Cellular plasticity in adult multicellular organisms is a protective mechanism that allows certain tissues to regenerate in response to injury. Considering that aging involves exposure to repeated injuries over a lifetime, it is conceivable that cell identity itself is more malleable—and potentially erroneous—with age. In this review, we summarize and critically discuss the available evidence that cells undergo age-related shifts in identity, with an emphasis on those that contribute to age-associated pathologies, including neurodegeneration and cancer. Specifically, we focus on reported instances of programs associated with dedifferentiation, biased differentiation, acquisition of features from alternative lineages, and entry into a preneoplastic state. As some of the most promising approaches to rejuvenate cells reportedly also elicit transient changes to cell identity, we further discuss whether cell state change and rejuvenation can be uncoupled to yield more tractable therapeutic strategies.

Solid tumors that arise in the body interact with neurons, which influences cancer progression and treatment response. Here, we discuss key questions in the field, including defining the nature of interactions between tumors and neural circuits and defining how neural signals shape the tumor microenvironment. This information will allow us to optimally target neural signaling to improve outcomes for cancer patients.

Thermoregulation, responsible for maintaining a stable core temperature during wide fluctuations in external and internal thermal environments, is an iconic homeostatic process. However, we suggest that despite its fundamental physiological significance, the potential for required cool housing temperatures and thermoregulatory mechanisms to influence the interpretation of experimental data is not sufficiently appreciated. Moreover, although it is generally assumed that the major thermoregulatory pathways are well understood, here we discuss new research that suggests otherwise and reveals the emergence of a new wave of exciting ideas for this "old" field of research.

Recent work has highlighted the central role the brain–body axis plays in not only maintaining organismal homeostasis but also coordinating the body's response to immune and inflammatory insults. Here, we discuss how science is poised to address the many ways that our brain is directly involved with disease. In particular, we feel that combining cutting-edge tools in neuroscience with translationally relevant models of cancer will be critical to understanding how the brain and tumors communicate and modulate each other's behavior.

The brain's capacity to predict and anticipate changes in internal and external environments is fundamental to initiating efficient adaptive responses, behaviors, and reflexes that minimize disruptions to physiology. In the context of feeding control, the brain predicts and anticipates responses to the consumption of dietary substances, thus driving adaptive behaviors in the form of food choices, physiological preparation for meals, and engagement of defensive mechanisms. Here, we provide an integrative perspective on the multisensory computation between exteroceptive and interoceptive cues that guides feeding strategy and may result in food-related disorders.

Neural reflexes occupy a central role in physiological homeostasis. The vagus nerve is a major conduit for transmitting afferent and efferent signals in homeostatic reflex arcs between the body and the brain. Recent advances in neuroscience, immunology, and physiology have revealed important vagus nerve mechanisms in suppressing inflammation and treating rheumatoid arthritis and other autoimmune conditions. Numerous clinical trials indicate that there is significant benefit to vagus nerve stimulation therapy. Although many questions are still unanswered, it will be important, even necessary, to pursue answers that will be useful in guiding interventions to modulate immunological and physiological homeostasis.

The world of cancer science is moving toward a paradigm shift in making connections with neuroscience. After decades of research on genetic instability and mutations or on the tumor microenvironment, emerging evidence suggests that a malignant tumor is able to hijack and use the brain and its network of peripheral and central neurons as disrupters of homeostasis in the body. Whole-body homeostasis requires brain–body circuits to maintain survival and health via the processes of interoception, immunoception, and nociception. It is now likely that cancer disturbs physiological brain–body communication in making bidirectional brain tumor connections.

The study of biological mechanisms, while crucial, cannot fully explain complex phenomena like the instinct to eat. The mind–body connection, as exemplified by the concept of "voodoo death," highlights the profound influence of belief and cultural context on physiology. Indigenous knowledge systems further emphasize the interconnectedness of humans with their environment. Recent discoveries in gut–brain communication reveal the intricate neural circuits that drive our visceral desires, but a holistic approach that integrates both physiological mechanisms and the subjective experience of life, informed by diverse cultural perspectives, will be essential to truly understand what it means to be alive.

The dorsal vagal complex contains three structures: the area postrema, the nucleus tractus solitarii, and the dorsal motor nucleus of the vagus. These structures are tightly linked, both anatomically and functionally, and have important yet distinct roles in not only conveying peripheral bodily signals to the rest of the brain but in the generation of behavioral and physiological responses. Reports on the new discoveries in these structures were highlights of the symposium. In this outlook, we focus on the roles of the area postrema in mediating brain–body interactions and its potential utility as a therapeutic target, especially in cancer cachexia.

