BSS + Thimerosal (0.0075 mg) and 1I. U. ~ 150I. U. Significant toxic effects were observed on the eye of rabbits treated with all doses of hyaluronidase (Wydase®) during the 7th day. In contrast, 1I. U. ~ 150I. U. No toxic effects were observed in the eyes of animals treated with hyaluronidase (ACS) in doses ranging from.

[Safety and efficacy of hyaluronidase (ACS) injected into the vitreous of rabbit eyeball]
In this example, twelve healthy New Zealand Cross rabbits were individually housed in a hanging basket with identification marks for identification. These animals were given a commercial pelletized rabbit diet daily and tap water without restriction.

  The animals were randomly divided into 4 treatment groups with 3 animals each.

  First, each animal's eyeball is instilled with 1-2 drops of a 10% tropicamide solution, followed by a visual examination, followed by an inverted fundus examination using a 20 diopter lens, and then in front of the eyeball. The anatomical structure of the part was examined by performing a slit lamp examination.

  Following the initial examination of the animal's eye, 100 μl or 10 μl of blood was injected into the vitreous of each eye of each animal.

  On the second day, animals in each treatment group were injected once intravitreally with BSS or hyaluronidase (ACS) according to the following treatment regime.

  The hyaluronidase (ACS) formulation used in this experiment is a formulation with the preferred formulation described above and shown in [Table 5].

  On days 3, 5, 7, 14, and 21, each animal's eyeball was again examined with a slit lamp to evaluate the cornea, anterior chamber, and iris. In addition, each animal's eyeball was mydriated with 10% tropicamide solution, and the retina was examined by the inverted fundus examination using a 20 diopter lens.

  The observed bleeding removal efficacy of hyaluronidase (ACS) is summarized in [Table 2]. Overall, the left eye of each animal in each treatment group (no treatment) contained a hazy vitreous and several clots due to the volume of blood injected therein. The right eye of group A (control) animals treated with BSS also contained a hazy vitreous and some blood clots, but the right eye of all animals in treatment groups BD treated with hyaluronidase. Had a transparent vitreous and was able to see the retina through the vitreous. Furthermore, the right eye retina of all animals in treatment groups B-D appeared normal and free of treatment-related toxicity.

  The results of this experiment showed that hyaluronidase (ACS) administered into the vitreous was 25-75 I.D. U. A single dose was effective in accelerating the rate at which blood was removed from the eyeball of the treated animal, and the single dose of such hyaluronidase (ACS) performed in this experiment was Show no observable toxic effects on the eyeballs of the rabbits treated in this experiment.

  The observations following each administration are consistent and are summarized in [Table 3]. Overall, the left eye of each animal in each treatment group (no treatment) had a vitreous humor and some clots due to the volume of blood injected into it. The right eye of group A (control) animals treated with BSS was also treated with all animals in treatment groups BE (ie, hyaluronidase (ACS)), although the vitreous was hazy and had some clots. The right eye of (animal) had a clear vitreous body and was able to see the retina through the vitreous body. Furthermore, the right eye retina of all animals in treatment groups B-E appeared normal and free of treatment-related toxicity even after multiple doses of hyaluronidase (ACS).

  The results of this experiment showed that hyaluronidase (ACS) administered into the vitreous was 25-75 I.D. U. That four doses were effective in accelerating the rate at which blood was removed from the rabbit eyeball, and that multiple doses of such hyaluronidase (ACS) could be This shows that even after four consecutive doses of hyaluronidase (ACS), it did not cause any observable toxic effects on the treated rabbit eyeballs.

[Safety and efficacy of hyaluronidase (ACS) injected into the vitreous of human eyeball]
The primary objective of this study was to use a buffered saline solution containing a highly purified hyaluronidase extract from sheep testis tissue in the vitreous body of the visually impaired eye without causing any serious eye damage. It was to check whether injection was possible.

<Materials and methods>
Buffered saline solution (BSS) was taken as a placebo control and was obtained from Allergan Pharmaceuticals (Irvine, CA, USA). This BSS consists of 0.64% sodium chloride, 0.075% potassium chloride, 0.048% calcium chloride dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% acetic acid. Sodium trihydrate, 0.17% sodium citrate dihydrate, sufficient sodium hydroxide / hydrochloric acid to adjust pH to 7.1-7.2 and water for injection (qs .100%). A 30 μl aliquot of BSS or hyaluronidase specific formulation X [Table 7] was filled into a 300 μl microsyringe fitted with a 0.5 inch long 29 gauge needle. The material was injected into the vitreous of the patient's eye using the filled syringe.

  Initially, 8 human subjects with at least one visually impaired eye were randomly assigned to receive hyaluronidase (ACS) 50I. U. 50 μl (3: 1 ratio) of either BSS containing BSS alone or BSS alone was administered to the vitreous. Followed for one month, 50I. U. After confirming that the dose of 2 was sufficiently tolerated, a second group of 6 visually impaired subjects was enrolled in the study to obtain a high dose group of hyaluronidase (ACS) (100 IU. ) Or BSS control group (2: 1 ratio).

  The procedures used to assess the safety of the specimens were completed at various time intervals throughout the study, and included procedures such as inverted fundus examination, fundus photography, fluorescein angiography. There are radiography, electroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, and autorefraction.

  This study included a placebo control group at the same time so that adverse events specifically associated with hyaluronidase (ACS) could be distinguished from adverse events that could be attributed to the vehicle (BSS) / injection procedure. In addition, because the test object was injected adjacent to the retina, it could potentially impair vision as an unfortunate response of critical nature, so only the visually impaired eye was treated. Patients were assigned and treated using a computer generated randomization scheme starting at number 601 as the first phase of the study and starting at number 701 as the second phase. Neither the patient nor the investigator knew at all whether it was a BSS vehicle or a hyaluronidase (ACS) / BSS solution that was injected into the vitreous.

  After confirming each patient's baseline, subjects were injected with either an enzyme or a placebo control. The patient was placed in a comfortable chair. Apply 1-2 drops of local anesthetic locally to the eye to be treated, then let the patient look down and place a sterile cotton swab dipped in the Proparacaine hydrochloride ophthalmic solution onto the sclera approximately 4-5 mm above the cornea In one area (upper / 12:00 meridian) for 10 seconds. The specimen was then injected into the vitreous through a 29 gauge needle attached to a 200 μl microsyringe. The needle was inserted to the full length of the needle at the site where the second anesthetic was applied.

<Test results>
Although statistical significance was rarely obtained, slit lamp biomicroscopy data showed that a fairly high proportion of patients treated with hyaluronidase (ACS) / BSS formulations were treated with BSS alone. In contrast to the patient, it was suggested that the anterior segment showed pathological changes, most notably the presence of cells and flares in the anterior chamber. However, after the sixth visit, one month after treatment, no difference between groups was observed for any variable assessed with slit lamps.

  One patient treated with BSS and 50I. U. The two patients who received 1 hyaluronidase (ACS) / BSS had a retinal / cortical response that decreased with time as measured by electroretinography / visual evoked potential. However, the electroretinogram pattern alternation is always bilateral and also in the treated eyes of patients assigned to high dose (100 IU) hyaluronidase (ACS) / BSS. None of the untreated eyes occurred, and the results of the fluorescein angiography study also indicated that retinal ischemia was not present in any eye regardless of treatment.

  Inversion fundus examination revealed that liquefaction was seen in the subject's eyeball, and posterior vitreal detachment (PVD) occurred. The vitreous was characterized by a high degree of motility and / or melting as soon as the test substance was injected, which is to be expected in the case of hyaluronidase (ACS) containing formulations. there were. One eyeball injected with the BSS control showed melting and PVD, which appeared to be present before treatment. This is because BSS has no enzyme activity and is only administered in a very small amount (30 μl).

  For PVD in the first group of patients, 4 out of 6 patients treated with hyaluronidase (ACS) (ie, # 601, 602, 604 and 606) do not show PVD in slit lamp microscopy (See [Table 8]). After treatment, each of these patients showed the presence of PVD. The test results obtained from the second group of patients were less clear because it was difficult to image the vitreous using slit lamp microscopy.

  In early preclinical studies, hyaluronidase (ACS) was reduced to 150 I.D. U. In view of the test results of Example 2 in which no significant histopathological changes were injected into the rabbit vitreous at various doses up to 150I. U. It was expected that less doses would be well tolerated in humans. Consistent with this expectation, intravitreal administration of hyaluronidase (ACS) / BSS to visually impaired eyes in this study caused few symptoms, all of which were comparable in frequency for each study group. The adverse sequelae associated with the treatment were relatively mild and short-lived.

  Furthermore, when the human eyeball was treated with hyaluronidase (ACS), an increased incidence of observed posterior vitreous detachment was observed. The observed increase in PVD in patients injected with hyaluronidase (ACS) into the vitreous indicates that the method described herein is effective in inducing vitreal fluid melting and detachment. Thus, the results of this study indicate that hyaluronidase (ACS) can be injected into the human vitreous without inducing severe or long-term ocular complications.

