Real-World Evidence to Support EU Regulatory Decision Making-Results From a Pilot of Regulatory Use Cases.

Studies using real-world data (RWD) can complement evidence from clinical trials and fill evidence gaps during different stages of a medicine's lifecycle. This review presents the experience resulting from the European Medicines Agency (EMA) pilot to generate RWE to support evaluations by EU regulators and down-stream decision makers from September 2021 to February 2023. A total of 61 research topics were identified for RWE generation during this period, covering a wide range of research questions, primarily generating evidence on medicines safety (22, 36%), followed by questions on the design and feasibility of clinical trials (11, 18%), drug utilization (10, 16%), clinical management (10, 16%), and disease epidemiology. A significant number of questions were related to the pediatric population and/or rare diseases. A total of 27 regulatory-led RWD studies have been conducted. Most studies were descriptive and aimed at estimating incidence and prevalence rates of clinical outcomes including adverse events or to evaluate medicines utilization. The review highlights key learnings to guide further efforts to enable the use and establish the value of real-world evidence (RWE) for regulatory decisions. For instance, there is a need to access additional fit-for-purpose and representative data, and to explore further means to provide timely evidence that meets regulatory timelines. The need for early interactions and close collaboration with study requesters, e.g., from the Agency's scientific Committees, to better understand the research question is equally important. Finally, the review provides our perspective on the way forward to maximize the potential of regulatory-led RWE generation.

European Medicines Agency review of post-authorisation studies with implications for the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance.

PURPOSE: A review of post-authorisation studies requested in 2007 by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) was undertaken to determine compliance and the need for research capacity in the European Union (EU), with implications for the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP). METHODS: Information from the EMA's electronic records management systems was analysed. RESULTS: As of 31 January 2010, of the 60 relevant studies identified, 52 had been progressed to being able to start data collection (including six merged into a single study). Of the remaining eight studies, the agreement of the CHMP that a proposed study was no longer required is documented for six, with a final decision having not been reached for another study and an acknowledgement by the CHMP that a further study would not be progressed. Of the 47 studies that could therefore have commenced data collection or extraction, 38 were ongoing, four were complete and five had not yet started. Most studies were conducted within the EU. CONCLUSION: Compliance with the request of the CHMP to conduct studies is very good. The review identified the need for careful consideration of the necessity of studies and of timely dialogue on protocols in advance of a CHMP opinion. The need for expertise and capacity within the EU for the conduct of post-authorisation studies is confirmed. ENCePP as a transparency and excellence network and as an initiative to build research capacity will enhance post-authorisation medicines research.

Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity.

Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M(2)-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M(2) selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM(2) cells disclosed pathway-specific signaling. Selective receptor activation (M(2)>M(1)>M(3)) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation.

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.

Allosteric interactions with muscarinic acetylcholine receptors: complex role of the conserved tryptophan M2422Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity.

In general, the M2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M5 subtype the lowest. The M2/M5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M2 177Tyr and M2 423Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M2 422Trp and the adjacent M2 423Thr in the binding of alkane-bisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M2 or M5 receptors or mutants thereof. It was found that M2 422Trp and M2 423Thr independently influenced allosteric agent binding. The presence of M2 423Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M5 counterpart 478His). Replacement of M2 422Trp and of the corresponding M5 477Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMS-bound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M2 receptor model revealed that the aromatic rings of M2 177Tyr and M2 422Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M2 422Trp and the spatially adjacent M2 177Tyr, as well as M2 423Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M2/M5 subtype selectivity and baseline affinity of allosteric agents.

Critical amino acid residues of the common allosteric site on the M2 muscarinic acetylcholine receptor: more similarities than differences between the structurally divergent agents gallamine and bis(ammonio)alkane-type hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide.

The structurally divergent agents gallamine and hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) are known to interact competitively at a common allosteric site on muscarinic receptors. Previous studies reported that the M2 selectivity of gallamine depended largely on the EDGE (172-175) sequence in the second outer loop (o2) and on 419Asn near the junction of o3 and the seventh transmembrane domain (TM7), whereas the selectivity of W84 depended on nearby residues 177Tyr and 423Thr. However, it has so far proven difficult to confer the high sensitivity for allosteric modulation of the M2 subtype onto the weakly sensitive M5 subtype by substituting these key residues. We now have found that M2 423Thr, not 419Asn, is the dominant residue in the o3/TM7 region for gallamine's high potency, although 419Asn can substitute for 423Thr in some contexts; in contrast, the presence of 419Asn reduces the potency of W84 in every context we have studied. In addition, the orientation of 177Tyr is crucial to high sensitivity toward W84, and it seems that the proline residue at position 179 in M5 (corresponding to M2 172Glu) may interfere with that orientation. Consistent with these observations, a mutant M5 receptor with these three key mutations, M5P179E, Q184Y, and H478T, showed dramatically increased sensitivity for W84 (>100-fold), compared with the wild-type M5 receptor. This same mutant receptor approached M2 sensitivity toward gallamine. Thus, gallamine and W84 derive high potency from the same receptor domains (epitopes in o2 and near the junction between o3 and TM7), even though these allosteric agents have quite different structures.