In Vitro and In Vivo Activities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae.

Gram-negative bacteria are evolving to produce ß-lactamases of increasing diversity that challenge antimicrobial chemotherapy. OP0595 is a new diazabicyclooctane serine ß-lactamase inhibitor which acts also as an antibiotic and as a ß-lactamase-independent ß-lactam "enhancer" against Enterobacteriaceae Here we determined the optimal concentration of OP0595 in combination with piperacillin, cefepime, and meropenem, in addition to the antibacterial activity of OP0595 alone and in combination with cefepime, in in vitro time-kill studies and an in vivo infection model against five strains of CTX-M-15-positive Escherichia coli and five strains of KPC-positive Klebsiella pneumoniae An OP0595 concentration of 4 µg/ml was found to be sufficient for an effective combination with all three ß-lactam agents. In both in vitro time-kill studies and an in vivo model of infection, cefepime-OP0595 showed stronger efficacy than cefepime alone against all ß-lactamase-positive strains tested, whereas OP0595 alone showed weaker or no efficacy. Taken together, these data indicate that combinational use of OP0595 and a ß-lactam agent is important to exert the antimicrobial functions of OP0595.

OP0595, a new diazabicyclooctane: mode of action as a serine ß-lactamase inhibitor, antibiotic and ß-lactam 'enhancer'.

OBJECTIVES: The production of a growing diversity of ß-lactamases by Gram-negative bacteria challenges antimicrobial chemotherapy. OP0595, discovered separately by each of Meiji Seika Pharma and Fedora Pharmaceuticals, is a new diazabicyclooctane serine ß-lactamase inhibitor that also acts as an antibiotic and as a ß-lactamase-independent ß-lactam 'enhancer'. METHODS: Inhibitory activity against serine ß-lactamases and affinity for PBPs were determined using nitrocefin and Bocillin FL, respectively. MICs alone and in combination with ß-lactam agents were measured according to CLSI recommendations. Morphological changes in Escherichia coli were examined by phase-contrast microscopy. RESULTS: IC50s of OP0595 for class A and C ß-lactamases were <1000 nM, with covalent binding demonstrated to the active-site serine of CTX-M-44 and AmpC enzymes. OP0595 also had direct antibiotic activity against many Enterobacteriaceae, associated with inhibition of PBP2 and conversion of the bacteria into spherical forms. Synergy between OP0595 and ß-lactam agents was seen against strains producing class A and C ß-lactamases vulnerable to inhibition. Lastly, OP0595 lowered the MICs of PBP3-targeted partner ß-lactam agents for a non-ß-lactamase-producing E. coli mutant that was resistant to OP0595 itself, indicating ß-lactamase-independent 'enhancer'-based synergy. CONCLUSIONS: OP0595 acts in three ways: (i) as an inhibitor of class A and C ß-lactamases, covalently binding at their active sites; (ii) as an antibacterial, by inhibiting PBP2 of several Enterobacteriaceae; and (iii) as an 'enhancer' of ß-lactam agents that bind to other PBPs besides PBP2 for several Enterobacteriaceae. OP0595 has considerable potential to overcome resistance when it is combined with various ß-lactam agents.

Interfacial electron-transfer kinetics in metal-free organic dye-sensitized solar cells: combined effects of molecular structure of dyes and electrolytes.

Electron diffusion coefficient, lifetime, and density in the TiO(2) electrode of dye-sensitized TiO(2) solar cells (DSCs) employing I(-)/I(3)(-) redox couples were measured with eight different metal-free organic dyes and three Ru complex dyes. At matched electron density, all DSCs using organic dyes (ODSCs) showed shorter electron lifetime with comparable or larger diffusion coefficients in comparison to the DSCs using the Ru dyes (RuDSC). The shorter lifetime was attributed partially to the slower dye cation reduction rate of the organic dyes by I(-), faster electron diffusion coefficient in the TiO(2), and mostly higher I(3)(-) concentration in the vicinity of the TiO(2) surface. Whereas a slight shift of the conduction band edge potential (E(cb)) of the TiO(2) was seen with a few organic dyes, no correlation was found with the dipole moment of the adsorbed dyes. This implies that the adsorbed dyes interact with cations in the electrolyte, so the direction of the dipole is altered or simply screened. The increase of [I(3)(-)] in the vicinity of the TiO(2) surface was interpreted with partial charge distribution of the dyes. Under one-sun conditions, less electron density due to shorter electron lifetime was found to be the main reason for the lower values of V(oc) for all ODSCs in comparison to that of RuDSCs. Among the organic dyes, having larger molecular size and alkyl chains showed longer electron lifetime, and thus higher V(oc). Toward higher open circuit voltage, a design guide of organic dyes controlling the electron lifetime is discussed.

