Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm.

BACKGROUND: Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. METHODS: Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6-10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. RESULTS: Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45-70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2-3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. CONCLUSIONS: The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering.

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P.aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.

Augmentation of Antipsychotic Medications with Low-Dose Clozapine in Treatment-Resistant Schizophrenia-Case Reports and Discussion.

Treatment resistance in schizophrenia is often encountered in clinical practice, with clozapine usually recommended as the appropriate therapy. However, where clozapine proves ineffective or cannot be tolerated due to side effects, treatment options are limited. In patients within forensic mental health services, residual symptomatology often presents a barrier to discharge and can have lasting effects on prospects for rehabilitation as well as risk to self and others. This paper presents a review of the relevant literature and three cases of a novel approach, utilising clozapine in doses usually considered subtherapeutic, in combination with the primary antipsychotic treatment. In all three patients, it improved clinical efficacy as well as tolerability, resulting in improvement that allowed discharge from the forensic hospital.

2-Heptylcyclopropane-1-Carboxylic Acid Disperses and Inhibits Bacterial Biofilms.

Fatty-acid signaling molecules can inhibit biofilm formation, signal dispersal events, and revert dormant cells within biofilms to a metabolically active state. We synthesized 2-heptylcyclopropane-1-carboxylic acid (2CP), an analog of cis-2-decenoic acid (C2DA), which contains a cyclopropanated bond that may lock the signaling factor in an active state and prevent isomerization to its least active trans-configuration (T2DA). 2CP was compared to C2DA and T2DA for ability to disperse biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa. 2CP at 125 µg/ml dispersed approximately 100% of S. aureus cells compared to 25% for C2DA; both 2CP and C2DA had significantly less S. aureus biofilm remaining compared to T2DA, which achieved no significant dispersal. 2CP at 125 µg/ml dispersed approximately 60% of P. aeruginosa biofilms, whereas C2DA and T2DA at the same concentration dispersed 40%. When combined with antibiotics tobramycin, tetracycline, or levofloxacin, 2CP decreased the minimum concentration required for biofilm inhibition and eradication, demonstrating synergistic and additive responses for certain combinations. Furthermore, 2CP supported fibroblast viability above 80% for concentrations below 1 mg/ml. This study demonstrates that 2CP shows similar or improved efficacy in biofilm dispersion, inhibition, and eradication compared to C2DA and T2DA and thus may be promising for use in preventing infection for healthcare applications.

In vitro evaluation of loaded chitosan membranes for pain relief and infection prevention.

Wounds resulting from surgeries, implantation of medical devices, and musculoskeletal trauma result in pain and can also result in infection of damaged tissue. Up to 80% of these infections are due to biofilm formation either on the surface of implanted devices or on surrounding wounded tissue. Bacteria within a biofilm have intrinsic growth and development characteristics that allow them to withstand up to 1,000 times the minimum inhibitory concentration of antibiotics, demonstrating the need for new therapeutics to prevent and treat these infections. Cis-2-decenoic acid (C2DA) is known to disperse preformed biofilms and can prevent biofilm formation entirely for some strains of bacteria. Additionally, local anesthetics like bupivacaine have been shown to have antimicrobial effects against multiple bacterial strains. This study sought to evaluate hexanoic acid-treated electrospun chitosan membranes (HA-ESCM) as wound dressings that release C2DA and bupivacaine to simultaneously prevent infection and alleviate pain associated with musculoskeletal trauma. Release profiles of both therapeutics were evaluated, and membranes were tested in vitro against Methicillin-resistant Staphylococcus aureus (MRSA) to determine efficacy in preventing biofilm infection and bacterial growth. Results indicate that membranes release both therapeutics for 72 hr, and release profile can be tailored by loading concentration. Membranes were effective in preventing biofilm growth but were toxic to fibroblasts when loaded with 2.5 or 5 mg of bupivacaine.

Staphylococcal infection prevention using antibiotic-loaded mannitol-chitosan paste in a rabbit model of implant-associated osteomyelitis.

