Evaluation of a Library of FDA-Approved Drugs for Their Ability To Potentiate Antibiotics against Multidrug-Resistant Gram-Negative Pathogens.

The Prestwick library was screened for antibacterial activity or "antibiotic resistance breaker" (ARB) potential against four species of Gram-negative pathogens. Discounting known antibacterials, the screen identified very few ARB hits, which were strain/drug specific. These ARB hits included antimetabolites (zidovudine, floxuridine, didanosine, and gemcitabine), anthracyclines (daunorubicin, mitoxantrone, and epirubicin), and psychoactive drugs (gabapentin, fluspirilene, and oxethazaine). These findings suggest that there are few approved drugs that could be directly repositioned as adjunct antibacterials, and these will need robust testing to validate efficacy.

Dose-linearity of the pharmacokinetics of an intravenous [14 C]midazolam microdose in children.

AIMS: Drug disposition in children may vary from adults due to age-related variation in drug metabolism. Microdose studies present an innovation to study pharmacokinetics (PK) in paediatrics; however, they should be used only when the PK is dose linear. We aimed to assess dose linearity of a [14 C]midazolam microdose, by comparing the PK of an intravenous (IV) microtracer (a microdose given simultaneously with a therapeutic midazolam dose), with the PK of a single isolated microdose. METHODS: Preterm to 2-year-old infants admitted to the intensive care unit received [14 C]midazolam IV as a microtracer or microdose, followed by dense blood sampling up to 36 hours. Plasma concentrations of [14 C]midazolam and [14 C]1-hydroxy-midazolam were determined by accelerator mass spectrometry. Noncompartmental PK analysis was performed and a population PK model was developed. RESULTS: Of 15 infants (median gestational age 39.4 [range 23.9-41.4] weeks, postnatal age 11.4 [0.6-49.1] weeks), 6 received a microtracer and 9 a microdose of [14 C]midazolam (111 Bq kg-1 ; 37.6 ng kg-1 ). In a 2-compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the microdose and microtracer, nor in the area under curve ratio [14 C]1-OH-midazolam/[14 C]midazolam, showing the PK of midazolam to be linear within the range of the therapeutic and microdoses. CONCLUSION: Our data support the dose linearity of the PK of an IV [14 C]midazolam microdose in children. Hence, a [14 C]midazolam microdosing approach may be used as an alternative to a therapeutic dose of midazolam to study developmental changes in hepatic CYP3A activity in young children.

Distinctive Roles for Periplasmic Proteases in the Maintenance of Essential Outer Membrane Protein Assembly.

Outer membrane protein (OMP) biogenesis in Escherichia coli is a robust process essential to the life of the organism. It is catalyzed by the ß-barrel assembly machine (Bam) complex, and a number of quality control factors, including periplasmic chaperones and proteases, maintain the integrity of this trafficking pathway. Little is known, however, about how periplasmic proteases recognize and degrade OMP substrates when assembly is compromised or whether different proteases recognize the same substrate at distinct points in the assembly pathway. In this work, we use well-defined assembly-defective mutants of LptD, the essential lipopolysaccharide assembly translocon, to show that the periplasmic protease DegP degrades substrates with assembly defects that prevent or impair initial contact with Bam, causing the mutant protein to accumulate in the periplasm. In contrast, another periplasmic protease, BepA, degrades a LptD mutant substrate that has engaged the Bam complex and formed a nearly complete barrel. Furthermore, we describe the role of the outer membrane lipoprotein YcaL, a protease of heretofore unknown function, in the degradation of a LptD substrate that has engaged the Bam complex but is stalled at an earlier step in the assembly process that is not accessible to BepA. Our results demonstrate that multiple periplasmic proteases monitor OMPs at distinct points in the assembly process.IMPORTANCE OMP assembly is catalyzed by the essential Bam complex and occurs in a cellular environment devoid of energy sources. Assembly intermediates that misfold can compromise this essential molecular machine. Here we demonstrate distinctive roles for three different periplasmic proteases that can clear OMP substrates with folding defects that compromise assembly at three different stages. These quality control factors help ensure the integrity of the permeability barrier that contributes to the intrinsic resistance of Gram-negative organisms to many antibiotics.

Productivity costs of work loss associated with osteoarthritis in Canada from 2010 to 2031.

