Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum ß-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to ß-lactam/ß-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum ß-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

Application of 1,1-ADEQUATE and DFT to correct 13 C misassignments of carbonyl chemical shifts for carbapenem antibiotics.

Prior to the development of sensitive proton-detected 2D NMR experiments, assigning 13 C signals could be a significant challenge, and mistakes have occurred even for prominent compound classes. In this study, 1,1-ADEQUATE data were used to unambiguously reassign the 13 C chemical shifts for the ß-lactam carbonyl at the C-7 position and the proximal carboxylate at the C-10 position of the carbapenems, meropenem and imipenem. Density functional theory (DFT) was then investigated to provide sufficiently accurate 13 C chemical shift predictions, allowing for the carbonyl signal reassignment of thienamycin.

Interplay between the Enamine and Imine Forms of the Hydrolyzed Imipenem in the Active Sites of Metallo-ß-lactamases and in Water Solution.

Deactivation of the ß-lactam antibiotics in the active sites of the ß-lactamases is among the main mechanisms of bacterial antibiotic resistance. As drugs of last resort, carbapenems are efficiently hydrolyzed by metallo-ß-lactamases, presenting a serious threat to human health. Our study reveals mechanistic aspects of the imipenem hydrolysis by bizinc metallo-ß-lactamases, NDM-1 and L1, belonging to the B1 and the B3 subclasses, respectively. The results of QM(PBE0-D3/6-31G**)/MM simulations show that the enamine product with the protonated nitrogen atom is formed as the major product in NDM-1 and as the only product in the L1 active site. In NDM-1, there is also another reaction pathway that leads to the formation of the (S)-enantiomer of the imine form of the hydrolyzed imipenem; this process occurs with the higher energy barriers. The absence of the second pathway in L1 is due to the different amino acid composition of the active site loop. In L1, the hydrophobic Pro226 residue is located above the pyrroline ring of imipenem that blocks protonation of the carbon atom. Electron density analysis is performed at the stationary points to compare reaction pathways in L1 and NDM-1. Tautomerization from the enamine to the imine form likely happens in solution after the dissociation of the hydrolyzed imipenem from the active site of the enzyme. Classical molecular dynamics simulations of the hydrolyzed imipenem in solution, both with the neutral enamine and the negatively charged N-C2-C3 fragment, demonstrate a huge diversity of conformations. The vast majority of conformations blocks the C3-atom from the side required for the (S)-imine formation upon tautomerization. Thus, according to our calculations, formation of the (R)-imine is more likely. QM(PBE0-D3/6-31G**)/MM molecular dynamics simulations of the hydrolyzed imipenem with the negatively charged N-C2-C3 fragment followed by the Laplacian bond order analysis demonstrate that the N═C2-C3- resonance structure is the most pronounced that facilitates formation of the imine form. The proposed mechanism of the enzymatic enamine formation and its subsequent tautomerization to the imine form in solution is in agreement with the recent spectroscopic and NMR studies.

Metallo-beta-lactamase CphA evolving into more efficient hydrolases through gene mutation is a novel pathway for the resistance of super bacteria.

The evolution of metallo-beta-lactamase CphA in discontinuous gradient concentration of imipenem was investigated in this work. The results suggested that single-base mutations K218R, K249T, K249M, Q253H, and a frameshift mutation M1 were observed. Compared with wild type, the minimum inhibitory concentration (MICs) of K249T, K249M, and M1 increased by at least 128 times and that of K218R increased by 64 times. And the catalytic efficiency increased by 312% and 653%, respectively. It is speculated from the details of the structural changes revealed by molecular dynamics simulations that the carbon skeleton migration caused by the outward motion of the loop 3 in the mutant may have significantly increased the cavity volume of the binding pocket, which is more conducive to the entry and expulsion of imipenem and its hydrolytic product. And the conformational change of the TDRAGGN (71-77) is located at the bottom of the binding pocket from order α-helix to disorder random coil enabled the binding pocket to be more conducive to accommodate and hold the imipenem respectively. All these indicated that during the repeated drug resistance, the wild-type achieved gene mutations and conformational change and evolved to the mutant enzymes with a more delicate structure and stronger hydrolysis ability. KEY POINTS: • The mutation and evolution of CphA under the selective pressure of imipenem. • The CphA evolved to the mutants with stronger hydrolysis capacity. • A novel pathway for the resistance of super bacteria.

