Drug–drug interaction (DDI) assessments are well defined in health authority guidelines for small molecule drugs, and US Food and Drug Administration (FDA) draft guidance is now available for therapeutic proteins. However, there are currently no regulatory guidelines outlining DDI assessments for therapeutic peptides, which poses significant uncertainty and challenges during drug development for this heterogenous class of molecules. A cross‐industry peptide DDI working group consisting of experts from 10 leading companies was formed under the sponsorship of the European Federation of Pharmaceutical Industries and Associations. We aimed to capture the range of DDI studies undertaken for peptide drugs by (i) anonymously surveying relevant companies involved in peptide drug development to better understand DDI study type/timing currently performed and (ii) compiling a database containing in vitro / clinical DDI data from submission packages for recently approved peptide drugs. Our analyses highlight significant gaps and uncertainty in the field. For example, the reported timing of in vitro peptide DDI studies, if performed, vary substantially across responding companies from early research to phase III. Nearly all in vitro cytochrome P450 / transporter inhibition studies reported in the survey were negative. For the few positive hits reported, no clinical follow‐up studies were performed, questioning the clinical relevance of these findings. Furthermore, available submission packages reveal DDI likelihood is low for peptides >2 kDa, making it reasonable to adopt a risk‐based approach during drug development for larger peptides. By benchmarking the landscape of peptide DDI activities across the industry, we set the stage for future discussions with health authorities on harmonizing peptide DDI approaches.

Pharmacokinetic drug-drug interactions (DDIs) are investigated to ensure safety for patients receiving concomitant medications. Here, we present a strategy to characterise the DDI potential of remibrutinib, as an inhibitor of drug-metabolising enzymes and drug transporters, and as an inducer. Initial in vitro studies were performed, followed by a biomarker-based assessment of induction in a first in human study, concluded by a clinical study to verify initial results. Remibrutinib is a covalent inhibitor of Bruton's Tyrosine kinase (BTKi) carrying a reactive acrylamide moiety (warhead), thus the potential contribution of covalent binding (off-target) to observed interactions was investigated as this could lead to prolonged and more potent drug interactions. DDI assessment was focused on the putative inhibition of key metabolic enzymes (Cytochrome P450, CYP), drug transporters and a potential effect on oral contraceptives (OC) by induction of enzymes that are involved in their clearance (CYP3A4). The impact of covalent binding was assessed by synthesising an identical reference molecule but with an inactivated warhead. An interaction potential of limited clinical relevance was revealed for remibrutinib for CYP enzymes and drug transporters. The reactive warhead of remibrutinib had no impact on CYP enzyme and transporter inhibition, including time-dependent inhibition of CYP3A4, but may increase the induction potential of remibrutinib. Observed inhibition of metabolic enzymes indicated that remibrutinib is a weak inhibitor of CYP3A4 and CYP2C9 and is not a clinically relevant inhibitor of uptake and efflux transporters, except for intestinal P-glycoprotein and breast cancer resistance protein inhibition. OC may be safely administered and are effective when given with pharmacologically relevant doses of remibrutinib.

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is ∼10-fold below the EC₅₀values for most purinergic receptors expressed in the heart (1.5–20 μM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP.

The risk of clinically relevant drug–drug interactions (DDIs) for therapeutic peptides remains unclear, mandating a comprehensive analysis for this modality. In our prior study, we analyzed DDIs for 31 peptide drugs approved between January 2008 and August 2021. Here, we analyze DDI data for an additional nine peptide drugs (trofinetide, nirmatrelvir, danicopan, odevixibat, rezafungin, motixafortide, zilucoplan, vosoritide, and tirzepatide) approved from September 2021 to September 2024, focusing on in vitro and clinical DDI data for metabolism‐ and transporter‐based interactions. All nine peptides investigated CYP inhibition in human liver microsomes (HLMs), with low risk identified for larger peptides (> 2 kDa). Likewise, all nine peptides assessed CYP induction in human hepatocytes, with one peptide showing a risk in vitro (danicopan). Phenotyping investigations varied from standard studies (e.g., HLMs with selective CYP inhibitors) to submission packages without classical phenotyping studies. All nine peptides included information related to in vitro transporter properties, but the level of detail varied between submitted packages. Clinical studies investigating metabolism‐ or transporter‐mediated DDIs were performed for four peptides (all < 2 kDa). Area under the curve changes attributed to the peptide drug were < 2.3 fold. Our expanded dataset now includes 40 therapeutic peptides approved since 2008, providing a unique resource for drug developers. The findings reinforce our previous conclusions regarding the low likelihood of DDIs for larger peptides and a higher risk for smaller peptides with xenobiotic structural properties. This collective data will be invaluable in developing clear and meaningful DDI guidelines for therapeutic peptides.