Polymyxin B and ethylenediaminetetraacetic acid act synergistically against Pseudomonas aeruginosa and Staphylococcus aureus.

Polymyxin B and ethylenediaminetetraacetic acid are antimicrobials possessing antibiofilm activity. They act by displacement and chelation, respectively, of divalent cations in bacterial membranes and may therefore act synergistically when applied in combination. If so, this combination of agents may be useful for the treatment of diseases like cystic fibrosis (CF), in which biofilms are present on the respiratory epithelium. We used checkerboard assays to investigate the synergy between these agents using reference strains Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 6538 in planktonic form. We then determined the efficacy of each agent against biofilms of both species grown on 96-pin lids and proceeded to combination testing against the P. aeruginosa reference strain and 10 clinical isolates from patients with CF. Synergism was observed for planktonic forms of both species and for biofilms of P. aeruginosa. The susceptibility of biofilms of P. aeruginosa clinical isolates to these agents was variable compared to the laboratory reference strain. This combination of agents may be useful in the management of biofilm-associated conditions, particularly those amenable to topical therapies. These results provide a basis upon which the antimicrobial and antibiofilm efficacy of preparations containing these agents may be enhanced.IMPORTANCEBacteria living in biofilms produce a protective matrix which makes them difficult to kill. Patients with severe respiratory disease often have biofilms. Polymyxin B is an antibiotic commonly used in topical medications, such as eye drops and nasal sprays. Ethylenediaminetetraacetic acid (EDTA) is used widely as a preservative in medication but also has antimicrobial properties. It has been hypothesized that Polymyxin B and EDTA could have a synergistic relationship: when used in combination their antimicrobial effect is enhanced. Here, we evaluated the levels at which Polymyxin B and EDTA work together to kill common pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We found that Polymyxin B and EDTA were synergistic. This synergy may be useful in the management of planktonic infection with P. aeruginosa and S. aureus, or biofilm infection with P. aeruginosa. This synergy may be beneficial in the treatment of respiratory biofilms, in which P. aeruginosa biofilms are common.

Thiol-ene enabled preparation of S-lipidated anti-HBV peptides.

Despite significant efforts made towards treatments for Hepatitis B virus (HBV), a long-term curative treatment has thus far eluded scientists. Recently, the Sodium Taurocholate Co-Transporting Polypeptide (NTCP) receptor has been identified as the entry pathway of HBV into hepatocytes. Myrcludex B, an N-terminally myristoylated 47-mer peptide mimic of the preS1 domain of the Hepatitis B virion, was identified as a potent protein-protein interaction (PPI) inhibitor blocking HBV fusion (IC50 = 140 pM). Herein we report an optimised chemical synthesis of Myrcludex B and a series of novel analogues. Employing a small modification to the Cysteine Lipidation of a Peptide or Amino acid (CLipPA) thiol-ene reaction, a library of S-lipidated Myrcludex B and truncated (21-mer) analogues were prepared, providing novel chemical space to probe for the discovery of novel anti-HBV peptides. The S-lipidated analogues showed an equivalent or a slight decrease (∼2-fold) in binding effectiveness to NTCP expressing hepatocytes compared to Myrcludex B. Three S-lipidated analogues were highly potent HBV inhibitors (IC50 0.97-3.32 nM). These results demonstrate that incorporation of heteroatoms into the lipid 'anchor' is tolerated by this antiviral scaffold and to the best of our knowledge constitutes the first report of potent S-lipidated antiviral peptides. Interestingly, despite only moderate reductions in binding effectiveness, truncated analogues possessed dramatically reduced inhibitory activity thus providing new insights into the structure activity relationship of these hitherto unreported antiviral S-lipopeptides.

Thiol-ene Enabled Chemical Synthesis of Truncated S-Lipidated Teixobactin Analogs.

Herein is described the introduction of lipid moieties onto a simplified teixobactin pharmacophore using a modified Cysteine Lipidation on a Peptide or Amino acid (CLipPA) technique, whereby cysteine was substituted for 3-mercaptopropionic acid (3-MPA). A truncated teixobactin analog was prepared with the requisite thiol handle, thus enabling an array of vinyl esters to be conveniently conjugated onto the simplified teixobactin pharmacophore to yield S-lipidated cyclic lipopeptides.

