The Role of MexCD-OprJ and MexEF-OprN Efflux Systems in the Multiple Antibiotic Resistance of Pseudomonas aeruginosa Isolated from Clinical Samples.

Increasing antimicrobial resistance and the development of multi-drug resistant (MDR) Pseudomonas aeruginosa is dependent on the expression of efflux pumps. This study aimed to investigate the role of overexpression of MexCD-OprJ and MexEF-OprN efflux pumps in reduced susceptibility to antimicrobial agents among P. aeruginosa strains. Totally, 100 clinical isolates of P. aeruginosa were collected from patients and the strains were identified by standard diagnostic tests. The MDR isolates were detected using the disk agar diffusion method. The expression levels of MexCD-OprJ and MexEF-OprN efflux pumps were evaluated by the real-time PCR. Forty-one isolates showed MDR phenotype, while piperacillin-tazobactam and levofloxacin were the most- and least-effective antibiotics, respectively. Also, all 41 MDR isolates showed a more than tenfold increase in the expression of mexD and mexF genes. In this study, a significant relationship was observed between the rate of antibiotic resistance, the emergence of MDR strains, and increasing the expression levels of MexEF-OprN and MexCD-OprJ efflux pumps (P < 0.05). Efflux systems mediated resistance was a noteworthy mechanism causative to multidrug resistance in P. aeruginosa clinical isolates. The study results demonstrated mexE and mexF overexpression as the primary mechanism conferring in the emergence of MDR phenotypes among P. aeruginosa strains. In addition, we also show that piperacillin/tazobactam exhibited a stronger ability in the management of infections caused by MDR P. aeruginosa in this area.

Quantitative Determination of Unbound Piperacillin and Imipenem in Biological Material from Critically Ill Using Thin-Film Microextraction-Liquid Chromatography-Mass Spectrometry.

ß-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction-LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.

Meropenem, Cefepime, and Piperacillin Protein Binding in Patient Samples.

BACKGROUND: The mortality rate of patients with a drug-resistant bacterial infection is high, as are the associated treatment costs. To overcome these issues, optimization of the available therapeutic options is required. Beta-lactams are time-dependent antibiotics and their efficacy is determined by the amount of time the free concentration remains above the minimum inhibitory concentration. Therefore, the aim of this study was to assess the extent and variability of protein binding for meropenem, cefepime, and piperacillin. METHODS: Plasma samples for the analysis of meropenem, cefepime, and piperacillin were collected from patients admitted to a tertiary care hospital as part of the standard care. The bound and unbound drug fractions in the samples were separated by ultrafiltration. Validated liquid chromatography-tandem mass spectrometry assays were used to quantify the total and free plasma concentrations, and the protein binding was determined. RESULTS: Samples from 95 patients were analyzed. The median (range) age of patients was 56 years (17-87) and the median (range) body mass index was 25.7 kg/m (14.7-74.2). Approximately 59% of the patients were men. The median (range) unbound fraction (fu) was 62.5% (41.6-99.1) for meropenem, 61.4% (51.6-99.2) for cefepime, and 48.3% (39.4-71.3) for piperacillin. In the bivariate analysis, as the total meropenem concentration increased, the fu increased (r = 0.37, P = 0.045). A decrease in piperacillin fu was observed as the albumin concentration increased (r = -0.56, P = 0.005). CONCLUSIONS: The average fu values were lower than those reported in the literature. There was also a large variability in fu; hence, it should be considered when managing patients administered with these drugs through direct measurements of free drug concentrations.

The Reaction Mechanism of Metallo-ß-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop.

Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.

Definition of the Nature and Hapten Threshold of the ß-Lactam Antigen Required for T Cell Activation In Vitro and in Patients.

Covalent modification of protein by drugs may disrupt self-tolerance, leading to lymphocyte activation. Until now, determination of the threshold required for this process has not been possible. Therefore, we performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the ß-lactam antibiotic piperacillin and the threshold required for T cell activation. A hydrolyzed piperacillin hapten was detected on four lysine residues of human serum albumin (HSA) isolated from tolerant patients. The level of modified Lys541 ranged from 2.6 to 4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1-10 d after the commencement of therapy. Piperacillin-responsive skin-homing CD4+ clones expressing an array of Vß receptors were activated in a dose-, time-, and processing-dependent manner; analysis of incubation medium revealed that 2.6% of Lys541 in HSA was modified when T cells were activated. Piperacillin-HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4+ clones, which expressed a more restricted Vß repertoire. To conclude, the levels of piperacillin-HSA modification that activated T cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug Ag are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors, such as the presence of T cells within an individual's repertoire that can be activated with the ß-lactam hapten and/or an imbalance in immune regulation.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 2: Stability Evaluation.

