Sequentially testing for a gene-drug interaction in a genomewide analysis

Assaying a large number of genetic markers from patients in clinical trials is now possible in order to tailor drugs with respect to efficacy. The statistical methodology for analysing such massive data sets is challenging. The most popular type of statistical analysis is to use a univariate test for each genetic marker, once all the data from a clinical study have been collected. This paper presents a sequential method for conducting an omnibus test for detecting gene-drug interactions across the genome, thus allowing informed decisions at the earliest opportunity and overcoming the multiple testing problems from conducting many univariate tests. We first propose an omnibus test for a fixed sample size. This test is based on combining F-statistics that test for an interaction between treatment and the individual single nucleotide polymorphism (SNP). As SNPs tend to be correlated, we use permutations to calculate a global p-value. We extend our omnibus test to the sequential case. In order to control the type I error rate, we propose a sequential method that uses permutations to obtain the stopping boundaries. The results of a simulation study show that the sequential permutation method is more powerful than alternative sequential methods that control the type I error rate, such as the inverse-normal method. The proposed method is flexible as we do not need to assume a mode of inheritance and can also adjust for confounding factors. An application to real clinical data illustrates that the method is computationally feasible for a large number of SNPs. Copyright (c) 2007 John Wiley & Sons, Ltd.

Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.

Evaluating explanations about drug prescriptions: effects of varying the nature of information about side effects and its relative position in explanations

This study evaluates computer-generated written explanations about drug prescriptions that are based on an analysis of both patient and doctor informational needs. Three experiments examine the effects of varying the type of information given about the possible side effects of the medication, and the order of information within the explanation. Experiment 1 investigated the effects of these two factors on people's ratings of how good they consider the explanations to be and of their perceived likelihood of taking the medication, as well as on their memory for the information in the explanation. Experiment 2 further examined the effects of varying information about side effects by separating out the contribution of number and severity of side effects. It was found that participants in this study did not “like” explanations that described severe side effects, and also judged that they would be less likely to take the medication if given such explanations. Experiment 3 therefore investigated whether information about severe side effects could be presented in such a way as to increase judgements of how good explanations are thought to be, as well as the perceived likelihood of adherence. The results showed some benefits of providing additional explanatory information.

Temperature-responsive properties and drug solubilization capacity of amphiphilic copolymers based on N-vinylpyrrolidone and vinyl propyl ether

A series of amphiphilic copolymers were synthesized by free-radical copolymerization of N-vinylpyrrolidone (NVP) with vinyl propyl ether (VPE), and the structure of the copolymers was characterized by elemental analysis and gel permeation chromatography. The reactivity of VPE in copolymerization was found to be significantly lower than the reactivity of NVP, which resulted in a decrease of copolymers’ yields and molecular weights with higher content of VPE in the feed mixture. An investigation of the behavior of the copolymers in aqueous solutions at different temperatures by dynamic light scattering revealed the presence of lower critical solution temperature, which depending on the content of VPE ranged within 23−38 °C. Aqueous solutions of these copolymers were studied by fluorescent spectroscopy with pyrene as a polarity probe to reveal the formation of hydrophobic domains. The copolymers were found to be useful for enhancing the solubility of riboflavin in water.

Efficient and cost-effective experimental determination of kinetic constants and data: the success of a Bayesian systematic approach to drug transport, receptor binding, continuous culture and cell transport kinetics

Details about the parameters of kinetic systems are crucial for progress in both medical and industrial research, including drug development, clinical diagnosis and biotechnology applications. Such details must be collected by a series of kinetic experiments and investigations. The correct design of the experiment is essential to collecting data suitable for analysis, modelling and deriving the correct information. We have developed a systematic and iterative Bayesian method and sets of rules for the design of enzyme kinetic experiments. Our method selects the optimum design to collect data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. The rules select features of the design such as the substrate range and the number of measurements. We show here that this method can be directly applied to the study of other important kinetic systems, including drug transport, receptor binding, microbial culture and cell transport kinetics. It is possible to reduce the errors in the estimated parameters and, most importantly, increase the efficiency and cost-effectiveness by reducing the necessary amount of experiments and data points measured. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

Objectives: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. Methods: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. Results: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. Conclusion: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.

A novel Bayesian approach to drug design with simple and complex enzymes: Use with lens aldehyde dehydrogenase and the glyoxalase pathway

Purpose: Acquiring details of kinetic parameters of enzymes is crucial to biochemical understanding, drug development, and clinical diagnosis in ocular diseases. The correct design of an experiment is critical to collecting data suitable for analysis, modelling and deriving the correct information. As classical design methods are not targeted to the more complex kinetics being frequently studied, attention is needed to estimate parameters of such models with low variance. Methods: We have developed Bayesian utility functions to minimise kinetic parameter variance involving differentiation of model expressions and matrix inversion. These have been applied to the simple kinetics of the enzymes in the glyoxalase pathway (of importance in posttranslational modification of proteins in cataract), and the complex kinetics of lens aldehyde dehydrogenase (also of relevance to cataract). Results: Our successful application of Bayesian statistics has allowed us to identify a set of rules for designing optimum kinetic experiments iteratively. Most importantly, the distribution of points in the range is critical; it is not simply a matter of even or multiple increases. At least 60 % must be below the KM (or plural if more than one dissociation constant) and 40% above. This choice halves the variance found using a simple even spread across the range.With both the glyoxalase system and lens aldehyde dehydrogenase we have significantly improved the variance of kinetic parameter estimation while reducing the number and costs of experiments. Conclusions: We have developed an optimal and iterative method for selecting features of design such as substrate range, number of measurements and choice of intermediate points. Our novel approach minimises parameter error and costs, and maximises experimental efficiency. It is applicable to many areas of ocular drug design, including receptor-ligand binding and immunoglobulin binding, and should be an important tool in ocular drug discovery.

