David W. Schulz

Pharmacological profile of the α4β2 nicotinic acetylcholine receptor partial agonist varenicline, an effective smoking cessation aid

The preclinical pharmacology of the α4β2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline, a novel smoking cessation agent is described. Varenicline binds with subnanomolar affinity only to α4β2 nAChRs and in vitro functional patch clamp studies in HEK cells expressing nAChRs show that varenicline is a partial agonist with 45% of nicotines maximal efficacy at α4β2 nAChRs. In neurochemical models varenicline has significantly lower (40–60%) efficacy than nicotine in stimulating [3H]-dopamine release from rat brain slices in vitro and in increasing dopamine release from rat nucleus accumbens in vivo, while it is more potent than nicotine. In addition, when combined with nicotine, varenicline effectively attenuates the nicotine-induced dopamine release to the level of the effect of varenicline alone, consistent with partial agonism. Finally, varenicline reduces nicotine self-administration in rats and supports lower self-administration break points than nicotine. These data suggest that varenicline can reproduce to some extent the subjective effects of smoking by partially activating α4β2 nAChRs, while preventing full activation of these receptors by nicotine. Based on these findings, varenicline was advanced into clinical development and recently shown to be an effective and safe aid for smoking cessation treatment.

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1‐Like Dopamine Receptor

Abstract: The compound [9‐3H]SCH23390 [R‐(+)‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐1H‐3‐benzazepine‐7‐ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4‐dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high‐affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and olfactory bulb. Specific binding in caudate‐putamen was found to be both temperature‐ and pH‐dependent, with optima at 25–30°C and pH 7.8–8.0. Scatchard or Woolf analyses of binding in caudate‐putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD= 0.7 ± 0.1 nM; Bmax= 347 ± 35 fmol/mg of protein). Both dopamine and cis‐flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis‐flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.

Combined Administration of a 5-Hydroxytryptamine (5-HT)1D Antagonist and a 5-HT Reuptake Inhibitor Synergistically Increases 5-HT Release in Guinea Pig Hypothalamus In Vivo

Abstract: In vivo microdialysis in guinea pig hypothalamus was used to study the effect of serotonin [5‐hydroxytryptamine (5‐HT)] subtype 1D autoreceptor blockade on the increase in extracellular 5‐HT levels produced by a selective 5‐HT reuptake inhibitor (SSRI). Administration of the selective 5‐HT1D antagonist GR127935 at 0.3 mg/kg had no effect, but 5 mg/kg significantly increased extracellular levels of 5‐HT and 5‐hydroxyindoleacetic acid to 135% of basal values. Moreover, at these doses GR127935 significantly attenuated the decrease in extracellular 5‐HT levels following local perfusion with the selective 5‐HT1D agonist CP‐135,807. The SSRI sertraline at 2 mg/kg increased 5‐HT levels to 130% of basal levels. The combination of this low dose of sertraline with either dose of GR127935 resulted in a pronounced, long‐lasting increase in 5‐HT levels to 230% of basal values. These results indicate that the effects of an SSRI on terminal 5‐HT are significantly enhanced by coadministration of a 5‐HT1D antagonist and confirm that in addition to somatodendritic 5‐HT1A autoreceptors, terminal 5‐HT1D autoreceptors mitigate the effect of SSRIs on terminal 5‐HT. As such, antagonists of the 5‐HT1D autoreceptor could be useful as rapidly acting antidepressants and may shorten the onset of antidepressant action when combined with SSRIs.

SCH23390 causes persistent antidopaminergic effects in vivo: Evidence for longterm occupation of receptors

SCH23390 has neurochemical properties characteristic of a specific D1 dopamine receptor antagonist. However, it is a potent inhibitor of dopamine-mediated behaviors which previously had been thought to be linked to D2 receptors. The metabolism of SCH23390 following parenteral administration to rats was much more rapid in the periphery than in brain, and SCH23390 had behavioral effects long after its circulating concentration had declined below detectable levels. Furthermore, the stimulation of adenylate cyclase by dopamine was attenuated in striatal homogenates taken from rats treated with SCH23390 as much as twelve hours before sacrifice. Pretreatment with cis-flupenthixol, a compound with equivalent D1 potency in vitro, failed to inhibit dopamine-stimulated adenylate cyclase activity one or four hours following injection, despite the fact that this dose produced significant behavioral effects. These data indicate that SCH23390 may act with unusual tenacity at certain sites in the central nervous system.

