Metabolism and transport of oxazphosphorines and the clinical implications

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO), and trofosfamide represent an important group of therapeutic agents due to their substantial antitumor and immuno-modulating activity. CPA is widely used as an anticancer drug, an immunosuppressant, and for the mobilization of hematopoetic progenitor cells from the bone marrow into peripheral blood prior to bone marrow transplantation for aplastic anemia, leukemia, and other malignancies. New oxazaphosphorines derivatives have been developed in an attempt to improve selectivity and response with reduced toxicity. These derivatives include mafosfamide (NSC 345842), glufosfamide (D19575, β-D-glucosylisophosphoramide mustard), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). This review highlights the metabolism and transport of these oxazaphosphorines (mainly CPA and IFO, as these two oxazaphosphorine drugs are the most widely used alkylating agents) and the clinical implications. Both CPA and IFO are prodrugs that require activation by hepatic cytochrome P450 (CYP)-catalyzed 4-hydroxylation, yielding cytotoxic nitrogen mustards capable of reacting with DNA molecules to form crosslinks and lead to cell apoptosis and/or necrosis. Such prodrug activation can be enhanced within tumor cells by the CYP-based gene directed-enzyme prodrug therapy (GDEPT) approach. However, those newly synthesized oxazaphosphorine derivatives such as glufosfamide, NSC 612567 and NSC 613060, do not need hepatic activation. They are activated through other enzymatic and/or non-enzymatic pathways. For example, both NSC 612567 and NSC 613060 can be activated by plain phosphodiesterase (PDEs) in plasma and other tissues or by the high-affinity nuclear 3'-5' exonucleases associated with DNA polymerases, such as DNA polymerases and ε. The alternative CYP-catalyzed inactivation pathway by N-dechloroethylation generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA). Various aldehyde dehydrogenases (ALDHs) and glutathione S-transferases (GSTs) are involved in the detoxification of oxazaphosphorine metabolites. The metabolism of oxazaphosphorines is auto-inducible, with the activation of the orphan nuclear receptor pregnane X receptor (PXR) being the major mechanism. Oxazaphosphorine metabolism is affected by a number of factors associated with the drugs (e.g., dosage, route of administration, chirality, and drug combination) and patients (e.g., age, gender, renal and hepatic function). Several drug transporters, such as breast cancer resistance protein (BCRP), multidrug resistance associated proteins (MRP1, MRP2, and MRP4) are involved in the active uptake and efflux of parental oxazaphosphorines, their cytotoxic mustards and conjugates in hepatocytes and tumor cells. Oxazaphosphorine metabolism and transport have a major impact on pharmacokinetic variability, pharmacokinetic-pharmacodynamic relationship, toxicity, resistance, and drug interactions since the drug-metabolizing enzymes and drug transporters involved are key determinants of the pharmacokinetics and pharmacodynamics of oxazaphosphorines. A better understanding of the factors that affect the metabolism and transport of oxazaphosphorines is important for their optional use in cancer chemotherapy.

A mechanistic study of the intestinal absorption of crypotanshinone, the major active constituent of Saliva miltiorrhiza

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models:1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein
(PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (P_lumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (P_blood). P_blood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 µM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

The omega-3 fatty acid, DHA, decreases neuronal cell death in association with altered zinc transport

Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid in neuronal cell membranes. We hypothesize that DHA induces a decrease in neuronal cell death through reduced ZnT3 expression and zinc uptake. Exposure of M17 cells to DHA-deficient medium increased the levels of active caspase-3, relative to levels in DHA-replete cells, confirming the adverse effects of DHA deficiency in promoting neuronal cell death. In DHA-treated M17 cells, zinc uptake was 65% less and ZnT3 mRNA and protein levels were reduced in comparison with DHA-depleted cells. We propose that the neuroprotective function of DHA is exerted through a reduction in cellular zinc levels that in turn inhibits apoptosis.

Process-based modelling of mass transport in the subsurface

Modern societies rely on natural water pathways that include subsurface flow of water and dissolved chemicals. The thesis presents a range of numerical and analytical models for simulating physical, chemical and biological processes in the subsurface, including coastal aquifers, the near-surface vadose zone, and solute transport in biogeochemically active aquifers.

