Pharmacokinetic Bioequivalence between Generic and Originator Orally Inhaled Drug Products: Validity of Administration of Doses above the Approved Single Maximum Dose.

Pharmacokinetic bioequivalence of orally inhaled drug products is a critical component of the US FDA's "weight of evidence" approach, and it can serve as the sole indicator of safety and effectiveness of follow-on inhalation products approved in Europe and some other geographic areas. The approved labels of the orally inhaled drug products recommend the maximum number of actuations that can be administered in a single dose on one occasion. This single maximum dose may consist of one or more inhalations depending upon the product. Bioequivalence studies for the inhalation drug product registrations in the US and EU have employed single and multiple actuation doses, in some cases over and above the approved single maximum labeled doses, thus, inconsistent with the approved labeling of the reference products. Pharmacokinetics of inhaled drug products after single and multiple doses may be different, with implications for bioequivalence determined at single and multiple doses. Scientific literature indicates that the relative bioavailability of the Test and Reference products may differ between administrations of doses in one and multiple inhalations. Multiple doses not only alter the pharmacokinetics but also may reduce the sensitivity of the bioassay to actual differences between the Test and Reference product performances. Ability of the pharmacokinetic bioassay to accurately determine the extent of difference between two products may also be substantially reduced at high doses. Therefore, in our opinion, pharmacokinetic bioequivalence to support regulatory approvals of inhalation products at doses above the recommended single maximum dose should be avoided. Furthermore, the bioequivalence of products (if any) established at doses exceeding the approved single maximum doses should be revisited to determine if the products maintain bioequivalence when evaluated at the clinically relevant single maximum doses.

iBCS: 3. A Biopharmaceutics Classification System for Orally Inhaled Drug Products.

In this article, we specify for the first time a quantitative biopharmaceutics classification system for orally inhaled drugs. To date, orally inhaled drug product developers have lacked a biopharmaceutics classification system like the one developed to navigate the development of immediate release of oral medicines. Guideposts for respiratory drug discovery chemists and inhalation product formulators have been elusive and difficult to identify due to the complexity of pulmonary physiology, the intricacies of drug deposition and disposition in the lungs, and the influence of the inhalation delivery device used to deliver the drug as a respirable aerosol. The development of an inhalation biopharmaceutics classification system (iBCS) was an initiative supported by the Product Quality Research Institute (PQRI). The goal of the PQRI iBCS working group was to generate a qualitative biopharmaceutics classification system that can be utilized by inhalation scientists as a "rule of thumb" to identify desirable molecular properties and recognize and manage CMC product development risks based on physicochemical properties of the drug and the deposited lung dose. Herein, we define the iBCS classes quantitatively according to the dose number and permeability. The proposed iBCS was evaluated for its ability to categorize marketed inhaled drugs using data from the literature. The appropriateness of the classification of each drug was assessed based on published development, clinical and nonclinical data, and mechanistic physiologically based biopharmaceutics modeling. The inhaled drug product development challenges for each iBCS classification are discussed and illustrated for different classes of marketed inhaled drugs. Finally, it is recognized that discriminatory laboratory methods to characterize regional lung deposition, dissolution, and permeability will be key to fully realizing the benefits of an iBCS to streamline and derisk inhaled drug development.

What causes cardiac mitochondrial failure at high environmental temperatures?

Although a mechanism accounting for hyperthermic death at critical temperatures remains elusive, the mitochondria of crucial active excitable tissues (i.e. heart and brain) may well be key to this process. Mitochondria produce ∼90% of the ATP required by cells to maintain cellular integrity and function. They also integrate into biosynthetic pathways that support metabolism as a whole, allow communication within the cell, and regulate cellular health and death pathways. We have previously shown that cardiac and brain mitochondria demonstrate decreases in the efficiency of, and absolute capacity for ATP synthesis as temperatures rise, until ultimately there is too little ATP to support cellular demands, and organ failure follows. Importantly, substantial decreases in ATP synthesis occur at temperatures immediately below the temperature of heart failure, and this suggests a causal role of mitochondria in hyperthermic death. However, what causes mitochondria to fail? Here, we consider the answers to this question. Mitochondrial dysfunction at high temperature has classically been attributed to elevated leak respiration suspected to result from increased movement of protons (H+) through the inner mitochondrial membrane (IMM), thereby bypassing the ATP synthases. In this Commentary, we introduce some alternative explanations for elevated leak respiration. We first consider respiratory complex I and then propose that a loss of IMM structure occurs as temperatures rise. The loss of the cristae folds of the IMM may affect the efficiency of H+ transport, increasing H+ conductance either through the IMM or into the bulk water phases of mitochondria. In either case, O2 consumption increases while ATP synthesis decreases.

