Drug development for children presents unique challenges and is highly regulated. Novel approaches, such as the use of extrapolation to address for example the need to avoid unethical studies, whilst supporting robust evidence generation have been developed in support of benefit/risk considerations by regulatory authorities. This is only one step in the decision-making process towards access, which in Europe also includes health technology assessment (HTA) bodies. Discussions related to evidentiary requirements in small populations through the use of evidence transfer has been identified as a priority action by EMA/ EUnetHTA 21. We describe the outcome of this work; reflect on the discussions taken place how to leverage prior knowledge through identifying and addressing uncertainties during life cycle management to support regulatory and HTA decision making. Through examples, we discussed the spectrum of use to support evidence generation, and developed regulatory and HTA reflections on general design considerations important for robust evidence generation; reflective of the joint ambition. Early interactions with all respective stakeholders, particularly between regulators and HTA bodies are key to optimise data generation and utility in children. In Europe, the HTA regulation will offer opportunities for collaborations, which are important for all development efforts. We collaboratively explored the unique specific challenges relating to paediatric drug development, ethically and in its ability to leverage prior knowledge, as exemplified using extrapolation. Learnings from these offers opportunities to further develop methodology how to leverage uncertainties across a product’s life cycle for small populations generally.

Background and purpose Diabetic nephropathy (DN) is a leading cause of chronic kidney diseases (CKD) characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signaling in diabetic kidney fibrosis to reveal potential therapeutic target. Experimental Approach Db/db mice were used to study DN. RNA sequence analyses, western blot and immunostaining were conducted to profile gene expression in kidneys. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signaling microdomains in MCs. Rolipram, a PDE4 inhibitor, was orally administrated in db/db mice to test its impact on the kidney. Key Results Db/db mice exhibited impaired kidney function with elevated adrenergic and fibrotic gene expression, including ADRB1, PDEs, ACTA2, and TGF-β. MCs with dysregulated YAP pathway played a pivotal role in extracellular matrix secretion in DN. TGF-β up-regulated β1-adrenergic receptors (β1ARs) and α-SMA in MCs. Diminished nuclear-specific cAMP signaling in MCs by TGF-β triggered profibrotic gene transcription via reducing PKA-dependent phosphorylation of YAP. The profibrotic formation was partially alleviated by norepinephrine administration and prevented by combined treatment of norepinephrine and rolipram. In db/db mice, rolipram treatment alleviated kidney fibrosis and restored kidney filtration function. Conclusion and Implications DN impairs nuclear-localized β1AR-cAMP signaling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in DN.

Background and purpose Currently, the clinical benefits of liver cancer treatment are still limited. Emerging evidences have highlighted that paraptosis may be an effective strategy to threapy liver cancer. In our previous studies, compound 4a was found to induce paraptosis in cancer cells. Here, the characteristics of compound 4a induced paraptosis were further revealed, and for the first time, the target and related molecular mechanisms of compound 4a induced paraptosis in liver cancer were explored. Study design and methods The effect and mechanism of compound 4a on liver cancer cells both in vitro and in vivo. And the targets of compound 4a that triggering paraptosis, were identified and confirmed by using the technology of mass spectrometry-based drug affinity responsive target stability(DARTS), siRNA, and Cellular thermal Shift Assay(CETSA). Intracellular calcium concentration and protein distribution were detected by Cal520-AM and immunofluorescence, respectively. Results We found that compound 4a can effectively induce paraptosis-like cell death in liver cancer both in vitro and in vivo, and its effect is comparable to the first-line anti-liver cancer drugs oxaliplatin while with higher safety. We identified that Calreticulin(CRT) protein as a target of compound 4a, which causing cellular endoplasmic reticulum(ER) stress and calcium overload. CRT knockdown increased cell proliferation and reduced cytoplasmic vacuoles, which was associated with inhibited ER stress and paraptosis. Conclusions Our study provides a potential safe and effective agent for the treatment of liver cancer. Moreover, we have clarified characteristics of compound 4a-triggered paraptosis and revealed a unique function of CRT in paraptosis.

