WO2005118799A1 - Ovine hyaluronidase - Google Patents

Ovine hyaluronidase Download PDF

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Publication number
WO2005118799A1
WO2005118799A1 PCT/US2004/034150 US2004034150W WO2005118799A1 WO 2005118799 A1 WO2005118799 A1 WO 2005118799A1 US 2004034150 W US2004034150 W US 2004034150W WO 2005118799 A1 WO2005118799 A1 WO 2005118799A1
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Prior art keywords
hyaluronidase
ohuase
amino acid
acid sequence
ovine
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French (fr)
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Donald J. Brown
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ISTA Pharmaceuticals Inc
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ISTA Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to a composition of matter comprising an isolated polynucleotide such as DNA or RNA encoding alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO:
  • Hyaluronidase is a versatile class of enzymes that are expressed in vertebrates and invertebrates alike.
  • the mammalian hyaluronidase catalyzes the random hydrolysis of 1,4- linkages between 2-acetamido-2-deoxy-b-D-glucose and D-glucuronic acid residues in hyaluronate.
  • the hyaluronidase from bovine testes has a reported molecular weight of 65,000
  • composition of matter comprising an isolated polynucleotide such as DNA or RNA encoding alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO: 1, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined, or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1 and having hyaluronidase activity.
  • Ovine hyaluronidase is an enzyme product purified from ovine testes and capable of hydrolyzing mucopolysaccharides of the type of hyaluronic acid.
  • Amino Acid Sequence - -form (SEQ ID NO: 1) The consensus sites for glycosylation are underlined. The site of cleavage that yields the ⁇ -form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined.
  • Asp Arg Leu Gly Tyr Tyr Pro His lie Asp Glu Lys Thr Gly Asn Thr 95 100 105 gta tat gga gga att ccc cag ttg gga aac tta aaa aat cat ttg gaa 628 val Tyr Gly Gly lie Pro Gin Leu Gly Asn Leu Lys Asn His Leu Glu 110 115 120 aaa gcc aaa aaa gac att gcc tat tat ata cca aat gac age gtg ggc 676
  • Lys Ala Lys Lys Asp lie Ala Tyr Tyr lie Pro Asn Asp ser Val Gly 125 130 135 ttg gcg gtc att gac tgg gaa aac tgg agg cct ace tgg gca aga aac 724 Leu Ala Val lie Asp Trp Glu Asn Trp Arg Pro Thr Trp Ala Arg Asn
  • Gin Lys Asn Pro Gin Leu Ser Phe Pro Glu Ala Ser Lys lie Ala Lys 175 180 185 gtg gat ttt gag aca gca gga aag agt ttc atg caa gag act tta aaa 868
  • Tyr lie lie lie Asn Val Thr Leu Ala Ala Lys Met cys ser Gin Val Leu 365 370 375 tgc cac gat gaa gga gtg tgt aca agg aaa caa tgg aat tea age gac 1444
  • oHUase that is folded in its naturally-occurring configuration (i.e., oHUase is not denatured).
  • native oHUase polypeptides are those polypeptides that, when folded, mimic a three-dimensional epitope of native, full- length oHUase such that antibodies that bind native oHUase bind to the oHUase polypeptide.
  • Native oHUase encompasses both oHUase naturally found in testes, as well as oHUase that is recombinantly produced.
  • ⁇ -form is meant a polypeptide chain of oHUase which is of higher molecular weight relative to ⁇ -form polypeptide of oHUase.
  • ⁇ -form as used herein is meant to encompass oHUase ⁇ -form polypeptides having the amino acid sequence of naturally- occurring oHUase ⁇ -form polypeptide, as well as all naturally-occurring allelic variants and modified oHUase ⁇ -form polypeptide which contains amino acid substitution(s), deletion(s), and/or addition(s) and the like relative to the naturally-occurring amino acid sequence.
  • " ⁇ -form polypeptide” encompasses ⁇ -form polypeptides that are biologically active (e.g., can bind anti-oHUase antibodies and/or exhibit hyaluronidase activity).
  • Ovine ⁇ -form polypeptide having the amino acid sequence of SEQ ID NO: 1 is an exemplary oHUase ⁇ -form polypeptide of the invention.
  • ⁇ -form polypeptide is meant a polypeptide chain of oHUase which is of lower molecular weight relative to ⁇ -form polypeptide of oHUase.
  • ⁇ -form polypeptide as used herein is meant to encompass oHUase ⁇ -form polypeptides having the amino acid sequence of naturally-occurring oHUase ⁇ -form polypeptide, as well as all naturally-occurring allelic variants and modified oHUase ⁇ -form polypeptide which contains amino acid substitution(s), deletion(s), and/or addition(s) and the like relative to the naturally-occurring amino acid sequence.
  • " ⁇ -form polypeptide” encompasses ⁇ -form polypeptides that are biologically active (e.g., can bind anti-oHUase antibodies and/or exhibit hyaluronidase activity).
  • Ovine ⁇ -form polypeptide having the amino acid sequence of SEQ ID NO: 1 is an exemplary oHUase ⁇ -form polypeptide of the invention, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta- form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined.
  • polypeptide is meant any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation, phosphorylation, or fatty acid chain modification).
  • substantially pure polypeptide is meant, for example, oHUase polypeptide that has been separated from components which naturally accompany it (e.g., a substantially pure oHUase polypeptide purifiable from ovine testes is substantially free of components normally associated with ovine testes).
  • the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, oHUase polypeptide.
  • a substantially pure oHUase polypeptide can be obtained, for example, by extraction from a natural source (e.g., ovine testes); by expression of a recombinant nucleic acid encoding oHUase polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • a protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • substantially pure polypeptides include those derived from eukaryotic organisms or synthesized in E. coli or other prokaryotes.
  • antibody is meant an immunoglobulin protein that is capable of binding an antigen.
  • Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g., F(ab')2, Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.
  • Antibodies of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to native oHUase polypeptide.
  • Anti-oHUase antibodies are preferably immunospecific (i.e., not substantially cross-reactive with related materials).
  • Antibodies may be polyclonal or monoclonal, preferably monoclonal.
  • purified antibody is meant one that is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated.
  • Such an antibody “preferentially binds” to an antigenic oHUase polypeptide, i.e., does not substantially recognize and bind to other antigenically-unrelated molecules.
  • binds specifically is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide i.e., epitope of oHUase.
  • Antibody binding to its epitope on this specific polypeptide is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest, e.g., binds more strongly to oHUase than to other components in ovine testes.
  • Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls.
  • anti-native oHUase antibody or “anti-oHUase antibody” is meant an antibody that specifically binds native (i.e., non-denatured) oHUase.
  • such antibodies can be used to immunopurify (e.g., by immunoprecipitation or immunoaffinity column chromatography) naturally-occurring oHUase from ovine testes and/or recombinant oHUase expressed by, for example, mammalian cells.
  • Polynucleotide refers to an oligonucleotide, nucleotide, and fragments or portions thereof, as well as to peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and to DNA or RNA of genomic or synthetic origin which can be single- or double-stranded, and represent the sense or antisense strand.
  • PNA peptide nucleic acids
  • polypeptide refers to an oligopeptide, peptide, or protein.
  • polypeptide is recited herein to refer to an amino acid sequence of a naturally- occurring protein molecule
  • polypeptide and like terms are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • antisense polynucleotide is meant polynucleotide having a nucleotide sequence complementary to a given polynucleotide sequence including polynucleotide sequences associated with the transcription or translation of the given polynucleotide sequence, where the antisense polynucleotide is capable of hybridizing to a oHUase polynucleotide sequence.
  • antisense polynucleotides capable of inhibiting transcription and/or translation of a oHUase polynucleotide either in vitro or in vivo.
  • substantially identical is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 100 amino acids, preferably at least 200 amino acids, more preferably at least 250 amino acids, more preferably at least 300 amino acids, more preferably at least 350 amino acids, more preferably at least 400 amino acids, and most preferably at least 450 amino acids.
  • the length of comparison sequences will generally be at least 300 nucleotides, preferably at least 600 nucleotides, more preferably at least 750 nucleotides, more preferably at least 900 nucleotides, more preferably at least 1050 nucleotides, more preferably at least 1200 nucleotides, and most preferably at least 1350 nucleotides.
  • Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, as
  • treatment generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • therapeutically effective amount of a substantially pure oHUase polypeptide is meant an amount of a substantially pure oHUase polypeptide effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated.
  • the degradation of hyaluronan is the desired therapeutic effect where oHUase is administered to the subject in the treatment of a condition associated with excess hyaluron, undesirable cell motility (e.g., tumor cell metastasis), and/or to enhance circulation of physiological fluids and/or therapeutic drugs at the site of administration and/or inhibit tumor growth or progression.
  • oHUase Coding Sequences any nucleic acid sequence which encodes the amino acid sequence of oHUase can be used to generate recombinant molecules which express oHUase.
  • the invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices (see table below). These combinations are made in accordance with the standard triplet genetic code as applied to the amino acid sequence of naturally occurring oHUase, and all such variations are ' to be considered as being specifically disclosed.
  • the nucleotide sequence encoding a oHUase can be isolated according to any one of a variety of methods well known to those of ordinary skill in the art.
  • DNA encoding oHUase can be isolated from either a cDNA library or from a genomic DNA library by either hybridization or expression cloning methods.
  • the DNA can be isolated using standard polymerase chain reaction (PCR) amplification of synthetic oligonucleotide primers, e.g., as described in Mullis et al., U.S. Pat. No. 4,800,159, or expression cloning methods well known in the art (see, e.g., Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual.
  • PCR polymerase chain reaction
  • oHUase polypeptide-encoding DNA can be determined using methods well known in the art (see, for example, Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Following sequence confirmation, the resulting clones can be used to, for example, identify homologs of oHUase (e.g., other ovine alleles encoding oHUase or a hyaluronidase of another mammalian species, and/or to transform a target host cell for expression of DNA encoding a polypeptide of oHUase.
  • homologs of oHUase e.g., other ovine alleles encoding oHUase or a hyaluronidase of another mammalian species
  • nucleotide sequences which encode oHUase and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring oHUase under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding oHUase or its variants possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence. It is now possible to produce a DNA sequence, or portions thereof, encoding a oHUase and its variants entirely by synthetic chemistry, after which the synthetic gene may be inserted into any of the many available DNA vectors and cell systems using reagents that are well known in the art at the time of the filing of this application. Moreover, synthetic chemistry may be used to introduce mutations into a oHUase sequence or any portion thereof.
  • polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of the naturally occurring oHUase under various conditions of stringency.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA).
  • “Maximum stringency” typically occurs at about T m -5°C. (5°C. below the T m of the probe); “high stringency” at about 5°C. to 10°C. below T m ; “intermediate stringency” at about 10°C. to 20°C.
  • hybridization as used herein shall include "any process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J. 1994 Dictionary of Biotechnology, Stockton Press, New York, NY). Amplification as carried out in the polymerase chain reaction technologies is described in Dieffenbach C.W. and G.S.
  • a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • An “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring oHUase.
  • a “substitution” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • Altered oHUase nucleic acid sequences which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent oHUase.
  • the protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent oHUase. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of oHUase is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydropliilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, and tyrosine.
  • amino acids with uncharged polar head groups having similar hydropliilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, and tyrosine.
  • Included within the scope of the present invention are alleles of oHUase.
  • an "allele” or "allelic sequence” is an alternative form of oHUase.
  • Alleles result from a mutation, i.e., a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • the polynucleotide sequences encoding oHUase may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • genomic DNA as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions.
  • genomic oHUase sequences may have non-contiguous open reading frames, where introns interrupt the protein coding regions.
  • Genomic sequences can also comprise the nucleic acid present between the initiation codon and the stop codon, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA.
  • the genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence.
  • the nucleic acid compositions of the subject invention may encode all or a part of the oHUase polypeptides as appropriate. Fragments maybe obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc.
  • DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than about 50 nt to 100 nt are useful for production of the encoded polypeptide.
  • a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.
  • the oHUase-encoding sequences are isolated and obtained in substantial purity, generally as other than an intact mammalian chromosome.
  • the DNA will be obtained substantially free of other nucleic acid sequences that do not include a oHUase- encoding sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
  • the DNA sequences are used in a variety of ways. They may be used as probes for identifying homologs of oHUase. Mammalian homologs have substantial sequence similarity to one another, i.e.
  • Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc.
  • a reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared.
  • Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. 1990 JMol Biol 215:403-10. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C.
  • sequence identity may be determined by hybridization under high stringency conditions, for example, at 50°C. or higher and O.lxSSC (9 mM saline/0.9 mM sodium citrate).
  • probes particularly labeled probes of DNA sequences, one can isolate homologous or related genes.
  • the source of homologous genes may be any species, for example, Primate species, particularly human; rodents, such as rats and mice, canines, felines, bovine, opines, equine, yeast, Drosophila, Caenhorabditis, etc.
  • oHUase-encoding DNA can also be used to identify expression of the gene in a biological specimen.
  • the manner in which one probes cells for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature and does not require elaboration here.
  • mRNA is isolated from a cell sample. mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g., nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe.
  • a suitable support e.g., nitrocellulose, nylon, etc.
  • oligonucleotide ligation assays such as in situ hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to a oHUase sequence is indicative of oHUase expression in the sample.
  • oHUase-encoding sequences may be modified for a number of purposes, particularly where they will be used intracellularly, for example, by being joined to a nucleic acid cleaving agent, e.g., a chelated metal ion, such as iron or chromium for cleavage of the gene; or the like. Techniques for in vitro mutagenesis of cloned genes are known.
  • DNA encoding hyaluronidases homologous to oHUase can be accomplished by screening various cDNA or genomic DNA libraries by hybridization or PCR using oligonucleotides based upon the DNA sequence and/or amino acid sequence of a oHUase.
  • the oligonucleotides used may be degenerate, e.g., based upon a selected amino acid sequence of oHUase or designed so as to allow detection or amplification of DNA encoding a oHUase-like amino acid sequence having conservative amino acid substitutions and/or to take into account the frequency of codon usage in the mammalian species DNA to be screened.
  • Such "degenerate oligonucleotide probes" can be used in combination in order to increase the sensitivity of the hybridization screen, and to identify and isolate oHUase analogs and orthologs in other species or variant alleles encoding oHUase.
  • oHUase polypeptides can be made by standard synthetic techniques, or by using recombinant DNA technology and expressed in bacterial, yeast, insect, or mammalian cells using standard techniques.
  • oHUase includes natural, recombinant, and modified forms of the protein unless the context in which the term is used clearly indicates otherwise.
  • Chemical Synthesis oHUase polypeptides can be synthesized based on the amino acid sequences described herein and variations thereof by standard solid-phase methods using the tert- butyloxy-carbonyl and benzyl protection strategy described in Clark-Lewis et al. 1993
  • the proteins After deprotection with hydrogen fluoride, the proteins are folded by air oxidation and purified by reverse-phase HPLC. Purity is determined by reverse-phase HPLC and isoelectric focusing. Amino acid incorporation is monitored during synthesis, and the final composition is determined by amino acid analysis. The correct covalent structure of the protein can be confirmed using ion-spray mass spectrometry (SCIEX API ).
  • Expression of a oHUase polypeptide is accomplished by inserting a nucleotide sequence encoding a oHUase polypeptide into a nucleic acid vector such that a promoter in the construct is operably linked to oHUase-encoding sequence.
  • the construct can then be used to transform a mammalian, insect, yeast, or bacterial host cell.
  • Numerous, commercially available vectors useful in recombinant polypeptide expression can be used.
  • the vector is capable of replication in both eukaryotic and prokaryotic hosts, and is generally composed of a bacterial origin of replication and a eukaryotic promoter operably linked to a DNA of interest.
  • a number of vectors suitable for stable transfection of mammalian, insect, yeast, and bacterial cells are available to the public from a wide variety of sources, for example, the American Type Culture Collection, Rockville, MD. Suitable host cells, as well as methods for constructing stably-transformed host cell lines, are also publicly available, e.g., Pouwels et al. 1985 Cloning Vectors: A Laboratory Manual; Ausubel et al. 1989 Current Protocols in Molecular Biology, John Wiley & Sons, New York; and Sambrook et al. 1989 Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.
  • recombinant oHUase polypeptide can be assayed by immunological procedures, such as Western blot or immunoprecipitation analysis of recombinant cell extracts, or by the HAse activity assay as described in for example, Tolksdorf, et al. 1949 JLab Clin Med 34:74; and Kass & Seastone, 1944 JExp Med 79:319.
  • oHUase polypeptides according to the invention can be produced by transformation of a suitable host cell (e.g., bacterial, yeast, insect or mammalian cell) with a oHUase polypeptide-encoding nucleotide sequence(s) in a suitable expression vehicle, and culturing the transformed cells under conditions that promote expression of the encoded polypeptide.
  • a suitable host cell e.g., bacterial, yeast, insect or mammalian cell
  • the vector is preferably designed to allow for secretion of oHUase into the culture medium.
  • the method of transformation and the choice of expression vehicle will depend on the host system selected.
  • oHUase polypeptides of the invention may be used to produce oHUase polypeptides of the invention.
  • the precise host cell used is not critical to the invention.
  • Expression Systems hi order to express a biologically active oHUase, the nucleotide sequence encoding oHUase or its functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a oHUase coding sequence and appropriate transcriptional or translational controls.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell
  • control elements or “regulatory sequences” of these systems vary in their strength and specificities and are those nontranslated regions of the vector, enhancers, promoters, and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, CA) or pSportl (Gibco BRL) and ptrp-lac hybrids and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a cell line that contains multiple copies of oHUase, vectors based on SV40 or EBV may be used with an appropriate selectable marker. hi bacterial systems, a number of expression vectors may be selected depending upon the use intended for oHUase.
  • vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the oHUase coding sequence may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced; pTN vectors (Van Heeke & Schuster 1989 JBiol Chem 264:5503-5509); and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST), hi general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • GST glutathione S- transferase
  • oHUase is an insect system.
  • Autographa californica nuclear polyhedrosis virus is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the oHUase coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • oHUase Successful insertion of oHUase will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat.
  • the recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which oHUase is expressed (Smith et al. 1983 J Virol 46:584; Engelhard E.K. et al. 1994 PNAS USA 91:3224-7).
  • a number of viral-based expression systems may be utilized.
  • a oHUase coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in a nonessential El or E3 region of the viral genome will result in a viable viras capable of expressing oHUase in infected host cells (Logan & Shenk 1984 PNAS USA 81:3655-59).
  • transcription enhancers such as the rous sarcoma viras (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV rous sarcoma viras
  • Specific initiation signals may also be required for efficient translation of a oHUase sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where oHUase, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed.
  • exogenous transcriptional control signals including the ATG initiation codon must be provided.
  • the initiation codon must be in the correct reading frame to ensure transcription of the entire insert.
  • Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf D. et al. 1994 Results Probl Cell Differ 20:125-62; Bittner et al. 1987 Methods Enzymol 153:516-544).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein. For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines which stably express oHUase may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex viras thymidine kinase (Wigler M. et al.
  • host cells which contain the coding sequence for oHUase and express oHUase may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of the nucleic acid or protein. The presence of the oHUase polynucleotide sequence can be detected by DNA-
  • oligonucleotides or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer.
  • a variety of protocols for detecting and measuring the expression of oHUase, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on oHUase is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R. et al. 1990 Serological Methods, a Laboratory Manual, APS Press, St Paul, MN; and Maddox D.E. et al.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to oHUase include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the oHUase sequence, or any portion of it may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos.
  • recombinant immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567.
  • Host cells transformed with a oHUase nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing oHUase can be designed with signal sequences which direct secretion of oHUase through a prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may join oHUase to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll D.J. et al. 1993 DNA Cell Biol 12:441-53).
  • oHUase may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle, WA.
  • the inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and oHUase is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein compromising a oHUase and contains nucleic acid encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site.
  • the histidine residues facilitate purification on LMIAC (immobilized metal ion affinity chromatography as described in Porath et al. 1992 Protein Express Purif 3:263-2$!), while the enterokinase cleavage site provides a means for purifying the chemokine from the fusion protein.
  • polypeptides oHUase, ⁇ -form, or ⁇ -form polypeptide-encoding DNAs can encode all or a portion of oHUase.
  • the expressed polypeptide is biologically active, e.g., exhibits hyaluronidase activity in the cleavage of hyaluronan and/or can be bound by an anti-native oHUase antibody, i general, once information regarding the ability of a protein to elicit antibodies and/or information regarding an enzymatic or other biological activity of a protein of interest is known, methods for identification of biologically active polypeptides of the full-length protein are routine to the ordinarily skilled artisan, particularly where the nucleotide sequence and/or amino acid sequence encoding the protein of interest (here oHUase, ⁇ -form polypeptide, or ⁇ -form polypeptide) is provided as in the present case.
  • oHUase activity assays for example, Tolksdorf, et al. 1949 J Lab Clin Med 34:74; and Kass & Seastone, 1944 J Exp Med 79:319.
  • biologically active oHUase polypeptides can be detected by binding of an anti-native oHUase antibody to a component of the transformed host cell supernatant and/or lysate.
  • oHUase polypeptides preferably exhibit at least 25%, more preferably 50%, still more preferably 75%, even more preferably 95% of the activity of native oHUase.
  • Embodiments also include polypeptides that comprise essentially full-length oHUase or fragments thereof.
  • fragments can be, for example, at least 100, 105, 110 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 ; 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470 : 475, 480, 485, 490, 495, 500, 505, 510, 515, 516, 517, 518, and 519 amino acids in length as long as
  • Embodiments also include nucleic acids encoding polypeptides that comprise essentially full-length oHUase or fragments thereof. These fragments of nucleic acids encoding oHUase can be, for example, at least 300, 315, 330, 345, 360, 375, 390, 405, 420 : 435, 450, 465, 480, 495, 510, 525, 540, 555, 570, 585, 600, 615, 630, 645, 660, 675, 690 705, 720, 735, 750, 765, 780, 795, 810, 825, 840, 855, 870, 885, 900, 915, 930, 945, 960 : 975, 990, 1005, 1020, 1035, 1050, 1065, 1080, 1095, 1110, 1125, 1140, 1155, 1170, 1185 1200, 1215, 1230, 1245, 1260, 1275, 1290
  • oHUase-specific antibodies are useful in various immunotechniqu.es, including immunopurification and immunodetection techniques, and for the diagnosis of conditions and diseases associated with expression of HUase.
  • oHUase for antibody induction does not require biological activity; however, the protein fragment, or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. They should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule.
  • oHUase amino acids Short stretches of oHUase amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to oHUase. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with oHUase or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response.
  • Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • Monoclonal antibodies to oHUase may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989 PNAS USA 86:3833-3837), and Winter G. and Milstein C. )1991 Nature 349:293-299). Antibody fragments which contain specific binding sites for oHUase may also be generated.
  • such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al. 1989 Science 256:1275-1281).
  • a variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the formation of complexes between oHUase and its specific antibody and the measurement of complex formation.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific oHUase protein is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D.E. et al. (1983 JExp ed 158:1211).
