Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1997-08-21
2001-02-27
Ungar, Susan (Department: 1642)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C424S094610, C424S094620, C514S002600, C514S012200
Reexamination Certificate
active
06193963
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of &bgr;-1,4-endoglycosidases, particularly hyaluronidases.
BACKGROUND OF THE INVENTION
Hyaluronidases (HAses; E. C. 3.1.25) are a group of neutral- and acid-active enzymes found throughout the animal kingdom in organisms as diverse as microbes (e.g,
Streptococcus pyogenes, Treponema palladium,
and nematodes), bees, wasps, hornet, spiders, scorpions, fish, snakes, lizards, and mammals. Hyaluronidases degrade hyaluronan (HA; also known as hyaluronic acid) and, to a lesser extent, chondroitin sulfates (for a review, see Kreil et al. 1995
Protein Sci.
4:1666-9). Vertebrate hyaluronidases are separated into two general classes: 1) the neutral hyaluronidases, such as the predominantly sperm-associated protein PH20 (Liu et al. 1996
Proc. Natl. Acad. Sci. USA
2:7832-7; Primakoff et al. 1985
J. Cell Biol.
101:2239-44; Lin et al. 1993
Proc. Natl. Acad. Sci. USA
90: 10071-5); and 2) the acid-active hyaluronidases, which have a distinct pH optimum between pH 3.5 to 4.0 and have been described in extracts of liver (Fiszer-Szafarz et al. 1995
Acta Biochim Pol.
42: 31-3), kidney (Komender et al. 1973
Bull. Acad. Pol. Sci. [Biol.]
21:637-41), lung (Thet et al. 1983
Biochem. Biophys. Res. Commun.
117:71-7), brain (Margolis et al. 1972
J. Neutrochem.
19:2325-32), skin (Cashman et al. 1969
Arch. Biochem. Biophys.
135:387-95), placenta, macrophages, fibroblasts (Lien et al. 1990
Biochim Biophys. Acta
1034:318-25; Ruggiero et al. 1987
J. Dent. Res.
66:1283-7), urine (Fiszer-Szafarz et al. supra) and human plasma (De Salegui et al. 1967
Arch. Biochem. Biophys.
120:60-67). Acid-active hyaluronidase activity has also been described in the sera of mammals, though some species exhibit no detectable activity at all (Fiszer-Szafarz et al. 1990
Biol. Cell
68:95-100; De Salegui et al. 1967 supra).
Hyaluronan, the main substrate for hyaluronidase, is a repeating disaccharide of [GlcNAc&bgr;1-4GlcUA&bgr;1-3]
n
that exists in vivo as a high molecular weight linear polysaccharide. Degradation of hyaluronan by hyaluronidase is accomplished by either cleavage at P-N-acetyl-hexosamine-[1-4]-glycosidic bonds or cleavage at &bgr;-gluconorate-[1→3]-N-acetylglucosamine bonds.
Hyaluronan is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid. Hyaluronan is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with hyaluronan creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. Hyaluronan plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis (Toole 1991
Cell Biol. Extracell. Matrix,
Hay (ed), Plenum Press, New York, 1384-1386; Bertrand et al. 1992
Int. J. Cancer
52:1-6; Knudson et al, 1993
FASEB J.
7:1233-1241). In addition, hyaluronan levels correlate with tumor aggressiveness (Ozello et al. 1960
Cancer Res.
20:600-604; Takeuchi et al. 1976,
Cancer Res.
36:2133-2139; Kimata et al. 1983
Cancer Res.
43:1347-1354).
Hyaluronidase is useful as a therapeutic in the treatment of diseases associated with excess hyaluronan and to enhance circulation of physiological fluids and/or therapeutic agents at the site of administration. For example, hyaluronidase has been used to reduce intraocular pressure in the eyes of glaucoma patients through degradation of hyaluronan within the vitreous humor (U.S. Pat. No. 4,820,516, issued Apr. 11, 1989). Hyaluronidase has also been used in cancer therapy as a “spreading agent” to enhance the activity of chemotherapeutics and/or the accessibility of tumors to chemotherapeutics (Schuller et al., 1991,
Proc. Amer. Assoc. Cancer Res.