Our approaches toward understanding cancer have evolved beyond cell-intrinsic and local microenvironmental changes within the tumor to encompass how the cancer interfaces with the entire host organism. The nervous system is uniquely situated at the interface between the brain and body, constantly receiving and sending signals back and forth to maintain homeostasis and respond to salient stimuli. It is becoming clear that various cancers disrupt this dialog between the brain and body via both neuronal and humoral routes, leading to aberrant brain activity and accelerated disease. In this outlook, I discuss this view of cancer as a homeostatic challenge, emphasize cutting-edge work, and provide outstanding questions that need to be answered to move the field forward.

Memory formation is contingent on molecular and structural changes in neurons in response to learning stimuli—a process known as neuronal plasticity. The initiation step of mRNA translation is a gatekeeper of long-term memory by controlling the production of plasticity-related proteins in the brain. The mechanistic target of rapamycin complex 1 (mTORC1) controls mRNA translation, mainly through phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and ribosomal protein S6 kinases (S6Ks). mTORC1 signaling decreases throughout brain development, starting from the early postnatal period. Here, we discovered that in mice, the age-dependent decrease in mTORC1 signaling occurs selectively in excitatory but not inhibitory neurons. Using a gene conditional knockout (cKO) strategy, we demonstrate that either up- or downregulating the mTORC1-4E-BP2 axis in GAD65 inhibitory interneurons, but not excitatory neurons, results in long-term object recognition and object location memory deficits. Our data indicate that the mTORC1 pathway in inhibitory but not excitatory neurons plays a key role in memory formation.

Binding of arbitrary information into distinct memory representations that can be used to guide behavior is a hallmark of relational memory. What is and is not bound into a memory representation and how those things influence the organization of that representation remain topics of interest. While some information is intentionally and effortfully bound—often the information that is consistent with task goals or expectations about what information may be required later—other information appears to be bound automatically. The present set of experiments sought to investigate whether spatial memory would be systematically influenced by the presence and absence of distinct categories of stimuli on a spatial reconstruction task. In this task, participants must learn multiple item-location bindings and place each item back in its studied location after a short delay. Across three experiments, participants made significantly more within-category errors (i.e., misassigning one item to the location of a different item from the same category) than between-category errors (i.e., misassigning one item to the location of an item from a different category) when categories were perceptually or semantically distinct. These data reveal that category information contributed to the organization of the memory representation and influenced spatial reconstruction performance. Together, these results suggest that categorical information can influence memory organization, and not always to the benefit of overall task performance.

Internal ribosomal entry sites (IRESs) recruit the ribosome to promote translation, typically in an m7G cap-independent manner. Although IRESs are well-documented in viral genomes, they have also been reported in mammalian transcriptomes, where they have been proposed to mediate cap-independent translation of mRNAs. However, subsequent studies have challenged the idea of these "cellular" IRESs. Current methods for screening and discovering IRES activity rely on a bicistronic reporter assay, which is prone to producing false positive signals if the putative IRES sequence has a cryptic promoter or cryptic splicing sites. Here, we report an assay for screening IRES activity using a genetically encoded circular RNA comprising a split nanoluciferase (nLuc) reporter. The circular split nLuc reporter is less susceptible to the various sources of false positives that adversely affect the bicistronic IRES reporter assay and provides a streamlined method for screening IRES activity. Using the circular split nLuc reporter, we find that nine reported cellular IRESs have minimal IRES activity. Overall, the circular split nLuc reporter offers a simplified approach for identifying and validating IRESs and exhibits reduced propensity for producing the types of false positives that can occur with the bicistronic reporter assay.

Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression and tissue patterning in animals. An invaluable tool in modern gene expression research is the presence of a high-resolution ribosome structure, though no such structure exists for C. elegans. Here, we present a high-resolution single-particle cryogenic electron microscopy (cryo-EM) reconstruction and molecular model of a C. elegans ribosome, revealing a significantly streamlined animal ribosome. Many facets of ribosome structure are conserved in C. elegans, including overall ribosomal architecture and the mechanism of cycloheximide, whereas other facets, such as expansion segments and eL28, are rapidly evolving. We identify uL5 and uL23 as two instances of tissue-specific ribosomal protein paralog expression conserved in Caenorhabditis, suggesting that C. elegans ribosomes vary across tissues. The C. elegans ribosome structure will provide a basis for future structural, biochemical, and genetic studies of translation in this important animal system.