OBJECTIVE: To estimate and project the productivity costs of work loss (PCWL) associated with osteoarthritis (OA) in Canada using the Population Health Model (POHEM). DESIGN: We integrated an employment module based on 2006 Canadian Census into the previously developed microsimulation model of OA. The Canadian Community Health Survey (CCHS) Cycle 2.1 with an OA sample aged 25-64 (n = 7067) was used to calibrate the results of the employment module and to estimate the fraction of non-employment associated with OA. Probabilities of non-employment together with attributable fractions were then implemented in POHEM to estimate PCWL associated with OA from 2010 to 2031. RESULTS: Among the OA population, 44.4% and 59.4% of non-employment due to illness was associated with OA for those not working full-year and part-year, respectively. According to POHEM projections, the size of the working age population with OA increased from 1.5 million in 2010 to 1.7 million in 2031. The PCWL associated with OA increased from $12 billion to $17.5 billion in constant 2008 Canadian dollars. Around 38% of this increase was due to the increase in OA prevalence and changes in demographics, while the rest was due to increase in real wage growth. Male and female OA patients between 55 and 64 years of age had the highest total projected PCWL, respectively. CONCLUSIONS: The total PCWL associated with OA in Canada is estimated to be substantial and increasing in future years. Results of this study could be used to inform policies aiming to increase employment sustainability among individuals with OA.

The OncoSim model: development and use for better decision-making in Canadian cancer control.

The Canadian Partnership Against Cancer was created in 2007 by the federal government to accelerate cancer control across Canada. Its OncoSim microsimulation model platform, which consists of a suite of specific cancer models, was conceived as a tool to augment conventional resources for population-level policy- and decision-making. The Canadian Partnership Against Cancer manages the OncoSim program, with funding from Health Canada and model development by Statistics Canada. Microsimulation modelling allows for the detailed capture of population heterogeneity and health and demographic history over time. Extensive data from multiple Canadian sources were used as inputs or to validate the model. OncoSim has been validated through expert consultation; assessments of face validity, internal validity, and external validity; and model fit against observed data. The platform comprises three in-depth cancer models (lung, colorectal, cervical), with another in-depth model (breast) and a generalized model (25 cancers) being in development. Unique among models of its class, OncoSim is available online for public sector use free of charge. Users can customize input values and output display, and extensive user support is provided. OncoSim has been used to support decision-making at the national and jurisdictional levels. Although simulation studies are generally not included in hierarchies of evidence, they are integral to informing cancer control policy when clinical studies are not feasible. OncoSim can evaluate complex intervention scenarios for multiple cancers. Canadian decision-makers thus have a powerful tool to assess the costs, benefits, cost-effectiveness, and budgetary effects of cancer control interventions when faced with difficult choices for improvements in population health and resource allocation.

The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain.

UNLABELLED: The periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity in Escherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the ß-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process. IMPORTANCE: This work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many ß-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenic Escherichia coli, E. coli O157:H7, Shigella, and Salmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.

Role for Skp in LptD assembly in Escherichia coli.

The periplasmic chaperone Skp has long been implicated in the assembly of outer membrane proteins (OMPs) in Escherichia coli. It has been shown to interact with unfolded OMPs, and the simultaneous loss of Skp and the main periplasmic chaperone in E. coli, SurA, results in synthetic lethality. However, a Δskp mutant displays only minor OMP assembly defects, and no OMPs have been shown to require Skp for their assembly. Here, we report a role for Skp in the assembly of the essential OMP LptD. This role may be compensated for by other OMP assembly proteins; in the absence of both Skp and FkpA or Skp and BamB, LptD assembly is impaired. Overexpression of SurA does not restore LptD levels in a Δskp ΔfkpA double mutant, nor does the overexpression of Skp or FkpA restore LptD levels in the ΔsurA mutant, suggesting that Skp acts in concert with SurA to efficiently assemble LptD in E. coli. Other OMPs, including LamB, are less affected in the Δskp ΔfkpA and Δskp bamB::kan double mutants, suggesting that Skp is specifically necessary for the assembly of certain OMPs. Analysis of an OMP with a domain structure similar to that of LptD, FhuA, suggests that common structural features may determine which OMPs require Skp for their assembly.

Lupus nephritis: a 15-year multi-centre experience in the UK.