Pharmacokinetic Considerations for Optimizing Inhaled Spray-Dried Pyrazinoic Acid Formulations.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death with 1.6 million deaths worldwide reported in 2021. Oral pyrazinamide (PZA) is an integral part of anti-TB regimens, but its prolonged use has the potential to drive the development of PZA-resistant Mtb. PZA is converted to the active moiety pyrazinoic acid (POA) by the Mtb pyrazinamidase encoded by pncA, and mutations in pncA are associated with the majority of PZA resistance. Conventional oral and parenteral therapies may result in subtherapeutic exposure in the lung; hence, direct pulmonary administration of POA may provide an approach to rescue PZA efficacy for treating pncA-mutant PZA-resistant Mtb. The objectives of the current study were to (i) develop novel dry powder POA formulations, (ii) assess their feasibility for pulmonary delivery using physicochemical characterization, (iii) evaluate their pharmacokinetics (PK) in the guinea pig model, and (iv) develop a mechanism-based pharmacokinetic model (MBM) using in vivo PK data to select a formulation providing adequate exposure in epithelial lining fluid (ELF) and lung tissue. We developed three POA formulations for pulmonary delivery and characterized their PK in plasma, ELF, and lung tissue following passive inhalation in guinea pigs. Additionally, the PK of POA following oral, intravenous, and intratracheal administration was characterized in guinea pigs. The MBM was used to simultaneously model PK data following administration of POA and its formulations via the different routes. The MBM described POA PK well in plasma, ELF, and lung tissue. Physicochemical analyses and MBM predictions suggested that POA maltodextrin was the best among the three formulations and an excellent candidate for further development as it has: (i) the highest ELF-to-plasma exposure ratio (203) and lung tissue-to-plasma exposure ratio (30.4) compared with POA maltodextrin and leucine (75.7/16.2) and POA leucine salt (64.2/19.3) and (ii) the highest concentration in ELF (CmaxELF: 171 nM) within 15.5 min, correlating with a fast transfer into ELF after pulmonary administration (KPM: 22.6 1/h). The data from the guinea pig allowed scaling, using the MBM to a human dose of POA maltodextrin powder demonstrating the potential feasibility of an inhaled product.

Dynamic defence? Intertidal triplefin species show better maintenance of mitochondrial membrane potential than subtidal species at low oxygen pressures.

Oxygen is essential for most eukaryotic lifeforms, as it supports mitochondrial oxidative phosphorylation to supply ∼90% of cellular adenosine triphosphate (ATP). Fluctuations in O2 present a major stressor, with hypoxia leading to a cascade of detrimental physiological changes that alter cell operations and ultimately induce death. Nonetheless, some species episodically tolerate near-anoxic environments, and have evolved mechanisms to sustain function even during extended hypoxic periods. While mitochondria are pivotal in central metabolism, their role in hypoxia tolerance remains ill defined. Given the vulnerability of the brain to hypoxia, mitochondrial function was tested in brain homogenates of three closely related triplefin species with varying degrees of hypoxia tolerance (Bellapiscis medius, Forsterygion lapillum and Forsterygion varium). High-resolution respirometry coupled with fluorometric measurements of mitochondrial membrane potential (mtMP) permitted assessment of differences in mitochondrial function and integrity in response to intermittent hypoxia and anoxia. Traditional steady-state measures of respiratory flux and mtMP showed no differences among species. However, in the transition into anoxia, the tolerant species B. medius and F. lapillum maintained mtMP at O2 pressures 7- and 4.4-fold lower, respectively, than that of the hypoxia-sensitive F. varium and exhibited slower rates of membrane depolarisation. The results indicate that dynamic oxic-hypoxic mitochondria transitions underlie hypoxia tolerance in these intertidal fish.