Bacterial regulatory RNAs (sRNAs) are important players to control gene expression. In Staphylococcus aureus, SprC is an antivirulent trans-acting sRNA known to base-pair with the major autolysin atl mRNA, preventing its translation. Using MS2-affinity purification coupled with RNA sequencing, we looked for its sRNA-RNA interactome and identified 14 novel mRNA targets. In vitro biochemical investigations revealed that SprC binds two of them, czrB and deoD, and uses a single accessible region to regulate its targets, including Atl translation. Unlike Atl regulation, the characterization of the SprC-czrB interaction pinpointed a destabilization of the czrAB cotranscript, leading to a decrease of the mRNA level that impaired CzrB zinc efflux pump expression. On a physiological standpoint, we showed that SprC expression is detrimental to combat against zinc toxicity. In addition, phagocyctosis assays revealed a significant, but moderate, increase of czrB mRNA levels in a sprC-deleted mutant, indicating a functional link between SprC and czrB upon internalization in macrophages, and suggesting a role in resistance to both oxidative and zinc bursts. Altogether, our data uncover a novel pathway in which SprC is implicated, highlighting the multiple strategies used by S. aureus to balance virulence using an RNA regulator.

The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases, including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.

The spliceosome is a eukaryotic multimegadalton RNA–protein complex that removes introns from transcripts. The spliceosome ensures the selection of each exon-intron boundary through multiple recognition events. Initially, the 5' splice site (5' SS) and branch site (BS) are bound by the U1 small nuclear ribonucleoprotein (snRNP) and the U2 snRNP, respectively, while the 3' SS is mostly determined by proximity to the branch site. A large number of splicing factors recognize the splice sites and recruit the snRNPs before the stable binding of the snRNPs occurs by base-pairing the snRNA to the transcript. Fidelity of this process is crucial, as mutations in splicing factors and U2 snRNP components are associated with many diseases. In recent years, major advances have been made in understanding how splice sites are selected in Saccharomyces cerevisiae and humans. Here, I review and discuss the current understanding of the recognition of splice sites by the spliceosome with a focus on recognition and binding of the branch site by the U2 snRNP in humans.

Background

Recently, cough reflex hypersensitivity has been proposed as a common underlying feature of chronic cough in adults. However, symptoms and clinical characteristics of cough hypersensitivity have not been studied amongst phenotypes of chronic cough. This study aimed to compare symptom features, such as cough triggers and associated throat sensations, of cough hypersensitivity in patients with asthmatic chronic cough and those with refractory chronic cough (RCC).

Background

Exacerbations of noncystic fibrosis bronchiectasis (bronchiectasis) are associated with reduced health-related quality of life and increased mortality, likelihood of hospitalisation and lung function decline. This study investigated patient clinical characteristics associated with exacerbation frequency.

Background

Long COVID is a heterogeneous clinical syndrome characterised by a variety of reported symptoms and signs. Its clinical management is expected to differ significantly worldwide.

Extract

It is of utmost importance that medicines are available at all times for our patients. Historically, medication unavailability has typically, if not exclusively, affected low- and middle-income countries [1]. More recently however, drug shortages have also been reported in high-income European countries [2]. Drug shortages have negative health consequences for patients [3], and a profound economic impact, with the need to resort to more expensive alternatives and demands on healthcare professionals’ time to find, prescribe and dispense alternatives [4].

Extract

With a disproportionate burden of tuberculosis (TB) amongst migrants in Europe [1], Burman et al. [2] have highlighted the pressing need for alternative approaches to make TB infection (TBI) screening comprehensive and accessible. Across high-income Organisation for Economic Co-operation and development countries, a median of 52% of TB cases occur in foreign-born individuals, who are at their highest risk of developing TB disease within the first 5 years of migration [3]. Molecular epidemiological studies indicate that the majority of these cases occur as a result of TBI reactivation, often acquired overseas [4]. Within the UK, overseas-born migrants have a 14-fold higher TB incidence than UK-born individuals [5]. The World Health Organization therefore recommends that migrants from countries with a high TB burden may be prioritised for TBI screening [6, 7].