  • oHUase polypeptides e.g., oHUase polypeptides that are not associated with the components of tissue from which oHUase is purified
  • the substantially pure oHUase polypeptides can be used in a variety of applications including human and veterinary therapies, either alone or in combination with other therapeutic agents.
  • Purified oHUase of the invention can generally be used in place of Hyaluronidase (ACS) or Hyaluronidase WYDASE® where the condition to be treated is associated with excess hyaluronic acid and/or therapy is designed to increase oHUase activity generally.
  • ACS Hyaluronidase
  • WYDASE® Hyaluronidase WYDASE®
  • compositions which may comprise nucleotides, proteins, antibodies, agonists, antagonists, or inhibitors, alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers, hi one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.
  • Administration of Pharmaceutical Compositions Administration of pharmaceutical compositions is accomplished orally or parenterally.
  • compositions for parenteral delivery include topical, intra-arterial (directly to the site of interest), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intravitreal, or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Maack Publishing Co, Easton PA).
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, .e., dosage.
  • Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD 5 o/ED 5 o.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, for example, tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drag combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art will employ different formulations for nucleotides than for proteins.
  • delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.
  • EXAMPLE 1 Ophthalmic Toxicities of Thimerosal, Hyaluronidase (ACS) and Hyaluronidase (Wydase®) in Rabbits
  • Certain types of enzymes when contacted with the vitreous humor following hemorrhage thereinto, will accelerate the rate at which the hemorrhagic blood is cleared from the vitreous humor.
  • a method is provided for accelerating clearance of hemorrhagic blood from the vitreous of the eye, said method generally comprising the step of contacting, with the vitreous humor, a quantity of hyaluronidase at a dose which is sufficient to accelerate the clearance of hemorrhagic blood from the vitreous without causing damage to the retina or other tissues of the eye.
  • the hyaluronidase is selected to have a molecular weight distribution which allows the hyaluronidase to be administered intravitreally at doses above 1 IU, and preferably above 15 IU, and advantageously above 75 IU, in the absence of thimerosal, without causing toxic damage to the retina or other tissues of the eye.
  • This hemorrhage-clearing method may be performed without any vitrectomy or other surgical manipulation or removal of the vitreous humor, thereby avoiding the potential risks and complications associated with such vitrectomy procedures.
  • the preferred route of administration of these hemorrhage-clearing enzymes is by intraocular injection directly into the vitreous body.
  • the hemorrhage-clearing enzyme(s) may be administered by any other suitable route of administration (e.g., topically) which results in sufficient distribution of the enzyme(s) to the vitreous body to cause the desired hemorrhage-clearing effect.
  • the preferred injectable solution may contain a hyaluronidase which has a molecular weight distribution which allows it to be administered intravitreally at doses above 1 IU, and preferably above 15 IU, and advantageously above 75 IU, without causing toxic damage to the eye, along with inactive ingredients which cause the solution to be substantially isotonic, and of a pH which is suitable for injection into the eye.
  • This preferred hyaluronidase preparation is preferably devoid of thimerosal.
  • Such solution for injection may be initially lyophilized to a dry state and, thereafter, may be reconstituted prior to use.
  • hyaluronidase (ACS) as used herein describes a hyaluronidase solution for intravitreal injection which is devoid of thimerosal and which is devoid of hyaluronidase molecular weight fractions above 100,000, between 50,000-60,000 and below 20,000, as determined by electrophoresis gel (4-20% gradient SDS-PAGE).
  • Such hyaluronidase may be derived from ovine testicles and is available commercially from Biozyme Laboratories Limited, San Diego, California, which source may be a starting material for the disclosed process for isolating and purifying ovine hyaluronidase.
  • This specific molecular weight distribution of the hyaluronidase (ACS) results in less ophthalmic toxicity than other hyaluronidase preparations, while exhibiting desirable therapeutic efficacy in a number of ophthalmic applications.
  • hyaluronidase may be injected directly into the posterior chamber of the eye at dosage levels which bring about desirable therapeutic affects, including but not necessarily limited to the intravitreal hemorrhage clearing effect, without causing significant toxicity to the eye or associated anatomical structures.
  • Fifty-Two (52) healthy rabbits of the New Zealand Cross variety (26 male, 26 female) weighing 1.5 kg to 2.5 kg, were individually marked for identification and were housed individually in suspended cages. The animals received a commercially available pelleted rabbit feed on a daily basis, with tap water available ad libitum. The animals were divided into thirteen groups of 4 animals each (2 male, 2 female).
  • the fundus photography was performed by restraining the animals and visualizing the optic nerve, retinal arcades and fundus with a KOWA® RC-3 Fundus Camera loaded with Kodak Gold 200 ASA film.
  • the fluorescein angiography involved a 1.5 ml injection of 2% sterile fluorescein solution via the marginal ear vein. Approximately 30 seconds post-injection the fluorescein was visualized upon localization of the optic nerve, retinal vessels and fundus. The following day, each animal was anesthetized by intravenous administration of a combination of 34 mg/kg of ketamine hydrochloride and 5 mg/kg xylazine.
  • one randomly selected rabbit was euthanized by pentobarbital injection but then fixed by intracardiac injection of the of the glutaraldehyde solution into the left ventricle to determine the effect of the fixation procedure on the histology findings within the enucleated eyes.
  • the 13 female rabbits that had been previously photographed and angiography performed were subjected to the same observations following the methods previously described.
  • the remaining 26 animals were euthanized as described above 7 days after dosing.
  • the eyes were fixed in the same manner as those which had been fixed on day 2.
  • one randomly selected rabbit was subjected to the same intracardiac glutaraldehyde fixation procedure described hereabove for the previously randomly selected animal.
  • the eyes of the animals treated in this example were examined grossly and microscopically for evidence of treatment-related toxicities.
  • the eyes of the BSS-treated control group were free of toxicity at 2 and 7 days post dose.
  • the eyes of the Group No. 2 animals treated with BSS+thimerosal (0.0075 mg) were free of toxicity at day 2, but exhibited evidence that there was a breakdown of the blood-retinal barrier at day 7.
  • the Group No. 3 animals treated with BSS+thimerosal (0.025 mg) exhibited severe treatment-related toxic effects, at days 2 and 7 post dose.
  • the hyaluronidase (ACS) preparation used in this experiment was the preferred formulation described hereabove and shown in Table 1.
  • the eyes of each animal were again examined by slit- lamp to evaluate the cornea, anterior chamber and iris.
  • the eyes of each animal were dilated with 10% tropicamide solution and the retina was examined by indirect ophthalmoscopy with a 20 diopter lens.
  • the observed hemorrhage-clearing efficacy of the hyaluronidase (ACS) is summarized in Table 4.
  • the left eye (untreated) of each animal in each treatment group contained hazy vitreous and some blood clots, due to the quantity of blood which had been injected therein.
  • the right eyes of the BSS treated (control) animals of Group A also contained hazy vitreous and some blood clots, while the right eyes of all hyaluronidase- treated animals in Treatment Groups B-D contained vitreous which was clear and through which transvitreal visualization of the retina was possible. Furthermore, the retinas of the rights eyes of all animals in Treatment Groups B-D appeared normal and free of treatment- related toxicity. Table 4. Hemorrhage clearing Efficacy of Single-Dose Intravitreal Hyaluronidase (ACS) in the Rabbits (12 New Zealand rabbits are injected with 10 ⁇ l or 100 ⁇ l of blood in both eyes intravitreally)
  • ACS Hemorrhage clearing Efficacy of Single-Dose Intravitreal Hyaluronidase
  • the right eyes of the BSS treated (control) animals of Group A also contained hazy vitreous and some blood clots, while the right eyes of all animals in treatment Groups B-E (i.e., the animals treated with hyaluronidase (ACS)) contained clear vitreous through which transvitreal visualization of the retina was possible. Furthermore, the retinas of the right eyes of all animals in treatment Groups B-D appeared to be normal and free of treatment-related toxicity, even after multiple doses of the hyaluronidase (ACS). Table 5. Safety and Efficacy of Multiple-Dose Intravitreal Hyaluronidase (ACS) in Rabbit
  • the BSS contained 0.64% sodium chloride, 0.075% potassium chloride, 0.048% calcium chloride dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% sodium acetate trihydrate, 0.17% sodium citrate dihydrate, sufficient sodium hydroxide/hydrochloric acid for adjustment of pH to 7.1-7.2, and water for injection (q.s. 100%).
  • Thirty microliter aliquots of BSS or hyaluronidase specific formulation X (Table 6) were loaded into a 300 ⁇ l microsyringe fitted with a 29 gauge needle 0.5 inches in length. The loaded microsyringes were then used to inject the material into the vitreous of the patient's eye. Table 6. Specific formulation X
  • Procedures used to evaluate the safety of the test articles were completed at various intervals throughout the study, and included indirect ophthalmoscopy, fundus photography, fluorescein angiography, elecfroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, and autorefraction.
  • a concurrent placebo control group was included in the study so that adverse events peculiarly related to hyaluronidase (ACS) could be distinguished from those attributable to the vehicle (BSS)/injection procedure.
  • ACS hyaluronidase
  • One or two drops of a local anesthetic were topically instilled into the eye that was to be treated, after which the patient was asked to look down and a sterile cotton swab soaked in Proparacaine Hydrochlori.de Ophthalmic solution was applied for 10 seconds to an area on the sclera approximately 4-5 mm above the cornea (superior position 12:00 meridian).
  • the test article was then injected into the vitreous through a 29 gauge needle attached to a 200 ⁇ l microsyringe that was inserted up to the full length of the needle at the site of application of the second anesthetic.
  • Example 2 Given the results from Example 2 where injection of hyaluronidase (ACS) into the vitreous of rabbits at various doses up to 150 LU. did not result in any significant histopathologic changes in an earlier preclinical study, it was expected that doses below 150 LU. would be well-tolerated in humans. Consistent with this expectation, the intravitreal administration of hyaluronidase (ACS)/BSS into visually impaired eyes in the current trial elicited few symptoms, all of which were believed attributable to the injection procedure itself as they occurred with comparable frequency in each of the study groups, and treatment-related adverse sequelae were relatively mild and of short duration.
  • ACS hyaluronidase
  • EXAMPLE 4 Use of Hyaluronidase to Accelerate the Clearance of Hemorrhagic Blood from the Vitreous of the Eye
  • the Example set forth herebelow describes cases in which intravitreal hyaluronidase (ACS) was used to accelerate the clearance of hemorrhagic blood from the vitreous of the eye.
  • the hyaluronidase used was the thimerosal-free hyaluronidase (ACS) formulation described above and shown in Table 8.
  • ACS thimerosal-free hyaluronidase
  • ACS thimerosal-free hyaluronidase
  • six (6) human patients (5 female, 1 male) who presented with vitreous hemorrhage were treated with single intravitreal injections of hyaluronidase (ACS) at dosages of 50-200 LU.
  • the hyaluronidase (ACS) administered in this experiment was prepared by the formulation, described hereabove and shown in Table 8. Table 8.
  • EXAMPLE 5 Use of Hyaluronidase to Treat Other Ophthalmological Disorders
  • ACS hyaluronidase
  • Hyaluronidase (ACS) is capable of being administered intravitreally at doses of or in excess of 1 LU.
  • hyaluronidase is particularly suitable for the treatment of many disorders (e.g., proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates and cystoid macular edema) which benefit from liquefaction disconnection of the vitreous and/or accelerated clearance of toxins or other deleterious substances (e.g., angiogenic factors, edema fluids, etc.) from the posterior chamber of the eye and/or from tissues adjacent the posterior chamber (e.g., the retina or macula).
  • disorders e.g., proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates and cystoid macular edema
  • toxins or other deleterious substances e.g., angiogenic factors, edema fluids, etc.
  • liquefaction of the vitreous is also believed to remove the matrix, in the form of the polymerized vitreous, necessary to support neovascularization.
  • the present method is useful in preventing or reducing the incidence of retinal neovascularization.
  • many ophthalmic disorders have as a causative component, a destabilization of the blood-retina membrane. This destabilization permits various components (e.g., serum components, lipids, proteins) of the choriocapillaries to enter the vifreal chamber and damage the retinal surface. This destabilization is also a precursor to vascular infiltration of the vifreal chamber, known as neovascularization.
  • embodiments of the present method are directed toward the prevention and treatment of various disorders of the mammalian eye which result from damage or pathology to the vascularization of the eye or which result in damage to the blood-retinal barrier.
  • diseases include but are not limited to proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates, and cystoid macular edema, and others in which the clinical symptoms of these disorders respond to the hyaluronidase (ACS) treatment.
  • ACS hyaluronidase
  • PDR Proliferative Diabetic Retinopathy
  • LRMA intraretinal microvascular abnormalities
  • Neovascularization may be easily seen on fluorescein angiogram by the profuse leakage of dye from these new vessels since they lack the tight endothelial junctions of the retinal vasculature. Impaired axoplasmic flow in areas of retinal hypoxia result in cotton wool spots.
  • Proliferative diabetic retinopathy requires careful screening of diabetics for early identification and treatment since PDR remains largely asymptomatic in the early stages.
  • Proliferative diabetic retinopathy can be classified into three subgroups: (1) nonprohferative retinopathy; (2) preproliferative retinopathy; (3) proliferative retinopathy. Each classification has certain morphological characteristics.
  • nonprohferative retinopathy include capillary microangiopathy (microvascular obstructions and permeability changes, nonperfusion of capillaries, retinal capillary microaneurysms, basement membrane thickening, and internal microvascular abnormalities (LRMA)); intraretinal hemorrhages; exudates; and macular changes.
  • Preproliferative retinopathy is indicated by any or all of the changes described for nonprohferative retinopathy and the following: significant venous beading, cotton-wool exudates, extensive LRMA and extensive retinal ischemia.
  • Proliferative retinopathy is indicated by extraretinal neovascularization and fibrous tissue proliferation, vifreous alterations and hemorrhage, macular disease, and retinal detachment.
  • the creation of fibrovascular tissue is an especially important complication of PDR since it often will lead to retinal damage mediated by the vitreous.
  • the fibrovascular tissue may form preretinal membranes that create dense adhesions with the posterior hyaloid membrane. These adhesions are responsible for transmitting the forces of vifreous fraction to the retina, which may result in retinal detachments.
  • the vitreous base is normally firmly attached to the adjacent retina and to the outer circumference of the optic nerve head, known as the ring of Martegiani.
  • Neovascularization from the retina leads to the formation of vascular strands extending into the vifreous from the nerve head or elsewhere in the fundus. Contraction of these strands may cause partial or complete retinal detachment. Retinal detachment at the macula is a major complication of PDR. Most retinal detachments resulting from PDR begin as fractional detachments without holes, but they may become rhegmatogenous by the formation of retinal holes at some later point in the disease.
  • the fractional detachments are caused by abnormal vifreoretinal adhesions or vifreal traction with subsequent shrinkage of the fibrous bands and elevation of the retina.
  • the present method contemplates treatment of PDR in the preproliferative and proliferative states using hyaluronidase (ACS) intravitreal injections. Without being limited to a particular mechanism, it is believed that the effect of intravitreal hyaluronidase (ACS) injection is to promote the clearance of the liquid phase of the vitreous.
  • ACS intravitreal hyaluronidase
  • the rate of transfer of intravitreally injected tritiated water from the mid vitreous to the choroid was significantly increased after depolymerization of vitreous hyaluronic acid by injected hyaluronidase (ACS).
  • ACS hyaluronidase
  • the present method capitalizes upon this observation to liquefy the vitreous, for example, in order to promote the clearance of various growth inducing factors and other serum products leaked into the vifreous due to the presence of PDR. It is further contemplated that the hyaluronidase (ACS) freatment of the present method may be performed alone or in combination with other treatments of PDR.
  • EXAMPLE 7 Treatment of Non-Proliferative Diabetic Retinopathy Purpose: To determine the effect of hyaluronidase (ACS) on progression of moderately severe to severe non-proliferative diabetic retinopathy (NPDR in the presence or absence of an induced posterior vifreous detachment (PVD). Methods: sixty patients evaluated by ulfrasonography and masked fundus photography were randomly assigned to: saline (0.05 ml), hyaluronidase (ACS) (75 I.U., 0.05 ml), SF6 gas (0.3 ml) or hyaluronidase (ACS) plus SF6 gas 4 weeks later. PVD was assessed through week 16; seven-field fundus.
  • ACS hyaluronidase
  • EXAMPLE 8 Treatment of Preproliferative Diabetic Retinopathy hi this Example, a diabetic patient manifesting preproliferative diabetic retinopathy is treated for this complication of diabetes mellitus through the intravitreal injection of hyaluronidase (ACS). The purpose of this treatment is to reduce or prevent the development of proliferative diabetic retinopathy manifested by extraretinal neovascularization and fibrous tissue proliferation, vitreous alterations and hemorrhage, macular disease, and retinal detachment.
  • ACS hyaluronidase
  • PDR proliferative diabetic retinopathy
  • This increased surveillance should include periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, LRMA, and retinal ischemia.
  • LRMA venous beading
  • ACS hyaluronidase
  • hyaluronidase (ACS) method of freatment is initiated.
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements.
  • ACS ACS
  • ACS ophthalmic solution described above intravitreally to promote the depolymerization of vitreous hyaluronic acid, resulting in the liquefaction of the vitreous.
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60).
  • the patient is monitored for vitreous liquefaction.
  • the patient is monitored for posterior vifreous detachments using indirect ophthalmoscopy with scleral depression.
  • the extent of PDR presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, LRMA, and retinal ischemia.
  • EXAMPLE 9 Treatment of Proliferative Retinopathy
  • a diabetic patient manifesting proliferative diabetic retinopathy is freated by the intravitreal injection of hyaluronidase (ACS).
  • the purpose of this treatment is to reduce the extent of proliferative diabetic retinopathy, to prevent further manifestations of the disease after removal of any exfraretinal neovascularized tissue, and to reduce the likelihood of retinal detachment.
  • a patient presenting proliferative diabetic retinopathy is to receive the hyaluronidase (ACS) method of treatment in combination with surgical treatment of the neovascularized tissue.
  • the proliferation usually begins with the formation of new vessels with very little fibrous tissue component.
  • the new vessels arise from primitive mesenchymal elements that differentiate into vascular endothelial cells.
  • the newly formed vascular channels then undergo fibrous metaplasia; that is, the angioblastic buds are transformed into fibrous tissue.
  • the new vessels leak fluorescein, so the presence of proliferation is especially noticeable during angiography.
  • the new vessels and fibrous tissue break through the internal limiting membrane and arborize at the interface between the internal limiting membrane and the posterior hyaloid membrane.
  • the fibrovascular tissue may form preretinal membranes that create dense adhesions with the posterior hyaloid membrane. These adhesions are extremely important because they are responsible for transmitting the forces of vitreous fraction to the retina during the later stage of vitreous shrinkage.
  • the proliferative stage of PDR is defined as the presence of three or more of the following characteristics: new vessels, new vessels on or within one disc diameter of the optic nerve, severe new vessels (as defined by one-third disc area neovascularization at the optic nerve or one-half disc area neovascularization at the optic nerve or one-half disc area neovascularization elsewhere), and preretinal or vitreous hemorrhage.
  • new vessels new vessels on or within one disc diameter of the optic nerve
  • severe new vessels as defined by one-third disc area neovascularization at the optic nerve or one-half disc area neovascularization at the optic nerve or one-half disc area neovascularization elsewhere
  • preretinal or vitreous hemorrhage Once diagnosed as entering the proliferative stage, the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected visual acuity) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements.
  • an intravitreal injection of hyaluronidase (ACS) is given to patient's affected eyes. If both eyes are affected, they may be treated separately. The eye is injected with 50 ⁇ l of 50 LU.
  • panretinal photocoagulation PRP
  • Panretinal photocoagulation may be used to treat patients presenting PDR in conjunction with the hyaluronidase (ACS) method of treatment.
  • Panretinal photocoagulation is a form of laser photocoagulation.
  • lasers such as the argon green (614 nm), argon blue-green (488 and 514 nm), krypton red (647 nm), tunable dye, diode and xenon arc lasers, are used for retinal surgery.
  • Laser energy is absorbed predominantly by tissues containing pigment (melanin, xanthophyll, or hemoglobin) producing thermal effects on adjacent structures.
  • Krypton red lasers are the preferred method of freatment, as they are better able to penetrate nuclear sclerotic cataracts and vitreous hemorrhage than the argon lasers, which require more energy to produce equal levels of penetration.
  • the parameters used during laser retinal surgery may be modified depending on the goal of the photocoagulation.
  • the laser has a coagulative effect on small vessels.
  • Focal laser photocoagulation is used in diabetes to stop leakage of microaneurysms.
  • the laser spot is place directly over the microaneurysm to achieve a slight whitening and closure of the aneurysm.
  • the laser may reduce microvascular leakage.
  • Panretinal photocoagulation is thought to be effective by destroying tissue, reducing the amount of ischemic tissue in the eye.
  • Confluent laser spots may be used over a neovascular membrane to obliterate the abnormal vessels.
  • the patient is first freated with hyaluronidase (ACS) and then laser freatment.
  • ACS hyaluronidase
  • the patient is first undergoes laser treatment followed by the hyaluronidase (ACS) treatment.
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60).
  • the patient is monitored for vifreous liquefaction.
  • the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression.
  • AMD retinal pigment epithelium
  • drasen the plural form of druse
  • Drasen may be categorized as hard, soft or basal laminar drasen. The present method is directed both to the freatment and prevention of wet and dry forms of AMD.
  • the choriocapillari.es are a component of the choroid which serves to vascularize the globe.
  • the choriocapillaries consists of a rich capillary network that supply most of the nutrition for the pigment epithelium and outer layers of the retina. Damage to the choriocapillaries is thought to result ultimately in neovascular complications, a cause of macular degeneration. In the dry form, nondisciform macular degeneration results from a partial or total obliteration of the underlying choriocapillaries. Ophthalmoscopically, degeneration of the retinal pigment epithelium and hole formation may be observed.
  • subpigment epithelial deposits of material such as calcium chelates or proteinaceous material and others may be observed, h dry AMD, secondary retinal changes generally occur gradually, resulting in the gradual loss of visual acuity. Nevertheless, in some percentage of patients, a severe loss of vision results.
  • the present method contemplates utility in treating dry AMD and preventing macular degeneration through liquefaction of the vifreous. It is contemplated that the liquefaction of the vitreous would result in an increase in the rate of clearance from the retina of deposited material that later results in macular degeneration. Wet AMD most frequently results from choriocapillary insufficiency, leading to subsequent subpigment epithelial neovascularization.
  • Neovascularization also is thought to occur as an adaptation of retinal vascularization to inadequate oxygenation as a result of vesicular damage. Neovascularization may also cause several other disorders such as detachment of the pigment epithelium and sensory retina. Typically the disease usually begins after 60 years of age, manifesting in both sexes equally and in patients presenting the disease, bilaterally. Perhaps the most important complication of age-related macular degeneration is the development of defects in Bruch's membranes of the globe through which new vessels grow. This epithelial neovascularization may result in the production of exudative deposits in and under the retina.
