Richard B. Mailman

Functional Selectivity and Classical Concepts of Quantitative Pharmacology

The concept of intrinsic efficacy has been enshrined in pharmacology for half of a century, yet recent data have revealed that many ligands can differentially activate signaling pathways mediated via a single G protein-coupled receptor in a manner that challenges the traditional definition of intrinsic efficacy. Some terms for this phenomenon include functional selectivity, agonist-directed trafficking, and biased agonism. At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor. Data illustrating this phenomenon are presented from serotonin, opioid, dopamine, vasopressin, and adrenergic receptor systems. A variety of mechanisms may influence this apparently ubiquitous phenomenon. It may be initiated by differences in ligand-induced intermediate conformational states, as shown for the β2-adrenergic receptor. Subsequent mechanisms that may play a role include diversity of G proteins, scaffolding and signaling partners, and receptor oligomers. Clearly, expanded research is needed to elucidate the proximal (e.g., how functionally selective ligands cause conformational changes that initiate differential signaling), intermediate (mechanisms that translate conformation changes into differential signaling), and distal mechanisms (differential effects on target tissue or organism). Besides the heuristically interesting nature of functional selectivity, there is a clear impact on drug discovery, because this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizing therapeutic action. It also may be timely to revise classic concepts in quantitative pharmacology and relevant pharmacological conventions to incorporate these new concepts.

Aripiprazole, a novel atypical antipsychotic drug with a unique and robust pharmacology

Atypical antipsychotic drugs have revolutionized the treatment of schizophrenia and related disorders. The current clinically approved atypical antipsychotic drugs are characterized by having relatively low affinities for D2-dopamine receptors and relatively high affinities for 5-HT2A serotonin receptors (5-HT, 5-hydroxytryptamine (serotonin)). Aripiprazole (OPC-14597) is a novel atypical antipsychotic drug that is reported to be a high-affinity D2-dopamine receptor partial agonist. We now provide a comprehensive pharmacological profile of aripiprazole at a large number of cloned G protein-coupled receptors, transporters, and ion channels. These data reveal a number of interesting and potentially important molecular targets for which aripiprazole has affinity. Aripiprazole has highest affinity for h5-HT2B-, hD2L-, and hD3-dopamine receptors, but also has significant affinity (5–30 nM) for several other 5-HT receptors (5-HT1A, 5-HT2A, 5-HT7), as well as α1A-adrenergic and hH1-histamine receptors. Aripiprazole has less affinity (30–200 nM) for other G protein-coupled receptors, including the 5-HT1D, 5-HT2C, α1B-, α2A-, α2B-, α2C-, β1-, and β2-adrenergic, and H3-histamine receptors. Functionally, aripiprazole is an inverse agonist at 5-HT2B receptors and displays partial agonist actions at 5-HT2A, 5-HT2C, D3, and D4 receptors. Interestingly, we also discovered that the functional actions of aripiprazole at cloned human D2-dopamine receptors are cell-type selective, and that a range of actions (eg agonism, partial agonism, antagonism) at cloned D2-dopamine receptors are possible depending upon the cell type and function examined. This mixture of functional actions at D2-dopamine receptors is consistent with the hypothesis proposed by Lawler et al (1999) that aripiprazole has ‘functionally selective’ actions. Taken together, our results support the hypothesis that the unique actions of aripiprazole in humans are likely a combination of ‘functionally selective’ activation of D2 (and possibly D3)-dopamine receptors, coupled with important interactions with selected other biogenic amine receptors—particularly 5-HT receptor subtypes (5-HT1A, 5-HT2A).

