Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-05
2004-05-18
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S069100, C435S252300, C435S320100, C436S501000, C530S350000, C536S023500
Reexamination Certificate
active
06737242
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for assaying GABA-modulatory compounds for activity as antidepressants, cognitive enhancers, sedative hypnotics, or non-sedating anxiolytics. In particular, the method includes determining efficacy (generally in vitro efficacy) and EC
50
values (as used herein incorporating IC
50
values) for the compounds at several different cloned (i.e., expressed in cells as directed by heterologous cloned receptor-encoding nucleic acid expression vector molecules) GABA
A
subtype receptors (each subtype made up of a defined set of specific receptor subunit isotypes). The method optionally includes determining binding affinity of compounds for GABA
A
receptors. The method results in the development of an activity profile for each compound. As an additional step, animal models predictive of such effects may be used to measure the ability of compounds to effect cognitive enhancement, to act as antidepressants, to mediate sedative hypnotic effects, or to effect anxiolysis in vivo without eliciting certain undesirable side effects.
2. Description of Related Art
Modern drug discovery methodology allows the testing of large numbers of compounds (often assembled into collections termed libraries) for functional characteristics that confer pharmaceutical utility. This “screening” of such libraries, using specific tests (assays) for functional activity properties, allows the rapid identification of promising compounds for further development as pharmaceutical agents. There has been a longstanding quest in the pharmaceutical industry for new means of identifying such promising compounds. Such new means may involve new assays, or may use old assays to generate data that can be analyzed and applied in new ways to identify compounds with new and useful characteristics.
In the field of psychopharmacology, the use of cloned neuronal receptors as substrates has, provided new, more specific assays with which compounds can be characterized. The use of such receptors has enabled the development of receptor binding profile criteria that are particularly beneficial in the identification of useful psychopharmacological agents. For example, such profiling can identify compounds that will be free of certain undesirable adverse effects (side effects).
The GABA
A
receptor superfamily represents one of the classes of receptors through which the major inhibitory neurotransmitter, &ggr;-aminobutyric acid, or GABA acts. In addition to being the site of neurotransmitter action a number of drugs including the anxiolytic and sedating benzodiazepines bind to this receptor. The GABA
A
receptor is a chloride channel that opens in response to GABA, allowing chloride to enter the cell. This effects a slowing of neuronal activity through hyperpolarization of the cell membrane potential. GABA
A
receptors are composed of several protein subunits and are generally pentameric in structure.
A number of cDNAs for GABA
A
receptor subunits have been cloned. While these subunits share a basic motif of 4 membrane-spanning helices, there is sufficient sequence diversity to classify them into several groups. To date at least 6&agr;, 3&bgr;, 3&ggr;, 1&egr;, 1&dgr;, 2&rgr;, and 1&pgr; subunit species have been identified; some representing alternatively spliced forms. Native GABA
A
receptors are typically composed of &agr;, &bgr;, and &ggr; subunits, most often in the ratio of two alphas, two betas, and one gamma, although other combinations (some comprising other subunits such as &egr;, &dgr;, &rgr;, or &pgr;) have been described. Even if restricted to only &agr;, &bgr;, and &ggr; subunits, however, an enormous diversity of GABA
A
subtype receptors are possible. Evidence such as message distribution, genome localization and biochemical studies suggests that the major naturally occurring receptor combinations are &agr;
1
&bgr;
2
&ggr;
2
, &agr;
2
&bgr;
3
&ggr;
2
, &agr;
3
&bgr;
3
&ggr;
2
, and &agr;
5
&bgr;
3
&ggr;
2
.
In the typical GABA
A
receptor, the binding sites for GABA (2 per receptor complex) are formed by amino acids from the &agr; and &bgr; subunits. Amino acids from the &agr; and &ggr; subunits contribute to form 1 benzodiazepine site per receptor complex. In a classic allosteric mechanism, the binding of a drug to the benzodiazepine site increases the affinity of GABA binding to the receptor. Benzodiazepines and related drugs that enhance the ability of GABA to open GABA
A
receptor channels are known as agonists or partial agonists depending on the level of enhancement. Other classes of drugs such as &bgr;-carboline derivatives that occupy the same site and negatively modulate the action of GABA are called inverse agonists. A third class of compounds exists that occupies the same site as both the agonists and inverse agonists (blocking access of these agents to the site) and yet has little or no direct effect on GABA activity. These compounds are referred to as antagonists.
The characterization of activities of different subtype receptors has been an area of intense pharmacological discovery. Agonists that act at the benzodiazepine site have long been known to know to exhibit anxiolytic, sedative, and hypnotic effects in animal behavior models, while compounds that act as inverse agonists at this site elicit anxiogenic, cognition enhancing, and proconvulsant effects. While benzodiazepines have long been used as anxiolytics, these compounds exhibit a number of undesirable side effects. These include cognitive impairment, sedation, ataxia, potentiation of ethanol effects, and a tendency for tolerance and drug dependence. Likewise the development of benzodiazepine site ligands for other indications has been thwarted by unfavorable side effect profiles for each indication. For example, compounds known to possess cognition enhancing properties have generally tended to be anxiogenic and proconvulsant, while compounds that produce anxiolytic effects tend to generate unwanted sedation, and do so more powerfully when taken in conjunction with the consumption of alcoholic beverages.
SUMMARY OF THE INVENTION
The present invention provides methods for characterizing compounds that act at the GABA
A
receptor benzodiazepine site. In particular, it provides methods for identifying compounds with characteristics indicating that the identified compounds will exhibit pharmacological properties consistent with their use as antidepressants, cognitive enhancers without anxiogenic or proconvulsant activity, sedative hypnotics without cognition-impairing activity, or non-sedating anxiolytics.
This invention is useful in screening libraries of compounds for therapeutic potential and in drug design efforts.
Measurement of GABA receptor binding affinity is a useful step in any of the methods of the invention. Alternatively, these assays may be performed without measuring the binding affinity of the compound. The assays may include an assessment of the ability of the compound to mediate the desired effects in vivo without eliciting side effects using animal models established to be predictive of the desired effects and animal models predictive of the undesired side effects that have been associated with other compounds acting at GABA
A
receptors.
As used herein, the term “efficacy” refers to amount of potentiation (represented as a % increase, e.g., 10%) or inhibition (represented as a % decrease, e.g., −10%) of GABA activated responses measured for GABA
A
receptors.
In addition to the ability of a compound to effect a specified magnitude of change in the GABA response at distinct subtype receptors, the EC
50
value of the compound at the pertinent receptors is also taken into consideration. As used herein, the term “EC
50
” or “EC
50
value” refers to the concentration of a compound needed to elicit half the maximal response (to the agonist or inverse agonist effects of a compound) that can be obtained with the compound. Thus, a compound that exhibits dissimilar EC
50
values at different subtype receptors can selectively potentiate one of t
Albaugh Pamela
Cassella James
Crandall Marci
Gallager Dorothy
Rajachandran Lavanya
Brannock Michael
Eyler Yvonne
McDonnell Boehnen & Hulbert & Berghoff LLP
Neurogen Corporation
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