  • neovascularization may also lead to hemorrhage into the vitreous, which may lead to degeneration of the retina's rods and cones, and cystoid macular edema (discussed below).
  • a macular hole may form which results in irreversible visual loss.
  • Risk factors include increasing age, soft drasen, nongeographic atrophy, family history, hyperopia, and retinal pigment epithelial detachments.
  • Symptoms of choroidal neovascularization in AMD include metamorphopsia, paracentral scotomas or diminished central vision.
  • Ophthalmoscopic findings include subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane.
  • Fluorescein angiography is often an effective method of diagnosis. During this diagnostic procedure, progressive pooling of the dye in the subretinal space, seen as blurring of the boundaries of the lesion or leakage from undetermined sources are indicators of the disease.
  • Other components of choroidal neovascular membranes as delineated by fluorescein angiography include elevated blocked fluorescence, flat blocked fluorescence, blood, and disciform scar.
  • neovascular AMD suggests that classic choroidal neovascularization is the lesion component most strongly associated with rapid visual deterioration. Accordingly, treatment of AMD must encompass all neovascular and fibrovascular components of the lesion. At present, freatment is only indicated when classic neovascularization has boundaries that are well demarcated, and photocoagulation has been shown to be beneficial. In eyes with extrafoveal choroidal neovascularization (>-200 microns from the foveal center), argon laser photocoagulation diminished the incidence of severe visual loss, ($6 lines) at 5 years from 64% to 46%. Recurrent neovascularization developed in one-half of laser-treated eyes, usually in the first year after treatment.
  • This freatment is to reduce or prevent the development of neovascularization, macular disease, and retinal damage.
  • increased ophthalmic surveillance is performed to detect the presence of AMD.
  • This increased surveillance should include periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane.
  • ACS hyaluronidase
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements.
  • an intravifreal injection of hyaluronidase (ACS) is given to the patient's affected eye manifesting AMD. If both eyes are affected, they may be freated separately. The eye to be freated is injected with 50 ⁇ l of 50 LU.
  • ACS hyaluronidase
  • ophthalmic solution (described above) intravifreally to promote the depolymerization of vifreous hyaluronic acid, resulting in the liquefaction of the vifreous.
  • Laser photocoagulation treatment of the hyaluronidase (ACS) injected eyes may be required.
  • the laser treatment protocol described in Examples 8 and 9 should be followed when treating AMD.
  • photocoagulation treatment occurs before the enzyme freatment of the present method. After freatment, the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). Because of the possibility of reoccurrence, the patient should return for periodic examinations on a monthly basis thereafter.
  • the patient On each examination day the patient is monitored for vifreous liquefaction. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression. Finally, the extent of AMD presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane. Additional hyaluronidase (ACS) and/or laser treatments may be required if indicia of reoccurring neovascularization are observed.
  • ACS hyaluronidase
  • laser treatments may be required if indicia of reoccurring neovascularization are observed.
  • amblyopia is derived from Greek and means dull vision (amblys— dull, ops— eye). Poor vision is caused by abnormal development in visual areas of the brain, which is in turn caused by abnormal visual stimulation during early visual development.
  • the pathology associated with amblyopia is not specific to the eye, rather, it is located in the visual areas of the brain including the lateral geniculate nucleus and the striate cortex. This abnormal development is caused by three mechanisms: (1) blurred retinal image called pattern distortion; (2) cortical suppression, or (3) both cortical suppression plus pattern distortion.
  • the present method is primarily concerned with pattern distortions caused by media opacity. More specifically, the present method addresses issues of vitreous opacity.
  • Amblyopic vision is usually defined as a difference of at least two Snellen lines of visual acuity.
  • Critical to the treatment of amblyopia is early detection and early intervention.
  • the strategy for treating amblyopia caused by vitreous opacity is to provide a clear retinal image by altering the opacity of the vitreous so that clear vision results.
  • a patient manifesting amblyopia resulting from vifreal opacity was treated with an intravitreal injection of hyaluronidase (ACS).
  • ACS hyaluronidase
  • the purpose of this treatment was to reduce the opacity of the vifreous by increasing the exchange rate of the liquid in the vifreous.
  • a forty (40) year old female human being having a history of amblyopia presented with uncorrected vision of 20:400 in her right eye and corrected vision in that eye of 20:200.
  • a single 100 LU. dose of the hyaluronidase (ACS) was injected intravitreally into her right eye. The other eye remained untreated. The patient was examined repeatedly post-dose and the vision in her left (untreated) eye remained unchanged while the vision in her right (treated) eye was observed to improve as follows:
  • RP Central acuity may also be affected earlier in the course of disease either by cystoid macular edema, macular atrophy, or development of a posterior subcapsular cataract.
  • RP represents a varied group of diseases whose common thread is the abnormal production of at least one protein in photoreceptor outer segments critical to light transduction.
  • One clinical result of RP is the destabilization of the blood-retinal barrier of the perifoveal capillaries and the optic nerve head. This destabilization results in leakage of fluorescein dye observed by angiography. In addition to leakage, accumulation of fluid as microcycts in the outer plexiform layer may occur and be observed. These fluid filled cysts may eventually burst, resulting in damage to the retinal layer.
  • the present method contemplates treating RP related damage to the retina by promoting the accelerated clearance of the tissue fluid accumulating in the microcycts.
  • a fifty-nine (59) year old male human being presented with a history of retinitis pigmentosa. The uncorrected vision in his left eye was 20:400 and with correction was also 20:400.
  • a single intravitreal injection of 100 LU. of the hyaluronidase (ACS) was administered to the left eye of the patient. The other eye remained untreated.
  • the patient was examined repeatedly following the dose of hyaluronidase (ACS) and the vision in the patient's right (unfreated) eye remained unchanged, while the vision in the patient's left (treated) eye was observed to improve as follows:
  • the posterior vifreal gel refreats from the retinal surface, the resulting gap between the two surfaces creates an area wherein movement of the vifreous humor may negatively interact with the retinal surface.
  • the tangential movement of the vitreous humor within the space of the posterior vifreous syneresis cavity is thought to promote tears of the retinal membrane, resulting in the creation of macular holes.
  • the present method contemplates the use of hyaluronidase (ACS) to depolymerize the vitreous so as to eliminate the conditions which result in macular hole formation.
  • ACS hyaluronidase
  • the posterior vitreous syneresis cavity is eliminated as a result of hyaluronidase (ACS)-mediated reorganization of the vitreous.
  • ACS hyaluronidase
  • the elimination of this cavity permits the fluid between the vitreous and the retina to move freely about the vifreal chamber, dispersing any harmful forces that would have otherwise have been directed against the retinal surface.
  • a patient presenting the early signs of macular hole formation is treated with an intravitreal injection of hyaluronidase (ACS).
  • the patient to be treated presents the various signs of premacular hole formation. These include loss of the foveal depression associated with a yellow foveal spot or ring.
  • the fovea has begun to thin in the region of hole formation and the lesion may obtain a reddish appearance. Fluorescein angiography at this stage may appear normal or show faint hyperfluorescence. The appearance of an eccentric full thickness dehiscence denotes an advanced early stage of the disease.
  • freatment is commenced.
  • the hyaluronidase (ACS) treatment of the present method is commenced when the formation of a macular hole is diagnosed. The patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A- scan measurements.
  • an intravitreal injection of hyaluronidase (ACS) is given to the patient's affected eye. If both eyes are affected, they may be treated separately.
  • the eye to be freated is injected with 50 ⁇ l of 50 LU.
  • ACS hyaluronidase
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60).
  • the patient is monitored for vitreous liquefaction.
  • Fluorescein angiography considered a particularly effect method of monitoring the course of the freatment, is also performed.
  • the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression.
  • Macular exudates are material that penetrates the blood-retina barrier and seeps through the macula into the vifreal chamber. There are two kinds, soft exudates and hard exudates. The soft exudates are actually not exudates but clusters of ganglion cell axons in the nerve fiber layer that have undergone a bulbous dilation at a site of ischemic damage or infarction. Hard exudates are commonly exuded as a result of microvascular changes in background retinopathy. Hard exudates appear yellow and waxy are often deposited in a circular fashion about the macula.
  • lipid and proteinaceous material derived from the exudation of serum components from leaking vessels or from the lipid products of degenerating neural elements within the retina. Adsorption of hard exudates is primarily mediated by macrophagic resorption, however, the rate of this process may be slow since exudation often occurs in the outer plexiform layer within the avascular zone of the retina.
  • the present method is particularly useful in reducing the extent of exudative accumulation resulting from the destabilization of the retinal membrane since hyaluronidase (ACS) depolymerization of the vitreous promotes an increased turn-over rate of the aqueous components of the vifreous.
  • ACS hyaluronidase
  • a patient presenting macular exudates is treated with hyaluronidase (ACS) injection method of treatment.
  • the patient is to receive a full ophthalmic examination to establish a baseline of ocular health.
  • the ophthalmic examination included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements.
  • ACS intravitreal injection of hyaluronidase
  • the eye to be treated is injected with 50 ⁇ l of 50 LU. of the hyaluronidase (ACS) ophthalmic solution described above intravitreally to promote the depolymerization of vitreous hyaluronic acid, resulting in the liquefaction of the vifreous.
  • ACS hyaluronidase
  • the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). On each examination day the patient is monitored for vitreous liquefaction. Fluorescein angiography, considered a particularly effect method of monitoring the course of the treatment, is also performed. Additionally, the patient is monitored for posterior vifreous detachments using indirect ophthalmoscopy with scleral depression.
  • Cystoid macular edema is a common ocular abnormality resulting form a diverse group of etiologies. Most the causes of this condition stem from a disturbance of the blood- retinal barrier of the perifoveal capillaries and the optic nerve head that result in fluid leakage which accumulates in microcysts of the outer plexiform layer. This region is a relatively thin and under vascularized area of the retina. Clinically, a cystoid macular edema produces a honey-comb appearance when examined with fluorescein angiography. As the edema progresses, the outer plexiform layer may rapture, producing a lamellar hole.
  • the hole may be confined to the inner layer of the retina or it may eventually progress to a complete macular hole.
  • the present method contemplates the freatment of cystoid macular edema and the prevention of macular hole formation through the hyaluronidase (ACS)-mediated depolymerization of the vitreous.
  • ACS hyaluronidase
  • a patient presenting the indicia of cystoid macular edema is treated with an intravifreal hyaluronidase (ACS) injection as described in Examples 13 and 14.
  • ACS intravifreal hyaluronidase
  • EXAMPLE 16 Other Pharmacological Uses of Hyaluronidase Hyaluronidase has been used therapeutically for many years now.
  • Hyaluronidase accelerates and enhances the absorbtion of injected drags (antibiotics, cytostatic agents, local anesthesia, chemotherapeutic agents, antivirals, etc.) by the tissue, even when large volumes of the medications are administered in solution, suspension or emulsion form.
  • Hyaluronidase has been successfully used in orthopedics, surgery, ophthalmology, internal medicine, oncology, gynecology, dermatology, etc. for many years. The experimental use of hyaluronidase was tested in numerous areas of medicine. The substance has been administered clinically in various indications and therapies.
  • Ophthalmology is now an important and well documented area of indication for hyaluronidase (Farr C. et al. 1997 Wien Med Wschr 147:1-8).
  • Hyaluronic acid is often applied during ophthalmic surgery (e.g., cataract surgery), for example, to keep the anterior chamber of the eye intact or to protect the corneal endothelium during lens implantation. This results in an increase in intraocular pressure. Measurements have shown that introduction of hyaluronidase in the anterior chamber of the eye can effectively decrease the intraocular pressure postoperatively.
  • Hyaluronidase was also found to be effective in reducing the intraocular pressure in patients who underwent trabeculectomy for treatment of wide-angle glaucoma (doses of 300 IU were administered as a subconjunctival injection). The authors concluded that hyaluronidase reduced the number of complications and improved the prognosis of trabeculectomy. Hyaluronidase can also be helpful in retro- and peribulbar anesthesia for cataract surgery when used in combination with local anesthetics such as lidocaine and bupivacaine (with or without adrenaline). The effects of hyaluronidase in local anesthesia of the eye were reported back in 1949.
  • Hyaluronidase makes is possible for lidocaine and bupivacaine to spread more rapidly within the peribulbar space.
  • the injection pressure of local anesthesia administered prior to cataract operations was investigated in 50 patients in a double-blind study. The study concluded that significant (sufficient) akinesia of the extraocular muscles can be achieved by administration of 1% etidocaine, 0.5% bupivacaine, and 50 IU hyaluronidase.
  • Glaucoma Hyaluronidase is useful for treatment of glaucoma or to alleviate intraocular pressure.
  • Orthopedics, diseases of the supportive and locomotive apparatus For many years now, hyaluronidase has been successfully used for treatment of various diseases of the supportive and locomotive apparatus, e.g., acute conditions of the synovial sheath, surrounding connective tissue and varied inflammations in these areas (paratendinitis crepitans, humeroscapular periarthritis, humeral epicondylitis, tibial condylitis, radial styloiditis, etc.).
  • Good freatment results can usually be achieved (especially in combination with exercise or physiotherapy) if hyaluronidase therapy is started as early as possible, even if the affected limb cannot be immobilized.
  • hyaluronidase depolymerizes mucopolysaccharides deposited in the connective tissue matrix during exfraosseous calcification processes that occur due to increased mesenchymal metabolism. If calcium salts are present, they can be depolymerized as long as calcium does not occur as tertiary calcium phosphate. This can restrict the formation of exfraosseous calcification.
  • the improvement of flexibility e.g., in the affected spinal segment) could be attributed to the presumed depolymerization resulting from the loosening of previously formed connective structures.
  • hyaluronidase is able to influence the composition and structure of the dermis, and that it can promote the re-synthesis of the proteoglycans. h the freatment of joint stiffness, which often occurs as a complication of supracondylar fractures, prior or simultaneous administration of hyaluronidase can provide the patient quicker relief from symptoms.
  • Treatment of malignant diseases When used as a supplement to chemotherapy of malignant tumors, hyaluronidase can dissolve hyaluronic acid-containing areas around tumor cells and tumor cell conglomerates, thereby enabling a higher concenfration of the cytostatic agent to take effect in the desired target area.
  • hyaluronidase may induce a related enhancement of immunological defensive processes, e.g., by creating direct contact between immunocompetent cells ("natural killer cells") and antigens on the tumor surface.
  • immunological defensive processes e.g., by creating direct contact between immunocompetent cells ("natural killer cells") and antigens on the tumor surface.
  • malignant diseases hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ENT region, adenocarcinomas of the lung and colon, and carcinomas of the bladder
  • malignant diseases hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ENT region, adenocarcinomas of the lung and colon, and carcinomas of the bladder
  • hyaluronidase was usually found to increase the response rate to cytostatic agents if high doses of the enzyme are administered prior to administration of the cytostatic agent.
  • Hyaluronidase supplements can improve the patient's response to chemotherapy when used in therapy-resistant patients with malignant hematological diseases.
  • hyaluronidase has proven to be particularly useful when administered as a supplement to cytostatic agents like doxorubicin and adriamycin.
  • the enzyme improves the penetration of doxorabicin in the cells and increases the activity of adriamycin in breast cancer.
  • the combined administration of cisplatin, vindesin, hyaluronidase and radiation therapy for treatment of advanced squamous cell carcinoma in the head and neck region was found to be highly effective: a high rate of remissions and improved tolerance of cytostatic therapy was observed in carcinoma patients.
  • Hyaluronidase led to a decrease in adhesion-related multicellular drug resistance in carcinomas of the breast. This mechanism of action is based on the reduction of cell- contact-dependent inhibition of growth and on the sensitization of cells for the cytostatic agents.
  • Hyaluronidase in tumor treatment increases the effects of cytostatic agents used for treatment of such malignant diseases as hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ear, nose and throat region, adenocarcinomas of the lung and colon, and carcinomas of the bladder. • In clinical studies, hyaluronidase was found to induce cessation of growth (remission) of various tumors. • Therapy-resistant patients respond better to cytostatic agents if an intravesical dose of the enzyme is instilled prior to the cytostatic drug. • Hyaluronidase can improve the subjective well-being and the quality of life of tumor patients.
  • hyaluronidase is useful in certain dermatological diseases, such as, for example, progressive scleroderma, which is a systemic disorder of the entire vascular connective tissue system, with its most important characteristic being the displacement of collagen fractions.
  • progressive scleroderma which is a systemic disorder of the entire vascular connective tissue system, with its most important characteristic being the displacement of collagen fractions.
  • histomorphological skin changes that occur in scleroderma begin with a dermal edema rich in acidic mucopolysaccharides (hyaluronic acid, chondroitin sulphate). Histopathological and chemical tests have shown that part of the ground substance occurs as cement in the collagen fibers. It would therefore appear that acidic mucopolysaccharides, soluble collagen, and polymeric collagen are responsible for the sclerosis.
  • hyaluronidase for treatment of acute myocardial infarction was first described in 1959. Studies have shown that the administration of hyaluronidase in the acute stage, i.e., in the early stage of fresh myocardial infarction (2 to 4 hours after the onset of infarction) can reduce the size of the necrotic area in the heart. Investigators studied medications that lead to a reduction in infarction size, e.g., beta blockers, nitrates, calcium antagonists, etc. Hyaluronidase was found to have a favorable effect on concomitantly administered thrombolytic agents such as streptokinase.
  • Hyaluronidase was found to improve the absorption of locally administered drags and to reduce the risk of progression of skin necrosis in patients treated with intravenously administered Vinca alkaloids.
  • the use of hyaluronidase as an antidote for the extravasation of chemotherapeutic agents has also been described.
  • Hyaluronidase is one of the few antidotes that can be used as an antidote for Vinca alkaloids or epipodophyllotoxins such as etoposide.
  • Gynecology is another area of application for hyaluronidase.
  • Hyaluronidase When injected in the perineal region prior to the expulsive stage of labor, hyaluronidase was found to soften the consistency of the birth canal of first-time mothers, which often eliminated the need for episiotomy. Hyaluronidase is also useful for facilitation of partial and complete aspiration of viscous joint effusions and pleural effusions, i.e., it liquefies the effusions. The enzyme is also used for treatment of edema of various origins and for treatment of arthritic joint changes. Hyaluronidase is a treatment for corneaplasty, corneal scars, opacification, and haze, and cornea in need of delamination.
  • Hyaluronidase can be used as an alternative or adjunct to conventional mechanical vitrectomy. Hyaluronidase is also useful for the induction of retinal detachments. Hyaluronidase is indicated as an adjuvant to increase the absorption and dispersion of other injected drugs; for hypodermoclysis; and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. Summary: The proven and diverse activity of hyaluronidase occurs mainly in the intercellular connective tissue. This action is clearly attributable to the breakdown of hyaluronic acid in the tissue. The therapeutically useful consequences of this action
  • the hyaluronidase-related enhancement of diffusion and increase in permeability that occurs after administration of liquids and/or radiopaque media is of therapeutic significance.
  • the substance is able to accelerate and increase the absorption of drags (antibiotics, cytostatics, local anesthetics, etc.) by the tissue, even when large volumes of the medication are injected in solution, suspension or emulsion form.
  • hyaluronidase can accelerate (by 200 to 300%) the absorption of subcutaneous or intramuscular doses of the drug in the bloodstream, which is of particular significance for internal medicine.
  • the efficacy of hyaluronidase in treatment of disorders of the supportive and locomotive system can be attributed to the so-called “softening effect" of the enzyme.
  • the "antiphlogistic" effect of hyaluronidase makes it possible to control acute symptoms involving the synovial sheaths and the surrounding connective tissue (peritendinitis crepitans, humeroscapular periarthritis, humeral epicondylitis, tibial condylitis, radial styloiditis, etc.) Joint stiffness (e.g., due to supracondylar fracture) can also be treated successfully.
  • hyaluronidase can also be used for freatment of posttraumatic hematomas or edemas of any origin, and for liquefaction of joint and pleural effusions in orthopedics.
  • Hyaluronidase is indicated to be useful as an anti-edema and anti-inflammatory agent in the prevention of transplant rejection. It has been shown in pre-clinical experiments to lend itself to this role, because it breaks down hyaluronan in damaged tissues.
  • Hyaluronan a glucosaminoglycan with unique water-binding capacity, draws water into some transplanted organs causing edema.
  • hyaluronidase freatment can be used to reduce edema and inflammation after organ transplantation.
  • a local anesthetic such as procaine
  • pre-injection a local anesthetic
  • the onset of effect of the anesthetic is quicker, the anesthetic region is larger, and the pain after completion of the procedure is significantly lower.
  • hyaluronidase and local anesthetics are now widely used, particularly in ophthalmology and especially in cataract surgery.
  • the preoperative administration of hyaluronidase with certain local anesthetics (procaine, lidocaine, bupivacaine, etc.) for retro- and peribulbar anesthesia is useful in various ophthalmologic operations.
  • the enzyme accelerates the onset of effect of the anesthetic agent and causes reliable blockade of the eye muscles which, in turn, creates excellent conditions for surgery.
  • vasopressors such as adrenaline
  • hyaluronidase increases the duration of anesthesia in the treated area and prevents the rapid diminishment of local anesthesia.
  • Hyaluronidase is also effective for treatment of postoperatively increased internal eye pressure due to the administration of viscoelastic substances such as sodium hyaluronate during ophthalmic surgery.
  • Hyaluronidase is also widely used in dermatology, i.e., in selected skin disorders involving the connective tissue system and characterized by degeneration of it (scleroderma, keloid formation, psoriasis, chronic varicose ulcer, etc.).
  • Hyaluronidase is also useful in gynecology, i.e., for prevention of episiotomy. The usefulness of hyaluronidase has been validated for freatment of myocardial infarction.
  • hyaluronidase is helpful as a supplement to chemotherapy in patients with cancer (myeloma, Hodgkin's disease, non- Hodgkin's lymphoma, breast cancer [also with concomitant cerebral metastasis], cerebral lymphomas, gliomas, squamous cell carcinomas in the ear, nose and throat region, and carcinomas of the bladder).
  • the enzyme not only increases the patient's response to the cytostatic agents, but also drastically improves the patient's overall subjective feeling of well-being and the remission rate.
  • EXAMPLE 17 Hyaluronidase for Injection In this Example for use of hyaluronidase as a spreading agent, hyaluronidase for injection dehydrated in the solid state under high vacuum with the inactive ingredients listed below, is supplied as a sterile, nonpreserved, white, odorless, amorphous solid. The product is to be reconstituted with Sodium Chloride Injection, USP, before use. Each vial of 6200 USP units contains 5 mg lactose, 1.92 mg potassium phosphate dibasic, and 1.22 mg potassium phosphate monobasic. The USP/NF hyaluronidase unit is equivalent to the turbidity-reducing (TR) unit and equal to 0.81 International Units (IU).
  • TR turbidity-reducing
  • the reconstituted solution is clear and colorless, with an approximate pH of 6.7 and osmolality of 290 to 310 mOsm.