Inhibition of OXA-1 beta-lactamase by penems.

The partnering of a beta-lactam with a beta-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to beta-lactam antibiotics mediated by serine beta-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D beta-lactamase that is resistant to inactivation by tazobactam. The K(i) of each penem inhibitor for OXA-1 was in the nM range (K(i) of penem 1, 45 +/- 8 nM; K(i) of penem 2, 12 +/- 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 beta-lactamase were 0.13 +/- 0.01 s(-1) and 0.11 +/- 0.01 s(-1), respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in

5,5,6-Fused tricycles bearing imidazole and pyrazole 6-methylidene penems as broad-spectrum inhibitors of beta-lactamases.

Beta-lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against beta-lactam antibiotics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital settings, have reduced activity against newly emerging beta-lactamases. Bacterial production of diverse beta-lactamases including class-A, class-C, and ESBLs has motivated several research groups to search for inhibitors with a broader spectrum of activity. Previously, several novel 6-methylidene penems bearing, [5,5] [5,6] and [5,5,5] heterocycles have been synthesized in our laboratory and were shown to be potent and broad-spectrum beta-lactamase inhibitors. As a continuation of our previous work and in order to extend the structure-activity relationships, in this paper, we describe herein the synthesis and in vitro, in vivo activities of several novel 5,5,6-fused tricyclic heterocycles attached to the 6-methylidene penem core. The compounds presented in the current paper are potent and broad-spectrum inhibitors of the TEM-1 and AmpC beta-lactamases. In combination with piperacillin, their in vitro activities showed enhanced susceptibility to class A- and C-resistant strains studied in various bacteria. Some of the newly synthesized compounds such as 12a-c were shown to have in vivo activity in the acute lethal infection model against TEM-1 producing organisms. The 5,5,6-fused heterocyclic ring cores such as 21, 25, and 35 reported here are hitherto unknown in the literature.

In vitro and in vivo activities of the diazabicyclooctane OP0595 against AmpC-derepressed Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common cause for healthcare-associated infections, which have been historically treated by antipseudomonal ß-lactam agents in the clinical setting. However, P. aeruginosa has evolved to overcome these ß-lactam agents via multiple endogenous resistance mechanisms, including derepression of the chromosomal cephalosporinase (AmpC). In this article, we investigated the effective concentration of OP0595 for combination with piperacillin, cefepime or meropenem in in vitro susceptibility tests, and the antibacterial activity of cefepime in combination with OP0595 in both in vitro time-kill studies and in vivo murine thigh infection model study with AmpC-derepressed P. aeruginosa. The sufficient combinational concentration of OP0595 was a 4 µg ml-1 with all these three ß-lactam agents. OP0595 increased the antibacterial activity of cefepime in both in vitro and in vivo studies against all strains tested. Taken together, OP0595 is the diazabicyclooctane serine ß-lactamase inhibitor with activity against AmpC-derepressed P. aeruginosa and its combinational use with a ß-lactam agent will provide a new approach for the treatment of P. aeruginosa infections.

Structure-activity relationship of 6-methylidene penems bearing 6,5 bicyclic heterocycles as broad-spectrum beta-lactamase inhibitors: evidence for 1,4-thiazepine intermediates with C7 R stereochemistry by computational methods.

