Development and application of a PBPK modeling strategy to support antimalarial drug development.

As part of a collaboration between Medicines for Malaria Venture (MMV), Certara UK and Monash University, physiologically-based pharmacokinetic (PBPK) models were developed for 20 antimalarials, using data obtained from standardized in vitro assays and clinical studies within the literature. The models have been applied within antimalarial drug development at MMV for more than 5 years. During this time, a strategy for their impactful use has evolved. All models are described in the supplementary material and are available to researchers. Case studies are also presented, demonstrating real-world development and clinical applications, including the assessment of the drug-drug interaction liability between combination partners or with co-administered drugs. This work emphasizes the benefit of PBPK modeling for antimalarial drug development and decision making, and presents a strategy to integrate it into the research and development process. It also provides a repository of shared information to benefit the global health research community.

DEC-7/SUSD2, a sushi domain-containing protein, regulates an ultradian behavior mediated by intestinal epithelial Ca2+ oscillations in Caenorhabditis elegans.

In Caenorhabditis elegans, rhythmic posterior body wall muscle contractions mediate the highly regular defecation cycle. These contractions are regulated by inositol-1,4,5-trisphosphate (InsP3) receptor-dependent Ca2+ oscillations in intestinal epithelial cells. Here, we find that mutations in dec-7, which encodes the nematode ortholog of the human Sushi domain-containing 2 protein (SUSD2), lead to an increase in InsP3 receptor-dependent rhythmic posterior body wall muscle contractions. DEC-7 is highly expressed in the intestinal epithelia and localizes to the cell-cell junction. The increase in rhythmic activity caused by the loss of dec-7 is dependent on the innexin gap junction protein INX-16. Moreover, DEC-7 is required for the clustering of INX-16 to the cell-cell junction of the intestinal epithelia. We hypothesize that DEC-7/SUSD2 regulates INX-16 activity to mediate the rhythmic frequency of the defecation motor program. Thus, our data indicate a critical role of a phylogenetically conserved cell-cell junction protein in mediating an ultradian rhythm in the intestinal epithelia of C. elegans.NEW & NOTEWORTHY The conserved complement group protein DEC-7/SUSD2 acts at the apical cell-cell junction of C. elegans intestinal epithelia to mediate gap junction protein organization and function to facilitate a Ca2+ wave-regulated ultradian behavior.

Development and verification of an endogenous PBPK model to inform hydrocortisone replacement dosing in children and adults with cortisol deficiency.

The goal of hormone replacement is to mirror physiology. Hydrocortisone granules and modified release formulations are being developed to optimise cortisol replacement in the rare disease of adrenal insufficiency. To facilitate clinical development, we built and verified a physiologically based pharmacokinetic (PBPK) model for the endogenous hormone cortisol (hydrocortisone) in healthy adults, and children and adults with adrenal insufficiency. The model predicted immediate-release hydrocortisone pharmacokinetics in adults across the dose range 0.5 to 20 mg, with predicted/observed AUCs within 0.8 to 1.25-fold.  The model also tightly predicted pharmacokinetic parameters for modified-release formulations, with AUCs within 0.8 to 1.25-fold after single and multiple dosing.  Predicted modified-release formulation pharmacokinetics (PK) in 12 to 18-year olds showed PK to be similar to adults. This hydrocortisone PBPK model is a useful tool to predict adult and paediatric pharmacokinetics of both immediate- and modified-release hydrocortisone formulations, and develop clinical dosing regimens.

Preclinical evaluation of the first intravenous small molecule MDM2 antagonist alone and in combination with temozolomide in neuroblastoma.

High-risk neuroblastoma, a predominantly TP53 wild-type (wt) tumour, is incurable in >50% patients supporting the use of MDM2 antagonists as novel therapeutics. Idasanutlin (RG7388) shows in vitro synergy with chemotherapies used to treat neuroblastoma. This is the first study to evaluate the in vivo efficacy of the intravenous idasanutlin prodrug, RO6839921 (RG7775), both alone and in combination with temozolomide in TP53 wt orthotopic neuroblastoma models. Detection of active idasanutlin using liquid chromatography-mass spectrometry and p53 pathway activation by ELISA assays and Western analysis showed peak plasma levels 1 h post-treatment with maximal p53 pathway activation 3-6 h post-treatment. RO6839921 and temozolomide, alone or in combination in mice implanted with TP53 wt SHSY5Y-Luc and NB1691-Luc cells showed that combined RO6839921 and temozolomide led to greater tumour growth inhibition and increase in survival compared to vehicle control. Overall, RO6839921 had a favourable pharmacokinetic profile consistent with intermittent dosing and was well tolerated alone and in combination. These preclinical studies support the further development of idasanutlin in combination with temozolomide in neuroblastoma in early phase clinical trials.