  • Hyaluronidase for injection is to be reconstituted in a vial to a concentration of 1000 Units/mL of Sodium Chloride Injection, USP by adding 6.2 mL of solution to the vial. Prior to administration, the reconstituted solution should be further diluted to the desired concentration, commonly 150 Units/mL, see table below. The resulting solution should be used immediately after preparation.
  • a ImL syringe and a 5-micron filter needle are supplied in a hyaluronidase for injection kit.

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Abstract

The invention relates generally to a composition of matter comprising an isolated polynucleotide such as DNA or RNA encoding alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO:1, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined, or encoding a hyaluronidase having an amino acid sequence at least 97, 98 or 99 % identical to SEQ ID NO:1 and having hyaluronidase activity.

Description

OVINE HYALURONIDASE Related Applications This application claims the benefit of US Provisional Application having Docket No. ISTA.097PR, filed May 25, 2004, which is hereby incorporated by reference in its entirety. Field of the Invention The invention relates generally to a composition of matter comprising an isolated polynucleotide such as DNA or RNA encoding alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO:
1, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined, or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1 and having hyaluronidase activity. Background of the Invention Hyaluronidase is a versatile class of enzymes that are expressed in vertebrates and invertebrates alike. The mammalian hyaluronidase catalyzes the random hydrolysis of 1,4- linkages between 2-acetamido-2-deoxy-b-D-glucose and D-glucuronic acid residues in hyaluronate. The hyaluronidase from bovine testes has a reported molecular weight of 65,000
(Lathrop et al. 1990 J Cell Biol 111:2939). The bovine testicular hyaluronidase hydrolyzes the endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin sulfates A and C
(but not B), primarily to tetrasaccharide residues (Ludovig et al. 1961 J Biol Chem 236:333). Typical purification protocols call for the use of standard chromatographic techniques to produce a purified solution possessing hyaluronidase activity. Tolksdorf, et al. 1949 J Lab Clin Med 34:74; and Kass & Seastone, 1944 J Exp Med 79:319 have articulated a generally accepted assay protocol to determine hyaluronidase activity. These purification protocols were sufficient to provide relatively crade hyaluronidase preparations. There is a need for purified active hyaluronidase that can be used in therapy and other applications. The present disclosure addresses this problem. Summary of the Invention Typical purification protocols call for the use of standard chromatographic techniques to produce a purified solution possessing hyaluronidase activity. Tolksdorf, et al. 1949 J Lab Clin Med 34:74; and Kass & Seastone, 1944 J Exp Med 79:319 have articulated a generally accepted assay protocol to determine hyaluronidase activity. These purification protocols were sufficient to provide relatively crude hyaluronidase preparations. There is a need for purified active hyaluronidase that can be used in therapy and other applications. The present disclosure addresses this problem by providing generally a composition of matter comprising an isolated polynucleotide such as DNA or RNA encoding alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO: 1, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined, or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1 and having hyaluronidase activity. Detailed Description of the Preferred Embodiment Ovine hyaluronidase is an enzyme product purified from ovine testes and capable of hydrolyzing mucopolysaccharides of the type of hyaluronic acid.
Amino Acid Sequence - -form (SEQ ID NO: 1) The consensus sites for glycosylation are underlined. The site of cleavage that yields the β-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined.
Ovine 1 LDFRAPPLISNTSFLWAWNAPAERCVKIFKLPPDLRLFSVKGSPQKSATG Ovine 51 QFITLFYADRLGYYPHIDEKTGNTVYGGPQLGNLKNHLEKAKKDIAYYI Ovine 101 PNDSVGLAVIDWENWRPTWARNWKPKOVYRDESVELVLQKNPQLSFPEA^ Ovine 151 K1AKVDFETAGKSFMQETLKLGKLLRPNHLWGYYLFPDCYNHNYNQPTYN Ovine 201 GNCSDLEKRRNDDLDWLV7KESTALFPSVYLNIKLKSTPKAAFYVRNRVQE Ovine 251 AΠU.SKIASVESPLPVFVYHRPVFTDGSSTYLSQGDLVNSVGEIVALGAS Ovine 301 GπMWGSLNLSLTMQSCMNLGNYLNTTLNPYIINNTLAAKMCSQVLCHDE Ovine 351 GVCTRKQWNSSDYLHLNPMNFAIQTGKGGKYTVPGKVTLEDLQTFSDKFY Ovine 401 CSCYANINCKXRVDIKNVHSVNVCMAEDICIEGPVKLQPSDHSSSQNEAS Ovine 451 TTTVSSISPSTTATTVSPCTPEKQSPECLKVRCLEAIANVTQTGCQGVKW Ovine 501 KNTSSQSQSSIQNTKNQTTY c DNA Sequence of Ovine Hyaluronidase - SEQ ID NO: 2, and Translated polypeptide Sequence - SEQ ID NO: 3 actcctgttt atctctgttc ttggtgagga gacagacaga attgactgct gtgctcatcc 60 accaggttac agatgaagca acttgcaaaa cattcctaaa tacgaaggaa gaagagtatt 120 taaacgtaaa tcatcattat tcatttttat ccatcaaagt agcttcattc tgtgttccta 180 tcttgcatca aatattaggt aaaccaaagt gtgtaggaga aaaaagtgct tttcatagtc 240 atcgctcttt gtgaagaga atg eta agg cgc cgc cat ate tec ttt agg age 292 Met Leu Arg Arg Arg His lie Ser Phe Arg ser 1 5 10 ttt gtt ggg gcc agt gga aca ccc cag gcg gcg etc gcc ttc ctt ctg 340
Phe val Gly Ala ser Gly Thr Pro Gin Ala Ala Leu Ala Phe Leu Leu 15 20 25 ctt cca tgt tgg ttg get ctg gac ttc aga gca ccc cct ctt att tea 388
Leu Pro cys Trp Leu Ala Leu Asp Phe Arg Ala Pro Pro Leu lie Ser 30 35 40 aac act tct ttc etc tgg gcc tgg aat gcc cca get gaa cgt tgt gtt 436
Asn Thr ser Phe Leu Trp Ala Trp Asn Ala Pro Ala Glu Arg cys val 45 50 55 aaa ate ttt aaa eta cct cca gat ctg aga etc ttc tct gta aaa gga 484 Lys lie Phe Lys Leu Pro Pro Asp Leu Arg Leu Phe ser Val Lys Gly 60 65 70 75 age ccc caa aaa agt get acg gga caa ttt att aca tta ttt tat get 532
Ser Pro Gin Lys Ser Ala Thr Gly Gin Phe lie Thr Leu Phe Tyr Ala 80 85 90 gat aga ctt ggc tac tat cct cat ata gat gaa aaa aca ggc aac ace 580
Asp Arg Leu Gly Tyr Tyr Pro His lie Asp Glu Lys Thr Gly Asn Thr 95 100 105 gta tat gga gga att ccc cag ttg gga aac tta aaa aat cat ttg gaa 628 val Tyr Gly Gly lie Pro Gin Leu Gly Asn Leu Lys Asn His Leu Glu 110 115 120 aaa gcc aaa aaa gac att gcc tat tat ata cca aat gac age gtg ggc 676
Lys Ala Lys Lys Asp lie Ala Tyr Tyr lie Pro Asn Asp ser Val Gly 125 130 135 ttg gcg gtc att gac tgg gaa aac tgg agg cct ace tgg gca aga aac 724 Leu Ala Val lie Asp Trp Glu Asn Trp Arg Pro Thr Trp Ala Arg Asn
140 145 150 155 tgg aaa cct aaa gat gtt tac agg gat gag tct gtt gag ttg gtt ctg 772
Trp Lys Pro Lys Asp val Tyr Arg Asp Glu Ser Val Glu Leu Val Leu 160 165 170 caa aaa aat cca caa etc agt ttc cca gag get tec aag att gca aaa 820
Gin Lys Asn Pro Gin Leu Ser Phe Pro Glu Ala Ser Lys lie Ala Lys 175 180 185 gtg gat ttt gag aca gca gga aag agt ttc atg caa gag act tta aaa 868
Val Asp Phe Glu Thr Ala Gly Lys ser Phe Met Gin Glu Thr Leu Lys 190 195 200 ctg gga aaa tta ctt egg cca aat cac tta tgg ggt tat tat ctt ttt 916
Leu Gly Lys Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe 205 210 215 cct gat tgt tac aat cat aat tat aac cag cct act tac aat gga aat 964
Pro Asp cys Tyr Asn His Asn Tyr Asn Gin Pro Thr Tyr Asn Gly Asn
220 225 230 l 235 tgc tct gat tta gaa aaa agg aga aat gat gat etc gac tgg ttg tgg 1012
Cys Ser Asp Leu Glu Lys Arg Arg Asn Asp Asp Leu Asp Trp Leu Trp 240 245 250 aag gaa age act gcc ctt ttc cct tct gtt tat ttg aat ate aag tta 1060
Lys Glu Ser Thr Ala Leu Phe Pro ser Val Tyr Leu Asn lie Lys Leu 255 260 265 aaa tct act cca aaa get gcc ttc tat gtt cgt aat cgt gtc cag gaa 1108 Lys Ser Thr Pro Lys Ala Ala Phe Tyr val Arg Asn Arg Val Gin Glu 270 275 280 gcc att egg ttg tct aaa ata gcg agt gtt gaa agt cca ctt ccg gtt 1156
Ala lie Arg Leu Ser Lys lie Ala ser Val Glu ser Pro Leu Pro Val 285 290 295 ttt gta tat cac cgt cca gtt ttt act gat ggg tct tea aca tac ctt 1204
Phe val Tyr His Arg Pro val Phe Thr Asp Gly ser Ser Thr Tyr Leu
300 305 310 315 tct cag ggt gac ctt gtg aat teg gtt ggt gag ate gtt get eta ggt 1252
Ser Gin Gly Asp Leu Val Asn Ser val Gly Glu lie val Ala Leu Gly 320 325 330 gcc tct ggg att ata atg tgg ggc agt etc aat eta age eta act atg 1300
Ala Ser Gly lie lie Met Trp Gly Ser Leu Asn Leu Ser Leu Thr Met 335 340 345 caa tct tgc atg aac eta ggc aat tac ttg aac act aca ctg aat cct 1348 Gin Ser Cys Met Asn Leu Gly Asn Tyr Leu Asn Thr Thr Leu Asn Pro 350 355 360 tac ata ate aac gtc ace eta gca gcc aaa atg tgc age caa gtg ctt 1396
Tyr lie lie Asn Val Thr Leu Ala Ala Lys Met cys ser Gin Val Leu 365 370 375 tgc cac gat gaa gga gtg tgt aca agg aaa caa tgg aat tea age gac 1444
Cys His Asp Glu Gly Val Cys Thr Arg Lys Gin Trp Asn Ser ser Asp
380 385 390 395 tat ctt cac ctg aac cca atg aat ttt get att caa act ggg aaa ggt 1492
Tyr Leu His Leu Asn Pro Met Asn Phe Ala lie Gin Thr Gly Lys Gly 400 405 410 gga aaa tac aca gta cct ggg aaa gtc aca ctt gaa gac ctg caa acg 1540
Gly Lys Tyr Thr Val Pro Gly Lys Val Thr Leu Glu Asp Leu Gin Thr 415 420 425 ttt tct gat aaa ttt tat tgc agt tgt tat gcc aac ate aac tgt aag 1588 Phe ser Asp Lys Phe Tyr cys ser cys Tyr Ala Asn lie Asn cys Lys 430 435 440 aag aga gtt gat ata aaa aat gtt cat agt gtt aat gta tgt atg gca 1636 Lys Arg val Asp lie Lys Asn Val His Ser Val Asn Val cys Met Ala 445 450 455 gaa gac att tgt ata gag ggc cct gtg aag tta caa ccc agt gat cat 1684 Glu Asp lie Cys lie Glu Gly Pro val Lys Leu Gin Pro ser Asp His 460 465 470 475 tec tct age cag aat gag gca tct act ace ace gtc age agt ate tea 1732 Ser ser Ser Gin Asn Glu Ala ser Thr Thr Thr Val Ser ser lie ser 480 485 490 ccc tct act aca gcc ace aca gta tct cca tgt act cct gag aaa cag 1780 Pro Ser Thr Thr Ala Thr Thr Val ser Pro cys Thr Pro Glu Lys Gin 495 500 505 tec cct gag tgc etc aaa gtc agg tgt ttg gaa gcc ate gcc aac gtc 1828 Ser Pro Glu Cys Leu Lys Val Arg cys Leu Glu Ala lie Ala Asn Val 510 515 520 ace caa acg ggg tgt caa ggt gtt aaa tgg aag aac act tec agt cag 1876 Thr Gin Thr Gly Cys Gin Gly Val Lys Trp Lys Asn Thr ser ser Gin 525 530 535 tea agt att caa aat att aaa aat caa aca ace tat t aaaatataaa 1923
Ser ser lie Gin Asn lie Lys Asn Gin Thr Thr Tyr
540 545 550 ttcagtgctt attaagaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1967 Definitions By "native oHUase" is meant oHUase that is folded in its naturally-occurring configuration (i.e., oHUase is not denatured). Where the native oHUase does not comprise the entire amino acid sequence of naturally-occurring oHUase, native oHUase polypeptides are those polypeptides that, when folded, mimic a three-dimensional epitope of native, full- length oHUase such that antibodies that bind native oHUase bind to the oHUase polypeptide. "Native oHUase" encompasses both oHUase naturally found in testes, as well as oHUase that is recombinantly produced. By "α-form" is meant a polypeptide chain of oHUase which is of higher molecular weight relative to β-form polypeptide of oHUase. "α-form" as used herein is meant to encompass oHUase α-form polypeptides having the amino acid sequence of naturally- occurring oHUase α-form polypeptide, as well as all naturally-occurring allelic variants and modified oHUase α-form polypeptide which contains amino acid substitution(s), deletion(s), and/or addition(s) and the like relative to the naturally-occurring amino acid sequence. Preferably, "α-form polypeptide" encompasses α-form polypeptides that are biologically active (e.g., can bind anti-oHUase antibodies and/or exhibit hyaluronidase activity). Ovine α-form polypeptide having the amino acid sequence of SEQ ID NO: 1 is an exemplary oHUase α-form polypeptide of the invention. By "β-form polypeptide" is meant a polypeptide chain of oHUase which is of lower molecular weight relative to α-form polypeptide of oHUase. "β-form polypeptide" as used herein is meant to encompass oHUase β-form polypeptides having the amino acid sequence of naturally-occurring oHUase β-form polypeptide, as well as all naturally-occurring allelic variants and modified oHUase β-form polypeptide which contains amino acid substitution(s), deletion(s), and/or addition(s) and the like relative to the naturally-occurring amino acid sequence. Preferably, "β-form polypeptide" encompasses β-form polypeptides that are biologically active (e.g., can bind anti-oHUase antibodies and/or exhibit hyaluronidase activity). Ovine β-form polypeptide having the amino acid sequence of SEQ ID NO: 1 is an exemplary oHUase β-form polypeptide of the invention, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta- form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined. By "polypeptide" is meant any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation, phosphorylation, or fatty acid chain modification). By a "substantially pure polypeptide" is meant, for example, oHUase polypeptide that has been separated from components which naturally accompany it (e.g., a substantially pure oHUase polypeptide purifiable from ovine testes is substantially free of components normally associated with ovine testes). Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, oHUase polypeptide. A substantially pure oHUase polypeptide can be obtained, for example, by extraction from a natural source (e.g., ovine testes); by expression of a recombinant nucleic acid encoding oHUase polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms or synthesized in E. coli or other prokaryotes. By "antibody" is meant an immunoglobulin protein that is capable of binding an antigen. Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g., F(ab')2, Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest. Antibodies of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to native oHUase polypeptide. Anti-oHUase antibodies are preferably immunospecific (i.e., not substantially cross-reactive with related materials). Antibodies may be polyclonal or monoclonal, preferably monoclonal. By "purified antibody" is meant one that is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody "preferentially binds" to an antigenic oHUase polypeptide, i.e., does not substantially recognize and bind to other antigenically-unrelated molecules. By "binds specifically" is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide i.e., epitope of oHUase. Antibody binding to its epitope on this specific polypeptide is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest, e.g., binds more strongly to oHUase than to other components in ovine testes. Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls. By "anti-native oHUase antibody" or "anti-oHUase antibody" is meant an antibody that specifically binds native (i.e., non-denatured) oHUase. Preferably, such antibodies can be used to immunopurify (e.g., by immunoprecipitation or immunoaffinity column chromatography) naturally-occurring oHUase from ovine testes and/or recombinant oHUase expressed by, for example, mammalian cells. "Polynucleotide" as used herein refers to an oligonucleotide, nucleotide, and fragments or portions thereof, as well as to peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and to DNA or RNA of genomic or synthetic origin which can be single- or double-stranded, and represent the sense or antisense strand. Similarly, "polypeptide" as used herein refers to an oligopeptide, peptide, or protein. Where "polypeptide" is recited herein to refer to an amino acid sequence of a naturally- occurring protein molecule, "polypeptide" and like terms are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. By "antisense polynucleotide" is meant polynucleotide having a nucleotide sequence complementary to a given polynucleotide sequence including polynucleotide sequences associated with the transcription or translation of the given polynucleotide sequence, where the antisense polynucleotide is capable of hybridizing to a oHUase polynucleotide sequence. Of particular interest are antisense polynucleotides capable of inhibiting transcription and/or translation of a oHUase polynucleotide either in vitro or in vivo. By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 100 amino acids, preferably at least 200 amino acids, more preferably at least 250 amino acids, more preferably at least 300 amino acids, more preferably at least 350 amino acids, more preferably at least 400 amino acids, and most preferably at least 450 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 300 nucleotides, preferably at least 600 nucleotides, more preferably at least 750 nucleotides, more preferably at least 900 nucleotides, more preferably at least 1050 nucleotides, more preferably at least 1200 nucleotides, and most preferably at least 1350 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. By "therapeutically effective amount of a substantially pure oHUase polypeptide" is meant an amount of a substantially pure oHUase polypeptide effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated. For example, the degradation of hyaluronan is the desired therapeutic effect where oHUase is administered to the subject in the treatment of a condition associated with excess hyaluron, undesirable cell motility (e.g., tumor cell metastasis), and/or to enhance circulation of physiological fluids and/or therapeutic drugs at the site of administration and/or inhibit tumor growth or progression. The oHUase Coding Sequences In accordance with the invention, any nucleic acid sequence which encodes the amino acid sequence of oHUase can be used to generate recombinant molecules which express oHUase. The invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices (see table below). These combinations are made in accordance with the standard triplet genetic code as applied to the amino acid sequence of naturally occurring oHUase, and all such variations are ' to be considered as being specifically disclosed.
Figure imgf000010_0001
Figure imgf000011_0001
The nucleotide sequence encoding a oHUase can be isolated according to any one of a variety of methods well known to those of ordinary skill in the art. For example, DNA encoding oHUase can be isolated from either a cDNA library or from a genomic DNA library by either hybridization or expression cloning methods. Alternatively, the DNA can be isolated using standard polymerase chain reaction (PCR) amplification of synthetic oligonucleotide primers, e.g., as described in Mullis et al., U.S. Pat. No. 4,800,159, or expression cloning methods well known in the art (see, e.g., Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The sequence of isolated oHUase polypeptide-encoding DNA can be determined using methods well known in the art (see, for example, Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Following sequence confirmation, the resulting clones can be used to, for example, identify homologs of oHUase (e.g., other ovine alleles encoding oHUase or a hyaluronidase of another mammalian species, and/or to transform a target host cell for expression of DNA encoding a polypeptide of oHUase. Although nucleotide sequences which encode oHUase and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring oHUase under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding oHUase or its variants possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding oHUase and its variants without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence. It is now possible to produce a DNA sequence, or portions thereof, encoding a oHUase and its variants entirely by synthetic chemistry, after which the synthetic gene may be inserted into any of the many available DNA vectors and cell systems using reagents that are well known in the art at the time of the filing of this application. Moreover, synthetic chemistry may be used to introduce mutations into a oHUase sequence or any portion thereof. Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of the naturally occurring oHUase under various conditions of stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). "Maximum stringency" typically occurs at about Tm-5°C. (5°C. below the Tm of the probe); "high stringency" at about 5°C. to 10°C. below Tm; "intermediate stringency" at about 10°C. to 20°C. below Tm; and "low stringency" at about 20°C. to 25°C. below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences. The term "hybridization" as used herein shall include "any process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J. 1994 Dictionary of Biotechnology, Stockton Press, New York, NY). Amplification as carried out in the polymerase chain reaction technologies is described in Dieffenbach C.W. and G.S. Dveksler 1995 PCR Primer, a Laboratory Manual Cold Spring Harbor Press, Plainview NY. A "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. An "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring oHUase. A "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. Altered oHUase nucleic acid sequences which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent oHUase. The protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent oHUase. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of oHUase is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydropliilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, and tyrosine. Included within the scope of the present invention are alleles of oHUase. As used herein, an "allele" or "allelic sequence" is an alternative form of oHUase. Alleles result from a mutation, i.e., a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. The polynucleotide sequences encoding oHUase may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host. The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. In contrast, genomic oHUase sequences may have non-contiguous open reading frames, where introns interrupt the protein coding regions. Genomic sequences can also comprise the nucleic acid present between the initiation codon and the stop codon, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' or 3' end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The nucleic acid compositions of the subject invention may encode all or a part of the oHUase polypeptides as appropriate. Fragments maybe obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than about 50 nt to 100 nt are useful for production of the encoded polypeptide. For PCR amplification, it is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other. The oHUase-encoding sequences are isolated and obtained in substantial purity, generally as other than an intact mammalian chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a oHUase- encoding sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome. The DNA sequences are used in a variety of ways. They may be used as probes for identifying homologs of oHUase. Mammalian homologs have substantial sequence similarity to one another, i.e. at least 75%, usually at least 90%, more usually at least 95% sequence identity. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. 1990 JMol Biol 215:403-10. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C. and lOxSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C. in lxSSC. Sequence identity may be determined by hybridization under high stringency conditions, for example, at 50°C. or higher and O.lxSSC (9 mM saline/0.9 mM sodium citrate). By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes may be any species, for example, Primate species, particularly human; rodents, such as rats and mice, canines, felines, bovine, opines, equine, yeast, Drosophila, Caenhorabditis, etc. oHUase-encoding DNA can also be used to identify expression of the gene in a biological specimen. The manner in which one probes cells for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature and does not require elaboration here. mRNA is isolated from a cell sample. mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g., nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe. Other techniques, such as oligonucleotide ligation assays, in situ hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to a oHUase sequence is indicative of oHUase expression in the sample. oHUase-encoding sequences may be modified for a number of purposes, particularly where they will be used intracellularly, for example, by being joined to a nucleic acid cleaving agent, e.g., a chelated metal ion, such as iron or chromium for cleavage of the gene; or the like. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al. 1993 Biotechniques 14:22; Barany 1985 Gene 37:111-23; Colicelli et al. 1985 Mol Gen Genet 199:537-9; and Prentki et al. 1984 Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108; Weiner et al. 1993 Gene 126:35-41; Sayers et al. 1992 Biotechniques 13:592-6; Jones and Winistorfer 1992 Biotechniques 12:528-30; Barton et al. 1990 Nucleic Acids Res 18:7349-55; Marotti and Tomich 1989 Gene Anal Tech 6:67-70; and Zhu 1989 Anal Biochem 177:120-4.