Interactions of the Novel Antipsychotic Aripiprazole (OPC-14597) with Dopamine and Serotonin Receptor Subtypes

OPC-14597 {aripiprazole; 7-(4-(4-(2,3-dichlorophenyl)-1-piperazinyl)butyloxy)-3,4-dihydro-2(1H)-quinolinone} is a novel candidate antipsychotic that has high affinity for striatal dopamine D2-like receptors, but causes few extrapyramidal effects. These studies characterized the molecular pharmacology of OPC-14597, DM-1451 (its major rodent metabolite), and the related quinolinone derivative OPC-4392 at each of the cloned dopamine receptors, and at serotonin 5HT6 and 5HT7 receptors. All three compounds exhibited highest affinity for D2L and D2S receptors relative to the other cloned receptors examined. Both OPC-4392 and OPC-14597 demonstrated dual agonist/antagonist actions at D2L receptors, although the metabolite DM-1451 behaved as a pure antagonist. These data suggest that clinical atypicality can occur with drugs that exhibit selectivity for D2L/D2S rather than D3 or D4 receptors, and raise the possibility that the unusual profile of OPC-14597 in vivo (presynaptic agonist and postsynaptic antagonist) may reflect different functional consequences of this compound interacting with a single dopamine receptor subtype (D2) in distinct cellular locales.

Behavioural assessment of mice lacking D1A dopamine receptors

Dopamine D1A receptor-deficient mice were assessed in a wide variety of tasks chosen to reflect the diverse roles of this receptor subtype in behavioural regulation. The protocol included examination of exploration and locomotor activity in an open field, a test of sensorimotor orienting, both place and cue learning in the Morris water maze, and assessment of simple associative learning in an olfactory discrimination task. Homozygous mice showed broad-based impairments that were characterized by deficiencies in initiating movement and/or reactivity to external stimuli. Data obtained from flash evoked potentials indicated that these deficits did not reflect gross visual impairments. The partial reduction in D1A receptors in the heterozygous mice did not affect performance in most tasks, although circumscribed deficits in some tasks were observed (e.g., failure to develop a reliable spatial bias in the water maze). These findings extend previous behavioural studies of null mutant mice lacking D1A receptors and provide additional support for the idea that the D1A receptor participates in a wide variety of behavioural functions. The selective impairments of heterozygous mice in a spatial learning task suggest that the hippocampal/cortical dopaminergic system may be uniquely vulnerable to the partial loss of the D1A receptor.

AF64A, a cholinergic neurotoxin, selectively depletes acetylcholine in hippocampus and cortex, and produces long-term passive avoidance and radial-arm maze deficits in the rat

The behavioral and biochemical effects of AF64A, a presynaptic cholinergic neurotoxin, were investigated. Bilateral administration of this compound into the lateral cerebral ventricles produced transient and dose-related effects on sensorimotor function and long-term impairments of cognitive behavior. Male Fischer-F344 rats dosed with either 15 or 30 nmol of AF64A reacted 29-62% faster than CSF-injected controls in a hot-plate test 14 (but not 1, 7, 21 or 28) days following dosing. The group administered 15 nmol of AF64A was also significantly more active (41%) than controls 28 days following dosing. The activity level of this group was comparable to that of controls at other times and hyperactivity was never observed in the 30 nmol group. Retention of a step-through passive avoidance task, assessed 35 days after dosing, was impaired in both the 15 and the 30 nmol groups. Their step-through latencies were significantly shorter than the control latencies, and they exhibited more partial entries during the 24-h retention test. Radial-arm maze performance, measured 60-80 days following treatment, was markedly impaired in the treated groups. Animals treated with AF64A made fewer correct responses in their first 8 choices, required more total selections to complete the task, and had an altered pattern of spatial responding in the maze. The neurochemical changes produced by AF64A, determined 120 days after dosing, were specific to the cholinergic system and consisted of decreases of ACh in both the hippocampus (15 and 30 nmol groups) and the frontal cortex (30 nmol group). The concentrations of catecholamines, indoleamines, their metabolites and choline in various brain regions were not affected by AF64A. Furthermore, histological analysis revealed that the doses of AF64A used in the present study did not damage the hippocampus, the fimbria-fornix, the septum or the caudate nucleus. These data support the contention that cholinergic processes in the hippocampus and/or frontal cortex play an important role in learning and memory processes. Furthermore, based upon the behavioral and biochemical data presented, it is suggested that AF64A could be a useful pharmacological tool for examining the neurobiological substrates of putative cholinergic disorders such as senile dementia of the Alzheimers type.