OBJECTIVES: Our aim was to audit the outcome of lupus nephritis (LN) at three East Midlands centres. METHODS: We undertook a retrospective review of all biopsy-proven LN types III-V 1995-2010. RESULTS: In total, 61 patients with LN were identified, with a median follow-up of 68 months. LN was present at the time of systemic lupus erythematosus (SLE) diagnosis in 20 patients. The median time from SLE diagnosis to the first LN episode was 5.3 years. Some 35 patients received IV cyclophosphamide and 17 received mycophenolate mofetil (MMF) as induction therapy; 81.8% of those treated with cyclophosphamide and 81.3% with MMF had at least 'improved' according to the ACR-response criteria 6 months from induction; 33.3% and 37.5%, respectively, had a 'complete' response. MMF and azathioprine were the most frequently used maintenance therapy. We found that 32.8% experienced a flare after a mean post-induction time of 3.5 years, irrespective of the maintenance therapy used, and 43.8% of partial responders flared compared with 4.8% of complete responders. End-stage renal failure developed in 8.2%. CONCLUSIONS: Overall, outcomes (response, flare-rate, end-stage renal failure) were comparable with European clinical studies. Partial responders are more likely to flare compared with complete responders. The results highlight that LN can occur, and flare, after many years of SLE, emphasizing the importance of continued vigilance for LN in all patients.

Parenting children with neurodevelopmental disorders and/or behaviour problems.

BACKGROUND: Parenting behaviours influence child well-being and development. However, much of the research on parenting behaviours and their correlates has focused on caregivers of healthy, typically developing children. Relatively less is known about the parenting behaviours of caregivers of children with chronic health conditions. OBJECTIVE: To examine and compare three parenting behaviours (positive interactions, consistency and ineffective parenting) among caregivers of children with neurodevelopmental disorders and/or externalizing behaviour problems, before and after accounting for child and family socio-demographic characteristics. METHODS: Participants (n= 14 226) were drawn from the National Longitudinal Survey of Children and Youth, a long-term study of Canadian children that follows their development and well-being from birth to early adulthood. Children (and their caregivers) were divided into four groups according to the presence of a neurodevelopmental disorder (NDD; n= 815), the presence of an externalizing behaviour problem (EBP; n= 1322), the presence of both conditions (BOTH; n= 452) or neither of these conditions (NEITHER; n= 11 376). RESULTS: Caregivers of children in the NEITHER group reported significantly higher positive interaction scores and lower ineffective parenting behaviours than caregivers of children in any of the other three groups. Caregivers of children in the EBP and BOTH groups reported similar levels of consistency, but significantly lower levels than caregivers of NDD or NEITHER children. These associations largely remained after accounting for child and family socio-demographic characteristics, with two exceptions: caregivers' reports of positive interactions were no longer significantly associated with child's NDD and BOTH conditions. CONCLUSIONS: Parenting children with multiple health conditions can be associated with less positive, less consistent and more ineffective parenting behaviours. Understanding the factors that are associated with the challenges of caring for these children may require additional research attention.

Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos.

Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.

Oncogenes in radioresistant, noncancerous skin fibroblasts from a cancer-prone family.

Li-Fraumeni syndrome is manifested in a variety of neoplasms that are transmitted in a dominantly inherited pattern. The noncancerous skin fibroblasts of family members exhibit a unique characteristic of being resistant to the killing effect of ionizing radiation. A three- to eightfold elevation in expression of c-myc and an apparent activation of c-raf-1 gene have been observed in these noncancerous skin fibroblasts. These results may provide insight into the heritable defect underlying the familial predisposition to a variety of cancers.

Light-activation of gene function in mammalian cells via ribozymes.

A ribozyme based gene control element enabled the spatio-temporal regulation of gene function in mammalian cell culture with light.

A pharmacokinetic evaluation of five H(1) antagonists after an oral and intravenous microdose to human subjects.

AIMS: To evaluate the pharmacokinetics (PK) of five H(1) receptor antagonists in human volunteers after a single oral and intravenous (i.v.) microdose (0.1 mg). METHODS: Five H(1) receptor antagonists, namely NBI-1, NBI-2, NBI-3, NBI-4 and diphenhydramine, were administered to human volunteers as a single 0.1-mg oral and i.v. dose. Blood samples were collected up to 48 h, and the parent compound in the plasma extract was quantified by high-performance liquid chromatography and accelerator mass spectroscopy. RESULTS: The median clearance (CL), apparent volume of distribution (V(d)) and apparent terminal elimination half-life (t(1/2)) of diphenhydramine after an i.v. microdose were 24.7 l h(-1), 302 l and 9.3 h, and the oral C(max) and AUC(0-infinity) were 0.195 ng ml(-1) and 1.52 ng h ml(-1), respectively. These data were consistent with previously published diphenhydramine data at 500 times the microdose. The rank order of oral bioavailability of the five compounds was as follows: NBI-2 > NBI-1 > NBI-3 > diphenhydramine > NBI-4, whereas the rank order for CL was NBI-4 > diphenhydramine > NBI-1 > NBI-3 > NBI-2. CONCLUSIONS: Human microdosing provided estimates of clinical PK of four structurally related compounds, which were deemed useful for compound selection.