On-Resin Preparation of Allenamidyl Peptides: A Versatile Chemoselective Conjugation and Intramolecular Cyclisation Tool.

The ability to modify peptides and proteins chemoselectively is of continued interest in medicinal chemistry, with peptide conjugation, lipidation, stapling, and disulfide engineering at the forefront of modern peptide chemistry. Herein we report a robust method for the on-resin preparation of allenamide-modified peptides, an unexplored functionality for peptides that provides a versatile chemical tool for chemoselective inter- or intramolecular bridging reactions with thiols. The bridging reaction is biocompatible, occurring spontaneously at pH 7.4 in catalyst-free aqueous media. By this "click" approach, a model peptide was successfully modified with a diverse range of alkyl and aryl thiols. Furthermore, this technique was demonstrated as a valuable tool to induce spontaneous intramolecular cyclisation by preparation of an oxytocin analogue, in which the native disulfide bridge was replaced with a vinyl sulfide moiety formed by thia-Michael addition of a cysteine thiol to the allenamide handle.

Direct synthesis of cyclic lipopeptides using intramolecular native chemical ligation and thiol-ene CLipPA chemistry.

Naturally occurring cyclic lipopeptides exhibit a diverse range of biological activities and possess several favourable properties. Chemically synthesising and modifying these natural compounds can alter their biological and physical properties. Cyclic lipopeptides are often difficult to synthesise, especially when the lipid moiety is directly attached to the cyclic scaffold. The construction of a series of cyclic lipopeptide analogues of the antifungal peptide iturin A is reported herein. The synthesis of the parent peptide macrocycle was achieved using native chemical ligation (NCL), whereupon the regenerated free thiol was used to attach a lipid moiety using Cysteine Lipidation on a Peptide or Amino acid (CLipPA) technology.

"CLipP"ing on lipids to generate antibacterial lipopeptides.

We herein report the synthesis and biological and computational evaluation of 12 linear analogues of the cyclic lipopeptide battacin, enabled by Cysteine Lipidation on a Peptide or Amino Acid (CLipPA) technology. Several of the novel "CLipP"ed lipopeptides exhibited low micromolar MICs and MBCs against both Gram-negative and Gram-positive bacteria. The mechanism of action was then simulated with the MIC data using computational methods.

Synthesis of Antimicrobial Lipopeptides Using the "CLipPA" Thiol-Ene Reaction.

Cysteine Lipidation on a Peptide or Amino acid (CLipPA) technology provides a facile method for the lipidation of unprotected peptides containing a free thiol group by using a "click" radical-initiated thiol-ene reaction to effect addition to a vinyl ester. The methodology is highly versatile, leading to high conversion rates while maintaining excellent chemoselectivity and tolerance for a large variety of peptide substrates and functional groups. Herein we describe the simple general procedure for the synthesis of a focused library of bioactive S-lipidated antimicrobial peptides via late-stage derivatization using solution-phase CLipPA lipidation.

Synthesis and SAR Analysis of Lipovelutibols B and D and Their Lipid Analogues.

The first syntheses of the cytotoxic peptides lipovelutibols B and D are described. While lipovelutibol D was prepared using solid-phase peptide synthesis followed by an O-N acyl migration to install the C-terminal amino alcohol, a different strategy was required to access lipovelutibol B and a series of N-terminal lipid analogues of the natural products. A cytotoxicity structure-activity relationship study revealed that the lipovelutibol D framework, whereby serine is substituted for alanine in the fifth position, provided the most potent analogues. Modification of the lipid tail was generally well tolerated, with longer alkyl chains enhancing analogue cytotoxicity.

Postmenopausal hormone therapy as a risk factor for gastroesophageal reflux symptoms among female twins.