Tyvaso DPI: Drug-device characteristics and patient clinical considerations.

Tyvaso DPI is a drug-device combination therapy comprised of a small, portable, reusable, breath-powered, dry powder inhaler (DPI) for the delivery of treprostinil. It is approved for the treatment of pulmonary arterial hypertension and pulmonary hypertension associated with interstitial lung disease. Tyvaso DPI utilizes single-use prefilled cartridges to ensure proper dosing. Unlike nebulizer devices, administration of Tyvaso DPI is passive and does not require coordination with the device. The low-flow rate design results in targeted delivery to the peripheral lungs due to minimal drug loss from impaction in the oropharynx. The inert fumaryl diketopiperazine (FDKP) excipient forms microparticles that carry treprostinil into the airways, with a high fraction of the particles in the respirable range. In a clinical study in patients with pulmonary arterial hypertension, Tyvaso DPI had similar exposure and pharmacokinetics, low incidence of adverse events, and high patient satisfaction compared with nebulized treprostinil solution. Tyvaso DPI may be considered as a first prostacyclin agent or for those that do not tolerate other prostacyclin formulations, patients with pulmonary comorbidities, patients with mixed Group 1 and Group 3 pulmonary hypertension, or those that prefer an active lifestyle and need a portable, non-invasive treatment. Tyvaso DPI is a patient-preferred, maintenance-free, safe delivery option that may improve patient compliance and adherence.

Succinate-mediated reactive oxygen species production in the anoxia-tolerant epaulette (Hemiscyllium ocellatum) and grey carpet (Chiloscyllium punctatum) sharks.

Anoxia/re-oxygenation (AR) results in elevated unchecked oxidative stress and mediates irreversible damage within the brain for most vertebrates. Succinate accumulation within mitochondria of the ischaemic brain appears to increase the production of reactive oxygen species (ROS) upon re-oxygenation. Two closely related elasmobranchs, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) repeatedly experience near anoxia and re-oxygenation in their habitats and have adapted to survive AR at tropical temperatures without significant brain injuries. However, these anoxia-tolerant species display contrasting strategies to survive AR, with only H. ocellatum having the capacity to supress metabolism and H. ocellatum mitochondria the capacity to depress succinate oxidation post-AR. We measured oxygen consumption alongside ROS production mediated by elevated succinate in mitochondria of permeabilized cerebellum from both shark species. Although mitochondrial respiration remained similar for both species, the ROS production in H. ocellatum was half that of C. punctatum in phosphorylating and non-phosphorylating mitochondria. Maximum ROS production in H. ocellatum was mediated by succinate loads 10-fold higher than in C. punctatum mitochondria. The contrasting survival strategies of anoxia-tolerant sharks reveal the significance of mitigating ROS production under elevated succinate load during AR, shedding light on potential mechanisms to mitigate brain injury.

Minimal adaptation of the molecular regulators of mitochondrial dynamics in response to unilateral limb immobilisation and retraining in middle-aged men.