The utility of microdosing over the past 5 years.

BACKGROUND: Microdosing studies (human Phase 0) are used to select drug candidates for Phase I clinical trials on the basis of their pharmacokinetic properties, using subpharmacologic doses (maximum 100 microg). There are questions as to whether pharmacokinetic data obtained at these low doses will predict those at the clinically relevant dose. OBJECTIVE: To review the current literature on microdosing and assess how well microdose data have predicted the pharmacokinetics obtained at a therapeutic dose. METHODS: All data published in the peer reviewed literature comparing pharmacokinetics at a microdose with a therapeutic dose were reviewed, excluding those studies aimed at imaging. CONCLUSIONS: Of the 18 drugs reported, 15 demonstrated linear pharmacokinetics within a factor of 2 between a microdose and a therapeutic dose. Therefore, data that support the utility of microdosing are beginning to emerge.

An activation-dependent, T-lymphocyte-specific transcriptional activator in the mouse mammary tumor virus env gene.

Transcription of the complete mouse mammary tumor virus (MMTV) proviral genome in mouse cells is controlled by a strong promoter in its long terminal repeat. In the mouse T lymphoma EL4, there is a second, activation-dependent transcriptional initiation site within the envelope (env) gene, from which a short mRNA is generated, encoding the open reading frame of the long terminal repeat. We now report the isolation of a segment of the MMTV env gene (called META, for MMTV env transcriptional activator) which has the expected transcription-activating properties seen in EL4.E1 cells. Namely, it induces activation-dependent, T-lymphocyte-specific transcription of a chloramphenicol acetyltransferase reporter gene. It is active in mouse or human T-helper lymphocyte lines when they are stimulated to transcribe lymphokine genes but is inactive in unstimulated T-helper cells, fibroblasts, a cytotoxic T-lymphocyte line, and a mastocytoma cell line. Its activity is inhibited by cyclosporin A, a specific inhibitor of lymphokine transcription. Several forms of the META have been isolated from EL4.E1 cells, a mouse T-helper cell hybridoma, and from BALB/c spleen cells. Linked to the heterologous thymidine kinase promoter, a 400-bp portion of it is an inducible, orientation-independent, and cyclosporin A-sensitive transcriptional activator in T-helper cells.

Big physics, small doses: the use of AMS and PET in human microdosing of development drugs.

The process of early clinical drug development has changed little over the past 20 years despite an up to 40% failure rate associated with inappropriate drug metabolism and pharmacokinetics of candidate molecules. A new method of obtaining human metabolism data known as microdosing has been developed which will permit smarter candidate selection by taking investigational drugs into humans earlier. Microdosing depends on the availability of two ultrasensitive 'big-physics' techniques: positron emission tomography (PET) can provide pharmacodynamic information, whereas accelerator mass spectrometry (AMS) provides pharmacokinetic information. Microdosing allows safer human studies as well as reducing the use of animals in preclinical toxicology.

Use of microdosing to predict pharmacokinetics at the therapeutic dose: experience with 5 drugs.

OBJECTIVES: A volunteer trial was performed to compare the pharmacokinetics of 5 drugs--warfarin, ZK253 (Schering), diazepam, midazolam, and erythromycin--when administered at a microdose or pharmacologic dose. Each compound was chosen to represent a situation in which prediction of pharmacokinetics from either animal or in vitro studies (or both) was or is likely to be problematic. METHODS: In a crossover design volunteers received (1) 1 of the 5 compounds as a microdose labeled with radioactive carbon (carbon 14) (100 microg), (2) the corresponding (14)C-labeled therapeutic dose on a separate occasion, and (3) simultaneous administration of an intravenous (14)C-labeled microdose and an oral therapeutic dose for ZK253, midazolam, and erythromycin. Analysis of (14)C-labeled drugs in plasma was done by use of HPLC followed by accelerator mass spectrometry. Liquid chromatography-tandem mass spectrometry was used to measure plasma concentrations of ZK253, midazolam, and erythromycin at therapeutic concentrations, whereas HPLC-accelerator mass spectrometry was used to measure warfarin and diazepam concentrations. RESULTS: Good concordance between microdose and therapeutic dose pharmacokinetics was observed for diazepam (half-life [t((1/2))] of 45.1 hours, clearance [CL] of 1.38 L/h, and volume of distribution [V] of 90.1 L for 100 microg and t((1/2)) of 35.7 hours, CL of 1.3 L/h, and V of 123 L for 10 mg), midazolam (t((1/2)) of 4.87 hours, CL of 21.2 L/h, V of 145 L, and oral bioavailability [F] of 0.23 for 100 microg and t((1/2)) of 3.31 hours, CL of 20.4 L/h, V of 75 L, and F of 0.22 for 7.5 mg), and development compound ZK253 (F = <1% for both 100 microg and 50 mg). For warfarin, clearance was reasonably well predicted (0.17 L/h for 100 microg and 0.26 L/h for 5 mg), but the discrepancy observed in distribution (67 L for 100 microg and 17.9 L for 5 mg) was probably a result of high-affinity, low-capacity tissue binding. The oral microdose of erythromycin failed to provide detectable plasma levels as a result of possible acid lability in the stomach. Absolute bioavailability for the 3 compounds examined yielded excellent concordance with data from the literature or data generated in house. CONCLUSION: Overall, when used appropriately, microdosing offers the potential to aid in early drug candidate selection.