Identification of Hyaluronidases Homologous to oHUase DNA encoding hyaluronidases homologous to oHUase (e.g., contain conservative amino acid substitutions relative to a native oHUase) can be accomplished by screening various cDNA or genomic DNA libraries by hybridization or PCR using oligonucleotides based upon the DNA sequence and/or amino acid sequence of a oHUase. Alternatively the oligonucleotides used may be degenerate, e.g., based upon a selected amino acid sequence of oHUase or designed so as to allow detection or amplification of DNA encoding a oHUase-like amino acid sequence having conservative amino acid substitutions and/or to take into account the frequency of codon usage in the mammalian species DNA to be screened. Such "degenerate oligonucleotide probes" can be used in combination in order to increase the sensitivity of the hybridization screen, and to identify and isolate oHUase analogs and orthologs in other species or variant alleles encoding oHUase. Methods for designing and using degenerate oligonucleotide probes to identify a protein for which an amino acid and/or nucleotide sequence, as well as methods for hybridization and PCR techniques for screening and isolation of homologous DNAs, are routine and well known in the art (see, for example, Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.). Methods of Making oHUase In addition to various purification procedures, oHUase polypeptides can be made by standard synthetic techniques, or by using recombinant DNA technology and expressed in bacterial, yeast, insect, or mammalian cells using standard techniques. As used herein, the term "oHUase" includes natural, recombinant, and modified forms of the protein unless the context in which the term is used clearly indicates otherwise.
Chemical Synthesis oHUase polypeptides can be synthesized based on the amino acid sequences described herein and variations thereof by standard solid-phase methods using the tert- butyloxy-carbonyl and benzyl protection strategy described in Clark-Lewis et al. 1993
PNAS USA 90:3574-3577; Clark-Lewis et al. 1991 Biochemistry 30:3128-3135; Stewart et al. 1969 Solid-Phase Peptide Synthesis, W H Freeman Co, San Francisco; and Merrifield J. 1963 J Am Chem Soc 85:2149-2154. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, CA) in accordance with the instructions provided by the manufacturer. Various fragments of oHUase may be chemically synthesized separately and combined using chemical methods to produce the full length molecule (α-form or β-form). After deprotection with hydrogen fluoride, the proteins are folded by air oxidation and purified by reverse-phase HPLC. Purity is determined by reverse-phase HPLC and isoelectric focusing. Amino acid incorporation is monitored during synthesis, and the final composition is determined by amino acid analysis. The correct covalent structure of the protein can be confirmed using ion-spray mass spectrometry (SCIEX API ).
Expression of oHUase Polypeptides Expression of a oHUase polypeptide is accomplished by inserting a nucleotide sequence encoding a oHUase polypeptide into a nucleic acid vector such that a promoter in the construct is operably linked to oHUase-encoding sequence. The construct can then be used to transform a mammalian, insect, yeast, or bacterial host cell. Numerous, commercially available vectors useful in recombinant polypeptide expression can be used. Preferably the vector is capable of replication in both eukaryotic and prokaryotic hosts, and is generally composed of a bacterial origin of replication and a eukaryotic promoter operably linked to a DNA of interest. A number of vectors suitable for stable transfection of mammalian, insect, yeast, and bacterial cells are available to the public from a wide variety of sources, for example, the American Type Culture Collection, Rockville, MD. Suitable host cells, as well as methods for constructing stably-transformed host cell lines, are also publicly available, e.g., Pouwels et al. 1985 Cloning Vectors: A Laboratory Manual; Ausubel et al. 1989 Current Protocols in Molecular Biology, John Wiley & Sons, New York; and Sambrook et al. 1989 Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y. Techniques for preparation of constructs containing nucleic acid sequence of interest and for obtaining expression of exogenous DNA or RNA sequences in a host cell are well known in the art (see, for example, Kormal et al. 1987 PNAS USA 84:2150-2154; Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY; each of which are hereby incorporated by reference with respect to methods and compositions for eukaryotic expression of a DNA of interest). Expression of recombinant oHUase polypeptide can be assayed by immunological procedures, such as Western blot or immunoprecipitation analysis of recombinant cell extracts, or by the HAse activity assay as described in for example, Tolksdorf, et al. 1949 JLab Clin Med 34:74; and Kass & Seastone, 1944 JExp Med 79:319. For example, oHUase polypeptides according to the invention can be produced by transformation of a suitable host cell (e.g., bacterial, yeast, insect or mammalian cell) with a oHUase polypeptide-encoding nucleotide sequence(s) in a suitable expression vehicle, and culturing the transformed cells under conditions that promote expression of the encoded polypeptide. Where the host cell is a mammalian cell, the vector is preferably designed to allow for secretion of oHUase into the culture medium. The method of transformation and the choice of expression vehicle will depend on the host system selected. Those skilled in the field of molecular biology will understand that any of a wide variety of prokaryotic and eukaryotic expression systems may be used to produce oHUase polypeptides of the invention. The precise host cell used is not critical to the invention. Expression Systems hi order to express a biologically active oHUase, the nucleotide sequence encoding oHUase or its functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a oHUase coding sequence and appropriate transcriptional or translational controls. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination or genetic recombination. Such techniques are described in Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY; and Ausubel F.M. et al. 1989 Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. A variety of expression vector/host systems may be utilized to contain and express a oHUase coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. The "control elements" or "regulatory sequences" of these systems vary in their strength and specificities and are those nontranslated regions of the vector, enhancers, promoters, and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, CA) or pSportl (Gibco BRL) and ptrp-lac hybrids and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a cell line that contains multiple copies of oHUase, vectors based on SV40 or EBV may be used with an appropriate selectable marker. hi bacterial systems, a number of expression vectors may be selected depending upon the use intended for oHUase. For example, when large quantities of oHUase are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the oHUase coding sequence may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β- galactosidase so that a hybrid protein is produced; pTN vectors (Van Heeke & Schuster 1989 JBiol Chem 264:5503-5509); and the like. pGEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST), hi general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will. In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. For reviews, see Ausubel et al. 1989 Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY; and Grant et al. 1987 Methods Enzymol 153:516-544. In cases where plant expression vectors are used, the expression of a sequence encoding oHUase may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al. 1984 Nature 310:511-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al. 1987 EMBO J 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al. 1984 EMBO J 3:1671-1680; Broglie et al. 1984 Science 224:838-843); or heat shock promoters (Winter J. & Sinibaldi R.M. 1991 Results Probl Cell Differ 17:85-105) may be used. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. For reviews of such techniques, see Hobbs S. or Murry L.E. in: McGraw Hill Yearbook of Science and Technology 1992 McGraw Hill New York, NY, pp. 191-196; or Weissbach and Weissbach 1988 Methods for Plant Molecular Biology, Academic Press, New York, NY, pp. 421-463. An alternative expression system which could be used to express oHUase is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The oHUase coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of oHUase will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which oHUase is expressed (Smith et al. 1983 J Virol 46:584; Engelhard E.K. et al. 1994 PNAS USA 91:3224-7). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a oHUase coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable viras capable of expressing oHUase in infected host cells (Logan & Shenk 1984 PNAS USA 81:3655-59). In addition, transcription enhancers, such as the rous sarcoma viras (RSV) enhancer, may be used to increase expression in mammalian host cells. Specific initiation signals may also be required for efficient translation of a oHUase sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where oHUase, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf D. et al. 1994 Results Probl Cell Differ 20:125-62; Bittner et al. 1987 Methods Enzymol 153:516-544). In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express oHUase may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex viras thymidine kinase (Wigler M. et al. 1977 Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy I. et al. 1980 Cell 22:817-23) genes which can be employed in tk' or aprf cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M. et al. 1980 PNAS USA 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin F. et al. 1981 J Mol Biol 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry L.E. in: McGraw Hill Yearbook of Science and Technology 1992 McGraw Hill New York, NY, pp. 191-196). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman S.C. & R.C. Mulligan 1988 PNAS USA 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase (GUS) and its substrate, and luciferase and its substrate, luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes CA. et al. 1995 Methods Mol Biol 55:121-131).
Identification of Transformants Containing the Polynucleotide Sequence Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed. For example, if the oHUase is inserted within a marker gene sequence, recombinant cells containing oHUase can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a oHUase sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem oHUase as well. Alternatively, host cells which contain the coding sequence for oHUase and express oHUase may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of the nucleic acid or protein. The presence of the oHUase polynucleotide sequence can be detected by DNA-
DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of oHUase. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the oHUase sequence to detect transformants containing oHUase DNA or RNA. As used herein "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer. A variety of protocols for detecting and measuring the expression of oHUase, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on oHUase is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R. et al. 1990 Serological Methods, a Laboratory Manual, APS Press, St Paul, MN; and Maddox D.E. et al. 1983 J Exp Med 158:1211). A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to oHUase include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the oHUase sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are coimnercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567. Host cells transformed with a oHUase nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing oHUase (α-form or β-form) can be designed with signal sequences which direct secretion of oHUase through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join oHUase to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll D.J. et al. 1993 DNA Cell Biol 12:441-53). oHUase may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and oHUase is useful to facilitate purification. One such expression vector provides for expression of a fusion protein compromising a oHUase and contains nucleic acid encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site. The histidine residues facilitate purification on LMIAC (immobilized metal ion affinity chromatography as described in Porath et al. 1992 Protein Express Purif 3:263-2$!), while the enterokinase cleavage site provides a means for purifying the chemokine from the fusion protein. Identification of Biologically Active oHUase and oHUase Polypeptides oHUase, α-form, or β-form polypeptide-encoding DNAs can encode all or a portion of oHUase. Preferably, the expressed polypeptide is biologically active, e.g., exhibits hyaluronidase activity in the cleavage of hyaluronan and/or can be bound by an anti-native oHUase antibody, i general, once information regarding the ability of a protein to elicit antibodies and/or information regarding an enzymatic or other biological activity of a protein of interest is known, methods for identification of biologically active polypeptides of the full-length protein are routine to the ordinarily skilled artisan, particularly where the nucleotide sequence and/or amino acid sequence encoding the protein of interest (here oHUase, α-form polypeptide, or β-form polypeptide) is provided as in the present case. Biologically active oHUase polypeptides can be identified by using conventional
HAse activity assays, for example, Tolksdorf, et al. 1949 J Lab Clin Med 34:74; and Kass & Seastone, 1944 J Exp Med 79:319. Alternatively, biologically active oHUase polypeptides can be detected by binding of an anti-native oHUase antibody to a component of the transformed host cell supernatant and/or lysate. oHUase polypeptides preferably exhibit at least 25%, more preferably 50%, still more preferably 75%, even more preferably 95% of the activity of native oHUase. Embodiments also include polypeptides that comprise essentially full-length oHUase or fragments thereof. These fragments can be, for example, at least 100, 105, 110 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200; 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470: 475, 480, 485, 490, 495, 500, 505, 510, 515, 516, 517, 518, and 519 amino acids in length as long as they have hyaluronidase activity. Embodiments also include nucleic acids encoding polypeptides that comprise essentially full-length oHUase or fragments thereof. These fragments of nucleic acids encoding oHUase can be, for example, at least 300, 315, 330, 345, 360, 375, 390, 405, 420: 435, 450, 465, 480, 495, 510, 525, 540, 555, 570, 585, 600, 615, 630, 645, 660, 675, 690 705, 720, 735, 750, 765, 780, 795, 810, 825, 840, 855, 870, 885, 900, 915, 930, 945, 960: 975, 990, 1005, 1020, 1035, 1050, 1065, 1080, 1095, 1110, 1125, 1140, 1155, 1170, 1185 1200, 1215, 1230, 1245, 1260, 1275, 1290, 1305, 1320, 1335, 1350, 1365, 1380, 1395 1410, 1425, 1440, 1455, 1470, 1485, 1500, 1515, 1530, 1545, 1548, 1551, 1554, and 1557 nucleotides in length as long as the polypeptides they encode have hyaluronidase activity. oHUase Antibodies oHUase-specific antibodies are useful in various immunotechniqu.es, including immunopurification and immunodetection techniques, and for the diagnosis of conditions and diseases associated with expression of HUase. oHUase for antibody induction does not require biological activity; however, the protein fragment, or oligopeptide must be antigenic. Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. They should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of oHUase amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to oHUase. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with oHUase or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Monoclonal antibodies to oHUase may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include but are not limited to the hybridoma technique originally described by
Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique
(Kosbor et al. 1983 Immunol Today 4:72; Cote et al. 1983 PNAS USA 80:2026-2030) and the EBV-hybridoma technique (Cole et al. 1985 Monoclonal Antibodies and Cancer
Therapy, Alan R Liss Inc, New York NY, pp 77-96). hi addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al. 1984 PNAS USA 81:6851-6855; Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985
Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce oHUase-specific single chain antibodies. Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989 PNAS USA 86:3833-3837), and Winter G. and Milstein C. )1991 Nature 349:293-299). Antibody fragments which contain specific binding sites for oHUase may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al. 1989 Science 256:1275-1281). A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between oHUase and its specific antibody and the measurement of complex formation. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific oHUase protein is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D.E. et al. (1983 JExp ed 158:1211).
Therapies Using oHUase Polypeptides The substantially pure oHUase polypeptides (e.g., oHUase polypeptides that are not associated with the components of tissue from which oHUase is purified) of the invention can be used in a variety of applications including human and veterinary therapies, either alone or in combination with other therapeutic agents. Purified oHUase of the invention can generally be used in place of Hyaluronidase (ACS) or Hyaluronidase WYDASE® where the condition to be treated is associated with excess hyaluronic acid and/or therapy is designed to increase oHUase activity generally. Pharmaceutical Compositions The present invention relates to pharmaceutical compositions which may comprise nucleotides, proteins, antibodies, agonists, antagonists, or inhibitors, alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers, hi one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert. Administration of Pharmaceutical Compositions Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the site of interest), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intravitreal, or intranasal administration. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton PA). Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, .e., dosage. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Manufacture and Storage The pharmaceutical compositions of the present invention may be manufactured in a manner that known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. After pharmaceutical compositions comprising a compound of the invention formulated in a acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of oHUase, such labeling would include amount, frequency and method of administration. Ηierapeutically Effective Dose Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors which ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD5o/ED5o. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, for example, tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drag combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art will employ different formulations for nucleotides than for proteins. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention. EXAMPLE 1 Ophthalmic Toxicities of Thimerosal, Hyaluronidase (ACS) and Hyaluronidase (Wydase®) in Rabbits Certain types of enzymes, when contacted with the vitreous humor following hemorrhage thereinto, will accelerate the rate at which the hemorrhagic blood is cleared from the vitreous humor. In this regard, a method is provided for accelerating clearance of hemorrhagic blood from the vitreous of the eye, said method generally comprising the step of contacting, with the vitreous humor, a quantity of hyaluronidase at a dose which is sufficient to accelerate the clearance of hemorrhagic blood from the vitreous without causing damage to the retina or other tissues of the eye. Preferably, the hyaluronidase is selected to have a molecular weight distribution which allows the hyaluronidase to be administered intravitreally at doses above 1 IU, and preferably above 15 IU, and advantageously above 75 IU, in the absence of thimerosal, without causing toxic damage to the retina or other tissues of the eye. This hemorrhage-clearing method may be performed without any vitrectomy or other surgical manipulation or removal of the vitreous humor, thereby avoiding the potential risks and complications associated with such vitrectomy procedures. The preferred route of administration of these hemorrhage-clearing enzymes is by intraocular injection directly into the vitreous body. Alternatively, , however, the hemorrhage-clearing enzyme(s) may be administered by any other suitable route of administration (e.g., topically) which results in sufficient distribution of the enzyme(s) to the vitreous body to cause the desired hemorrhage-clearing effect. The preferred injectable solution may contain a hyaluronidase which has a molecular weight distribution which allows it to be administered intravitreally at doses above 1 IU, and preferably above 15 IU, and advantageously above 75 IU, without causing toxic damage to the eye, along with inactive ingredients which cause the solution to be substantially isotonic, and of a pH which is suitable for injection into the eye. This preferred hyaluronidase preparation is preferably devoid of thimerosal. Such solution for injection may be initially lyophilized to a dry state and, thereafter, may be reconstituted prior to use. Under USP 6,610,292 and 6,551,590, which are hereby expressly incorporated by reference in their entireties, the term "hyaluronidase (ACS)" as used herein describes a hyaluronidase solution for intravitreal injection which is devoid of thimerosal and which is devoid of hyaluronidase molecular weight fractions above 100,000, between 50,000-60,000 and below 20,000, as determined by electrophoresis gel (4-20% gradient SDS-PAGE). Such hyaluronidase may be derived from ovine testicles and is available commercially from Biozyme Laboratories Limited, San Diego, California, which source may be a starting material for the disclosed process for isolating and purifying ovine hyaluronidase. This specific molecular weight distribution of the hyaluronidase (ACS) results in less ophthalmic toxicity than other hyaluronidase preparations, while exhibiting desirable therapeutic efficacy in a number of ophthalmic applications. As described in the following examples, hyaluronidase (ACS) may be injected directly into the posterior chamber of the eye at dosage levels which bring about desirable therapeutic affects, including but not necessarily limited to the intravitreal hemorrhage clearing effect, without causing significant toxicity to the eye or associated anatomical structures. Fifty-Two (52) healthy rabbits of the New Zealand Cross variety (26 male, 26 female) weighing 1.5 kg to 2.5 kg, were individually marked for identification and were housed individually in suspended cages. The animals received a commercially available pelleted rabbit feed on a daily basis, with tap water available ad libitum. The animals were divided into thirteen groups of 4 animals each (2 male, 2 female). Two animals in each group (1 male, 1 female) were selected for pretreatment fundus photography and fluorescein angiography. The fundus photography was performed by restraining the animals and visualizing the optic nerve, retinal arcades and fundus with a KOWA® RC-3 Fundus Camera loaded with Kodak Gold 200 ASA film. The fluorescein angiography involved a 1.5 ml injection of 2% sterile fluorescein solution via the marginal ear vein. Approximately 30 seconds post-injection the fluorescein was visualized upon localization of the optic nerve, retinal vessels and fundus. The following day, each animal was anesthetized by intravenous administration of a combination of 34 mg/kg of ketamine hydrochloride and 5 mg/kg xylazine. The eyelids were retracted using a lid speculum, and the eyes were disinfected with an iodine-providone wash. Experimental treatments of either balanced salt solution (BSS), BSS+thimerosal, hyaluronidase (Wydase®) or hyaluronidase (ACS) were admimstered by injection using a 1 cc tuberculin syringe with a 30 gauge, 0.5 inch needle attached thereto. The hyaluronidase (ACS) solution utilized in this example was free of thimerosal and constituted the specific formulation set forth in Table 1. Table 1. Specific Formulation
Figure imgf000033_0001
Table 2. The experimental treatments administered to each animal group were as follows:
Figure imgf000033_0002
The day following the injections (Day 1), the 26 animals which were subjected to the fundus photography and fluorescein angiography were observed using the same methods as for the pre-dose examination. On Day 2 following the injections, the 13 male rabbits that had received the fundus photography and fluorescein angiography at pre-dose and Day 1, as well as the 13 female rabbits that were not selected for photography were euthanized with a sodium pentobarbital based drug. The eyes were then surgically removed and placed in a fixture solution of 2.5% glutaraldehyde with 0.1 M phosphate buffered saline at pH 7.37. Alternatively, one randomly selected rabbit was euthanized by pentobarbital injection but then fixed by intracardiac injection of the of the glutaraldehyde solution into the left ventricle to determine the effect of the fixation procedure on the histology findings within the enucleated eyes. On Day 7, the 13 female rabbits that had been previously photographed and angiography performed were subjected to the same observations following the methods previously described. The remaining 26 animals were euthanized as described above 7 days after dosing. The eyes were fixed in the same manner as those which had been fixed on day 2. Also, one randomly selected rabbit was subjected to the same intracardiac glutaraldehyde fixation procedure described hereabove for the previously randomly selected animal. The eyes of the animals treated in this example were examined grossly and microscopically for evidence of treatment-related toxicities. A table setting forth a summary of the histological evidence of toxicity or non-toxicity in each treatment group, is set forth in Table 3. In summary, the eyes of the BSS-treated control group were free of toxicity at 2 and 7 days post dose. The eyes of the Group No. 2 animals treated with BSS+thimerosal (0.0075 mg) were free of toxicity at day 2, but exhibited evidence that there was a breakdown of the blood-retinal barrier at day 7. The Group No. 3 animals treated with BSS+thimerosal (0.025 mg) exhibited severe treatment-related toxic effects, at days 2 and 7 post dose. The Group No. 4 animals treated with Wydase® at the 1 LU. dose were free of toxicity at days 2 and 7, however, the eyes of the animals in Group Nos. 5-8 treated with Wydase® at dosages ranging from 15 I.U.-150 LU. exhibited generally dose-related toxic effects at days 2 and 7 post dose. The eyes of animals in treatment Groups Nos. 9-13 treated with the hyaluronidase (ACS) at dosages ranging from 1 LU. through 150 I.U., were free of evidence of toxic effects at days 2 and 7 post dose. Accordingly, it is concluded that thimerosal and the thimerosal-containing
Wydase® formulation do cause toxic effects in the eyes of rabbits at the dosages tested, however, the hyaluronidase (ACS) caused no toxic effects in these animals at the dosages tested. The results of the examinations conducted on day 7 are summarized in Table 3. As shown, in Table 3, significant toxic effects were observed on day 7 in the eyes of rabbits treated with BSS plus thimerosal (0.0075 mg.) and hyaluronidase (Wydase®) at all doses between 1 I.U.-150 LU. In contrast, no toxic effects were observed in the eyes of animals treated with the hyaluronidase (ACS) at doses between 1 and 150 IU. Table 3. Toxic Effect of Single Dose Intravitreal Injection of BSS*, BSS + Thimerosal, Hyaluronidase (ACS), and Hyaluronidase (Wydase®) in Rabbits
Figure imgf000035_0001
*BSS - Balanced Salt Solution EXAMPLE 2 Safety and Efficacy of the Hyaluronidase (ACS) Injected Intravitreally in Rabbit Eyes In this example, 12 healthy rabbits of the New Zealand Cross variety were marked for identification and individually housed in suspended cages. The animals received commercially pelleted rabbit feed on a daily basis and tap water was available ad libitum. The animals were randomly divided into four (4) treatment groups of three (3) animals each. Initially, the eyes of each animal were examined by dilation with 1-2 drops of 10% Tropicanide followed by gross examination, indirect ophthalmoscopy using a 20 diopter lens, and slit lamp examination of the anterior anatomy of the eye. Following the initial examination of the animals eyes, 100 μl or 10 μl of blood was injected intravitreally into each eye of each animal. On day 2, the animals of each treatment group received a single intravitreal injection of either BSS or the hyaluronidase (ACS) into the right eye, in accordance with the following treatment schedule:
Figure imgf000036_0001
