Janus PEG-based dendrimers for use in combination therapy: controlled multi-drug loading and sequential release

The increasing use of drug combinations to treat disease states, such as cancer, calls for improved delivery systems that are able to deliver multiple agents. Herein, we report a series of novel Janus dendrimers with potential for use in combination therapy. Different generations (first and second) of PEG-based dendrons containing two different “model drugs”, benzyl alcohol (BA) and 3-phenylpropionic acid (PPA), were synthesized. BA and PPA were attached via two different linkers (carbonate and ester, respectively) to promote differential drug release. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of BA and PPA, namely, (BA)2-G1-G1-(PPA)2, (BA)4-G2-G1-(PPA)2, (BA)2-G1-G2-(PPA)4, and (BA)4-G2-G2-(PPA)4. Release studies in plasma showed that the dendrimers provided sequential release of the two model drugs, with BA being released faster than PPA from all of the dendrons. The different dendrimers allowed delivery of increasing amounts (0.15–0.30 mM) and in exact molecular ratios (1:2; 2:1; 1:2; 2:2) of the two model drug compounds. The dendrimers were noncytotoxic (100% viability at 1 mg/mL) toward human umbilical vein endothelial cells (HUVEC) and nontoxic toward red blood cells, as confirmed by hemolysis studies. These studies demonstrate that these Janus PEG-based dendrimers offer great potential for the delivery of drugs via combination therapy.

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

Aims: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracyclineresistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. Methods and Results: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l(-1) and a 10% increase in E. coli with an MIC > 50 mg l(-1) during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 mug g(-1) faeces during treatment. Conclusion: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. Significance and Impact of the Study: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

Sources of variation in the ampicillin-resistant Escherichia coli concentration in the feces of organic broiler chickens

Currently, there are limited published data for the population dynamics of antimicrobial-resistant commensal bacteria. This study was designed to evaluate both the proportions of the Escherichia coli populations that are resistant to ampicillin at the level of the individual chicken on commercial broiler farms and the feasibility of obtaining repeated measures of fecal E. coli concentrations. Short-term temporal variation in the concentration of fecal E. coli was investigated, and a preliminary assessment was made of potential factors involved in the shedding of high numbers of ampicillin-resistant E. coli by growing birds in the absence of the use of antimicrobial drugs. Multilevel linear regression modeling revealed that the largest component of random variation in log-transformed fecal E. coli concentrations was seen between sampling occasions for individual birds. The incorporation of fixed effects into the model demonstrated that the older, heavier birds in the study were significantly more likely (P = 0.0003) to shed higher numbers of ampicillin-resistant E. coli. This association between increasing weight and high shedding was not seen for the total fecal E. coli population (P = 0.71). This implies that, in the absence of the administration of antimicrobial drugs, the proportion of fecal E. coli that was resistant to ampicillin increased as the birds grew. This study has shown that it is possible to collect quantitative microbiological data on broiler farms and that such data could make valuable contributions to risk assessments concerning the transfer of resistant bacteria between animal and human populations.

Effects of neuropathy on high-voltage-activated Ca2+ current in sensory neurones

Voltage-dependent Ca2+ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the CaV2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca2+ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from CaV2.3(−/−) knock-out and wild-type (WT) mice. DRG neurones from CaV2.3(−/−) mice had significantly reduced sensitivity to SNX-482 versusWTmice. DRGs from CaV2.3(−/−) mice also had increased sensitivity to the CaV2.2 VDCC blocker -conotoxin. In WT mice, PSNL caused a significant increase in -conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In CaV2.3(−/−) mice, PSNL caused a significant reduction in -conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca2+ current were not accompanied by effects on voltagedependence of activation in either CaV2.3(−/−) or WT mice. These data suggest that CaV2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca2+ current in these cells. In WT mice, PSNL caused adaptive changes in CaV2.2- and CaV2.3-mediated Ca2+ currents, supporting roles for these VDCCs in nociception during neuropathy. In CaV2.3(−/−) mice, PSNL-induced changes in CaV1 and CaV2.2 Ca2+ current, consistent with alternative adaptive mechanisms occurring in the absence of CaV2.3 subunits.

Farm-level risk factors for fluoroquinolone resistance in E-coli and thermophilic Campylobacter spp. on finisher pig farms

Logistic regression, supported by other statistical analyses was used to explore the possible association of risk factors with the fluoroquinolone (FQ)-resistance status of 108 pig finisher farms in Great Britain. The farms were classified as 'affected' or 'not affected' by FQ-resistant E. coli or Campylobacter spp. on the basis of isolation of organisms from faecal samples on media containing 1 mg/l FQ. The use of FQ was the most important factor associated with finding resistant E. coli and/or Campylobacter, which were found on 79% (FQ-resistant E. coli) and 86% (FQ-resistant Campylobacter) of farms with a history of FQ use. However, resistant bacteria were also found on 19% (FQ-resistant E. coli) and 54% (FQ-resistant Campylobacter) of farms with no history of FQ use. For FQ-resistant E. coli, biosecurity measures may be protective and there was strong seasonal variation, with more farms found affected when sampled in the summer. For FQ-resistant Campylobacter, the buying-in of grower stock may increase risk and good on-farm hygiene may be protective. The findings suggest that resistant organisms, particularly Campylobacter, may spread between pig farms.