2-aryloxy-4-alkylaminopyridines: discovery of novel corticotropin-releasing factor 1 antagonists.

An orally active clinical candidate of corticotropin-releasing factor 1 (CRF 1) antagonist 1 showed a significant positive food effect in dog and human after oral administration. Efforts to address the food effect issue led us to explore and discover compounds in series 2 as orally active CRF 1 receptor antagonists, in which some compounds showed improved physicochemical properties while retaining desired pharmacological properties. Compound 3a (CP-376395) was selected for further development, due not only to its reduced food effects but also its greater efficacy in CNS models. Compound 3a was advanced to the clinic. The synthesis of representative potential candidates and their in vitro, ex vivo, and in vivo data are described.

Synthesis and SAR of 2-aryloxy-4-alkoxy-pyridines as potent orally active corticotropin-releasing factor 1 receptor antagonists.

A series of 2-aryloxy-4-alkoxy-pyridines ( 1) was identified as novel, selective, and orally active antagonists of the corticotropin-releasing factor 1 (CRF 1) receptor. Among these, compound 2 (CP-316311) is a potent and selective CRF 1 receptor antagonist with an IC 50 value of 6.8 nM in receptor binding and demonstrates oral efficacy in central nervous system (CNS) in vivo models. The regiochemistry of compounds in this series was determined by an X-ray structural analysis. A method to control regioselectivity via pyridine- N-oxides was developed. The synthesis of compounds in series 1 (Figure ) and [ (3)H]- 2 as well as the structure-activity relationship (SAR) are discussed. The in vitro, ex vivo, and in vivo properties of representative compounds are described herein. Compound 2 was advanced to phase II depression trials to test the hypothesis that CRF 1 antagonists could be used clinically as antidepressant drugs.

An Improved, Automated Adenylate Cyclase Assay Utilizing Preparative HPLC: Effects of Phosphodiesterase Inhibitors

Abstract: Analysis of adenylate cyclase (ACase) activity in broken cell preparations usually involves conversion of [α‐32P]ATP to [32P]cyclic AMP (cAMP) followed by purification of cAMP by liquid chromatographic methods. An automated, preparative reverse‐phase HPLC procedure was developed that purifies cAMP rapidly and decreases variability and background. It permits the separation procedure to be validated rapidly prior to use with actual samples, and is readily adaptable for assaying guanylate cyclase, phosphodiesterases (PDE), or a variety of other related nucleotide‐metabolizing enzymes. For ACase assays, 4.5% ZnSO4‐10% Ba(OH)2 is added to the incubation mixture, and following centrifugation, the supernatant is injected on an HPLC apparatus fitted with a Waters Z‐Module containing a 10‐μ C18 reverse‐phase cartridge. Using a mobile phase of 0.15 M sodium acetate‐20% methanol (pH 5.0) at a flow rate of 4 ml/min, cAMP is eluted at k′ > 1.25, whereas k′ < 0.5 for all other adenine nucleotides, permitting collection of the cAMP fraction after running the other nucleotides to waste. The method was validated by characterizing dopamine‐sensitive ACase in homogenates of striatum from Sprague‐Dawley rats. Basal activity (177 ± 16 pmol/mg protein/min), the stimulation by dopamine (186 ± 19 pmol/mg/min), the apparent Km for dopamine (5.0 ± 1.5 μM), and expected effects of varying magnesium, EGTA, and GTP were similar to available data. However, it was found that isobutylmethylxanthine (IBMX) or theophylline, usually included in the incubation mixture as PDE inhibitors, markedly inhibited the synthesis of cAMP in both the presence and absence of dopamine. A consequence of this inhibition was a marked change in the apparent Km of dopamine calculated from a Lineweaver‐Burk plot. The use of IBMX to inhibit PDEs was compared with an alternate strategy, the addition of excess exogenous cAMP. Simultaneous analysis of PDE and ACase activity was accomplished by including [3H]cAMP in the incubation and quantifying the amounts of [3H]cAMP hydrolyzed and [32P]cAMP synthesized. Without IBMX, a concentration of 1 mM exogenous cAMP was sufficient to prevent significant loss of [3H]cAMP. In the absence of exogenous cAMP, 0.5 mM IBMX did not completely prevent the breakdown of [3H]cAMP, whereas 2.5 mM IBMX did. Although there was 25% less [3H]cAMP recovered in the presence of 0.5 mM IBMX than with 2.5 mM IBMX, there was no difference in the amount of [32P]cAMP formed (either with or without dopamine). Moreover, in the presence of IBMX, there was a 20–30% lower synthesis of [32P]cAMP compared with incubations in which only 1 mM cAMP was used to prevent breakdown of [32P]cAMP. These data suggest that alkylxanthines, possibly through effects on adenosine receptors, may cause unexpected effects on estimations of dopamine‐stimulated ACase. The use of exogenous cAMP as an alternate substrate for PDEs may be one way to obviate these problems.

