Conducting dermatology clinical trials during the COVID-19 pandemic.

The clinical trials industry faces a number of challenges during the evolving coronavirus disease 2019 (COVID-19) pandemic, for example, site closures, mandatory self-isolation, travel restrictions, interruptions to delivery of investigational product, or staff or participants becoming infected with severe acute respiratory syndrome coronavirus 2. These challenges can pose difficulties in adhering to visit schedules, performing laboratory testing, conducting protocol-specified procedures, and administration of investigational product. As a result, clinical trial sites, contract research organizations, and sponsors have had to act swiftly to ensure that trial patients are safe and systems are in place for continuing trials. Protocols should also be amended to reflect changes and approved by an ethics board. The authors provide practical considerations and recommendations for dermatology clinical trial operations during the COVID-19 pandemic.

Convergent Balancing Selection on the Mu-Opioid Receptor in Primates.

The mu opioid receptor is involved in many natural processes including stress response, pleasure, and pain. Mutations in the gene also have been associated with opiate and alcohol addictions as well as with responsivity to medication targeting these disorders. Two common and mutually exclusive polymorphisms have been identified in humans, A118G (N40D), found commonly in non-African populations, and C17T (V6A), found almost exclusively in African populations. Although A118G has been studied extensively for associations and in functional assays, C17T is much less well understood. In addition to a parallel polymorphism previously identified in rhesus macaques (Macaca mulatta), C77G (P26R), resequencing in additional non-human primate species identifies further common variation: C140T (P47L) in cynomolgus macaques (Macaca fascicularis), G55C (D19H) in vervet monkeys (Chlorocebus aethiops sabeus), A111T (L37F) in marmosets (Callithrix jacchus), and C55T (P19S) in squirrel monkeys (Saimiri boliviensis peruviensis). Functional effects on downstream signaling are observed for each of these variants following treatment with the endogenous agonist ß-endorphin and the exogenous agonists morphine, DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin), and fentanyl. In addition to demonstrating the importance of functional equivalency in reference to population variation for minority health, this also shows how common evolutionary pressures have produced similar phenotypes across species, suggesting a shared response to environmental needs and perhaps elucidating the mechanism by which these organism-environment interactions are mediated physiologically and molecularly. These studies set the stage for future investigations of shared functional polymorphisms across species as a new genetic tool for translational research.

Trace amine associated receptor 1 signaling in activated lymphocytes.

Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.

Normal thermoregulatory responses to 3-iodothyronamine, trace amines and amphetamine-like psychostimulants in trace amine associated receptor 1 knockout mice.

3-Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine-associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co-transfected cells, and wild-type (WT) and TAAR1 knock-out (KO) mice. The IC(50) of T1AM competition for binding of the DAT-specific radio-ligand [(3)H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72-0.81 microM). T1AM inhibition of 10 nM [(3)H]dopamine uptake (IC(50): WT, 1.4 + or - 0.5 microM; KO, 1.2 + or - 0.4 microM) or 50 nM [(3)H]serotonin uptake (IC(50): WT, 4.5 + or - 0.6 microM; KO, 4.7 + or - 1.1 microM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE-luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT -6.0 + or - 0.4, KO -5.6 + or - 1.0; and 25 mg/kg at 30 min: WT -2.7 + or - 0.4, KO -3.0 + or - 0.2). Other TAAR1 agonists including beta-phenylethylamine (beta-PEA), MDMA (3,4-methylenedioxymethamphetamine) and methamphetamine also induced significant, time-dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co-expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [(3)H]monoamine uptake ex vivo, and TAAR1 agonist-induced thermoregulatory responses are TAAR1-independent. Accordingly, TAAR1-directed compounds will likely not affect thermoregulation nor are they likely to be cryogens.

Modafinil occupies dopamine and norepinephrine transporters in vivo and modulates the transporters and trace amine activity in vitro.