Background and Purpose Emerging evidence has suggested that somatic stimulation, including electroacupuncture (EA), could produce autonomic reflexes to modulate visceral functions. However, the efficacy and underlying mechanisms for EA on allergic pulmonary inflammation (API) remain elusive. Experimental Approach Papain-induced API mice were treated with distinct current-intensity EA at the back BL13, hindlimb ST36, and forelimb LU5 acupoints. The change in API was determined by whole-body plethysmograph (WBP), immunohistochemistry, flow cytometry, and HE staining. We employed lung functional testing combined with pharmacological inhibition and optogenetic activation approaches to examine the underlying mechanisms for EA effects on API. Key Results EA at the back BL13 region, but not limb regions, via a current-intensity-dependent manner, exacerbated the API. Pharmacological blocking of the local thoracic sensory or the lung-innervated autonomic nerve eliminates the EA-produced detrimental effects. Chemical pulmonary sympathectomy was further enhanced, but inhibition of the upregulated muscarinic M3 acetylcholine receptor within the pulmonary was sufficient to protect from the inflammation-exacerbation effect of EA. Conclusions and Implications Our findings suggest that BL13 EA evokes a somatic-autonomic-pulmonary M3 pathway to enhance API. The revelation of somatotopic organization and intensity-dependency in driving the pulmonary autonomic pathways could help optimize stimulation parameters to improve both efficacy and safety in various forms of physical therapy, including bioelectronic medicine and traditional as well as modern forms of moxibustion and EA, to modulate API.

This brief review highlights some of the structure-activity relationships of classic serotonergic psychedelics. In particular, we discuss structural features of three chemotypes: phenethylamines, ergolines, and certain tryptamines, which possess psychedelic activity in humans. Where it is known, we point out the underlying molecular mechanisms utilized by each of the three chemotypes of psychedelic molecules. With a focus on the serotonin 5-HT2A receptor subtype, a G-protein coupled receptor known to be the primary target of psychedelics, we reference several x-ray and cryoEM structures with various ligands bound to illustrate the underlying atomistic basis for some of the known pharmacological observations of psychedelic drug actions.

Mechanopharmacology is an emerging interdisciplinary field that investigates drug action using biomechanically appropriate in vitro systems to the relevant (patho)physiology. This review outlines emerging technologies and techniques which aim to bridge the gap between mechanical cues influencing cellular biology and conventional pharmacology. We delve into the impact of mechanopharmacology on drug development in cancers and fibrotic diseases. Mechanical cues such as stretch, stiffness, circadian rhythms, fluid flow, intercellular signalling cascades and cytoskeletal structures can modulate drug interactions with molecular targets with implications for drug discovery and development. Models incorporating mechanopharmacological cues to investigate pharmacokinetics, pharmacodynamics and therapeutic outcomes are outlined. Furthermore, this review discusses innovations in the use of biomaterials and microfluidics, to enable further the emulation of the mechanical microenvironment. We advocate for the application of mechanopharmacological considerations to improve the physiological relevance of methods used in the drug discovery pipeline.

Background and purpose: Several analgosedative drugs used in the management of critically ill patients impair gastrointestinal (GI) propulsion and thereby carry a risk for developing sepsis. The gut microbiota has emerged as a factor that can influence GI motility, but whether GI microbial disruption modifies GI peristalsis impairment by analgosedative drugs has not yet been analysed. This question was addressed in the guinea-pig small intestine following antibiotic-induced depletion of the GI microbiome. Experimental approach: Guinea-pigs were enorally pretreated with meropenem, neomycin and vancomycin, and antibiotic-induced depletion of the GI microbiome was confirmed by 16S rDNA sequencing. Peristalsis in the isolated guinea-pig small intestine was evaluated by assessing the peristaltic pressure threshold at which a peristaltic wave is triggered. The expression of factors that may be relevant to communication between GI microbiota and motor system was examined at the mRNA (qPCR) and/or protein (ELISA) level. Key results: Antibiotic treatment disturbed the small intestinal microbiome as shown by a decrease of bacterial load and α-diversity. Microbial disruption did not affect peristalsis at baseline but blunted the ability of α2-adrenoceptor (ADRA2) agonists to inhibit peristalsis, while the anti-peristaltic effects of sufentanil, midazolam, neostigmine and propofol were inconsistently affected. These functional alterations were complemented by a decreased expression of ADRA2, TLR3, TLR4 and TLR7, IFN-γ and NOS2. Conclusions and implications: Antibiotic-induced disturbance of the GI microbiome selectively blunts the ability of ADRA2 agonists to impair peristalsis. This effect is explained by decreased ADRA2 expression, which may arise from TLR downregulation in the dysbiotic gut.