Presenilin mutations deregulate mitochondrial Ca2+ homeostasis and metabolic activity causing neurodegeneration in Caenorhabditis elegans.

Mitochondrial dysfunction and subsequent metabolic deregulation is observed in neurodegenerative diseases and aging. Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) cause most cases of familial Alzheimer's disease (AD); however, the underlying mechanism of pathogenesis remains unclear. Here, we show that mutations in the C. elegans gene encoding a PSEN homolog, sel-12 result in mitochondrial metabolic defects that promote neurodegeneration as a result of oxidative stress. In sel-12 mutants, elevated endoplasmic reticulum (ER)-mitochondrial Ca2+ signaling leads to an increase in mitochondrial Ca2+ content which stimulates mitochondrial respiration resulting in an increase in mitochondrial superoxide production. By reducing ER Ca2+ release, mitochondrial Ca2+ uptake or mitochondrial superoxides in sel-12 mutants, we demonstrate rescue of the mitochondrial metabolic defects and prevent neurodegeneration. These data suggest that mutations in PSEN alter mitochondrial metabolic function via ER to mitochondrial Ca2+ signaling and provide insight for alternative targets for treating neurodegenerative diseases.

Development of a physiologically based pharmacokinetic model of actinomycin D in children with cancer.

AIMS: Use of the anti-tumour antibiotic actinomycin D is associated with development of hepatotoxicity, particularly in young children. A paucity of actinomycin D pharmacokinetic data make it challenging to develop a sound rationale for defining dosing regimens in younger patients. The study aim was to develop a physiologically based pharmacokinetic (PBPK) model using a combination of data from the literature and generated from experimental analyses. METHODS: Assays to determine actinomycin D Log P, blood:plasma partition ratio and ABCB1 kinetics were conducted. These data were combined with physiochemical properties sourced from the literature to generate a compound file for use within the modelling-simulation software Simcyp (version 14 release 1). For simulation, information was taken from two datasets, one from 117 patients under the age of 21 and one from 20 patients aged 16-48. RESULTS: The final model incorporated clinical renal and biliary clearance data and an additional systemic clearance value. The mean AUC0-26h of simulated subjects was within 1.25-fold of the observed AUC0-26h (84 ng h ml(-1) simulated vs. 93 ng h ml(-1) observed). For the younger age ranges, AUC predictions were within two-fold of observed values, with simulated data from six of the eight age/dose ranges falling within 15% of observed data. Simulated values for actinomycin D AUC0-26h and clearance in infants aged 0-12 months ranged from 104 to 115 ng h ml(-1) and 3.5-3.8 l h(-1) , respectively. CONCLUSIONS: The model has potential utility for prediction of actinomycin D exposure in younger patients and may help guide future dosing. However, additional independent data from neonates and infants is needed for further validation. Physiological differences between paediatric cancer patients and healthy children also need to be further characterized and incorporated into PBPK models.

Does age affect gastric emptying time? A model-based meta-analysis of data from premature neonates through to adults.

PURPOSE: Gastric emptying (GE) is often reported to be slower and more irregular in premature neonates than in older children and adults. The aim of this study was to investigate the impact of age and other covariates on the rate of GE. METHODS: The effect of age on the mean gastric residence times (MGRT) of liquid and solid food was assessed by analysing 49 published studies of 1457 individuals, aged from 28 weeks gestation to adults. The data were modelled using the nonlinear mixed-effects approach within NONMEM version 7.2 (ICON, Dublin, Ireland), with evaluation of postnatal age, gestational age and meal type as covariates. A double Weibull function was selected as a suitable model since it could account for the typical biphasic nature of GE. RESULTS: Age was not a significant covariate for GE but meal type was. Aqueous solutions were associated with the fastest emptying time (mean simulated gastric residence time of 45 min) and solid food was associated with the slowest (98 min). CONCLUSIONS: These findings challenge the assertion that GE is different in neonates, as compared with older children and adults due to age, and they reinforce the significance of food type in modulating GE.

Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Activation of Wnt signaling using lithium chloride: inquiry-based undergraduate laboratory exercises.

Zebrafish provide researchers and students alike with an excellent model of vertebrate nervous system development due to a high degree of conserved developmental mechanisms and transparent embryos that develop in synchrony. In these laboratory exercises, undergraduate students explore cell biological concepts while performing hypothesis-driven novel research utilizing methodologies such as immunofluorescence, confocal microscopy, image analysis, pharmacology, and basic statistics. In the first block of exercises, students perform anti-acetylated tubulin (anti-AT) immunofluorescence, identify spinal tracts and neuronal subtypes, and perform conventional and confocal microscopy. Building on knowledge acquired in the first block of exercises, during the second block, students subsequently perform pharmacological activation of Wnt signaling through lithium chloride treatments, and assess nervous system integrity through anti-AT immunofluorescence. Students perform various quantitative methods and apply statistics to determine outcomes of Wnt activation. In their final laboratory report, students contextualize their results with foundations of molecular mechanisms of nervous system development. In sum, these exercises offer undergraduate students a model of independent research at the graduate level.

Exploring community gardens in a health disparate population: findings from a mixed methods pilot study.

BACKGROUND: Despite recommendations, there have been few efforts to apply the community-based participatory research (CBPR) approach in the development, implementation, and evaluation of community gardens. OBJECTIVES: As guided by the CBPR approach and grounded in a social-ecological model and behavioral theory, the purpose of this mixed methods study was to understand opinions and interests in developing and implementing a community garden and to understand factors impacting fruit, vegetable, and gardening behaviors. METHODS: Community and academic members collaborated to develop and execute this study. The qualitative phase- targeting regional key informants-was designed to elicit perceived benefits and challenges of community gardens at the environmental, community, and individual levels. The quantitative phase targeted low resourced youth and parents and included a variety of validated theory-based questionnaires to understand factors impacting fruit, vegetable, and gardening behaviors. RESULTS: Major benefits of community gardens that emerged from the 10 qualitative interviews included increasing community cohesion and improving nutrition and physical activity factors. The quantitative phase included 87 youth and 67 parents. Across 16 items for fruits and vegetables, the average willingness to try was 1.32 (standard deviation [SD] = 0.40) on a 2-point scale. The majority of youth indicated they would work in a garden (n = 59; 68%) and eat food grown in their garden (n = 71; 82%). Among parents, gardening attitude, belief, and self-efficacy scores were all above average; however, gardening intentions were neutral. CONCLUSION: This research illustrates the successful partnering a community-academic team and has provided the partnership with a clearer lens to conceptualize and launch future regional community garden efforts.

Large isoforms of UNC-89 (obscurin) are required for muscle cell architecture and optimal calcium release in Caenorhabditis elegans.

Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction.

Midline crossing is not required for subsequent pathfinding decisions in commissural neurons.

BACKGROUND: Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. While midline crossing is achieved through coordinated signaling of attractive and repulsive cues, this has never been demonstrated at the single cell level. Further, though growth cone responsiveness to guidance cues changes after crossing the midline, it is unclear whether midline crossing itself is required for subsequent guidance decisions in vivo. In the zebrafish, spinal commissures are initially formed by a pioneer neuron called CoPA (Commissural Primary Ascending). Unlike in other vertebrate models, CoPA navigates the midline alone, allowing for single-cell analysis of axon guidance mechanisms. RESULTS: We provide evidence that CoPA expresses the known axon guidance receptors dcc, robo3 and robo2. Using loss of function mutants and gene knockdown, we show that the functions of these genes are evolutionarily conserved in teleosts and that they are used consecutively by CoPA neurons. We also reveal novel roles for robo2 and robo3 in maintaining commissure structure. When midline crossing is prevented in robo3 mutants and dcc gene knockdown, ipsilaterally projecting neurons respond to postcrossing guidance cues. Furthermore, DCC inhibits Robo2 function before midline crossing to allow a midline approach and crossing. CONCLUSIONS: Our results demonstrate that midline crossing is not required for subsequent guidance decisions by pioneer axons and that this is due, in part, to DCC inhibition of Robo2 function prior to midline crossing.

