WO1999064861A2 - Method of identifying antidepressant compounds - Google Patents

Abstract

There is disclosed a method of identifying compounds having antidepressant activity which method comprises: a) contacting a cell expressing an alpha-1b-adrenergic receptor and transport-P protein with said compound to be tested; and b) prior to or after step a) contacting said compound with a cell expressing an alpha-1b-adrenergic receptor but not transport-P protein; and c) selecting a compound which preferentially binds transport P. Compounds identified using the method of the invention are also provided in addition to pharmaceutical compositions containing them and their use in treating depression.

Description

METHOD OP IDENTIFYING ANTIDEPRESSANT COMPOUNDS

The present invention is concerned with a method of identifying compounds having antidepressant activity, any compounds identified and their inclusion in pharmaceutical compositions which may advantageously be useful in treating clinical depression. Also provided by the present invention is a nucleic acid sequence encoding a peptidergic neurone receptor and its corresponding amino acid sequence.

Endogenous depression is a mental illness of unknown aetiology, causing a disorder of mood or affect. It is distinguishable from exogenous or reactive depression in which sadness is precipitated by some identifiable external event, such as a loss or bereavement . Endogenous depression is a common disease with significant morbidity and mortality.

The aetiology of endogenous depression is unknown. Genetic factors are probably more important in the aetiology of bipolar disorder than in depression alone, as patients with bipolar disorder have a far greater frequency of positive family history than patients with depression alone. In families afflicted with bipolar disease, the risk of depressive illness is 100% in monozygotic twins and approximately 30% in first degree relatives of index patients. However, no genetic mutations have been linked with certainty to the disease phenotype . Depression is a feature of some endocrine diseases including Cushing ' s syndrome, hypothyroidism and hypercalcaemia. Depression may also be an adverse effect of some medications such as reserpine, clonidine, progestogens and glucocorticoids . However, in the vast majority of depressed patients, the aetiology of the disease is unknown. The hypothesis which has attracted the greatest attention is the biogenic amine hypothesis of affective disorders. According to this hypothesis, depression is due to deficiency of the action of the neurotransmitter amines serotonin or noradrenaline at post-synaptic receptors in the brain (Schildkraut , 1965;).

Presynaptic nerve terminals possess transporters for reuptake of amine and amino acid neurotransmitters. These carrier molecules are located in the plasma membrane and serve to recapture released neurotransmitters from the extracellular synaptic space into the cytoplasm. Other transporters, located in membranes of neurosecretory vesicles, then transport the recaptured neurotransmitters from the cytoplasm for storage in the vesicles (Iversen, 1967;; Lester et al . , 1994). Neurotransmitters may also be accumulated by transporters into some non-neuronal cells, including myocytes, glia and endothelial cells (Iversen, 1965;). Tricyclic compounds and their newer derivatives are the main pharmacological treatment for depressive disease. They are used in the treatment of both unipolar and bipolar disease; in the latter, they may be combined with lithium, in order to prevent tricyclic-induced exacerbation of mania (Baldessarini, 1996) . These compounds are also used in long term prophylaxis, to prevent recurrence of depressive episodes (Montgomery, 1994) . The mechanism of the therapeutic effect of these compounds is unknown, but they are believed to act by inhibiting the presynaptic re-uptake of neurotransmitter amines in the brain. According to the pre-synaptic re-uptake hypothesis, antidepressants exert their therapeutic effect by blocking the pre-synaptic plasma membrane transporters for noradrenaline and serotonin, resulting in an increase in synaptic concentrations of these neurotransmitter amines and restoration of their excitatory post-synaptic action ( Schildkraut, 1965;). However, there are objections to this hypothesis:

1. Inhibition of pre-synaptic re-uptake is immediate but the therapeutic antidepressant effect is delayed for 2-6 weeks (Oswald et al , 1972) . This applies both to the tricyclic antidepressants and to the newer serotonin-selective re-uptake inhibitors (SSRIs; Leonard, 1993) . These findings indicate that inhibition of pre-synaptic amine re-uptake is insufficient for appearance of the therapeutic effect, and that other factors which have a slower time course may be involved. The delay is a problem as these patients become non-compliant and are prone to suicide risk.

2. There is no correlation between potency at inhibition of pre-synaptic re-uptake and clinical therapeutic potency (Morris & Beck, 1974; Leonard, 1993) . Some antidepressants are highly potent in inhibiting the re-uptake of noradrenaline or serotonin whereas others are weak and non-selective; however, the clinical potency of all the antidepressants is very similar and they are all used in similar clinical doses, usually ranging from 20 to 200 mg/day (Leonard, 1993; Baldessarini, 1996;). This applies both to the tricyclic compounds and to the SSRIs.

3. Some compounds which inhibit pre-synaptic re-uptake have no antidepressant activity. Such compounds include cocaine and amphetamines (Hare et al, 1962; Overall et al , 1962; Post et al, 1974) . These compounds exert acute behavioural stimulant effects in normal subjects and in depressed patients but they are not useful in the treatment of depression (Hare et al , 1962; Overall et al , 1962; Post et al , 1974) . Cocaine inhibits the human pre-synaptic plasma membrane transporters for both serotonin and noradrenaline whereas amphetamines are more potent in inhibiting uptake of noradrenaline (Giros et al , 1994; Barker et al, 1994). Some SSRIs have no antidepressant activity . These observations indicate that inhibition of pre- synaptic re-uptake is insufficient for appearance of the antidepressant effect. 4. "Atypical antidepressants": bupropion, trazodone, nefazodone, trimipramine and mianserin are clinically effective antidepressants which have weak effects on the pre-synaptic re-uptake of amines, in comparison to the tricyclics and SSRIs. Trimipramine and mianserin are structurally related to the tricyclics whereas the others are a heterogeneous group. Trazodone and nefazodone are of particular interest : these two compounds are much more potent at antagonising post- synaptic adrenergic and serotonergic receptors than at inhibiting the pre-synaptic re-uptake of noradrenaline and serotonin; their overall effect is therefore to block noradrenergic and serotonergic neurotransmission. Mianserin was thought to act as an antagonist of pre-synaptic alpha-2 adrenergic receptors which inhibit noradrenaline release; however, this compound is more potent as an antagonist of post-synaptic alpha-1 adrenergic receptors, so its overall effect is therefore to block noradrenergic neurotransmission. The mechanism of the therapeutic action of the atypical antidepressants is unknown

(Baldessarini, 1996). Thus, inhibition of pre-synaptic re-uptake is not a pre-requisite for the antidepressant effect of such compounds. In addition to the amine uptake processes in presynaptic nerve terminals and in non-neuronal cells, recent evidence has suggested the existence of a novel uptake process for amines in postsynaptic neurones. Primary hypothalamic cell cultures and a cell line of hypothalamic neurones were found to accumulate amines by a desipramine-blockable process (Al-Damluji & Krsmanovic, 1992; Al-Damluji et al . , 1993). These cells accumulate amines which are present in nanomolar concentrations in the extracellular space by an energy-dependent process that is linked to a vacuolar- type ATPase (V-ATPase) and requires an electrochemical gradient of protons for its source of energy (Al- Damluji & Kopin, 1996) . Uptake is due to a carrier which is activated by increasing concentrations of prazosin, resulting in a paradoxical increase in the apparent binding of [³H] -prazosin. While it resembles the presynaptic plasma membrane transporters in that it is blocked by desipramine, it differs from these transporters by its independence of sodium and reliance on protons for a source of energy. Uptake of prazosin differs from the vesicular transporters by its insensitivity to reserpine and blockade by antidepressants (Al-Damluji & Kopin, 1996). Thus, this carrier is distinguishable from other neuronal transporters both by its anatomical location and by its functional properties. Uptake of prazosin is insensitive, to steroid hormones and is not absolutely dependent on sodium ions (Al-Damluji & Kopin, 1996), which distinguishes it from non-neuronal uptake process, such as uptake₂ in myocytes and the uptake process in pulmonary endothelial cells (Salt, 1972;) . The postsynaptic uptake process is a site of action of drugs which act on c -adrenoceptors (Al- Damluji et al . , 1993) . The present inventor has suggested that the physiological significance of postsynaptic uptake may be to remove neurotransmitter from the vicinity of postsynaptic receptors, thus preventing desensitization of the receptors and maintaining the responsiveness of postsynaptic neurones to repeated bursts of neurotransmitter released from presynaptic nerve terminals. In contrast, the presynaptic transporters are presumably less effective in removing neurotransmitter from postsynaptic receptors, as they would rely on diffusion of neurotransmitter back across the synapse, against its concentration gradient (Al-Damluji et al . , 1993) . Clearly, a concentration gradient must exist across the synapse to enable forward diffusion of the transmitter.

Hypothalamic peptidergic neurons are innervated by noradrenergic nerve terminals and noradrenaline plays an important role in regulating the physiological functions of these cells (for review, see Al-Damluji, 1993) .

Peptidergic neurones have previously been identified as possessing alpha-1 adrenergic receptors and an unusual uptake process which has been called transport-P. As the concentration of unlabelled alpha-1-adrenergic receptor prazosin is increased, t³H] prazosin is displaced from the alpha-1 adrenergic receptors. Uptake of prazosin is evident at nanomolar concentrations, but uptake becomes activated by increasing concentrations of prazosin, resulting in the paradoxical increase in accumulation of [³H]prazosin (Al-Damluji & Krsmanovic, 1992). This model is supported by evidence which showed that in the presence of desipramine, [³H] prazosin was displaced by unlabelled prazosin (Al-Damluji et al, 1993) . The affinity of prazosin for these binding sites was similar to its affinity for alpha-1 adrenergic receptors expressed in cultured cells which have been transfected with alpha-1 adrenergic receptor cDNA (Al-Damluji & Kopin, 1996a) .