Background and Purpose: Given the recent rise in cannabis legalization, accessibility and consumption by pregnant individuals, there are unintended developmental consequences considering the key role that the endocannabinoid system plays in fetal development and later-life energy homeostasis. Whether perinatal cannabis exposure (PCE) affects energy homeostasis positively or adversely in adulthood is unknown. We explored the long-term effects of maternal voluntary oral cannabis consumption on the metabolic outcomes of high fat diet (HFD) in adult offspring. Experimental Approach: Pregnant mice voluntarily consumed cannabis from gestational day 1.5 until postnatal day (PD) 10. Pregnancy and pup outcomes and active maternal behavior were recorded. Male and female offspring (PD49) were placed on a 12-week HFD or control diet; their weight gain, adiposity, glucose tolerance, insulin sensitivity, circulating hormones, and pancreas structure were measured. Key Results: PCE pup weight was reduced but was restored by PD16. PCE did not influence weight gain or metabolic characteristics of male mice on a HFD. However, PCE female offspring on a HFD had reduced accumulation of adipose tissue and lower insulin, leptin, and resistin independent of body weight and while PCE females on control diet showed altered basal insulin sensitivity likely due to increased glucagon levels in parallel with reduced islets of Langerhans size and enhanced gene expression of cannabinoid 2 receptors in white adipose tissue. Conclusion and Implications: PCE influences metabolic outcomes in female offspring; it adversely affected glycemic control in female offspring on control diet while it mitigated HFD-induced metabolic dysfunction. This raises concerns about the long-term effects of PCE on the metabolic health of offspring.

Background and Purpose: Hyoscine butylbromide (HBB) has a low oral (PO) bioavailability. Further, limited data on its activity on non-gastrointestinal (GI) smooth muscle spasms after oral dosing are available, meaning its effect outside the GI tract has been questioned. This pharmacokinetic/pharmacodynamic (PK/PD) study, conducted using female rats, aims to cover this gap. Experimental Approach: PK study: HBB and atropine (comparator) were administered PO and IV to rats, and concentrations in plasma, and colon, uterus and urinary bladder (CUB) were measured. PD study-1: Concentration–response (C-R) curves of HBB and atropine (10-9–10-4M) were obtained for carbachol (CCH)-induced (10-5 M) pre-contracted CUB; PD study-2: CUB were pre-incubated with HBB and atropine at maximum concentrations (Cmax) from PK study, and CCH C-R curves (10-9–10-4 M) were obtained; PD study-3: HBB and atropine were administered PO and IV to rats as for PK study, CUB were collected at 0.5 h (IV) and 4 h (PO), and CCH C-R curves (10-9–10-4 M) were obtained. Key Results: PO HBB showed higher Cmax in CUB (192.5, 3.70 and 1.85 ng∙g-1, respectively) than plasma (0.008 ng∙mL-1). HBB and atropine reduced (concentration dependently) CCH-induced contractions in CUB. PO HBB showed highest spasmolytic activity in colon (40%), followed by uterus (30%) and urinary bladder (10%). Conclusion and Implications: This is the first comparative study investigating PO and IV HBB and atropine in GI and non-GI tissues. Despite low bioavailability, PO HBB accumulates and exerts spasmolytic effects outside the GI tract.

Background and Purpose: Fentanyls and nitazenes are μ opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we compared the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues and compared them to morphine and DAMGO. Experimental Approach: In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Key Results: The rate of agonist dissociation from the μ receptor varied, with morphine, DAMGO, alfentanil and fentanyl dissociating rapidly whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow rate of dissociation was not due to G protein receptor kinase-mediated arrestin recruitment as its rate of dissociation was not affected by inhibition of GRKs with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. Conclusions and Implications: With fentanyls and nitazenes, that slowly dissociate from the opioid receptor, antagonism by naloxone is pseudo competitive. In overdoses involving fentanyls and nitazenes higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose.