Determination of the bioavailability of [14C]-hexaminolevulinate using accelerator mass spectrometry after intravesical administration to human volunteers.

Hexaminolevulinate (HAL) is a diagnostic agent that allows the visualization of tumor tissue in the bladder by fluorescence cystoscopy. It is administered intravesically via a catheter for 1 hour, followed by blue light bladder inspection to induce selective red tumor fluorescence. Hexaminolevulinate should ideally be confined to the bladder only, but it is likely that some absorption occurs during administration, and therefore the systemic bioavailability is of interest. The bioavailability of HAL was determined by intravesical and intravenous administration of [14C]-HAL hydrochloride to 8 human volunteers. To reduce the radiation dose as low as possible, the ultrasensitive analytical technique of accelerator mass spectrometry was used to measure [14C]-HAL. The bioavailability of [14C]-HAL after intravesical and intravenous administration was determined from the respective area under the curve based on total radioactivity and was determined to be 7% (range, 5%-10%; 90% confidence interval). The systemic absorption of [14C]-HAL after intravesical administration is low and supports previous clinical experience with HAL showing no systemic side effects.

Novel use of accelerator mass spectrometry for the quantification of low levels of systemic therapeutic recombinant protein.

Although 14C-labelling has been routinely used for small molecules, this technique is not routinely applied to therapeutic proteins due to difficulties of incorporating the label into the protein to a sufficiently high specific activity. An analytical method known as accelerator mass spectrometry (AMS) offers an extremely sensitive method of 14C quantification, thereby enabling (14)C-labeling methods to be applied to therapeutic protein detection. The therapeutic protein CAT-192 (metelimumab), a human anti-TGFss1 monocloncal antibody was manufactured in the presence of 14C-precursors resulting in a low specific activity product (1.4% 14C incorporation). [14C]-CAT-192 was administered to rats (1mg/kg and 222, 22 and 2.2 dpm/kg) and serum samples were collected. 14C in serum samples from the 2.2 dpm dosing was not detectable but samples from the 22 and 2220 dpm doses were measured by AMS and by ELISA for comparison. By both ELISA and AMS bioassay, the half-lives approximated 140 h (S.E.M. 15 h). The estimates of clearance were also comparable, 7.3 and 4.6 x 10(-4)ml/h/g (S.E.M. 6.6 and 5.1 x 10(-5)) for ELISA and AMS, respectively. The estimated limit of quantification (LOQ) was approximately 1 ng/ml, about 15 times lower than the ELISA LOQ of 15.6 ng/ml.

The use of isotopes in the determination of absolute bioavailability of drugs in humans.

Absolute bioavailability studies in humans are not routinely performed as part of the drug registration process. They tend to be reasonably demanding, not least because toxicology data are required to support intravenous administration of a drug. Moreover, the classical crossover design of an absolute bioavailability study can suffer from artefacts caused by concentration-dependent pharmacokinetics. Many of the problems associated with absolute bioavailability studies can be alleviated using isotopically labelled drugs. Stable isotopes have been used in the performance of absolute bioavailability studies in humans for > 30 years. More recently, the advantages of using radiolabelled drugs have been expanded by using the ultrasensitive technology of accelerator mass spectrometry. Isotopic labelling not only allows for the accurate and efficient determination of absolute bioavailability, but can also provide information on first-pass effects and other pharmacokinetic parameters.