Tricyclic antidepressants were previously shown to be active at transport-P (Al-Damluji & Kopin, 1996b) . However, tricyclic antidepressants have many pharmacological actions, inhibition of noradrenaline re-uptake, inhibition of dopamine re-uptake, inhibition of serotonin re-uptake, histamine release, histamine H-l receptor blockade, histamine H-2 receptor blockade, muscarinic receptor blockade, nicotinic receptor blockade, alpha-1 adrenergic receptor blockade, alpha-2 adrenergic receptor blockade, dopaminergic D-l receptor blockade, and dopaminergic D-2 receptor blockade and which actions are not responsible for their therapeutic effects, the action of these compounds on transport-P was thought to be yet another pharmacological effect.

The present inventor has now surprisingly found that antidepressants such as tricyclic antidepressants and related compounds exert their therapeutic effect by virtue of their action on transport-P on postsynaptic neurones. These compounds are internalised by transport-P in post-synaptic neurones, where they accumulate in acidified intracellular vesicles. The normal function of these acidified vesicles is to degrade internalised post-synaptic receptors. Because of their basic amine groups, the antidepressants tend to neutralise the acidity of the vesicles. The rise of vesicular pH slows the rate of degradation of post-synaptic receptors. The increase in availability of post-synaptic receptors makes postsynaptic neurones more responsive to the excitatory effects of the neurotransmitter amines and improves the clinical features of endogenous depression. Thus, inhibition of pre-synaptic amine re-uptake is not the primary site of action antidepressants as was previously hypothesised; but in post-synaptic neurones. This post-synaptic effect results in an increase in the density of post-synaptic receptors, leading to increased responsiveness of post-synaptic neurones. Although inhibition of pre-synaptic re- uptake increases the synaptic concentration of the neurotransmitter amines, the post-synaptic neurones would not be more responsive; in fact, increasing the synaptic concentrations of amines may result in desensitisation and down-regulation of post-synaptic receptors. In contrast, compounds which are internalised by transport-P can be expected to reduce the rate of receptor down-regulation, which would increase the sensitivity of post-synaptic neurones to a given concentration of neurotransmitter. This mode of action which has never previously been provided thus opens the possibility for identification of novel antidepressant compounds which exert their biochemical effects by acting on transport-P. Compounds which are selective in activating transport-P are likely to have therapeutic utility. They can be administered in smaller doses than currently used antidepressants, resulting in a lower incidence of peripheral unwanted effects such as cardiotoxicity. Such compounds should be accumulated in large amounts in post-synaptic neurones by virtue of their activation of transport-P.

Therefore, according to a first aspect of the present invention there is provided a method of identifying compounds having antidepressant activity which method comprises (a) contacting a cell expressing an alpha-lb-adrenergic receptor and transport-P protein with said compound to be tested; and (b) prior to or after step a) contacting said compound with a cell expressing an alpha-ib-adrenergic receptor but not transport-P protein; and (c) selecting a compound which preferentially binds transport P. Thus, advantageously, it is possible to identify compounds which preferentially bind transport-P and which compounds exert an antidepressant activity.

Also encompassed by the present invention are compounds identifiable by the method according to the invention, which compounds may advantageously be used as a medicament, or in the preparation of a medicament for treating depression. Such compounds may advantageously be included in a. pharmaceutical composition, together with a pharmaceutically acceptable carrier, diluent or excipient therefor. As mentioned above, the post-synaptic peptidergic neurones of the hypothalamus possess an unusual uptake process, referred to as transport-P. This uptake became evident while hypothalamic neurones were being examined for the presence of _x adrenoceptors . The x_λ- adrenoceptor ligand [³H] -prazosin bound to the cells and was displaced by unlabelled prazosin in concentrations up to 10^"7 M. However, at concentrations of unlabelled prazosin greater than 10^" M, there was a paradoxical increase in the accumulation of [³H] -prazosin which could be abolished by desipramine; in the presence of desipramine, only displacement of [³H] -prazosin by unlabelled prazosin was seen. These findings were interpreted as indicating the presence in peptidergic neurones of o^- adrenoceptors and an unusual uptake process. As the concentration of unlabelled prazosin is increased, [³H] -prazosin is displaced from the receptors the present inventors subsequently cloned alpha-1- adrenoceptor cDNA from these neurones, providing further evidence for this part of the hypothesis (White & Al-Damluji, 1997) . The uptake process is evident at nanomolar concentrations of prazosin, but when the concentration of unlabelled prazosin is increased, there is a further activation of the uptake process, manifested by the paradoxical increase in accumulation of the radioligand (Al-Damluji & Krsmanovic, 1992; Al-Damluji et al . , 1993). The paradoxical increase requires the electrochemical gradient of protons and is linked to V-ATPase (Al- Damluji & Kopin, 1996a) .

A further assay which may advantageously be used to identify compounds having antidepressant activity may be based upon the apparent paradox which is observed when amines which bind to the alpha-lb- adrenergic receptor are contacted with a cell expressing both the adrenergic receptor and transport- P and subsequently with increasing concentrations of the compound to be tested for antidepressant activity. Therefore, according to a further aspect of the invention there is provided a method of identifying compounds having antidepressant activity which method comprises (a) contacting a cell expressing an alpha-lb- adrenergic receptor and transport-P with a labelled ligand of said receptor; and (b) subsequently contacting said cell with said compound to be tested at progressively increasing concentrations; and (c) monitoring for increased binding of said ligand at increasing concentrations of said compound to be tested and selecting a compound which increases binding of said ligand upon addition of increasing concentrations of said compound to be tested.

Increased accumulation or binding of the labelled amine in the presence of increasing concentrations of the compounds to be tested and which compounds displace the labelled amine from the receptor, provides evidence that the compound reacts with the alpha- lb-adrenergic receptor and may also be a ligand for transport-P.

Preferably, the labelled amine comprises [³H] - prazosin, an amine which is known to bind the receptor and to be taken up into the cell- by virtue of transport-P. Preferably, the cell comprises a peptidergic neuronal cell which is preferably a GnRH cell . Preferably, the method according to the invention further comprises the step of contacting the selected compound from step c with a cell expressing said alpha-lb-adrenergic receptor and transport P and monitoring the level of accumulation of said compound in said cell as compared with the concentration of the applied compound and selecting a compound which activates uptake by transport P.

Preferred compounds having an antidepressant activity are those which are internalised in the post- synaptic neurone and which are maintained for a longer period within intracellular vesicles in the peptidergic neuron. Compounds which are released slowly are likely to have therapeutic utility; they can be administered in smaller doses thus reducing the risks of their unwanted effects. The therapeutic effect of such compounds is likely to be exerted for a longer period of time as they are retained in postsynaptic neurones for longer periods following each dose. Therefore, preferably the method according to the invention further comprises the step of contacting said selected compound with a cell expressing alpha-lb adrenergic receptors and transport P and monitoring the rate of release of said compound from intracellular vesicles in said cell and selecting compounds having a relatively longer retention time in said vesicles.

The antidepressant compounds are therefore internalised by Transport-P in post-synaptic neurones, where they accumulate in acidified intracellular vesicles. The normal function of these acidified vesicles is to degrade internalised post-synaptic receptors. Because of their basic amine groups, the antidepressants tend to neutralise the acidity of the vesicles. The rise of vesicular pH slows the rate of degradation of post-synaptic receptors. The increase in availability of post-synaptic receptors makes postsynaptic neurones more responsive to the excitatory effects of the neurotransmitter amines and improves the clinical features of endogenous depression. Neurotransmitters down-regulate their postsynaptic receptors, so the density of post-synaptic receptors should be considered in relation to the concentration of the neurotransmitter in the extracellular synaptic space. Thus, chronic administration of antidepressants which inhibit the pre-synaptic re-uptake of noradrenaline causes a consistent reduction in the density of post-synaptic beta adrenergic receptors (Banerjee et al, 1977) . However, the density of post-synaptic alpha-1 adrenergic receptors is either increased or unaffected by chronic administration of antidepressants (Stockmeier et al , 1987;) . Unchanged density of postsynaptic alpha-1 adrenergic receptors despite the increase in neurotransmitter concentration can be regarded as a relative increase in the availability of post-synaptic receptors. This increase in relative availability of alpha-1 adrenergic receptors may be due to the fact that they are colocalised with Transport-P in post-synaptic neurones (White & Al- Damluji, 1997). Antidepressants also progressively increase the responsiveness of post-synaptic serotonergic receptors by an unknown mechanism (reviewed by Blier & DeMontigny, 1994) . The interaction of Transport-P with the serotonergic system has not been investigated, but it is possible that Transport-P may modulate post-synaptic serotonergic receptors in a manner which is analogous to its proposed effect on alpha-1 adrenergic receptors.

Tricyclic antidepressants and their derivatives are clearly effective in increasing extracellular concentrations of the neurotransmitter amines by inhibition of pre-synaptic re-uptake, and this increase in the availability of neurotransmitters is likely to contribute to the activation of postsynaptic neurones. However, in view of the objections described above, this inhibition of pre-synaptic re-uptake is unlikely to be the primary mechanism of the therapeutic action of these compounds. In any case, if the aim of therapy is to increase the activation of post-synaptic neurones, then inhibition of presynaptic re-uptake is not the best therapeutic strategy. This is because increasing the extracellular concentration of neurotransmitters is followed by receptor down-regulation and reduced responsiveness of post-synaptic neurones. Thus, chronic administration of antidepressants reduces the sensitivity of postsynaptic neurones to the stimulant effect of noradrenaline on the production of c-AMP, due to down- regulation of post-synaptic beta adrenergic receptors (Banerjee et al, 1977) . In contrast, compounds which are internalised by Transport-P can be expected to increase the sensitivity of post-synaptic neurones to a given concentration of neurotransmitter, due to reduced degradation of post-synaptic receptors. A mechanism which acts via Transport-P can be expected to have a synergistic effect with inhibition of presynaptic re-uptake, resulting in more sustained activation of post-synaptic neurones.