N-Aryl Mercaptopropionamides as Broad-Spectrum Inhibitors of Metallo-ß-Lactamases.

Drug-resistant pathogens pose a global challenge to public health as they cause diseases that are extremely difficult to cure. Metallo-ß-lactamases (MBLs) are a diverse set of zinc-containing enzymes that catalyze the hydrolysis of ß-lactam drugs, including carbapenems, which are considered as the last resort to fight severe infections. To restore the activity of current ß-lactam antibiotics and to offer an orthogonal strategy to the discovery of new antibiotics, we have identified a series of polar N-aryl mercaptopropionamide derivatives as potent inhibitors of several class B1 MBLs. We have identified a hit structure with high selectivity restoring the effect of imipenem and reducing minimum inhibitory concentration (MIC) values up to 256-fold in resistant isolates from Escherichia coli. Furthermore, the combination of imipenem with our inhibitor showed in vivo efficacy in a Galleria mellonella model, increasing the survival rate of infected larvae by up to 31%.

Exploring the Role of L10 Loop in New Delhi Metallo-ß-lactamase (NDM-1): Kinetic and Dynamic Studies.

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

Simultaneous determination of meropenem and imipenem in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

An efficient and reliable method using LC-MS/MS was established and validated for the simultaneous quantification of meropenem and imipenem in rat plasma. An electronic spray ion source in the positive multiple reaction monitoring mode was used for the detection and the transitions were m/z 384.6 → m/z 141.2 for meropenem, m/z 300.1 → m/z 141.8 for imipenem and m/z 423.4 → m/z 207.1 for matrine (IS). The calibration curves of meropenem and imipenem were linear in the range of 0.50-200 µg/mL. Satisfactory separation was achieved with a total run time of 3.0 min, the injection volume was 3 µl. The retention times of meropenem, imipenem and IS were 1.19, 1.14 and 1.13 min, respectively. Meropenem and imipenem are easily hydrolyzed in plasma. HEPES was used as a stabilizer and added to the plasma samples immediately after centrifugation. Extractions of meropenem, imipenem and IS were carried out by protein precipitation with acetonitrile. The specificity, precision and accuracy, stability, recovery and matrix effects were within acceptance limits. This method was successfully applied to investigate the pharmacokinetics of intravenous injection of meropenem and imipenem single administration or combined with sulbactam in rats. We found that sulbactam has no influence on the pharmacokinetics behavior of meropenem or imipenem.

Novel Multifunctional Silver Nanocomposite Serves as a Resistance-Reversal Agent to Synergistically Combat Carbapenem-Resistant Acinetobacter baumannii.

In the face of the abundant production of various types of carbapenemases, the antibacterial efficiency of imipenem, seen as "the last line of defense", is weakening. Following, the incidence of carbapenem-resistant Acinetobacter baumannii (CRAB), which can generate antibiotic-resistant biofilms, is increasing. Based on the superior antimicrobial activity of silver nanoparticles against multifarious bacterial strains compared with common antibiotics, we constructed the IPM@AgNPs-PEG-NOTA nanocomposite (silver nanoparticles were coated with SH-PEG-NOTA as well as loaded by imipenem) whose core was a silver nanoparticle to address the current challenge, and IPM@AgNPs-PEG-NOTA was able to function as a novel smart pH-sensitive nanodrug system. Synergistic bactericidal effects of silver nanoparticles and imipenem as well as drug-resistance reversal via protection of the ß-ring of carbapenem due to AgNPs-PEG-NOTA were observed; thus, this nanocomposite confers multiple advantages for efficient antibacterial activity. Additionally, IPM@AgNPs-PEG-NOTA not only offers immune regulation and accelerates tissue repair to improve therapeutic efficacy in vivo but also can prevent the interaction of pathogens and hosts. Compared with free imipenem or silver nanoparticles, this platform significantly enhanced antibacterial efficiency while increasing reactive oxygen species (ROS) production and membrane damage, as well as affecting cell wall formation and metabolic pathways. According to the results of crystal violet staining, LIVE/DEAD backlight bacterial viability staining, and real-time quantitative polymerase chain reaction (RT-qPCR), this silver nanocomposite downregulated the levels of ompA expression to prevent formation of biofilms. In summary, this research demonstrated that the IPM@AgNPs-PEG-NOTA nanocomposite is a promising antibacterial agent of security, pH sensitivity, and high efficiency in reversing resistance and synergistically combatting carbapenem-resistant A. baumannii. In the future, various embellishments and selected loads for silver nanoparticles will be the focus of research in the domains of medicine and nanotechnology.

