Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1998-03-18
2001-12-04
Jarvis, William R. A. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
active
06326370
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the use of a specific class of compounds for the prevention of glutamate-evoked cytotoxicity.
STATE OF THE ART
Glutamate constitute the major excitatory neurotransmitter of the central nervous system (Hollmann M., Heinemann S., Annu. Rev. Neurosci. 17, 31-108, 1994) and the ubiquitous distribution of glutamate receptors throughout the CNS proves that glutamate plays a central role in a wide range of physiological as well as pathological events (Watkins J. C., Collingridge G. L., The NMDA receptor, IRL Oxford, 1989).
By most plausible theories and several experimental findings it is suggested a central role for glutamate-dependent neurotransmission in functions such as learning, pattern recognition, and memory (Bliss T. V. P. Collingridge G. L., Nature 361, 31-39, 1993).
It has also been known for decades that glutamate is toxic to neurons in vivo and in culture and that glutamate receptor functioning is crucial in a number of brain diseases and injuries (Appel S. H., Trends Neurosci. 16, 3-5, 1993). Many neurological illnesses involving strokes or epileptic seizures result in fact in brain damage just because of over-stimulation by glutamate, and degenerative diseases among which Alzheimer's, Huntington's, Parkinson's and amyotrophic lateral sclerosis (ALS) involve neuronal cell death caused by excessive activation of the glutamate receptors.
OBJECTS OF THE INVENTION
Object of the present invention is to provide a specific class of compounds for the modulation of glutamate-evoked neurotransmission and neurotoxicity, which allows the possible treatment of acute and chronic neurodegenerative diseases.
Another object of the present invention is to provide a specific class of compounds which allows the modulation of glutamate-related physiological functions, among which pain, hormonal balance, blood pressure, thermoregulation, respiration, learning, pattern recognition and memory.
Still an object of the present invention is to provide a specific class of compounds which can be used as pharmacological tools for the prevention of glutamate-evoked cytotoxicity.
A further object of the present invention is to provide a specific class of compounds which represents a valid pharmacological alternative to previously described compounds, such as competitive and non-competitive glutamate antagonists,, gangliosides and growth factors, for the treatment of acute and chronic glutamate-related neurological diseases.
DESCRIPTION OF THE INVENTION
These and still other objects and related advantages which will be more clearly stressed by the following description are achieved by the use of compounds which are agonists or antagonists of P
2
purinoceptors for the prevention of glutamate-evoked cytotoxicity.
The fundamental novelty of this invention is the correlation between glutamate-evoked biological events and P2 purinoceptor modulators (agonists or antagonists). Both glutamate receptors and P2 purinoceptors share in fact the property of being ionotropic as well as metabotropic kinds of receptors.
As an example, we have chosen the compounds Basilen Blue E 3G (also called Reactive Blue 2) and Cibacron Blue 3GA, which are antagonists of P
2
purinoceptors. These compounds can be purchased, for example, from Sigma and their molecular structures and main characteristics are described on the 1995 Sigma catalog distributed in Italy, respectively on pages 149 for Basilen Blue E-3G and 266 for Cibacron Blue 3GA. The other compound we have chosen is 5-adenylylimidodiphosphate (AMPPNP), which is an agonist of P2 purinoceptors. This compound can be also purchased from Sigma and its molecular structure and main characteristics are described in page 52 of the Sigma catalog published in Italy in 1995.
Always according to the present invention, these compounds are used to prevent glutamate-evoked cytotoxicity in the nervous system's cells, particularly in CNS neurons. As a cellular model system for CNS neurons we have adopted postnatal rat cerebellar neurons. These cells, which are among the best characterized primary neuronal cultures, when isolated from postnatal rat cerebellum (Lasher R. S., and Zagon I. S., Brain Res. 41, 428-438, 1972), develop in vitro their mature phenotype as interneurons which use glutamate as a neurotransmitter and furthermore constitute an excellent model system for the study of glutamate-mediated cytotoxicity.
By exposing granule neurons to 100 &mgr;M glutamate for 15-30 min is obtained (after 15-20 hours) 80-100% of total cell death. We find that the P
2
purinoceptor antagonist basilen blue, also called reactive blue 2 (an anthraquinone sulfonic acid derivative), when somministered to granule neurons at 100 &mgr;M in the simultaneous presence of glutamate, completely sustains cellular survival, thereby abolishing the cytotoxic action of glutamate. The effects of basilen blue on cerebellar granule cell morphology reveal, despite the exposure to glutamate, which otherwise induces complete cell death, apparently healty-looking cell bodies that do bear a dense network of highly branching processes. Adhesion and neurites fasciculation are also preserved by basilen blue. The acute response characterized by rapid swelling of the cell body and loss of brightness, generally observed in granule neurons within the first 5 min of treatment with glutamate, is furthermore prevented by addition of basilen blue, suggesting that the compound probably acts very early in the chain of events immediately downstream of the EAA-receptor interaction.
It is important to stress that basilen blue per se, up to the highest concentration tested of 300 &mgr;M, is not toxic to the cells and, when somministered to granule neurons for 0.5-26 hours, does not affect plasma membrane permeability (as measured by ethidium bromide uptake), or cellular metabolism (as measured by conversion of MTT into formazan by mitochondrial dehydrogenase activities). Basilen blue prevents glutamate-evoked cell death with an IC50 in the 10-20 &mgr;M range, value generally in agreement with the concentrations of the compound reported for P2 purinoceptor antagonism. The other commercially available isomer of the sulfonic derivatives of anthraquinone (cibacron blue) is effective in this regard. Caffeine, a P1 purinoceptor antagonist, up to 100 &mgr;M does not abolish the cytotoxic action of glutamate.
The effects of basilen blue on protection from cytotoxicity are linear with time and depend on the modality of somministration of the compound. When basilen blue is added to the cells 10 min after glutamate, and then incubated with granule neurons for only 15 min, it protects from cell death 60-70% of the entire neuronal population; if somministered only for the last 5 min of treatment of the cells with glutamate, basilen blue sustains survival of 25-40% of total neurons. When instead it is added 1-2 min or 30 min or 2 hours after exposure of granule neurons to glutamate (and then incubated with the cells for the next 20 hours) basilen blue protects from cell death respectively 55-70%, 30% and 10% of the total neuronal population. When it is added before (not during or after) exposure of granule neurons to glutamate, basilen blue requires a pretreatment of at least 20-25 hours in order to prevent by 70-80% the cytotoxic action of glutamate. Inhibition of aspartate uptake is not obtained as a consequence of these same treatments. Independently from the modality of somministration to granule neurons, the prevention of cytotoxicity induced by basilen blue does not depend on new protein synthesis, since it is insensitive to inhibitors such as actinomycin D (used at 10 &mgr;M) or anisomycin (used at 100 &mgr;M).
Basilen blue inhibits binding of [
3
H] ATP to membranes of granule neurons with an IC50 of about 10 &mgr;M, that corresponds to the IC50 that prevents glutamate-evoked cytotoxicity. Binding studies with [
3
H] ATP have been performed also directely with intact cells and basilen blue was shown to be as effective.
We have cultured the ce
Merlo Daniela
Volonte' Cinzia
Collard & Roe P.C.
Consiglio Nazionale Delle Recerche
Jarvis William R. A.
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