Extract

In cystic fibrosis (CF), Pseudomonas aeruginosa acquisition represents a turning point in disease progression. The presence of chronic P. aeruginosa infection is associated with worsening lung function and increased risk of earlier death, whereas treatment substantially improves lung function and survival [1, 2]. Efforts to diagnose and eradicate early P. aeruginosa provide lasting benefits for children with CF [3, 4]. However, the timing of infection varies considerably between individuals with CF, treatment centres [5, 6], and different birth cohorts of people with the disease [7, 8].

Background

Computer-aided detection (CAD) systems hold promise for improving tuberculosis (TB) detection on digital chest radiographs. However, data on their performance in exclusively paediatric populations are scarce.

Background

The real-world consequences of a Philips Respironics recall for positive airway pressure (PAP) devices distributed between 2009 and 2021 are unknown.

The management of chylothorax remains challenging given the limited evidence and significant heterogeneity in practice. In addition, there are no practical guidelines on the optimal approach to manage this complex condition. We convened an international group of 27 experts from 20 institutions across five countries and four specialties (pulmonary, interventional radiology, thoracic surgery and nutrition) with experience and expertise in managing adult patients with chylothorax. We performed a literature and internet search for reports addressing seven clinically relevant PICO (Patient, Intervention, Comparison and Outcome) questions pertaining to the management of adult patients with chylothorax. This consensus statement, consisting of best practice statements based on expert consensus addressing these seven PICO questions, was formulated by a systematic and rigorous process involving the evaluation of published evidence, augmented with provider experience. Panel members participated in the development of the final best practice statements using the modified Delphi technique. Our consensus statement aims to offer guidance in clinical decision making when managing patients with chylothorax while also identifying gaps in knowledge and informing future research.

Background

Pseudomonas aeruginosa is a common pathogen that contributes to progressive lung disease in cystic fibrosis (CF). Genetic factors other than CF-causing CFTR (CF transmembrane conductance regulator) variations contribute ~85% of the variation in chronic P. aeruginosa infection age in CF according to twin studies, but the susceptibility loci remain unknown. Our objective is to advance understanding of the genetic basis of host susceptibility to P. aeruginosa infection.

Background

Halm's clinical stability criteria have long guided antibiotic treatment and hospital discharge decisions for patients hospitalised with community-acquired pneumonia (CAP). Originally introduced in 1998, these criteria were established based on a relatively small and select patient population. Consequently, our study aims to reassess their applicability in the management of CAP in a contemporary real-world setting.

Background

Control of latent tuberculosis infection (LTBI) is a priority in the World Health Organization strategy to eliminate TB. Many high-income, low TB incidence countries have prioritised LTBI screening and treatment in recent migrants. We tested whether a novel model of care, based entirely within primary care, was effective and safe compared to secondary care.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are important hepatic transporters. We previously identified OATP1B3 being critically implicated in the disposition of abiraterone. We aimed to further investigate the effects of abiraterone on the activities of OATP1B1 and OATP1B3 utilizing a validated endogenous biomarker coproporphyrin I (CP-I). We used OATP1B-transfected cells to characterize the inhibitory potential of abiraterone against OATP1B-mediated uptake of CP-I. Inhibition constant (K_i) was incorporated into our physiologically based pharmacokinetic (PBPK) modeling to simulate the systemic exposures of CP-I among cancer populations receiving either our model-informed 500 mg or clinically approved 1000 mg abiraterone acetate (AA) dosage. Simulated data were compared with clinical CP-I concentrations determined among our nine metastatic prostate cancer patients receiving 500 mg AA treatment. Abiraterone inhibited OATP1B3-mediated, but not OATP1B1-mediated, uptake of CP-I in vitro, with an estimated K_i of 3.93 μM. Baseline CP-I concentrations were simulated to be 0.81 ± 0.26 ng/ml and determined to be 0.72 ± 0.16 ng/ml among metastatic prostate cancer patients, both of which were higher than those observed for healthy subjects. PBPK simulations revealed an absence of OATP1B3-mediated interaction between abiraterone and CP-I. Our clinical observations confirmed that CP-I concentrations remained comparable to baseline levels up to 12 weeks post 500 mg AA treatment. Using CP-I as an endogenous biomarker, we identified the inhibition of abiraterone on OATP1B3 but not OATP1B1 in vitro, which was predicted and observed to be clinically insignificant. We concluded that the interaction risk between AA and substrates of OATP1Bs is low.

Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2’,7’-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1’s complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17β-glucuronide (E17βG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17βG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called adverse analytical finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e., samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility.

Compound probiotics have been widely used and commonly coadministered with other drugs for treating various chronic illnesses, yet their effects on drug pharmacokinetics remain underexplored. This study elucidated the impact of VSL#3 on the metabolism of probe drugs for cytochrome P450 enzymes (P450s), specifically omeprazole, tolbutamide, midazolam, metoprolol, phenacetin, and chlorzoxazone. Male Wistar rats were administered drinking water containing VSL#3 or not for 14 days and then intragastrically administered a P450 probe cocktail; this was done to investigate the host P450’s metabolic phenotype. Stool, liver/jejunum, and serum samples were collected for 16S ribosomal RNA sequencing, RNA sequencing, and bile acid profiling. The results indicated significant differences in both α and β diversity of intestinal microbial composition between the probiotic and vehicle groups in rats. In the probiotic group, the bioavailability of omeprazole increased by 269.9%, whereas those of tolbutamide and chlorpropamide decreased by 28.1% and 27.4%, respectively. The liver and jejunum exhibited 1417 and 4004 differentially expressed genes, respectively, between the two groups. In the probiotic group, most of P450 genes were upregulated in the liver but downregulated in the jejunum. The expression of genes encoding metabolic enzymes and drug transporters also changed. The serum-conjugated bile acids in the probiotic group were significantly reduced. Shorter duodenal villi and longer ileal villi were found in the probiotic group. In summary, VSL#3 administration altered the gut microbiota, host drug–processing gene expression, and intestinal structure in rats, which could be reasons for pharmacokinetic changes.

Solute carrier family 6 member 19 (SLC6A19) inhibitors are being studied as therapeutic agents for phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7 ± 7.7 compared with vehicle 0.3 ± 0.1 (P = 0.01) as determined by tissue mass spectrometry imaging analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters such as organic anion transporter (OAT)1, OAT3, and breast cancer resistance protein (BCRP). RA836 inhibits BCRP with an IC₅₀ of 0.045 μM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using liquid chromatography–mass spectrometry. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, whereas compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma.

Exercise significantly alters human physiological functions, such as increasing cardiac output and muscle blood flow and decreasing glomerular filtration rate (GFR) and liver blood flow, thereby altering the absorption, distribution, metabolism, and excretion of drugs. In this study, we aimed to establish a database of human physiological parameters during exercise and to construct equations for the relationship between changes in each physiological parameter and exercise intensity, including cardiac output, organ blood flow (e.g., muscle blood flow and kidney blood flow), oxygen uptake, plasma pH and GFR, etc. The polynomial equation P = a_iHRⁱ was used for illustrating the relationship between the physiological parameters (P) and heart rate (HR), which served as an index of exercise intensity. The pharmacokinetics of midazolam, quinidine, digoxin, and lidocaine during exercise were predicted by a whole-body physiologically based pharmacokinetic (WB-PBPK) model and the developed database of physiological parameters following administration to 100 virtual subjects. The WB-PBPK model simulation results showed that most of the observed plasma drug concentrations fell within the 5^th–95^th percentiles of the simulations, and the estimated peak concentrations (C_max) and area under the curve (AUC) of drugs were also within 0.5–2.0 folds of observations. Sensitivity analysis showed that exercise intensity, exercise duration, medication time, and alterations in physiological parameters significantly affected drug pharmacokinetics and the net effect depending on drug characteristics and exercise conditions. In conclusion, the pharmacokinetics of drugs during exercise could be quantitatively predicted using the developed WB-PBPK model and database of physiological parameters.

In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection was used. The results showed rapid clearance from plasma (0.5 to 1.5 hours of half-life) and predominant distribution to the liver and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 hours was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration.

