A given G protein-coupled receptor can signal through a range of downstream transducers depending on the stimulating ligand, enabling biased signalling towards different biological outcomes. Lefkowitz and colleagues describe the latest advances in the field, including efforts to harness biased signalling for improved therapeutic outcomes. G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and some of the most common drug targets. It is now well established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, GPCR kinases and β-arrestins. While these signalling pathways can be activated or blocked by 'balanced' agonists or antagonists, they can also be selectively activated in a 'biased' response. Biased responses can be induced by biased ligands, biased receptors or system bias, any of which can result in preferential signalling through G proteins or β-arrestins. At many GPCRs, signalling events mediated by G proteins and β-arrestins have been shown to have distinct biochemical and physiological actions from one another, and an accurate evaluation of biased signalling from pharmacology through physiology is crucial for preclinical drug development. Recent structural studies have provided snapshots of GPCR–transducer complexes, which should aid in the structure-based design of novel biased therapies. Our understanding of GPCRs has evolved from that of two-state, on-and-off switches to that of multistate allosteric microprocessors, in which biased ligands transmit distinct structural information that is processed into distinct biological outputs. The development of biased ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.

/pdf/biased-signalling-from-simple-switches-to-allosteric-1plq6dv06q.pdf

Biased signalling: from simple switches to allosteric microprocessors

Role of the development scientist in compound lead selection and optimization

Sex differences in neural mechanisms mediating reward and addiction.

https://www.cell.com/neuron/pdf/S0896-6273(14)00208-6.pdf

Dynorphin Acts as a Neuromodulator to Inhibit Itch in the Dorsal Horn of the Spinal Cord

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.

https://dmd.aspetjournals.org/content/dmd/early/2009/04/09/dmd.109.026716.full.pdf

The Conduct of in Vitro Studies to Address Time-Dependent Inhibition of Drug-Metabolizing Enzymes: A Perspective of the Pharmaceutical Research and Manufacturers of America

Species and tissue differences in the activity of three major classes of esterases, carboxylesterase (CE), butyrylcholinesterase (BChE) and paraoxonase (PON), were studied. Substantial species differences in activity of these esterases were observed between the mouse, rat, dog monkey and human. Such species differences must be considered when using these preclinical species to optimize the pharmacokinetic properties of ester compounds intended for human use.

Esterase Activities in the Blood, Liver and Intestine of Several Preclinical Species and Humans

The relationship between time-dependent inactivation (TDI) and IC50 is examined using a consolidated method for evaluating CYP450 inhibition during drug discovery. An IC50 fold-shift of >1.5 indicated significant TDI potency. Further, the "shifted IC50" could be used to estimate, the K(I) and TDI potency ratio k(inact)/K(I) to within 2-fold in most cases.

An examination of IC50 and IC50-shift experiments in assessing time-dependent inhibition of CYP3A4, CYP2D6 and CYP2C9 in human liver microsomes.

Nor-BNI, GNTI and JDTic induce κ opioid antagonism that is delayed by hours and can persist for months. Other effects are transient. It has been proposed that these drugs may be slowly absorbed or distributed, and may dissolve in cell membranes, thus slowing elimination and prolonging their effects. Recent evidence suggests, instead, that they induce prolonged desensitization of the κ opioid receptor. To evaluate these hypotheses, we measured relevant physicochemical properties of nor-BNI, GNTI and JDTic, and the timecourse of brain and plasma concentrations in mice after intraperitoneal administration (using LC-MS-MS). In each case, plasma levels were maximal within 30 min and declined by >80% within four hours, correlating well with previously reported transient effects. A strong negative correlation was observed between plasma levels and the delayed, prolonged timecourse of κ antagonism. Brain levels of nor-BNI and JDTic peaked within 30 min, but while nor-BNI was largely eliminated within hours, JDTic declined gradually over a week. Brain uptake of GNTI was too low to measure accurately, and higher doses proved lethal. None of the drugs were highly lipophilic, showing high water solubility (> 45 mM) and low distribution into octanol (log D7.4 7% unbound). JDTic showed P-gp-mediated efflux; nor- BNI and GNTI did not, but their low unbound brain uptake suggests efflux by another mechanism. The negative plasma concentration-effect relationship we observed is difficult to reconcile with simple competitive antagonism, but is consistent with desensitization. The very slow elimination of JDTic from brain is surprising given that it undergoes active efflux, has modest affinity for homogenate, and has a shorter duration of action than nor-BNI under these conditions. We propose that this persistence may result from entrapment in cellular compartments such as lysosomes.

/pdf/long-acting-k-opioid-antagonists-nor-bni-gnti-and-jdtic-2aaauj9ayj.pdf

Long-acting κ opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity

Sex differences in sensitivity to the depressive-like effects of the kappa opioid receptor agonist U-50488 in rats.

Prediction of human volume of distribution at steady state (V(ss)) before first administration of a new drug candidate to humans has become an important part of the drug development process. This study examines the assumptions behind interspecies scaling techniques used to predict human V(ss) from preclinical data, namely the equivalency of V(ss,u) and/or f(ut) across species. In addition, several interspecies scaling techniques are evaluated side by side using a set of 67 reference compounds where observed V(ss) from rats, dogs, monkeys, and humans were compiled from the literature and where plasma protein binding was determined across species using an ultracentrifugation technique. Species similarity in V(ss,u) or f(ut) does not appear to be the norm among rats, dogs, monkeys, or humans. Despite this, interspecies scaling from rats, dogs, and monkeys is useful and can provide reasonably accurate predictions of human V(ss), although some interspecies scaling approaches were better than others. For example, the performance of the common V(ss,u) or f(ut) equivalency approaches using average V(ss,u) or f(ut) across three preclinical species was superior to allometric scaling techniques. In addition, considering data from several preclinical species, using the equivalency approach, was superior to scaling from any single species. Although the mechanistic tissue composition equations available in the Simcyp population-based pharmacokinetic simulator did not necessarily provide the most accurate predictions, and the equations used likely need refinement, they still provide the best opportunity for a mechanistic understanding and prediction of human V(ss).

https://dmd.aspetjournals.org/content/dmd/early/2011/08/18/dmd.111.040766.full.pdf

Zhiyang Zhao

Papers

Esterase Activities in the Blood, Liver and Intestine of Several Preclinical Species and Humans

An examination of IC50 and IC50-shift experiments in assessing time-dependent inhibition of CYP3A4, CYP2D6 and CYP2C9 in human liver microsomes.

Long-acting κ opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity

Sex differences in sensitivity to the depressive-like effects of the kappa opioid receptor agonist U-50488 in rats.

Species differences in distribution and prediction of human V(ss) from preclinical data.