Simultaneous quantification of dopamine, 5-hydroxytryptaine and four metabolically related compounds by means of reversed-phase high-performance liquid chromatography with electrochemical detection

A method for simultaneously quantifying dopamine, 5-hydroxytryptamine (5-HT) and four metabolically related compounds has been developed, permitting more efficient neurochemical examination of these often interrelated biogenic amine systems. The method uses high-performance liquid chromatographic separation of these compounds on a C18 reversed-phase column with a buffered mobile phase containing methanol as an organic modifier and heptanesulfonate as an ion-pair reagent. Using 5-hydroxy-N-methyltryptamine as an internal standard and electrochemical detection, chromatography time is less than 12 min. Sample preparation simply involves the addition of internal standard, homogenization in the mobile phase, centrifugation and injection of the supernatant into the chromatograph. The method is sensitive to a tissue content of these compounds of less than 1 ng. The utility of this method for neuropharmacological--neurochemical studies is illustrated with studies using inhibitors of monoamine oxidase (pargyline) and aromatic amino acid decarboxylase (RO 4-4602).

Aripiprazole has Functionally Selective Actions at Dopamine D2 Receptor-Mediated Signaling Pathways

Aripiprazole is a unique atypical antipsychotic drug with an excellent side-effect profile presumed, in part, to be due to lack of typical D2 dopamine receptor antagonist properties. Whether aripiprazole is a typical D2 partial agonist, or a functionally selective D2 ligand, remains controversial (eg D2-mediated inhibition of adenylate cyclase is system dependent; aripiprazole antagonizes D2 receptor-mediated G-protein-coupled inwardly rectifying potassium channels and guanosine triphosphate nucleotide (GTP)γS coupling). The current study examined the D2L receptor binding properties of aripiprazole, as well as the effects of the drug on three downstream D2 receptor-mediated functional effectors: mitogen-activated protein kinase (MAPK) phosphorylation, potentiation of arachidonic acid (AA) release, and D2 receptor internalization. Unlike quinpirole (a full D2 agonist) or (−)3PPP (S(−)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride, a D2 partial agonist), the apparent D2 affinity of aripiprazole was not decreased significantly by GTP. Moreover, full or partial agonists are expected to have Hill slopes <1.0, yet that of aripiprazole was significantly >1.0. Whereas aripiprazole partially activated both the MAPK and AA pathways, its potency vs MAPK phosphorylation was much lower relative to potencies in assays either of AA release or inhibition of cyclic adenosine 3′,5′-cyclic monophosphate accumulation. In addition, unlike typical agonists, neither aripiprazole nor (−)3PPP produced significant internalization of the D2L receptor. These data are clear evidence that aripiprazole affects D2L-mediated signaling pathways in a differential manner. The results are consistent with the hypothesis that aripiprazole is a functionally selective D2 ligand rather than a simple partial agonist. Such data may be useful in understanding the novel clinical actions of this drug.

Third generation antipsychotic drugs: partial agonism or receptor functional selectivity?

Functional selectivity is the term that describes drugs that cause markedly different signaling through a single receptor (e.g., full agonist at one pathway and antagonist at a second). It has been widely recognized recently that this phenomenon impacts the understanding of mechanism of action of some drugs, and has relevance to drug discovery. One of the clinical areas where this mechanism has particular importance is in the treatment of schizophrenia. Antipsychotic drugs have been grouped according to both pattern of clinical action and mechanism of action. The original antipsychotic drugs such as chlorpromazine and haloperidol have been called typical or first generation. They cause both antipsychotic actions and many side effects (extrapyramidal and endocrine) that are ascribed to their high affinity dopamine D(2) receptor antagonism. Drugs such as clozapine, olanzapine, risperidone and others were then developed that avoided the neurological side effects (atypical or second generation antipsychotics). These compounds are divided mechanistically into those that are high affinity D(2) and 5-HT(2A) antagonists, and those that also bind with modest affinity to D(2), 5-HT(2A), and many other neuroreceptors. There is one approved third generation drug, aripiprazole, whose actions have been ascribed alternately to either D(2) partial agonism or D(2) functional selectivity. Although partial agonism has been the more widely accepted mechanism, the available data are inconsistent with this mechanism. Conversely, the D(2) functional selectivity hypothesis can accommodate all current data for aripiprazole, and also impacts on discovery compounds that are not pure D(2) antagonists.