A further problem associated with compounds which inhibit pre-synaptic re-uptake is that the increase in synaptic neurotransmitter concentrations activates pre-synaptic autoreceptors which inhibit the discharge rate of pre-synaptic neurones, resulting in reduced synthesis and release of neurotransmitter (Langer, 1977; Charney et al , 1981; Langer & Lehmann, 1988; Blier & DeMontigny, 1994; Romero et al , 1996) . During chronic treatment with antidepressants, these inhibitory pre-synaptic autoreceptors gradually become desensitised, resulting in a partial recovery in the rate of synthesis and release of neurotransmitter (Crews & Smith, 1978; Svensson & Usdin, 1978; Spyraki & Fibiger, 1980; McMillen et al , 1980; Smith et al , 1981; Kreiss & Lucki, 1995). Nevertheless, these inhibitory autoreceptor effects are clearly undesirable, as they diminish the activation of postsynaptic neurones. In contrast, compounds which act selectively on Transport-P can be expected to have no inhibitory effect on the release of neurotransmitters, and this should result in more effective activation of post-synaptic receptors.

According to a further aspect of the present invention there is provided an alpha- lb adrenergic receptor from GnRH neurones or a functional equivalent, derivative or bioprecursor of said receptor having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1. Further provided by the invention is an alpha- lb-adrenergic receptor or a functional equivalent, derivative or bioprecursor therefor having an amino acid sequence illustrated in figure 2 or an amino acid sequence which differs from said amino acid sequence in one or more conservative amino acid changes. The nucleic acid molecule encoding said alpha-lb- adrenergic receptor according to the invention is preferably a cDNA molecule having the sequence identified in figure 1. Also provided by the invention is an antisense molecule capable of hybridising to the cDNA sequence according to the invention under high stringency conditions.

Stringency of hybridisation as used herein refers to conditions under which polynucleic acids are stable. The stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. Tm can be approximated by the formula :

81 . 5 °C- 16 . 6 ( logl O [Na⁺] +0 . 41 ( %G&C) - 600 /1

wherein 1 is the length of the hybrids in nucleotides. Tm decreases approximately by 1-1.5°C with every 1% decrease in sequence homology.

Nucleic acid molecules or polynucleotides of theinvention capable of selectively hybridising to the cDNA according to the invention will generally be at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the cDNA according to the invention. It is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not effect the polypeptide sequence encoded to reflect the codon usage in any particular host organism in which the polypeptides of the invention are to be expressed. The cDNA may advantageously be included in an expression vector for subsequent transformation or transfection into a host cell or the like. Preferably, the vector includes an alpha-lb-adrenergic receptor promoter, and even more preferably said vector further comprises a reporter molecule, which may be a fluorophore, or the like. The host cell transfected with the vector according to the invention also forms part of the present invention, and which host cell is preferably a COS-7 cell.

Advantageously, the nucleic acid molecule according to the invention may be used to express the receptor according to the invention, in a host cell or the like using an appropriate expression vector.

An expression vector according to the invention includes vectors capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments.

Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art.

Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that upon introduction into an appropriate host cell results in expression of the DNA or RNA fragments. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

The antisense molecule capable of hybridising to the nucleic acid according to the invention may be used as a probe or as a medicament or in a pharmaceutical composition. Nucleic acid molecules according to the invention may be inserted into the vectors described in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense nucleic acids may be produced by synthetic means.

A further aspect of the invention comprises the host cell transformed, ^• transfected or infected with the expression vector according to the invention, which cell preferably comprises a eukaryotic cell and more preferably a mammalian cell.

Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al (1989) Molecular Cloning, A Laboratory manual, Cold Spring Harbour Laboratory Press .

A further aspect of the present invention comprises a nucleic acid molecule having at least 15 nucleotides of the nucleic acid molecule according to the invention and preferably from 15 to 50 nucleotides .

These sequences may, advantageously be used as probes or primers to initiate replication or the like. Such nucleic acid molecules may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or devices or the like for detecting for the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with a sample under hybridising conditions and detecting for the presence of any duplex formation between the probe and any nucleic acid in the sample.

According to the present invention these probes may be anchored to a solid support. Preferably, they are present on an array so that multiple probes can simultaneously hybridize to a single biological sample . The probes can be spotted onto the array or synthesised in si tu on the array. (See Lockhart et al . , Nature Biotechnology, vol. 14, December 1996 "Expression monitoring by hybridisation into high density oligonucleotide arrays" . A single array can contain more than 100, 500 or even 1,000 different probes in discrete locations.

Nucleic acid molecules according to the invention may also be produced using such recombinant or synthetic means, such as, for example, using PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA. Generally, such techniques as defined herein are well known in the art, such as described in Sambrook et al (Molecular Cloning: a Laboratory Manual, 1989). The present invention also provides for a transgenic cell, tissue or organism comprising a transgene capable of expressing a receptor according to the invention.

The cells expressing said receptor are particularly advantageous when used in an assay or kit for identifying compounds having antidepressant activity according to the invention.

The term "transgene capable of expressing a receptor" according to the invention as used herein, means a suitable nucleic acid sequence which leads to the expression of the receptor. The transgene may include, for example, genomic nucleic acid or synthetic nucleic acid, including cDNA. The term "transgenic organism, tissue or cell, as used herein means any suitable organism, and/or part of an organism, tissue or cell, that contains exogenous nucleic acid either stably integrated in the genome or in an extrachromosomal state .

Preferably, the transgenic cell comprises a COS-7 cell. Preferably, the transgene comprises the nucleic acid or cDNA sequence encoding said receptor according to the invention.

In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including in particular, substitutions in bases which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term "nucleic acid molecule" also includes the complementary sequence to any single stranded sequence given regarding base variations.

Proteins or polypeptides according to the invention further include variants of such sequences, including naturally occurring allelic variants which are substantially homologous to said proteins or polypeptides. In this context, substantial homology is regarded as a sequence which has at least 70%, and preferably 80%, 90% or 95% amino acid homology with the proteins or polypeptides encoded by the nucleic acid molecules according to the invention. Antibodies to the receptor according to the invention may be produced according to known techniques as would be known to those skilled in the art and these also form part of the present invention. For example, polyclonal antibodies may be prepared by inoculating a host animal, such as rat or the like, with said receptor or an epitope thereof, preferably haptenised to another polypeptide for use as an immunogen, and recovering immune serum. Monoclonal antibodies may be prepared according to the method of Kohler R. and Milstein C, (1975) Nature 256, pp495 to 497. For the purposes of the present invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab')₂ fragments as well as single chain antibodies.

According to a further aspect of the invention, it may be possible to identify proteins, such as transport-P, which interact with said receptor, using techniques known to those skilled in the art, such as the yeast-two hybrid vector system first proposed by Chien et al . , (1991) Proc. Nat. Acad. Sci. USA 88, 9578-9582.

Transport-P is a site of action of psychoactive compounds such as cocaine, amphetamines and ephedrine .

Compounds or molecules which block transport-P such as an anti-transport-P antibody or the like may act as antidotes to the action of these drugs of abuse. The present invention may be more clearly understood from the following examples, which are purely exemplary, with reference to the accompanying drawings wherein

Figure 1: is a cDNA sequence of the alpha-lb- adrenergic receptor in accordance with the invention;

Figure 2 : is an illustration of the amino acid sequence of the receptor of figure 1 from mouse GnRH cells, compared to hamster smooth P muscle and rate whole brain,•

Figure 3 : is a graphic representation of the effects of fluoxetine and fluroxamine on the uptake of prazosin in GT1-1 GnRH cells,^•

Figure 4 : is a graphic representation of uptake of prazosin in GT1-1 GnRH cells.

Figure 5: is a graphic representation of uptake of noradrenaline in SK-N-SH cells;

Figure 6: is a graphic representation of the effect of temperature on prazosin release from GT1-1 GnRH cells;

Figure 7: is an illustration of aniline derivatives and their potency at Transport-P;

Figure 8: is a graphic representation of the results obtained in an assay to determine the binding effect of compounds using cells transfected with the alpha- lb sequence according to Figure 1. Figures 9 to 14 : illustrate the effects of various substitutions of phenylethylamine derivatives at inhibition of uptake of prazosin 10^"6 in immortalised peptidergic neurones.

Identification of Compounds Which Are Internalized By Virtue of Interaction With Transport-P

MATERIALS:

Fetal rat hypothalamic cells in primary culture or GT1-1 GnRH cells [7-methoxy- ³H]prazosin (eg., Amersham TRK.843; specific activity 73 Ci/mmol; store at -20 °c) were used. Unlabelled prazosin. HCl (Sigma P- 7791) FW 420 Compound under study (eg., fluoxetine hydrochloride) FW 345.79, Uptake buffer: DMEM (Sigma) with 25 M HEPES (5.6 g/1) , pH '7.4 Before use, add sodium ascorbate 10 mg/100 ml (0.5 mM) L-Ascorbic acid sodium salt (Sigma A-4034) FW 198.1 Solubilisation solution:

0.1 % sodium dodecyl sulphate (Sigma) 0.1 M sodium hydroxide (4 g/1) Cocktail T scintillation liquid (BDH 14509 6B)Bicinchoninic Acid Protein Assay, Reagent kit (Pierce Y 23225X) .

PROCEDURE :

1. Fetal rat hypothalamic cells or GT1-1 GnRH cells where dispersed in culture medium at a density of 10^"s cells/ml and incubated in 12 -well plates (2 ml/well; 2X10^"6 cells/well) . The culture media was changed at 48 -hour intervals and studies were carried out four days after dispersion.

