Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimizing administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterized for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumors from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumors, CTPS2 was the gene with the highest probability of differential expression (p = 0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analog, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p = 0.072), while those with high expression did (p = 0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

Patient selection for oncology phase I trials: a multi-institutional study of prognostic factors.

PURPOSE The appropriate selection of patients for early clinical trials presents a major challenge. Previous analyses focusing on this problem were limited by small size and by interpractice heterogeneity. This study aims to define prognostic factors to guide risk-benefit assessments by using a large patient database from multiple phase I trials. PATIENTS AND METHODS Data were collected from 2,182 eligible patients treated in phase I trials between 2005 and 2007 in 14 European institutions. We derived and validated independent prognostic factors for 90-day mortality by using multivariate logistic regression analysis. Results The 90-day mortality was 16.5% with a drug-related death rate of 0.4%. Trial discontinuation within 3 weeks occurred in 14% of patients primarily because of disease progression. Eight different prognostic variables for 90-day mortality were validated: performance status (PS), albumin, lactate dehydrogenase, alkaline phosphatase, number of metastatic sites, clinical tumor growth rate, lymphocytes, and WBC. Two different models of prognostic scores for 90-day mortality were generated by using these factors, including or excluding PS; both achieved specificities of more than 85% and sensitivities of approximately 50% when using a score cutoff of 5 or higher. These models were not superior to the previously published Royal Marsden Hospital score in their ability to predict 90-day mortality. CONCLUSION Patient selection using any of these prognostic scores will reduce non-drug-related 90-day mortality among patients enrolled in phase I trials by 50%. However, this can be achieved only by an overall reduction in recruitment to phase I studies of 20%, more than half of whom would in fact have survived beyond 90 days.

Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia

One common mechanism of resistance against antimicrobial peptides in Gram-negative bacteria is the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l-Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l-Ara4N synthesis and transfer to the LPS. The absence of l-Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi-protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l-Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.

Novel semi-interpenetrating hydrogel networks with enhanced mechanical properties and thermoresponsive engineered drug delivery, designed as bioactive endotracheal tube biomaterials

Thermoresponsive polymeric platforms are used to optimise drug delivery in pharmaceutical systems and bioactive medical devices. However, the practical application of these systems is compromised by their poor mechanical properties. This study describes the design of thermoresponsive semi-interpenetrating polymer networks (s-IPNs) based on cross-linked p(NIPAA) or p(NIPAA-co-HEMA) hydrogels containing poly(e-caprolactone) designed to address this issue. Using DSC, the lower critical solution temperature of the co-polymer and p(NIPAA) matrices were circa 34 °C and 32 °C, respectively. PCL was physically dispersed within the hydrogel matrices as confirmed using confocal scanning laser microscopy and DSC and resulted in marked changes in the mechanical properties (ultimate tensile strength, Young's modulus) without adversely compromising the elongation properties. P(NIPAA) networks containing dispersed PCL exhibited thermoresponsive swelling properties following immersion in buffer (pH 7), with the equilibrium-swelling ratio being greater at 20 °C than 37 °C and greatest for p(NIPAA)/PCL systems at 20 °C. The incorporation of PCL significantly lowered the equilibrium swelling ratio of the various networks but this was not deemed practically significant for s-IPNs based on p(NIPAA). Thermoresponsive release of metronidazole was observed from s-IPN composed of p(NIPAA)/PCL at 37 °C but not from p(NIPAA-co-HEMA)/PCL at this temperature. In all other platforms, drug release at 20 °C was significantly similar to that at 37 °C and was diffusion controlled. This study has uniquely described a strategy by which thermoresponsive drug release may be performed from polymeric platforms with highly elastic properties. It is proposed that these materials may be used clinically as bioactive endotracheal tubes, designed to offer enhanced resistance to ventilator associated pneumonia, a clinical condition associated with the use of endotracheal tubes where stimulus responsive drug release from biomaterials of significant mechanical properties would be advantageous. © 2012 Elsevier B.V. All rights reserved.