[Use of hyaluronidase to accelerate the clearance of bleeding blood from the vitreous of the eyeball]
In the examples described below, a case where hyaluronidase (ACS) is used in the vitreous body to accelerate the clearance (removal rate) of bleeding blood from the vitreous body of the eyeball will be described. The hyaluronidase used was the thimerosal-free hyaluronidase (ACS) formulation described above and shown in [Table 9].

  In this experiment, hyaluronidase (ACS) was 50-200 I.D. to 6 human patients (5 females, 1 male) who had vitreous hemorrhage. U. Intravitreal injection at a dose of

  The hyaluronidase (ACS) administered in this experiment was prepared as described above and shown in [Table 9].

  All patients treated in this experiment had a history of diabetic retinopathy and were known to have different periods of vitreous hemorrhage. In each patient, the amount of blood present in the vitreous was sufficient to prevent standard fundus examination means from seeing the retina.

  Each patient was injected once with hyaluronidase (ACS) into the vitreous. Four patients had 50I. U. One patient received 70I. U. And one patient received 200 I.D. U. Of administration.

  The observation results of this experiment are summarized in [Table 4].

  In the six patients treated in this example, the hemorrhagic vitreous was sufficient to allow the retina to be seen through the vitreous between 6 and 16 days after a single injection of hyaluronidase (ACS) into the vitreous. It became transparent. It was confirmed that this clearing of the vitreous occurred significantly faster than expected in these patients without treatment with hyaluronidase.

  It should be noted that unlike the fluorescein leakage observed when high doses of hyaluronidase (ACS) were administered to rabbits, no toxicity was observed in this human-based study.

[Use of hyaluronidase to treat other ophthalmic disorders]
Hyaluronidase (ACS) is effective at treating some ophthalmic disorders, even at a single dose in the vitreous at a test dose. Patients who have been previously diagnosed with eye disorders including proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular hole, macular exudation, and cystoid macular edema are treated with hyaluronidase ( Treatment with ACS) improved clinical symptoms of these disorders.

  Hyaluronidase (ACS) is 1I. U. Or, at doses exceeding this, it can be administered to the vitreous without causing toxic damage to the eyeball, so that the vitreous is rapidly melted and the vitreous is retina and other tissues (e.g., epiretinal membrane, It can be used to separate or exfoliate from the macula). When the vitreous melts and detaches in this manner, the physical force by which the vitreous pulls the retina and other tissues is minimized, and the natural rotational displacement rate of the liquid in the vitreous is accelerated. Thus, hyaluronidase (ACS) can be used for thawing / separation of the vitreous and / or toxins and other harmful substances from the posterior chamber of the eyeball and / or tissue adjacent to the posterior chamber (eg, retina or macular) (eg, Many disorders (eg proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular hole, macular effusion and ameliorated by accelerated clearance of angiogenic factors, edema fluid, etc.) It is particularly suitable for the treatment of cystoid macular edema). Furthermore, it is believed that the melting of the vitreous removes the matrix in the form of the polymerized vitreous necessary to support angiogenesis. Therefore, this method is useful for preventing or reducing the phenomenon of vascularization in the retina.

  In addition, many ophthalmic disorders have blood-retinal membrane instability as a causal factor. Due to this destabilization, various components of the choroidal capillary layer (for example, serum components, lipids, proteins) enter the vitreous cavity and damage the surface of the retina. This destabilization is a precursor of blood vessel infiltration into the vitreous cavity, known as angiogenesis.

  Accordingly, embodiments of this method are directed to preventing and treating various disorders of the mammalian eye that result in ocular neovascularization or damage to the blood-retinal barrier from ocular damage or lesions. Examples of such diseases include, but are not limited to, proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular hole, macular exudation and cystoid macular edema, and clinical manifestations of these diseases There are other diseases that respond to treatment with hyaluronidase (ACS).

[Treatment of proliferative diabetic retinopathy (PDR) with hyaluronidase]
Diabetic retinopathy is a leading cause of blindness in working-age Americans. The incidence of retinopathy increases with the duration of the diabetic state, increasing from an approximately 50% incidence level in diabetes with a diabetes period of 7 years to an approximately 90% incidence level in diabetes with a diabetes duration of more than 20 years. It is estimated that approximately 700,000 Americans are affected by PDAs.

  The retinal vascular events caused by diabetes include, in part, microvascular leakage and capillary non-perfusion resulting from chronic hyperglycemia. Microvascular leakage then causes retinal edema, lipid leaching and intraretinal hemorrhage. Capillary non-perfusion leads to the formation of microvascular abnormalities (IRMA) in the retina. These abnormalities are arteriovenous shunts formed to perfuse into retinal areas where angiogenesis has been blocked by diabetes-mediated arteriolar degeneration.

  It is believed that extraretinal neovascularization occurs due to the expression of vascular endothelial growth factor from the hypoxic retina in the region of capillary non-perfusion. Such angiogenesis and the associated fibrous components disappear spontaneously or are exacerbated by vitreous hemorrhage or traction retinal detachment. Angiogenesis can be easily seen with fluorescein angiography, as a large amount of dye leaks from these new blood vessels. This is because these new blood vessels lack a solid endothelial connection of the retinal vasculature. Impaired axonal flow in the area of retinal hypoxia results in fluffy vitiligo.

  Proliferative diabetic retinopathy (PDR) has few subjective symptoms at an early stage, so it is necessary to carefully examine diabetes for early confirmation and treatment. Proliferative diabetic retinopathy can be divided into three subgroups: That is, (1) nonproliferative retinopathy, (2) preproliferative retinopathy, and (3) proliferative retinopathy. Each class of disease has specific morphological characteristics. Non-proliferative retinopathy is characterized by capillary microvessel failure (microvascular occlusion and permeability changes, capillary non-perfusion, retinal capillary microaneurysm, basement membrane thickening and internal microvascular abnormalities (IRMA)), bleeding in the retina, exudation, and macular changes. Preproliferative retinopathy is manifested by any or all of the changes described for nonproliferative retinopathy, and the following: marked venous beading, cottony exudates, extensive IRMA, and extensive retinal ischemia It is. Proliferative retinopathy is manifested by extraretinal neovascularization and fibrous tissue growth, vitreous changes and bleeding, macular disease, and retinal detachment.

  Fibrous vascular tissue generation is a particularly significant complication of PDR because it often results in vitreous-mediated retinal damage. This fibrous vascular tissue may form an anterior retinal membrane that forms a tight adhesion with the posterior vitreous membrane. These adhesion parts are the main cause of the possibility of transmitting the traction force of the vitreous body to the retina to cause retinal detachment.

  The bottom of the vitreous is usually firmly attached to the adjacent retina and the outer periphery of the optic disc known as the Martegiani ring. The connection of the vitreous to the retina at all other sites between the Martageani ring and the vitreous base is much less rigid. New blood vessel generation from the retina forms vascular strands that extend into the vitreous from the optic disc or other sites in the fundus. Shrinkage of these strands can cause partial or complete retinal detachment.

  Retinal detachment in the macula is a major complication of PDR. The retinal detachment that occurs from PDR mostly begins as traction detachment without through-holes, but becomes ruptured by the formation of retinal holes at some point later in the disease. This traction detachment occurs due to abnormal adhesion between the vitreous and the retina, or subsequent retraction of the vitreous by contraction of the fibrous band, and retinal elevation.

  This method is intended to treat pre-proliferative and proliferative PDR by injecting hyaluronidase (ACS) into the vitreous. Without being limited to a particular mechanism, intravitreal injection of hyaluronidase (ACS) is believed to promote the clearing of the vitreous fluid phase. The rate of transfer of tritiated water injected into the vitreous from the central vitreous to the choroid was significantly increased after the hyaluronic acid in the vitreous was depolymerized by the injected hyaluronidase (ACS). This method uses this observation to melt the vitreous to promote clearance of various growth-inducing factors and other serum products that leak into the vitreous, for example due to the presence of PDR. It is further contemplated that this method of treatment with hyaluronidase (ACS) may be performed alone or in combination with other treatments of PDR.

[Treatment of nonproliferative diabetic retinopathy]
Objective: To confirm the effect of hyaluronidase (ACS) on the progression of moderately severe to severe nonproliferative diabetic retinopathy (NPDR with or without induced posterior vitreous detachment (PVD)).

  Methods: Sixty patients evaluated by ultrasonography and occlusion fundus photography were analyzed after 4 weeks with saline (0.05 ml), hyaluronidase (ACS) (75 IU, 0.05 ml), SF6 gas. (0.3 ml) or hyaluronidase (ACS) + SF6 gas was randomly assigned. PVD was evaluated throughout 16 weeks and 7-field fundus photographs were obtained after 12 months as surrogate baselines.