Although the stability of ß-lactam antibiotics is a known issue, none of the previously reported bioanalytical methods had an adequate evaluation of the stability of these drugs. In the current study, the stability of cefepime, meropenem, piperacillin, and tazobactam under various conditions was comprehensively evaluated. The evaluated parameters included stock solution stability, short-term stability, long-term stability, freeze-thaw stability, processed sample stability, and whole-blood stability. When stored at -20°C, the stock solution of meropenem in methanol was stable for up to 3 weeks, and the stock solutions of cefepime, piperacillin, and tazobactam were stable for up to 6 weeks. All four antibiotics were stable in human plasma for up to 3 months when stored at -80°C and were stable in whole blood for up to 4 h at room temperature. Short-term stability results indicated that all four ß-lactams were stable at room temperature for 2 h, but substantial degradation was observed when the plasma samples were stored at room temperature for 24 h, with the degradation rates for cefepime, meropenem, piperacillin, and tazobactam being 30.1%, 75.6%, 49.0%, and 37.7%, respectively. Because the stability information is method independent, our stability results can be used as a reference by other research groups that work with these antibiotics.

Computationally Assessing the Bioactivation of Drugs by N-Dealkylation.

Cytochromes P450 (CYPs) oxidize alkylated amines commonly found in drugs and other biologically active molecules, cleaving them into an amine and an aldehyde. Metabolic studies usually neglect to report or investigate aldehydes, even though they can be toxic. It is assumed that they are efficiently detoxified into carboxylic acids and alcohols. Nevertheless, some aldehydes are reactive and escape detoxification pathways to cause adverse events by forming DNA and protein adducts. Herein, we modeled N-dealkylations that produce both amine and aldehyde metabolites and then predicted the reactivity of the aldehyde. This model used a deep learning approach previously developed by our group to predict other types of drug metabolism. In this study, we trained the model to predict N-dealkylation by human liver microsomes (HLM), finding that including isozyme-specific metabolism data alongside HLM data significantly improved results. The final HLM model accurately predicted the site of N-dealkylation within metabolized substrates (97% top-two and 94% area under the ROC curve). Next, we combined the metabolism, metabolite structure prediction, and previously published reactivity models into a bioactivation model. This combined model predicted the structure of the most likely reactive metabolite of a small validation set of drug-like molecules known to be bioactivated by N-dealkylation. Applying this model to approved and withdrawn medicines, we found that aldehyde metabolites produced from N-dealkylation may explain the hepatotoxicity of several drugs: indinavir, piperacillin, verapamil, and ziprasidone. Our results suggest that N-dealkylation may be an under-appreciated bioactivation pathway, especially in clinical contexts where aldehyde detoxification pathways are inhibited. Moreover, this is the first report of a bioactivation model constructed by combining a metabolism and reactivity model. These results raise hope that more comprehensive models of bioactivation are possible. The model developed in this study is available at http://swami.wustl.edu/xenosite/ .

Targeted Metabolomics Analysis Identifies Intestinal Microbiota-Derived Urinary Biomarkers of Colonization Resistance in Antibiotic-Treated Mice.

Antibiotics excreted into the intestinal tract may disrupt the microbiota that provide colonization resistance against enteric pathogens and alter normal metabolic functions of the microbiota. Many of the bacterial metabolites produced in the intestinal tract are absorbed systemically and excreted in urine. Here, we used a mouse model to test the hypothesis that alterations in levels of targeted bacterial metabolites in urine specimens could provide useful biomarkers indicating disrupted or intact colonization resistance. To assess in vivo colonization resistance, mice were challenged with Clostridium difficile spores orally 3, 6, and 11 days after the completion of 2 days of treatment with piperacillin-tazobactam, aztreonam, or saline. For concurrent groups of antibiotic-treated mice, urine samples were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the concentrations of 11 compounds targeted as potential biomarkers of colonization resistance. Aztreonam did not affect colonization resistance, whereas piperacillin-tazobactam disrupted colonization resistance 3 days after piperacillin-tazobactam treatment, with complete recovery by 11 days after treatment. Three of the 11 compounds exhibited a statistically significant and >10-fold increase (the tryptophan metabolite N-acetyltryptophan) or decrease (the plant polyphenyl derivatives cinnamoylglycine and enterodiol) in concentrations in urine 3 days after piperacillin-tazobactam treatment, followed by recovery to baseline that coincided with the restoration of in vivo colonization resistance. These urinary metabolites could provide useful and easily accessible biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.

Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant.

Beta-lactam resistant clinical isolates of Streptococcus pneumoniae contain altered penicillin-binding protein (PBP) genes and occasionally an altered murM, presumably products of interspecies gene transfer. MurM and MurN are responsible for the synthesis of branched lipid II, substrate for the PBP catalyzed transpeptidation reaction. Here we used the high-level beta-lactam resistant S. oralis Uo5 as donor in transformation experiments with the sensitive laboratory strain S. pneumoniae R6 as recipient. Surprisingly, piperacillin-resistant transformants contained no alterations in PBP genes but carried murEUo5 encoding the UDP-N-acetylmuramyl tripeptide synthetase. Codons 83-183 of murEUo5 were sufficient to confer the resistance phenotype. Moreover, the promoter of murEUo5 , which drives a twofold higher expression compared to that of S. pneumoniae R6, could also confer increased resistance. Multiple independent transformations produced S. pneumoniae R6 derivatives containing murEUo5 , pbp2xUo5 , pbp1aUo5 and pbp2bUo5 , but not murMUo5 sequences; however, the resistance level of the donor strain could not be reached. S. oralis Uo5 harbors an unusual murM, and murN is absent. Accordingly, the peptidoglycan of S. oralis Uo5 contained interpeptide bridges with one L-Ala residue only. The data suggest that resistance in S. oralis Uo5 is based on a complex interplay of distinct PBPs and other enzymes involved in peptidoglycan biosynthesis.

Sub-Inhibitory Concentration of Piperacillin-Tazobactam May be Related to Virulence Properties of Filamentous Escherichia coli.

Sub-inhibitory concentrations of antibiotics are always generated as a consequence of antimicrobial therapy and the effects of such residual products in bacterial morphology are well documented, especially the filamentation generated by beta-lactams. The aim of this study was to investigate some morphological and pathological aspects (virulence factors) of Escherichia coli cultivated under half-minimum inhibitory concentration (1.0 µg/mL) of piperacillin-tazobactam (PTZ sub-MIC). PTZ sub-MIC promoted noticeable changes in the bacterial cells which reach the peak of morphological alterations (filamentation) and complexity at 16 h of antimicrobial exposure. Thereafter the filamentous cells and a control one, not treated with PTZ, were comparatively tested for growth curve; biochemical profile; oxidative stress tolerance; biofilm production and cell hydrophobicity; motility and pathogenicity in vivo. PTZ sub-MIC attenuated the E. coli growth rate, but without changes in carbohydrate fermentation or in traditional biochemical tests. Overall, the treatment of E. coli with sub-MIC of PTZ generated filamentous forms which were accompanied by the inhibition of virulence factors such as the oxidative stress response, biofilm formation, cell surface hydrophobicity, and motility. These results are consistent with the reduced pathogenicity observed for the filamentous E. coli in the murine model of intra-abdominal infection. In other words, the treatment of E. coli with sub-MIC of PTZ suggests a decrease in their virulence.

Comparison of the risk of acquiring in vitro resistance to doripenem and tazobactam/piperacillin by CTX-M-15-producing Escherichia coli.

To compare the risk of acquiring in vitro resistance between doripenem and tazobactam/piperacillin by CTX-M-15-producing Escherichia coli, the in vitro frequency of resistance was determined. Four strains carrying multiple ß-lactamases such as blaOXA-1 or blaCTX-M-27 as well as blaCTX-M-15 and blaTEM-1 were used. No resistant colonies appeared on doripenem-containing plates, whereas resistant colonies were obtained from three of four test strains against tazobactam/piperacillin using agar plate containing 8- to 16-fold MIC of each drug. These three acquired tazobactam/piperacillin-resistant strains were not cross-resistant to doripenem, and they showed 1.9- to 3.1-fold higher piperacillin-hydrolysis activity compared to those of each parent strain. The change of each ß-lactamase mRNA expression measured by real-time PCR varied among three resistant strains. One of three tazobactam/piperacillin-resistant strains with less susceptibility to ceftazidime overexpressed both blaCTX-M-15 and blaTEM-1, and the other two strains showed higher mRNA expression of either blaTEM-1 or blaOXA-1. These results demonstrate that multiple ß-lactamases carried by CTX-M-15-producing E. coli contributed to the resistance to tazobactam/piperacillin. On the other hand, these resistant strains maintained susceptibility to doripenem. The risk of acquiring in vitro resistance to doripenem by CTX-M-15-producing E. coli seems to be lower than that to tazobactam/piperacillin.

Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido ß-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido ß-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of ß-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Relationship between ceftolozane-tazobactam exposure and drug resistance amplification in a hollow-fiber infection model.

In an era of rapidly emerging antimicrobial-resistant bacteria, it is critical to understand the importance of the relationships among drug exposure, duration of therapy, and selection of drug resistance. Herein we describe the results of studies designed to determine the ceftolozane-tazobactam exposure necessary to prevent the amplification of drug-resistant bacterial subpopulations in a hollow-fiber infection model. The challenge isolate was a CTX-M-15-producing Escherichia coli isolate genetically engineered to transcribe a moderate level of blaCTX-M-15. This organism's blaCTX-M-15 transcription level was confirmed by relative quantitative reverse transcription-PCR (qRT-PCR), ß-lactamase hydrolytic assays, and a ceftolozane MIC value of 16 mg/liter. In these studies, the experimental duration (10 days), ceftolozane-tazobactam dose ratio (2:1), and dosing interval (every 8 h) were selected to approximate those expected to be used clinically. The ceftolozane-tazobactam doses studied ranged from 125-62.5 to 1,500-750 mg. Negative- and positive-control arms included no treatment and piperacillin-tazobactam at 4.5 g every 6 h, respectively. An inverted-U-shaped function best described the relationship between bacterial drug resistance amplification and drug exposure. The least- and most-intensive ceftolozane-tazobactam dosing regimens, i.e., 125-62.5, 750-375, 1,000-500, and 1,500-750 mg, did not amplify drug resistance, while drug resistance amplification was observed with intermediate-intensity dosing regimens (250-125 and 500-250 mg). For the intermediate-intensity ceftolozane-tazobactam dosing regimens, the drug-resistant subpopulation became the dominant population by days 4 to 6. The more-intensive ceftolozane-tazobactam dosing regimens (750-375, 1,000-500, and 1,500-750 mg) not only prevented drug resistance amplification but also virtually sterilized the model system. These data support the selection of ceftolozane-tazobactam dosing regimens that minimize the potential for on-therapy drug resistance amplification.

Ultrasound-responsive catalytic microbubbles enhance biofilm elimination and immune activation to treat chronic lung infections.

Efficient treatment of chronic lung infections caused by Pseudomonas aeruginosa biofilms is a great challenge because of drug tolerance and immune evasion issues. Here, we develop ultrasound-responsive catalytic microbubbles with biofilm elimination and immune activation properties to combat chronic lung infection induced by P. aeruginosa biofilms. In these microbubbles, piperacillin and Fe3O4 nanoparticles form a drug-loaded shell surrounding the air core. Under ultrasound stimulation, the microbubbles can physically disrupt the structure of biofilms and enhance the penetration of both Fe3O4 nanoparticles and piperacillin into the biofilm. Then, Fe3O4 nanoparticles chemically degrade the biofilm matrix and kill the bacteria with the assistance of piperacillin. Fe3O4 nanoparticles can activate the immune response for biofilm elimination by polarizing macrophages into a pro-inflammatory phenotype. These ultrasound-responsive catalytic microbubbles efficiently treat chronic lung infections in a mouse model by combining physical/chemical/antibiotic biofilm elimination and immune activation, thus providing a promising strategy for combating bacterial biofilm infections.

Interactions Between Meropenem and Renal Drug Transporters.

BACKGROUND: Meropenem is a carbapenem antibiotic and is commonly used with other antibiotics for the treatment of bacterial infections. It is primarily eliminated renally by glomerular filtration and renal tubular secretion. OBJECTIVE: This study aimed to evaluate the roles of renal uptake and efflux transporters in the excretion of meropenem and potential drug interactions mediated by renal drug transporters. METHODS: Uptake and inhibition studies were conducted in human embryonic kidney 293 cells stably transfected with Organic Anion Transporter (OAT) 1, OAT3, Multidrug and Toxin Extrusion Protein (MATE) 1, and MATE2K, as well as membrane vesicles containing breast cancer resistance-related protein (BCRP), multidrug resistance protein 1 (MDR1), and Multidrug Resistance-associated Protein 2 (MRP2). Probenecid and piperacillin were used to assess potential drug interactions with meropenem in rats. RESULTS: We observed that meropenem was a low-affinity substrate of OAT1/3 and had a weak inhibitory effect on OAT1/3 and MATE2K. BCRP, MDR1, MRP2, MATE1, and MATE2K could not mediate renal excretion of meropenem. Moreover, meropenem was not an inhibitor of BCRP, MDR1, MRP2, or MATE1. Among five tested antibiotics, moderate inhibition on OAT3-mediated meropenem uptake was observed for linezolid (IC50 value was 69.2 µM), weak inhibition was observed for piperacillin, benzylpenicillin, and tazobactam (IC50 values were 282.2, 308.0 and 668.1 µM, respectively), and no inhibition was observed for sulbactam. Although piperacillin had a relatively high drug-drug interaction index (ratio of maximal unbound plasma concentration to IC50 was 1.42) in vitro, no meaningful impact was reported on the pharmacokinetics of meropenem in rats. CONCLUSION: Our results indicated that clinically significant interactions between meropenem and these five antibiotics are low.