Antibiotic-loaded chitosan pastes have shown advantages in the treatment and coverage of complex musculoskeletal defects. We added mannitol, previously shown to increase antibiotic susceptibility of biofilm, to an injectable chitosan/polyethylene glycol paste for delivery of antibiotics. Ground sponges (0.85% acetic acid solution, 1% chitosan, 0% or 2% mannitol, 1% polyethylene glycol) were hydrated using phosphate-buffered saline with 10 mg/ml amikacin and 10 mg/ml vancomycin added to form pastes. We inoculated rabbit radial defects with 105 colony-forming units of Staphylococcus aureus (UAMS-1) and inserted titanium pins into the cortical bone. Groups compared included mannitol blend pastes, non-mannitol blends, antibiotic-loaded bone cement, vancomycin powder, and no treatment controls. We harvested tissue samples and retrieved the pins retrieved at 3 weeks. All antibiotic-loaded groups lowered bacterial growth and colony-forming unit counts in soft and bone tissue and on titanium pins in in vivo studies. The results indicate this biomaterial is capable of eluting active antibiotics at concentrations that reduce bacterial growth on biomaterials and tissue, which, in turn, may prevent biofilm formation. Blends of chitosan and mannitol may be useful in prevention and treatment of osteomyelitis and implant-associated infections.

Characterization and Antibiofilm Activity of Mannitol-Chitosan-Blended Paste for Local Antibiotic Delivery System.

Mannitol, a polyalcohol bacterial metabolite, has been shown to activate dormant persister cells within bacterial biofilm. This study sought to evaluate an injectable blend of mannitol, chitosan, and polyethylene glycol for delivery of antibiotics and mannitol for eradication of Staphylococcal biofilm. Mannitol blends were injectable and had decreased dissociation and degradation in the enzyme lysozyme compared to blends without mannitol. Vancomycin and amikacin eluted in a burst response, with active concentrations extended to seven days compared to five days for blends without mannitol. Mannitol eluted from the paste in a burst the first day and continued through Day 4. Eluates from the mannitol pastes with and without antibiotics decreased viability of established S. aureus biofilm by up to 95.5% compared to blends without mannitol, which only decreased biofilm when loaded with antibiotics. Cytocompatibility tests indicated no adverse effects on viability of fibroblasts. In vivo evaluation of inflammatory response revealed mannitol blends scored within the 2-4 range at Week 1 (2.6 ± 1.1) and at Week 4 (3.0 ± 0.8), indicative of moderate inflammation and comparable to non-mannitol pastes (p = 0.065). Clinically, this paste could be loaded with clinician-selected antibiotics and used as an adjunctive therapy for musculoskeletal infection prevention and treatment.

A computational triage approach to the synthesis of novel difluorocyclopentenes and fluorinated cycloheptadienes using thermal rearrangements.

Electronic structure calculations have been used for the effective triage of substituent effects on difluorinated vinylcyclopropane precursors and their ability to undergo vinyl cyclopropane rearrangements (VCPR). Groups which effectively stabilised radicals, specifically heteroarenes, were found to result in the lowest energy barriers. Ten novel precursors were synthesised to test the accuracy of computational predictions; the most reactive species which contained heteroarenes underwent thermal rearrangements at room temperature to afford novel difluorocyclopentenes and fluorinated benzocycloheptadienes through competing VCPR and [3,3]-rearrangement pathways, respectively. More controlled rearrangement of ethyl 3-(1'(2'2'-difluoro-3'-benzo[d][1,3]dioxol-5-yl)cyclopropyl)propenoate (22) allowed these competing pathways to be monitored at the same time and activation energies for both reactions were determined; Ea(VCPR) = (23.4 ± 0.2) kcal mol-1 and Ea([3,3]) = (24.9 ± 0.3) kcal mol-1. Comparing our calculated activation energies with these parameters showed that no single method stood out as the most accurate for predicting barrier heights; (U)M05-2X/6-31+G* methodology remained the best for VCPR but M06-2X/6-31G* was better for the [3,3]-rearrangement. The consistency observed with (U)B3LYP/6-31G* calculations meant that it came closest to a universal method for dealing with these systems. The developed computational design model correctly predicted the observed selectivity of rearrangement pathways for both our system and literature compounds.