  Test results: Of all treated eyeballs, those with stable ETDRS scores, regardless of PVD, had 38% saline (6/16), 67% hyaluronidase (ACS) (10/15), SF6 was 40% (6/15) and hyaluronidase (ACS) + SF6 was 43% (6/14). The ETDRS score was worse for saline 38% (6/16), hyaluronidase (ACS) 13% (2/15), SF6 20% (3/15), and hyaluronidase (ACS) + SF6 21 % (3/14). The proportion of eyes with complete PVD and stable ETDRS score by 16 weeks was 0% (0/14) for saline, 50% (6/12) for hyaluronidase (ACS), 30% for SF6 (3 / 10) and hyaluronidase (ACS) + SF6 was 40% (4/10). Visual loss across all groups was the most frequently reported ocular adverse event.

  Conclusion: Eyes treated with hyaluronidase (ACS) had a stable ETDRS retinopathy score compared to eyes treated with saline. Eyes treated with saline had a stable score with the same number of EDTRS scores worsening. The percentage of eyeballs with complete PVD and stable ETDRS score was highest in the hyaluronidase (ACS) group. These data indicate that induction of PVD and / or enzymatic vitreal melting has properties that affect the progression of diabetic retinopathy.

[Treatment of preproliferative diabetic retinopathy]
In this example, a diabetic patient with preproliferative diabetic retinopathy was treated for this complication of diabetes mellitus by injecting hyaluronidase (ACS) intravitreally. The purpose of this treatment is to reduce or prevent the development of extraretinal neovascularization and fibrous tissue growth, vitreous changes and hemorrhage, macular disease and proliferative diabetic retinopathy as indicated by retinal detachment. is there.

  Once a patient is diagnosed with diabetes, ophthalmic surveillance is monitored in view of the high proportion of individuals affected by the disease that subsequently develop proliferative diabetic retinopathy (PDR). Increase and implement. This increased surveillance should include periodic retinal examination and fluorescein angiography to monitor vitreous beading, IRMA and the extent of retinal ischemia.

  Treatment with hyaluronidase (ACS) begins when preproliferative diabetic retinopathy begins to reach the proliferative stage. This stage may include the presence of venous beading in two or more quadrants (quarter quarter), the presence of IRMA in one or more quadrants, and / or small aneurysms and points in all quadrants. Defined as the presence of hemorrhagic bleeding. Once these signs appear, treatment with hyaluronidase (ACS) is started.

  Patients should undergo a full ophthalmic examination to establish a baseline for eye health. This ophthalmic examination includes inversion fundus examination, slit lamp biomicroscopy, peripheral retina examination, measurement of intraocular pressure, symptomatic examination of visual acuity (naked eye and best correction), fundus photography, fluorescein angiography, There are electroretinography and A-scan measurements.

  Following the preliminary examination, hyaluronidase (ACS) is injected into the vitreous of the affected eye of the patient. If both eyes are affected, they may be treated separately. In the vitreous of the eye to be treated, the 50I. U. Inject 50 μl of hyaluronidase (ACS) ophthalmic solution to promote depolymerization of hyaluronic acid in the vitreous and melt the vitreous.

  After treatment, the patient's eyeball will be examined on days 1, 2, 7, 15, 30, and 60. On each examination day, the patient is monitored for vitreous thawing. In addition, the patient is monitored for posterior vitreous detachment by an inverted fundus examination of the scleral depression. Finally, the patient's degree of PDR is monitored continuously with periodic retinal examination and fluorescein angiography to monitor the degree of vitreous beading, IRMA and retinal ischemia.

[Treatment of proliferative retinopathy]
In this example, a diabetic patient with proliferative diabetic retinopathy is treated by injecting hyaluronidase (ACS) into the vitreous. The purpose of this treatment is to reduce the extent of proliferative diabetic retinopathy, to prevent further development of the disease after removal of angiogenic tissue in the outer retina, and to reduce the likelihood of retinal detachment It is.

  Patients with proliferative diabetic retinopathy will receive hyaluronidase (ACS) therapy in combination with angiogenic tissue surgery. Its growth usually begins with the formation of new blood vessels with very small fibrous components. New blood vessels arise from primitive mesenchymal elements that differentiate into vascular endothelial cells. This newly formed vascular channel then undergoes fibrosis, ie, the angioblastic bud is transformed into fibrous tissue.

  Since neovascular vessels leak fluorescein, the presence of proliferation is particularly noticeable during angiography. New blood vessels and fibrous tissue protrude from the inner limiting membrane and become dendritic at the interface between the inner limiting membrane and the posterior vitreous membrane. The fibrous vascular tissue can form a preretinal membrane that creates a tight adhesion with the posterior vitreous membrane. These adhesion parts are extremely important because they are the main cause of transmitting the traction force of the vitreous body to the retina at a later stage of the vitreous contraction.

  The growth phase of PDR is defined as the presence of three or more of the following characteristics: That is, new blood vessels, new blood vessels on or in one disc diameter of the optic nerve, severe new blood vessels (1/3 disc area angiogenesis in the optic nerve or 1/2 disc area angiogenesis in the optic nerve or As defined by angiogenesis of 1/2 disc area elsewhere), and preretinal or vitreous hemorrhage.

  Once diagnosed as entering the proliferative stage, the patient will undergo a full ophthalmic examination to confirm a baseline eye health. The ophthalmological examination includes inversion fundus examination, slit lamp biomicroscopy, peripheral retina examination, intraocular pressure measurement, symptomatic examination of visual acuity (naked eye and best correction), fundus photography, fluorescein angiography, There are electroretinography and A-scan measurements.

  Following the preliminary examination, hyaluronidase (ACS) is injected into the vitreous of the affected eyeball of the patient. If both eyes are affected, the eyes may be treated separately. In the vitreous body of the eyeball, 50I. U. Is injected with 50 μl of hyaluronidase (ACS) ophthalmic solution to promote depolymerization of hyaluronic acid in the vitreous and melt the vitreous. In addition to vitreous depolymerization, angiogenic tissue is also treated directly using panretinal photocoagulation to minimize subsequent damage to the retina.

  Panretinal photocoagulation (PRP) can be used in combination with hyaluronidase (ACS) therapy to treat patients with PDR. Panretinal photocoagulation is a form of laser photocoagulation. Currently, lasers such as argon green laser (614 nm), argon blue green laser (488 nm and 514 nm), krypton red laser (647 nm), tunable dye laser, diode laser and xenon arc laser are used in retinal surgery. The energy of the laser is significantly absorbed by the tissue containing the pigment (melanin, xanthophyll or hemoglobin) and causes thermal effects on adjacent structures. The krypton red laser is the preferred treatment because it can better penetrate nuclear sclerosing cataracts and vitreous hemorrhage than an argon laser that requires more energy to obtain an equal level of penetration.

  The parameters used in laser retinal surgery may be varied depending on the purpose of the photocoagulation method. A low power laser that provides long spot treatment and produces a large spot size has a coagulation effect on small blood vessels. Focused laser photocoagulation is used in diabetes to stop leakage of small aneurysms. The laser spot is placed directly over the small aneurysm and the aneurysm is slightly whitened and closed. When the laser is applied in a grid over the edema-like area of the retina, the leakage of microvessels can be reduced. At higher energy levels, it is possible to ablate tissue with a laser. Panretinal photocoagulation is believed to be effective by destroying tissue in the eyeball and reducing the amount of ischemic tissue. Abnormal blood vessels can also be removed using a confluent laser spot on the neovascular membrane.

  This method should be understood as not requiring a specific treatment sequence. In one embodiment, the patient is first treated with hyaluronidase (ACS) and then treated with a laser. In another embodiment, the patient is first treated with a laser and then treated with hyaluronidase (ACS).

  Following treatment, the patient's eye will be examined on days 1, 2, 7, 15, 30, and 60. On each examination day, the patient is monitored for vitreous thawing. In addition, the patient is monitored for posterior vitreous detachment using an inverted fundus examination of the scleral depression. Finally, the degree of PDR exhibited by the patient is continuously monitored by periodic retinal examination and fluorescein angiography to monitor the degree of venous beading, IRMA, retinal ischemia, angiogenesis and vitreous hemorrhage. To monitor. If there is a sign of a new neopolymerization or incomplete depolymerization of the vitreous, the patient will be treated repeatedly as described above.

[Treatment of age-related macular degeneration with hyaluronidase (ACS)]
This method is also intended for use in the treatment of age-related macular degeneration (AMD). Age-related macular degeneration is a disease in which visual acuity gradually decreases on both sides. This is the most common cause of legal blindness in adults. It is probably caused by aging of choroidal capillaries or imported retinal blood vessels and vascular disease. There are basically two types of AMD: dry and wet.

  An abnormality underlying AMD is the occurrence of an involutional change in the level of the Brook membrane and retinal pigment epithelium (RPE). A characteristic lesion of such changes is druse. Clinically, drusen (druse) appears as a small pale yellow deposit at the level of RPE. Drusen may be classified as hard-layer drusen, soft-layer drusen or basal layer drusen.

  This method is directed to both the treatment and prevention of wet and dry AMD. In the wet form of the disease, the symptom is thought to affect choroidal capillaries. Choroidal capillaries are choroidal elements that have the function of creating blood vessels in the eyeball. Choroidal capillaries consist of an abundant capillary network that supplies most of the nutrients to the pigment epithelium and the outer layer of the retina. Choroidal capillary damage is thought to ultimately lead to angiogenic complications that cause macular degeneration.