Boronic Acids as Prospective Inhibitors of Metallo-ß-Lactamases: Efficient Chemical Reaction in the Enzymatic Active Site Revealed by Molecular Modeling.

Boronic acids are prospective compounds in inhibition of metallo-ß-lactamases as they form covalent adducts with the catalytic hydroxide anion in the enzymatic active site upon binding. We compare this chemical reaction in the active site of the New Delhi metallo-ß-lactamase (NDM-1) with the hydrolysis of the antibacterial drug imipenem. The nucleophilic attack occurs with the energy barrier of 14 kcal/mol for imipenem and simultaneously upon binding a boronic acid inhibitor. A boron atom of an inhibitor exhibits stronger electrophilic properties than the carbonyl carbon atom of imipenem in a solution that is quantified by atomic Fukui indices. Upon forming the prereaction complex between NDM-1 and inhibitor, the lone electron pair of the nucleophile interacts with the vacant p-orbital of boron that facilitates the chemical reaction. We analyze a set of boronic acid compounds with the benzo[b]thiophene core complexed with the NDM-1 and propose quantitative structure-sroperty relationship (QSPR) equations that can predict IC50 values from the calculated descriptors of electron density. These relations are applied to classify other boronic acids with the same core found in the database of chemical compounds, PubChem, and proposed ourselves. We demonstrate that the IC50 values for all considered benzo[b]thiophene-containing boronic acid inhibitors are 30-70 µM.

KPC-2 ß-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate.

Serine active-site ß-lactamases hydrolyze ß-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most ß-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 ß-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase ß-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 ß-lactamase.

The Efficacy of AgNO3 Nanoparticles Alone and Conjugated with Imipenem for Combating Extensively Drug-Resistant Pseudomonas aeruginosa.

INTRODUCTION: The extensive drug-resistant (XDR) Pseudomonas aeruginosa (P. aeruginosa) causes a range of infections with high mortality rate, which inflicts additional costs on treatment. The use of nano-biotechnology-based methods in medicine has opened a new perspective against drug-resistant bacteria. The aim of this study was to evaluate the effectiveness of the AgNO3 nanoparticles alone and conjugated with imipenem (IMI) to combat extensively drug-resistant P. aeruginosa. METHODS: Antibiotic susceptibility was carried out using disc diffusion method. Detection of different resistant genes was performed using standard polymerase chain reaction (PCR). The chemically synthesized AgNO3 particles were characterized using scanning electron microscope (SEM), dynamic light scattering (DLS) and X-ray diffraction (XRD) methods. Fourier transform infrared spectroscopy (FTIR) was accomplished to confirm the binding of AgNO3 with IMI. The microdilution broth method was used to obtain minimum inhibitory concentration (MIC) of AgNO3 and IMI-conjugated AgNO3. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out on L929 cell line to study the cytotoxicity of nanoparticles. The data were analyzed by Eta correlation ratio and chi-square (X 2) test. RESULTS: Analysis of the antibiotic resistance pattern showed that 12 (24%) isolates were XDR, and MIC values of IMI were between 64 and 128 µg/mL. Frequency of SHV, TEM, CTX M, IMP, VIM, OPR, SIM, SPM, GIM, NDM, VEB, PER, KPC, OXA, intI, intII, and intIII genes were 29 (58%), 26 (52%), 26 (52%), 32 (64%), 23 (46%), 43 (86%), 3 (6%), 6 (12%), 3 (6%), 4 (8%), 7 (14%), 6 (12%), 18 (36%), 4 (8%), 19 (38%), 16 (32%), and 2 (4%), respectively. The XRD, SEM, DLS, and FTIR analysis confirmed the synthesis of AgNO3 nanoparticles and their conjugation with IMI. The AgNO3 nanoparticles had antimicrobial activity, and their conjugation with IMI showed enhanced effectiveness against XDR isolates. The synthesized AgNO3 showed no cytotoxic effects. CONCLUSION: The results suggest that IMI-conjugated AgNO3 has a strong potency as a powerful antibacterial agent against XDR P. aeruginosa.

