WO2005118799A1 - Hyaluronidase ovine - Google Patents

Hyaluronidase ovine 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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WIPO (PCT)
Prior art keywords
hyaluronidase
ohuase
amino acid
acid sequence
ovine
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PCT/US2004/034150
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English (en)
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Donald J. Brown
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Ista Pharmaceuticals, Inc.
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Application filed by Ista Pharmaceuticals, Inc. filed Critical Ista Pharmaceuticals, Inc.
Publication of WO2005118799A1 publication Critical patent/WO2005118799A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • 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

L'invention concerne, d'une manière générale, une composition de substance comprenant un polynucléotide isolé, tel qu'un ADN ou ARN codant une forme alpha ou béta de la hyaluronidase ovine purifiable à partir de tests ovins possédant la séquence d'acides aminés de SEQ ID NO:1, les sites de consensus destinés à la glycosylation étant soulignés et le site de clivage produisant la forme béta de la hyaluronidase étant attribué par homologie avec la séquence bovine et indiqué en gras et souligné, ou codant une hyaluronidase possédant une séquence d'acides aminés à au moins 97, 98 ou 99 % identique à la SEQ ID NO:1 et possédant une activité d'hyaluronidase.
PCT/US2004/034150 2004-04-15 2004-10-15 Hyaluronidase ovine WO2005118799A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2217276A2 (fr) * 2007-12-05 2010-08-18 UCL Business PLC Composition renfermant un agent inhibiteur de sulfate de chondroitine protéoglycane et un agent anti-inflammatoire
WO2012012300A2 (fr) 2010-07-20 2012-01-26 Halozyme, Inc. Effets secondaires indésirables associés à l'administration d'un agent anti-hyaluronane et procédés pour améliorer ou prévenir ces effets secondaires
WO2013040501A1 (fr) 2011-09-16 2013-03-21 Pharmathene, Inc. Compositions et combinaisons d'accepteurs biologiques organophosphorés et d'enzymes dégradant le hyaluronane, et leurs utilisations
WO2012174478A3 (fr) * 2011-06-17 2013-04-11 Halozyme, Inc. Formulations stables d'enzyme de dégradation d'hyaluronane
WO2013063155A2 (fr) 2011-10-24 2013-05-02 Halozyme, Inc. Diagnostic compagnon pour le traitement avec un agent anti-hyaluronane et procédés d'utilisation dudit diagnostic
WO2013151774A1 (fr) 2012-04-04 2013-10-10 Halozyme, Inc. Polythérapie par un agent anti-hyaluronane et un taxane ciblant une tumeur
WO2014062856A1 (fr) 2012-10-16 2014-04-24 Halozyme, Inc. Hypoxie et hyaluronane et leurs marqueurs pour le diagnostic et la surveillance de maladies et de pathologies, et méthodes associées
WO2016033555A1 (fr) 2014-08-28 2016-03-03 Halozyme, Inc. Thérapie combinée associant une enzyme dégradant le hyaluronane et un inhibiteur des postes de contrôle immunitaire
WO2016061286A2 (fr) 2014-10-14 2016-04-21 Halozyme, Inc. Compositions d'adénosine désaminase-2 (ada2), variants de cette dernière et leurs procédés d'utilisation
US9562223B2 (en) 2003-03-05 2017-02-07 Halozyme, Inc. Methods for reducing intraocular pressure by administering a soluble hyaluronidase glycoprotein (sHASEGP)
US9993529B2 (en) 2011-06-17 2018-06-12 Halozyme, Inc. Stable formulations of a hyaluronan-degrading enzyme
WO2019222435A1 (fr) 2018-05-16 2019-11-21 Halozyme, Inc. Procédés de sélection de sujets pour une polythérapie anticancéreuse avec un ph20 soluble conjugué à un polymère
US20220233830A1 (en) * 2021-01-28 2022-07-28 Prostalund Ab Kit and method intended for prostate surgery
US11654185B2 (en) * 2014-07-16 2023-05-23 New York University Use of hyaluronidase for treatment of muscle stiffness

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997018835A1 (fr) * 1995-11-22 1997-05-29 Advanced Corneal Systems, Inc. Procede enzymatique et compositions pour le traitement de sang d'hemorragie intravitreenne

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997018835A1 (fr) * 1995-11-22 1997-05-29 Advanced Corneal Systems, Inc. Procede enzymatique et compositions pour le traitement de sang d'hemorragie intravitreenne

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 12 June 2003 (2003-06-12), "Bos taurus hyaluronidase PH-20 mRNA, partial cds.", XP002332359, retrieved from EBI accession no. EM_PRO:AY297029 Database accession no. AY297029 *
DATABASE UniProt [online] 1 October 2003 (2003-10-01), "Hyaluronidase PH-20 (Fragment).", XP002332358, retrieved from EBI accession no. UNIPROT:Q7YS45 Database accession no. Q7YS45 *
FARR C ET AL: "[Clinical pharmacology and possible applications of hyaluronidase with reference to Hylase "Dessau"]. Klinische Pharmakologie und Anwendungsmoglichkeiten von Hyaluronidase unter Beruck-sichtigung von Hylase "Dessau".", WIENER MEDIZINISCHE WOCHENSCHRIFT , 147 (15) 347-55. REF: 77 JOURNAL CODE: 8708475. ISSN: 0043-5341., 1997, XP009049267 *
MENZEL E J ET AL: "HYALURONIDASE AND ITS SUBSTRATE HYALURONAN: BIOCHEMISTRY, BIOLOGICAL ACTIVITIES AND THERAPEUTIC USES", CANCER LETTERS, NEW YORK, NY, US, vol. 131, no. 1, 1998, pages 3 - 11, XP001073484, ISSN: 0304-3835 *
MORTON D B: "PURIFICATION AND PROPERTIES OF OVINE TESTICULAR HYALURONIDASE", BIOCHEMICAL SOCIETY TRANSACTIONS, COLCHESTER, ESSEX, GB, vol. 1, no. 2, 1973, pages 385, XP009035210, ISSN: 0300-5127 *