2. Ascorbate 10 mg/100 ml uptake buffer added. 3. [³H] prazosin 0.7 μl to 40 ml buffer (approximately 0 . 2 nM) added .

4. Unlabelled prazosin 10^"3 M (4.2 mg in 10 ml water) prepared.

5. A dilution of 1:1000 to the 40 ml of [³H] prazosin = 10^"6 M unlabelled prazosin was prepared.

6. Fluoxetine 10^"1 M in dimethyl sulphoxide (DMSO) was prepared.

7. Diluted 10^"1 M 1:1000 to 6 ml

[³H] prazosin/unlabelled prazosin = 10^{" 4} M fluoxetine 8. 34 ul DMSO was added to the remaining 34 ml

[³H] prazosin/unlabelled prazosin = 1:1000 DMSO

9. A dilution of 1.5 ml fluoxetine 10^"4 M + 3 ml

[³H] prazosin/unlabelled prazosin = 3.33 x 10^"5 M fluoxetine was prepared. 10. A serial dilution of 1:10 to 4 ml

[³H] prazosin/unlabelled prazosin was prepared to obtain

Fluoxetine 10^"4 M

3.33X10^"5 M

10^"5 M

3.33X10^"6 M

10^"6 M 3.33X10^"7 M

10^"7 M

11. The cells carried out were washed twice with uptake buffer at room temp., 1 ml each wash. 12. 1 ml of [³H] prazosin/unlabelled prazosin or fluoxetine dilutions in [³H] praZosin/unlabelled prazosin were added.

13. The cells were incubated at 37°C for 60 minutes.

14. The culture plates were placed on ice. 15. The buffer was removed after 30 seconds and washed twice with ice cold buffer, 1 ml each wash.

16. 1 ml of warm solubilisation solution was added and left for 15 minutes.

17. The extract was removed to scintillation vials. 18. The above step was repeated.

19. Vials were vortexed thoroughly to shear the viscous cell extract.

20. 50 μl aliquots were removed into 12X75 mm tubes for protein assay. 21. Prepare total counts', 1 ml, in triplicate, were provided.

22. Add 10 ml Cocktail-T, vortex and count in a scintillation counter.

ANALYSIS OF DATA:

Concentration-response curves are constructed as demonstrated in Figure 3. IC₅₀ values can be used to examine the effects of various modifications of the chemical structures on the relative potency of the compounds .

Identification of Compounds Interacting with Transport-P

Immortalized GT1-1 GnRH neuronal cells were cultured as previously described in detail (Al-Damluji et al . , 1993) . Briefly, the cells were grown in Corning 75 cm² 150 CM² flasks in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) and Ham's F- 12 (ratio 1:1) containing 10% FBS, sodium bicarbonate 3.7gl^"** and gentamicin 100 mgl^"1, in a humidified atmosphere containing 5% C0₂ in air. Culture media were changed at 48 h intervals. When the cells reached confluence, they were dispersed in the presence of trypsin, DNAsel and EDTA and incubated in Costar or Corning 12-well plates (2xl0^"6 cells/well) . Culture media were changed at 48 h intervals and the experiments were carried out four days following culture . Uptake studies were performed on intact cells, as previously described in detail (Al-Damluji et al . , 1993) . Drugs were dissolved in uptake buffer consisting of DMEM with 25 mM HEPES and 0.5X10^"3 M sodium ascorbate, pH7.4. The cells were washed twice with 1 ml of buffer and then incubated for 60 min at 37°C in buffer containing [³H] -prazosin 2xl0^"9 M and unlabelled prazosin 10^"6 M, with or without phenylethylamine derivatives in concentrations of 10^"δ M to 10^"3 M. Accumulation of prazosin and antidepressants reaches equilibrium within 60 min (Al- Damluji δ. Kopin, 1996b) . At the end of the incubation period, the wells were placed on ice and the cells were washed twice with 1 ml volumes of ice-cold buffer. The cells were then solubilized with two ml of a warm solution of 0.1% sodium dodecyl sulphate and

0.1 M NaOH. Fifty microlitre aliquots were removed for protein assay and 10 ml of scintillation liquid was then added to the cell extract, mixed and radioactivity was measured in a scintillation spectrometer with an efficiency of 50%. Protein content was measured by the bicinchoninic acid modification of the biuret reaction (Smith et al . , 1985) using albumin standards and reagents supplied by Pierce (Rockford, Illinois or Chester, Cheshire) . A representative member of each group of compounds was tested for its ability to inhibit the uptake of prazosin competitively. This was done by examining the effects of different concentrations of the phenylethylamine derivative in the presence of different concentrations of unlabelled prazosin, while the concentration of [³H] -prazosin was kept constant at 2x10^{" 9} M.

Efficacy was defined as % inhibition of the uptake of prazosin 10^"6 M when the test compound was used in a maximal concentration (10^"4 M or 10^"3 M) .

Efficacy was expressed as % of the effect of a maximal inhibitory concentration of desipramine (10^"5 M) Typically, desipramine 10^"5 M inhibited the accumulation of prazosin 10^"6 M by 80% (Al-Damluji & Kopin, 1996b) ; the remaining 20% was regarded as nonspecific uptake for the purposes of this study. Half- maximal inhibitory concentrations (IC₅₀ values) were calculated from the concentration-response curves. IC₅₀ values were calculated only for compounds which achieved a maximal inhibitory response, defined as the inhibitory effect of desipramine 10^"5 M. When a compound did not achieve the maximal inhibitory response, IC₅₀ values were not calculated and the data were expressed only as efficacy (% inhibition relative to desipramine 10^"5 M) ; for these compounds, the concentrations from which the efficacy values were derived are indicated in Table 1 or in the relevant figures. Relative potencies of compounds were calculated as follows: relative potency = (IC₅₀ drug A/IC₅₀ drug B) -1.

It is necessary to subtract: ' 1' from the quotient to take account of the fact that equipotent compounds have equal IC₅₀ values. IC₅₀ values were used to examine the effects of structural modifications on the ability of phenylethylamine analogues to inhibit the accumulation of prazosin 10^"6 M in GT1-1 GnRH cells. The mathematical basis for using IC₅₀ values for such purposes has been described by De Lean et al . (1978) . Each experimental point was carried out in triplicate and each experiment was replicated at least once; the minimum number of estimations for each experimental point was therefore six. The data are expressed as the means + s.e.mean. S.e.mean are not shown where they are smaller than the sizes of the symbols.

Immortalized GnRH neuronal cells (GT1-1 cells; Mellon et al . , 1990) were generously provided by Dr R.I. Weiner. Heat-inactivated foetal bovine serum (FBS) was from Life Technologies (Gaithersburg,

Maryland or Paisley, Scotland) . Culture media were from Sigma-Aldrich (St. Louis, Missouri or Poole, Dorset). t³H] -prazosin was from Amersham (TRK.843; specific activity 74-83 Ci mmol^"1) . Unlabelled compounds were from Sigma-Aldrich, Research

Biochemicals International (Natick, Massachusetts or St Albans, Hertfordshire) or Tocris-Cookson (Bristol, Avon) .

Identification of compounds Which Stimulate Uptake by Transport-P

MATERIALS :

Fetal rat hypothalamic cells in primary culture or GT1-1 GnRH cells [7-methoxy- ³H] Prazosin (eg.,

Amersham TRK.843; specific activity 73 Ci/mmol; store at -20OC)

Compound under study eg., unlabelled prazosin. HCl

(Sigma P-7791) FW 420 Uptake buffer: DMEM (Sigma) with 25 mM HEPES (5.6g/l), pH 7.4. Before use, add sodium ascorbate 10 mg/100 ml (0.5 mM) L-Ascorbic acid sodium salt (Sigma A-4034) FW

198.1 Solubilisation solution:

0.1 % sodium dodecyl sulphate (Sigma) 0.1 M sodium hydroxide (4 g/1) Cocktail T scintillation liquid (BDH 14509 6B) Bicinchoninic Acid Protein Assay Reagent kit (Pierce 23225X) .

PROCEDURE :

1. Disperse fetal rat hypothalamic cells or GTl-1 GnRH cells in culture medium at a density of 10^"6 cells/ml. Incubate in 12 -well plates (2 ml/well; 2X10^"6 cells/well) . Change the culture media at 48 -hour intervals. Studies should be carried out four days after dispersion.

2. Add ascorbate 10 mg/100 ml uptake buffer.

3. Add [³H] prazosin 1.0 μl to 60 ml uptake buffer (final approx 0.2 nM) . 4. Prepare unlabelled prazosin 10^"3 M (4.2 mg in 10 mis water) .

5. Dilute 1:1000 to 15 ml [³H] , prazosin = 10^"6 M unlabelled prazosin.

6. Dilute the above 7 ml + 3.5 ml [³H] prazosin = 6.66X10^"7 M unlabelled prazosin.

7. Dilute the above 3.5 ml + 3.5 ml [³H] prazosin = 3.33X10^"7 M unlabelled prazosin.

8. Dilute serially 1:10 to 7 ml [³H] prazosin to obtain:

Unlabel led prazosin

10 ^"6 M

6 . 66X10 ^"7 M

3 . 33X10 ^"7 M

10^"7 M

10^"8 M

10 ^"9 M

9. Place the cells at room temperature or on ice. 10. Wash the cells twice with 1 ml buffer at room temp or on ice .

11. Add 1 ml of [³H] prazosin or unlabelled prazosin dilutions in [³H] prazosin at room temperature or on ice. 12. Incubate at 37°C or on ice for 60 minutes.

13. Place the cells which were at 37°C on ice.

14. Remove the buffer after 30 seconds and wash twice with ice cold buffer, 1 ml each wash.

15. Add 1 ml of warm solubilisation solution and leave for 15 minutes.

16. Remove the extract to scintillation vials.

17. Repeat the above step.

18. Vortex thoroughly to shear the viscous cell extract . 19. Remove 50 μl aliquots into 12X75 mm tubes for protein assay.