Liposomes and skin: From drug delivery to model membranes

The early eighties saw the introduction of liposomes as skin drug delivery systems, initially promoted primarily for localised effects with minimal systemic delivery. Subsequently, a novel ultradeformable vesicular system (termed "Transfersomes" by the inventors) was reported for transdermal delivery with an efficiency similar to subcutaneous injection. Further research illustrated that the mechanisms of liposome action depended on the application regime and the vesicle composition and morphology. Ethical, health and supply problems with human skin have encouraged researchers to use skin models. 'IYaditional models involved polymer membranes and animal tissue, but whilst of value for release studies, such models are not always good mimics for the complex human skin barrier, particularly with respect to the stratum corneal intercellular lipid domains. These lipids have a multiply bilayered organization, a composition and organization somewhat similar to liposomes, Consequently researchers have used vesicles as skin model membranes. Early work first employed phospholipid liposomes and tested their interactions with skin penetration enhancers, typically using thermal analysis and spectroscopic analyses. Another approach probed how incorporation of compounds into liposomes led to the loss of entrapped markers, analogous to "fluidization" of stratum corneum lipids on treatment with a penetration enhancer. Subsequently scientists employed liposomes formulated with skin lipids in these types of studies. Following a brief description of the nature of the skin barrier to transdermal drug delivery and the use of liposomes in drug delivery through skin, this article critically reviews the relevance of using different types of vesicles as a model for human skin in permeation enhancement studies, concentrating primarily on liposomes after briefly surveying older models. The validity of different types of liposome is considered and traditional skin models are compared to vesicular model membranes for their precision and accuracy as skin membrane mimics. (c) 2008 Elsevier B.V. All rights reserved.

Causes of preventable drug-related hospital admissions: a qualitative study

Objective: To explore the causes of preventable drug-related admissions (PDRAs) to hospital. Design: Qualitative case studies using semi-structured interviews and medical record review; data analysed using a framework derived from Reason's model of organisational accidents and cascade analysis. Participants: 62 participants, including 18 patients, 8 informal carers, 17 general practitioners, 12 community pharmacists, 3 practice nurses and 4 other members of healthcare staff, involved in events leading up to the patients' hospital admissions. Setting: Nottingham, UK. Results: PDRAs are associated with problems at multiple stages in the medication use process, including prescribing, dispensing, administration, monitoring and help seeking. The main causes of these problems are communication failures ( between patients and healthcare professionals and different groups of healthcare professionals) and knowledge gaps ( about drugs and patients' medical and medication histories). The causes of PDRAs are similar irrespective of whether the hospital admission is associated with a prescribing, monitoring or patient adherence problem. Conclusions: The causes of PDRAs are multifaceted and complex. Technical solutions to PDRAs will need to take account of this complexity and are unlikely to be sufficient on their own. Interventions targeting the human causes of PDRAs are also necessary - for example, improving methods of communication.

Cannabinoid influences on palatability: microstructural analysis of sucrose drinking after Delta(9)-tetrahydrocannabinol, anandamide, 2-arachidonoyl glycerol and SR141716

Rationale: Central cannabinoid systems have been implicated in appetite control through the respective hyperphagic and anorectic actions of CB1 agonists and antagonists. The motivational changes underlying these actions remain to be determined, but may involve alterations to food palatability. Objectives: The mode of action of cannabinoids on ingestion was investigated by examining the effects of exogenous and endogenous agonists, and a selective CB1 receptor antagonist, on licking microstructure in rats ingesting a palatable sucrose solution. Methods: Microstructural analyses of licking for a 10% sucrose solution was performed over a range of agonist and antagonist doses administered to non-deprived, male Lister hooded rats. Results: Delta(9)-tetrahydrocannabinol (0.5, 1 and 3 mg/kg) and anandamide (1 mg/kg and 3 mg/kg) significantly increased total number of licks. This was primarily due to an increase in bout duration rather than bout number. There was a nonsignificant increase in total licks following administration of 2-arachidonoyl glycerol (0.2, 1.0 and 2.0 mg/kg), whereas administration of the CB1 antagonist SR141716 (1 mg/kg and 3 mg/kg) significantly decreased total licks. All drugs, with the exception of anandamide, significantly decreased the intra-bout lick rate. An exponential function fitted to the cumulative lick rate curves for each drug revealed that all compounds altered the asymptote of this function without having any marked effects on the exponent. Conclusions: These data are consistent with endocannabinoid involvement in the mediation of food palatability.