Relationships of land use mix with walking for transport : do land uses and geographical scale matter?

Physical activity and public health recommendations now emphasize the creation of activity-friendly neighborhoods. Mixed land use in a neighborhood is important in this regard, as it reflects the availability of destinations to which residents can walk or ride bicycles, and thus is likely to contribute to residents’ active lifestyles that in turn will influence their overall health. Relationships between land use mix (LUM) and physical activity have not been apparent in some studies, which may be because geographical scale and the specificity of hypothesized environment–behavior associations are not taken into account. We compared the strength of association of four Geographic Information Systems-derived LUM measures with walking for transport and perceived proximity to destinations. We assessed physical activity behaviors of 2,506 adults in 154 Census Collection Districts (CCDs) in Adelaide, Australia, for which ‘‘original’’ LUM measures were calculated, and then refined by either: accounting for the geographic scale of measurement; including only the most-relevant land uses; or, both. The refined (but not the ‘‘original’’) LUM measures had significant associations with the frequency of walking for transport (p < 0.05) and area-corrected measures had significant associations with the duration of walking for transport. All LUM measures had significant associations with perceived proximity to destinations, but stronger associations were seen when using the refined measures compared with the original LUM. Identifying the LUM attributes most strongly associated with walking for transport is a priority and can inform environmental and policy initiatives that are needed to promote health-enhancing physical activity.

Active, healthy cities ? how does population physical activity vary between Australian cities?

Issue addressed: Despite recognition that urban infrastructure influences physical activity, there have been no comparisons between Australian city-level patterns of physical activity. This study ranked Australian cities in terms of adults? participation in leisure-time physical activity and examined city-level variations in activity trends between 2001 and 2009. Methods: Data on participation in leisure-time physical activity in adults (=15 years) between 2001 and 2009 were obtained from the Exercise Recreation and Sport Survey (ERASS), a computer-assisted telephone interview conducted to collect population-level sport participation information by the Australian Sports Commission. Data were analysed for respondents residing in the eight capital cities of Australia. The prevalence of meeting recommended ?health-enhancing physical activity? (HEPA) and levels of walking were calculated by age, gender and survey year. Multiple linear logistic regression analyses were used to compare cities. Results: Pooled data from 174,323 adults across years showed that Melbourne, Brisbane, Perth and Canberra residents were significantly more active than Sydney, Adelaide and Hobart residents in terms of HEPA. Hobart, Perth and Melbourne residents were significantly more likely to walk =5 sessions a week compared with their counterparts in other cities. HEPA and walking increased across most cities between 2001 and 2009. Conclusion: There are significant differences between Australian cities in physical activity and walking levels, over and above differences attributable to age, gender or educational levels. While this may be due to infrastructure differences, comparative information on indicators of the built environment and transport infrastructure are not available.

Perceived neighborhood environmental attributes associated with adults’ transport-related walking and cycling: Findings from the USA, Australia and Belgium

Background : Active transportation has the potential to contribute considerably to overall physical activity levels in adults and is likely to be influenced by neighborhood-related built environment characteristics. Previous studies that examined the associations between built environment attributes and active transportation, focused mainly on transport-related walking and were conducted within single countries, limiting environmental variability. We investigated the direction and shape of relationships of perceived neighborhood attributes with transport-related cycling and walking in three countries; and examined whether these associations differed by country and gender.

Methods Data from the USA (Baltimore and Seattle), Australia (Adelaide) and Belgium (Ghent) were pooled. In total, 6,014 adults (20–65 years, 55.7% women) were recruited in high-/low-walkable and high-/low-income neighborhoods. All participants completed the Neighborhood Environmental Walkability Scale and the International Physical Activity Questionnaire. Generalized additive mixed models were used to estimate the strength and shape of the associations.

Results Proximity to destinations, good walking and cycling facilities, perceiving difficulties in parking near local shopping areas, and perceived aesthetics were included in a ‘cyclability’ index. This index was linearly positively related to transport-related cycling and no gender- or country-differences were observed. The ‘walkability’ index consisted of perceived residential density, land use mix access, proximity of destinations and aesthetics. A non-linear positive relationship with transport-related walking was found. This association was stronger in women than in men, and country-specific associations were identified: the strongest association was observed in Seattle, the weakest in Adelaide. In Ghent, the association weakened at higher levels of walkability.