PURPOSE: Mitochondrial dynamics are regulated by the differing molecular pathways variously governing biogenesis, fission, fusion, and mitophagy. Adaptations in mitochondrial morphology are central in driving the improvements in mitochondrial bioenergetics following exercise training. However, there is a limited understanding of mitochondrial dynamics in response to inactivity. METHODS: Skeletal muscle biopsies were obtained from middle-aged males (n = 24, 49.4 ± 3.2 years) who underwent sequential 14-day interventions of unilateral leg immobilisation, ambulatory recovery, and resistance training. We quantified vastus lateralis gene and protein expression of key proteins involved in mitochondrial biogenesis, fusion, fission, and turnover in at baseline and following each intervention. RESULTS: PGC1α mRNA decreased 40% following the immobilisation period, and was accompanied by a 56% reduction in MTFP1 mRNA, a factor involved in mitochondrial fission. Subtle mRNA decreases were also observed in TFAM (17%), DRP1 (15%), with contrasting increases in BNIP3L and PRKN following immobilisation. These changes in gene expression were not accompanied by changes in respective protein expression. Instead, we observed subtle decreases in NRF1 and MFN1 protein expression. Ambulatory recovery restored mRNA and protein expression to pre-intervention levels of all altered components, except for BNIP3L. Resistance training restored BNIP3L mRNA to pre-intervention levels, and further increased mRNA expression of OPA-1, MFN2, MTFP1, and PINK1 past baseline levels. CONCLUSION: In healthy middle-aged males, 2 weeks of immobilisation did not induce dramatic differences in markers of mitochondria fission and autophagy. Restoration of ambulatory physical activity following the immobilisation period restored altered gene expression patterns to pre-intervention levels, with little evidence of further adaptation to resistance exercise training.

A SPLUNC1 Peptidomimetic Inhibits Orai1 and Reduces Inflammation in a Murine Allergic Asthma Model.

Orai1 is a plasma membrane Ca2+ channel that mediates store-operated Ca2+ entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier that inhibits Orai1 and SOCE via its C-terminal α6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled α6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate α6-Orai1 interactions, we used fluorescent assays to measure Ca2+ signaling, Förster resonance energy transfer, fluorescent recovery after photobleaching, immunostaining, total internal reflection microscopy, and Western blotting. To test whether α6 peptidomimetics inhibited SOCE and decreased inflammation in vivo, wild-type and SPLUNC1-/- mice were exposed to house dust mite (HDM) extract with or without α6 peptide. We also performed nebulization, jet milling, and scanning electron microscopy to evaluate α6 for inhalation. SPLUNC1-/- mice had an exaggerated response to HDM. In BAL-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1-/- but not wild-type mice. Inhaled α6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, α6 dose-dependently reduced eosinophilia and neutrophilia. In vitro, α6 inhibited SOCE in multiple immune cell types, and α6 could be nebulized or jet milled without loss of function. These data suggest that α6 peptidomimetics may be a novel, effective antiinflammatory therapy for patients with asthma.

iBCS: 1. Principles and Framework of an Inhalation-Based Biopharmaceutics Classification System.

For oral drugs, the formulator and discovery chemist have a tool available to them that can be used to navigate the risks associated with the selection and development of immediate release oral drugs and drug products. This tool is the biopharmaceutics classification system (giBCS). Unfortunately, no such classification system exists for inhaled drugs. The perspective outlined in this manuscript provides the foundational principles and framework for a classification system for inhaled drugs. The proposed classification system, an inhalation-based biopharmaceutics classification system (iBCS), is based on fundamental biopharmaceutics principles adapted to an inhalation route of administration framework. It is envisioned that a classification system for orally inhaled drugs will facilitate an understanding of the technical challenges associated with the development of new chemical entities and their associated new drug products (device and drug formulation combinations). Similar to the giBCS, the iBCS will be based on key attributes describing the drug substance (solubility and permeability) and the drug product (dose and dissolution). This manuscript provides the foundational aspects of an iBCS, including the proposed scientific principles and framework upon which such a system can be developed.

Cardiac mitochondrial energetics of the Australasian red spiny lobster, Jasus edwardsii, when exposed to isoeugenol within the commercial anaesthetic AQUI-S.