  In the dry form, non-discular macular degeneration results from partial or total occlusion of the underlying choroidal capillaries. Ophthalmoscopic examination can observe retinal pigment epithelium degeneration and pore formation. Also, deposits under the pigment epithelium of substances such as calcium chelate compounds or proteinaceous substances can be observed. In dry AMD, secondary retina changes generally occur gradually and vision is gradually lost. However, some percent of patients lead to severe vision loss.

  This method is intended for use in treating dry AMD and preventing macular degeneration by melting the vitreous. By melting the vitreous, it is intended to increase the rate at which deposits that later become macular degeneration are removed from the retina.

  Wet AMD most frequently results from choroidal capillary dysfunction followed by neovascularization beneath the pigment epithelium. Angiogenesis is also thought to occur by the formation of blood vessels in the retina in response to inadequate oxygen supply caused by damaged blood vessels. Angiogenesis can also cause several other disorders, such as detachment of the pigment epithelium and the sensory retina. Typically, the disease usually begins after age 60, appears equally in both men and women, and appears in both eyes of patients presenting with the disease.

  Perhaps the most serious complication of age-related macular degeneration is a defect in the Brook's membrane of the eyeball where new blood vessels grow. This epithelial neovascularization can result in the formation of exudates in and under the retina. This angiogenesis results in bleeding into the vitreous, which can lead to degeneration of the retinal rods and cones and saccular macular edema (discussed below). A macular hole may be formed that results in irreversible vision loss.

  Although only 10% of patients are affected by AMD, AMD angiogenic complications account for the overwhelming majority of cases of severe vision loss. Risk factors include aging, soft layer drusen, nongeographic atrophy, family history, hyperopia and detachment of the retinal pigment epithelium. Symptoms of choroidal neovascularization in AMD include osteopathia, paracentric dark spots or reduced central vision. Ophthalmoscopic examination findings include subretinal fluid, blood, exudate, RPE detachment, cystic retinal changes or the presence of grey-green angiogenic membrane under the retina. Fluorescein angiography is often an effective diagnostic method. During this diagnostic procedure, the progressive accumulation of pigment in the space below the retina, seen as blurred borders of the lesion or leakage from unidentified sources, is an indicator of the disease. Other elements of the choroidal neovascularization depicted by fluorescein angiography include elevated blocked fluorescenece, flat blocked fluorescence, blood and disc-shaped scars.

  This understanding of angiogenic AMD suggests that classical choroidal neovascularization is the focal component most strongly associated with rapid visual deterioration. Therefore, treatment of AMD must cover all the neovascular and fibrous elements of the lesion. Currently, treatment is only indicated when classical angiogenesis has a clearly demarcated boundary and photocoagulation is found to be beneficial.

  Eyeballs with extrafoveal choroidal neovascularization (≧ 200 μm from the fovea) have a severe visual loss of 5 years ($ 6 line) from 64% to 46 by photocoagulation with an argon laser. %. Usually, within one year after treatment, angiogenesis recurred in half of the eyes treated with laser. When angiogenesis recurred, it was always accompanied by severe visual loss.

  Eyeballs with choroidal neovascularization (1 to 199 μm from the fovea) near the pits reduced the occurrence of severe visual loss from 45% to 31% in the first year by photocoagulation with krypton laser At 5 years, the difference between the untreated group and the treated group was not very clear.

  Although treatment with laser is still an essential treatment for treating AMD, the treatment of this invention will increase the value of laser treatment by reducing the recurrence of angiogenesis.

[Treatment of age-related macular degeneration]
In this example, a patient exhibiting age-related macular degeneration is treated by injecting hyaluronidase (ACS) into the vitreous. The purpose of this treatment is to reduce or prevent the occurrence of angiogenesis, macular disease and retinal damage.

  When the patient reaches 60 years of age, ophthalmic monitoring is increased to detect the presence of AMD. This increased monitoring includes periodic monitoring to monitor subretinal fluid, blood, exudate, RPE detachment, the presence of cystic retinal changes, or the presence of a grey-green angiogenic membrane beneath the retina. Retinal examination and fluorescein angiography must be included.

  Once AMD is diagnosed, hyaluronidase (ACS) therapy is started with or without other therapies such as photocoagulation. As a first step in treatment, patients must undergo a full ophthalmic examination to confirm baseline ocular health. The ophthalmologic examination includes inversion fundus examination, slit lamp biomicroscopy, peripheral retina examination, intraocular pressure measurement, visual (naked or best correction) symptomatic examination, fundus photography, fluorescein angiography, There are electroretinography and A-scan measurements.

  Following the preliminary examination, hyaluronidase (ACS) is injected into the vitreous of the affected eye of a patient with AMD. If both eyes are affected, they may be treated separately. To the vitreous of the eye to be treated, 50I. U. Is injected with 50 μl of hyaluronidase (ACS) ophthalmic solution (described above) to promote depolymerization of hyaluronic acid in the vitreous and melt the vitreous.

  It may be necessary to treat the eyeball injected with hyaluronidase (ACS) with laser photocoagulation. When treating AMD, the laser therapy protocol described in Examples 8 and 9 must be followed. In another embodiment, photocoagulation is performed prior to treatment with the enzyme of the method of the invention.

  After treatment, the patient's eyes will be examined on day 1, day 2, day 7, day 15, day 30 and day 60. Because of the possibility of recurrence, the patient must then have a periodic check again every month. On each examination day, patients are monitored for vitreous thawing. In addition, the patient is monitored for posterior vitreous detachment using an inverted fundus examination of the scleral cavity. Finally, the extent of AMD exhibited by the patient is continuously monitored by periodic retinal examination and fluorescein angiography to determine subretinal fluid, blood, exudate, RPE detachment, and cystic retinal changes. Monitor for presence or presence of grey-green angiogenic membrane under the retina. When signs of revascularization are observed, additional hyaluronidase (ACS) and / or laser treatment may be necessary.

  The following examples demonstrate the effectiveness of this method without using a photocoagulation method.

[Improvement of macular degeneration symptoms after intravitreal injection of hyaluronidase]
One male person over 79 years of age appeared as a patient, had a history of age-related macular degeneration, and the naked eye vision of the right eye was 20: 400. The vitreous body of the right eye of this patient was given 100I. U. Of hyaluronidase (ACS). The left eye remained untreated.

  When this patient was examined repeatedly after administration, the visual acuity of the left eye (untreated) did not change, but it was observed that the visual acuity of the right eye (treated) was improved as follows.

  In this experiment, no adverse effects of hyaluronidase (ACS) were observed.

[Treatment of amblyopia with hyaluronidase]
The term “amblyopia” is a Greek term and means weak vision (amblys = weak, ops = eye). Inferior visual acuity is caused by abnormal development of the visual cortex of the brain, and this abnormal development is due to abnormal visual stimulation during the early stages of visual development. The pathology associated with amblyopia is not unique to the eyeball, but rather exists in the visual cortex of the brain, including the lateral knee nucleus and the wired cortex. This abnormal development occurs by the following three mechanisms. That is, (1) blurred retinal image called pattern distortion, (2) cortical suppression, or (3) cortical suppression + pattern distortion. The method of the invention is primarily concerned with pattern distortions that occur due to turbidity of the medium. More specifically, the method of the present invention addresses the problem of vitreous opacity.

  Amblyopia is usually defined as the difference between at least two Snellen lines of vision. Critical and important for treating amblyopia are early detection and early intervention. A strategy for treating amblyopia caused by vitreous opacity is to provide a clear retinal image by changing the vitreous opacity so that clear vision is obtained.

  In this example, patients with amblyopia due to vitreous opacity were treated by injecting hyaluronidase (ACS) into the vitreous. The purpose of this treatment was to reduce vitreous turbidity by increasing the exchange rate of liquid in the vitreous.

  A 40-year-old female human with a history of one amblyopia appeared as a patient with a right eye naked eye vision of 20: 400 and a corrected eye vision of 20: 200. In the vitreous body of her right eye, 100 I.D. U. A single dose of hyaluronidase (ACS) was administered. The other eye remained untreated.

  When this patient was examined repeatedly after administration, it was observed that the vision of the left eye (untreated) remained unchanged, but that of the right eye (treated) was improved as follows: .

  No adverse effects of hyaluronidase (ACS) were observed in this patient.

[Treatment of retinitis pigmentosa with hyaluronidase]
Retinitis pigmentosa (RP) is the name given to a group of inherited disorders of progressive retinal degeneration characterized by bilateral night blindness with constricted visual field and electroretinogram abnormalities. Early symptoms include difficulty in dark adaptation and loss of central retinal visual field. As the disease progresses, vision loss progresses, typically leaving the central vision small and eventually affecting the central vision. Early in the course of the disease, central vision is also affected by the development of cystoid macular edema, macular atrophy, or posterior subcapsular cataract. RP represents a variant disease group that is commonly characterized by the abnormal production of at least one protein in the outer segment of the photoreceptor important for photoconversion.

