Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-02-22
2003-04-01
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C800S003000, C800S013000, C800S022000
Reexamination Certificate
active
06541193
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the development of a novel test model and assay for screening the activity of drugs for central nervous system (CNS). More particularly, the invention identifies the use of the fruit fly as a model to test the neuroactivity of test drugs/samples.
BACKGROUND OF THE INVENTION
Drugs that stimulate or depress CNS play an important role in human therapeutics. They act as anesthetics, analgesics, sedatives, psychostimulants, analeptics, antidepressants, anticonvulsants etc. and are used in the treatment of conditions such as narcolepsy, depression, hyperactivity disorders, epilepsy and drug addiction in human (Wood-Smith and Stewart, 1964, in Drugs in anesthetic practice, Butterworth; Beckman, 1958, in Drugs, their nature, action and use, W. B. Saunder, Green and Levy, 1976, Drug misuse, human abuse, Dekker). Undesirable side effects and ineffectiveness of currently available CNS stimulants/depressants in many situations call for development of novel drugs.
Animal models currently used in neuroactive drug screening mostly include higher animals such as nonhuman primates and rodents. Although their indispensability in advanced stages of drug development can not be denied, using these models in primary rug screening is time, cost and labor intensive. Many components of neuronal signaling are conserved between the fruit fly
Drosophila melanogaster
and human (Rubin e al, 2000, Science 287:2204-2215; Littleton and Ganetzky, 2000, Neuron 26:35-43). Moreover, many CNS stimulant/depressant drugs used in human therapy also exhibit their neuroactivity in fly (Shaw et al, 2000, Science 287:1834-1837; Hendricks et al, 2000, Neuron 25:129-138; Andretic et al, 1999, Science 285:1066-1068; Li et al, 2000, Curr. Biol. 10:211-214; Baintaon et al, 2000, Curr. Biol. 10:187-194; McClung and Hirsh, 1999, Curr. Biol. 9:853-860). Considering these, the applicants thought of evaluating of potential of Drosophila to serve as a simple, rapid and inexpensive model for screening of CNS active agents.
A variety of CNS stimulant/depressant drugs have originated from plants (Wood-Smith and Stewart, 1964, in Drugs in anesthetic practice, Butterworth; Beckman, 1958, in Drugs, their nature, action and use, W. B. Saunder; Green and Levy, 1976, Drug misuse, human abuse, Dekker, Plotkin, 2000, in Medicine quist in search of nature's healing secrets, Viking; Gratzer, 2000, Nature 406:235-236). There are several reasons why neuroactive compounds made by plants work on receptors in human brain is also (Lam et al, 1998, Nature 396:125-126). Keeping the above in view, the applicant used the fly model plant samples and developed a simple assay for screening of substances and evaluate their utility in drug screening.
OBJECTS OF THE INVENTION
The main object of the invention is to provide a novel method for screening of neuroactive drugs using the fruit fly
Drosophila melanogaster
as an in vivo model.
Another object is to develop a method for simultaneous screening of antiepileptic and central nervous system stimulant/depressant classes of drugs.
Yet another object is to provide a simple and cost-effective method for studying the neuroactivity of a substance using
Drosophila melanogaster
as a model.
SUMMARY OF THE INVENTION
Accordingly, the invention provides a novel method for screening of neuroactive drugs using
Drosophila melanogaster
as an in vivo test model. The invention also provides methods for studying the neuroactivity of a substance using
Drosophila melanogaster
as a model.
DETAILED DESCRIPTION
The invention relates to a method for screening of neuroactive drugs using the fruit fly
Drosophila melanogaster
, which comprises the steps of:
a) generating a double mutant line of K
+
channel genes in
Drosophila melanogaster;
b) culturing the Sh
5
eag
1
mutant flies on Dorsophila medium under standard conditions; and
c) anesthetizing flies with diethyl ether and observing the time taken by flies to recover from anesthesia.
In an embodiment, decreased arousal time in flies treated with normal fly food mixed with the agent being screened, compared to flies fed on normal fly food, is indicative of analeptic activity of the agent.
In another embodiment, an early arousal in flies treated with normal fly food mixed with both the agent being screened as well as the drug phenobarbital, compared to flies fed on normal fly food mixed with phenobarbital alone, is indicative of an analeptic activity of the agent.
In yet another embodiment, spontaneous locomotor activity is observed.
In still another embodiment, increased locomotor activity in flies treated with normal fly food mixed with both the agent being screened as well as ethanol, compared to flies fed on normal fly food mixed with ethanol alone, is indicative of an analeptic activity of the agent.
In an embodiment, an increased locomotor activity in flies treated with normal fly food mixed with the agent being screened, compared to flies fed on normal fly food, is indicative of a psychostimulant activity of the agent.
Thus, the present invention relates to a novel method for screening of CNS active agents. Ether-sensitive leg shaking phenotype of
Drosophila melanogaster
K
+
channel double mutant Sh
5
eag
1
has been earlier evaluated by the applicants as a target for antiepileptic drug screening (Sharma and Kumar, 2000, U.S. patent application Ser. No. 09/535,517). To further enhance the usefulness of Sh
5
eag
1
in neuroactive drug screening the applicants studied the effect of a CNS depressant drug, phenobarbital, on time taken by flies to recover from ether anesthesia. The drug was found to delay recovery. This suggested that a change in recovery time could be exploited as a screening criterion for CNS active drugs. A comparison between Sh
5
eag
1
and Oregon-R wild-type flies revealed that recovery is faster in the former. This indicated the advantage of mutant over wild-type in speeding up the process of drug screening. The position of Sh
5
eag
1
as an efficient neuroactive drug testing model was therefore further consolidated. The model thus evolved was applied for screening of the targeted activities in plant extracts. Out of 50 plant species screened, one was found to test positive. Further experiments confirmed that the plant substance screened has analeptic and psychostimulant properties (Sharma et al, a substance from
Acorus calamus
plant with analeptic and psychostimulant properties, a separate patent filed). This demonstrates the practical usefulness of the fly model developed. Accordingly, the present invention provides a CNS drug screening method, which comprises use of the fruit fly
Drosophila melanogaster
as a whole organism in vivo model for drug testing.
Vertebrate animal models currently used for screening of neuroactive agents are time, labor and cost intensive. The applicants therefore thought of using fruit fly
Drosophila melanogaster
as a simple, rapid and inexpensive in vivo whole organism behavioral model for primary drug screening. In this context, we previously validated the ether-sensitive leg shaking phenotype in K
+
channel double mutant Sh
5
eag
1
as a target for antiepileptic drug screening (Sharma and Kumar, 2000, U.S. patent application Ser. No. 09/535,517). To add value to this model, it was desirous to exploit it for simultaneous screening of other CNS active drugs. Anesthetics produce a profound depression of CNS and, therefore, we wondered if recovery from anesthesia in flies could serve as a simple means to rapidly screen CNS depressants/stimulants. To explore this possibility, the applicants studied the effect of known CNS depressant, phenobarbital, on time taken by Sh
5
eag
1
flies to recover from ether anesthesia. It turned out that the drug delays recovery. A change in recovery time was therefore validated as a criterion for screening CNS active agents. While applying Sh
5
eag
1
in antiepileptic drug screening, the applicants noticed that the mutant flies recover from ether anesthesia earlier than wild-type ones. If it is so, we thought,
Kumar Sushil
Sharma Abhay
Council of Scientific & Industrial Research
Srivastava Kailash C.
Weber Jon P.
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