The anaesthetic isoeugenol has been used as metabolic suppressant for commercial transport of live lobsters in order to decrease energy expenditure and improve survival. Given the central role of mitochondria in metabolism and structural similarities of isoeugenol to the mitochondrial electron carrier coenzyme Q, we explored the influence on mitochondrial function of isoeugenol. Mitochondrial function was measured using high-resolution respirometry and saponin-permeabilised heart fibres from the Australasian red spiny lobster, Jasus edwardsii. Relative to vehicle (polysorbate), isoeugenol inhibited respiration supported by complex I (CI) and cytochrome c oxidase (CCO). While complex II (CII), which also reduces coenzyme Q, was largely unaffected by isoeugenol, respiration supported by CII when uncoupled was depressed. Titration of isoeugenol indicates that respiration through CI has a half-maximal inhibitory concentration (IC50) of 2.4±0.1 µmol l-1, and a full-maximal inhibitory concentration (IC100-) of approximately 6.3 µmol l-1. These concentrations are consistent with those used for transport and euthanasia of J. edwardsii and indicate that CI is a possible target of isoeugenol, like many other anaesthetics with quinone-like structures.

Optimization and Scale Up of Spray Dried CPZEN-45 Aerosol Powders for Inhaled Tuberculosis Treatment.

PURPOSE: Tuberculosis (TB) remains one of the most serious diseases caused by a single organism. Multiple (MDR) and extensively (XDR) drug resistant disease poses a threat to global health and requires new drugs and/or innovative approaches to treatment. A number of drugs have been proposed as inhaled therapy for TB, frequently prepared by spray drying. CPZEN-45 is a novel anti-tubercular drug that has poor oral bioavailability but has shown promise when administered via inhalation. METHODS: Excipient-free CPZEN-45 HCl has been spray dried into a powder with physicochemical characteristics, aerodynamic particle size distribution, and delivered dose suitable for consideration as an inhaled product. RESULTS: The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the powder delivered using a RS01 inhaler were 2.62 ± 0.04 µm and 1.76 ± 0.09, respectively. Additionally, the powder was physically and chemically stable after storage at ambient conditions for >1.5 years with particle size similar to freshly manufactured product. Overages in spray dried powder were recycled the powder and resprayed into drug product likewise resulting in negligible change in quality thus allowing for further preclinical characterization as necessary. CPZEN-45 was scaled up using pilot-scale manufacturing equipment where the density of the powder was increased to facilitate larger delivered doses without affecting the aerodynamic performance properties. CONCLUSION: The spray dried powders were suitable for pharmacokinetics, efficacy and preclinical toxicology studies. The final method of manufacture may be used directly for CGMP particle manufacture to support IND and Phase I clinical trials and beyond.

Multiple approaches to repurposing drugs for neuroblastoma.

Neuroblastoma (NB) is the second leading extracranial solid tumor of early childhood with about two-thirds of cases presenting before the age of 5, and accounts for roughly 15 percent of all pediatric cancer fatalities in the United States. Treatments against NB are lacking, resulting in a low survival rate in high-risk patients. A repurposing approach using already approved or clinical stage compounds can be used for diseases for which the patient population is small, and the commercial market limited. We have used Bayesian machine learning, in vitro cell assays, and combination analysis to identify molecules with potential use for NB. We demonstrated that pyronaridine (SH-SY5Y IC50 1.70 µM, SK-N-AS IC50 3.45 µM), BAY 11-7082 (SH-SY5Y IC50 0.85 µM, SK-N-AS IC50 1.23 µM), niclosamide (SH-SY5Y IC50 0.87 µM, SK-N-AS IC50 2.33 µM) and fingolimod (SH-SY5Y IC50 4.71 µM, SK-N-AS IC50 6.11 µM) showed cytotoxicity against NB. As several of the molecules are approved drugs in the US or elsewhere, they may be repurposed more readily for NB treatment. Pyronaridine was also tested in combinations in SH-SY5Y cells and demonstrated an antagonistic effect with either etoposide or crizotinib. Whereas when crizotinib and etoposide were combined with each other they had a synergistic effect in these cells. We have also described several analogs of pyronaridine to explore the structure-activity relationship against cell lines. We describe multiple molecules demonstrating cytotoxicity against NB and the further evaluation of these molecules and combinations using other NB cells lines and in vivo models will be important in the future to assess translational potential.

Exploiting machine learning for end-to-end drug discovery and development.

