Genetic and Non-Genetic Determinants of Raltegravir Penetration into Cerebrospinal Fluid: A Single Arm Pharmacokinetic Study

Background

Antiretroviral drugs vary in their central nervous system penetration, with better penetration possibly conferring neurocognitive benefit during human immunodeficiency virus (HIV) therapy. The efflux transporter gene ABCB1 is expressed in the blood-brain barrier, and an ABCB1 variant (3435C→T) has been reported to affect ABCB1 expression. The integrase inhibitor raltegravir is a substrate for ABCB1. We examined whether ABCB1 3435C→T affects raltegravir disposition into cerebrospinal fluid (CSF), and explored associations with polymorphisms in other membrane transporter genes expressed in the blood-brain barrier.

Methods

Forty healthy, HIV-negative adults of European descent (20 homozygous for ABCB1 3435 C/C, 20 homozygous for 3435 T/T, each group divided equally between males and females) were given raltegravir 400 mg twice daily for 7 days. With the final dose, plasma was collected for pharmacokinetic analysis at 9 timepoints over 12 hours, and CSF collected 4 hours post dose.

Results

The 4-hour CSF concentration correlated more strongly with 2-hour (r²=0.76, P=1.12x10^-11) than 4-hour (r²=0.47, P=6.89x10^-6) single timepoint plasma concentration, and correlated strongly with partial plasma area-under-the-curve values (AUC_0-4h r²=0.86, P=5.15x10^-16). There was no significant association between ABCB1 3435C→T and ratios of CSF-to-plasma AUC or concentration (p>0.05 for each comparison). In exploratory analyses, CSF-to-plasma ratios were not associated with 276 polymorphisms across 16 membrane transporter genes.

Conclusions

Among HIV-negative adults, CSF raltegravir concentrations do not differ by ABCB1 3435C→T genotype but strongly correlate with plasma exposure.

Trial Registration

ClinicalTrials.gov NCT00729924 http://clinicaltrials.gov/show/NCT00729924     

Oral but Not Intravenous Glucose Acutely Decreases Circulating Interleukin-6 Concentrations in Overweight Individuals

Background

Plasma interleukin-6 (IL-6) concentrations decrease acutely 1 h after ingestion of a glucose load or mixed meals and this may be mediated by an anti-inflammatory effect of insulin. The aim of the present study was to compare the effect of higher versus lower insulin levels on plasma IL-6 concentrations following oral compared with intravenous glucose administration in overweight/obese subjects.

Methods and Findings

Fifteen subjects (12 women and 3 men) with BMI >28 kg/m² were given an oral glucose load (75g) followed a week later by an intravenous infusion of glucose aimed at matching plasma glucose concentrations during the oral glucose load. A week later, they drank a volume of water equivalent to the volume consumed with the oral glucose load. Plasma glucose, insulin, nonesterified fatty acids, and IL-6 concentrations and blood hematocrit were measured at 30 minute intervals for 2 h following each intervention. Plasma IL-6 decreased (13–20%) significantly (P = 0.009) at 30 min to 90 min following the oral glucose load and did not change significantly following the other two interventions. The incremental area under the curve for plasma IL-6 concentrations following oral intake of glucose was significantly lower compared with concentrations following intravenous glucose (P = 0.005) and water control (P = 0.02). Circulating insulin concentrations were significantly (P<0.001) and 2.8 fold higher following oral compared with intravenous glucose administration.