  One clinical result of RP is the destabilization of the blood-retinal barrier and the optic nerve head of the peri-focal capillary. This destabilization causes leakage of the fluorescein dye, which is observed with angiography. In addition to this leakage, liquid accumulation may occur and be observed as small cysts in the outer epidermal layer. Cysts filled with these fluids may eventually rupture and damage the retinal layer. The method of the invention is intended to treat RP-related retinal damage by accelerating the removal of tissue fluid that accumulates in microcysts.

  A 59-year-old male human appeared as a patient and had a history of retinitis pigmentosa. His left eye had a naked eye vision of 20: 400 and a corrected vision of 20: 400. The patient's left eye vitreous had 100 I.D. U. Of hyaluronidase (ACS). The other eye remained untreated.

  When this patient was repeatedly examined after administration of hyaluronidase (ACS), the visual acuity of the patient's right eye (untreated) remained unchanged, but the visual acuity of the patient's left eye (treated) was as follows: An improvement was observed.

  The test results demonstrate that there was a significant improvement in the subject's visual ability, with naked eye vision (from 20: 400 to 20:80) and best corrected vision (from 20: 400 to 20:40). There were improvements on both. In addition, changes in the intraocular pressure of the subjects were observed during the treatment period, and the intraocular pressure appeared to return to the baseline level about 2 weeks after the injection. These test results were obtained from a single patient, but seem to be expected to fully warrant future trials.

[Treatment of macular holes with hyaluronidase]
The macular rupture or explosive opening is known as the macular hole. Interestingly, this symptom usually occurs in women in their 50s to 70s or after trauma such as flash injury, sunlight damage, scleral twist, etc. Or in the eyes of uveoma. Symptoms include osteopathia and decreased vision.

  The formation of macular holes is thought to be due to tangential traction across the retinal surface induced by posterior cortical vitreous, involving the movement of fluid in the synergetic cavity of the posterior vitreous. It has been. A posterior vitreous syneresis cavity is present in most patients with macular holes. This is probably because when the posterior vitreous gel retracts from the retinal surface, the movement of the vitreous humor has a negative interaction with the retinal surface in the region of the gap formed between the two surfaces. It is thought that the tangential movement of the vitreous humor in the plug space of the posterior vitreous body promotes tearing of the retina and leads to the formation of a macular hole.

  The method of the invention contemplates using hyaluronidase (ACS) to depolymerize the vitreous so as to eliminate symptoms that result in the formation of macular holes. When the vitreous is depolymerized, the plug-like cavities of the posterior vitreous are eliminated as a result of hyaluronidase (ACS) -mediated reconstitution of the vitreous. This elimination of the cavity allows the fluid between the vitreous and the retina to move freely around the vitreous cavity, and any harmful forces that would otherwise be directed against the retina surface. Also dissipate.

  Patients who show early signs of macular hole formation are treated by injecting hyaluronidase (ACS) into the vitreous. The patient to be treated shows various signs before the macular hole is formed. These signs include the loss of a foveal depression involving a yellow foveal spot or ring. In the fovea, the area where the macular hole is formed begins to thin, and the lesion becomes reddish. Fluorescein angiography results at this stage may appear normal or show weak hyperfluorescence. The appearance of an eccentric, thick enough dehiscence means that the early stages of the disease have progressed further. Treatment with hyaluronidase (ACS) begins when these signs are observed.

  Treatment of the method of the invention with hyaluronidase (ACS) begins when it is diagnosed that a macular hole has formed. The patient will undergo a full ophthalmic examination to confirm the baseline eye health. The ophthalmologic examination includes inversion fundus examination, slit lamp biomicroscopy, peripheral retina examination, intraocular pressure measurement, symptomatic examination of visual acuity (naked eye or best correction), fundus photography, fluorescein angiography, There are electroretinography and A-scan measurements.

  Following the preliminary examination, hyaluronidase (ACS) is injected into the vitreous of the affected eye of the patient. If both eyes are affected, they may be treated separately. For the eye to be treated, the 50I. U. 50 μl of hyaluronidase (ACS) ophthalmic solution is injected into the vitreous to promote depolymerization of the hyaluronic acid in the vitreous and melt the vitreous.

  After treatment, the patient's eyes will be examined on the 1st, 2nd, 7th, 15th, 30th and 60th days. On each examination day, patients are monitored for vitreous thawing. Fluorescein angiography is also performed, which is considered to be a particularly effective way to monitor the course of treatment. In addition, the patient is monitored for posterior vitreous detachment using an inverted fundus examination of the scleral cavity.

[Treatment of macular exudation with hyaluronidase]
Macular exudate is a substance that passes through the blood-retinal barrier, passes through the macula, and penetrates into the vitreous chamber. There are two types of soft exudate and hard exudate. This soft exudate is not actually exudate, but a cluster of ganglion cell axons in a spherically expanded nerve fiber layer at the site of ischemic injury or infarct. Hard exudate usually exudes due to changes in the underlying retinopathy microvessels. Hard exudates are yellow and waxy and often accumulate in a circle around the macula. Hard exudates consist of lipids and proteinaceous substances derived from the exudation of serum components from leaking blood vessels or from the lipid products of degenerating neural elements in the retina. Adsorption of hard exudate is primarily mediated by macrophage reabsorption, but the process appears to be slow because exudation often occurs in the outer plexiform layer in areas without retinal blood vessels. The method of this invention is particularly useful in reducing the extent of exudate accumulation resulting from retinal destabilization. This is because depolymerization of the vitreous with hyaluronidase (ACS) increases the turnover rate of the aqueous component of the vitreous.

  Patients presenting with macular exudate are treated with a therapy that injects hyaluronidase (ACS). The patient will undergo a full ophthalmic examination to confirm the baseline eye health. The ophthalmologic examination includes inversion fundus examination, slit lamp biomicroscopy, peripheral retina examination, intraocular pressure measurement, symptomatic examination of visual acuity (naked eye or best correction), fundus photography, fluorescein angiography, There are electroretinography and A-scan measurements.

  Following the preliminary examination, hyaluronidase (ACS) is injected into the vitreous of the affected eye of the patient. If both eyes are affected, they may be treated separately. For the eye to be treated, the 50I. U. 50 μl of hyaluronidase (ACS) ophthalmic solution is injected into the vitreous to promote depolymerization of the hyaluronic acid in the vitreous and melt the vitreous.

  After treatment, the patient's eyes will be examined on the 1st, 2nd, 7th, 15th, 30th and 60th days. On each examination day, patients are monitored for vitreous thawing. Fluorescein angiography is also performed, which is considered to be a particularly effective way to monitor the course of treatment. In addition, the patient is monitored for posterior vitreous detachment using an inverted fundus examination of the scleral cavity.

[Treatment of Cystoid Macular Edema]
Cystoid macular edema is a common ocular abnormality that arises from various groups of etiology. The majority of this symptom stems from damage to the blood-retinal barrier and the optic papilla of the capillary around the fovea, resulting in leaking fluid that accumulates in the microcysts of the outer reticular layer. This region is in a relatively thin region of the retina and less angiogenesis. Clinically, cystoid macular edema exhibits a honeycomb-type appearance when examined with fluorescein angiography. As this edema progresses, the outer reticulated layer ruptures, creating a layered through hole. This through hole may be confined to the inner layer of the retina or may eventually progress to a complete macular hole.

  The method of the present invention is intended to treat cystoid macular edema to prevent the formation of macular holes by depolymerizing the vitreous with hyaluronidase (ACS).

  Patients showing signs of cystoid macular edema are treated by injecting hyaluronidase (ACS) into the vitreous as described in Examples 13 and 14.

[Other pharmacological uses of hyaluronidase]
Hyaluronidase has been used therapeutically for many years. The various actions that have been demonstrated mainly occur in connective tissues between cells, and it is thought that the main cause is degradation of hyaluronic acid in the tissues. The therapeutically useful consequences of this effect, namely the decrease in intercellular cementum viscosity and increased membrane and blood vessel permeability, are based on its “diffusion effect”. The effect of hyaluronidase to increase permeability after administration of a fluid and / or radiopaque medium is therapeutically significant.

[Hyaluronidase-its diffusion effect]:
Reduces intercellular cementum viscosity Increases membrane and vascular permeability → Injection through intradermal, subcutaneous, intradermal, intravenous or intramuscular route enhances spatial processes Cause temporary acceleration.
[Results]: Improves the diffusion, penetration and penetration of solutions, suspensions and emulsions into surrounding tissues.

  Hyaluronidase can be used to inject drugs (antibiotics, cytostatics, local anesthetics, chemotherapeutics, antivirals, etc.) even when large volumes are administered in the form of solutions, suspensions or emulsions. ) To accelerate and enhance absorption by tissues.

  Hyaluronidase has been used successfully for many years in orthopedics, surgery, ophthalmology, internal medicine, oncology, gynecology, dermatology and the like.

  The experimental use of hyaluronidase has been tested in a number of medical fields. This substance has been clinically administered for various indications and treatments.

  [Ophthalmology / periocular anesthesia and post-ocular anesthesia]: Ophthalmology is currently an important and well-reported field of various indications for hyaluronidase (Farr C. et al., 1997, Wien Med Wschr, 147: 1-8).