Simultaneous administration of imipenem/cilastatin/relebactam with selected intravenous antimicrobials, a stewardship approach.

Imipenem/cilastatin/relebactam is a ß-lactam/ß-lactamase inhibitor that has been recently FDA approved for complicated intra-abdominal and urinary tract infections under the brand name Recarbrio®. It has activity against imipenem non-susceptible Pseudomonas species as well as KPC-producing Enterobacteriaceae. Optimization of PK/PD of antimicrobials particularly in critically-ill patients is essential, but unfortunately, is hindered by separate administration that requires significant resources. The objective of the study is to investigate the compatibility of Y-site administration of imipenem/cilastatin/relebactam with a wide range of antimicrobials. After admixture, physical characteristics, pH changes and turbidity were measured for each 2-drug combination at a time. With the exception of amphotericin B deoxycholate, and posaconazole, imipenem/cilastatin/relebactam was compatible with a variety of antimicrobial agents. The compatibility profile described, will facilitate incorporation into hospital protocols, contribute to therapy optimization and guide clinicians to avoid successive administration, consequently resulting in reduction of total infusion time, optimization of PK/PD, economizing nursing time and cost containment.

Y-site Administration of Imipenem/Cilastatin/Relebactam With Common Intravenous Medications.

PURPOSE: Imipenem/cilastatin/relebactam has shown efficacy in complicated intra-abdominal and urinary tract infections in the RESTORE IMI-1 study, and it was recently approved by the US Food and Drug Administration. A press release announced that another Phase III study (RESTORE IMI-2) in patients with hospital-acquired and ventilator-associated pneumonia has met the primary end point. Critically ill patients with multidrug-resistant infections are expected to receive several pharmaceutical intravenous drugs while admitted in hospitals, warranting the need for Y-site compatibility studies. This study was conducted to evaluate the physical compatibility of imipenem/cilastatin/relebactam for injection during Y-site administration with common injectable intravenous medications. METHODS: Imipenem/cilastatin/relebactam was prepared to the concentration of 5 mg/mL, and other intravenous tested drugs were reconstituted as per the package inserts. Y-site was simulated as a 2-drug combination by mixing 5 mL of each in a glass tube, with reversing of the order of mixing; physical characteristics were recorded, and pH changes and turbidity were measured at time intervals. FINDINGS: Imipenem/cilastatin/relebactam was found to be compatible with a wide range of intravenous medications, facilitating co-administration with various IV medications. IMPLICATIONS: The compatibility reported is limited to a 2-h observation period in this study to adequately cover imipenem/cilastatin/relebactam infusion time. In addition, it is based on the measured turbidity with no chemical assay of the components of the admixture.

Chemiluminescent Carbapenem-Based Molecular Probe for Detection of Carbapenemase Activity in Live Bacteria.