A survey of fluoroquinolone resistance in Escherichia coli and thermophilic Campylobacter spp. on poultry and pig farms in Great Britain

Aims: To estimate the proportions of farms on which broilers, turkeys and pigs were shedding fluoroquinolone (FQ)-resistant Escherichia coli or Campylobacter spp. near to slaughter. Methods and Results: Freshly voided faeces were collected on 89 poultry and 108 pig farms and cultured with media containing 1.0 mg l(-1) ciprofloxacin. Studies demonstrated the specificity of this sensitive method, and both poultry and pig sampling yielded FQ-resistant E. coli on 60% of farms. FQ-resistant Campylobacter spp. were found on around 22% of poultry and 75% of pig farms. The majority of resistant isolates of Campylobacter (89%) and E. coli (96%) tested had minimum inhibitory concentrations for ciprofloxacin of >= 8 mg l(-1). The proportion of resistant E. coli and Campylobacter organisms within samples varied widely. Conclusions: FQ resistance is commonly present among two enteric bacterial genera prevalent on pig and poultry farms, although the low proportion of resistant organisms in many cases requires a sensitive detection technique. Significance and Impact of the Study: FQ-resistant bacteria with zoonotic potential appear to be present on a high proportion of UK pig and poultry farms. The risk this poses to consumers relative to other causes of FQ-resistant human infections remains to be clarified.

Decision-making in a phase II clinical trial: a new approach combining Bayesian and frequentist concepts

The aim of a phase H clinical trial is to decide whether or not to develop an experimental therapy further through phase III clinical evaluation. In this paper, we present a Bayesian approach to the phase H trial, although we assume that subsequent phase III clinical trials will hat,e standard frequentist analyses. The decision whether to conduct the phase III trial is based on the posterior predictive probability of a significant result being obtained. This fusion of Bayesian and frequentist techniques accepts the current paradigm for expressing objective evidence of therapeutic value, while optimizing the form of the phase II investigation that leads to it. By using prior information, we can assess whether a phase II study is needed at all, and how much or what sort of evidence is required. The proposed approach is illustrated by the design of a phase II clinical trial of a multi-drug resistance modulator used in combination with standard chemotherapy in the treatment of metastatic breast cancer. Copyright (c) 2005 John Wiley & Sons, Ltd.

The staphylococcus aureus surface protein IsdA mediates resistance to the innate defenses of human skin

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.

Bacterial pathogen evolution: breaking news

The immense social and economic impact of bacterial pathogens, from drug-resistant infections in hospitals to the devastation of agricultural resources, has resulted in major investment to understand the causes and conse- quences of pathogen evolution. Recent genome se- quencing projects have provided insight into the evolution of bacterial genome structures; revealing the impact of mobile DNA on genome restructuring and pathogenicity. Sequencing of multiple genomes of relat- ed strains has enabled the delineation of pathogen evo- lution and facilitated the tracking of bacterial pathogens globally. Other recent theoretical and empirical studies have shown that pathogen evolution is significantly influenced by ecological factors, such as the distribution of hosts within the environment and the effects of co- infection. We suggest that the time is ripe for experi- mentalists to use genomics in conjunction with evolu- tionary ecology experiments to further understanding of how bacterial pathogens evolve.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform bursting induced by 100M4-AP or removal of external Mg2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. It may also uncover additional modes of action that contribute to anti-epileptiform drug effects

Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines.

The discovery of new molecular targets and the subsequent development of novel anticancer agents are opening new possibilities for drug combination therapy as anticancer treatment. Polymer-drug conjugates are well established for the delivery of a single therapeutic agent, but only in very recent years their use has been extended to the delivery of multi-agent therapy. These early studies revealed the therapeutic potential of this application but raised new challenges (namely, drug loading and drugs ratio, characterisation, and development of suitable carriers) that need to be addressed for a successful optimisation of the system towards clinical applications.

Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform, bursting induced by 100 mu M 4-AP or removal of external Mg2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. it may also uncover additional modes of action that contribute to anti-epileptiform drug effects. (C) 2009 Elsevier B.V. All rights reserved.

Complete sequence and molecular epidemiology of IncK epidemic plasmid encoding bla(CTX-M-14)

Antimicrobial drug resistance is a global challenge for the 21st century with the emergence of resistant bacterial strains worldwide. Transferable resistance to beta-lactam antimicrobial drugs, mediated by production of extended-spectrum beta-lactamases (ESBLs), is of particular concern. In 2004, an ESBL-carrying IncK plasmid (pCT) was isolated from cattle in the United Kingdom. The sequence was a 93,629-bp plasmid encoding a single antimicrobial drug resistance gene, bla(CTX-M-14). From this information, PCRs identifying novel features of pCT were designed and applied to isolates from several countries, showing that the plasmid has disseminated worldwide in bacteria from humans and animals. Complete DNA sequences can be used as a platform to develop rapid epidemiologic tools to identify and trace the spread of plasmids in clinically relevant pathogens, thus facilitating a better understanding of their distribution and ability to transfer between bacteria of humans and animals.