Experimental strategies to discover and develop the next generation of psychedelics and entactogens as medicines.

Research on classical psychedelics (psilocybin, LSD and DMT) and entactogen, MDMA, has produced a renaissance in the search for more effective drugs to treat psychiatric, neurological and various peripheral disorders. Psychedelics and entactogens act though interaction with 5-HT2A and other serotonergic receptors and/or monoamine reuptake transporters. 5-HT, which serves as a neurotransmitter and hormone, is ubiquitously distributed in the brain and peripheral organs, tissues and cells where it has vasoconstrictor, pro-inflammatory and pro-nociceptive actions. Serotonergic psychedelics and entactogens have known safety and toxicity risks. For these drugs, the risks been extensively researched and empirically assessed through human experience. However, novel drug-candidates require thorough non-clinical testing not only to predict clinical efficacy, but also to address the risks they pose during clinical development and later after approval as prescription medicines. We have defined the challenges researchers will encounter when developing novel serotonergic psychedelics and entactogens. We describe screening techniques to predict clinical efficacy and address the safety/toxicity risks emerging from our knowledge of the existing drugs: 1) An early-stage, non-clinical screening cascade to pharmacologically characterise novel drug-candidates. 2) Models to detect hallucinogenic activity. 3) Models to differentiate hallucinogens from entactogens. 4) Non-clinical preclinical lead optimisation technology (PLOT) screening to select drug-candidates. 5) Modified animal models to evaluate the abuse and dependence risks of novel psychedelics in Safety Pharmacology testing. Our intention has been to design non-clinical screening strategies that will reset the balance between benefits and harms to deliver more effective and safer novel psychedelics for clinical use. This article is part of the Special Issue on 'National Institutes of Health Psilocybin Research Speaker Series'.

A review of late-stage CNS drug candidates for the treatment of obesity.

Obesity is an important causative factor in morbidity, disability and premature death. Increasing levels of obesity will impose enormous health, financial and social burdens on worldwide society unless effective interventions are implemented. For many obese individuals, diet and behavioural modification need to be supplemented by pharmacotherapy. Preclinical research has revealed a greater understanding of the complex nature of the hypothalamic regulation of food intake and has generated a wide range of new molecular targets for the development of drug candidates for obesity treatment. As shown by the clinical results that have been obtained with this next generation of therapies, some approaches, for example, fixed-dose drug combinations, have already demonstrated an ability to deliver levels of efficacy that are not achievable with the current antiobesity drug therapies. The regulatory and marketing landscape for development, registration and commercialisation of novel centrally acting drugs for treatment of obesity and related metabolic disorders has changed substantially in recent years. Now a much greater emphasis is placed on tolerability and safety, as well as efficacy. In this review we briefly describe the therapeutic approaches to tackle obesity that are in late-stage clinical development. We then discuss drugs in late-stage development for the treatment of obesity and also future directions.

Lisdexamfetamine and immediate release d-amfetamine - differences in pharmacokinetic/pharmacodynamic relationships revealed by striatal microdialysis in freely-moving rats with simultaneous determination of plasma drug concentrations and locomotor activity.

Lisdexamfetamine mesylate (Vyvanse(®)) is a novel prodrug approved for attention deficit hyperactivity disorder (ADHD). It is metabolised to d-amfetamine and l-lysine. In drug-experienced humans, lisdexamfetamine evoked lower "Drug liking" scores on Drug Rating Questionnaire (DRQ) scales than immediate-release (IR) d-amfetamine. This study investigated why lisdexamfetamine may have lower abuse potential and a better therapeutic window than d-amfetamine. We compared the pharmacokinetic/pharmacodynamic relationships of lisdexamfetamine and IR d-amfetamine in freely-moving rats by measuring simultaneously extracellular concentrations of striatal dopamine, plasma concentrations of d-amfetamine and lisdexamfetamine, and locomotor activity. At equivalent doses (1.5 mg/kg d-amfetamine base), lisdexamfetamine produced smaller, but more sustained, increases in striatal dopamine efflux than d-amfetamine and substantially less locomotor activation. Consistent with it being a prodrug, increased striatal dopamine and locomotion correlated with plasma concentration of its metabolite, d-amfetamine, but not the parent compound. Compared with IR d-amfetamine, lisdexamfetamine produced an identical AUC for plasma d-amfetamine, but a 50% lower C(max) and significantly delayed t(max). Where a hysteresis relationship did exist between plasma concentrations of d-amfetamine and striatal dopamine or locomotor activity, they were anticlockwise in direction for lisdexamfetamine and IR d-amfetamine. For extracellular striatal dopamine (neurochemical mediator) and locomotor activity (functional outcome), it was anticlockwise for lisdexamfetamine, but clockwise for IR d-amfetamine. This shows that lisdexamfetamine produced less pronounced behavioural activation as dopamine concentrations increased, but activity was maintained for longer when they declined. These findings help explain why the unusual pharmacokinetics of lisdexamfetamine evoked lower "Drug liking" scores than IR d-amfetamine and also suggest therapeutic window between efficacy and stimulant side-effects will be larger.

Uncultured blood samples can be labeled by PRINS and ready for chromosome enumeration analysis 1 H after collection.

We describe a rapid technique to determine numerical abnormalities of chromosomes that can be applied to slides prepared from fresh, uncultured blood samples. Primed in situ synthesis is a method that has previously been utilized as a rapid alternative to conventional fluorescence in situ hybridization for localizing repeated DNA sequences on metaphase chromosomes and interphase nuclei prepared from cultured lymphocytes. By applying the technique to uncultured preparations from fresh blood, aneuploidy analysis can be completed in less than 3 h from the collection of the blood sample.