The organic cation transporter (OCT)-1 mediates hepatic uptake of cationic endogenous compounds and xenobiotics. To date, limited information exists on how Oct1/OCT1 functionally develops with age in rat and human livers and how this would affect the pharmacokinetics of OCT substrates in children or juvenile animals. The functional ontogeny of rOct/hOCT was profiled in suspended rat (2–57 days old) and human hepatocytes (pediatric liver tissue donors: age 2–12 months) by determining uptake clearance of 4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide (ASP+) as a known rOct/hOCT probe substrate. mRNA expression was determined in rat liver tissue corresponding to rat ages used in the functional studies, while hOCT1 mRNA expressions were determined in the same hepatocyte batches as those used for uptake studies. Maturation of rOct/hOCT activity and expression were evaluated by comparing values obtained at the various ages to the adult values. Relative to adult values (at 8 weeks), ASP⁺ uptake clearance in suspended rat hepatocytes aged 0, 1, 2, 3, 4, 5, and 6 weeks reached 26%, 29%, 33%, 37%, 72%, 63%, and 71%, respectively. Hepatic Oct1 mRNA expression was consistent with Oct activity (correlation coefficient of 0.92). In human hepatocytes, OCT1 activity was age dependent and also correlated with mRNA levels (correlation coefficient of 0.88). These data show that Oct1/OCT1 activities and expression mature gradually in rat/human liver, thereby mirroring the expression pattern of organic anion transporting polypeptide in rat. These high-resolution transporter ontogeny profiles will allow for more accurate prediction of the pharmacokinetics of OCT1/Oct1 substrates in pediatric populations and juvenile animals.

Organic anion transporting polypeptides (OATP, gene symbol SLCO) are well-recognized key determinants for the absorption, distribution, and excretion of a wide spectrum of endogenous and exogenous compounds including many antineoplastic agents. It was therefore proposed as a potential drug target for cancer therapy. In our previous study, it was found that low-dose X-ray and carbon ion irradiation both upregulated the expression of OATP family member OATP1A2 and in turn, led to a more dramatic killing effect when cancer cells were cotreated with antitumor drugs such as methotrexate. In the present study, the underlying mechanism of the phenomenon was explored in breast cancer cell line MCF-7. It was found that the nonreceptor tyrosine kinase v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES-1) was temporally coordinated with the change of OATP1A2 after irradiation. The overexpression of YES-1 significantly increased OATP1A2 both at the mRNA and protein level. The signal transducer and activator of transcription 3 (STAT3) pathway is likely the downstream target of YES-1 because phosphorylation and nuclear accumulation of STAT3 were both enhanced after overexpressing YES-1 in MCF-7 cells. Further investigation revealed that there are two possible binding sites of STAT3 localized at the upstream sequence of SLCO1A2, the encoding gene of OATP1A2. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis suggested that these two sites bound to STAT3 specifically and the overexpression of YES-1 significantly increased the association of the transcription factor with the putative binding sites. Finally, inhibition or knockdown of YES-1 attenuated the induction effect of radiation on the expression of OATP1A2.

Sterol 12α-hydroxylase (CYP8B1) is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12α-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12α-hydroxylation of C₂₇ BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12α-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12α-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7α-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12α-hydroxylation of both DHCA and C4, with substrate concentration occupying half of the binding sites of 3.0 and 1.9 μM and k_cat of 3.2 and 2.6 minutes^–1, respectively. In summary, the confirmed role of CYP8B1 in 12α-hydroxylation of C₂₇ BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases.

The CYP3A7 enzyme accounts for ~50% of the total cytochrome P450 (P450) content in fetal and neonatal livers and is the predominant P450 involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-sulfate. The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk that drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We used three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC₅₀ and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC₅₀ values and P450 inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that clobetasol propionate (CP) was not an MDI of CYP3A5 but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP’s ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7, and our results provide confidence of CYP3cide’s and CP’s ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms.

Early detection of drug-drug interactions (DDIs) can facilitate timely drug development decisions, prevent unnecessary restrictions on patient enrollment, resulting in clinical study populations that are not representative of the indicated study population, and allow for appropriate dose adjustments to ensure safety in clinical trials. All of these factors contribute to a streamlined drug approval process and enhanced patient safety. Here we describe a new approach for early prediction of the magnitude of change in exposure for cytochrome P450 (P450) CYP3A4-related DDIs of small-molecule anticancer drugs based on the model-based extrapolation of human-CYP3A4-transgenic mice pharmacokinetics to humans. Victim drugs brigatinib and lorlatinib were evaluated with the new approach in combination with the perpetrator drugs itraconazole and rifampicin. Predictions of the magnitude of change in exposure deviated at most 0.99- to 1.31-fold from clinical trial results for inhibition with itraconazole, whereas exposure predictions for the induction with rifampicin were less accurate, with deviations of 0.22- to 0.48-fold. Results for the early prediction of DDIs and their clinical impact appear promising for CYP3A4 inhibition, but validation with more victim and perpetrator drugs is essential to evaluate the performance of the new method.