The hyaluronidase (ACS) preparation used in this experiment was the preferred formulation described hereabove and shown in Table 1. On days 3, 5, 7, 14 and 21 the eyes of each animal were again examined by slit- lamp to evaluate the cornea, anterior chamber and iris. In addition, the eyes of each animal were dilated with 10% tropicamide solution and the retina was examined by indirect ophthalmoscopy with a 20 diopter lens. The observed hemorrhage-clearing efficacy of the hyaluronidase (ACS) is summarized in Table 4. In general, the left eye (untreated) of each animal in each treatment group contained hazy vitreous and some blood clots, due to the quantity of blood which had been injected therein. The right eyes of the BSS treated (control) animals of Group A also contained hazy vitreous and some blood clots, while the right eyes of all hyaluronidase- treated animals in Treatment Groups B-D contained vitreous which was clear and through which transvitreal visualization of the retina was possible. Furthermore, the retinas of the rights eyes of all animals in Treatment Groups B-D appeared normal and free of treatment- related toxicity. Table 4. Hemorrhage clearing Efficacy of Single-Dose Intravitreal Hyaluronidase (ACS) in the Rabbits (12 New Zealand rabbits are injected with 10 μl or 100 μl of blood in both eyes intravitreally)
Figure imgf000037_0001
*BSS - Balanced Salt Solution The results of this experiment indicate that intravitreally administered hyaluronidase (ACS) was effective at single doses of 25-75 IU. to accelerate the rate at which blood was cleared from the eyes of the treated animals and further that such single doses of hyaluronidase (ACS) administered in this experiment did not cause observable toxic effects in the eyes of the rabbits treated in this experiment. The observations following each dose were consistent and are summarized in Table 5. In general, the left eye (untreated) of each animal in each treatment group, contained hazy vitreous humor and some blood clots, due to the quantity of blood which had been injected therein. The right eyes of the BSS treated (control) animals of Group A also contained hazy vitreous and some blood clots, while the right eyes of all animals in treatment Groups B-E (i.e., the animals treated with hyaluronidase (ACS)) contained clear vitreous through which transvitreal visualization of the retina was possible. Furthermore, the retinas of the right eyes of all animals in treatment Groups B-D appeared to be normal and free of treatment-related toxicity, even after multiple doses of the hyaluronidase (ACS). Table 5. Safety and Efficacy of Multiple-Dose Intravitreal Hyaluronidase (ACS) in Rabbit
Figure imgf000038_0001
The results of this experiment indicate that intravitreally administered hyaluronidase (ACS) was effective, at single doses of 25-75 LU. x 4, to accelerate the rate at which blood was cleared from the eyes of rabbits and that such doses of the hyaluronidase (ACS) did not cause observable toxic effects in the eyes of the treated rabbits, even after four (4) consecutive doses of the hyaluronidase (ACS) administered at 2 week intervals. EXAMPLE 3 Safety and Efficacy of the Hyaluronidase (ACS) Injected Intravitreally in Human Eyes The primary objective of this study was to deteπnine if a balanced salt solution containing a highly purified hyaluronidase extract from ovine testicular tissue could be injected into the vitreous of visually impaired eyes without eliciting any serious ocular adverse effects. Materials and Methods Balanced Salt Solution (BSS) was employed as the placebo control, and was obtained from Allergan Pharmaceuticals (Irvine, Calif.). The BSS contained 0.64% sodium chloride, 0.075% potassium chloride, 0.048% calcium chloride dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% sodium acetate trihydrate, 0.17% sodium citrate dihydrate, sufficient sodium hydroxide/hydrochloric acid for adjustment of pH to 7.1-7.2, and water for injection (q.s. 100%). Thirty microliter aliquots of BSS or hyaluronidase specific formulation X (Table 6) were loaded into a 300 μl microsyringe fitted with a 29 gauge needle 0.5 inches in length. The loaded microsyringes were then used to inject the material into the vitreous of the patient's eye. Table 6. Specific formulation X
Figure imgf000039_0001
Initially, eight human subjects with at least one visually impaired eye were randomly assigned to receive intravitreally either 50 μl of 50 LU. of the hyaluronidase (ACS) in BSS or BSS alone (3:1 ratio). After one month of follow-up to assure the 50 LU. dosage was well-tolerated, a second group of six visually impaired subjects were enrolled in the study and randomly assigned to a higher hyaluronidase (ACS) dosage group (100 LU.) or the BSS control in a 2:1 ratio. Procedures used to evaluate the safety of the test articles were completed at various intervals throughout the study, and included indirect ophthalmoscopy, fundus photography, fluorescein angiography, elecfroretinography, external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, and autorefraction. A concurrent placebo control group was included in the study so that adverse events peculiarly related to hyaluronidase (ACS) could be distinguished from those attributable to the vehicle (BSS)/injection procedure. Only visually impaired eyes were treated, moreover, since the test articles were injected proximate to the retina and any untoward responses of a serious nature could have been sight threatening. Patients were assigned to treatment using a computer generated randomization scheme beginning with the number 601 for the first phase of the study, and 701 for the second. Neither the patients nor investigators were aware of whether it was the BSS vehicle or hyaluronidase (ACS)/BSS solution that was being injected intravitreally. Following establishment of a baseline for each patient, the subjects were injected with either the enzyme or the placebo control. Patients were placed in a sitting position on a comfortable chair. One or two drops of a local anesthetic were topically instilled into the eye that was to be treated, after which the patient was asked to look down and a sterile cotton swab soaked in Proparacaine Hydrochlori.de Ophthalmic solution was applied for 10 seconds to an area on the sclera approximately 4-5 mm above the cornea (superior position 12:00 meridian). The test article was then injected into the vitreous through a 29 gauge needle attached to a 200 μl microsyringe that was inserted up to the full length of the needle at the site of application of the second anesthetic. Results Although only infrequently attaining statistical significance, the slit lamp biomicroscopy data suggested that a substantially higher proportion of patients treated with the hyaluronidase (ACS)/BSS preparations as opposed to BSS alone exhibited anterior segment pathologic changes, the most prominent being the presence of cells and flare in the anterior chamber. After the sixth (one month post treatment) visit, however, no intergroup differences were observed for any of the slit lamp assessed variables. Retinal/cortical responses, as measured by elecfroretinography/visual evoked potential, deteriorated over time in one patient treated with BSS and two who were given 50 LU. of hyaluronidase (ACS)ZBSS. However, alterations in electroretinographic patterns were always bilateral and did not occur in either the treated or untreated eyes of the patients assigned to high dose (100 LU.) hyaluronidase (ACS)/BSS, nor did fluorescein angiographic test results indicate that retinal ischemia was present in any eye irrespective of treatment. The indirect ophthalmoscopic exams revealed liquefaction and the establishment of posterior vifreal detachment (PVD) in the eyes of the test subjects. The vitreous was characterized as exhibiting a high degree of motility and/or liquefaction soon after injecting the test articles, which was expected for the hyaluronidase (ACS)-containing preparations. Certain test eyes injected with BSS control showed liquefaction and PVD, which was likely present before treatment, since the latter did not possess any enzymatic activity and was given in very small volume (30 μl). Concerning PVD, in the first group of patients, four of the six patients to be freated with hyaluronidase (ACS) displayed the absence of PVD by slit lamp biomicroscopy (i.e., 601, 602, 604, and 606) (See Table 7 below). After treatment, each of these subjects showed the presence of PVD. The results from the second group of patients were less clear, due to difficulties in imaging the vitreous using slit lamp microscopy. Table 7. Human Safety Study with 50 μl and 100 μl Hyaluronidase (ACS) Injection Intravitreall
Figure imgf000041_0001
Given the results from Example 2 where injection of hyaluronidase (ACS) into the vitreous of rabbits at various doses up to 150 LU. did not result in any significant histopathologic changes in an earlier preclinical study, it was expected that doses below 150 LU. would be well-tolerated in humans. Consistent with this expectation, the intravitreal administration of hyaluronidase (ACS)/BSS into visually impaired eyes in the current trial elicited few symptoms, all of which were believed attributable to the injection procedure itself as they occurred with comparable frequency in each of the study groups, and treatment-related adverse sequelae were relatively mild and of short duration. Furthermore, freatment of human eyes with hyaluronidase (ACS) was observed to increase the incidence of observed posterior vifreal detachment. The observed increase in PVD in patients injected intravitreally with hyaluronidase (ACS) shows that the methods described herein are effective in inducing liquefaction and detachment of the vifreal humor. Thus, the results of the present study indicate that hyaluronidase (ACS) can be injected into the vitreous of humans without eliciting any serious or long-term ocular complications. EXAMPLE 4 Use of Hyaluronidase to Accelerate the Clearance of Hemorrhagic Blood from the Vitreous of the Eye The Example set forth herebelow describes cases in which intravitreal hyaluronidase (ACS) was used to accelerate the clearance of hemorrhagic blood from the vitreous of the eye. The hyaluronidase used was the thimerosal-free hyaluronidase (ACS) formulation described above and shown in Table 8. In this experiment, six (6) human patients (5 female, 1 male) who presented with vitreous hemorrhage were treated with single intravitreal injections of hyaluronidase (ACS) at dosages of 50-200 LU. The hyaluronidase (ACS) administered in this experiment was prepared by the formulation, described hereabove and shown in Table 8. Table 8. Specific Formulation Z
Figure imgf000042_0001
All of the patients treated in this experiment had a history of diabetic retinopathy, and were found to have vitreous hemorrhages of varying duration. In each patient, the amount of blood present in the vitreous was sufficient to prevent viewing of the retina by standard funduscopic means. Each patient received a single intravitreal injection of hyaluronidase (ACS). Four (4) patients received a dose of 50 I.U., one (1) patient received a dose of 70 I.U., and one (1) patient received a dose of 200 LU. The observed results of this experiment are summarized in Table 9. Table 9. Hemorrhage Clearing Efficacy of Single Intravitreal Injection of Hyaluronidase (ACS) in Human Patients with Diabetic Retinopathy
Figure imgf000043_0001
In the six (6) patients treated in this example, the hemorrhagic vitreous became sufficiently clear to permit frans-vifreal viewing of the retina within 6-16 days of the single intravifreal injection of the hyaluronidase (ACS). Such clearing of the vitreous was subjectively determined to have occurred significantly faster than that which would have been expected to occur in these patients without hyaluronidase treatment. It should be noted that unlike the fluorescein leakage observed at higher doses of hyaluronidase (ACS) in rabbits, no toxicity was observed in the present human based study. EXAMPLE 5 Use of Hyaluronidase to Treat Other Ophthalmological Disorders Even a single intravitreal administration of hyaluronidase (ACS), at an experimental dose, is efficacious in treating certain ophthalmological disorders. Patients suffering from previously diagnosed disorders of the eye, including proliferative diabetic retinopathy, age- related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates and cystoid macular edema, have exhibited improvement in the clinical symptoms of these disorders upon freatment with hyaluronidase (ACS). Hyaluronidase (ACS) is capable of being administered intravitreally at doses of or in excess of 1 LU. without causing toxic damage to the eye and thus is useable to effect prompt liquefaction of the vitreous body and concomitantly the disconnection or detachment of the vifreous body from the retina and other tissues (e.g., epiretinal membranes, macula). As a result of this vifreal liquefaction and detachment, the physical pulling forces of the vitreous on the retina and other tissues are minimized and the rate of natural turnover of fluids within the vitreous is accelerated. Accordingly, hyaluronidase (ACS) is particularly suitable for the treatment of many disorders (e.g., proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates and cystoid macular edema) which benefit from liquefaction disconnection of the vitreous and/or accelerated clearance of toxins or other deleterious substances (e.g., angiogenic factors, edema fluids, etc.) from the posterior chamber of the eye and/or from tissues adjacent the posterior chamber (e.g., the retina or macula). Moreover, liquefaction of the vitreous is also believed to remove the matrix, in the form of the polymerized vitreous, necessary to support neovascularization. Thus, the present method is useful in preventing or reducing the incidence of retinal neovascularization. Furthermore, many ophthalmic disorders have as a causative component, a destabilization of the blood-retina membrane. This destabilization permits various components (e.g., serum components, lipids, proteins) of the choriocapillaries to enter the vifreal chamber and damage the retinal surface. This destabilization is also a precursor to vascular infiltration of the vifreal chamber, known as neovascularization. Accordingly, embodiments of the present method are directed toward the prevention and treatment of various disorders of the mammalian eye which result from damage or pathology to the vascularization of the eye or which result in damage to the blood-retinal barrier. Examples of such diseases include but are not limited to proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, macular exudates, and cystoid macular edema, and others in which the clinical symptoms of these disorders respond to the hyaluronidase (ACS) treatment. EXAMPLE 6 Hyaluronidase Treatment of Proliferative Diabetic Retinopathy (PDR) Diabetic retinopathy is the leading cause of blindness in working age Americans. The incidence of retinopathy increases with the time of the disease state, from a level of about 50% manifestation in diabetics with the disease for 7 years to approximately 90% of those with the disease for more than 20 years. It is estimated that PDR affects an estimated 700,000 Americans. The retinovascular consequences of diabetes essentially consist, in part, of microvascular leakage and capillary nonperfusion resulting from chronic hyperglycemia. Microvascular leakage may in turn result in retinal edema, lipid exudates and intraretinal hemorrhages. Capillary nonperfusion results in the formation of intraretinal microvascular abnormalities (LRMA). These abnormalities are arteriovenous shunts formed to perfuse retinal regions deprived of vascularization by diabetes-mediated arteriole degeneration. Expression of vascular endothelial growth factor from a hypoxic retina in areas of capillary nonperfusion is thought to result in the development of extraretinal neovascularization. Such neovascularization and its associated fibrous components may spontaneously involute or be complicated by vitreous hemorrhage or traction retinal detachment. Neovascularization may be easily seen on fluorescein angiogram by the profuse leakage of dye from these new vessels since they lack the tight endothelial junctions of the retinal vasculature. Impaired axoplasmic flow in areas of retinal hypoxia result in cotton wool spots. Proliferative diabetic retinopathy (PDR) requires careful screening of diabetics for early identification and treatment since PDR remains largely asymptomatic in the early stages. Proliferative diabetic retinopathy can be classified into three subgroups: (1) nonprohferative retinopathy; (2) preproliferative retinopathy; (3) proliferative retinopathy. Each classification has certain morphological characteristics. Features of nonprohferative retinopathy include capillary microangiopathy (microvascular obstructions and permeability changes, nonperfusion of capillaries, retinal capillary microaneurysms, basement membrane thickening, and internal microvascular abnormalities (LRMA)); intraretinal hemorrhages; exudates; and macular changes. Preproliferative retinopathy is indicated by any or all of the changes described for nonprohferative retinopathy and the following: significant venous beading, cotton-wool exudates, extensive LRMA and extensive retinal ischemia. Proliferative retinopathy is indicated by extraretinal neovascularization and fibrous tissue proliferation, vifreous alterations and hemorrhage, macular disease, and retinal detachment. The creation of fibrovascular tissue is an especially important complication of PDR since it often will lead to retinal damage mediated by the vitreous. The fibrovascular tissue may form preretinal membranes that create dense adhesions with the posterior hyaloid membrane. These adhesions are responsible for transmitting the forces of vifreous fraction to the retina, which may result in retinal detachments. The vitreous base is normally firmly attached to the adjacent retina and to the outer circumference of the optic nerve head, known as the ring of Martegiani. The attachment of the vifreous to the retina in all other sites between the ring of Martegiani and the vifreous base is much less firm. Neovascularization from the retina leads to the formation of vascular strands extending into the vifreous from the nerve head or elsewhere in the fundus. Contraction of these strands may cause partial or complete retinal detachment. Retinal detachment at the macula is a major complication of PDR. Most retinal detachments resulting from PDR begin as fractional detachments without holes, but they may become rhegmatogenous by the formation of retinal holes at some later point in the disease. The fractional detachments are caused by abnormal vifreoretinal adhesions or vifreal traction with subsequent shrinkage of the fibrous bands and elevation of the retina. The present method contemplates treatment of PDR in the preproliferative and proliferative states using hyaluronidase (ACS) intravitreal injections. Without being limited to a particular mechanism, it is believed that the effect of intravitreal hyaluronidase (ACS) injection is to promote the clearance of the liquid phase of the vitreous. The rate of transfer of intravitreally injected tritiated water from the mid vitreous to the choroid was significantly increased after depolymerization of vitreous hyaluronic acid by injected hyaluronidase (ACS). The present method capitalizes upon this observation to liquefy the vitreous, for example, in order to promote the clearance of various growth inducing factors and other serum products leaked into the vifreous due to the presence of PDR. It is further contemplated that the hyaluronidase (ACS) freatment of the present method may be performed alone or in combination with other treatments of PDR. EXAMPLE 7 Treatment of Non-Proliferative Diabetic Retinopathy Purpose: To determine the effect of hyaluronidase (ACS) on progression of moderately severe to severe non-proliferative diabetic retinopathy (NPDR in the presence or absence of an induced posterior vifreous detachment (PVD). Methods: sixty patients evaluated by ulfrasonography and masked fundus photography were randomly assigned to: saline (0.05 ml), hyaluronidase (ACS) (75 I.U., 0.05 ml), SF6 gas (0.3 ml) or hyaluronidase (ACS) plus SF6 gas 4 weeks later. PVD was assessed through week 16; seven-field fundus. photographs were obtained as surrogate baseline and 12 months later. Results: Of all eyes treated, without regard to PVD, those with stable ETDRS scores were: saline 38% (6/16), hyaluronidase (ACS) 67% (10/15), SF6 40% (6/15) and hyaluronidase (ACS) plus SF6 43% (6/14). Worsening of ETDRS scores were: saline 38% (6/16), hyaluronidase (ACS) 13% (2/15), SF6 20% (3/15) and hyaluronidase (ACS) plus SF6 21% (3/14). Percent of eyes with a complete PVD on or prior to 16 weeks and stable ETDRS scores were: saline 0% (0/14), hyaluronidase (ACS) 50%| (6/12), SF6 30% (3/10) and hyaluronidase (ACS) plus SF6 40%) (4/10). Reduced visual acuity across all groups was the most frequently reported ocular adverse event. Conclusions: Eyes treated with hyaluronidase (ACS) had stable ETDRS retinopathy scores compared to saline. The same number of saline-treated eyes had stable ETDRS scores as worsened scores. The percent of eyes with a complete PVD and stable ETDRS scores was highest in hyaluronidase (ACS) group. These data indicate that induction of a PVD and/or enzymatic liquefaction of the vitreous has the ability to affect the progression of diabetic retinopathy. EXAMPLE 8 Treatment of Preproliferative Diabetic Retinopathy hi this Example, a diabetic patient manifesting preproliferative diabetic retinopathy is treated for this complication of diabetes mellitus through the intravitreal injection of hyaluronidase (ACS). The purpose of this treatment is to reduce or prevent the development of proliferative diabetic retinopathy manifested by extraretinal neovascularization and fibrous tissue proliferation, vitreous alterations and hemorrhage, macular disease, and retinal detachment. Once a patient has been diagnosed with diabetes, increased ophthalmic surveillance is performed, given the high percentage of individuals suffering from this disease later developing proliferative diabetic retinopathy (PDR). This increased surveillance should include periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, LRMA, and retinal ischemia. When preproliferative diabetic retinopathy begins reaching the proliferative stage, the hyaluronidase (ACS) freatment is commenced. This stage is defined as the presence of venous beading in 2 or more quadrants, LRMA in one or more quadrants, and/or microaneurysm and dot hemorrhages in all quadrants. Once these indicia are present, hyaluronidase (ACS) method of freatment is initiated. The patient is to receive a full ophthalmic examination to establish a baseline of ocular health. The ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements. Following the preliminary examination, an intravifreal injection of hyaluronidase
(ACS) is given to the patient's affected eye. If both eyes are affected, they may be freated separately. The eye to be treated is injected with 50 μl of 50 LU. of the hyaluronidase
(ACS) ophthalmic solution described above intravitreally to promote the depolymerization of vitreous hyaluronic acid, resulting in the liquefaction of the vitreous. After freatment, the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). On each examination day the patient is monitored for vitreous liquefaction. Additionally, the patient is monitored for posterior vifreous detachments using indirect ophthalmoscopy with scleral depression. Finally, the extent of PDR presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, LRMA, and retinal ischemia. EXAMPLE 9 Treatment of Proliferative Retinopathy In this Example, a diabetic patient manifesting proliferative diabetic retinopathy is freated by the intravitreal injection of hyaluronidase (ACS). The purpose of this treatment is to reduce the extent of proliferative diabetic retinopathy, to prevent further manifestations of the disease after removal of any exfraretinal neovascularized tissue, and to reduce the likelihood of retinal detachment. A patient presenting proliferative diabetic retinopathy is to receive the hyaluronidase (ACS) method of treatment in combination with surgical treatment of the neovascularized tissue. The proliferation usually begins with the formation of new vessels with very little fibrous tissue component. They arise from primitive mesenchymal elements that differentiate into vascular endothelial cells. The newly formed vascular channels then undergo fibrous metaplasia; that is, the angioblastic buds are transformed into fibrous tissue. The new vessels leak fluorescein, so the presence of proliferation is especially noticeable during angiography. The new vessels and fibrous tissue break through the internal limiting membrane and arborize at the interface between the internal limiting membrane and the posterior hyaloid membrane. The fibrovascular tissue may form preretinal membranes that create dense adhesions with the posterior hyaloid membrane. These adhesions are extremely important because they are responsible for transmitting the forces of vitreous fraction to the retina during the later stage of vitreous shrinkage. The proliferative stage of PDR is defined as the presence of three or more of the following characteristics: new vessels, new vessels on or within one disc diameter of the optic nerve, severe new vessels (as defined by one-third disc area neovascularization at the optic nerve or one-half disc area neovascularization at the optic nerve or one-half disc area neovascularization elsewhere), and preretinal or vitreous hemorrhage. Once diagnosed as entering the proliferative stage, the patient is to receive a full ophthalmic examination to establish a baseline of ocular health. The ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected visual acuity) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements. Following the preliminary examination, an intravitreal injection of hyaluronidase (ACS) is given to patient's affected eyes. If both eyes are affected, they may be treated separately. The eye is injected with 50 μl of 50 LU. of the hyaluronidase (ACS) ophthalmic solution intravitreally to promote the depolymerization of vitreous hyaluronic acid, resulting in the liquefaction of the vifreous. In addition to depolymerization of the vifreous, the neovascularized tissue is also treated directly to minimize subsequent damage to the retina using panretinal photocoagulation. Panretinal photocoagulation (PRP) may be used to treat patients presenting PDR in conjunction with the hyaluronidase (ACS) method of treatment. Panretinal photocoagulation is a form of laser photocoagulation. Currently lasers such as the argon green (614 nm), argon blue-green (488 and 514 nm), krypton red (647 nm), tunable dye, diode and xenon arc lasers, are used for retinal surgery. Laser energy is absorbed predominantly by tissues containing pigment (melanin, xanthophyll, or hemoglobin) producing thermal effects on adjacent structures. Krypton red lasers are the preferred method of freatment, as they are better able to penetrate nuclear sclerotic cataracts and vitreous hemorrhage than the argon lasers, which require more energy to produce equal levels of penetration. The parameters used during laser retinal surgery may be modified depending on the goal of the photocoagulation. At lower power setting, using longer durations of treatment and producing larger spot sizes, the laser has a coagulative effect on small vessels. Focal laser photocoagulation is used in diabetes to stop leakage of microaneurysms. The laser spot is place directly over the microaneurysm to achieve a slight whitening and closure of the aneurysm. When applied as a grid over an edematous area of retina, the laser may reduce microvascular leakage. At higher energy levels, laser ablation of tissue is possible. Panretinal photocoagulation is thought to be effective by destroying tissue, reducing the amount of ischemic tissue in the eye. Confluent laser spots may be used over a neovascular membrane to obliterate the abnormal vessels. It should be understood that the present method does not require a particular order of treatment. In one embodiment, the patient is first freated with hyaluronidase (ACS) and then laser freatment. In. another embodiment the patient is first undergoes laser treatment followed by the hyaluronidase (ACS) treatment. After treatment, the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). On each examination day the patient is monitored for vifreous liquefaction. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression. Finally, the extent of PDR presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor the extent of venous beading, LRMA, retinal ischemia, neovascularization, and vifreal hemorrhage. Evidence of new neopolymerization or incomplete depolymerization of the vitreous would warrant a repeat treatment of the patient as described above. EXAMPLE 10 Hyaluronidase Treatment of Age-Related Macular Degeneration The present methodology also contemplates utility in the freatment of age-related macular degeneration (AMD). Age-related macular degeneration consists of a gradual, often bilateral decrease of vision. It is the most common cause of legal blindness in adults.
