Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

(R)-11-Hydroxyaporphine (2) and (R)-11-hydroxy-10-methylaporphine (3) were synthesized from natural morphine by using new, short, and efficient synthetic sequences. The dopaminergic and serotonergic effects of 2 and 3 were evaluated by use of in vitro and in vivo test systems. The results indicate that 3 is a potent, selective, and efficacious 5-HT1A receptor agonist. In contrast, 2 is a partial 5-HT1A receptor agonist of low potency which has affinity also for central D1 and D2A receptors. The differences in pharmacological profiles were rationalized by modeling of ligand-receptor interactions using homology-based receptor models of the 5-HT1A and D2A receptor binding site. The selective and pronounced serotonergic effects of 3 appear to be due to the C10-methyl group, which is accommodated by a lipophilic pocket in the 5-HT1A receptor. In contrast, the C10-methyl group of 3 is not accommodated by the binding site model of the D2A receptor.

Hence, we evaluated in vivo D 2 occupancy of these agents in rats and correlated it to observed effects in a series of behavioral, neurochemical, and endocrine models relevant to the dopamine system and antipsychotic effect. Both (Ϫ)-OSU6162 and ACR16 showed robust dose-dependent striatal D 2 occupancy with ED 50 values of 5.27 and 18.99 mg/kg s.c., respectively, and functional assays showed no partial agonism. Over an occupancy range of 37 to 87% (3-60 mg/kg) for (Ϫ)-OSU6162 and 35 to 74% (10 -60 mg/kg) for ACR16, we observed both inhibitory (amphetamine-induced locomotor activity) and stimulatory effects (in habituated rats). Haloperidol, over a similar occupancy range (33-78%), potently inhibited psychostimulant activity and induced catalepsy, but it failed to activate habituated animals. In the conditioned avoidance response assay, ACR16 was clearly more efficacious than (Ϫ)-OSU6162. In addition, both these compounds demonstrated significant preferential Fos induction in the nucleus accumbens compared with the dorsolateral striatum, a strong predictor of atypical antipsychotic efficacy. The results suggest that dopamine stabilizers exhibit locomotor stabilizing as well as antipsychoticlike effects, with low motor side effect liability, in a dose range that corresponds to high D 2 in vivo occupancy.

The two enantiomers of the putative centrally acting dopamine (DA) autoreceptor agonist 3-(3-hydroxyphenyl)-N-n-propylpiperidine, 3-PPP (Hjorth et al. 1981), were pharmacologically evaluated. An extensive series of biochemical and behavioural experiments unexpectedly revealed that both (+)- and (-)-3-PPP showed clear, but differential, effects on the DA receptors. Thus, (+)-3-PPP is a DA agonist with autoreceptor as well as postsynaptic receptor stimulatory properties. In contrast, although (-)-3-PPP similarly activates DA autoreceptors it acts concomitantly as an antagonist at postsynaptic DA receptors. Moreover, both behavioural and biochemical data on motor activity and DA synthesis and turnover suggest a preferential limbic action for the (-)-enantiomer. These results are discussed in terms of the dual antidopaminergic action of (-)-3-PPP coupled with anatomical differences in the feedback organisation in central (viz, limbic vs striatal) DA systems. It is suggested that compounds like (-)-3-PPP may be of potential clinical utility in the treatment of psychotic disorders, whilst lacking the seriously incapacitating motor dysfunctions produced by current neuroleptic therapy.

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K. Rasmussen, K.G. Welinder and J. Hejgaard (1991) Plant Mol Biol 16: 317-327). A cDNA library was prepared from mRNA isolated from seeds 15 days after flowering. Full-length clones were obtained and showed 3' end length variants, a G+C content of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1 is the first characterized plant peroxidase which is not blocked by pyroglutamate. BP 1 polymorphism was observed. BP 1 is less than 50% identical to other plant peroxidases which, taken together with its developmentally dependent expression in the endosperm 15-20 days after flowering, suggests a unique biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical plant modified-type structure, Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc. The BP 1 gene was RFLP-mapped on barley chromosome 3, and we propose Prx5 as the name for this new peroxidase locus.

Dopaminergic stabilizers can be defined as drugs that stimulate or inhibit dopaminergic signalling depending on the dopaminergic tone. (-)-OSU6162 and ACR16 appear to possess such a profile. They have been proposed to act as partial dopamine receptor agonists or as antagonists with preferential action on dopaminergic autoreceptors. Previous studies have shown either stimulation or inhibition of behaviour in response to (-)-OSU6162 and ACR16, which has been suggested to reflect their dual effects on dopaminergic signalling. The aims of the present work are to (1) examine the relation between behavioural response to these drugs and activity baseline, and (2) test the suggested mechanisms of action by means of close comparisons with the known partial D2-receptor agonists (-)-3-PPP and aripiprazole, and the D2 autoreceptor preferring antagonist amisulpride with respect to effects on behaviour. From the results of these experiments it can be concluded that: (1) The direction of the response to (-)-OSU6162 and ACR16 is dependent on activity baseline, which in turn, under physiological conditions, is determined primarily by test arena size of and degree of habituation to the environment. (2) The effects of (-)-OSU6162 and ACR16 cannot be explained on the basis of either partial dopamine receptor agonism or preferential dopamine autoreceptor antagonism. Nevertheless, the current data suggest at least two different D2-receptor-associated targets which mediate opposite effects on activity. This result fits in with a mechanism proposed from a recent in vitro study, according to which (-)-OSU6162 has a dual action on dopamine D2 receptors, (a) an allosteric effect causing an enhanced response to dopamine, and (b) the previously proposed orthosteric effect antagonizing the action of dopamine.

Background: The presently available pharmaceutical aids in smoking cessation possess a rather limited effectiveness. Therefore, we have synthesized a series of immunoconjugates that stimulate the induction of antibodies which may bind nicotine in the blood, thereby preventing it from passing the blood-brain barrier. Thus, the reinforcing action of nicotine in the brain, which is the driving force in tobacco smoking, should be abolished. Objective: The present study was undertaken to test this notion in a long-term relapse model in rats, measuring the reinstatement of nicotine-seeking behavior, following active immunization with IP18-KLH, one of our immunoconjugates. Methods: Male Wistar rats were immunized with a nicotine-KLH conjugate (nicotine immunogen) and Freund’s adjuvant after having been trained to meet the criteria of stable nicotine self-administration on a fixed ratio (FR3) schedule. The rats were subsequently extinguished from nicotine self-administration behavior and finally, as extinction was completed, they were exposed to small, priming doses of nicotine, which previously have been shown to reinstate the nicotine-seeking behavior. The antibody titers were measured by ELISA. Results: It was found that rats with high titers (>1:10,000) of antibodies against nicotine, in contrast to those with low/no nicotine selective antibodies, do not reinstate nicotine self-administration behavior when they are exposed to nicotine. Conclusions: Our findings indicate that active immunization against nicotine may effectively abolish the reinforcing action of nicotine in brain, an effect which is critical for relapse in nicotine dependence. These data suggest the potential utility of active immunization in smoking cessation programs.

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