The design and synthesis of a series of 6-methylidene penems containing [6,5]-fused bicycles (thiophene, imidazole, or pyrazle-fused system) as novel class A, B, and C beta-lactamase inhibitors is described. These penems proved to be potent inhibitors of the TEM-1 (class A) and AmpC (class C) beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. Their in vitro and in vivo activities in combination with piperacillin are discussed. On the basis of the crystallographic structures of a serine-bound reaction intermediate of 2 with SHV-1 (class A) and GC1 (class C) enzymes, compounds 14a-l were designed and synthesized. Penems are proposed to form a seven-membered 1,4 thiazepine ring in both class A and C beta-lactamases. The interaction energy calculation for the enzyme-bound intermediates favor the formation of the C7 R enantiomer over the S enantiomer of the 1,4-thiazepine in both beta-lactamases, which is consistent with those obtained from the crystal structure of 2 with SHV-1 and GC1.

Syntheses and pharmacokinetic studies of prodrug esters for the development of oral carbapenem, L-084.

We discovered an orally active carbapenem, L-084, through pharmacokinetic studies on various prodrug esters of (1R,5S,6S)-6-[(R)-1-hydroxyethyl]-l-methyl-2-[1-(1,3-thiazolin-2-yl)azetidin-3-yl]thio-1-carbapen-2-em-3-carboxylic acid (LJC11,036). L-084 showed a strong antimicrobial activity against Gram-positive and Gram-negative bacteria and exhibited the highest intestinal absorption among synthesized prodrugs of LJC11,036.

Contamination status and accumulation features of persistent organochlorines in pet dogs and cats from Japan.

Concentrations of persistent organochlorines (OCs) such as polychlorinated dibenzo-p-dioxin (PCDDs), dibenzofurans (PCDFs), biphenyls, dichlorodiphenyltrichloroethane and their metabolites (DDTs), hexachlorocyclohexane isomers, hexachlorobenzene, and chlordane compounds were determined in genital organs of pet dogs and cats and pet foods from Japan. Levels of OCs in dogs were relatively lower than those in cats, while residue levels in their diets were almost similar, implying that accumulation and elimination mechanisms of these contaminants are different between dogs and cats. When bioconcentration factors (BCFs) were estimated from concentrations of OCs in dogs, cats, and their diets, BCFs of all the OCs except PCDD/DFs exceeded 1.0 in cats. On the other hand, in all the dogs, BCFs of DDTs were below 1.0, suggesting that dogs do not bioconcentrate DDTs. Furthermore, BCFs of all the OCs except PCDD/DFs in dogs were notably lower than those in cats, suggesting that dogs have higher metabolic and elimination capacity for these contaminants than cats. When residue levels of OCs in livers, adipose tissue, and genital organs of two pet dogs were examined, hepatic sequestration of PCDD/DFs and oxychlordane was observed.

Mechanism of inactivation of beta-lactamases by novel 6-methylidene penems elucidated using electrospray ionization mass spectrometry.

The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.

Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates.

The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine beta-lactamase inhibitors is described. These compounds proved to be very potent inhibitors of the TEM-1 and AmpC beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria. Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A, respectively, and refined to R-factors equal 0.163 and 0.145. In both beta-lactamases, a seven-membered 1,4-thiazepine ring has formed. The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate. Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes. The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.

Synthesis of pure colloidal silver nanoparticles with high electroconductivity for printed electronic circuits: the effect of amines on their formation in aqueous media.

This paper describes a practical and convenient method to prepare stable colloidal silver nanoparticles for use in printed electronic circuits. The method uses a dispersant and two kinds of reducing agents including 2-(dimethylamino) ethanol (DMAE), which play important roles in the reduction of silver ions in an aqueous medium. The effect of DMAE and dispersant, as well as the factors affecting particle size and morphology are investigated. In the formation of the silver nanoparticles, reduction occurs rapidly at room temperature and the silver particles can be separated easily from the mixture in a short time. In addition, organic solvents are not used. Pure, small and relatively uniform particles with a diameter less than 10 nm can be obtained that exhibit high electroconductivity. The silver nanoparticles are stable, and can be isolated as a dried powder that can be fully redispersed in deionized water. This method of producing colloidal silver nanoparticles will find practical use in electronics applications.