Carbapenemase-producing organisms (CPOs) pose a severe threat to antibacterial treatment due to the acquisition of antibiotic resistance. This resistance can be largely attributed to the antibiotic-hydrolyzing enzymes that the bacteria produce. Current carbapenem "wonder drugs", such as doripenem, ertapenem, meropenem, imipenem, and so on, are resistant to regular ß-lactamases, but susceptible to carbapenemases. Even worse, extended exposure of bacteria to these drugs accelerates the spread of resistance genes. In order to preserve the clinical efficacy of antibacterial treatment, carbapenem drugs should be carefully regulated and deployed only in cases of a CPO infection. Early diagnosis is therefore of paramount importance. Herein, we report the design, synthesis, and activity of the first carbapenemase-sensitive chemiluminescent probe, CPCL, which may be used to monitor CPO activity. The design of our probe enables enzymatic cleavage of the carbapenem core, which is followed by a facile 1,8-elimination process and the emission of green light through rapid chemical excitation. We have demonstrated the ability of the probe to detect a number of clinically relevant carbapenemases and the successful identification of CPO present in bacterial cultures, such as those used for clinical diagnosis. We believe that our use of "turn-on" chemiluminescence activation will find significant application in future diagnostic assays and improve antibacterial treatment.

Structural Basis for Substrate Specificity and Carbapenemase Activity of OXA-48 Class D ß-Lactamase.

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are a diverse family of enzymes that are rapidly becoming the predominant cause of bacterial resistance against ß-lactam antibiotics in many regions of the world. OXA-48, an atypical member of CHDLs, is one of the most frequently observed in the clinic and exhibits a unique substrate profile. We applied X-ray crystallography to OXA-48 complexes with multiple ß-lactam antibiotics to elucidate this enzyme's carbapenemase activity and its preference of imipenem over meropenem and other substrates such as cefotaxime. In particular, we obtained acyl-enzyme complexes of OXA-48 with imipenem, meropenem, faropenem, cefotaxime, and cefoxitin, and a product complex with imipenem. Importantly, the product complex captures a key reaction milestone with the newly generated carboxylate group still in the oxyanion hole, and represents the first such complex with a wild-type serine ß-lactamase. A potential hydrogen bond is observed between the two carboxylate groups from the product and the carbamylated Lys73, representing the stage immediately after the breakage of the acyl-enzyme bond where the product carboxylate would be neutral. The placement of the product carboxylate also illustrates the approximate transient location of the deacylation water that has long eluded structural characterization in class D ß-lactamases. Additionally, comparing the product complex with the acyl-enzyme intermediates provides new insights into the various mechanisms by which specific side chain groups hinder the access of the deacylation water to the acyl-enzyme linkage, especially in meropenem. Taken together, these data offer valuable information on the substrate specificity of OXA-48 and the catalytic mechanism of CHDLs.

Heteroaryl Phosphonates as Noncovalent Inhibitors of Both Serine- and Metallocarbapenemases.

Gram-negative pathogens expressing serine ß-lactamases (SBLs) and metallo-ß-lactamases (MBLs), especially those with carbapenemase activity, threaten the clinical utility of almost all ß-lactam antibiotics. Here we describe the discovery of a heteroaryl phosphonate scaffold that exhibits noncovalent cross-class inhibition of representative carbapenemases, specifically the SBL KPC-2 and the MBLs NDM-1 and VIM-2. The most potent lead, compound 16, exhibited low nM to low µM inhibition of KPC-2, NDM-1, and VIM-2. Compound 16 potentiated imipenem efficacy against resistant clinical and laboratory bacterial strains expressing carbapenemases while showing some cytotoxicity toward human HEK293T cells only at concentrations above 100 µg/mL. Complex structures with KPC-2, NDM-1, and VIM-2 demonstrate how these inhibitors achieve high binding affinity to both enzyme classes. These findings provide a structurally and mechanistically new scaffold for drug discovery targeting multidrug resistant Gram-negative pathogens and more generally highlight the active site features of carbapenemases that can be leveraged for lead discovery.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.

Stability Studies of Antipyocyanic Beta-Lactam Antibiotics Used in Continuous Infusion.