20. Prepare 'total counts', 1 ml, in triplicate

21. Add 10 ml Cocktail-T, vortex and count in a scintillation counter.

ANALYSIS OF DATA:

In Figure 4, the lower panel is a log-linear plot which describes the effect of unlabelled prazosin on the accumulation of [³H] prazosin (expressed as dpm) in GnRH neurones. The upper panel is a log-log plot which describes the same data, but the vertical axis represents the total amount of prazosin accumulated (ie, [³H] prazosin and unlabelled prazosin) by correcting for specific activity.

In the lower panel, the paradoxical increase in accumulation of [³H] prazosin becomes evident when the concentration of unlabelled prazosin exceeds 10^"7 M. Cooling the cells abolished the prazosin paradox. Accumulation of prazosin in the cold can be regarded as non-specific uptake which is described by a straight line (upper panel) .

The upper panel demonstrates that accumulation of prazosin in GnRH cells is a non-linear process; at prazosin concentrations greater than 10^"7 M, specific accumulation of prazosin is described by a sigmoidal function. This is a surprising finding; it contrasts with the accumulation of noradrenaline in pre-synaptic noradrenergic neurones (SK-N-SH cells) which were studied in an identical manner (Figure 5) .

The lowest concentration of prazosin which activates transport-P is approximately 3X10^"7 M. Other compounds may be more effective than prazosin in one of two ways: they may have greater affinity for transport-P, ie, they will activate transport-P at lower concentrations than prazosin. Alternatively, such compounds may activate transport-P with the same affinity but with greater efficacy than prazosin; in that case, activation starts at 3X10^"7 M, but the compound then accumulates in greater quantities than prazosin, as its extracellular concentration exceeds 3X10" 7 M. Some compounds may be more effective than prazosin in both affinity and efficacy at transport-P. The following method can be used to analyse these effects :

Relative slope of accumulation = SS6:7 / SNS6:7 where: SS6:7 is the slope of specific accumulation at 10^"7 M to 10^"6 M

SNS6:7 is the slope of non-specific accumulation at 10^"7 M to 10^"6 M

Expression of the data as relative slope has the advantage that the values are independent of the number of cells being studied.

Accumulation index = relative slope X negative log of higher concentration. For example the accumulation index at 10^"6 M (A16) is calculated as follows: A16 = (SS6:7 / SNS6:7) X 6

Interpretation : 1. If the value of A16 is equal to 6:

The relative slope of accumulation = 1. Therefore the uptake process is not saturated by its ligand in this concentration range.

2. If the value of A16 is less than 6:

The relative slope of accumulation is less than 1. Therefore the uptake process is saturated by its ligand in this concentration range.

3. If the value of A16 is greater than 6:

The relative slope of accumulation is greater than 1.

Therefore the uptake process is activated by its ligand in this concentration range.

A compound which has greater affinity than prazosin may have A17 greater than 7, whereas in the case of prazosin, A17 = 7.

A compound which has the same affinity but greater efficacy than prazosin will have A16 greater than prazosin.

The accumulation index can be used to examine the effects of various modifications of the chemical structures on the relative affinity and efficacy of compounds in activating transport-P.

The Rate of Release of Compounds Internalized by Transport-P

MATERIALS :

Fetal rat hypothalamic cells in primary culture or

GTl-1 GnRH cells Radio-labelled compound eg, [³H] Prazosin

Uptake buffer: DMEM (Sigma) with 25 mM HEPES (5.6 g/1) , pH 7.4

Before use, add sodium ascorbate 10 mg/100 ml (0.5 mM)

L-Ascorbic acid sodium salt (Sigma A-4034) FW 198.1 Solubilisation solution:

0.1 % sodium dodecyl sulphate (Sigma) 0.1 M sodium hydroxide (4 g/l)

Cocktail T scintillation liquid (BDH 14509 6B)

Bicinchoninic Acid Protein Assay Reagent kit (Pierce 23225X)

PROCEDURE :

1. Disperse fetal rat hypothalamic cells or GTl-1 GnRH cells in culture medium at a density of 10^"5 cells/ml. Incubate in 12 -well plates (2 ml/well; 2

XlO^"6 cells/well) . Change the culture media at 48 -hour intervals. Studies should be carried out four days after dispersion.

2. Add ascorbate 10 mg/100 ml uptake buffer 3. Add [³H] prazosin 1.0 μl to 60 ml uptake buffer (final approx 0.2 nM) .

4. Prepare unlabelled prazosin 10^"3 M (4.2 mg in 10 mis water) .

5. Dilute 1:1000 to the 60 ml [³H] prazosin = 10^"6 M unlabelled prazosin. 6. Wash the cells twice with 1 ml buffer at room temperature .

7. Add 1 ml of [³H] prazosin/unlabelled prazosin 10^"6 M.

8. Incubate at 37°C for 60 minutes. 9. Place the cells at the desired temperature.

10. Remove the incubation buffer.

11. Wash the cells once with 1 ml buffer at the appropriate temperature .

12. Add 1 ml buffer at the appropriate temperature 13. Incubate at the appropriate temperature for 0, 10, 20 or 30 minutes.

14. Remove the buffer at the indicated times

15. Add 1 ml of warm solubilisation solution and leave for 15 minutes. 16. Remove the extract to scintillation vials.

17. Repeat the above step.

18. Vortex thoroughly to shear the viscous cell extract .

19. Remove 50 μl aliquots into 12X75 mm tubes for protein assay.

20. Prepare 'total counts', 1 ml , in triplicate.

21. Add 10 ml Cocktail-T, vortex and count in a scintillation counter.

ANALYSIS OF DATA:

Figure 6 demonstrates the rate of release of prazosin from GnRH cells, following accumulation by transportP. It is clear that release is temperature-dependent , requiring cellular energy. The rate of release at 37°C can be used to identify compounds which are retained for longer periods of time. Such compounds can be expected to have a longer duration of action following administration of a single dose.

In this example, the compound under study was labelled with radioactivity. The compounds of interest can be labelled with alternative means such as fluorescence.

CLONING OF ALPHA-IB-ADRENERGIC RECEPTOR

Three sub-types of alpha-1 adrenergic receptor cDNA have been cloned. These sub-types share significant sequence homology in the first half of the molecule. Poly (A+) RNA was prepared from GTl-1 cells and a cDNA library, was constructed in the expression plasmid pSVSPORTl Life Technologies, Paisley, Glasgow, UK. Sets of polymerase chain reaction (PCR) primers were designed from the cDNA sequence of the hamster smooth muscle receptor published in Cotecchia, S et al., 1988, Proc. Natl. Acad. Sci. (USA) 85,7159- 7163 which spanned the coding regions of the sequences. Using GTl-1 GnRH cell RNA or the cDNA library as templates, we detected specific PCR products which we cloned into the pCR2.1 (Invitrogen, San Diego, USA) plasmid by the technique of TA cloning. We sequenced the PCR products and found that the nucleotide sequence is 95% identical with the alpha-ib sub-type of adrenergic receptors.

Structural Properties of Compounds Which Interact With Transport-P

Phenylethylamines are the class of compounds which includes the neurotransmitters adrenaline, noradrenaline and dopamine . The alpha-1 adrenergic agonist methoxamine is a phenylethylamine derivative which has prominent effects on hypothalamic neuroendocrine function (for review, see Al-Damluji, 1993) . The structural similarity, between methoxamine and prazosin prompted an examination of the effects of phenylethylamine derivatives. The study examined phenylethylamine analogues for their ability to antagonise competitively the uptake of prazosin 10^"6 M in GnRH neurones. As prazosin and methoxamine are ligands for alpha-1 adrenoceptors, the findings were compared to the structural properties of phenylethylamine derivatives which are active at alpha adrenoceptors and at other amine uptake processes . The following procedure was carried out to establish the structural properties of phenylethylamine derivatives which are substrates for transport-P, as judged by competitive inhibition of the uptake of prazosin 10^"6 M in immortalised hypothalamic peptidergic neurones.

THE AMINE GROUP

Absence of an alkyl amine abolished the ability of phenylethylamines to inhibit the uptake of prazosin 10^"6 M. Thus, phenylethanolamine was fully active in inhibiting the uptake of prazosin (efficacy 99%; IC_S0 54X10^"6 M) whereas its analogue phenylethylalcohol was inactive (efficacy 4%;) . Table :L lists a series of phenylalkyl compounds which lack an alkyl amine, all of which were essentially inactive in inhibiting the accumulation of prazosin 10^"6 M.

Presence of a carboxyl group on the alpha carbon abolished the ability of phenylethylamines to inhibit the uptake of prazosin; phenylethylamine (efficacy 100%; IC₅₀ 16X10^"6 M) and tyraminne (efficacy 100%; IC₅₀ 800X10^"6 M) were fully active whereas their respective carboxylated analogues phenylalanine (efficacy 2%) and tyrosine (efficacy 0%) were inactive (Table 1) . Table 1 lists other carboxylated compounds which were inactive in inhibiting the accumulation of prazosin. Presence of an aminomethyl group slightly reduced potency. Thus, amphetamine (IC₅₀ 6X10^"6 M) was 1.5 fold more potent than methylamphet mine (IC₅₀ 15X10^"6 M) and norephedrine (IC₅₀ 37 XlO^"6 M) was 0.2 fold more potent than ephedrine (IC₅₀ 43X10^"6 M) . However, these secondary amines retained full efficacy in inhibiting the uptake of prazosin, as did tertiary amines (Table 1) . In contrast, quaternary amines and guanidines were inactive. Thus, l-methyl-4-phenyl-l-l, 2 , 3 , 6- tetrahydropyridine (MPTP) , a tertiary amine, was fully active (efficacy 100%; IC₅₀ 16X10^"6 M) ' in contrast to its quaternary amine analogue 1-methyl-4- phenylpyridinium (MPP⁺; efficacy, 14%;) . Similarly, tyramine (efficacy 100%; IC₅₀ 800X10^"6 M) was fully active in contrast to N-guanyl-tyramine (efficacy

18%;). Table 1 lists further quaternary and guanyl- amines which were inactive in inhibiting the uptake of prazosin 10^"6 M.