It is probably caused by aging and vascular disease in the choriocapillari.es or the afferent retinal vessels. There are basically two morphologic types of AMD: "dry" and "wet". The underlying abnormality of AMD is the development of involutional changes at the level of Bruch's membrane and the retinal pigment epithelium (RPE). The hallmark lesion of such changes is the druse. Clinically, drasen (the plural form of druse) appear as small, yellow-white deposits at the level of the RPE. Drasen may be categorized as hard, soft or basal laminar drasen. The present method is directed both to the freatment and prevention of wet and dry forms of AMD. hi the wet form the disease, the condition is thought to affect the choriocapillaries. The choriocapillari.es are a component of the choroid which serves to vascularize the globe. The choriocapillaries consists of a rich capillary network that supply most of the nutrition for the pigment epithelium and outer layers of the retina. Damage to the choriocapillaries is thought to result ultimately in neovascular complications, a cause of macular degeneration. In the dry form, nondisciform macular degeneration results from a partial or total obliteration of the underlying choriocapillaries. Ophthalmoscopically, degeneration of the retinal pigment epithelium and hole formation may be observed. Also, subpigment epithelial deposits of material such as calcium chelates or proteinaceous material and others may be observed, h dry AMD, secondary retinal changes generally occur gradually, resulting in the gradual loss of visual acuity. Nevertheless, in some percentage of patients, a severe loss of vision results. The present method contemplates utility in treating dry AMD and preventing macular degeneration through liquefaction of the vifreous. It is contemplated that the liquefaction of the vitreous would result in an increase in the rate of clearance from the retina of deposited material that later results in macular degeneration. Wet AMD most frequently results from choriocapillary insufficiency, leading to subsequent subpigment epithelial neovascularization. Neovascularization also is thought to occur as an adaptation of retinal vascularization to inadequate oxygenation as a result of vesicular damage. Neovascularization may also cause several other disorders such as detachment of the pigment epithelium and sensory retina. Typically the disease usually begins after 60 years of age, manifesting in both sexes equally and in patients presenting the disease, bilaterally. Perhaps the most important complication of age-related macular degeneration is the development of defects in Bruch's membranes of the globe through which new vessels grow. This epithelial neovascularization may result in the production of exudative deposits in and under the retina. The neovascularization may also lead to hemorrhage into the vitreous, which may lead to degeneration of the retina's rods and cones, and cystoid macular edema (discussed below). A macular hole may form which results in irreversible visual loss. Although affecting only 10% of patients with AMD, neovascular complications of AMD account for the overwhelming majority of cases of severe visual loss. Risk factors include increasing age, soft drasen, nongeographic atrophy, family history, hyperopia, and retinal pigment epithelial detachments. Symptoms of choroidal neovascularization in AMD include metamorphopsia, paracentral scotomas or diminished central vision. Ophthalmoscopic findings include subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane. Fluorescein angiography is often an effective method of diagnosis. During this diagnostic procedure, progressive pooling of the dye in the subretinal space, seen as blurring of the boundaries of the lesion or leakage from undetermined sources are indicators of the disease. Other components of choroidal neovascular membranes as delineated by fluorescein angiography include elevated blocked fluorescence, flat blocked fluorescence, blood, and disciform scar. The present understanding of neovascular AMD suggests that classic choroidal neovascularization is the lesion component most strongly associated with rapid visual deterioration. Accordingly, treatment of AMD must encompass all neovascular and fibrovascular components of the lesion. At present, freatment is only indicated when classic neovascularization has boundaries that are well demarcated, and photocoagulation has been shown to be beneficial. In eyes with extrafoveal choroidal neovascularization (>-200 microns from the foveal center), argon laser photocoagulation diminished the incidence of severe visual loss, ($6 lines) at 5 years from 64% to 46%. Recurrent neovascularization developed in one-half of laser-treated eyes, usually in the first year after treatment. Recurrent neovascularization was invariably associated with the development of severe visual loss. In eyes with juxtafoveal choroidal neovascularization (1 to 199 microns from the foveal center), krypton laser photocoagulation diminished the incidence of severe visual loss from 45% to 31% at 1 year, although the difference between untreated and freated groups was less marked at 5 years. Laser treatment remains an essential therapeutic method for the freatment of AMD, however, the present method would augment this approach by reducing the reoccurrence of neovascularization. Treatment of Age-Related Macular Degeneration In this Example, a patient manifesting age-related macular degeneration is freated with an intravitreal injection of hyaluronidase (ACS). The purpose of this freatment is to reduce or prevent the development of neovascularization, macular disease, and retinal damage. Once a patient reaches the age of 60, increased ophthalmic surveillance is performed to detect the presence of AMD. This increased surveillance should include periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane. When AMD is diagnosed, a regime of hyaluronidase (ACS) treatment is commenced coupled with or without other treatments such as photocoagulation. As the first step of treatment, the patient is to receive a full ophthalmic examination to establish a baseline of ocular health. The ophthalmic examination includes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements. Following the preliminary examination, an intravifreal injection of hyaluronidase (ACS) is given to the patient's affected eye manifesting AMD. If both eyes are affected, they may be freated separately. The eye to be freated is injected with 50 μl of 50 LU. of the hyaluronidase (ACS) ophthalmic solution (described above) intravifreally to promote the depolymerization of vifreous hyaluronic acid, resulting in the liquefaction of the vifreous. Laser photocoagulation treatment of the hyaluronidase (ACS) injected eyes may be required. The laser treatment protocol described in Examples 8 and 9 should be followed when treating AMD. In an alternative embodiment, photocoagulation treatment occurs before the enzyme freatment of the present method. After freatment, the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). Because of the possibility of reoccurrence, the patient should return for periodic examinations on a monthly basis thereafter. On each examination day the patient is monitored for vifreous liquefaction. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression. Finally, the extent of AMD presented by the patient is continuously monitored through periodic retinal examinations and fluorescein angiograms to monitor for the presence of subretinal fluid, blood, exudates, RPE detachment, cystic retinal changes, or the presence of grayish green subretinal neovascular membrane. Additional hyaluronidase (ACS) and/or laser treatments may be required if indicia of reoccurring neovascularization are observed. The following Example demonstrates the efficacy of the present method, even without the use of photocoagulation. Improvement in Symptoms of Macular Degeneration Following Intravitreal Hyaluronidase Infection A greater than seventy-nine (79) year old male human being presented with a history of age-related macular degeneration, and uncorrected vision of 20:400 in his right eye. A single dose of 100 LU. of the hyaluronidase (ACS) was injected intravitreally into his right eye. The other eye remained untreated. The patient was repeatedly examined post-dose and the vision in his left (untreated) eye remained unchanged, while the vision in his right (treated) eye was observed to improve as follows: .
Figure imgf000054_0001
*cf denotes finger counting No adverse effects of the hyaluronidase (ACS) were observed in this experiment. EXAMPLE 11 Hyaluronidase Treatment of Amblyopia The term amblyopia is derived from Greek and means dull vision (amblys— dull, ops— eye). Poor vision is caused by abnormal development in visual areas of the brain, which is in turn caused by abnormal visual stimulation during early visual development. The pathology associated with amblyopia is not specific to the eye, rather, it is located in the visual areas of the brain including the lateral geniculate nucleus and the striate cortex. This abnormal development is caused by three mechanisms: (1) blurred retinal image called pattern distortion; (2) cortical suppression, or (3) both cortical suppression plus pattern distortion. The present method is primarily concerned with pattern distortions caused by media opacity. More specifically, the present method addresses issues of vitreous opacity. Amblyopic vision is usually defined as a difference of at least two Snellen lines of visual acuity. Critical to the treatment of amblyopia is early detection and early intervention. The strategy for treating amblyopia caused by vitreous opacity is to provide a clear retinal image by altering the opacity of the vitreous so that clear vision results. In this Example, a patient manifesting amblyopia resulting from vifreal opacity was treated with an intravitreal injection of hyaluronidase (ACS). The purpose of this treatment was to reduce the opacity of the vifreous by increasing the exchange rate of the liquid in the vifreous. A forty (40) year old female human being having a history of amblyopia presented with uncorrected vision of 20:400 in her right eye and corrected vision in that eye of 20:200. A single 100 LU. dose of the hyaluronidase (ACS) was injected intravitreally into her right eye. The other eye remained untreated. The patient was examined repeatedly post-dose and the vision in her left (untreated) eye remained unchanged while the vision in her right (treated) eye was observed to improve as follows:
Figure imgf000055_0001
No adverse effects of the hyaluronidase (ACS) were observed in this patient. EXAMPLE 12 Hyaluronidase Treatment of Retinitis Pigmentosa Retinitis pigmentosa (RP) is the name given to a group of heritable disorders of progressive retinal degeneration characterized by bilateral nyctalopia constricted visual fields and abnormality of the elecfroretinogram. Early symptoms include difficulty with dark adaptation and midperipheral visual field loss. As the disease progresses, visual field loss advances, typically leaving a small central field of vision until eventually even central vision is affected. Central acuity may also be affected earlier in the course of disease either by cystoid macular edema, macular atrophy, or development of a posterior subcapsular cataract. RP represents a varied group of diseases whose common thread is the abnormal production of at least one protein in photoreceptor outer segments critical to light transduction. One clinical result of RP is the destabilization of the blood-retinal barrier of the perifoveal capillaries and the optic nerve head. This destabilization results in leakage of fluorescein dye observed by angiography. In addition to leakage, accumulation of fluid as microcycts in the outer plexiform layer may occur and be observed. These fluid filled cysts may eventually burst, resulting in damage to the retinal layer. The present method contemplates treating RP related damage to the retina by promoting the accelerated clearance of the tissue fluid accumulating in the microcycts. A fifty-nine (59) year old male human being presented with a history of retinitis pigmentosa. The uncorrected vision in his left eye was 20:400 and with correction was also 20:400. A single intravitreal injection of 100 LU. of the hyaluronidase (ACS) was administered to the left eye of the patient. The other eye remained untreated. The patient was examined repeatedly following the dose of hyaluronidase (ACS) and the vision in the patient's right (unfreated) eye remained unchanged, while the vision in the patient's left (treated) eye was observed to improve as follows:
Figure imgf000057_0001
HM* denotes "hand movement" cf** denotes "finger counting" The study results demonstrate that there were significant improvements in the visual performance of the subject, both with respect to Unaided Visual Acuity (improving from 20:400 to 20:80) and Best Corrected Visual Acuity (improving from 20:400 to 20:40). Also, while changes in the intraocular pressure of the subject during the treatment period were observed, the intraocular pressure appeared to return to baseline levels approximately two weeks after the time of injection. Although these results are from a single patient, they appear sufficiently promising to warrant further studies. EXAMPLE 13 Hyaluronidase Treatment of Macular Holes A rupture or bursting open of the macula is known as a macular hole. Interestingly, this condition usually occurs in women in their sixth through eighth decades, or after trauma such as lightening injury, solar injury, scleral buckling, or in staphylomatous eyes. Symptoms include metamorphopsia and diminished visual acuity. Macular hole formation is thought to result from tangential traction across the retinal surface induced by the posterior cortical vitreous with involvement of fluid movement within a posterior vifreous synergetic cavity. The posterior vifreous syneresis cavity is present in the vast majority of patients presenting macular holes. It is thought that as the posterior vifreal gel refreats from the retinal surface, the resulting gap between the two surfaces creates an area wherein movement of the vifreous humor may negatively interact with the retinal surface. The tangential movement of the vitreous humor within the space of the posterior vifreous syneresis cavity is thought to promote tears of the retinal membrane, resulting in the creation of macular holes. The present method contemplates the use of hyaluronidase (ACS) to depolymerize the vitreous so as to eliminate the conditions which result in macular hole formation. Upon depolymerization of the vitreous, the posterior vitreous syneresis cavity is eliminated as a result of hyaluronidase (ACS)-mediated reorganization of the vitreous. The elimination of this cavity permits the fluid between the vitreous and the retina to move freely about the vifreal chamber, dispersing any harmful forces that would have otherwise have been directed against the retinal surface. A patient presenting the early signs of macular hole formation is treated with an intravitreal injection of hyaluronidase (ACS). The patient to be treated presents the various signs of premacular hole formation. These include loss of the foveal depression associated with a yellow foveal spot or ring. The fovea has begun to thin in the region of hole formation and the lesion may obtain a reddish appearance. Fluorescein angiography at this stage may appear normal or show faint hyperfluorescence. The appearance of an eccentric full thickness dehiscence denotes an advanced early stage of the disease. Upon observance of these symptoms hyaluronidase (ACS) freatment is commenced. The hyaluronidase (ACS) treatment of the present method is commenced when the formation of a macular hole is diagnosed. The patient is to receive a full ophthalmic examination to establish a baseline of ocular health. The ophthalmic examination included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A- scan measurements. Following the preliminary examination, an intravitreal injection of hyaluronidase (ACS) is given to the patient's affected eye. If both eyes are affected, they may be treated separately. The eye to be freated is injected with 50 μl of 50 LU. of the hyaluronidase (ACS) ophthalmic solution described above intravitreally to promote the depolymerization of vifreous hyaluronic acid, resulting in the liquefaction of the vifreous. After freatment, the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). On each examination day the patient is monitored for vitreous liquefaction. Fluorescein angiography, considered a particularly effect method of monitoring the course of the freatment, is also performed. Additionally, the patient is monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression. EXAMPLE 14 Hyaluronidase Treatment of Macular Exudates Macular exudates are material that penetrates the blood-retina barrier and seeps through the macula into the vifreal chamber. There are two kinds, soft exudates and hard exudates. The soft exudates are actually not exudates but clusters of ganglion cell axons in the nerve fiber layer that have undergone a bulbous dilation at a site of ischemic damage or infarction. Hard exudates are commonly exuded as a result of microvascular changes in background retinopathy. Hard exudates appear yellow and waxy are often deposited in a circular fashion about the macula. They consist of lipid and proteinaceous material derived from the exudation of serum components from leaking vessels or from the lipid products of degenerating neural elements within the retina. Adsorption of hard exudates is primarily mediated by macrophagic resorption, however, the rate of this process may be slow since exudation often occurs in the outer plexiform layer within the avascular zone of the retina. The present method is particularly useful in reducing the extent of exudative accumulation resulting from the destabilization of the retinal membrane since hyaluronidase (ACS) depolymerization of the vitreous promotes an increased turn-over rate of the aqueous components of the vifreous. A patient presenting macular exudates is treated with hyaluronidase (ACS) injection method of treatment. The patient is to receive a full ophthalmic examination to establish a baseline of ocular health. The ophthalmic examination included indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal examination, intraocular pressure measurements, visual acuity (unaided and best corrected) symptomatology, fundus photography, fluorescein angiography, elecfroretinography and A-scan measurements. Following the preliminary examination, an intravitreal injection of hyaluronidase (ACS) is given to the patient's affected eye. If both eyes are affected, they may be treated separately. The eye to be treated is injected with 50 μl of 50 LU. of the hyaluronidase (ACS) ophthalmic solution described above intravitreally to promote the depolymerization of vitreous hyaluronic acid, resulting in the liquefaction of the vifreous. After treatment, the patients' eyes are to be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60). On each examination day the patient is monitored for vitreous liquefaction. Fluorescein angiography, considered a particularly effect method of monitoring the course of the treatment, is also performed. Additionally, the patient is monitored for posterior vifreous detachments using indirect ophthalmoscopy with scleral depression. EXAMPLE 15 Treatment of Cystoid Macular Edema Cystoid macular edema is a common ocular abnormality resulting form a diverse group of etiologies. Most the causes of this condition stem from a disturbance of the blood- retinal barrier of the perifoveal capillaries and the optic nerve head that result in fluid leakage which accumulates in microcysts of the outer plexiform layer. This region is a relatively thin and under vascularized area of the retina. Clinically, a cystoid macular edema produces a honey-comb appearance when examined with fluorescein angiography. As the edema progresses, the outer plexiform layer may rapture, producing a lamellar hole. The hole may be confined to the inner layer of the retina or it may eventually progress to a complete macular hole. The present method contemplates the freatment of cystoid macular edema and the prevention of macular hole formation through the hyaluronidase (ACS)-mediated depolymerization of the vitreous. A patient presenting the indicia of cystoid macular edema is treated with an intravifreal hyaluronidase (ACS) injection as described in Examples 13 and 14. EXAMPLE 16 Other Pharmacological Uses of Hyaluronidase Hyaluronidase has been used therapeutically for many years now. Its proven and diverse effects, which occur mainly in intercellular connective tissue, are primarily attributable to the breakdown of hyaluronic acid in the tissue. The therapeutically useful consequences of this action, i.e. reduced viscosity of intercellular cement and increased membrane and vascular permeability, are due to its "spreading effect". The permeability- enhancing effect of hyaluronidase after administration of fluids and/or radiopaque media is of therapeutic significance. Hyaluronidase - The Spreading Effect: • Reduces the viscosity of intercellular cement. • Increases membrane and vascular permeability. => Enhances spatial processes and leads to temporal acceleration when injected via intracutaneous, subcutaneous, intradermal, intravenous or intramuscular route. Results: Improves diffusion and penetration of solutions, suspensions and emulsions into the surrounding tissue. Hyaluronidase accelerates and enhances the absorbtion of injected drags (antibiotics, cytostatic agents, local anesthesia, chemotherapeutic agents, antivirals, etc.) by the tissue, even when large volumes of the medications are administered in solution, suspension or emulsion form. Hyaluronidase has been successfully used in orthopedics, surgery, ophthalmology, internal medicine, oncology, gynecology, dermatology, etc. for many years. The experimental use of hyaluronidase was tested in numerous areas of medicine. The substance has been administered clinically in various indications and therapies. Ophthalmology / peri- and retro-bulbar anesthesia: Ophthalmology is now an important and well documented area of indication for hyaluronidase (Farr C. et al. 1997 Wien Med Wschr 147:1-8). Hyaluronic acid is often applied during ophthalmic surgery (e.g., cataract surgery), for example, to keep the anterior chamber of the eye intact or to protect the corneal endothelium during lens implantation. This results in an increase in intraocular pressure. Measurements have shown that introduction of hyaluronidase in the anterior chamber of the eye can effectively decrease the intraocular pressure postoperatively. Hyaluronidase was also found to be effective in reducing the intraocular pressure in patients who underwent trabeculectomy for treatment of wide-angle glaucoma (doses of 300 IU were administered as a subconjunctival injection). The authors concluded that hyaluronidase reduced the number of complications and improved the prognosis of trabeculectomy. Hyaluronidase can also be helpful in retro- and peribulbar anesthesia for cataract surgery when used in combination with local anesthetics such as lidocaine and bupivacaine (with or without adrenaline). The effects of hyaluronidase in local anesthesia of the eye were reported back in 1949. Later research studied the effects of the combined use of procaine + adrenaline + hyaluronidase for retrobulbar anesthesia in patients. Improvement of akinesia of the eye muscles was observed in subjective assessments. The hyaluronidase-induced improvement of the action of anesthesia may be due to the fact that the orbita is an optimal injection site. In a double-blind comparison of bupivacaine versus lidocaine with and without hyaluronidase in groups of 50 patients each, investigators observed a significantly larger number of patients with complete anesthesia in the hyaluronidase group. It was also found that none of these patients required additional anesthetics, because the induced muscular blockade persisted tliroughout the entire operation. These findings were unequivocally confirmed in a later study. The onset of effect of local anesthesia in conjunction with hyaluronidase was investigated in 1990. Investigators found that, in all patients in whom the enzyme supplement was injected together with an anesthetic, the anesthetic effect required for surgery occurred within 5 minutes after the injection, hi the control groups, on the other hand, an additional dose of anesthesia was required, and the onset of the anesthetic effect occurred later. The addition of hyaluronidase to local anesthesia improved the conditions for surgery. Hyaluronidase was found to be highly effective when added to lidocaine and noradrenaline in retrobulbar anesthesia in senile cataract operations. h one clinical study, the addition of 75 IU of hyaluronidase to 0.75% bupivacaine and 2% mepivacaine lead to improved motor blockade, analgesia and hypotonus of the eyeball. Studies on the use of hyaluronidase in strabotomies demonstrated that a sufficient anesthetic effect of mepivacaine and lidocaine can be achieved if supplemented with hyaluronidase. Even small volumes of hyaluronidase displayed the desired spreading effect. The findings for retrobulbar anesthesia generally apply for peribulbar and subconjunctival applications as well. The combination of local anesthetics with hyaluronidase leads to a reliable blockade of the ocular muscles and, thus, to an improvement of operation conditions. The effect of hyaluronidase on local anesthesia was again studied in another prospective, randomized, controlled, double-blind study. This study also showed that the addition of hyaluronidase can lead to additive effects. These results were confirmed when 80 patients with senile cataracts were administered 150 JU of hyaluronidase as a supplement to anesthesia. Their results were later confirmed in another group of 70 patients who received 250 IU of hyaluronidase. Investigators demonstrated that ocular circulatory changes occur during the combined use of lidocaine / bupivacaine and hyaluronidase. They detected significant reduction in the ocular pulsation volume and a significant reduction in intraocular pressure. Local anesthesia with the addition of 150 IU of hyaluronidase proved to be a reliable alternative for cataract operations. Regarding peribulbar anesthesia, investigators recommended that a mixture of 2% lidocaine, 0.5% bupivacaine, and 1550 IU hyaluronidase be warmed to body temperature to eliminate symptoms of pain in the target areas. In a separate study, two concentrations of hyaluronidase were used as a supplement to peribulbar anesthesia. Three groups received either 0, 50 or 150 IU of the hyaluronidase supplement, and the onset of effect, analgesia and akinesia were assessed. No statistically significant differences between the 50 IU and 150 IU hyaluronidase groups could be detected. The authors concluded that the addition of hyaluronidase did not lead to any complications. After comparing the results of peribulbar anesthesia with retrobulbar anesthesia, other authors concluded that the addition of hyaluronidase is useful in both cases, that hyaluronidase is extremely effective in preventing the vitreous body from bulging into the posterior chamber of the eye, and that it significantly reduces the occurrence of vis a tergo. Hyaluronidase makes is possible for lidocaine and bupivacaine to spread more rapidly within the peribulbar space. The injection pressure of local anesthesia administered prior to cataract operations was investigated in 50 patients in a double-blind study. The study concluded that significant (sufficient) akinesia of the extraocular muscles can be achieved by administration of 1% etidocaine, 0.5% bupivacaine, and 50 IU hyaluronidase. Glaucoma: Hyaluronidase is useful for treatment of glaucoma or to alleviate intraocular pressure. Combination with local anesthetics: When hyaluronidase was combined with a local anesthetic, e.g., procaine or lidocaine, the onset of effect was quicker, the analgesic region was enlarged, and postoperative pain was significantly reduced. One study showed that the anesthetized area after subcutaneous injection of a combination of procaine and hyaluronidase was almost twice as large as that of procaine alone. This study also demonstrated that the duration of anesthesia after administration of combined preparations of hyaluronidase and procaine / adrenaline led to a 6-fold extension of the anesthetic effect. The effects of bupivacaine plus adrenaline with or without the addition of hyaluronidase on blockade of the axillary plexus brachialis were compared in a double- blind study. The enzyme neither influenced the time of occurrence of anesthesia, nor the occurrence of inadequate blockade of the plexus, nor the level of the plasma bupivacaine level. The study demonstrated that the duration of anesthesia was reduced. Orthopedics, diseases of the supportive and locomotive apparatus: For many years now, hyaluronidase has been successfully used for treatment of various diseases of the supportive and locomotive apparatus, e.g., acute conditions of the synovial sheath, surrounding connective tissue and varied inflammations in these areas (paratendinitis crepitans, humeroscapular periarthritis, humeral epicondylitis, tibial condylitis, radial styloiditis, etc.). Good freatment results can usually be achieved (especially in combination with exercise or physiotherapy) if hyaluronidase therapy is started as early as possible, even if the affected limb cannot be immobilized. Investigators successfully freated patients with acute tendovaginitis crepitans with hyaluronidase in 1968. Combination therapy with hyaluronidase (300 IU) and Vitamin B12 injections for treatment of peritendinitis and periarthritis humeroscapularis has been reported. The efficacy of hyaluronidase in the treatment of epicondylitis and tendovaginitis was studied in a total of 53 patients. Daily intravenous doses of 1500 to 3000 units of hyaluronidase led to a decrease in symptoms or clear relief from complaints. The findings from other studies show the same treatment success in paratendinitis, humeral epicondylitis, humeroscapular periarthritis, and radial and ulnar styloiditis; a dose of 3000 IU of hyaluronidase every two days was used in most cases. Hyaluronidase was successfully used in combination with mephenesin for freatment of arthrosis, primarily in patients with gonarthrosis. The success rate in chronic cases was particularly impressive. The findings that i.v. administration of hyaluronidase in patients with Bechterew's disease produced long-lasting effects is of great therapeutic significance. Administration of an average 1500 to 9000 IU of hyaluronidase i.v., every 3 days increased the mobility of patients studied. Regarding its mode of action, investigators assume that hyaluronidase depolymerizes mucopolysaccharides deposited in the connective tissue matrix during exfraosseous calcification processes that occur due to increased mesenchymal metabolism. If calcium salts are present, they can be depolymerized as long as calcium does not occur as tertiary calcium phosphate. This can restrict the formation of exfraosseous calcification. The improvement of flexibility (e.g., in the affected spinal segment) could be attributed to the presumed depolymerization resulting from the loosening of previously formed connective structures. It is assumed that hyaluronidase is able to influence the composition and structure of the dermis, and that it can promote the re-synthesis of the proteoglycans. h the freatment of joint stiffness, which often occurs as a complication of supracondylar fractures, prior or simultaneous administration of hyaluronidase can provide the patient quicker relief from symptoms. Treatment of malignant diseases: When used as a supplement to chemotherapy of malignant tumors, hyaluronidase can dissolve hyaluronic acid-containing areas around tumor cells and tumor cell conglomerates, thereby enabling a higher concenfration of the cytostatic agent to take effect in the desired target area. Moreover, hyaluronidase may induce a related enhancement of immunological defensive processes, e.g., by creating direct contact between immunocompetent cells ("natural killer cells") and antigens on the tumor surface. The usefulness of hyaluronidase (i.v., i.m. and intravesical) for treatment of malignant diseases (hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ENT region, adenocarcinomas of the lung and colon, and carcinomas of the bladder) is the subject of various clinical studies, hi isolated cases, hyaluronidase was usually found to increase the response rate to cytostatic agents if high doses of the enzyme are administered prior to administration of the cytostatic agent. Hyaluronidase supplements can improve the patient's response to chemotherapy when used in therapy-resistant patients with malignant hematological diseases. In oncology, hyaluronidase has proven to be particularly useful when administered as a supplement to cytostatic agents like doxorubicin and adriamycin. The enzyme improves the penetration of doxorabicin in the cells and increases the activity of adriamycin in breast cancer. The combined administration of cisplatin, vindesin, hyaluronidase and radiation therapy for treatment of advanced squamous cell carcinoma in the head and neck region was found to be highly effective: a high rate of remissions and improved tolerance of cytostatic therapy was observed in carcinoma patients. Hyaluronidase led to a decrease in adhesion-related multicellular drug resistance in carcinomas of the breast. This mechanism of action is based on the reduction of cell- contact-dependent inhibition of growth and on the sensitization of cells for the cytostatic agents.
