Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2000-03-24
2001-09-18
Nguyen, Dave T. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S013000, C800S022000
Reexamination Certificate
active
06291739
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for the screening of anti-epileptic drugs using an animal model. More particularly, the present invention relates to a method for the screening of anti-epileptic drugs using neurological mutants of the fruit fly
Drosophila melanogaster.
By studying the effect of various established anti-epileptic drugs (AEDs) on neuronal hyperexcitability phenotype of these mutants, the applicants demonstrate that animal models, particularly fruit fly could serve as a simple, rapid and inexpensive whole organism in vivo phenotype-based model for screening of drugs, compounds, natural products etc. for anti-epileptic-like activities. The development of an animal model and the associated process for neuroactive drug screening is of immense value in the development and identification of drugs and their potential for treatment of neurological disorders such as epilepsy.
BACKGROUND OF THE INVENTION
Epilepsy refers to a collection of disorders affecting 1-2 % of the global population. It is a brain disorder characterized by recurrent seizures, brief changes in behaviour caused by disordered, synchronous and rhythmic firing of populations of neurons in the central nervous system. Epilepsies are caused both by genetic factors and by cortical damage. They have been classified into more than 40 distinct types on the basis of characteristic symptoms and signs, cause, seizure types, electroencephalographic patterns and age of onset. The single common feature of epilepsy syndromes is a persistent increase of neuronal excitability that occasionally and unpredictably results in a seizure. With the identification of mutations in genes encoding voltage- and ligand- gated ion channels as molecular aetiology of some forms of inherited human epilepsy, and with the understanding of altered synapse function as the causal factor in epilepsies caused by cortical damage, it has been learnt that the common mechanisms underlying the hyperexcitability in diverse forms of epilepsy is alteration of intrinsic properties of neurons and/or synaptic function (McNamara, 1999,
Nature
399: A15-A22; Puranam and McNamara, 1999,
Curr. Op. Neurobiol.
9: 281-287; Steinlein, 1998,
Clin. Genet.
54: 169-175).
In many epileptic patients, seizures can be controlled with the established AEDs such as phenobarbital, phenytoin, carbamazepine and valproate (Brodie and Dichter, 1996,
N. Eng. J. Med
334: 168-175; Marson and Chadwick, 1996,
Curr. Op. Neurol
9:103-106). However, around 25-30 % of patients continue to have seizures despite optimal therapy and others have unacceptable side effects (Brodie and Dichter, 1996,
N. Eng. J. Med
334: 168-175). In recent years, a number of new AEDs such as gabapentin, lamotrigine, felbamate and clobazam have been developed (Macdonald and Grenfield, 1997,
Curr. Op. Neurobiol.
10: 121-128). Adequate data on the possible teratogenic effects of these AEDs, however, is not available. In addition, these new drugs have limited efficacy and have the potential for serious side effects. Clinical trials have shown that some patients respond to one drug in a better way than to another, even when they have similar types of seizures and the drugs used have similar mechanisms of action; the frequency and severity of side effects also vary substantially. In view of the above, it is clear that there is a need to develop more AEDs.
As with other human disease research, epilepsy research has been dependent upon the use of animal models, notably in the screening of thousands of compounds for possible anticonvulsant activity. AEDs that are commonly used to treat generalized epilepsies in humans have been shown to respond to animal models of epilepsy (Batini et al, 1996,
Trends Neuosci.
19: 246-252). Putnam and Merritt (1937,
Science
85: 525-526) were the first to show that an experimental model of epilepsy could be used to screen chemical compounds systematically for anticonvulsant activity. Since then the search for new AEDs has followed three approaches (Laird II, 1985,
FASEB
44: 2627-2628). The majority of the currently used AEDs were discovered by the first approach in which new chemical entities synthesized as potential AEDs are screened for anticonvulsant efficacy using classical experimental models (electroshock and chemoshock) of epilepsy. The second approach has been to search for the molecular mechanism(s) by which clinically effective anticonvulsant agents work so that more efficacious and selective agents may be developed. In the third, information derived from an understanding of the molecular defect(s) responsible for the epileptic state has formed the basis of drug development.