A variety of machine learning methods such as naive Bayesian, support vector machines and more recently deep neural networks are demonstrating their utility for drug discovery and development. These leverage the generally bigger datasets created from high-throughput screening data and allow prediction of bioactivities for targets and molecular properties with increased levels of accuracy. We have only just begun to exploit the potential of these techniques but they may already be fundamentally changing the research process for identifying new molecules and/or repurposing old drugs. The integrated application of such machine learning models for end-to-end (E2E) application is broadly relevant and has considerable implications for developing future therapies and their targeting.

Voluntary wheel running prevents the acidosis-induced decrease in skeletal muscle mitochondrial reactive oxygen species emission.

Decreases in pH (acidosis) in vitro can alter skeletal muscle mitochondrial function [respiration and reactive oxygen species (ROS) emission]. However, because skeletal muscles readily adapt to exercise, the effects of acidosis may be different on sedentary vs. trained muscle. The aim of this work was to compare the effects of pH on skeletal muscle mitochondrial function between sedentary vs. exercise-trained male Sprague-Dawley rats ( n = 10 in each cohort). Rates of mitochondrial respiration and ROS emission were determined from the soleus muscle of both cohorts over a physiologic range of pH values (pH 6.2-7.1). Exercise-trained rats had 14% higher mean muscle buffering capacities; 46 and 40% greater enzyme activity of citrate synthase and lactate dehydrogenase, respectively; and greater activity of respiratory complexes I-IV. ADP-stimulated respiration with complex I and II substrates was ∼25% greater in exercise-trained rats but was unaffected by pH in either cohort. In both cohorts, lowering pH decreased respiration only in complex I- and complex II-supported nonphosphorylating (leak) state. However, as pH decreased, ROS emissions in complex I- and complex II-supported leak state decreased only in sedentary rats; in exercise-trained rats, ROS emissions in this state remained constant. We hypothesize that this effect may result from modulation at complex III, which declined 47% per unit pH in sedentary rats, in comparison to 23% in exercise-trained rats. Taken together, these data suggest that pH regulates mitochondrial respiratory complexes and that exercise training can decrease the effects of pH on skeletal muscle mitochondrial function.-Hedges, C. P., Bishop, D. J., Hickey, A. J. R. Voluntary wheel running prevents the acidosis-induced decrease in skeletal muscle mitochondrial reactive oxygen species emission.

MitoQ and CoQ10 supplementation mildly suppresses skeletal muscle mitochondrial hydrogen peroxide levels without impacting mitochondrial function in middle-aged men.

PURPOSE: Excess production of reactive oxygen species (ROS) from the mitochondria can promote mitochondrial dysfunction and has been implicated in the development of a range of chronic diseases. As such there is interest in whether mitochondrial-targeted antioxidant supplementation can attenuate mitochondrial-associated oxidative stress. We investigated the effect of MitoQ and CoQ10 supplementation on oxidative stress and skeletal muscle mitochondrial ROS levels and function in healthy middle-aged men. METHODS: Skeletal muscle and blood samples were collected from twenty men (50 ± 1 y) before and following six weeks of daily supplementation with MitoQ (20 mg) or CoQ10 (200 mg). High-resolution respirometry was used to determine mitochondrial respiration and H2O2 levels, markers of mitochondrial mass and antioxidant defences were measured in muscle samples and oxidative stress markers in urine and blood samples. RESULTS: Both MitoQ and CoQ10 supplementation suppressed mitochondrial net H2O2 levels during leak respiration, while MitoQ also elevated muscle catalase expression. However, neither supplement altered urine F2-isoprostanes nor plasma TBARS levels. Neither MitoQ nor CoQ10 supplementation had a significant impact on mitochondrial respiration or mitochondrial density markers (citrate synthase, mtDNA/nDNA, PPARGC1A, OXPHOS expression). CONCLUSION: Our results suggest that neither MitoQ and CoQ10 supplements impact mitochondrial function, but both can mildly suppress mitochondrial ROS levels in healthy middle-aged men, with some indication that MitoQ may be more effective than CoQ10.

Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

Muscle mass drives cost in sexually selected arthropod weapons.