Tcf3 inhibits spinal cord neurogenesis by regulating sox4a expression.

The Lef/Tcf factor Tcf3 is expressed throughout the developing vertebrate central nervous system (CNS), but its function and transcriptional targets are uncharacterized. Tcf3 is thought to mediate canonical Wnt signaling, which functions in CNS patterning, proliferation and neurogenesis. In this study, we examine Tcf3 function in the zebrafish spinal cord, and find that this factor does not play a general role in patterning, but is required for the proper expression of Dbx genes in intermediate progenitors. In addition, we show that Tcf3 is required to inhibit premature neurogenesis in spinal progenitors by repressing sox4a, a known mediator of spinal neurogenesis. Both of these functions are mediated by Tcf3 independently of canonical Wnt signaling. Together, our data indicate a novel mechanism for the regulation of neurogenesis by Tcf3-mediated repression.

Proliferation and patterning are mediated independently in the dorsal spinal cord downstream of canonical Wnt signaling.

Canonical Wnt signaling can regulate proliferation and patterning in the developing spinal cord, but the relationship between these functions has remained elusive. It has been difficult to separate the distinct activities of Wnts because localized changes in proliferation could conceivably alter patterning, and gain and loss of function experiments have resulted in both types of defects. To resolve this issue we have investigated canonical Wnt signaling in the zebrafish spinal cord using multiple approaches. We demonstrate that Wnt signaling is required initially for proliferation throughout the entire spinal cord, and later for patterning dorsal progenitor domains. Furthermore, we find that spinal cord patterning is normal in embryos after cell division has been pharmacologically blocked. Finally, we determine the transcriptional mediators of Wnt signaling that are responsible for patterning and proliferation. We show that tcf7 gene knockdown results in dorsal patterning defects without decreasing the mitotic index in dorsal domains. In contrast, tcf3 gene knockdown results in a reduced mitotic index without affecting dorsal patterning. Together, our work demonstrates that proliferation and patterning in the developing spinal cord are separable events that are regulated independently by Wnt signaling.

Reiterated Wnt signaling during zebrafish neural crest development.

While Wnt/beta-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/beta-catenin signaling, and inhibited endogenous Wnt/beta-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest, which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, we established that wnt8 is expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development.

The tibial-1 pioneer pathway: an in vivo model for neuronal outgrowth and guidance.

As neurons extend axons to their targets during development, growth cones must reorient their direction of migration in response to extracellular guidance cues. A variety of model systems have been employed in order to dissect the cellular and molecular mechanisms that underlie this complex process. One preparation, the developing grasshopper limb bud, has proved to offer a number of advantages in which to examine mechanisms of growth cone guidance and motility in vivo. First, the relatively large size of the embryonic nervous system allows for straightforward imaging of both fixed and live neurons in vivo. Second, the peripheral nerves generated in the limb bud are highly stereotyped. Third, intact embryos can be cultured for a period of days, allowing for fairly easy perturbations at precise developmental stages. Fourth, due to the ease of dissection, numerous cell biological and molecular techniques can be utilized in the limb bud. Finally, axon guidance molecules and mechanisms are conserved between grasshoppers and other organism, including vertebrates.

Migrating mesoderm establish a uniform distribution of laminin in the developing grasshopper embryo.

The basal lamina is composed of molecules which physically interact to form a network that serves as a migrational scaffold for many cell types. In the developing peripheral nervous system of the grasshopper, neuronal growth cones are intimately associated with the basal lamina as they migrate. Laminin is a major component of the basal lamina and is a potent promoter of neurite outgrowth in vitro. However, it is unclear what the source of laminin is or how the distribution of laminin within the basal lamina is established. To address this question, grasshopper laminin subunit genes were cloned. As expected, laminin was found within the basal lamina throughout the embryo, in particular in the limb bud, where its expression is coincident with the outgrowth and guidance of the Tibial (Til) pioneer neurons. Surprisingly, the synthesis of beta and gamma chains of laminin was restricted to migratory mesodermal cells, while in other nonmigratory tissues, such as epithelium and presumptive muscle, beta and gamma chains of laminin were not detected. In spite of this, laminin immunoreactivity in the basal lamina appears uniform and is available as a substrate for axonal outgrowth.