Optimization of Novel Indazoles as Highly Potent and Selective Inhibitors of Phosphoinositide 3-Kinase δ for the Treatment of Respiratory Disease.

Optimization of lead compound 1, through extensive use of structure-based design and a focus on PI3Kδ potency, isoform selectivity, and inhaled PK properties, led to the discovery of clinical candidates 2 (GSK2269557) and 3 (GSK2292767) for the treatment of respiratory indications via inhalation. Compounds 2 and 3 are both highly selective for PI3Kδ over the closely related isoforms and are active in a disease relevant brown Norway rat acute OVA model of Th2-driven lung inflammation.

Evaluating the thermal vinylcyclopropane rearrangement (VCPR) as a practical method for the synthesis of difluorinated cyclopentenes: experimental and computational studies of rearrangement stereospecificity.

Vinyl cyclopropane rearrangement (VCPR) has been utilised to synthesise a difluorinated cyclopentene stereospecifically and under mild thermal conditions. Difluorocyclopropanation chemistry afforded ethyl 3-(1'(2'2'-difluoro-3'-phenyl)cyclopropyl) propenoate as all four stereoisomers (18a, 18b, 22a, 22b) (all racemic). The trans-E isomer (18a), prepared in 70 % yield over three steps, underwent near quantitative VCPR to difluorocyclopentene 23 (99 %). Rearrangements were monitored by (19) F NMR (100-180 °C). While cis/trans cyclopropane stereoisomerisation was facile, favouring trans-isomers by a modest margin, no E/Z alkene isomerisation was observed even at higher temperatures. Neither cis nor trans Z-alkenoates underwent VCPR, even up to much higher temperatures (180 °C). The cis-cyclopropanes underwent [3,3]-rearrangement to afford benzocycloheptadiene species. The reaction stereospecificity was explored by using electronic structure calculations, and UB3LYP/6-31G* methodology allowed the energy barriers for cyclopropane stereoisomerisation, diastereoisomeric VCPR and [3,3]-rearrangement to be ranked in agreement with experiment.

Single-step microwave-mediated synthesis of oxazoles and thiazoles from 3-oxetanone: a synthetic and computational study.

The direct microwave-mediated condensation between 3-oxetanone and primary amides and thioamides has delivered moderate to good yields of (hydroxymethyl)oxazoles and (hydroxymethyl)thiazoles. The reactions use a sustainable solvent and only require short reaction times. These are highly competitive methods for the construction of two classes of valuable heteroarenes, which bear a useful locus for further elaboration. Electronic structure calculations have shown that the order of events involves chalcogen atom attack at sp(3) carbon and alkyl-oxygen cleavage. The critical role of acid catalysis was shown clearly, and the importance of acid strength was demonstrated. The calculated barriers were also fully consistent with the observed order of thioamide and amide reactivity. Spontaneous ring opening involves a modest degree of C-O cleavage, moderating the extent of strain relief. On the acid-catalysed pathway, C-O cleavage is less extensive still, but proton transfer to the nucleofuge is well advanced with the carboxylic acid catalysts, and essentially complete with methanesulfonic acid.

Photophysical properties of platinum(II)-acetylide complexes: the effect of a strongly electron-accepting diimine ligand on excited-state structure.