Hyaluronidase in tumor treatment: • Hyaluronidase increases the effects of cytostatic agents used for treatment of such malignant diseases as hematological systemic diseases, carcinomas of the breast, cerebral metastases, glioma, squamous cell carcinomas in the ear, nose and throat region, adenocarcinomas of the lung and colon, and carcinomas of the bladder. • In clinical studies, hyaluronidase was found to induce cessation of growth (remission) of various tumors. • Therapy-resistant patients respond better to cytostatic agents if an intravesical dose of the enzyme is instilled prior to the cytostatic drug. • Hyaluronidase can improve the subjective well-being and the quality of life of tumor patients. Dermatology: Investigators have concluded that hyaluronidase is useful in certain dermatological diseases, such as, for example, progressive scleroderma, which is a systemic disorder of the entire vascular connective tissue system, with its most important characteristic being the displacement of collagen fractions. Of pathomechanistic significance is the fact that the histomorphological skin changes that occur in scleroderma begin with a dermal edema rich in acidic mucopolysaccharides (hyaluronic acid, chondroitin sulphate). Histopathological and chemical tests have shown that part of the ground substance occurs as cement in the collagen fibers. It would therefore appear that acidic mucopolysaccharides, soluble collagen, and polymeric collagen are responsible for the sclerosis. Investigators published further findings on high-dose i.v. hyaluronidase therapy in patients with primary diseases such as keloids and localized scleroderma. The substance achieved good results in both diseases. The same applies for progressive scleroderma, but the increase in active flexibility of the joint was not permanent. High-dose i.v. administration of hyaluronidase for freatment of progressive scleroderma was therefore recommended only as a supplementary medication, as no influence on older areas of sclerosis could be demonstrated. Assessment of the selected routes of administration showed that local administration of the substance to the affected areas of the skin did not lead to treatment success. Only intravenous injection or infusion achieved, at minimum, improvement of symptoms in most patients. Treatment of myocardial infarction: The use of hyaluronidase for treatment of acute myocardial infarction was first described in 1959. Studies have shown that the administration of hyaluronidase in the acute stage, i.e., in the early stage of fresh myocardial infarction (2 to 4 hours after the onset of infarction) can reduce the size of the necrotic area in the heart. Investigators studied medications that lead to a reduction in infarction size, e.g., beta blockers, nitrates, calcium antagonists, etc. Hyaluronidase was found to have a favorable effect on concomitantly administered thrombolytic agents such as streptokinase. This appears to be attributable to the ability of hyaluronidase to entrap oxygen radicals. Statisticians performed meta-analyses in which they studied the role of hyaluronidase and other cardiovascular drags with a potential for reducing the size of myocardial infarction. They found that hyaluronidase supplements reduced the mortality rate by 15 to 20 percent. Investigators also recommended the use of hyaluronidase, in addition to conventional agents for freatment of acute myocardial infarction (nitrates, beta receptor blockers, calcium antagonists). Despite differences in the data and, in some case, contradictory findings, it appears that the use of hyaluronidase for freatment of myocardial infarction has been definitively proven and confirmed. Miscellaneous indications: Another indication is for freatment of submucosal fibrosis. Experience with 150 patients over a 10-year period has shown that the combination of hyaluronidase and dexamethasone is able to reduce symptoms over a long period of time in most cases. Local injection of chymotrypsin, hyaluronidase and dexamethasone was also reported to induce good treatment results. Surgical excision was, however, performed in therapy-resistant patients. 326 patients with oral submucosal fibrosis were randomized into 2 groups and freated with either steroids and hyaluronidase or with topical vitamin A steroid and oral iron preparations. Treatment with steroids and hyaluronidase was found to be much less problematic. Hyaluronidase has also been used in hair transplants and in therapeutic and cosmetic surgery of the scalp. It was found effective in improving the diffusion of local anesthetics. The use of hyaluronidase for prevention of postoperative adhesions following surgery has also been reported. hi cerebral abscess, hyaluronidase was alternatively combined with dexamethasone or antibiotics, primarily to eliminate edema in high-risk patients. Hyaluronidase was found to improve the absorption of locally administered drags and to reduce the risk of progression of skin necrosis in patients treated with intravenously administered Vinca alkaloids. The use of hyaluronidase as an antidote for the extravasation of chemotherapeutic agents has also been described. Hyaluronidase is one of the few antidotes that can be used as an antidote for Vinca alkaloids or epipodophyllotoxins such as etoposide. Gynecology is another area of application for hyaluronidase. When injected in the perineal region prior to the expulsive stage of labor, hyaluronidase was found to soften the consistency of the birth canal of first-time mothers, which often eliminated the need for episiotomy. Hyaluronidase is also useful for facilitation of partial and complete aspiration of viscous joint effusions and pleural effusions, i.e., it liquefies the effusions. The enzyme is also used for treatment of edema of various origins and for treatment of arthritic joint changes. Hyaluronidase is a treatment for corneaplasty, corneal scars, opacification, and haze, and cornea in need of delamination. Hyaluronidase can be used as an alternative or adjunct to conventional mechanical vitrectomy. Hyaluronidase is also useful for the induction of retinal detachments. Hyaluronidase is indicated as an adjuvant to increase the absorption and dispersion of other injected drugs; for hypodermoclysis; and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. Summary: The proven and diverse activity of hyaluronidase occurs mainly in the intercellular connective tissue. This action is clearly attributable to the breakdown of hyaluronic acid in the tissue. The therapeutically useful consequences of this action
(reduced viscosity of intercellular cement, increased the permeability of membranes and vessels) occur due to a "spreading effect." The hyaluronidase-related enhancement of diffusion and increase in permeability that occurs after administration of liquids and/or radiopaque media is of therapeutic significance. The substance is able to accelerate and increase the absorption of drags (antibiotics, cytostatics, local anesthetics, etc.) by the tissue, even when large volumes of the medication are injected in solution, suspension or emulsion form. If it is not possible to administer a certain drug intravenously in cases where a rapid onset of effect is necessary, a "pre-injection" of hyaluronidase can accelerate (by 200 to 300%) the absorption of subcutaneous or intramuscular doses of the drug in the bloodstream, which is of particular significance for internal medicine. The efficacy of hyaluronidase in treatment of disorders of the supportive and locomotive system can be attributed to the so-called "softening effect" of the enzyme. When administered early and, especially, when coupled with additional exercise and/or physiotherapy measures, the "antiphlogistic" effect of hyaluronidase makes it possible to control acute symptoms involving the synovial sheaths and the surrounding connective tissue (peritendinitis crepitans, humeroscapular periarthritis, humeral epicondylitis, tibial condylitis, radial styloiditis, etc.) Joint stiffness (e.g., due to supracondylar fracture) can also be treated successfully. Due to its diffusion potential, hyaluronidase can also be used for freatment of posttraumatic hematomas or edemas of any origin, and for liquefaction of joint and pleural effusions in orthopedics. Hyaluronidase is indicated to be useful as an anti-edema and anti-inflammatory agent in the prevention of transplant rejection. It has been shown in pre-clinical experiments to lend itself to this role, because it breaks down hyaluronan in damaged tissues. Hyaluronan, a glucosaminoglycan with unique water-binding capacity, draws water into some transplanted organs causing edema. This in turn impairs organ function which may lead to the transplanted organ failure and being rejected, hi addition to attracting water, hyaluronan attracts certain cells of the immune system and therefore may be instrumental in initiating inflammatory reactions. Studies have confirmed that hyaluronidase freatment can be used to reduce edema and inflammation after organ transplantation. When hyaluronidase is administered prior to administration of a local anesthetic such as procaine ("pre-injection"), the onset of effect of the anesthetic is quicker, the anesthetic region is larger, and the pain after completion of the procedure is significantly lower. Optimal and efficient combinations of hyaluronidase and local anesthetics are now widely used, particularly in ophthalmology and especially in cataract surgery. The preoperative administration of hyaluronidase with certain local anesthetics (procaine, lidocaine, bupivacaine, etc.) for retro- and peribulbar anesthesia is useful in various ophthalmologic operations. The enzyme accelerates the onset of effect of the anesthetic agent and causes reliable blockade of the eye muscles which, in turn, creates excellent conditions for surgery. In combination with vasopressors such as adrenaline, hyaluronidase increases the duration of anesthesia in the treated area and prevents the rapid diminishment of local anesthesia. Hyaluronidase is also effective for treatment of postoperatively increased internal eye pressure due to the administration of viscoelastic substances such as sodium hyaluronate during ophthalmic surgery. Hyaluronidase is also widely used in dermatology, i.e., in selected skin disorders involving the connective tissue system and characterized by degeneration of it (scleroderma, keloid formation, psoriasis, chronic varicose ulcer, etc.). Hyaluronidase is also useful in gynecology, i.e., for prevention of episiotomy. The usefulness of hyaluronidase has been validated for freatment of myocardial infarction. The most recent clinical studies have shown that hyaluronidase is helpful as a supplement to chemotherapy in patients with cancer (myeloma, Hodgkin's disease, non- Hodgkin's lymphoma, breast cancer [also with concomitant cerebral metastasis], cerebral lymphomas, gliomas, squamous cell carcinomas in the ear, nose and throat region, and carcinomas of the bladder). The enzyme not only increases the patient's response to the cytostatic agents, but also drastically improves the patient's overall subjective feeling of well-being and the remission rate. Preliminary evidence of the usefulness of this therapy principal has also been found in colon carcinoma, adenocarcinoma of the lung, bronchial carcinoma, hypernephroma, carcinomas of the stomach, pancreas and ovaries, myelosarcoma, and neurinoma. In different tumor types, the administration of hyaluronidase was found to induce temporary cessation of tumor growth, and it improved the response of therapy-resistant patients to the cytostatic agent. Furthermore, it is assumed that hyaluronidase stimulates the immune system. Positive findings after administration of the enzyme as a supplement to chemotherapy of malignant tumors give one reason to expect that hyaluronidase may one day be able to expand the potentials of anti-tumor chemotherapy and improve the results of therapy. From a toxicological point of view and in light of the wide range of available data on the various applications of hyaluronidase, there does not seem to be any reason to prohibit the use of the enzyme in humans. No serious pathological organ changes were detected in an acute toxicity test with administration of a single dose of hyaluronidase, nor after long-term administration of the substance. However, one should always remember that the enzyme is antigenic in nature, when used alone or mixed with other substances. These effects can never be completely excluded due to the complicated nature of the process used to isolate the enzyme. Thus, there is a need for a hyaluronidase preparation suitable for pharmaceutical applications, which need is met by the disclosed process for isolating and purifying ovine hyaluronidase. hi light of the positive clinical findings on hyaluronidase, one may conclude that hyaluronidase is a therapeutically versatile enzyme that promises to be a therapeutically useful agent in new areas of medical practice now and in the future. EXAMPLE 17 Hyaluronidase for Injection In this Example for use of hyaluronidase as a spreading agent, hyaluronidase for injection dehydrated in the solid state under high vacuum with the inactive ingredients listed below, is supplied as a sterile, nonpreserved, white, odorless, amorphous solid. The product is to be reconstituted with Sodium Chloride Injection, USP, before use. Each vial of 6200 USP units contains 5 mg lactose, 1.92 mg potassium phosphate dibasic, and 1.22 mg potassium phosphate monobasic. The USP/NF hyaluronidase unit is equivalent to the turbidity-reducing (TR) unit and equal to 0.81 International Units (IU). The reconstituted solution is clear and colorless, with an approximate pH of 6.7 and osmolality of 290 to 310 mOsm. Hyaluronidase for injection is to be reconstituted in a vial to a concentration of 1000 Units/mL of Sodium Chloride Injection, USP by adding 6.2 mL of solution to the vial. Prior to administration, the reconstituted solution should be further diluted to the desired concentration, commonly 150 Units/mL, see table below. The resulting solution should be used immediately after preparation. A ImL syringe and a 5-micron filter needle are supplied in a hyaluronidase for injection kit. Following reconstitution of hyaluronidase for injection, as described above, apply the 5-micron filter needle to the ImL syringe. Draw the desired amount of hyaluronidase for injection into the syringe, and dilute according to the table below. Remove the filter needle and apply a needle appropriate for the intended injection.
Figure imgf000072_0001
*** While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS: 1. A composition of matter comprising an isolated polynucleotide such as
DNA or RNA encoding alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO: 1, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined, or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1, or fragment of said hyaluronidase being at least 100 amino acids in length, said hyaluronidase or fragment thereof having hyaluronidase activity.
2. A method of making a hyaluronidase comprising providing conditions for expression of the composition of matter of Claim 1, such that a hyaluronidase is made.
3. A composition of matter comprising a polypeptide that is recombinant and is substantially free of components normally associated with ovine testes having an amino acid sequence of the alpha-form or beta-form of ovine hyaluronidase purifiable from ovine testes having the amino acid sequence of SEQ ID NO: 1, where the consensus sites for glycosylation are underlined and the site of cleavage that yields the beta-form of hyaluronidase is assigned by homology with the bovine sequence and is indicated as bold and underlined, or having an amino acid sequence of a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ JD NO: 1, or fragment of said hyaluronidase being at least 100 amino acids in length, said hyaluronidase or fragment thereof having hyaluronidase activity.
4. A method for accelerating the clearance of hemorrhagic blood from the vitreous humor of a mammalian eye, said method comprising contacting with the vitreous humor an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to accelerate the clearance of hemorrhagic blood from said vifreous humor.
5. A method for inducing liquefaction of a vitreous humor to treat a disorder of a mammalian eye, said method comprising contacting with said vitreous humor of said mammalian eye an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to liquefy said vifreous humor whereby said disorder is freated.
6. The method of Claim 5, wherein said method is carried out for the purpose of treating nonprohferative diabetic retinopathy.
7. The method of Claim 5, wherein said method is carried out for the purpose of treating preproliferative diabetic retinopathy.
8. The method of Claim 5, wherein said method is carried out for the purpose of treating proliferative diabetic retinopathy.
9. The method of Claim 5, where in said method is carried out for the purpose of treating age-related macular degeneration.
10. The method of Claim 5, wherein said method is carried out for the purpose of treating amblyopia.
11. The method of Claim 5, wherein said method is carried out for the purpose of treating retinitis pigmentosa.
12. The method of Claim 5, wherein said method is carried out for the purpose of treating macular holes.
13. The method of Claim 5, wherein said method is carried out for the purpose of treating macular exudates.
14. The method of Claim 5, wherein said method is carried out for the purpose of treating cystoid macular edema.
15. The method of Claim 5, wherein said liquefaction achieves posterior vifreal detachment (PVD).
16. A method for treating glaucoma comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to treat glaucoma.
17. A method for treating malignant disease comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to treat malignant disease.
18. A method for treating myocardial infarction comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to treat myocardial infarction.
19. A method for reducing edema or inflammation after organ transplantation comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to reduce edema or inflammation after organ transplantation.
20. A method for treating corneal scar, opacification, or haze comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to treat said corneal scar, opacification, or haze.
21. A method for enhancing diffusion of a radiopaque media comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1
International Unit effective to enhance the diffusion of a radiopaque media.
22. A method for enhancing permeability of a fluid into surrounding tissues of a human comprising administering an amount of the hyaluronidase of Claim 3 to provide a dose of at least 1 International Unit effective to enhance the permeability of a fluid into surrounding tissues of a human.
23. The composition of matter of Claim 3 in a solution form further comprising lactose and phosphate.
24. The composition of matter of Claim 3 in a lyophilized form.
25. The composition of matter of Claim 1, wherein the polynucleotide encoding alpha-form or beta-form of ovine hyaluronidase or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ LD NO: 1 is a polynucleotide encoding a hyaluronidase having an amino acid sequence at least 97% identical to SEQ D NO: l.
26. The composition of matter of Claim 1, wherein the polynucleotide encoding alpha-form or beta-form of ovine hyaluronidase or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1 is a polynucleotide encoding a hyaluronidase having an amino acid sequence at least 98% identical to SEQ JD NO: 1.
27. The composition of matter of Claim 1, wherein the polynucleotide encoding alpha-form or beta-form of ovine hyaluronidase or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ JD NO: 1 is a polynucleotide encoding a hyaluronidase having an amino acid sequence at least 99% identical to SEQ ID NO: l.
28. The composition of matter of Claim 1, wherein the polynucleotide encoding alpha-form or beta-form of ovine hyaluronidase or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1 is a polynucleotide encoding alpha-form or beta-form of ovine hyaluronidase.
29. The composition of matter of Claim 1, wherein the polynucleotide encoding alpha-form or beta-form of ovine hyaluronidase or encoding a hyaluronidase having an amino acid sequence at least 97, 98, or 99% identical to SEQ ID NO: 1 is a polynucleotide encoding beta-form of ovine hyaluronidase.
30. A polynucleotide encoding ovine hyaluronidase comprising SEQ ID NO: 2.
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