Background and Purpose: Fibroblast Growth Factor (FGF), VEGFR2, and CSF1R signalling pathways play a key role in the pathogenesis of multiple sclerosis (MS). Selective inhibition of FGFR by infigratinib in MOG35-55-induced EAE prevented severe first clinical episodes by 40%; inflammation and neurodegeneration were reduced, and remyelination was enhanced. Multi-kinase inhibition of FGFR1-3, CSF1R and VEGFR2 by AZD4547 may be more efficient in reducing inflammation, neurodegeneration and regeneration in the disease model. Experimental Approach: Female C57BL/6J mice were treated with AZD4547 (6.25 mg kg-1 or 12.5 mg kg-1) orally or placebo over 10 days either from time of EAE induction (prevention experiment) or onset of symptoms (suppression experiment). Effects on inflammation, neurodegeneration and remyelination were assessed at the peak of the disease (day 18/20 p.i.) and the chronic phase of EAE (day 41/42 p.i.). Key Results: In the prevention experiment, treatment with AZD4547 prevented severe first clinical episodes by 66.7 or 84.6% respectively. Mice treated with 12.5 mg kg-1 of AZD4547 hardly showed any symptoms in the chronic phase of EAE. In the suppression experiment, treatment with AZD4547 resulted in a long-lasting reduction of severe symptoms by 91 or 100%. Inflammation and demyelination were reduced, and axonal density, numbers of oligodendrocytes and their precursor cells, and remyelinated axons were increased in both experimental approaches. Conclusion and Implications: Multi-kinase inhibition by AZD4547 in a well-tolerated dose of 1 mg kg-1 in humans may be a promising approach to reduce inflammation and neurodegeneration, to slow down disease progression and support remyelination in patients.

The total iridoid glycoside extract of Lamiophlomis rotata (IGLR) has been proven to be the main pharmacological ingredient for wound healing; however, it is still unclear, which compounds in the extract are responsible for this effect. We developed and validated a mass spectrometry imaging (MSI) method to reveal the relationship between phytochemical components and the pharmacological efficacy in IGLR. Shanzhiside methyl (SM) ester, 8-O-acetylshanzhiside methyl ester (ASM), and Phlorigidoside C (PhC) have been identified as quality markers in IGLR to accelerate wound healing by MSI method. As per MSI data with UMAP manifold analysis, these compounds overlapped in the same class with endogenous metabolites (lactate, citrate and succinate). In addition, UPLC-Q/TOF-MSn coupled with multivariate analyses for skin tissues in the normal and wound groups further confirmed the results. Furthermore, MSI data from livers and kidneys revealed that SM, ASM, and PhC absorption significantly increased, whereas their elimination rates remarkably decreased in the injured group. Collectively, the overall data demonstrated that SM, ASM, and PhC selectively increased in the new granulation tissue and that the bioavailability of these three quality markers was remarkably improved in the wound group after continuous gavage for 7 days. This protocol can be applied to reveal the relationship between phytochemical components and the pharmacological efficacy of other iridoid glycoside extracts.

Background and Purpose: In diabetes, elevated blood sugar (BS) levels contribute to the overproduction of reactive oxygen species (ROS), leading to endothelial progenitor cell (EPC) dysfunction. 2-hydroxy hispolon (2HH), a derivative of Hispolon (HIS), shows potential in reversing high glucose-induced EPC dysfunction. Experimental Approach: In vitro, we inspected the effect of 2HH on three different angiogenic cells, including outgrowth endothelial cell (OEC), circulating angiogenic cell (CAC) and endothelial cell (EC). In vivo, High-fat diet and streptozotocin induced diabetic mice and hindlimb ischemic model were utilized to evaluate the effect of 2HH on angiogenesis and CAC mobilization. Key Results: Treatment with 2HH significantly improved the proliferation, migration, and tube formation, NO synthesis and ROS diminishing of OEC under high glucose conditions. Moreover, 2HH administration potentiates the AMP-activated protein kinase (AMPK)/heme oxygenase-1 (HO-1) and phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling pathways in OECs exposed to high glucose levels. Additionally, 2HH reverses high glucose-induced impairment of CAC function but fails to restore EC dysfunction under high glucose conditions. Hindlimb ischemic assay demonstrated that 2HH treatment enhances blood flow recovery in ischemic hindlimbs and increases the number of circulating CACs in diabetic mice after femoral ligation. Conclusion and Implications: Our study has demonstrated that 2HH effectively reduces oxidative stress, thereby preventing EPC dysfunction induced by high glucose levels, primarily through the PI3K/AKT/eNOS and AMPK/HO-1 pathways. These findings offer a promising therapeutic avenue for attenuating the susceptibility to critical limb ischemia in patients with diabetes.