Differential effects of clozapine and haloperidol on ketamine-induced brain metabolic activation

Subanesthetic doses of N-methyl-d-aspartate (NMDA) receptor antagonists such as ketamine and phencyclidine precipitate psychotic symptoms in schizophrenic patients. In addition, these drugs induce a constellation of behavioral effects in healthy individuals that resemble positive, negative, and cognitive symptoms of schizophrenia. Such findings have led to the hypothesis that decreases in function mediated by NMDA receptors may be a predisposing, or even causative, factor in schizophrenia. The present study examined the effects of the representative atypical (clozapine) and typical (haloperidol) antipsychotic drugs on ketamine- induced increases in [14C]-2-deoxyglucose (2-DG) uptake in the rat brain. As previously demonstrated, administration of subanesthetic doses of ketamine increased 2-DG uptake in specific brain regions, including medial prefrontal cortex, retrosplenial cortex, hippocampus, nucleus accumbens, basolateral amygdala, and anterior ventral thalamic nucleus. Pretreatment of rats with 5 or 10 mg/kg clozapine alone produced minimal or no change in 2-DG uptake, yet clozapine completely blocked ketamine-induced changes in 2-DG uptake in all brain regions studied. In striking contrast, a dose of haloperidol (0.5 mg/kg) that produces a substantial cataleptic response, potentiated, rather than blocked, ketamine-induced activation of 2-DG uptake. These results demonstrate, in a model with potential relevance to schizophrenia, a striking neurobiological difference between the actions of prototypical typical and atypical antipsychotic drugs. The dramatic blockade by clozapine of ketamine-induced brain metabolic activation suggests that antagonism of the consequences of reduced NMDA receptor function could contribute to the superior therapeutic effects of this atypical antipsychotic agent. The results also suggest that this model of ketamine-induced alterations in 2-DG uptake may be extremely useful for understanding the complex neural mechanisms of atypical antipsychotic drug action.

Smoking duration, intensity, and risk of Parkinson disease

Objective: To evaluate the relative importance of smoking duration vs intensity in reducing the risk of Parkinson disease (PD). Methods: The study included 305,468 participants of the NIH-AARP Diet and Health cohort, of whom 1,662 had a PD diagnosis after 1995. We estimated odds ratios (OR) and 95% confidence intervals from multivariate logistic regression models. Results: Compared with never smokers, the multivariate ORs were 0.78 for past smokers and 0.56 for current smokers. Among past smokers, a monotonic trend toward lower PD risk was observed for all indicators of more smoking. Stratified analyses indicated that smoking duration was associated with lower PD risk within fixed intensities of smoking. For example, compared with never smokers, the ORs among past smokers who smoked >20 cigarettes/day were 0.96 for 1–9 years of smoking, 0.78 for 10–19 years, 0.64 for 20–29 years, and 0.59 for 30 years or more (p for trend = 0.001). In contrast, at fixed duration, the typical number of cigarettes smoked per day in general was not related to PD risk. Close examination of smoking behaviors in early life showed that patients with PD were less likely to be smokers at each age period, but if they smoked, they smoked similar numbers of cigarettes per day as individuals without PD. Conclusions: This large study suggests that long-term smoking is more important than smoking intensity in the smoking–Parkinson disease relationship.