Over the past 20 years, quantitative proteomics has contributed a wealth of protein expression data, which are currently used for a variety of systems pharmacology applications, as a complement or a surrogate for activity of the corresponding proteins. A symposium at the 25th North American International Society for the Study of Xenobiotics meeting, in Boston, in September 2023, was held to explore current and emerging applications of quantitative proteomics in translational pharmacology and strategies for improved integration into model-informed drug development based on practical experience of each of the presenters. A summary of the talks and discussions is presented in this perspective alongside future outlook that was outlined for future meetings.

ATP-binding cassette transporter subfamily G member 2 (ABCG2) is a membrane-bound transporter responsible for the efflux of various xenobiotics and endobiotics, including protoporphyrin IX (PPIX), an intermediate in the heme biosynthesis pathway. Certain genetic mutations and chemicals impair the conversion of PPIX to heme and/or increase PPIX production, leading to PPIX accumulation and toxicity. In mice, deficiency of ABCG2 protects against PPIX-mediated phototoxicity and hepatotoxicity by modulating PPIX distribution. In addition, in vitro studies revealed that ABCG2 inhibition increases the efficacy of PPIX-based photodynamic therapy by retaining PPIX inside target cells. In this review, we discuss the roles of ABCG2 in modulating the tissue distribution of PPIX, PPIX-mediated toxicity, and PPIX-based photodynamic therapy.

The regulation of drug-metabolizing enzymes and transporters by cytokines has been extensively studied in vitro and in clinic. Cytokine-mediated suppression of cytochrome P450 (CYP) or drug transporters may increase or decrease the systemic clearance of drug substrates that are primarily cleared via these pathways; neutralization of cytokines by therapeutic proteins may thereby alter systemic exposures of such drug substrates. The Food and Drug Administration recommends evaluating such clinical drug interactions during clinical development and has provided labeling recommendations for therapeutic proteins. To determine the clinical relevance of these drug interactions to dose adjustments, trends in steady-state exposures of CYP-sensitive substrates coadministered with cytokine modulators as reported in the University of Washington Drug Interaction Database were extracted and examined for each of the CYPs. Coadministration of cytochrome P450 family 3 subfamily A (CYP3A) (midazolam/simvastatin), cytochrome P450 subfamily 2C19 (omeprazole), or cytochrome P450 subfamily 1A2 (caffeine/tizanidine) substrates with anti-interleukin-6 and with anti-interleukin-23 therapeutics led to changes in systemic exposures of CYP substrates ranging from ~ –58% to ~35%; no significant trends were observed for cytochrome P450 subfamily 2D6 (dextromethorphan) and cytochrome P450 subfamily 2C9 (warfarin) substrates. Although none of these changes in systemic exposures have been reported as clinically meaningful, dose adjustment of midazolam for optimal sedation in acute care settings has been reported. Simulated concentration-time profiles of midazolam under conditions of elevated cytokine levels when coadministered with tocilizumab, suggest a ~six- to sevenfold increase in midazolam clearance, suggesting potential implications of cytokine–CYP drug interactions on dose adjustments of sensitive CYP3A substrates in acute care settings. Additionally, this article also provides a brief overview of nonclinical and clinical assessments of cytokine–CYP drug interactions in drug discovery and development.

The precision medicine initiative has driven a substantial change in the way scientists and health care practitioners think about diagnosing and treating disease. While it has long been recognized that drug response is determined by the intersection of genetic, environmental, and disease factors, improvements in technology have afforded precision medicine guided dosing of drugs to improve efficacy and reduce toxicity. Pharmacometabolomics aims to evaluate small molecule metabolites in plasma and/or urine to help evaluate mechanisms that predict and/or reflect drug efficacy and toxicity. In this mini review, we provide an overview of pharmacometabolomic approaches and methodologies. Relevant examples where metabolomic techniques have been used to better understand drug efficacy and toxicity in major depressive disorder and cancer chemotherapy are discussed. In addition, the utility of metabolomics in drug development and understanding drug metabolism, transport, and pharmacokinetics is reviewed. Pharmacometabolomic approaches can help describe factors mediating drug disposition, efficacy, and toxicity. While important advancements in this area have been made, there remain several challenges that must be overcome before this approach can be fully implemented into clinical drug therapy.