2-[(Diphenylmethyl) sulfinyl]acetamide (modafinil), prescribed principally to treat narcolepsy, is undergoing assessment for other neuropsychiatric disorders and medical conditions. The neurochemical substrates of modafinil are unresolved. We postulated that modafinil enhances wakefulness by modulating dopamine (DAT), norepinephrine (NET), or serotonin (SERT) transporter activities. In vivo, we determined DAT and NET occupancy by modafinil by positron emission tomography imaging; in vitro, we determined modafinil activity at the DAT, NET, SERT, and rhesus monkey trace amine receptor 1 (TA1). In rhesus monkey, modafinil occupancy of striatal DAT was detected by [(11)C]2beta-carbomethoxy-3beta-4-(fluorophenyl)tropane and of thalamic NET by [(11)C](S,S)-2-(alpha-(2-methoxyphenoxy)-benzyl)morpholine. In vitro, modafinil effects in DAT-human embryonic kidney (HEK), NET-HEK, and SERT-HEK cells were investigated alone or combined with the TA1 receptor. Modafinil (i.v.) occupied striatal DAT sites (5 mg/kg: 35 +/- 12%, n = 4; 8 mg/kg: 54 +/- 3%, n = 3). In thalamus, modafinil occupied NET sites (5 mg/kg: 16 +/- 7.8%, n = 6; 8 mg/kg: 44 +/- 12%; n = 2). In vitro, modafinil inhibited [(3)H]dopamine (IC(50) = 6.4 microM), [(3)H]norepinephrine (IC(50) = 35.6 microM), and [(3)H]serotonin (IC(50) > 500 microM) transport via the human DAT, NET, and SERT. Modafinil did not activate the TA1 receptor in TA1-HEK cells, but it augmented a monoamine transporter-dependent enhancement of phenethylamine activation of TA1 in TA1-DAT and TA1-NET cells, but not in TA1-SERT cells. The present data provide compelling evidence that modafinil occupies the DAT and NET in living brain of rhesus monkeys and raise the possibility that modafinil affects wakefulness by interacting with catecholamine transporters in brain.

Variants of the primate vesicular monoamine transporter-2.

The vesicular monoamine transporter-2 (VMAT2) sequesters monoamine neurotransmitters into vesicles and prevents neurotoxicity. Human or monkey striatum generated three VMAT2 immunoreactive proteins of approximately 75 kDa, approximately 52-55 kDa, and approximately 45 kDa. The approximately 55-kDa band is considered the unglycosylated native protein. Deglycosylation of the VMAT2 from striatum or human VMAT2 expressed in HEK293 cells yielded a approximately 45-kDa, but not a 55-kDa immunoreactive band. We investigated this apparent mismatch between observed molecular size and predicted size.

Primate trace amine receptor 1 modulation by the dopamine transporter.

Recently identified trace amine receptors are potential direct targets for drugs of abuse, including amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). We cloned full-length rhesus monkey trace amine receptor 1 (rhTA(1)) that was 96% homologous to human TA(1). The trace amines tyramine and beta-phenylethylamine (PEA) and the monoamine transporter substrates (+/-)-amphetamine and (+/-)-MDMA stimulated cAMP accumulation in rhTA(1)-expressing cell lines, as measured by a cAMP response element-luciferase assay. Cocaine did not stimulate cAMP accumulation in rhTA(1) cells, but it blocked [(3)H]PEA transport mediated by the dopamine transporter. Cotransfection with the human dopamine transporter enhanced PEA-, amphetamine-, and MDMA-mediated rhTA(1) receptor activation, but it diminished tyramine activation of rhTA(1). Because TA(1) (EGFP-rhTA(1) chimera) was largely intracellular, conceivably the dopamine transporter can facilitate access of specific agonists to intracellular TA(1). rhTA(1) mRNA expression was detected in rhesus monkey substantia nigra, implying that TA(1) may be colocalized with the dopamine transporter in dopamine neurons. In summary, primate TA(1) receptors are direct targets of trace amines, amphetamine, and MDMA. These receptors could also be indirect targets of amphetamine, MDMA, and cocaine through modification of monoamine transporter function. Conceivably, rhTA(1) receptors may be located on pre- or postsynaptic membranes. Interference with the carrier function of monoamine transporters with a consequent rise of extracellular levels of trace amines could activate these receptors. The cloning of a highly homologous TA(1) from rhesus monkey and demonstration that rhTA(1) receptors are activated by drugs of abuse, indicate that nonhuman primates may serve to model physiological and pharmacological TA(1)-mediated responses in humans.