In intensive care, beta-lactams can be reconstituted in 50 mL polypropylene syringes with NaCl 0.9 % and administered for 8 to 12 h at various concentrations with motor-operated syringe pumps. The feasibility and/or the stability of these antibiotic therapies are often poorly known by clinicians. The purpose of this study was to determine the stability of seven antipyocyanic beta-lactam antibiotics and cilastatin under real-life conditions. Stability indicating HPLC methods allowing quantification in pharmaceutical preparations and subsequent stability studies were performed. The stability studies showed that continuous infusion of piperacillin/tazobactam 80/10 mg/mL, of cefepime 20 and 40 mg/mL and of aztreonam 40 and 120 mg/mL can be used over 12 h. Moreover, continuous infusion of cefepime 120 mg/mL can be used over 10 h, whereas meropenem 10 and 20 mg/mL and ceftazidime 40 mg/mL remained stable only over 8 h, and meropenem 40 mg/mL was significantly degraded after 6 h. Finally, imipenem/cilastatin 5/5 mg/mL and piperacillin/tazobactam 320/40 mg/mL should not be used as continuous infusion. These data allow the establishment of protocols of administration of antipyocyanic beta-lactams by continuous infusion. Some of them are not appropriate to this mode of administration (imipenem/cilastatin, piperacillin/ tazobactam 320/40 mg/mL) or must be avoided if possible (ceftazidime 40 mg/mL).

Recognition and interaction of CphA from Aeromonas hydrophila with imipenem and biapenem by spectroscopic analysis in combination with molecular docking.

The molecular recognition and interaction of CphA from Aeromonas hydrophila with imipenem (Imip) and biapenem (Biap) were studied by means of the combined use of fluorescence spectra and molecular docking. The results showed that both the fluorescence quenching of CphA by Imip and Biap were caused through the combined dynamic and static quenching, and the latter was dominating in the process; the microenvironment and conformational of CphA were altered upon the addition of Imip and Biap from synchronous and three-dimensional fluorescence. The binding of CphA with Imip or Biap caused a conformational change in the loop of CphA, and through the conformational change, the loop opened the binding pocket of CphA to allow for an induced fit of the newly introduced ligand. In the binding of CphA with Imip, the whole molecule entered into the active pocket of CphA. The binding was driven by enthalpy change, and the binding force between them was mainly hydrogen bonding and Van der Waals force; whereas in the binding of CphA with Biap, only the beta-lactam ring of Biap entered into the binding pocket of CphA while the side chain was located outside the active pocket. The binding was driven by the enthalpy change and entropy change together, and the binding force between them was mainly electrostatic interaction. This study provided an insight into the recognition and binding of CphA with antibiotics, which may be helpful for designing new substrate for beta-lactamase and developing new antibiotics resistant to superbugs.

A novel reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of imipenem and meropenem in human plasma and its application in TDM.

A rapid and specific reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with UV detection has been developed and validated for the simultaneous determination of imipenem and meropenem in human plasma. The extraction process was performed through protein precipitation method using acetonitrile and dichloromethane, and the recoveries of quality controls (QCs) were > 91.5%. Isocratic elution followed by gradient elution of acetonitrile and water was employed over a C18 analytical column for separation. The detection was performed at 298 nm. This method was accurate and reproducible (coefficient of variation, CV < 8%), allowing quantification of carbapenem at the plasma-level ranges from 0.1 to 100 µg/ml without interference of any of the 30 frequently prescribed drugs. Stabilities of imipenem and meropenem were determined with or without stabilizer solutions at -80°C, -20°C, +4 °C and room temperature 20°C. These two drugs showed higher stability at the low temperatures. Addition of 3-(N-morpholino) propanesulfonic acid (MOPS) might also increase their stability. The results of therapeutic drug monitoring (TDM) in neonates and adults showed high inter- and intra- individual variabilities in the trough concentrations of imipenem and meropenem, thus confirming the importance and necessity of TDM. For neonatal patients, imipenem 20 mg/kg, q12h (40mg/kg/day) failed to produce significant therapeutic effects, and either the dose or the frequency was adjusted to achieve 60mg/kg/day or above to maintain the trough concentration required for the curative effect. The low operational cost and good separation efficiency would help implement this assay for the routine therapeutic drug monitoring of imipenem and meropenem in hospitals.