Tunable pK of amino acid residues at the air-water interface gives an L-zyme (langmuir enzyme).

Various amino acid-carrying amphiphiles were synthesized, and the pK values of the attached amino acid residues were investigated at the air-water interface and in aqueous vesicles using pi-A isotherm measurements, (1)H NMR titration, and IR spectroscopy in reflection-adsorption mode. The epsilon-amino group of the Lys residue embedded at the air-water interface displays a significant pK shift (4 or 5 unit) compared with that observed in bulk water, while the pK shift in aqueous vesicles was not prominent (ca. 1 unit). Moreover, pK values of the amino acids at the air-water interface can be tuned simply by control of the subphase ionic strength as well as by molecular design of the amphiphiles. A simple equation based on the dominant contribution by the electrostatic energy to the pK shift reproduces well the surface pressure difference between protonated and unprotonated species, suggesting a reduction in the apparent dielectric constant at the air-water interface. Hydrolysis of a p-nitrophenyl ester derivative was used as a model reaction to demonstrate the use of the Lys-functionalized monolayer. Efficient hydrolysis was observed, even at neutral pH, after tuning of pK for the Lys residue in the monolayer, which is a similar case to that occurring in biological catalysis.

Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate.

A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases. The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM). High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively. The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively. Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety. The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent. In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.

Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors.

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.

A novel, mild, and facile method to prepare 6-methylidene penem derivatives.

A novel and mild method was established to synthesize 6-methylidene penem compounds. This method entails a MgBr(2)/Et(3)N-promoted aldol-type condensation on 6-bromopenem 12 with an appropriately substituted aldehyde to yield the intermediate acetylated bromohydrin, which was smoothly converted to the final product with simultaneous deprotection of C3 carboxylic acid ester using activated zinc dust and phosphate buffer at pH 6.5. This process provides a useful variation of C-C bond formation method for penem derivatives and also serves as a practical synthetic method to prepare 6-exomethylenepenem derivatives without racemization at the C5 position.

In vitro and in vivo activities of novel 6-methylidene penems as beta-lactamase inhibitors.

Novel penem molecules with heterocycle substitutions at the 6 position via a methylidene linkage were investigated for their activities and efficacy as beta-lactamase inhibitors. The concentrations of these molecules that resulted in 50% inhibition of enzyme activity were 0.4 to 3.1 nM for the TEM-1 enzyme, 7.8 to 72 nM for Imi-1, 1.5 to 4.8 nM for AmpC, and 14 to 260 nM for a CcrA metalloenzyme. All the inhibitors were more stable than imipenem against hydrolysis by hog and human dehydropeptidases. Piperacillin was combined with a constant 4-microg/ml concentration of each inhibitor for MIC determinations. The combinations reduced piperacillin MICs by 2- to 32-fold for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae strains. The MICs for piperacillin-resistant (MIC of piperacillin, >64 microg/ml) strains of Enterobacter spp., Citrobacter spp., and Serratia spp. were reduced to the level of susceptibility (MIC of piperacillin, < or =16 microg/ml) when the drug was combined with 4, 2, or 1 microg of these penem inhibitors/ml. Protection against acute lethal bacterial infections with class A and C beta-lactamase- and ESBL-producing organisms in mice was also demonstrated with piperacillin plus inhibitor. Median effective doses were reduced by approximately two- to eightfold compared to those of piperacillin alone when the drug was combined with the various inhibitors at a 4:1 ratio. Pharmacokinetic analysis after intravenous administration of the various inhibitors showed mean residence times of 0.1 to 0.5 h, clearance rates of 15 to 81 ml/min/kg, and volumes of distribution between 0.4 and 2.5 liters/kg. The novel methylidene penem molecules inhibit both class A and class C enzymes and warrant further investigation for potential as therapeutic agents when used in combination with a beta-lactam antibiotic.