Sexually selected weapons often function as honest signals of fighting ability. If poor-quality individuals produce high-quality weapons, then receivers should focus on other, more reliable signals. Cost is one way to maintain signal integrity. The costs of weapons tend to increase with relative weapon size, and thereby restrict large weapons to high-quality individuals who can produce and maintain them. Weapon cost, however, appears to be unpredictably variable both within and across taxa, and the mechanisms underlying this variation remain unclear. We suggest variation in weapon cost may result from variation in weapon composition-specifically, differences in the amount of muscle mass directly associated with the weapon. We test this idea by measuring the metabolic cost of sexually selected weapons in seven arthropod species and relating these measures to weapon muscle mass. We show that individuals with relatively large weapon muscles have disproportionately high resting metabolic rates and provide evidence that this trend is driven by weapon muscle mass. Overall, our results suggest that variation in weapon cost can be partially explained by variation in weapon morphology and that the integrity of weapon signals may be maintained by increased metabolic cost in species with relatively high weapon muscle mass.

Mitochondrial plasticity in the cerebellum of two anoxia-tolerant sharks: contrasting responses to anoxia/re-oxygenation.

Exposure to anoxia leads to rapid ATP depletion, alters metabolic pathways and exacerbates succinate accumulation. Upon re-oxygenation, the preferential oxidation of accumulated succinate most often impairs mitochondrial function. Few species can survive prolonged periods of hypoxia and anoxia at tropical temperatures and those that do may rely on mitochondria plasticity in response to disruptions to oxygen availability. Two carpet sharks, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) display different adaptive responses to prolonged anoxia: while H. ocellatum enters energy-conserving metabolic depression, C. punctatum temporarily elevates its haematocrit, prolonging oxygen delivery. High-resolution respirometry was used to investigate mitochondrial function in the cerebellum, a highly metabolically active organ that is oxygen sensitive and vulnerable to injury after anoxia/re-oxygenation (AR). Succinate was titrated into cerebellar preparations in vitro, with or without pre-exposure to AR, then the activity of mitochondrial complexes was examined. As in most vertebrates, C. punctatum mitochondria significantly increased succinate oxidation rates, with impaired complex I function post-AR. In contrast, H. ocellatum mitochondria inhibited succinate oxidation rates and both complex I and II capacities were conserved, resulting in preservation of oxidative phosphorylation capacity post-AR. Divergent mitochondrial plasticity elicited by elevated succinate post-AR parallels the inherently divergent physiological adaptations of these animals to prolonged anoxia, namely the absence (C. punctatum) and presence (H. ocellatum) of metabolic depression. As anoxia tolerance in these species also occurs at temperatures close to that for humans, examining their mitochondrial responses to AR could provide insights for novel interventions in clinical settings.

Insights on the impact of mitochondrial organisation on bioenergetics in high-resolution computational models of cardiac cell architecture.

Recent electron microscopy data have revealed that cardiac mitochondria are not arranged in crystalline columns but are organised with several mitochondria aggregated into columns of varying sizes spanning the cell cross-section. This raises the question-how does the mitochondrial arrangement affect the metabolite distributions within cardiomyocytes and what is its impact on force dynamics? Here, we address this question by employing finite element modeling of cardiac bioenergetics on computational meshes derived from electron microscope images. Our results indicate that heterogeneous mitochondrial distributions can lead to significant spatial variation across the cell in concentrations of inorganic phosphate, creatine (Cr) and creatine phosphate (PCr). However, our model predicts that sufficient activity of the creatine kinase (CK) system, coupled with rapid diffusion of Cr and PCr, maintains near uniform ATP and ADP ratios across the cell cross sections. This homogenous distribution of ATP and ADP should also evenly distribute force production and twitch duration with contraction. These results suggest that the PCr shuttle and associated enzymatic reactions act to maintain uniform force dynamics in the cell despite the heterogeneous mitochondrial organization. However, our model also predicts that under hypoxia activity of mitochondrial CK enzymes and diffusion of high-energy phosphate compounds may be insufficient to sustain uniform ATP/ADP distribution and hence force generation.