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US9562223B2 (en) 2003-03-05 2017-02-07 Halozyme, Inc. Methods for reducing intraocular pressure by administering a soluble hyaluronidase glycoprotein (sHASEGP)
JP2011506304A (ja) * 2007-12-05 2011-03-03 ユーシーエル ビジネス ピーエルシー 組成物
EP2217276A2 (fr) * 2007-12-05 2010-08-18 UCL Business PLC Composition renfermant un agent inhibiteur de sulfate de chondroitine protéoglycane et un agent anti-inflammatoire
WO2012012300A2 (fr) 2010-07-20 2012-01-26 Halozyme, Inc. Effets secondaires indésirables associés à l'administration d'un agent anti-hyaluronane et procédés pour améliorer ou prévenir ces effets secondaires
US9878046B2 (en) 2010-07-20 2018-01-30 Halozyme, Inc. Adverse side-effects associated with administration of an anti-hyaluronan agent and methods for ameliorating or preventing the side-effects
US10265410B2 (en) 2010-07-20 2019-04-23 Halozyme, Inc. Adverse side-effects associated with administration of an anti-hyaluronan agent and methods for ameliorating or preventing the side-effects
US9993529B2 (en) 2011-06-17 2018-06-12 Halozyme, Inc. Stable formulations of a hyaluronan-degrading enzyme
WO2012174478A3 (fr) * 2011-06-17 2013-04-11 Halozyme, Inc. Formulations stables d'enzyme de dégradation d'hyaluronane
WO2013040501A1 (fr) 2011-09-16 2013-03-21 Pharmathene, Inc. Compositions et combinaisons d'accepteurs biologiques organophosphorés et d'enzymes dégradant le hyaluronane, et leurs utilisations
EP2915542A1 (fr) 2011-10-24 2015-09-09 Halozyme, Inc. Diagnostic accompagnant une thérapie d'agent anti-hyaluronan et procédés d'utilisation associés
US8846034B2 (en) 2011-10-24 2014-09-30 Halozyme, Inc. Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof
WO2013063155A2 (fr) 2011-10-24 2013-05-02 Halozyme, Inc. Diagnostic compagnon pour le traitement avec un agent anti-hyaluronane et procédés d'utilisation dudit diagnostic
US9458442B2 (en) 2011-10-24 2016-10-04 Halozyme, Inc. Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof
WO2013151774A1 (fr) 2012-04-04 2013-10-10 Halozyme, Inc. Polythérapie par un agent anti-hyaluronane et un taxane ciblant une tumeur
US10137104B2 (en) 2012-04-04 2018-11-27 Halozyme, Inc. Combination therapy with an anti-hyaluronan agent and therapeutic agent
US9913822B2 (en) 2012-04-04 2018-03-13 Halozyme, Inc. Combination therapy with an anti-hyaluronan agent and therapeutic agent
US9278124B2 (en) 2012-10-16 2016-03-08 Halozyme, Inc. Hypoxia and hyaluronan and markers thereof for diagnosis and monitoring of diseases and conditions and related methods
WO2014062856A1 (fr) 2012-10-16 2014-04-24 Halozyme, Inc. Hypoxie et hyaluronane et leurs marqueurs pour le diagnostic et la surveillance de maladies et de pathologies, et méthodes associées
US11654185B2 (en) * 2014-07-16 2023-05-23 New York University Use of hyaluronidase for treatment of muscle stiffness
US11414489B2 (en) 2014-08-28 2022-08-16 Halozyme, Inc. Combination therapy with a hyaluronan-degrading enzyme and an immune checkpoint inhibitor
WO2016033555A1 (fr) 2014-08-28 2016-03-03 Halozyme, Inc. Thérapie combinée associant une enzyme dégradant le hyaluronane et un inhibiteur des postes de contrôle immunitaire
WO2016061286A2 (fr) 2014-10-14 2016-04-21 Halozyme, Inc. Compositions d'adénosine désaminase-2 (ada2), variants de cette dernière et leurs procédés d'utilisation
US11584923B2 (en) 2014-10-14 2023-02-21 Halozyme, Inc. Compositions of adenosine deaminase-2 (ADA2), variants thereof and methods of using same
US9969998B2 (en) 2014-10-14 2018-05-15 Halozyme, Inc. Compositions of adenosine deaminase-2 (ADA2), variants thereof and methods of using same
WO2019222435A1 (fr) 2018-05-16 2019-11-21 Halozyme, Inc. Procédés de sélection de sujets pour une polythérapie anticancéreuse avec un ph20 soluble conjugué à un polymère
US20220233830A1 (en) * 2021-01-28 2022-07-28 Prostalund Ab Kit and method intended for prostate surgery
US11938289B2 (en) * 2021-01-28 2024-03-26 Prostalund Ab Kit and method intended for prostate surgery

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