Conclusions For cycling, consistent correlates were found in the three countries, but associations were less straightforward for transport-related walking. Moreover, the identified neighborhood environmental correlates were different for walking compared to cycling. In order to further clarify the shape of these associations and reach more specific international guidelines for developing walkable and bikeable neighborhoods, future studies should include even more countries to maximize environmental variability.

Key influences on motivations for utility cycling (cycling for transport to and from places)

Issue addressed: 

Understanding water and ion transport behaviour and permeability through poly(amide) thin film composite membrane

Molecular dynamics (MD) together with the adaptive biasing force (ABF) and metadynamics free energy calculation methods was used to investigate the permeation properties of salt water through poly(amide) thin film composite reverse osmosis membranes. The thin films were generated by annealing an amorphous cell of poly(amide) chains through an MD method. The MD results showed they have typical structural properties of the active layer of thin film composite membranes and comparable water diffusivity (2.13×10^-5cm²/s for the film with a density of 1.06g/cm³) and permeability (9.27×10^-15cm³cm/cm²sPa) to experimental data. The simulations of water permeation through the films under different transmembrane pressures revealed the behaviours of water molecules in the thin films and the dynamic regimes of water permeation, including Brownian diffusion, flush and jump diffusion regimes. The intermolecular interactions of water and ions with poly(amide) chains showed a strong dependence on the local structure of films. The attraction between water and ploy(amide) molecules can be up to 8.5kcal/mol in dense polymer regions and 5kcal/mol in the pores of about 3nm. The ABF and metadynamics simulations produced the profiles of free energy potential of water and ions along the depth of the thin films, which provided important information for quantitatively determining the barrier energy required for water permeation and rejection of ions. The thin film with a density of 1.06g/cm³ and a thickness of 6nm offers a rejection to Na⁺ but a slight absorption of Cl^- (0.25kcal/mol) at 0.3-0.4nm distance to its surface. Water molecules must overcome 63kcal/mol energy to move to the centre of the film. The dependences of the barrier energy and the water-polymer interaction energy on the local free volume size in the thin film were analysed. The simulations of water permeation under high transmembrane pressures showed a nonlinear response of the concentration and distribution of water molecules in the film to the imposed pressure. Compaction of the film segments close to the porous substrate and water congestion in dense regions significantly influenced the water permeation when the membrane was operated under pressures of more than 3.0MPa.

Associations of public transport accessibility with walking, obesity, metabolic syndrome and diabetes

Objective: To examine associations of public transport system accessibility with walking, obesity, metabolic syndrome and diabetes/impaired glucose regulation. Methods: Associations of public transport accessibility with self-reported walking for transport or recreation and measured biomarkers of chronic disease risk were estimated in 5241 adult residents of 42 randomly selected areas in Australia in 2004/05, drawn from the second wave of a population-based cohort study (AusDiab). Public transport accessibility was objectively measured using an adaptation of the Public Transport Accessibility Levels (PTAL) methodology, comprising both GIS derived spatial and temporal accessibility measures. Logistic regression models were adjusted for individual and environmental level covariates and clustering within areas. Results: Above median public transport accessibility was positively associated with a walking time of more than the median 90 min per week (OR=1.28, 95%CI 1.03, 1.60) and walking above the recommended 150 min per week (OR=1.35, 95%CI 1.11, 1.63). There were no associations of public transport accessibility with obesity (OR=1.05, 95%CI 0.85, 1.30), the metabolic syndrome (OR=1.09, 95%CI 0.91, 1.31) nor diabetes/impaired glucose regulation (OR=1.11, 95%CI 0.94, 1.30). Findings were similar for a subgroup reporting no vigorous recreational physical activity. Conclusions: In this Australian sample, public transport accessibility was positively associated with walking at recommended levels, including for people who are not otherwise vigorously active. Significance: Walking is crucial for increasing physical activity levels and population health, as well as maximising public transport system efficiency. Building evidence on public transport accessibility and walking will enable governments to exploit this important synergy.

