Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-11
2001-06-05
Owens, Amelia (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C549S424000
Reexamination Certificate
active
06242582
ABSTRACT:
This invention relates to diagnostic methods, and in particular to methods for diagnosis of influenza virus infection. The invention provides a novel diagnostic method which can be used for detection of all strains of influenza virus Type A and Type B. The method is based on the use of compounds able to bind specifically to the active site of influenza virus neuraminidase, or functionalised derivatives of such compounds, as binding and/or detecting agents to identify influenza virus in clinical specimens. The term “neuraminidase binders” is used hereinafter to refer to these compounds and their functionalized derivatives. The method and compounds of the invention can function either in the presence or the absence of compounds binding non-specifically to influenza virus neuraminidase.
The invention particularly relates to a new class of chemical compounds and their use as diagnostic agents for the visual or instrumental detection of influenza A and B. More specifically the invention concerns a group of novel neuraminic acid (sialic acid) derivatives which bind to influenza A and B neuraminidase, and which also have a functionality which allows the compounds to be bound to a surface or to a detectable label.
BACKGROUND OF THE INVENTION
Influenza A and B viruses are major causes of acute respiratory disease, with an estimated 30-50 million infections annually in the United States alone. Influenza A has been responsible for major epidemics, such as the “Spanish Flu” of 1919 which killed millions of people. Many viral and bacterial infections may present with symptoms similar to those of influenza. The rapid identification of respiratory viruses would enable physicians to use the most appropriate therapy early in the illness. For example, an early and accurate diagnosis would allow decisions regarding the use of antibacterial therapy and hospitalisation of children and the elderly.
Laboratory tests for the identification of viruses in clinical material are widely used, and a variety of different detection methodology is available. The textbook, “Laboratory Diagnosis of Viral Infections”, Marcel Dekker 1992, Ed E. H. Lennette generally discusses methods which are used for a wide range of viruses, including influenza virus.
A nester of tests are available for the diagnosis of influenza A and B. The traditional method of identifying influenza viruses has been the use of cell culture, which is highly sensitive and specific. Unfortunately, the time required for culture, isolation and identification of influenza virus can range between 2 and 10 days, thus making it virtually useless in guiding the physician to an appropriate therapy. Since influenza virus infection is normally self-limited, diagnosis must be rapid if therapy is to be effective. In other words, such cell culture methods are normally only of value in providing retrospective epidemiological information.
In addition to the cell culture methods for detecting influenza, there have recently become available a few rapid direct tests, which are specific for influenza A. Thus, a monoclonal immunofluorescence assay (IFA) has been reported (Spada, B. et al, J. Virol. Methods, 1991 ×305) and at least one enzyme Immunoassay (EIA) is available (Ryan-Poirier, K. A. et al, J. Clin. Microbiol., 1992 30 1072). A nxxuer of comparisons of these rapid detection methods for influenza A have been reported; see for example Leonardi, G. P. et al, J. Clin. Microbiol., 1994 32 70, who recommended that direct specimen testing be used together with culture isolation, so as to permit both identification of the virus, in time to institute therapy and infection control measures, and to monitor the antigenic constitution of influenza strains prevalent in the cotunity for epidemiological purposes. The IFA method is reported to be labor-intensive, and requires considerable technical expertise, with the results often being difficult to interpret. On the other hand, the EIA method (Directigen FLU-A; Becton Dickinson Microbiology Systems) gave a high level of false-positive results, and it has been recmmended that this assay should be used in laboratories only in addition to or as a substitute for direct immunofluorescence tests (Waner, J. L. et al, J. Clin. Microbiol., 1991 29 479).
As well as the problems mentioned above with the available rapid assays for influenza, there are other fundamental deficiencies in some of these methods. Firstly, none of the available assays can detect influenza B, which means that even a negative test result would leave the physician uncertain about the type of therapy that should be used. Secondly, if a rapid immunoassay method depends on the use of antibodies to one of the influenza A proteins, there may be a serious problem in detecting new strains of the virus which have undergone a drift or shift in the structure of the antigenic proteins. Influenza A is notorious for its propensity to undergo such changes.
Another type of rapid assay for influenza viruses has been described in a series of patent specifications (see for example Liav, A. et al, International Patent Application No. WO 92/12256). The method involves the use of a chromogenic substrate for the influenza neuraminidase enzyme. In other words the assay depends on visualising a dye, which is formed when the influenza neuraminidase cleaves a special sialic acid-dye conjugate molecule. This technique appears to offer limited specificity, because it could not readily distinguish between the presence of viral neuraminidase and other forms of the enzyme, particularly bacterial neuraminidase. It may also have low sensitivity because of the relatively slow activity of viral neuraminidase.
Neuraminidase is one of the key proteins present on the surface of the influenza virus, and it plays an important role in the ability of the virus to infect humn cells. It has long been thought that agents which bind to the neuraminidase enzyme might prevent infection by influenza, and much effort has gone into seeking such binders. Whilst many compounds have shown in vitro activity against influenza neuraminidase, only recently has it been established that it is possible to achieve protection from influenza infection in vivo by the use of a powerful neuram idase binder which binds to the active site of the enzyme (see von Itzstein, M. et al, Nature 1993 363 418 and our earlier patent applications, International Patent Applications No. WO 92/06691 and No. WO 91/16320, the entire disclosures of which are herein incorporated by this reference). In particular it has been found that 2,3-didehydro-2,4-dideoxy-4-guanidinyl-N-acetylneuraminic acid (Compound I, designated GG167) is a potent binder of influenza neuraminidase, and also shows potent in vivo antiviral activity in aniinl s (Ryan, D. M. et al, Antimicrobial Agents and Chemotherapy, 1994 38 2270) and in human volunteers (Hayden, P. G. et al, J. American Medical Assoc., 1996 275 295).
More recently it has been found that certain substituted cyclohexene derivatives of sialic acid are also potent binders of influenza virus neuraminidase (Kim, C. U. et al, J. Amer. Chem. Soc., 1997 119 681), and specifically the compound (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (GS 4071)
It is the purpose of the present invention to overcome some of the problems inherent in the prior art methods and to provide a simple and sensitive means of detecting influenza viruses.
Accordingly, we have now found that biologically active substances which possess stereochemical complementarity to the active site of influenza virus neuraminidase, as disclosed in the publication by von Itzstein et al referred to above, such that the IC
50
for binding is 10
−6
M or less, and in particular certain derivatives of Compound I can be used to detect influenza virus through their ability both to selectively bind the influenza virus and at the same time to be attached to a surface or to a detectable linking group.
SUMMARY OF THE INVENTION
The invention therefore provides in a first aspect a method of detection of influenza v
Jin Betty
Krippner Guy Y.
Reece Phillip A.
Watson Keith Geoffrey
Wu Wen-Yang
Biota Scientific Management Pty Ltd.
Foley & Lardner
Owens Amelia
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