At physiological pH, the amine exists in a protonated form (Lentzen & Philippu, 1981; Maxwell et al, 1970) and this presumably enables interaction with a negatively charged group in the transport-P site, allowing entry into the cells. An amino-methyl group causes steric hindrance which may reduce potency. It is possible that the permanent positive charge in quaternary and guanyl amines may prohibit interaction with a strongly hydrophobic residue in the transport-P site .

Amino-methyl groups also reduced the affinity of phenylethylamines for the pre-synaptic plasma membrane dopamine and noradrenaline uptake sites but they enhanced affinity for noradrenaline uptake2 and for alpha-1 and alpha-2 adrenoceptors (Burgen & Iversen, 1965; Grohman & Trendelenburg, 1984; Horn, 1973; Nichols & Ruffolo, 1991; Ruffolo et al , 1988) . Amino methyl groups had no effect on affinity for the vesicular uptake process in rat brain or in bovine adrenal medulla (Peter et al, 1994; Slotkin & Kirshner, 1971; Slotkin et al , 1979) .

THE ALKYL SIDE CHAIN, ALPHA METHYL AND BETA

HYDROXYL GROUPS

Aniline was essentially inactive in inhibiting the uptake of prazosin 10^"6 M but lengthening the alkyl side chain progressively increased potency. This indicated that ligands for transport-P must have a side chain which separates the amine from the phenyl group . Presence of a methyl group on the alpha carbon enhanced potency at transport-P. This effect was observed in the following series of compounds: amphetamine (IC_S0 6X10-6 M) was 1.7 fold more potent than phenylethylamine (IC₅₀ 16X10^"6 M) ; norephedrine (IC₅₀ 37X10^"6 M) was 0.5 fold more potent than phenylethanolamine (IC_S0 54X10^"6 M) .

Presence of a hydroxyl group on the beta carbon reduced potency at transport-P. This effect was observed in the following series of compounds: phenylethylamine (IC₅₀ 16X10^"6 M) was 2.4 fold more potent than phenylethanolamine (IC₅₀ 54 XlO^"6 M) ; amphetamine (IC₅₀ 6X10^"6 M) was 5.2 fold more potent than norephedrine (IC₅₀ 37X10^"6 M) ; and methylamphetamine (IC₅₀ 15X10^"6 M) was 1.9 fold more potent than ephedrine (IC₅₀ 43X10^"6 M) . Further, tyramine was more potent than octopamine (efficacy 100% vs 21%) and dopamine was more potent than noradrenaline (efficacy 41% vs 28%;). The combined effect of an alpha methyl group and absence of a beta hydroxyl group increased potency 8 fold (amphetamine IC₅₀ 6X10^"6 M vs phenylethanolamine IC₅₀ 54X10^"6 M;) .

The effects on potency of a beta hydroxyl group and of an alpha methyl group may be attributable to their effects on lipophilicity (see below) . An alternative explanation is that these substitutions alter the molecular conformation of the alkylamine side chain; presence of a beta hydroxyl group exerts an electrostatic pull on the positively charged amine, resulting in preponderance of a conformational form in which the amine is folded towards the beta hydroxyl group (Ison et al, 1973; Pullman et al , 1972) . Conversely, presence of an alpha methyl group causes steric hindrance which reduces the likelihood of such folding (Ison et al , 1973) . Phenolic hydroxyl groups exert no significant electrostatic effect on the rotational conformation of the amine (Ison et al, 1973; Pullman et al , 1972) . The enhancement of potency by an alpha methyl group and reduction by a beta hydroxyl group suggest that folding of the side chain does not favour interaction with the transport-P site, which presumably favours a conformation in which the side chain is fully extended away from the phenyl group. In support of this suggestion, shortening the side chain progressively reduced potency whereas lengthening the side chain increased potency at transport-P .

Beta hydroxyl and alpha methyl groups influence the potencies of phenylethylamines at transport-P in a similar manner to their effects on the affinities of these compounds for the pre-synaptic plasma membrane transporters for dopamine and noradrenaline uptake and the vesicular transporters in brain and adrenal medulla (Burgen & Iversen, 1965; Giros et al, 1994; Horn, 1973; Pacholczyk et al , 1991; Slotkin et al , 1975 & 1979) . Studies which used rigid analogues confirmed that the side chain of phenylethylamines is in a fully extended conformation when these compounds interact with the pre-synaptic plasma membrane dopamine and noradrenaline uptake sites (Horn, 1974; Horn & Snyder, 1972; Miller et al , 1973) . In contrast, a beta hydroxyl group is essential for agonist activity at alpha-1 and alpha-2 adrenoceptors (Nichols & Ruffolo, 1991; Ruffolo et al , 1988) . However, an alpha methyl group enhances affinity of phenylethylamines for alpha-2 adrenoceptors but reduces affinity for alpha-1 adrenoceptors (Nichols & Ruffolo, 1991; Ruffolo et al , 1988) . These receptors presumably require different conformations of the side chain for maximal agonist binding (DeMarinis et al , 1981) . As in the case of alpha-1 adrenoceptors, affinity for uptake₂ is enhanced by a beta hydroxyl group and reduced by an alpha methyl group (Burgen & Iversen, 1965; Grohmann & Trendelenburg 1984).

Amphetamine possesses a single chiral centre around the alpha carbon whereas ephedrine and norephedrine possess two chiral centres around the alpha and beta carbons. R- (-) -Amphetamine was equipotent to S- (+) -amphetamine (IC_S0 6.7X10^"6 M and 6X10^"6 M, respectively). IR, 2S- (-) -Ephedrine was equipotent to IS, 2R- (+) -ephedrine (IC₅₀ 3.4X10^"5 M and 4 . 3X10^"5 M, respectively). IR, 2S- (-) -Norephedrine was equipotent to IS, 2R- (+) -norephedrine (IC₅₀ 40X10^"5 M and 3.7X10^"5 M, respectively) . The equipotent effects of these three sets of enantiomers suggest that transport-P may not distinguish between stereochemical arrangements of a methyl group at the alpha carbon or a hydroxyl group at the beta carbon of phenylethylamines. The pre-synaptic plasma membrane dopamine transporter in rat brain recognises asymmetry of a methyl group on the alpha carbon but does not distinguish asymmetry of a hydroxyl group on the beta carbon (Ferris et al, 1972; Giros et al , 1994; Harris & Baldessarini, 1973; Iversen et al , 1971; Koe, 1976; Meiergerd & Schenk, 1994; Thornburg & Moore, 1973). In contrast, alpha-1 adrenoceptors, the pre-synaptic plasma membrane noradrenaline transporter and uptake₂ distinguish asymmetry ot a hydroxyl group at the beta carbon but not a methyl group at the alpha carbon (Bryan & O'Donnell, 1984; Ferric; et al 1972; Grohman & Trendelenburg, 1984; Iversen et al , 1971; Ruffolo et al, 1988). Alpha-2 adrenoceptors and pre-synaptic vesicular monoamine transporters distinguish assymetry of both hydroxyl group at the beta carbon and a methyl group at the alpha carbon (Ferris & Tang, 1979; Peter et al, 1994; Ruffolo et al, 1988; Slotkin et al,

1979) . Hydroxyl and methyl groups are small entities; it is possible that larger substitutions at the alpha or beta carbons may be recognised stereospecifically by transport-P.

THE PHENYL GROUP

Absence of the phenyl group abolished the ability of phenylalkylamines to inhibit the uptake of prazosin. Thus, methylamine, ethylamine, propylamine and butylamine (efficacy 14%, 10%, 18% and 20%, respectively) were essentially inactive in comparison to their respective phenyl-alkyl analogues (phenylmethylamine efficacy 100%, IC₅₀ 37X10^"6 M; phenylethylamine efficacy 100%, IC₅₀ 16X10^"6 M; phenylpropylamine efficacy 100%, IC₅₀ 12X10^"6 M; phenylbutylamine efficacy 100%, IC₅₀ 6X10^"6 M) .

Surprisingly, presence of a single phenolic hydroxyl group in the para position strongly reduced potency at transport-P. Thus, phenylethylamine (IC₅₀ 16X10^"6 M) was 49 fold more potent than tyramine (IC_S0 800X10^"6 M) in inhibiting the uptake of prazosin. Presence of a second phenolic hydroxyl group in the meta position further reduced potency at transport-P (dopamine efficacy 41%) . The effect of the phenolic para hydroxyl group was also seen in the following series of compounds: phenylethanolamine and octopamine; norephedrine and alpha-methyl-octopamine

Phenolic chlorine atoms increased potency (Figure 6). Thus, 2,4-dιchlorophenylethylamine (IC₅₀ 4X10^"6 M) was 3 fold more potent than phenylethylamine (IC_S0 16X1 0^"6 M) ; .3,4-dichloro-phenylethanolamine (IC₅₀ 4X10^"6 M) was 12.5 fold more potent than phenylethanolamine (IC₅₀ 54X10^"6 M) ; 3,4- dichloromethylamphetamine (IC₅₀ 3X10^"6 M) was 3.7 fold more potent than methylamphetamine (IC₅₀ 14X10-6 M) . Substitution of chlorine atoms with hydroxyl groups in the same positions reduced potency (3 , 4-dichloro- phenylethanolamine efficacy 100% vs noradrenaline efficacy 28%) .