Perceived neighborhood environmental attributes sssociated with walking and cycling for transport among adult residents of 17 cities in 12 countries: the IPEN study

INTRODUCTION: Prevalence of walking and cycling for transport is low and varies greatly across countries. Few studies have examined neighborhood perceptions related to walking and cycling for transport in different countries. Therefore, it is challenging to prioritize appropriate built-environment interventions. OBJECTIVES: The aim of this study was to examine the strength and shape of the relationship between adults' neighborhood perceptions and walking and cycling for transport across diverse environments. METHODS: As part of the International Physical activity and Environment Network (IPEN) adult project, self-reported data were taken from 13,745 adults (18-65 years) living in physically and socially diverse neighborhoods in 17 cities across 12 countries. Neighborhood perceptions were measured using the Neighborhood Environment Walkability Scale, and walking and cycling for transport were measured using the International Physical Activity Questionnaire-Long Form. Generalized additive mixed models were used to model walking or cycling for transport during the last seven days with neighborhood perceptions. Interactions by city were explored. RESULTS: Walking-for-transport outcomes were significantly associated with perceived residential density, land use mix-access, street connectivity, aesthetics, and safety. Any cycling for transport was significantly related to perceived land use mix-access, street connectivity, infrastructure, aesthetics, safety, and perceived distance to destinations. Between-city differences existed for some attributes in relation to walking or cycling for transport. CONCLUSIONS: Many perceived environmental attributes supported both cycling and walking; however, highly walkable environments may not support cycling for transport. People appear to walk for transport despite safety concerns. These findings can guide the implementation of global health strategies.

Role of phosphate transport system component pstB1 in phosphate internalization by Nostoc punctiforme

In bacteria, limited phosphate availability promotes the synthesis of active uptake systems, such as the Pst phosphate transport system. To understand the mechanisms that facilitate phosphate accumulation in the cyanobacterium Nostoc punctiforme, phosphate transport systems were identified, revealing a redundancy of Pst phosphate uptake systems that exists across three distinct operons. Four separate PstB system components were identified. pstB1 was determined to be a suitable target for creating phenotypic mutations that could result in the accumulation of excessive levels of phosphate through its overexpression or in a reduction of the capacity to accumulate phosphate through its deletion. Using quantitative real-time PCR (qPCR), it was determined that pstB1 mRNA levels increased significantly over 64 h in cells cultured in 0 mM added phosphate and decreased significantly in cells exposed to high (12.8 mM) phosphate concentrations compared to the level in cells cultured under normal (0.8 mM) conditions. Possible compensation for the loss of PstB1 was observed when pstB2, pstB3, and pstB4 mRNA levels increased, particularly in cells starved of phosphate. The overexpression of pstB1 increased phosphate uptake by N. punctiforme and was shown to functionally complement the loss of PstB in E. coli PstB knockout (PstB(-)) mutants. The knockout of pstB1 in N. punctiforme did not have a significant effect on cellular phosphate accumulation or growth for the most part, which is attributed to the compensation for the loss of PstB1 by alterations in the pstB2, pstB3, and pstB4 mRNA levels. This study provides novel in vivo evidence that PstB1 plays a functional role in phosphate uptake in N. punctiforme IMPORTANCE: Cyanobacteria have been evolving over 3.5 billion years and have become highly adept at growing under limiting nutrient levels. Phosphate is crucial for the survival and prosperity of all organisms. In bacteria, limited phosphate availability promotes the synthesis of active uptake systems. The Pst phosphate transport system is one such system, responsible for the internalization of phosphate when cells are in phosphate-limited environments. Our investigations reveal the presence of multiple Pst phosphate uptake systems that exist across three distinct operons in Nostoc punctiforme and functionally characterize the role of the gene product PstB1 as being crucial for the maintenance of phosphate accumulation. We demonstrate that the genes pstB2, pstB3, and pstB4 show alterations in expression to compensate for the deletion of pstB1 The overall outcomes of this work provide insights as to the complex transport mechanisms that exist in cyanobacteria like N. punctiforme, allowing them to thrive in low-phosphate environments.