BACKGROUND & AIMS: Female sex hormones have been suggested to increase the risk of gastroesophageal reflux symptoms via a relaxing effect on the lower esophageal sphincter. We investigated the relationship of oral contraceptives and postmenopausal hormone therapy (HT) to risk of reflux symptoms, controlling for genetic factors and body mass. METHODS: Information on exposures and reflux symptoms was obtained by telephone interviews conducted in 1998-2002 among women in the Swedish Twin Registry. Use of oral contraceptives was also assessed in 1973 by questionnaires. Both cross-sectional and prospective nested case-control designs were used, each with external control analysis. The cross-sectional design was further submitted to monozygotic co-twin control analysis. RESULTS: The cross-sectional study design comprised 4365 twins with reflux and 17,321 without. In ever users of estrogen HT, the risk of reflux symptoms was increased by 32% (odds ratio, 1.32; 95% confidence interval, 1.18-1.47). This association remained in the nested case-control analyses and increased slightly with higher body mass index. A similar pattern was observed for the use of progestin in the cross-sectional design, but no association remained in the nested case-control analysis. Use of oral contraceptives was not associated with an increased risk of reflux symptoms. Generally, the risk estimates remained virtually unchanged after adjustments for potential confounding factors, including genetic factors. CONCLUSIONS: This population-based twin study indicates that estrogen HT is an independent risk factor for reflux symptoms, while the influence of progestin HT and oral contraceptives is less consistent.

Endoscopic treatment of high-grade dysplasia and early cancer in Barrett's oesophagus.

Barrett's oesophagus is the premalignant precursor of oesophageal adenocarcinoma. Non-dysplastic metaplasia can progress to low-grade dysplasia, high-grade dysplasia, and finally to invasive cancer. Although the frequency of adenocarcinoma in patients with Barrett's oesophagus is low, surveillance is justified because the outcome of adenocarcinoma is poor. Oesophagectomy remains the standard treatment for patients with high-grade dysplasia and superficial carcinoma. However, it has been associated with substantial morbidity and mortality and some patients are judged unfit for surgery. In this review, the present status of less invasive procedures is discussed. Endotherapy preserves the integrity of the oesophagus and allows a better quality of life to patients at low risk of developing lymph-node metastases. Opposition to endoscopic treatment is based mainly on the identification of undetected foci of cancer and high-grade dysplasia in oesophagectomy samples. The current ablative techniques used are photodynamic therapy, argon plasma coagulation, laser treatment, and endoscopic mucosal resection.

Small adenocarcinomas of the esophagogastric junction: association with intestinal metaplasia and dysplasia.

OBJECTIVES: Intestinal metaplasia in Barrett's esophagus predisposes to esophageal adenocarcinoma. Intestinal metaplasia of the cardia is a common finding in persons without cancer. Many adenocarcinomas of the esophagogastric junction are large enough to obliterate any underlying intestinal metaplasia. To estimate how often adenocarcinoma of the esophagogastric junction arises in intestinal metaplasia, we studied small adenocarcinomas of the esophagogastric junction. METHODS: Resection patients had adenocarcinomas 2 cm or smaller, within 2 cm of the esophagogastric junction. Age- and sex-matched controls had resection for squamous carcinoma. Saved and new histological slides from the esophagogastric junction were examined, with additional stains. RESULTS: Intestinal metaplasia was found in 86% (19/22) of adenocarcinoma cases, versus 32% (7/22) of controls (p < 0.001). Intestinal metaplasia with high or low grade dysplasia was associated with 64% (14/22) of adenocarcinomas and with 5% (1/22) of controls (p < 0.001). Excluding four cases with long and three with short Barrett's esophagus, 80% (12/15) of adenocarcinomas had associated intestinal metaplasia, 53% (8/15) with dysplasia. Most adenocarcinoma cases had the incomplete type of intestinal metaplasia with a Barrett type cytokeratin 7/20 staining pattern. Helicobacter pylori were seen in one adenocarcinoma and five control cases. CONCLUSIONS: Most adenocarcinomas of the esophagogastic junction arise in the background of intestinal metaplasia, sometimes in an endoscopically visible Barrett's esophagus, more often in small areas of intestinal metaplasia of the cardia. In cases of adenocarcinoma, the intestinal metaplasia resembled that found in Barrett's esophagus, and was not associated with H. pylori.

Barrett's esophagus: prevalence in symptomatic relatives.