Characterization of a chromosome-specific chimpanzee alpha satellite subset: evolutionary relationship to subsets on human chromosomes.

Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- and 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p.

Rapid bright-field detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections.

We describe a new application of a bright-field microscopic procedure for rapid enzyme cytochemical detection of repeated DNA sequences in metaphase preparations and frozen tissue sections. Various chromosome-specific oligonucleotide primers were used in up to three sequential primed in situ (PRINS) labeling reactions together with Taq DNA polymerase and biotin, digoxigenin and/or fluorescein isothiocyanate (FITC)-modified nucleotides. DNA target sequences were localized simultaneously by the precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) and horseradish peroxidase-teramethylbenzidine (PO-TMB, green color) reaction in hematoxylin counterstained metaphases and interphase nuclei using a standard bright-field microscope. In addition, a protocol is reported for the application of PRINS to frozen tissue sections from normal colon and bladder epithelium. Methanol/acetic acid fixation in combination with a pepsin digestion before performing the PRINS reaction proved to be critical steps in the total procedure that permits access of the PRINS reactants, while preserving the morphology of the nuclei in the tissue. Quantification of PRINS signals showed the majority of epithelial cells with the expected two chromosome copies. The described procedures can be considered valuable tools for application in molecular cytogenetics, cell biology and pathology.

Receptor binding and functional evidence suggest that postsynaptic alpha 2-adrenoceptors in rat brain are of the alpha 2D subtype.

This study has determined the subtype(s) of postsynaptic alpha 2-adrenoceptors in rat brain. This question has been addressed by using two separate approaches, i.e. ligand displacement of [3H]2-(2-methoxy)-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX 821002) from membranes prepared from rat cortex after noradrenergic denervation and, secondly, by antagonism of clonidine-induced mydriasis. After rats had been lesioned using N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 100 mg/kg i.p., 30 min after zimeldine 10 mg/kg i.p.), noradrenaline was undetectable in the cortex 3 days later. Displacement of [3H]RX 821002 with a range of agonists and antagonists which distinguish between the known alpha 2-adrenoceptor subtypes (alpha 2A-2D) yielded pKi values which correlated very well with reported values for the alpha 2D-adrenoceptor (r = 0.929; P < 0.001), but not the alpha 2A (r = 0.450; P = 0.192), alpha 2B (r = 0.280, P = 0.434) or alpha 2C (r = 0.283; P = 0.460) subtypes. Similarly, the potencies of various alpha 2-adrenoceptor antagonists to inhibit clonidine (0.03 mg/kg i.p.)-induced mydriasis in conscious rats correlated strongly with their pKi values for alpha 2D-adrenoceptors (r = 0.899; P = 0.015) but not alpha 2A-(r = 0.369; P = 0.472), alpha 2B-(r = -0.224; P = 0.670) or alpha 2C-adrenoceptors (r = 0.253; P = 0.584). These data are, therefore, consistent and argue strongly that postsynaptic alpha 2-adrenoceptors in the rat cortex and Edinger-Westphal nucleus are of the alpha 2D subtype.

Catch-linker + PCR labeling: a simple method to generate fluorescence in situ hybridization probes from yeast artificial chromosomes.

A simple and efficient method to generate hapten-labeled DNA fragments from a trace amount of YAC DNA isolated by PFGE is described. After agarase digestion of the gel slice containing the resolved YAC recombinant, the purified DNA is digested with Sau3Al and a compatible CL oligonucleotide duplex ligated on. A probe is generated by PCR amplification using a primer complementary to the CL with a single biotin moiety incorporated at the 5' end. When used as a FISH probe, this material yields mapping results superior to Alu-PCR or whole YAC labeling methods and allows sensitive detection of chimerism.

Multi-PRINS: multiple sequential oligonucleotide primed in situ DNA synthesis reactions label specific chromosomes and produce bands.

A fast method for identifying several chromosomes with chromosome-specific oligonucleotide primers directing an in situ labeling reaction is described. Up to three reactions distinguished by different fluorochromes (fluorescein isothiocyanate, rhodamine/Texas red, p-amino-methyl-cyclohexane carboxylic acid) can currently be performed. Prospects for increasing this up to seven colors, and for the future of the process in prenatal diagnosis are discussed.

Instant PRINS: a rapid method for chromosome identification by detecting repeated sequences in situ.

We describe a method for labeling specific chromosomes in situ by oligonucleotide-primed synthesis and incorporation of fluorochrome-labeled dUTP, together with an accelerated protocol for the reaction. The combination of the two developments produces extremely rapid results, in some cases taking less than 5 min for the complete process. The significance of this development for clinical diagnosis is discussed.

Rapid chromosome identification by oligonucleotide-primed in situ DNA synthesis (PRINS).

We describe a method for rapid identification of chromosomes at metaphase, and quantification of chromosomes in interphase, by annealing oligonucleotide primers, derived from chromosome-specific subsets of repeated DNA families, to the DNA of cytological preparations, and enzymatic extension with the incorporation of labelled nucleotides. The method is equally applicable to normal cells or those from somatic cell hybrids. Where the labelled product is too small or of too low a copy number to be readily seen after the single extension reaction, we have developed a method for cyclic amplification of the labelled DNA, enabling clear visualization of the signal.