Conclusions

These data show that plasma IL-6 concentrations did not decrease during isoglycemic, intravenous glucose administration suggesting that the markedly higher circulating insulin levels and/or gut-related factors may mediate the acute decrease in plasma IL-6 after oral glucose intake in overweight/obese subjects.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12612000491864     

Strength in numbers? Cytotype frequency mediates effect of reproductive barriers in mixed-ploidy arrays

When differentiated lineages come into contact, their fates depend on demographic and reproductive factors. These factors have been well-studied in taxa of the same ploidy, but less is known about sympatric lineages that differ in ploidy, particularly with respect to demographic factors. We assessed prezygotic, postzygotic, and total reproductive isolation in naturally pollinated arrays of diploid-tetraploid and tetraploid-hexaploid population mixes of Campanula rotundifolia by measuring pollinator transitions, seed yield, germination rate, and proportion of hybrid offspring. Four frequencies of each cytotype were tested, and pollinators consistently overvisited rare cytotypes. Seed yield and F1 hybrid production were greater in 4X-6X arrays than 2X-4X arrays, whereas germination rates were similar, creating two distinct patterns of reproductive isolation. In 2X-4X arrays, postzygotic isolation was near complete (3% hybrid offspring), and prezygotic isolation associated with pollinator preference is expected to facilitate the persistence of minority cytotypes. However, in 4X-6X arrays where postzygotic isolation permitted hybrid formation (44% hybrids), pollinator behavior drove patterns of reproductive isolation, with rare cytotypes being more isolated and greater gene flow expected from rare into common cytotypes. In polyploid complexes, both the specific cytotypes in contact and local cytotype frequency, likely reflecting spatial demography, will influence likelihood of gene exchange.     

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from Methanobrevibacter ruminantium

Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C₁ unit carrier where N⁵‐formyl‐tetrahydromethanopterin is converted to methenyl‐tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N‐terminal domain of six α‐helices encompassing a series of four β‐sheets and a predominantly anti‐parallel β–sheet at the C‐terminus flanked on one side by α‐helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation. Proteins 2013; 81:2064–2070. © 2013 Wiley Periodicals, Inc.     

Towards an evolutionary theory of the origin of life based on kinetics and thermodynamics

A sudden transition in a system from an inanimate state to the living state—defined on the basis of present day living organisms—would constitute a highly unlikely event hardly predictable from physical laws. From this uncontroversial idea, a self-consistent representation of the origin of life process is built up, which is based on the possibility of a series of intermediate stages. This approach requires a particular kind of stability for these stages—dynamic kinetic stability (DKS)—which is not usually observed in regular chemistry, and which is reflected in the persistence of entities capable of self-reproduction. The necessary connection of this kinetic behaviour with far-from-equilibrium thermodynamic conditions is emphasized and this leads to an evolutionary view for the origin of life in which multiplying entities must be associated with the dissipation of free energy. Any kind of entity involved in this process has to pay the energetic cost of irreversibility, but, by doing so, the contingent emergence of new functions is made feasible. The consequences of these views on the studies of processes by which life can emerge are inferred.     

Reassessing the Role of DotF in the Legionella pneumophila Type IV Secretion System

Legionella pneumophila, the causative agent of a severe pneumonia termed Legionnaires’ Disease, survives and replicates within both protozoan hosts and human alveolar macrophages. Intracellular survival is dependent upon secretion of a plethora of protein effectors that function to form a replicative vacuole, evade the endocytic pathway and subvert host immune defenses. Export of these factors requires a type IV secretion system (T4SS) called Dot/Icm that is composed of twenty-seven proteins. This report focuses on the DotF protein, which was previously postulated to have several different functions, one of which centered on binding Dot/Icm substrates. In this report, we examined if DotF functions as the T4SS inner membrane receptor for Dot/Icm substrates. Although we were able to recapitulate the previously published bacterial two-hybrid interaction between DotF and several substrates, the interaction was not dependent on the Dot/Icm substrates’ signal sequences as predicted for a substrate:receptor interaction. In addition, binding did not require the cytoplasmic domain of DotF, which was anticipated to be involved in recognizing substrates in the cytoplasm. Finally, inactivation of dotF did not abolish intracellular growth of L. pneumophila or translocation of substrates, two phenotypes dependent on the T4SS receptor. These data strongly suggest that DotF does not act as the major receptor for Dot/Icm substrates and therefore likely performs an accessory function within the core-transmembrane subcomplex of the L. pneumophila Dot/Icm type IV secretion system.     