  Hyaluronic acid is often applied during ophthalmic surgery (eg, during cataract surgery), eg, to keep the anterior chamber of the eyeball intact or to protect the corneal endothelium during lens implantation . This results in an increase in intraocular pressure.

  The measurement results show that introduction of hyaluronidase into the anterior chamber of the eyeball can effectively reduce intraocular pressure after surgery.

  Hyaluronidase has also been shown to be effective in reducing intraocular pressure in patients who have undergone trabeculectomy to treat wide-angle glaucoma (a dose of 300 IU was administered by subconjunctival injection). The authors concluded that hyaluronidase reduces the number of complications and improves the prognosis of trabeculectomy. Hyaluronidase is also useful for post-ocular and periocular anesthesia during cataract surgery when used in combination with local anesthetics (with or without adrenaline) such as lidocaine and bupivacaine. The effect of hyaluronidase on local anesthesia of the eye was long reported in 1949. Subsequent studies investigated the combined effects of procaine + adrenaline + hyaluronidase on the patient's post-ocular anesthesia.

  An improvement in eye muscle ataxia was observed by subjective assessment. The improvement of the anesthetic effect by hyaluronidase is because the orbit is the optimal injection site.

  In a double-blind comparative study of bupivacaine versus lidocaine with and without hyaluronidase in groups of 50 patients each, researchers found a significantly larger number of fully anesthetized patients in the group with hyaluronidase. Observed. It has also been found that none of these patients require additional anesthetics because the induced muscle blockage persisted throughout the surgery. These findings were clearly confirmed in subsequent tests.

  The onset of the effect of local anesthesia combined with hyaluronidase was studied in 1990. Researchers have found that in all patients injected with an enzyme supplement with an anesthetic, the anesthetic effect required for surgery occurred within 5 minutes after the injection. In contrast, the control group required an additional dose of anesthetic and the onset of the anesthetic effect started later.

  The addition of hyaluronidase to local anesthesia improved the surgical conditions. Hyaluronidase has been found to be very effective when added to lidocaine and noradrenaline for retrobulbar anesthesia in senile cataract surgery.

  In one clinical trial, 75I. U. Of hyaluronidase in 0.75% bupivacaine and 2% mepivacaine resulted in improved ocular blockade, analgesia and hypotonia.

  Studies using hyaluronidase in strabismus demonstrated that sufficient anesthetic effects of mepivacaine and lidocaine can be achieved when supplemented with hyaluronidase. Even small volumes of hyaluronidase showed the desired diffusion effect.

  The findings regarding post-ocular anesthesia generally apply to periocular and subconjunctival anesthesia. When hyaluronidase is used in combination with local anesthesia, reliable ocular muscle blockage is achieved and surgical conditions are improved.

  The effect of hyaluronidase on local anesthesia was tested again in a double-blind study with another promising random control. This test also showed that the addition of hyaluronidase provided additional effects.

  These results were confirmed when 80 senile cataract patients were administered 150 IU hyaluronidase as an adjunct to anesthesia. Their results were confirmed at a later date in another group of 70 patients who received 250 IU hyaluronidase.

  Researchers have demonstrated that intraocular circulation changes during the combined use of lidocaine / bupivacaine and hyaluronidase. They detected that the pulsatile volume of the eyeball was significantly reduced and the intraocular pressure was significantly reduced.

  Local anesthesia with 150 IU hyaluronidase has proven to be a reliable option for cataract surgery.

  For periocular anesthesia, the researchers recommended warming a mixture of 2% lidocaine, 0.5% bupivacaine and 1550 IU hyaluronidase to body temperature to eliminate pain in the area of interest. In another study, two concentrations of hyaluronidase were used as adjuncts to periocular anesthesia. Three groups were administered 0 IU, 50 IU or 150 IU hyaluronidase adjuvant to assess onset of anesthetic action, analgesia and ataxia. No statistically significant difference was detected between the 50 IU and 150 IU hyaluronidase groups. The authors concluded that adding hyaluronidase does not cause any complications.

  Other authors found that adding hyaluronidase is useful in both cases after comparing the results of periocular and post-ocular anesthesia testing, hyaluronidase causes the vitreous to protrude into the posterior chamber of the eyeball It was concluded that it is extremely effective in preventing and significantly reduces the occurrence of vis a tergo.

  Hyaluronidase allows lidocaine and bupivacaine to diffuse more rapidly into the periocular space.

  The injection pressure of local anesthetic administered prior to performing cataract surgery was measured in a double-blind test method in 50 patients. The study concluded that significant (sufficient) ataxia of the extraocular muscles could be achieved with administration of 1% etidocaine, 0.5% bupivacaine and 50 IU hyaluronidase.

  [Glaucoma]: Hyaluronidase is effective in treating glaucoma or improving intraocular pressure.

  [Combination with local anesthetic]: When hyaluronidase was used in combination with a local anesthetic such as procaine or lidocaine, the onset of anesthetic action was faster, the analgesic area was enlarged, and pain after surgery was significantly reduced.

  In one study, it was found that the area of anesthesia after subcutaneous injection with a combination of procaine and hyaluronidase was almost twice that of procaine alone. This study also demonstrated that the duration of anesthesia after administration of a combination of hyaluronidase and procaine / adrenaline is 6 times longer than the anesthetic effect.

  The effect of bupivacaine plus adrenaline on axillary brachial plexus blockade with or without the addition of hyaluronidase was compared in a double-blind manner. Hyaluronidase did not affect the time of anesthesia, the occurrence of inappropriate blockage of this plexus, or the plasma concentration of bupivacaine. This study demonstrated that the duration of anesthesia was shortened.

  [Orthopedics, supportive gait disorders]: For many years, hyaluronidase has been associated with various diseases of supportive gait organs such as synovial sheath, surrounding connective tissue and various inflammations in these areas Acute symptoms such as paratendinitis crepitans, humeroscapular periarthritis, humeral epicondylitis, tibial condylitis, radial styloititis, etc. Has been used successfully to treat. If treatment with hyaluronidase is started as soon as possible, good treatment results can usually be achieved (especially in conjunction with exercise or physical therapy) even if the affected limb cannot be fixed. Researchers succeeded in treating patients with acute tendovaginitis crepitans in 1968 with hyaluronidase.

 Combination therapy has been reported injecting hyaluronidase (300 IU) and vitamin B12 to treat peri-tendonitis and brachio-scapuloarthritis. The efficacy of hyaluronidase in treating external epicondylitis and tendonitis was tested in a total of 53 patients. Daily intravenous injections of 1500-3000 units of hyaluronidase reduced symptoms or clearly excluded complaints.

  Findings from other trials have shown that the same treatments for peritonitis, extrahumeral epicondylitis, scapuloscapuloarthritis and calcaneal inflammation of the radius and ulna were successful, Were administered hyaluronidase 3000 IU every 2 days.

  Hyaluronidase has been successfully used in combination with mefenesin to treat arthropathy primarily in patients with knee arthropathy. The success rate in chronic cases was particularly impressive.

  The finding that hyaluronidase was administered intravenously to patients with Bechteref's disease, the effect continued for a long time, is very therapeutically important. When an average of 1500-9000 IU hyaluronidase was administered intravenously every 3 days, the mobility of the test patient increased.

  With regard to its mode of action, researchers believe that hyaluronidase depolymerizes mucopolysaccharides that accumulate in the connective tissue matrix during the process of calcium deposition outside the bone that occurs due to increased mesenchymal metabolism. If calcium salts are present, they can be depolymerized as long as calcium is not present as tricalcium phosphate. This can suppress extra-bone calcium deposition. Improvements in flexibility (eg, of affected spinal segments) may be due to depolymerization presumed to result from loosening of the previously formed connecting structure. Hyaluronidase is thought to be able to affect the composition and structure of the dermis and promote the resynthesis of proteoglycans. In the treatment of joint stiffness, which often occurs as a complication of epicondylar fractures, administration of hyaluronidase first or simultaneously can release the patient from symptoms more quickly.

  [Treatment of malignant diseases]: When used as an adjunct to chemotherapy for malignant tumors, hyaluronidase can lyse hyaluronic acid-containing regions around tumor cells and tumor cell aggregates, so that higher concentrations of cytostatic agents Efficacy can be exerted in the desired target area. In addition, hyaluronidases may be associated with enhanced immune defense processes, for example, by contacting immune competent cells (“natural killer cells”) and antigen directly on the tumor surface.

  Hyaluronidase (intravenous, intramuscular and intravesical administration) for the treatment of malignant diseases (systemic blood disease, breast cancer, brain metastases, glioma, squamous cell carcinoma of the ENT region, lung and colon adenocarcinoma and bladder cancer) ) Is a challenge for various clinical trials. In sporadic cases, hyaluronidase was found to increase its response rate to cytostatic agents when administered at high doses prior to administering cytostatic agents. When hyaluronidase is used as an adjunct to a patient suffering from a malignant blood disease and the treatment effect is difficult to improve, the patient's response to chemotherapy can be improved.

