Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2002-08-15
2004-01-27
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S029000, C435S201000
Reexamination Certificate
active
06682904
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to hyaluronidase 2 (HYAL2) inhibition and, more specifically, to molecules that specifically inhibit HYAL2 activity, to methods of identifying HYAL2-specific inhibitors, and to methods for ameliorating a condition associated with the pro-inflammatory activity of HYAL2, including, for example, chronic inflammation or vasculitis, by specifically inhibiting HYAL2 activity and, therefore, HYAL2 mediated generation of pro-inflammatory and pro-angiogenic hyaluronan intermediate fragments.
2. Background Information
Inflammation and necrosis of blood vessels (vasculitis), including arteries, veins, and capillaries, can occur in connection with exposure to infectious agents, mechanical trauma, radiation, or toxins, and in association with immunological responses or disorders. In many cases, however, no etiology can be determined for a vasculitis (Rubin and Farber, “Pathology” 3d ed. (Lippincott-Raven 1999); pages 514-520).
Numerous vasculitis disorders have been identified, and have been classified based on the size of the blood vessels that primarily are affected, i.e., small vessel, medium vessel, or large vessel, or based on general similarities of the disorders, e.g hypersensitivity vasculitis, which includes serum sickness and some, but not all, other disorders that involve, at least in part, an undesirable immune response. Despite these attempted classifications of vasculitis disorders, however, there is considerable overlap of signs and symptoms associated with the vasculitis disorders in different classification groups, and, therefore, it has been difficult to prescribe general treatment protocols.
The occurrence of vasculitis has variously been attributed to an involvement of immune mechanisms and to viral infection. A potential involvement of immune mechanisms originally was based on the identification of immune complexes in serum sickness, which was one of the first human immunological disorders associated with vasculitis. However, evidence is lacking for a role of the immune response in most cases of vasculitis. Anti-neutrophil cytoplasmic antibodies (ANCA) have been identified in association with Wegener's granulomatosis and microscopic polyarteritis, which are small vessel vasculitis disorders. However, it is not clear whether the appearance of ANCA is causal for these disorders or merely an effect that is observable. In fact, the difficulty in classifying and treating vasculitis is due to a lack of understanding of the etiology of the disorder. Thus, a need exists to identify a general etiology of vasculitis such that methods for early diagnosis and prevention or treatment of this disorder can be developed. The present invention satisfies this need and provides additional advantages.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that structurally related hyaluronidase polypeptides have opposing mechanisms of action, including pro-inflammatory or anti-inflammatory activity, such that broad spectrum hyaluronidase inhibitors can cause undesirable effects when administered to an individual. As disclosed herein, the use of agents that specifically inhibit hyaluronidase 2 (HYAL2), which has pro-inflammatory activity, provides a means to ameliorate pathologic conditions associated an undesirable inflammatory response due to generation of an intermediate hyaluronan catabolite by HYAL2.
Accordingly, the present invention relates to a method of identifying an agent that specifically inhibits HYAL2 activity, without substantially affecting the activity of non-inflammatory and anti-inflammatory hyaluronidases. Such a method can be performed, for example, by contacting HYAL2 and hyaluronan (HA) with a test agent, under conditions sufficient for HYAL2 activity, and detecting a decrease in HYAL2 activity. In the same reaction, or in a different reaction, which can be run in parallel, the activity of one or more non-inflammatory hyaluronidases such as HYAL1 or PH20 can be determined to confirm that an agent that inhibits HYAL2 activity has substantially less, if any, effect on the non-inflammatory (or anti-inflammatory) hyaluronidase(s) and, therefore, is a specific inhibitor of HYAL2.
A screening assay of the invention can be performed using cells that express HYAL2, either naturally or due to introduction of a HYAL2 encoding transgene, or using a detergent extract of such cells, including the membrane fraction, which contains the associated HYAL2. Where a cell that is genetically modified to express HYAL2 is used, or a detergent extract of such a cell, the transgene, which encodes HYAL2, can be transiently contained in the cell, or can be stably maintained by integration into the cell genome. Such a stably transfected cell can provide a standardized source of membrane associated HYAL2 useful for performing a screening assay of the invention. A screening assay of the invention also can be practiced using HYAL2 associated with a synthetic membrane, for example, a lipid bilayer or a unilamellar or multilamellar vesicle such as a liposome.
A decrease in HYAL2 activity can be detected directly by measuring a decrease in the amount or rate of generation of the pro-inflammatory 20 kDa intermediate HA catabolite following addition of the test agent, or can be detected indirectly by detecting decreased expression of a reporter gene regulated from a chemokine promoter, which exhibits induced expression in the presence of the 20 kDa intermediate. Such a reporter gene includes the chemokine promoter, for example, a RANTES, promoter, operatively linked to a nucleotide sequence encoding a detectable polypeptide as an enzyme; a fluorescent or luminescent polypeptide; a ligand (or receptor) that specifically binds a particular receptor (or ligand); or the like.
An agent that specifically inhibits HYAL2 activity can be any type of molecule, including, for example, a peptide (or polypeptide), a polynucleotide, a peptidomimetic, a peptoid, or a small organic molecule. For example, the agent can be an anti-HYAL2 antibody, or a HYAL2 binding fragment of said antibody. It will be recognized that the screening assays of the invention are readily adaptable to a high throughput format. As such, the methods allow for the screening of large numbers of test agents in parallel, and further allow for control reactions to be run in parallel, for example, reactions containing non-inflammatory or anti-inflammatory hyaluronidases, thus providing internal controls useful for confirming that an agent specifically inhibits HYAL2 activity without substantially affecting the activity of hyaluronidases that are not pro-inflammatory.
The present invention further relates to a HYAL2 specific inhibitor identified using such a screening assay. The HYAL2 specific inhibitor can be useful as a purified reagent, for example, as a material to be added to cells in culture to specifically inhibit HYAL2 activity, thus providing a standard for a screening assay to identify agents that can specifically inhibit HYAL2 activity, or can be formulated as a composition, which, for example, can be in a form suitable for administration to a subject, including a vertebrate subject such as a mammal, particularly a human. Such a composition containing a HYAL2 specific inhibitor can be useful for treating an inflammatory disorder associated with HYAL2 activity, for example, a vasculitis, by reducing or inhibiting HYAL2 activity, thereby reducing the generation of the pro-inflammatory 20 kDa intermediate HA breakdown product. Accordingly, the present invention provides a medicament useful for treating a subject having such an inflammatory disorder.
The present invention also relates to a method of ameliorating an inflammatory condition associated with HYAL2 mediated generation of a 20 kDa intermediate HA breakdown product in a subject. Such a method can be performed, for example, by administering a HYAL2 specific inhibitor to the subject, whereby HYAL2 activity is reduced or inhibited, thereby ameliorating the inflammatory
Deliatroph Pharmaceuticals, Inc.
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Imbra Richard J.
Leary Louise N.
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