Development of an HPLC method for the determination of doripenem in human and mouse serum

A HPLC method utilizing solid phase extraction was developed to analyze doripenem (formerly S-4661) in human and mouse serum. A reversed-phase column was used with a UV detector set at 295 nm. The mobile phase consisted of methanol and phosphate buffer at a flow rate of 1.5 ml/min. Meropenem was used as the internal standard. The standard curve was linear over a range of 0.5-40 microg/ml. The assay is simple, reproducible, and accurate and has been used successfully to analyze doripenem concentrations from a murine pharmacokinetic study.     

Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene

Non-somatic synaptic and axonal compartments of neurons are primary pathological targets in many neurodegenerative conditions, ranging from Alzheimer disease through to motor neuron disease. Axons and synapses are protected from degeneration by the slow Wallerian degeneration (Wld(s)) gene. Significantly the molecular mechanisms through which this spontaneous genetic mutation delays degeneration remain controversial, and the downstream protein targets of Wld(s) resident in non-somatic compartments remain unknown. In this study we used differential proteomics analysis to identify proteins whose expression levels were significantly altered in isolated synaptic preparations from the striatum of Wld(s) mice. Eight of the 16 proteins we identified as having modified expression levels in Wld(s) synapses are known regulators of mitochondrial stability and degeneration (including VDAC1, Aralar1, and mitofilin). Subsequent analyses demonstrated that other key mitochondrial proteins, not identified in our initial screen, are also modified in Wld(s) synapses. Of the non-mitochondrial proteins identified, several have been implicated in neurodegenerative diseases where synapses and axons are primary pathological targets (including DRP-2 and Rab GDP dissociation inhibitor beta). In addition, we show that downstream protein changes can be identified in pathways corresponding to both Ube4b (including UBE1) and Nmnat1 (including VDAC1 and Aralar1) components of the chimeric Wld(s) gene, suggesting that full-length Wld(s) protein is required to elicit maximal changes in synaptic proteins. We conclude that altered mitochondrial responses to degenerative stimuli are likely to play an important role in the neuroprotective Wld(s) phenotype and that targeting proteins identified in the current study may lead to novel therapies for the treatment of neurodegenerative diseases in humans.     

Determination of the processes driving the acquisition of immunity to malaria using a mathematical transmission model

Acquisition of partially protective immunity is a dominant feature of the epidemiology of malaria among exposed individuals. The processes that determine the acquisition of immunity to clinical disease and to asymptomatic carriage of malaria parasites are poorly understood, in part because of a lack of validated immunological markers of protection. Using mathematical models, we seek to better understand the processes that determine observed epidemiological patterns. We have developed an age-structured mathematical model of malaria transmission in which acquired immunity can act in three ways (“immunity functions”): reducing the probability of clinical disease, speeding the clearance of parasites, and increasing tolerance to subpatent infections. Each immunity function was allowed to vary in efficacy depending on both age and malaria transmission intensity. The results were compared to age patterns of parasite prevalence and clinical disease in endemic settings in northeastern Tanzania and The Gambia. Two types of immune function were required to reproduce the epidemiological age-prevalence curves seen in the empirical data; a form of clinical immunity that reduces susceptibility to clinical disease and develops with age and exposure (with half-life of the order of five years or more) and a form of anti-parasite immunity which results in more rapid clearance of parasitaemia, is acquired later in life and is longer lasting (half-life of >20 y). The development of anti-parasite immunity better reproduced observed epidemiological patterns if it was dominated by age-dependent physiological processes rather than by the magnitude of exposure (provided some exposure occurs). Tolerance to subpatent infections was not required to explain the empirical data. The model comprising immunity to clinical disease which develops early in life and is exposure-dependent, and anti-parasite immunity which develops later in life and is not dependent on the magnitude of exposure, appears to best reproduce the pattern of parasite prevalence and clinical disease by age in different malaria transmission settings. Understanding the effector mechanisms underlying these two immune functions will assist in the design of transmission-reducing interventions against malaria.