Simulating Intestinal Transporter and Enzyme Activity in a Physiologically Based Pharmacokinetic Model for Tenofovir Disoproxil Fumarate

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0–24; 3,045 ng · h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0–24 (3,036 ng · h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Multitarget Stool DNA Test Performance in an Average-Risk Colorectal Cancer Screening Population

INTRODUCTION: We set out to evaluate the performance of a multitarget stool DNA (MT-sDNA) in an average-risk colonoscopy-controlled colorectal cancer (CRC) screening population. MT-sDNA stool test results were evaluated against fecal immunochemical test (FIT) results for the detection of different lesions, including molecularly defined high-risk adenomas and several other tumor characteristics. METHODS: Whole stool samples (n = 1,047) were prospectively collected and subjected to an MT-sDNA test, which tests for KRAS mutations, NDRG4 and BMP3 promoter methylation, and hemoglobin. Results for detecting CRC (n = 7), advanced precancerous lesions (advanced adenoma [AA] and advanced serrated polyps; n = 119), and non-AAs (n = 191) were compared with those of FIT alone (thresholds of 50, 75, and 100 hemoglobin/mL). AAs with high risk of progression were defined by the presence of specific DNA copy number events as measured by low-pass whole genome sequencing. RESULTS: The MT-sDNA test was more sensitive than FIT alone in detecting advanced precancerous lesions (46% (55/119) vs 27% (32/119), respectively, P < 0.001). Specificities among individuals with nonadvanced or negative findings (controls) were 89% (791/888) and 93% (828/888) for MT-sDNA and FIT testing, respectively. A positive MT-sDNA test was associated with multiple lesions (P = 0.005), larger lesions (P = 0.03), and lesions with tubulovillous architecture (P = 0.04). The sensitivity of the MT-sDNA test or FIT in detecting individuals with high-risk AAs (n = 19) from individuals with low-risk AAs (n = 52) was not significantly different. DISCUSSION: In an average-risk screening population, the MT-sDNA test has an increased sensitivity for detecting advanced precancerous lesions compared with FIT alone. AAs with a high risk of progression were not detected with significantly higher sensitivity by MT-sDNA or FIT

Explaining telecoms and electricity internationalization in the European Union: a political economy perspective

One consequence of the liberalization of certain services in the European Union was that a number of formerly inward-looking incumbents in telecommunications and electricity rapidly transformed themselves into some of the world’s leading Multinationals. However, the precise relationship between liberalization and incumbent internationalization is contested. This article tests three persuasive arguments derived from the political economy literature on this relationship. The first claims that those incumbents most exposed to domestic liberalization would internationalise most. The second asserts the opposite: incumbents operating where liberalization was restricted could exploit monopolistic rents to finance their aggressive internationalisation. The third argument claims that a diversity of paths will be adopted by countries and incumbents vis-à-vis liberalization and internationalization. Using correlation and cluster analysis of the sample of all major EU telecoms and electricity incumbent Multinationals evidence is found in favour of the third hypothesis. Internationalization as a response to liberalization took diverse forms in terms of timing and extent and this is best explained using a country, sector and firm logic

Direct observation of topoisomerase IA gate dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA

Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

Validation of a Multi-Purpose Depletion Chain for Burnup Calculation through TRIPOLI-4 Calculations and IFP Perturbation Method

International audienceThe current standard depletion chain used by the CEA, called CEA-V5, was validated to treat only LWRs in 2008. This single depletion chain must correctly model the global loss of reactivity for both LWRs and Fast Reactors for future APOLLO3 work on Generation III and IV reactors such as EPR and ASTRID projects milestones. In order to verify the loss of reactivity of the standard CEA-V5 chain, a reference chain with 885 Fission Products (FPs) has been defined and used with the French Monte Carlo code TRIPOLI-4 and its depletion module for comparisons with the standard CEA-V5 chain containing 126 FPs. Three test cases are modeled, a UOX PWR cell, a MOX PWR cell and a MOX SFR cell, to validate the standard chain against the reference one. Results obtained from TRIPOLI-4 can then be used to calculate a loss of reactivity for each case to verify that the standard chain takes into account the majority (ideally 99.9%) of the anti-reactivity of the reference chain. In addition, this loss of reactivity has been decomposed by isotope to rank the FPs by importance using the Iterated Fission Probability (IFP) method recently implemented in TRIPOLI-4