  Oncology has demonstrated that hyaluronidase is particularly useful when administered as an adjunct to cytostatics such as doxorubicin and adriamycin. This enzyme improves the penetration of doxorubicin into the cells, and increases the activity of adriamycin in breast cancer.

  Combined administration of cisplatin, vindesine, hyaluronidase and radiation therapy to treat advanced squamous cell carcinoma of the head and neck has been found to be very effective. That is, rapid remission was seen in cancer patients, and improved tolerance of cytostatic therapy was observed.

  Hyaluronidase reduced adhesion-related multicellular drug resistance in breast cancer. This mechanism of action is based on a decrease in growth inhibition dependent on cell contact and sensitization of cells to cytostatic agents.

[Hyaluronidase in treating tumors]:
Hyaluronidase is used to treat malignant diseases such as systemic blood diseases, breast cancer, brain metastases, glioma, squamous cell carcinoma of the ear, nose and throat area, lung and colon adenocarcinoma and bladder cancer Increases the effects of cytostatics ・ Clinical studies have found that hyaluronidase stops (remits) the growth of various tumors. ・ Hyaluronidase is instilled into the bladder before administering cytostatics. Then, patients who are difficult to improve the therapeutic response respond well to cytostatics • Hyaluronidase can improve the health of subjects and the quality of life of tumor patients.

  [Dermatology]: Researchers have found that hyaluronidase is, for example, certain types of progressive scleroderma, a systemic disorder of the entire vascular connective tissue system where the most important feature is the displacement of collagen fragments. It is concluded that it is useful for dermatological diseases. What is important as a pathological mechanism is that the histomorphological skin changes that occur in scleroderma begin with cutaneous edema that is rich in acidic mucopolysaccharides (hyaluronic acid, chondroitin sulfate). Histopathological and chemical tests show that part of the matrix is produced as cement in collagen fibers. Therefore, acidic mucopolysaccharides, soluble collagen and polymer collagen are considered to be the culprits of scleroderma.

  Researchers have released further findings on how to administer high doses of hyaluronidase intravenously to patients affected by primary diseases such as keloids and localized scleroderma. Hyaluronidase achieved good results for both diseases. The same is true for progressive scleroderma, but the increase in joint operational flexibility was not permanent. Therefore, intravenous administration of high doses of hyaluronidase to treat progressive scleroderma was only recommended as an adjunct because no effect on older areas of sclerosis could be demonstrated. When the chosen route of administration was evaluated, treatment with topical administration of hyaluronidase to the affected area of the skin was not successful. Intravenous injection or infusion only improved symptoms at a minimum in most patients.

  Treatment of myocardial infarction: The use of hyaluronidase to treat acute myocardial infarction was first described in 1959. Research papers show that administration of hyaluronidase early in the acute phase of new myocardial infarction (2-4 hours after the onset of infarction) can reduce the size of the necrotic area in the heart.

  Researchers have tested agents that reduce infarct size, such as beta blockers, nitrates, calcium antagonists, and the like. Hyaluronidase has been found to have a favorable effect on thrombolytic drugs such as streptokinase administered together. This appears to be due to the ability of hyaluronidase to trap oxygen radicals.

  Statistical experts conducted a meta-analysis to study the role of hyaluronidase and other cardiovascular agents that have the potential to reduce myocardial infarct size.

  Statistical experts have found that the adjuvant hyaluronidase reduced mortality by 15-20%.

  Researchers also recommended the use of hyaluronidase in addition to the traditional drugs used to treat acute myocardial infarction (nitrates, beta-receptor blockers, calcium antagonists).

  It appears that the use of hyaluronidase to treat myocardial infarction was finally proved and confirmed, despite the differences in data and, in some cases, conflicting findings.

  Various indications: Another indication is the treatment of submucosal fibrosis. Over 10 years of experience with 150 patients, it was found that the combination of hyaluronidase and dexamethasone can reduce symptoms over a long period of time in most cases.

  It has been reported that local treatment with chymotrypsin, hyaluronidase and dexamethasone also gives excellent therapeutic results. However, surgical excision was performed for patients who were difficult to improve.

  326 patients affected by intraoral submucosal fibrosis were randomly divided into two groups and treated with either steroids and hyaluronidase or topical vitamin A steroids and oral iron formulations. Treatment with steroids and hyaluronidase has proven to be less problematic.

  Hyaluronidase is also used in hair transplantation and scalp treatment and cosmetic surgery. Hyaluronidase has been found to be effective in improving local anesthetic diffusion.

  The use of hyaluronidase to prevent post-surgical adhesion after surgery has also been reported.

  In the case of brain abscesses, hyaluronidase was used in combination with dexamethasone or antibiotics to remove primarily edema in high-risk patients.

  Hyaluronidase has been found to improve absorption of locally administered drugs and reduce the risk of developing skin necrosis in patients being treated with intravenous vinca alkaloid injections.

  The use of hyaluronidase as an antidote to chemotherapeutic agents that migrated out of the blood vessel is also described. Hyaluronidase is one of a few antidotes that can be used as an antidote to epipodophyllotoxins such as vinca alkaloids or etoposide.

Gynecology is another area of application of hyaluronidase. Hyaluronidase, when injected into the perineum before the parturition stage of labor, has been found to soften the maternal rigidity of the first-born mother and often eliminate the need for perineal incision. It is also useful for facilitating the partial and complete aspiration of exudates and pleural exudates, ie hyaluronidase melts these exudates. Hyaluronidase is also used to treat edema of various origins and to treat joint changes in arthritis.

  Hyaluronidase is a therapeutic agent for keratoplasty, corneal scarring, opacification and haze, and the cornea that needs to be delaminated.

  Hyaluronidase can be used as an alternative or accessory to conventional mechanical vitrectomy.

  Hyaluronidase is also useful for causing retinal detachment.

  Hyaluronidase has been indicated as an adjunct to enhance the absorption and dispersion of other drugs injected for subcutaneous injection and as an adjunct to subcutaneous urography to improve absorption of radiopaque agents.

  [Summary]: The proven diverse activities of hyaluronidase occur mainly in connective tissue between cells. This effect is apparently due to the degradation reaction of hyaluronic acid in the tissue. The therapeutically useful consequences of this effect (decrease in intercellular cement viscosity, increase in membrane and blood vessel permeability) are caused by “diffusion”.

  The increased diffusion and increased permeability associated with hyaluronidase that occurs after administration of solutions and / or radiopaque vehicles has important therapeutic implications. Hyaluronidase accelerates the absorption of these drugs by tissues even when large volumes of drugs (antibiotics, cytostatics, local anesthetics, etc.) are injected in the form of solutions, suspensions or emulsions Can be increased. In cases where it is necessary to start working quickly, and certain drugs cannot be administered intravenously, hyaluronidase can be “pre-injected” into the subcutaneous or intramuscular dose of the drug into the bloodstream. Can be accelerated (200-300%), which is particularly important for internal medicine.

  The efficacy of hyaluronidase in treating support / walking system disorders is due to the so-called “softening action” of hyaluronidase.

  Hyaluronidase, when administered early, particularly in combination with additional exercise and / or physical therapy, causes acute symptoms that affect the synovial sheath and surrounding connective tissue due to its “anti-inflammatory” action (ciliac peritonitis, upper arm) Scapuloperiarthritis, extrahumeral epicondylitis, tibial condylaritis, calcaneal palsy, etc.)

  Joint stiffness (eg, due to epicondylar fractures) can also be successfully treated.

  Hyaluronidase, due to its ability to diffuse, can be used to treat post-traumatic hematoma or edema of any origin and can also be used to melt joint and pleural exudates in orthopedics.

  It has been pointed out that hyaluronidase is useful as an anti-edema agent and anti-inflammatory agent in preventing transplant organ rejection. Since hyaluronidase degrades hyaluronan in damaged tissues, it has been found to play a role in pre-clinical experiments. Hyaluronan, a glucosaminoglycan with unique water-binding properties, draws water into certain transplanted organs, causing edema. This can impair the function of the organ and lead to damage and rejection of the transplanted organ. In addition to attracting water, hyaluronan attracts certain cells of the immune system and can therefore be a means of initiating an inflammatory response. Studies have confirmed that treatment with hyaluronidase can be used to reduce edema and inflammation after organ transplantation.

  If hyaluronidase is administered prior to the administration of a local anesthetic such as procaine ("pre-injection"), the anesthetic action will start earlier, the area of anesthesia will be larger, and pain after the procedure will be significant It becomes weaker.

  The optimal and effective combination of hyaluronidase and local anesthetic is now widely used, especially in ophthalmology and especially in cataract surgery. Administration of hyaluronidase along with certain local anesthetics (such as procaine, lidocaine, bupivacaine, etc.) for post-ocular and periocular anesthesia is useful for various ophthalmic surgeries. Hyaluronidase accelerates the onset of anesthetics and causes a reliable blockage of the eye muscle, thereby creating excellent conditions for surgery. Hyaluronidase, when used in combination with a vasopressor such as adrenaline, prolongs the duration of anesthesia in the treatment area and prevents rapid decline in local anesthesia. Hyaluronidase is also useful for treating post-operative increases in intraocular pressure caused by the administration of viscoelastic substances such as sodium hyaluronate during ophthalmic surgery.