A qualitative exploration of the underlying causes of preventable drug-related morbidity in primary care, resulting in hospitalisation

This study has explored the underlying causes of preventable drug-related admissions to hospital, from primary care through semi-structured interviews and review of patients’ medical records. Analysis of the data has revealed that communication failures between different groups of healthcare professionals and between healthcare professionals and patients contribute to preventable drug-related admissions, as do knowledge gaps about medication in both healthcare professionals and patients. In addition, working conditions for community pharmacists severely limit their ability to effectively act as a safety barrier to patients receiving inappropriate medication. Limitations include heavy workloads, lack of access to patients’ clinical information, poor relationships with general practitioners and time restrictions. The results of this study represent an important addition to our understanding of the contribution of human error as an underlying cause of preventable drug-related morbidity, and the factors which contribute to errors occurring in the primary healthcare setting.

Stochiometrically governed molecular interactions in drug: Poloxamer solid dispersions

This study probes the molecular interactions between model drugs and poloxamers that facilitate dissolution rate improvements using solid dispersions. Ibuprofen and ketoprofen solid dispersions were prepared at different mole ratios using poloxamers 407 and 188. The carbonyl stretching vibration of the ibuprofen dimer shifted to higher wavenumber in the infrared spectra of 2:1 drug:carrier mole ratio solid dispersions, indicating disruption of the ibuprofen dimer concomitant with hydrogen bond formation between the drug and carrier. Solid dispersions with mole ratios >2:1 drug:carrier (up to 29:1) showed both ibuprofen hydrogen-bonded to the poloxamer, and excess drug present as dimers. X-ray diffraction studies confirmed these findings with no evidence of crystalline drug in 2:1 mole ratio systems whereas higher drug loadings retained crystalline ibuprofen. Similar results were found with ketoprofen-poloxamer solid dispersions. Thermal analysis of ibuprofen-poloxamer 407 solid dispersions and their resultant phase diagram suggested solid solutions and a eutectic system were formed, depending on drug loading. Dissolution studies showed fastest release from the solid solutions; dissolution rates from solid solutions were 12-fold greater than the dissolution of ibuprofen powder whereas the eutectic system gave a 6-fold improvement over the powder. When designing solid dispersions to improve the delivery of poorly-water soluble drugs, the nature of drug:carrier interactions, which are governed by the stochiometry of the composition, can affect the dissolution rate improvement.

Evaluation of drug physical form during granulation, tabletting and storage

An active pharmaceutical ingredient (API) was found to dissociate from the highly crystalline hydrochloride form to the amorphous free base form, with consequent alterations to tablet properties. Here, a wet granulation manufacturing process has been investigated using in situ Fourier transform (FT)-Raman spectroscopic analyses of granules and tablets prepared with different granulating fluids and under different manufacturing conditions. Dosage form stability under a range of storage stresses was also investigated. Despite the spectral similarities between the two drug forms, low levels of API dissociation could be quantified in the tablets; the technique allowed discrimination of around 4% of the API content as the amorphous free base (i.e. less than 1% of the tablet compression weight). API dissociation was shown to be promoted by extended exposure to moisture. Aqueous granulating fluids and manufacturing delays between granulation and drying stages and storage of the tablets in open conditions at 40◦C/75% relative humidity (RH) led to dissociation. In contrast, non-aqueous granulating fluids, with no delay in processing and storage of the tablets in either sealed containers or at lower temperature/humidity prevented detectable dissociation. It is concluded that appropriate manufacturing process and storage conditions for the finished product involved minimising exposure to moisture of the API. Analysis of the drug using FT-Raman spectroscopy allowed rapid optimisation of the process whilst offering quantitative molecular information concerning the dissociation of the drug salt to the amorphous free base form.

Control and analysis of oriented thin films of lipid inverse bicontinuous cubic phases using grazing incidence small-angle X‑ray scattering

Lipid cubic phases are complex nanostructures that form naturally in a variety of biological systems, with applications including drug delivery and nanotemplating. Most X-ray scattering studies on lipid cubic phases have used unoriented polydomain samples as either bulk gels or suspensions of micrometer-sized cubosomes. We present a method of investigating cubic phases in a new form, as supported thin films that can be analyzed using grazing incidence small-angle X-ray scattering (GISAXS). We present GISAXS data on three lipid systems: phytantriol and two grades of monoolein (research and industrial). The use of thin films brings a number of advantages. First, the samples exhibit a high degree of uniaxial orientation about the substrate normal. Second, the new morphology allows precise control of the substrate mesophase geometry and lattice parameter using a controlled temperature and humidity environment, and we demonstrate the controllable formation of oriented diamond and gyroid inverse bicontinuous cubic along with lamellar phases. Finally, the thin film morphology allows the induction of reversible phase transitions between these mesophase structures by changes in humidity on subminute time scales, and we present timeresolved GISAXS data monitoring these transformations.

Impact of the enhanced permeability and retention (EPR) effect and cathepsins levels on the activity of polymer-drug conjugates

Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i) the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR) effect) and (ii) the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B). Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates). An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer), although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.