Background and Purpose Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuro-inflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Experimental Approach Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcome were assessed. Abundance of target genes, proteins and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting and immunofluorescence. Key Results Stroke upregulates PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduces infarct size, apoptotic markers, macrophage accumulation and interleukin-1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Conclusions and Implications Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

A document by Mickaël Machicoane. Click on the document to view its contents.

Background and Purpose: Treatment of osteoarthritis (OA) pain often yields unsatisfactory results, making the search for new pain-relieving options essential. Vortioxetine, an antidepressant with a unique mechanism of action, has recently shown analgesic properties. We aimed to investigate its effects in the OA model and gain insight into the underlying molecular mechanisms. Duloxetine (a second-line drug for pain relief in OA) was studied as a reference. Experimental Approach: In the monoiodoacetate (MIA)-induced model of knee OA in rats of both sexes, pain-related behaviour was assessed in weight-bearing and von Frey tests. Antidepressants were administered orally once daily for 28 days. Gene expression of pain-related mediators (Ngf, Il-1β, Tnf-α, Bdnf, and Tac1 encoding substance P) and oxidative stress parameters were determined after completion of the treatment/behavioural testing protocol. Key results: Vortioxetine dose-dependently reduced weight-bearing asymmetry and mechanical allodynia of the paw ipsilateral to the MIA-injected knee. Duloxetine was also effective. Vortioxetine reduced the increased Ngf mRNA expression in the MIA-injected knees to the level observed in the sham-injected counterparts. It also reduced oxidative stress parameters in the affected knees, more effectively in females than in males. Duloxetine showed no effect on locally increased Ngf mRNA expression and oxidative stress. Both antidepressants decreased mRNA expression of pain-related mediators in the lumbar L3-L5 ipsilateral DRGs and spinal cords, which were upregulated in MIA-injected rats. This effect was male-specific. Conclusion and Implications: Vortioxetine may be effective against chronic pain in OA. This effect appears to be mediated, at least in part, by normalization of NGF expression in the affected joint.

RNA therapeutics are emerging as a unique opportunity to drug currently “undruggable” molecules and diseases. While their advantages over conventional, small molecule drugs, their therapeutic implications and the tools for their effective in vivo delivery have been extensively reviewed, little attention has been so far paid to the technological platforms exploited for the discovery of RNA therapeutics. Here, we provide an overview of the existing platforms and ex vivo assays for RNA discovery, their advantages and disadvantages, as well as their main fields of application, with specific focus on RNA therapies that have reached either phase 3 or market approval.

Background and Purpose: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with limited treatment options. ALS pathogenesis involves intricate processes within motor neurons (MNs), characterized by dysregulated Ca2+ influx and buffering in early ALS-affected MNs. This study proposes the modulation of ryanodine receptors (RyRs), key mediators of intracellular Ca2+, as a therapeutic target. Experimental Approach: A novel class of novel FKBP12 ligands that show activity as cytosolic calcium modulators through stabilizing RyR channel activity, were tested in the SOD1G93A mouse model of ALS. Different outcomes were used to assess treatment efficacy including electrophysiology, histopathology, neuromuscular function, and survival. Key Results: Among the novel FKBP12 ligands, MP-010 was chosen for its central nervous system availability. Chronic administration of MP-010 to SOD1G93A mice produced a dose-dependent preservation of motor nerve conduction, with the 61 mg/kg dose significantly delaying the onset of motor impairment. This was accompanied by improved motor coordination, increased innervated endplates, and significant preservation of MNs in the spinal cord of treated mice. Notably, MP-010 treatment significantly extended lifespan by an average of 10 days compared to vehicle. Conclusions and Implications: FKBP12 ligands, particularly MP-010, exhibit promising neuroprotective effects in ALS, highlighting their potential as novel therapeutic agents. Further investigations into the molecular mechanisms and clinical translatability of these compounds are needed for their application in ALS treatment.