Evaluation of anticonvulsant activity is possible at various biological levels such as subcellular, cellular, cerebral and spinal neuronal, whole brain, normal intact animal and modified intact animal (Swinyard and Kupferberg, 1985,
FASEB
44: 2629-2633). Methods like receptor pharmacology in vitro, single neuron recording in vitro and in vivo, electrophysiology, neurochemistry, histochemistry, electroencephalography etc. are used for the study of anticonvulsant activity. Many of these methods are highly valuable, particularly for differentiating drug mechanisms and as penultimate tests before clinical study of potential AEDs. Many of them are however time-intensive and expensive. They are therefore not suitable for routine use in the screening for anticonvulsant agents. It is clear that there is a need to develop new models. These models should be validated by testing against the prototype AEDs.
In the fruit fly
Drosophila melanogaster,
several mutations in genes encoding voltage- and ligand- gated ion channels and genes involved in synaptic function are known (Brunner and O'Kane, 1997,
Trend Genet.
13: 85-87; Lindsley and Zimm, 1992, The Genome of Drosophila melanogaster, Acad. Press; Wu and Bellen, 1997,
Curr. Op. Neurobiol.
7: 624-630; Zhou et al, 1999,
Neuron
22: 809-818). Some of these mutations produce behavioral hyperexcitability in flies. Of particular interests are the K
+
channel mutants Shaker (Sh), Hyprkinetic (Hk) and ether a go-go (eag) whose most distinctive phenotype is a rapid leg shaking under ether anesthesia (Choumnard et al, 1995,
Proc. Natl. Acad. Sci. USA
92: 6763-6767; Ganetzky, 1989,
Genetics
21 : 201-204; Kaplan and Trout, 1968,
Genetics
61: 399-409). Voltage-dependent K
+
channels are conserved from bacteria to man (Baumann et al, 1988,
EMBO J.
7: 2457-2463; Doyle et al, 1998,
Science
280: 69-77; Doyle and Stubbs, 1998,
Trends Genet.
14: 92-98; MacKinnon et al, 1998,
Science
280: 106-108Stansfeld et al, 1997,
Trends Neurosci.
20: 13-14; Trudeau et al, 1995,
Science
269: 92-95; Wang et al, 1998,
Science
282: 1890-1893). Prompted by the finding that a human counterpart of Drosophila K
+
channel gene is mutated in a human epilepsy (Biervert et al, 1998,
Science
274: 403-406; Singh et al, 1998,
Nature Genet.
18: 25-29), and fascinated by the simple, quick and inexpensive method of visualizing neuronal hyperexcitability phenotype in Sh, Hk and eag flies (Ganetzky, 1989,
Genetics
21: 201-204), we attempted to evaluate these mutants as a model to screen drugs, compounds, natural products etc. for antiepiletic-like activities. Most of the established AEDs act by preventing repetitive firing of action potentials in depolarized neurons through voltage- and use- dependent blockade of Na
+
channels (Brodie and Dichter, 1996,
New Eng. J. Med.
334: 168-175; Macdonald and Greenfield, 1997,
Curr. Op. Neurobiol.
10: 121-128; Rogawsky and Porter, 1990,
Pharmacol. Rev.
42: 223-286). The knowledge that a relatively minor reduction of K
+
current may produce epilepsy, and the possibility that a drug modestly enhancing the K
+
current could effectively inhibit seizures (McNamara, 1999, Nature 399: A15-A22), further strengthened our idea of evaluating K
+
channel mutants of
Drosophila melanogaster
as a phenotype-based whole organism in vivo model for antiepilep
Kumar Sushil
Sharma Abhay
Council of Scientific and Industrial Research
Darby & Darby
Nguyen Dave T.
Nguyen Quang
LandOfFree
Method for screening of potential anti-epileptic drugs using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for screening of potential anti-epileptic drugs using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for screening of potential anti-epileptic drugs using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2509249