The reduction of potency by phenolic hydroxyl groups suggests that the phenyl group should be hydrophobic for optimum activity at transport-P. This surprising suggestion is strengthened by the finding that hydrophobic chlorine atoms in the phenyl ring increased potency. Substitution of chlorine atoms with hydroxyl groups in the same positions reduced potency, suggesting that the enhancing effect of chlorine is unlikely to be due to an electronegative effect, but is more likely due to the hydrophobic nature of chlorine. These surprising findings are in striking contrast to the structural properties of phenylethylamines which bind alpha-1 and alpha-2 adrenoceptors, the pre-synaptic plasma membrane dopamine and noradrenaline uptake₂ transporters, and the vesicular transporters in rat brain and adrenal medulla, where phenolic hydroxyl groups strongly increased affinity (Nichols & Ruffolo, 1991; Ruffolo et al, 1988; Burgen & Iversen, 1965; Horn, 1973; Peter et al, 1994; Slotkin et al, 1975 & 1979) . These findings explain our previous observation that very small amounts of noradrenaline are accumulated in hypothalamic cells (fmoles noradrenaline/mg protein vs pmoles prazosin/mg protein; Al-Damluji et al, 1993); potency at transport-P is reduced by the phenolic and beta hydroxyl groups in noradrenaline.

Surprisingly, analogues of phenylethylamine which possessed one phenolic methoxyl group were equipotent to the parent compound, regardless of whether the methoxyl group was in the ortho, meta or para position. Further, methoxyphenamine was equipotent to methylamphetamine. Of the three compounds which possessed a dimethoxyphenyl group, 2,5- dimethoxyphenylethylamine was equipotent to phenylethylamine but 3 , 4-dimethoxyphenylethylamine

(IC₅₀ 69X10^{" 6} M) was 3.3 fold less potent than phenyl ethy lamine (IC50 16X10^{" 6} M) and methoxamine (IC_S0 68X10^"6 M) was 0.7 fold less potent than norephedrine IC₅₀ 40X10^"6 M; ) . Methoxyl groups are neutral entities, so their lack of effect is compatible with the suggestion that the phenyl ring of these compounds should be hydrophobic for optimum activity at transport-P. In clear contrast, a single phenolic methoxyl group reduced the affinity of phenylethylamines for the presynaptic plasma membrane dopamine and noradrenaline uptakel sites (Burgen & Iversen, 1965; Horn, 1973) Phenolic methoxyl groups enhanced the affinity of phenylethylamines for noradrenaline uptake ₂ (Burgen & Iversen, 1965; Grohman & Trendelenburg, 1984) and for alpha-1 adrenoceptors (DeMarinis et al, 1981) , but they had no effect on the affinity of these compounds for alpha-2 adrenoceptors (Ruffolo et al, 1988). The present conclusions are based on the potencies of compounds at transport-P, as indicated by competitive inhibition of the uptake of prazosin. Inhibition of uptake does not necessarily indicate that these compounds are themselves internalised by the uptake process; that would require direct measurement of the accumulation of radioactively labelled compounds in the cells. We therefore studied the accumulation of [³H] verapamil, which is a phenylethylamine derivative which possesses the structural properties which enable interaction with transport-P, as identified in this study.

[³H] verapamil was internalised by GnRH cells in a similar manner to prazosin. Phenylethylamine and its derivatives inhibited the uptake of prazosin competitively, suggesting that these compounds and prazosin act on the same transport-P carrier molecule in GnRH neurones. It therefore seems likely that the structural properties which are described herein may define some of the requirements for interaction of phenylethylamines with the transport-P carrier molecule in peptidergic neurones. An alternative explanation is that these amines may have inhibited the uptake of prazosin by dint of their lipophilic nature, which may have enabled them to diffuse across cell membranes, resulting in neutralisation of the acidified intracellular compartment in which prazosin is accumulated. However, this seems an unlikely explanation as uptake of prazosin was unaffected by some highly lipophilic amines, including reserpine, phenoxybenzamine and vesamicol (Al-Damluji & Kopin, 1996a; Table 1) . The most likely explanation for the findings is that these compounds compete with prazosin for binding to a carrier molecule in peptidergic neurones .

The basicity of ligands for Transport-P:

A positively charged amine had been thought to be an essential requirement for activity at Transport-P, as removal of the amine or neutralisation by a carboxyl group abolished activity (Al-Damluji & Kopin, 1995, Journal of Endocrinology 147, suppl . , P66) . However, compounds which possess a permanent positive charge on the amine (eg. quaternary and guanyl amines) are inactive at Transport-P. The reason for this lack of activity was unknown.

Aniline is inactive at Transport-P (Figure 7) . The amine group in aniline is neutral at physiological pH because the electron pair of the nitrogen atom becomes incorporated in the pi electron system of the phenyl ring. It is unknown whether the lack of activity of aniline at Transport-P is due to this weak basicity or to the fact that the amine is in close proximity to the phenyl ring, regardless of basicity.

This problem was addressed by examining the effect of an additional hydrophobic phenyl group which is located at a distance from the amine which is appropriate for activity at Transport-P. The distance chosen was three carbon atoms, which is the distance between the amine and the phenyl ring in phenylpropylamine, which is full active at Transport-P (Figure 7) . Surprisingly, compounds in which a phenyl group is located three carbon atoms from the anilino- amine group are inactive at Transport-P (eg, 2- benzylaniline; Figure 7) . Further compounds in which a phenoxyl group is located in the ortho, meta or para positions were also inactive. Thus, the lack of effect of aniline is not due simply to the proximity of the amine to the benzene ring. The lack of effect of aniline is likely to be due to the neutral charge of its amine at physiological pH.

In conclusion, compounds which are active at Transport-P must have an amine group which is neither permanently neutral nor permanently positively charged. The amine group must be able to acquire and to shed its charge.

A method for studying the interaction of compounds with receptors and Transport-P simultaneously:

It is proposed that an ideal antidepressant compound would be a substrate for Transport-P but it would not act as an agonist for post-synaptic receptors. This is because receptor agonists are likely to cause down-regulation of the receptor with consequent loss of responsiveness of the post-synaptic neurones. It is therefore important to be able to study the interaction of proposed antidepressants with both Transport-P and the post-synaptic receptors. The method described below makes it possible to study simultaneously the interaction of compounds with Transport-P and post-synaptic receptors.

The principle of the method is to express postsynaptic receptors in GTl-1 GnRH cells which endogenously express Transport-P. The post-synaptic receptor is introduced into GnRH cells as a cDNA ligated to an appropriate plasmid vector. Electroporation is a method which uses electric shocks to introduce plasmid DNA into mammalian cells.

Method

Electroporation of GTl-1 GnRH cells with alpha-lb adrenergic receptor cDNA

Materials : GTl-1 GnRH ^"cells grown in a 175cm² flask, approximately 20X10⁶ cells in the flask at initiation. cDNA for post-synaptic receptor (eg. alpha-lb adrenergic receptor cDNA in plasmid vector; 1 ug/ul in TE buffer) Electroporation apparatus (eg, BioRad Gene Pulser II with capacitance extender and pulse controller)

Electroporation cuvetts (BioRad 165-2088; inter- electrode distance 0.4 cam mechanism)

12-well culture plates coated with poly-D-lysine 1.4 ug/cm² and laminin 0.14 ug/cm²

Procedure :

1. Detach the cells from the flask using the trypsin/EDTA dispersion solution described above 2. Wash the cells once with culture medium

3. Disperse the cells in 10 ml culture medium. Count the cells in a haemacytometer

4. Centrifuge the cells and disperse in 600 ul culture medium to a density of 10⁸ cells/ml 5. Place 250 ul of the cell suspension (2.5X10⁷ cells) in each of two cuvettes

6. Add 10 ul DNA to each cuvette and mix well by pipetting

7. Electroporate using the following parameters: 190 Volts, 1300 uF 8. After 60 seconds, add culture medium to the cuvette. Transfer the cells to a total of 50 ml culture medium (5X10⁵ electroporated cells/ml)

9. Place 2 ml of the cell suspension in each well of the coated 12 -well plates

10. Incubate at 37°C in a humidified atmosphere containing 5% C0₂

11. Perform the assay 48 hours after electroporation

Assay for Transport-P and for binding to postsynaptic receptor

Materials : Electroporated GTl-1 GnRH cells

Binding buffer: DMEM (Sigma D-5648) with 25 mM HEPES (Sigma H-9136; 5.6 g/1), pH 7.4

Before use, add sodium ascorbate 10 mg/100 ml (0.5 mM) L-Ascorbic acid sodium salt (Sigma A-4034) FW 198.1, stored RT

Radiolabelled ligand (eg, [7-methoxy-³H] Prazosin; Amersham TRK.843)

Test compound (eg, unlabelled prazosin. HCl ; Sigma P-7791)

Solubilistation solution: 0.1% sodium dodecyl sulphate (Sigma L-4509; 1 g/1)

0.1 M sodium hydroxide (Sigma S-0899; 4 g/1) Warm before use Cocktail T scintillation liquid (BDH 14509 6B)

PROCEDURE :

1. Add ascorbate 28 mg to 280 ml buffer

2. Add t³H] prazosin 1.0 ul to 60 ml buffer (final -0.2 nM) 3. Dilute unlabelled prazosin 10^"3 M 1:1000 to 15 ml of [³H] prazosin = 10^"6 M unlabelled prazosin

4. Dilute the above serially to 7 ml [³H] prazosin to obtain:

Unlabelled prazosin

10^"6 M

6 . 66X10 ^"7 M

3 . 33X10 ^"7 M

10^"7 M

10^"8 M

10^"9 M

10^{" 10} M

5. Wash the cells twice with 1 ml buffer at room temperature

6. Add 1 ml of [³H] prazosin or unlabelled prazosin dilutions in [³H] prazosin

7. Incubate at 37°C for 60 minutes 8. Place the cells on ice

9. Remove the buffer after 30 seconds and wash twice with ice cold buffer, 1 ml each wash

10. Add 1 ml of warm solubilisation solution and leave for 15 minutes 11. Remove the extract to scintillation vials and repeat the above step

12. Vortex thoroughly to shear the viscous cell extract

13. Remove 50 ul aliquots into 12X75 mm tubes for protein assay

14. Prepare TC's, 1 ml in triplicate

15. Add 10 ml of scintillation liquid, vortex and count

Figure 8 Simultaneous assay for Transport-P and for binding to post-synaptic receptor

Transfection of GT-1 GnRH cells with alpha-lb adrenergic receptor cDNA results in over-expression of the receptor in the cells. In the absence of unlabelled prazosin, the radioligand binds to alpha-1 adrenergic receptors. In the presence of unlabelled prazosin greater than 10^"7 M, the radioligand is accumulated via Transport-P. Cells over-expressing the receptor accumulate prazosin via Transport-P in an identical manner to control cells, indicating that binding of prazosin to alpha-1 adrenergic receptors and accumulation of the compound via Transport-P are separate functions. Alpha-1 adrenergic receptors are shown as an illustrative example; to study other postsynaptic receptors (eg, serotonergic receptors) , appropriate radioligands should be used.