The compounds [Pt(MesBIAN)(C[triple bond]CR)2] (R = C6H4-CN-p, 1; SiMe3, 2; C6H4-CF3-p, 3; C6H5, 4; C6H4-CH3-p 5) {MesBIAN = bis(mesitylimino)acenaphthene} have been synthesized; the X-ray crystal structure determinations of 4 and 5 and the starting material [Pt(MesBIAN)Cl2] are reported. Chemical oxidation of 4 with diiodine leads to generation of an intermediate platinum(IV) bis(acetylide) diiodide complex, which then couples and reductively eliminates the acetylide ligands as a diyne, leading to the generation of [Pt(MesBIAN)I2] 6. Compound 2 readily forms an adduct 2a with copper(I) chloride, in which the copper atom is bonded to the two acetylide triple bonds. 1-5 each undergo an irreversible oxidation, and a reversible one-electron reduction to generate a stable anion. ESR studies of 1(-)-5(-) show that the unpaired electron is localized mainly on the pi* orbital of the coordinated MesBIAN ligand, with about 10% platinum contribution to the singly occupied molecular orbital (SOMO). The compounds show a strong absorption at around 500 nm in the UV/visible spectrum, which is assigned to a "mixed metal-ligand to ligand charge transfer" (MMLL'CT) transition; this assignment is supported by time-dependent density-functional theory (TD-DFT) calculations on 5. 1-5 emit in the near-infrared region from a (3)MMLL'CT excited state, with lifetimes ranging from 8 to 36 ns. Picosecond and nanosecond time-resolved infrared (TRIR) spectroscopy has been used to probe directly the nature and dynamics of the excited state of 5. The TRIR data show a decrease of the energy of the C[triple bond]C vibration upon excitation, by about 90 cm(-1) in comparison to the ground state, and formation of a new, very intense, and very broad band at 1820 cm(-1). We propose that the excited-state structure contains some contribution from a pseudo-cumulenic form of the platinum-acetylide moiety, which is supported by TD-DFT calculations. Picosecond TRIR allowed determination of the rate of vibrational relaxation (14 ps) of the vibrationally "hot" electronic excited state of 5 formed upon initial laser excitation.

Bulky 4-phosphacyclohexanones: diastereoselective complexations, orthometallations and unprecedented [3.1.1]metallabicycles.

The 4-phosphacyclohexanones, 2,2,6,6-tetramethyl-1-phenyl-4-phosphorinanone (La), 1,2,6-triphenyl-4-phosphorinanone ((Ph)Lb), 1-cyclohexyl-2,6-diphenyl-4-phosphorinanone ((Cy)Lb) and 1-tert-butyl-2,6-diphenyl-4-phosphorinanone ((Bu)Lb) have been made by modifications of literature methods. Phosphines (R)Lb are each formed as mixtures of meso- and rac-diastereoisomers. Isomerically pure rac-(Ph)Lb, rac-(Cy)Lb and meso-(Bu)Lb can be isolated by recrystallisation from MeCN. Heating mixtures of isomers of (R)Lb with TsOH leads to isomerisations to give predominantly the meso-(R)Lb. The complex trans-[PdCl2(La)2] (1) is readily made from [PdCl2(NCPh)2] but the analogous platinum complex 2 has not been detected and instead, cyclometallation at the 3-position (alpha to the ketone) in the phosphacycle occurs to give trans-[PtCl(La)(La-3H)] (3) (where La-3H = La deprotonated at the 3-position) featuring a [3.1.1]metallabicycle as confirmed by X-ray crystallography. The analogous palladabicycle 4 has been detected upon treatment of 1 with Et3N in refluxing toluene. The type of complex formed by (R)Lb depends on which diastereoisomer (meso or rac) is involved. rac-(Ph)Lb (a mixture of R,R- and S,S-enantiomers, labelled alpha and beta) forms trans-[MCl2(rac-(Ph)Lb)2], M = Pd (5) or Pt (6), as mixtures of diastereoisomers (alphaalpha/betabeta and alphabeta forms). The structure of alphaalpha-6 has been determined by X-ray crystallography. Ligand competition experiments monitored by 31P NMR showed that Pd(II) and Pt(II) have a significant preference to bind rac-(Ph)Lb over meso-(Ph)Lb. meso-(Bu)Lb reacts with [PtCl2(NCBu(t))2] under ambient conditions to give the binuclear complex [Pt2Cl2(meso-(Bu)Lb-2'H)2] (7) where orthometallation has occurred on one of the exocyclic phenyl substituents as confirmed by X-ray crystallography. rac-(Bu)Lb reacts with [PtCl2(NCBu(t))2] to give a mononuclear cyclometallated species assigned the structure trans-[PtCl(rac-(Bu)Lb-2'H)((Bu)Lb)] (8) on the basis of its 31P NMR spectrum. rac-(Cy)Lb reacts with [PtCl2(NCBu(t))2] in refluxing toluene to give trans-[PtCl2(rac-(Cy)Lb)2] (9) and the crystal structure of alphabeta-9 has been determined.