32:173, abstract no. 1034; Czejka et al., 1990,
Pharmazie
45:H.9) and has been used in combination with other chemotherapeutic agents in the treatment of a variety of cancers including urinary bladder cancer (Horn et al., 1985,
J. Surg. Oncol.,
2:304-307), squamous cell carcinoma (Kohno et al., 94,
J. Cancer Res. Oncol.,
120:293-297), breast cancer (Beckenlehner et al., 1992,
J. Cancer Res. Oncol.
118:591-596), and gastrointestinal cancer (Scheithauer et al., 1988,
Anticancer Res.
8:391-396). Administration of hyaluronidase also induces responsiveness of previously chemotherapy-resistant tumors of the pancreas, stomach, colon, ovaries, and breast (Baumgartner et al., 1988,
Reg. Cancer Treat.
1:55-58; Zänker et al., 1986,
Proc. Amer. Assoc. Cancer Res.
27:390). Serum hyaluronidase prevents growth of tumors transplanted into mice (De Maeyer et al., 1992,
Int. J. Cancer
51:657-660), while injection of hyaluronidase inhibits tumor formation caused by exposure to carcinogens (Pawlowsli et al., 1979,
Int. J. Cancer
23:105-109; Haberman et al., 1981,
Proceedings of the
17
th Annual Meeting of the American Society of Clinical Oncology
, Washington, D.C., 22:105, abstract no. 415). Intravenous or intramuscular injection of hyaluronidase is effective in the treatment of brain cancer (gliomas) (PCT published application no. WO88/02261, published Apr. 7, 1988).
Hyaluronidase expression, and levels of hyaluron, have been associated with tumor development and progression. Levels of a secreted neutral hyaluronidase activity in carcinomas derived from ovary (Miura et al. 1995
Anal. Biochem.
225:333-40), prostate (Lokeshwar et al. 1996
Cancer Res
56:651-7), brain, melanocyte, and colon (Liu et al. 1996
Proc. Natl. Acad. Sci. USA
93:7832-7837) are higher than in normal tissue. This secreted neutral hyaluronidase activity appears similar or identical to the neutral hyaluronidase activity of the sperm hyaluronidase PH20. In contrast to neutral activity, the acid active serum hyaluronidase activity is significantly decreased in metastatic carcinomas of the lung, breast, and colon (Northrup et al. 1973
Clin. Biochem.
6:220-8; Kolarova et al. 1970
Neoplasma
17:641-8). Further, mice having an allele of the hyal-1 locus that is associated with lower levels of serum hyaluronidase activity exhibit faster rates of growth of transplanted tumors than mice having an hyal-1 allele that is associated with 3-fold higher hyaluronidase activity levels (Fiszer-Szafarz et al. 1989
Somat. Cell. Mol. Genet.
15:79-83; De Maeyer et al. supra).
At present, the only hyaluronidase activity available for clinical use is a hyaluronidase isolated from a testicular extract from cattle (WYDAS®, Wyeth-Ayerst). The bovine extract is not optimum not only because of its non-human source, but also because the extract contains multiple types of hyaluronidases and other as yet undefined components. While the human serum acid-active hyaluronidase activity would be preferred for administration, this hyaluronidase has not been previously isolated or purified. Although previous studies were able to determine that the serum acid-active hyaluronidase activity is not a component of platelets since hyaluronidase activity levels in plasma are comparable to those found in serum (De Salegui et al. 1967 supra), attempts to isolate this acid active hyaluronidase activity from human serum have met with limited success due in part to the stability of the purified activity and the inability to obtain an adequately high specific activity. Immunopurification attempts have been hindered by the inability to identify and isolate antibodies that specifically bind the activity in its native form in plasma. Monoclonal antibodies identified by conventional ELISA techniques bind denatured human plasma hyaluronidase in the ELISA screening assay do not bind the native, folded protein (Harrison et al. 1988
J Reprod Fertil
82:777-85).
Conventional methods for hyaluronidase activity include ELISA-like assays (Delpech et al. 1987
J. Immunol. Methods
104:223-9; Stern et al. 1992
Matrix
12:397-403; Afify et al. 1993
Arch. Biochem. Biophys.
30:434-41; Reissig
Csoka Anthony
Frost Gregory I.
Stern Robert
Wong Tim M.
Borden Paula A.
Bozicevic Field & Francis LLP
Francis Carol L.
The Regents of the University of California
Ungar Susan
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