Purification and biochemical characterization of IMP-13 metallo-beta-lactamase.

The IMP-13 metallo-ß-lactamase was overproduced in Escherichia coli BL21(DE3) and purified by chromatography. Analysis of kinetic parameters revealed some notable differences with other IMP-type enzymes, noteworthily a higher catalytic efficiency toward ticarcillin and piperacillin and a marked preference for imipenem over meropenem.

Noncovalent complexes of an inactive mutant of CTX-M-9 with the substrate piperacillin and the corresponding product.

We determined the crystal structure of an inactive Ser70Gly mutant of CTX-M-9 in complex with the bulky penicillin piperacillin at precovalent and posthydrolytic stages in the catalytic process. The structures obtained at high resolution were compared with the corresponding structures for the small penicillin benzylpenicillin and the bulky cephalosporin cefotaxime. The findings highlight the key role of the configuration of the carbon adjacent to the acylamino group of the side chain of ß-lactams in the precovalent recognition of substrates.

Carboxylesterase catalyzed 18O-labeling of carboxylic acid and its potential application in LC-MS/MS based quantification of drug metabolites.

LC-MS quantification of drug metabolites is sometimes impeded by the availability of internal standards that often requires customized synthesis and/or extensive purification. Although isotopically labeled internal standards are considered ideal for LC-MS/MS based quantification, de novo synthesis using costly isotope-enriched starting materials makes it impractical for early stage of drug discovery. Therefore, quick access to these isotope-enriched compounds without chemical derivatization and purification will greatly facilitate LC-MS/MS based quantification. Herein, we report a novel 18O-labeling technique using metabolizing enzyme carboxylesterase (CES) and its potential application in metabolites quantification study. Substrates of CES typically undergo a two-step oxygen exchange with H218O in the presence of the enzyme, generating singly- and doubly-18O-labeled carboxylic acids; however, unexpected hydrolytic behavior was observed for three of the test compounds - indomethacin, piperacillin and clopidogrel. These unusual observations led to the discovery of several novel hydrolytic mechanisms. Finally, when used as internal standard for LC-MS/MS based quantification, these in situ labeled compounds generated accurate quantitation comparable to the conventional standard curve method. The preliminary results suggest that this method has potential to eliminate laborious chemical synthesis of isotope-labeled internal standards for carboxylic acid-containing compounds, and can be developed to facilitate quantitative analysis in early-stage drug discovery.

Interactions of some commonly used drugs with human α-thrombin.

Adverse side effects of drugs are often caused by the interaction of drug molecules to targets other than the intended ones. In this study, we investigated the off-target interactions of some commercially available drugs with human α-thrombin. The drugs used in the study were selected from Super Drug Database based on the structural similarity to a known thrombin inhibitor argatroban. Interactions of these drugs with thrombin were initially checked by in silico docking studies and then confirmed by thrombin inhibition assay using a fluorescence microplate-based method. Results show that the three commonly used drugs piperacillin (anti-bacterial), azlocillin (anti-bacterial), and metolazone (anti-hypertensive and diuretic) have thrombin inhibitory activity almost similar to that of argatroban. The Ki values of piperacillin, azlocillin, and metolazone with thrombin are .55, .95, and .62 nM, respectively. The IC50 values of piperacillin, azlocillin, and metolazone with thrombin are 1.7, 2.9, and 1.92 nM, respectively. This thrombin inhibitory activity might be a reason for the observed side effects of these drugs related to blood coagulation and other thrombin activities. Furthermore, these compounds (drugs) may be used as anti-coagulants as such or with structural modifications.

The place of temocillin in the treatment of hospital infections.

Infection due to Gram-negative bacteria continues to be a common problem in the hospital environment, for which a wide variety of antibiotics is available. Among Enterobacteria, resistance is expressed most commonly via beta-lactamases. Temocillin is the first penicillin to show stability to beta-lactamases of Gram-negative bacteria, therefore it is in this type of infection that the initial assessments of temocillin should be made.