Anthelmintic drug residues in beef: UPLC-MS/MS method validation, European retail beef survey, and associated exposure and risk assessments

Anthelmintic drugs are widely used to control parasitic infections in cattle. The ProSafeBeef project addressed the need for data on the exposure of European consumers of beef to potentially harmful drug residues. A novel analytical method based on matrix solid-phase dispersive extraction and ultra-performance liquid chromatography-tandem mass spectrometry was validated for 37 anthelmintic drugs and metabolites in muscle (assay decision limits, CCa, = 0.15-10.2 µg kg -1). Seven European countries (France, Spain, Slovenia, Ireland, Italy, Belgium and Portugal) participated in a survey of retail beef purchased in local shops. Of 1061 beef samples analysed, 26 (2.45%) contained detectable residues of anthelmintic drugs (0.2-171 µg kg -1), none above its European Union maximum residue limit (MRL) or action level. Residues detected included closantel, levamisole, doramectin, eprinomectin, moxidectin, ivermectin, albendazole and rafoxanide. In a risk assessment applied to mean residue concentrations across all samples, observed residues accounted for less than 0.1% of the MRL for each compound. An exposure assessment based on the consumption of meat at the 99th percentile of consumption of adults in 14 European countries demonstrated that beef accounted for less than 0.02% of the acceptable daily intake for each compound in each country. This study is the first of its kind to apply such a risk-based approach to an extensive multi-residue survey of veterinary drug residues in food. It has demonstrated that the risk of exposure of the European consumer to anthelmintic drug residues in beef is negligible, indicating that regulation and monitoring is having the desired effect of limiting residues to non-hazardous concentrations. © 2012 Copyright Taylor and Francis Group, LLC.

Stability during cooking of anthelmintic veterinary drug residues in beef

Anthelmintic drugs are widely used for treatment of parasitic worms in livestock, but little is known about the stability of their residues in food under conventional cooking conditions. As part of the European Commissionfunded research project ProSafeBeef, cattle were medicated with commercially available anthelmintic preparations, comprising 11 active ingredients (corresponding to 21 marker residues). Incurred meat and liver were cooked by roasting (40 min at 190°C) or shallow frying (muscle 8-12 min, liver 14-19 min) in a domestic kitchen. Raw and cooked tissues and expressed juices were analysed using a novel multi-residue dispersive solid-phase extraction method (QuEChERS) coupled with ultra-performance liquid chromatography-tandem mass spectrometry. After correction for sample weight changes during cooking, no major losses were observed for residues of oxyclozanide, clorsulon, closantel, ivermectin, albendazole, mebendazole or fenbendazole. However, significant losses were observed for nitroxynil (78% in fried muscle, 96% in roast muscle), levamisole (11% in fried muscle, 42% in fried liver), rafoxanide (17% in fried muscle, 18% in roast muscle) and triclabendazole (23% in fried liver, 47% in roast muscle). Migration of residues from muscle into expressed cooking juices varied between drugs, constituting 0% to 17% (levamisole) of total residues remaining after cooking. With the exception of nitroxynil, residues of anthelmintic drugs were generally resistant to degradation during roasting and shallow frying. Conventional cooking cannot, therefore, be considered a safeguard against ingestion of residues of anthelmintic veterinary drugs in beef. © 2011 Taylor & Francis.

Solvent induced phase inversion-based in situ forming controlled release drug delivery implants

In situ forming (ISF) drug delivery implants have gained tremendous levels of interest over the last few decades. This is due to their wide range of biomedical applications such as in tissue engineering, cell encapsulation, microfluidics, bioengineering and drug delivery. Drug delivery implants forming upon injection has shown a range of advantages which include localized drug delivery, easy and less invasive application, sustained drug action, ability to tailor drug delivery, reduction in side effects associated with systemic delivery and also improved patient compliance and comfort. Different factors such as temperature, pH, ions, and exchange of solvents are involved in in situ implant formation. This review especially focuses on ISF implants that are formed through solvent induced phase inversion (SPI) technique. The article critically reviews and compares a wide range of polymers, solvents, and co-solvents that have been used in SPI implant preparation for control release of a range of drug molecules. Major drawback of SPI systems has been their high burst release. In this regard, the article exhaustively discusses factors that affect the burst release and different modification strategies that has been utilised to reduce the burst effect from these implants. Performance and controversial issues associated with the use of different biocompatible solvents in SPI systems is also discussed. Biodegradation, formulation stability, methods of characterisation and sterilisation techniques of SPI systems is comprehensively reviewed. Furthermore, the review also examines current SPI-based marketed products, their therapeutic application and associated clinical data. It also exemplifies the interest of multi-billion dollar pharma companies worldwide for further developments of SPI systems to a range of therapeutic applications. The authors believe that this will be the first review article that extensively investigate and discusses studies done to date on SPI systems. In so doing, this article will undoubtedly serve as an enlightening tool for the scientists working in the concerned area.

Efficient Drug Delivery and Induction of Apoptosis in Colorectal Tumors Using a Death Receptor 5-Targeted Nanomedicine

Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.Molecular Therapy (2014); doi:10.1038/mt.2014.137.