OBJECTIVES: Relatives of patients with Barrett's esophagus have an increased prevalence of reflux symptoms. Our aim was to find if these relatives were at increased risk of having Barrett's esophagus. METHODS: First degree relatives of patients with Barrett's esophagus completed the Reflux Symptom Questionnaire. Relatives with reflux symptoms, never previously investigated. were invited for endoscopy. Controls were patients with similar reflux symptoms and no family histories of Barrett's esophagus. RESULTS: We found previously undiagnosed Barrett's esophagus (>3 cm) in eight of 100 relatives (8%) from 53 families and in five of 100 controls (5%) (adjusted OR = 1.58, 95% CI = 0.46-5.45). Including another 27 previously investigated cases, 10 of the 53 families had two or more cases of Barrett's esophagus. Barrett's esophagus prevalence increased with age (p = 0.014) and was associated with reflux symptoms of >10 yr (p = 0.020), and Barrett's esophagus was twice as common in males (p = 0.28). Reflux esophagitis was found in 74% of relatives and 57% of controls without Barrett's (p = 0.04). CONCLUSIONS: The risk of Barrett's esophagus in any one symptomatic relative of a patient with Barrett's esophagus was not statistically higher than in other persons with reflux symptoms. However, more relatives of Barrett's esophagus patients have reflux symptoms, so the overall prevalence of Barrett's esophagus and reflux esophagitis in relatives may also be greater than in the general population. In considering whether to screen patients with reflux symptoms for Barrett's esophagus, age and duration of symptoms are stronger predictors than having a relative with Barrett's esophagus.

Barrett's esophagus and reflux esophagitis: is there a missing link?

OBJECTIVES: Barrett's esophagus (BE) is associated with esophageal reflux. The development stage of BE is not well described. Epidemiological evidence indicates that the columnar epithelium in BE is acquired and reaches its full length rapidly. We tested the hypothesis that BE might result from direct replacement of erosions in reflux esophagitis (RE). METHODS: At endoscopy, we compared the length and distribution of esophageal erosions in 50 patients with RE with the length and distribution of columnar epithelium in 50 patients with BE. RESULTS: The median length of erosions in RE was 2 cm, less than the median length of columnar epithelium in BE, 5 cm (p < 0.001). Erosions in RE were usually multiple and scattered, involving the entire circumference of the esophagus in only 10% of cases, but circumferential involvement by columnar epithelium was found in 68% of BE cases (p < 0.001). Circumferential involvement, 3 cm or longer, was found in 0% of cases of RE versus 56% of BE cases (p < 0.001). Two patients without RE or BE had large areas of epithelial loss of uncertain etiology. CONCLUSIONS: The length and distribution of erosions in RE differ greatly from the length and distribution of columnar epithelium in BE. It is unlikely that BE arises directly from areas of esophagitis. We suggest that BE may develop after loss of a long segment of squamous epithelium, with columnar replacement in the presence of continuing acid reflux.

Gastroesophageal reflux disease in monozygotic and dizygotic twins.

BACKGROUND & AIMS: Gastroesophageal reflux disease (GERD) interferes with the quality of life and carries an increased risk for esophageal adenocarcinoma. We investigated genetic influence in the development of reflux. METHODS: We compared concordance for reflux in monozygotic (MZ) and dizygotic (DZ) twins. All twins age 55 and older in the nationwide Swedish Twin Registry were invited to participate. Data were collected by computer-assisted telephone interviews. Reflux disease was defined by symptomatic heartburn or acid regurgitation occurring at least weekly. RESULTS: A total of 2178 monozygotic, 3219 same-sex dizygotic, and 3014 unlike-sex dizygotic twin pairs provided information. Overall, 15.3% of the twins had reflux. In men, the intraclass correlation for reflux was 0.29 (95% confidence interval [CI], 0.15-0.43) for monozygotic and 0.13 (95% CI, 0.02-0.25) for dizygotic pairs. In women, the correlation was 0.33 (95% CI, 0.22-0.44) for monozygotic and 0.14 (95% CI, 0.04-0.24) for dizygotic pairs. For unlike-sex dizygotic pairs, the correlation was 0.06 (95% CI, -0.01 to 0.14). Concordance for reflux was not caused by inherited obesity or alcohol use; inherited smoking may be a minor factor. CONCLUSIONS: The increased concordance for reflux in monozygotic pairs, compared with dizygotic pairs, indicates genetic rather than shared environmental effects. Heritability accounted for 31% (23%-39%) of the liability to reflux disease in this population.