A STING (stimulator of interferon genes) agonist GSK3996915 under investigation in early discovery for hepatitis B was orally dosed to a mouse model for understanding the parent drug distribution in liver, the target organ. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to quantify the distribution of GSK3996915 in liver collected from mice administered a single oral dose at 90 mg/kg. GSK3996915 was detected with a zonal distribution localized in the portal triad and highly concentrated in the main bile ducts, indicating clearance through biliary excretion. High spatial resolution imaging showed the distribution of the parent drug localized to the cellular populations in the sinusoids, including the Kupffer cells. Additionally, a series of drug-related metabolites were observed to be localized in the central zones of the liver. These results exemplify the potential of utilizing MALDI IMS for measuring not only quantitative drug distribution and target exposure but also drug metabolism and elimination in a single suite of experiments.

Drug–drug interaction (DDI) assessment of therapeutic peptides is an evolving area. The industry generally follows DDI guidelines for small molecules, but the translation of data generated with commonly used in vitro systems to in vivo is sparse. In the current study, we investigated the ability of advanced human hepatocyte in vitro systems, namely HepatoPac, spheroids, and Liver-on-a-chip, to assess potential changes in regulation of CYP1A2, CYP2B6, CYP3A4, SLCO1B1, and ABCC2 in the presence of selected therapeutic peptides, proteins, and small molecules. The peptide NN1177, a glucagon and GLP-1 receptor co-agonist, did not suppress mRNA expression or activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac, spheroids, or Liver-on-a-chip; these findings were in contrast to the data obtained in sandwich cultured hepatocytes. No effect of NN1177 on SLCO1B1 and ABCC2 mRNA was observed in any of the complex systems. The induction magnitude differed across the systems (e.g., rifampicin induction of CYP3A4 mRNA ranged from 2.8-fold in spheroids to 81.2-fold in Liver-on-a-chip). Small molecules, obeticholic acid and abemaciclib, showed varying responses in HepatoPac, spheroids, and Liver-on-a-chip, indicating a need for EC₅₀ determinations to fully assess translatability data. HepatoPac, the most extensively investigated in this study (3 donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential. This study establishes an excellent foundation toward the establishment of more clinically-relevant in vitro tools for evaluation of potential DDIs with therapeutic peptides.

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are useful for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics that relies on synthetic stable isotope-labeled surrogate peptides as calibrators is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA does not consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage, which was applied to quantify 54 DMETs for characterization of 1) differential tissue DMET abundance in the human liver, kidney, and intestine, and 2) interindividual variability of DMET proteins in individual intestinal samples (n = 13). Uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7), microsomal glutathione S-transferases (MGST1, MGST2, and MGST3) carboxylesterase 2 (CES2), and multidrug resistance-associated protein 2 (MRP2) were expressed in all three tissues, whereas, as expected, four cytochrome P450s (CYP3A4, CYP3A5, CYP2C9, and CYP4F2), UGT1A1, UGT2B17, CES1, flavin-containing monooxygenase 5, MRP3, and P-glycoprotein were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic prediction of systemic and tissue concentration of drugs.

Hydrolases represent an essential class of enzymes indispensable for the metabolism of various clinically essential medications. Individuals exhibit marked differences in the expression and activation of hydrolases, resulting in significant variability in the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs metabolized by these enzymes. The regulation of hydrolase expression and activity involves both genetic polymorphisms and nongenetic factors. This review examines the current understanding of genetic and nongenetic regulators of six clinically significant hydrolases, including carboxylesterase (CES)-1 CES2, arylacetamide deacetylase (AADAC), paraoxonase (PON)-1 PON3, and cathepsin A (CTSA). We explore genetic variants linked to the expression and activity of the hydrolases and their effects on the PK and PD of their substrate drugs. Regarding nongenetic regulators, we focus on the inhibitors and inducers of these enzymes. Additionally, we examine the developmental expression patterns and gender differences in the hydrolases when pertinent information was available. Many genetic and nongenetic regulators were found to be associated with the expression and activity of the hydrolases and PK and PD. However, hydrolases remain generally understudied compared with other drug-metabolizing enzymes, such as cytochrome P450s. The clinical significance of genetic and nongenetic regulators has not yet been firmly established for the majority of hydrolases. Comprehending the mechanisms that underpin the regulation of these enzymes holds the potential to refine therapeutic regimens, thereby enhancing the efficacy and safety of drugs metabolized by the hydrolases.