Multidrug transporter proteins of the major facilitator superfamily: not only for drug efflux

Multidrug resistance in prokaryotes is due primarily to efflux of offending antimicrobials from the cell by representatives of several different families of integral membrane transporter proteins. Clearly, in evolutionary terms, these proteins did not arise specifically to pump human-made antimicrobials out of the cell and thereby confer resistance. Despite this, often only their role in antibiotic resistance is characterised and highlighted.
In recent years, however, a transition from the traditional anthropocentric perception of antibiotic resistance mechanisms in microorganisms has occurred, with naturally produced antimicrobials now generally regarded as physiologically important signalling molecules or sources of nutrition for bacteria rather than antimicrobial agents, and bacterial multidrug efflux proteins not merely as a defensive response to antimicrobials but as important players in fundamental physiological processes such as cellular homeostasis.
This emerging perspective supports the notion that a better understanding of the complexities of infection and multidrug resistance in bacteria can be achieved via a more detailed understanding of those physiological processes. In this chapter, we review the ‘true’ physiological roles of multidrug efflux proteins of the largest non-ATP-hydrolysing family of membrane transporters, the major facilitator superfamily, and explore the evidence for their function in processes such as pH and metal homeostasis, import and export of metabolites and biofilm formation

Pan-retinal photocoagulation and other forms of laser treatment and drug therapies for non-proliferative diabetic retinopathy: systematic review and economic evaluation

BACKGROUND: Diabetic retinopathy is an important cause of visual loss. Laser photocoagulation preserves vision in diabetic retinopathy but is currently used at the stage of proliferative diabetic retinopathy (PDR).

OBJECTIVES: The primary aim was to assess the clinical effectiveness and cost-effectiveness of pan-retinal photocoagulation (PRP) given at the non-proliferative stage of diabetic retinopathy (NPDR) compared with waiting until the high-risk PDR (HR-PDR) stage was reached. There have been recent advances in laser photocoagulation techniques, and in the use of laser treatments combined with anti-vascular endothelial growth factor (VEGF) drugs or injected steroids. Our secondary questions were: (1) If PRP were to be used in NPDR, which form of laser treatment should be used? and (2) Is adjuvant therapy with intravitreal drugs clinically effective and cost-effective in PRP?

ELIGIBILITY CRITERIA: Randomised controlled trials (RCTs) for efficacy but other designs also used.

REVIEW METHODS: Systematic review and economic modelling.

RESULTS: The Early Treatment Diabetic Retinopathy Study (ETDRS), published in 1991, was the only trial designed to determine the best time to initiate PRP. It randomised one eye of 3711 patients with mild-to-severe NPDR or early PDR to early photocoagulation, and the other to deferral of PRP until HR-PDR developed. The risk of severe visual loss after 5 years for eyes assigned to PRP for NPDR or early PDR compared with deferral of PRP was reduced by 23% (relative risk 0.77, 99% confidence interval 0.56 to 1.06). However, the ETDRS did not provide results separately for NPDR and early PDR. In economic modelling, the base case found that early PRP could be more effective and less costly than deferred PRP. Sensitivity analyses gave similar results, with early PRP continuing to dominate or having low incremental cost-effectiveness ratio. However, there are substantial uncertainties. For our secondary aims we found 12 trials of lasers in DR, with 982 patients in total, ranging from 40 to 150. Most were in PDR but five included some patients with severe NPDR. Three compared multi-spot pattern lasers against argon laser. RCTs comparing laser applied in a lighter manner (less-intensive burns) with conventional methods (more intense burns) reported little difference in efficacy but fewer adverse effects. One RCT suggested that selective laser treatment targeting only ischaemic areas was effective. Observational studies showed that the most important adverse effect of PRP was macular oedema (MO), which can cause visual impairment, usually temporary. Ten trials of laser and anti-VEGF or steroid drug combinations were consistent in reporting a reduction in risk of PRP-induced MO.

LIMITATION: The current evidence is insufficient to recommend PRP for severe NPDR.

CONCLUSIONS: There is, as yet, no convincing evidence that modern laser systems are more effective than the argon laser used in ETDRS, but they appear to have fewer adverse effects. We recommend a trial of PRP for severe NPDR and early PDR compared with deferring PRP till the HR-PDR stage. The trial would use modern laser technologies, and investigate the value adjuvant prophylactic anti-VEGF or steroid drugs.

STUDY REGISTRATION: This study is registered as PROSPERO CRD42013005408.

FUNDING: The National Institute for Health Research Health Technology Assessment programme.