  Hyaluronidase is also widely used in dermatology, a selected skin disorder that affects the connective tissue system and is characterized by its degeneration (scleroderma, keloid formation, psoriasis, chronic varicose ulcers, etc.).

  Hyaluronidase is also useful in gynecology, i.e. preventing perineal incision.

  It has been demonstrated that hyaluronidase is effective in treating myocardial infarction.

  In the most recent clinical trials, hyaluronidase has been detected in cancer (myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, breast cancer (with accompanying brain metastases), brain lymphoma, glioma, squamous cells in the ear, nose and throat areas Cancer, as well as bladder cancer) has proven useful as an adjunct to chemotherapy. Hyaluronidase not only increases the patient's response to cytostatics, but also dramatically improves the patient's overall subjective health and remission rate.

  Preliminary evidence of the usefulness of this treatment is also found in colon cancer, lung adenocarcinoma, bronchial cancer, adrenal gland, stomach, pancreatic and ovarian cancer, myelosarcoma, and neuroma. In different types of tumors, administration of hyaluronidase was found to induce a temporary cessation of tumor growth, and improved the response to cytostatics in patients who were less likely to be effective in treating hyaluronidase. Furthermore, hyaluronidase is thought to stimulate the immune system. The positive findings after administering hyaluronidase as an adjunct to chemotherapy for malignant tumors suggests that hyaluronidase may one day expand the potential of anti-tumor chemotherapy and improve treatment outcomes, This is the reason for expectation.

  There is no reason to prohibit humans from using hyaluronidase from a toxicological point of view and in the light of the wide range of available data on the various uses of hyaluronidase.

  Neither a single dose of hyaluronidase nor long-term administration detected any significant pathological changes in the organ in acute toxicity studies.

  However, it must always be remembered that hyaluronidase is actually antigenic, whether used alone or mixed with other substances. These effects can never be completely eliminated due to the complexity of the methods used to isolate hyaluronidase. Accordingly, there is a need for a hyaluronidase formulation suitable for pharmaceutical use, which is satisfied by the method of isolation and purification of sheep hyaluronidase disclosed herein.

  In light of the positive clinical findings regarding hyaluronidase, it can be concluded that hyaluronidase is a therapeutically versatile enzyme that has the potential to become a therapeutically useful drug in new areas of current and future medicine.

[Hyaluronidase for injection]
In this example using hyaluronidase as a diffusing agent, injectable hyaluronidase dehydrated to a solid state under high vacuum together with the inert ingredients listed below is sterilized, nonpreserved, white, odorless Provide as an amorphous solid. The product will be reconstituted with sodium chloride injection (USP) before use.

  Each vial of 6200 USP units contains 5 mg lactose, 1.92 mg dibasic potassium phosphate and 1.22 mg monobasic potassium phosphate.

  USP / NF hyaluronidase units correspond to turbidity reduction (TR) units and are equal to 0.81 international units (IU).

  The reconstituted solution is clear and colorless, has a pH of about 6.7, and an osmolarity of 290 to 310 mOsm (milliosmol).

  Hyaluronidase for injection will be reconstituted in the vial to a concentration of 1000 units / mL sodium chloride injection (USP) by adding 6.2 mL of solution to the vial. Prior to administration, the reconstituted solution is further diluted to the desired concentration, usually 150 units / mL (see table below). The resulting solution must be used immediately after preparation.

  A 1 mL syringe and a 5 μm filter needle are provided in the hyaluronidase kit for injection. Following reconstitution of injectable hyaluronidase as described above, a 5 μm filter needle is attached to a 1 mL syringe. The desired amount of hyaluronidase for injection is drawn into the syringe and diluted according to the table below. Remove the filter needle and attach an appropriate needle for the intended injection.

  Efforts have been made to describe the invention above with reference to only some presently preferred embodiments and examples, and to cover all embodiments in which the invention can take or be implemented in physical form. Those skilled in the art will understand that nothing has been paid. Indeed, the specific embodiments and examples described above may be varied in many ways without departing from the intended spirit and scope of the invention. Accordingly, all such reasonable variations to the above are intended to be included within the scope of the following claims.

Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 5a and FIG. 5b is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 6a and FIG. 6b is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 7a and FIG. 7b is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 8a and FIG. 8b is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 9a and FIG. 9b is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 10a, FIG. 10b, and FIG. 10c is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. The relationship between FIG. 11a, FIG. 11b, and FIG. 11c is shown. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. Fig. 2 shows a flow chart of a method for preparing the described hyaluronidase. A typical chromatogram for a standard is shown. A typical chromatogram for the sample is shown.

Claims (24)

Hyaluronidase purified from sheep testis and capable of hydrolyzing hyaluronic acid type mucopolysaccharides, with a molecular weight based on mobility in a 4-20% gradient SDS polyacrylamide gel of 70-74 kDa for α form and A composition containing hyaluronidase in the β form of 60-63 kDa and a specific activity in the range of 1.2 × 10 ^{4 to} 1.9 × 10 ⁴ USP units / mg protein and no thimerosal . a) providing a source of testis;
b) extracting the testis source to prepare a crude testis extract;
c) precipitating the crude testis extract to form a precipitate and suspending the precipitate to produce a crude testis suspension;
d) dialyzing the crude testis suspension with a food grade dialysate to prepare a crude dialysate;
e) subjecting the crude dialysate to ion exchange chromatography to isolate the crude eluate;
f) lyophilizing the crude eluate to produce a crude lyophilizate;
g) suspending the crude lyophilizate to produce a crude formulation;
h) subjecting the crude formulation to ion exchange chromatography to isolate the purified eluate;
i) dialyzing the purified eluate with a food grade dialysate to prepare a purified dialysate;
j) performing viral filtration of the purified dialysate to prepare purified mammalian hyaluronidase, comprising preparing purified mammalian hyaluronidase.   A product produced by the method of claim 2. A method of accelerating the clearance of bleeding blood from the vitreous humor of a mammalian eyeball,
A method comprising contacting the hyaluronidase of claim 1 in an amount that provides a dosage of at least one international unit effective to accelerate the clearance of bleeding blood from the vitreous humor. A method of inducing thawing of vitreous humor to treat a disorder of a mammalian eyeball,
Contacting the hyaluronidase of claim 1 with the vitreous humor of the mammalian eyeball to provide a dosage of at least one international unit effective to melt the vitreous humor such that the disorder is treated. A method comprising steps. The method of claim 5, wherein
A method characterized in that the method is carried out to treat nonproliferative diabetic retinopathy. The method of claim 5, wherein
A method characterized in that the method is carried out to treat preproliferative diabetic retinopathy. The method of claim 5, wherein
A method characterized in that the method is carried out to treat proliferative diabetic retinopathy. The method of claim 5, wherein
A method characterized in that the method is carried out to treat age-related macular degeneration. The method of claim 5, wherein
A method characterized in that the method is implemented to treat amblyopia. The method of claim 5, wherein
A method characterized in that the method is carried out to treat retinitis pigmentosa. The method of claim 5, wherein
A method characterized in that the method is carried out to treat macular holes. The method of claim 5, wherein
A method characterized in that the method is carried out to treat macular exudation. The method of claim 5, wherein
A method characterized in that the method is carried out to treat cystoid macular edema. The method of claim 5, wherein
A method wherein posterior vitreous detachment (PVD) is achieved by the melting.   A method of treating glaucoma comprising the step of administering a hyaluronidase according to claim 1 in an amount that provides a dosage of at least one international unit effective to treat glaucoma.   A method of treating a malignancy comprising the step of administering a hyaluronidase according to claim 1 in an amount that provides a dose of at least one international unit effective for treating the malignancy.   A method of treating myocardial infarction comprising the step of administering a hyaluronidase according to claim 1 in an amount that provides a dose of at least one international unit effective for treating myocardial infarction.   2. Reducing edema or inflammation after organ transplant comprising the step of administering a hyaluronidase according to claim 1 in an amount that provides a dosage of at least one international unit effective to reduce edema or inflammation after organ transplantation. how to.   A corneal scar, turbidity or blurred vision comprising the step of administering a hyaluronidase according to claim 1 in an amount providing a dosage of at least one international unit effective for treating corneal scarring, turbidity or blurred vision Treatment methods.   2. Facilitating diffusion of a radiopaque medium comprising administering a hyaluronidase according to claim 1 in an amount that provides a dosage of at least one international unit effective to facilitate diffusion of the radiopaque medium. how to.   The absorption and dispersion of an injected drug comprising the step of administering an amount of hyaluronidase according to claim 1 in an amount that provides a dose of at least one international unit effective to enhance absorption and dispersion of the injected drug. How to enhance. A composition according to claim 1, comprising:
A composition comprising lactose and phosphate and in the form of a solution. A composition according to claim 1, comprising:
A composition characterized in that it is in a lyophilized form.

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