The Multiplicity of the D1 Dopamine Receptor

Dopaminergic neurotransmission is known to modulate a variety of behaviors, including ambulation (Ungerstedt and Arbuthnott, 1970; Pijnenburg et al., 1976), stereotyped behaviors (Creese and Iversen, 1973), self-stimulation (Phillips and Fibiger, 1973), conditioned avoidance responding (Seiden and Carlsson, 1963), stimulus control (Ho and Huang, 1975), and feeding and drinking (Ungerstedt, 1971; Fitzsimons and Setler, 1975). It is not surprising, therefore, that drugs which are believed to act primarily as dopamine receptor agonists or antagonists have important clinical utility. Our work has sought to address two questions of some neuropharmacological importance. First, what is the nature of mechanisms by which dopamine initiates many of these psychopharmacological effects, and second, is it possible to design highly specific drugs targeted only at a selected subpopulation of dopamine receptors?

Bombesin-induced locomotor hyperactivity: evaluation of the involvement of the mesolimbic dopamine system

The neuropeptide bombesin was administered centrally to conscious, unrestrained rats. Bombesin caused a dose-dependent increase in locomotor behavior, as well as licking and grooming. The effect on locomotion was most readily elicited when bombesin was infused into the nucleus accumbens, and was attenuated by pretreatment with haloperidol but not atropine or diphenhydramine. Centrally administered bombesin did not change regional levels of dopamine or its principal metabolites.

Ascorbic acid decreases [3H]dopamine binding in striatum without inhibiting dopamine-sensitive adenylate cyclase

Ascorbic acid is found in very high concentrations in cells of neural crest origin such as the central nervous system and the adrenal gland. A variety of evidence has been marshalled to support a role for ascorbate as a chemical messenger. One of the first non-biosynthetic biochemical effects ascribed to ascorate in the CNS was its ability to inhibit dopamine-stimulated adenylate cyclase (DA-ACase) in homogenates from striata of Long Evans rats (J. Neurochem.28, 663, 1977). Using an adenylate cyclase assay based on preparative HPLC, we were unable to detect any inhibition of DA-ACase by ascorbate at concentrations as high as 1 mM. Moreover, this failure to find inhibitory effects of ascorbate on DA-ACase occurred not only when striatal homogenates from Long-Evans rats were used, but also when tissue from Sprague-Dawley rats of N.C. Board of Health mice was tested. Although ascorbate may play a neuromodulatory role, it does not appear that its effects are mediated through effects on cAMP biosynthesis. Despite our inability to detect effects of ascorbate on DA-ACase, we did confirm that ascorbate significantly altered the binding of [(3)H]dopamine to striatal membranes. Thus, it is clear that the sites binding [(3)H]dopamine that are affected by ascorbate are unlikely to be the same ones coupled to aenylate cyclase.