TABLE 1

Effects of selected substitutions on the efficacy of phenylethylamine derivatives and related compounds in inhibiting the uptake of prazosin (10^~6 M) in GTl-1 GnRH cells .

Compound Efficacy ABSENCE OF ALKYL AMINE 2, 5-Dimethoxyacetophenone 10"³ M 0%

3, 4-Dimethoxyacetophenone 10"³ M 5%

2 , 5-Dimethoxybenzaldehyde 10"³ M 0%

1, 4-Dimethoxybenzene 10^"3 M 0%

3, 4-Dimethoxyphenylacetic acid 10^"3 M 3%

PRESENCE OF ALPHA CARBOXYL GROUP

L-Phenylalanine 10"³ M 2%

L-Tyrosine 10^"3 M 0%

L-DOPA 10^"3 M 0% L-Serine lO^"4 M 0%

SECONDARY, TERTIARY, QUATERNARY AND GUANYL AMINES HEAT (4-hydroxyphenylethylaminomethyltetralone; secondary) 10^"3 M 100% Fluoxetine (secondary) lO^"4 M 100%

Verapamil (tertiary) 10^"4 M 100%

MPTP (l-methyl-4-phenyl-l,2, 3, 6,

-tetrahydropyridine; teritary) 10^"" M 100%

MPP⁺ ( l-methyl-4-phenylpyridinium; quaternary) 10^"3 M 14%

Bretylium (quarternary) 10^"4 M 7%

Meta-iodo-benzyl-guanidine 10^"3 M 25%

N-guanyltyramine 10^"3 M 18%

Guanethidine 10^"4 M 8%

PHENOLIC HYDROXYL AND METHOXYL GROUPS L-Metaraminol (1- [ 3-hydroxyphenyl] -2-amino-1- propanol) 10^"3 M 13%

L-Adrenaline (1- [3, 4-dihydroxyphenyl] -2- methylamino-ethanol 10^"" M 14% L-Phenylephrine (1- [3-hydroxyphenyl] -2- methylamino-ethanol 10^"3 M 37% DL-Normetanephrine (1- [3-methoxy-4-hydroxyphenyl] - ethanolamine 10^"3 M 14% L-Isoprenaline (1- [3, 4-dihydroxyphenyl] -2- isopropylamino-ethanol 10^"4 M 18%

MISCELLANEOUS AMINES AND NEUROTRANSMITTERS

Serotonin 10^"4 M 0% L-tryptophan 10-⁴ M 3%

Histamine 10^"4 M 4%

L-Histidine 10^"4 M 8%

Acetylcholine 10^"4 M 5%

Choline 10^-4 M 9% DL-Vesamicol 10^-5 M 29%

L-Glutamate 10^"4 M 0%

L-Aspartate 10^"4 M 0%

Glycine 10"^* M 0%

GABA 10^"4 M 0% ATP 10^"4 M 20%

Adenosine 10^"3 M 17%

Cocaine 10^'4 M 100%

Phenoxybenzamine 10^"4 M 22%

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Claims

1. A method of identifying compounds having antidepressant activity which method comprises (a) contacting a cell expressing or overexpressing an alpha- lbadrenergic receptor and transport-P protein with said compound to be tested; and

(b) prior to or after step a) contacting said compound with a cell expressing an alpha- lb-adrenergic receptor but not transport - P protein; and

(c) selecting a compound which preferentially binds transport P.

2. A method of identifying compounds having antidepressant activity which method comprises a) contacting a cell expressing or overexpressing an alpha- lbadrenergic receptor and transport-P with a labelled ligand of said receptor; b) subsequently contacting said cell with said compound to be tested at progressively increasing concentrations ; and c) monitoring for increased binding of said ligand at increasing concentrations of said compound to be tested and selecting a compound which increases binding of said ligand upon addition of increasing concentrations of said compound to be tested.

3. A method according to claim 2 wherein said label comprises a radiolabel .

4. A method according to claim 2 or 3 wherein said ligand comprises [³H] -prazosin.

5. A method according to any of claims 1 to 4 which further comprises the step of contacting the selected compound from step c with a cell expressing said alpha-lb-adrenergic receptor and transport P and monitoring the level of accumulation of said compound in said cell as ^"compared with the concentration of the applied compound and selecting a compound which activates uptake by transport-P.

6. A method according to any of claims 1 to 5 which further comprises the step of contacting said selected compound with a cell expressing alpha-lb adrenergic receptors and transport P and monitoring the rate of release of said compound from intracellular vesicles in said cell and selecting compounds having a relatively longer retention time in said vesicles .

7. A method according to claim 1 or 2 wherein said cell is a peptidergic neuronal cell.

8. A method according to claim 1 wherein said cell in step b is a COS-7 cell.

9. A compound which is identifiable as an antidepressant according to the method of any of claims 1 to 8.

10. A compound according to claim 9 which comprises a phenylalkylamine having a substituted hydrophobic phenyl group.

11. A compound according to claim 9 or 10 which comprises a compound of the formula:

wherein n is 1, 2, 3 or 4

R_x is selected from H, CH₃, OH

R₂ is selected from H, CH₃

R₃ is selected from H, Cl, OCH₃, and the NH group is neither permanently positively charged nor neutral

12. A compound according to claim 11 which comprises any of a D-amphetamine, methylampetamine, phenylethanolamine, D or L-norephedrine, D-ephedrine, Tyramine, 2 , 4-Dicholoro-PEA, 3 , 4-Dicholoro-PEOLA, 3,4- Dicholoromethylamphetamine, 2,3 or 4 methoxy-PEA, 2,- 5-dimethoxy-PEA, 3 , 4-Dimethoxy-PEA, methoxamine.

13. A compound according to any of claims 9 to 12 for use as a medicament.

14. Use of a compound according to any of claims 9 to 12 in the preparation of a medicament for treating depression.

15. A pharmaceutical composition comprising a compound according to any of claims 9 to 12 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

16. An alpha- lb-adrenergic receptor from GnRH neurones or a functional equivalent derivative or bioprecursor of said receptor having an amino acid sequence encoded by the nucleotide sequence illustrated in figure 1.

17. An alpha-lb-adrenergic receptor according to claim 16 having an amino acid sequence illustrated in figure 2 or an amino acid sequence which differs from said amino acid sequence in one or more conservative amino acid changes.

18. A nucleic acid molecule comprising a sequence of nucleotides encoding an alpha- lb- adrenergic receptor having an amino acid sequence illustrated in Figure 2 , or a functional equivalent, derivative or bioprecursor thereof, or one which differs from the sequence in Figure 2 in one or more conservative amino acid changes.

19. A nucleic acid molecule according to claim

18 which comprises any of a genomic DNA or a cDNA molecule.

20. A nucleic acid molecule according to claim

19 comprising a nucleotide sequence according to Figure 1.

21. A nucleic acid molecule capable of hybridising to the DNA sequence according to any of claims 18 to 20 under high stringency conditions.

22. A DNA expression vector which comprises a cDNA as claimed in claim 19.

23. An expression vector according to claim 22 which comprises a promoter of said alpha-lb-adrenergic receptor.

24. A vector according to claim 22 or 23 which further comprises a sequence encoding a reporter molecule .

25. A vector according to claim 24 wherein said reporter molecule comprises a flurophore.

26. A host cell transformed or transfected with the vector of any claims 22 to 25.

27. A host cell according to claim 26 which cell comprises a COS-7 cell or a cell expressing an alpha- lb-adrenergic receptor.

28. A transgenic cell, tissue or organism comprising a transgene capable of expressing a receptor according to claims 16 or 17.

29. A transgenic cell, tissue or organism according to claim 28, wherein said transgenic cell is a COS-7 cell or a cell expressing an alpha-lb- adrenergic receptor.

30. A transgenic cell, tissue or organism, according to claims 28 or 29 wherein said transgene comprises a vector according to any of claims 22 to

25.

31. A method of identifying compounds which inhibit transport-P uptake in a cell which method comprises contacting a cell expressing transport-P with said compound to be tested in the presence of a compound which is known to be internalised by transport-P and which compound has a label thereon, monitoring for the presence of said known compound in said cell and selecting compounds which substantially inhibit uptake of said known compound.

32. A method according to claim 31 wherein said known compound comprises [³H] prazosin.

33. A compound identifiable according to the method of claims 31 and 32.

34. A compound according to claim 33 for use as a medicament.

35. Use of a compound according to claim 33 in the preparation of a medicament for treating the effects of addictive drug abuse or depression.

36. Use according to claim 35 wherein said drug comprises any of cocaine, amphetamine or ephedrine, or compounds derived therefrom.