Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-30
2001-05-01
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S252300, C536S023500
Reexamination Certificate
active
06225115
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application various publications are referred to by partial citations or by number within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Chemical neurotransmission is a multi-step process which involves release of neurotransmitter from the presynaptic terminal, diffusion across the synaptic cleft, and binding to receptors resulting in an alteration in the electrical properties of the postsynaptic neuron. For most neurotransmitters, transmission is terminated by the rapid uptake of neurotransmitter via specific, high-affinity transporters located in the presynaptic terminal and/or surrounding glial cells (39). Since inhibition of uptake by pharmacologic agents increases the levels of neurotransmitter in the synapse, and thus enhances synaptic transmission, neurotransmitter transporters provide important targets for therapeutic intervention.
The amino acid GABA is the major inhibitory neurotransmitter in the vertebrate central nervous system and is thought to serve as the neurotransmitter at approximately 40% of the synapses in the mammalian brain (22,37). GABAergic transmission is mediated by two classes of GABA receptors. The more prevalent is termed GABA
A
, which is a multi-subunit protein containing an intrinsic ligand-gated chloride channel in addition to binding sites for a variety of neuroactive drugs including benzodiazepines and barbiturates (46,105). In contrast, GABA
B
receptors couple to G-proteins and thereby activate potassium channels (5,46) and possibly alter levels of the second messenger cyclic AMP (46). Positive modulation of GABA
A
receptors by diazepam, and related benzodiazepines has proven extremely useful in the treatment of generalized anxiety (116) and in certain forms of epilepsy (86).
Inhibition of GABA uptake provides a novel therapeutic approach to enhance inhibitory GABAergic transmission in the central nervous system (47,92). Considerable evidence indicates that GABA can be taken up by both neurons and glial cells, and that the transporters on the two cell types are pharmacologically distinct (24,47,92). A GABA transporter with neuronal-type pharmacology designated GAT-1 has previously been purified and cloned (31), but the molecular properties of other GABA transporters including glial transporter(s) have not yet been elucidated. We now report the cloning of two additional GABA transporters (GAT-2 and GAT-3) with distinct pharmacology and localization, revealing previously unsuspected heterogeneity in GABA transporters.
Termination of GABAergic neurotransmission is accomplished by uptake of neurotransmitter into the presynaptic terminal and the surrounding astroglial cells, which is mediated by high-affinity, sodium dependent transporters. Pharmacologic inhibition of uptake provides a novel mechanism for sustaining levels of neurotransmitter in the synapse and thereby increasing synaptic transmission.
Determining the efficacy of GABA transport inhibitors such as nipecotic acid in vivo has been hampered by their poor penetration of the blood-brain barrier, a property attributable to their high degree of hydrophilicity. In an effort to overcome this problem, Ali et al. (1985) examined the effect of adding lipophilic side chains to the nitrogen atom of various GABA transport blockers. Surprisingly, the addition of 4,4-diphenyl-3-butenyl side chains to nipecotic acid and guvacine (SK&F 89976-A and SK&F 100330-A, respectively) resulted in a 20-fold increase in potency when tested in brain synaptosomes. Since the original report, a number of other groups have synthesized similar derivatives such as CI-966 (Taylor et al., 1990) and Tiagabine (Nielsen et al., 1991). Importantly, these compounds all display anticonvulsive activity in laboratory animals (Yunger et al., 1984; Swinyard et al., 1991; Nielsen et al., 1991; Taylor et al., 1990; Suzdak et al., 1992).
Determining the site of action of the lipophilic GABA transport inhibitors is essential to understanding their mechanism of action, but is complicated by the heterogeneity of GABA transport. Early studies with cell culture systems suggested the existence of distinct neuronal and glial GABA transport systems (reviewed in Krogsgaard-Larsen et al., 1987). More recently, molecular cloning has identified four distinct GABA transporters termed GAT-1 (Guastella et al., 1990), GAT-2 and GAT-3 (disclosed herein), and BGT-1, the latter transporting both GABA and the osmolyte betaine (Yamauchi et al., 1992). A clone identical to GAT-3 was described by Clark et al. (1992) and termed GAT-B, and clones for all four GABA transporters have been identified in mice, though a different terminology was employed (Liu et al., 1993).
The specificities of known anticonvulsive agents were determined in order to examine the potency of four lipophilic transport inhibitors at each of the four cloned GABA transporters. We found that they all show a high degree of selectivity for GAT-1, indicating that their anticonvulsive activity is mediated via inhibition of GABA transport by this site.
In contrast to GAT-1, the relationship of GAT-2, GAT-3, and BGT-1 to the pharmacologically characterized neuronal and glial transport systems is not understood. While GAT-2 and GAT-3 display high affinity for &bgr;-alanine and low affinity for ACHC, suggesting a similarity to the glial transporter, their overall pharmacological profile indicates that they are distinct from this site. Additionally, the observation that GAT-3/GAT-B is located in neurons (Clark et al., 1992) indicates that neuronal transport is not limited to GAT-1, and that &bgr;-alanine sensitivity is not unique to glial GABA transport.
An additional level of complexity results from the heterogeneity of astrocytes. Two types of astrocytes, termed Type 1 and Type 2, have been described in cell cultures (for reviews see Raff, 1989; Miller et al., 1989). Interestingly, it has been suggested that Type 2 astrocytes and their precursor, termed O-2A, display a “neuronal-like” GABA transport pharmacology (Levi et al., 1983; Reynolds and Herschkowitz, 1984; Johnstone et al., 1986), suggesting that GAT-1 may not be restricted to neurons. A failure to recognize and/or distinguish these cell types in many older studies complicates their interpretation.
Taurine (2-aminoethanesulfonic acid) is a sulfur containing amino acid present in high concentrations in mammalian brain as well as various non-neural tissues. Many functions have been ascribed to taurine in both the nervous system and peripheral tissues. The best understood (and phylogenetically oldest) function of taurine is as an osmoregulator (36,107). Osmoregulation is essential to normal brain function and may also play a critical role in various pathophysiological states such as epilepsy, migraine, and ischemia. The primary mechanism by which neurons and glial cells regulate osmolarity is via the selective accumulation and release of taurine. Taurine influx is mediated via specific, high-affinity transporters which may contribute to efflux as well. Since taurine is slowly degraded, transport is an important means of regulating extracellular taurine levels.
Taurine is structurally related to the inhibitory amino acid &ggr;-aminobutyric acid (GABA) and exerts inhibitory effects on the brain, suggesting a role as a neurotransmitter or neuromodulator. Taurine can be released from both neurons and glial cells by receptor-mediated mechanisms as well as in response to cell volume changes (94). Its effects in the CNS may be mediated by GABA
A
and GABA
B
receptors (45,80) and by specific taurine receptors (109). Additionally, taurine can also regulate calcium homeostasis in excitable tissues such as the brain and heart (36,55), via an intracellular site of action. Together, the inhibitory and osmoregulatory pro
Borden Laurence A.
Hartig Paul R.
Smith Kelli E.
Weinshank Richard L.
Cooper & Dunham LLP
Synaptic Pharmaceutical Corporation
Ulm John
White John P.
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