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Cystic fibrosis (CF) is a genetic disorder affecting nearly 105,000 patients worldwide and is characterized by poor respiratory function due to accumulation of thick mucus in the lungs, which not just acts as a physical barrier, but also provides a breeding ground for bacterial infections. These infections can be controlled with the help of antibiotics which can be delivered directly into the lungs for amplifying the local anti-bacterial effect. More than 50 % of CF patients are associated with Pseudomonas aeruginosa infection in their lungs which requires antibiotics such as Aztreonam (AZT). In this study, we prepared inhalable AZT-loaded lipid nanoparticles using Hot-melt extrusion (HME) coupled with probe sonication to target Pseudomonas aeruginosa infection in the lungs. The optimized nanoparticles were tested for physicochemical properties, stability profile, in-vitro aerosolization, and antimicrobial activity against Pseudomonas aeruginosa. The optimized nanoparticles with a PEI concentration of 0.1 % demonstrated a uniform particle size of <50 nm, a spherical shape observed under a transmission electron microscope, and >70 % drug entrapment. Incorporating cationic polymer, PEI, resulted in sustained drug release from the lipid nanoparticles. The in-vitro aerosolization studies exhibited a mass median aerodynamic diameter (MMAD) of <4.3 μm, suggesting deposition of the nanoparticles in the respirable airway. The antimicrobial activity against Pseudomonas aeruginosa showed the minimum inhibitory concentration of the formulation is 2-fold lower than plain AZT. Stability profile showed the formulations are stable after exposure to accelerated conditions. In conclusion, hot-melt extrusion in combination with probe sonication can be used as a potential method for the continuous production of AZT-loaded lipid nanoparticles with enhanced anti-bacterial activity., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The synthesis of structurally new haptens and the development of suitable antigens are essential for boosting the sensitivity of drug allergy diagnostic testing. Unprecedented structural antigens for benzylpenicillin and amoxicillin are characterised and evaluated in a cohort of 70 subjects with a turnkey solution based on consumer electronics.

New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for β-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty β-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-β-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-β-lactam IgE antibodies with high sensitivity (< 0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of β-lactam antibiotics renders improved molecular recognition events when the 4-member β-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.

Purpose: The objective of this communication was to determine the intravenous compatibility of ceftazidime/avibactam and aztreonam using simulated and actual Y-site administration., Methods: Ceftazidime-avibactam was reconstituted and diluted to concentrations of 8, 25, and 50 mg/mL in 0.9% sodium chloride. Aztreonam was reconstituted and diluted to concentrations of 10 and 20 mg/mL. Each combination of concentrations was tested for compatibility using visual, Tyndall beam, microscopy, turbidity, and pH assessments. Microscopy results were compared to those from sodium chloride 0.9% in water, pH was compared to that at time 0, and turbidity of combinations was compared to that of individual agents. Actual Y-site mixing was conducted over 2-h infusions with samples collected at 0, 1, and 2 h. Test results were evaluated at 0, 1, 2, 4, 8, and 12 h after mixing. All experiments were completed in triplicate., Findings: Across simulated and actual Y-site experiments, no evidence of incompatibility between combinations of ceftazidime-avibactam + aztreonam was observed. Visual and microscopic tests revealed no particulate matter, color changes, or turbidity. Tyndall beam tests were negative with all combinations. No evidence of incompatibility was observed in turbidity testing. The pH values were consistent across each of the 6 combinations, from immediately after mixing until 12 h after mixing. When the addition of agents was reversed in simulated Y-site experiments, no differences in compatibility were observed. No differences in compatibility between actual and simulated Y-site administration were observed, and there was minimal variability across all replicate experiments., Implications: Ceftazidime-avibactam, at concentrations of 8, 25, and 50 mg/mL, appeared compatible with aztreonam at concentrations of 10 and 20 mg/mL., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recently, fluorenylmethyloxycarbonyl (Fmoc) conjugated amino acids (Fmoc-AA), especially Fmoc-phenylalanine (Fmoc-F), have been discovered to have antimicrobial properties specific to Gram-positive bacteria including MRSA. Their weak antibacterial activity against Gram-negative bacteria is due to their inability to cross the bacterial membrane. Here in order to increase the antibacterial spectrum of Fmoc-F, we prepared a formulation of Fmoc-F with the Gram-negative specific antibiotic aztreonam (AZT). This formulation displayed antibacterial activity against both Gram-positive and Gram-negative bacteria and significantly reduced the bacterial load in a mouse wound infection model. The combination produced a synergistic effect and higher efficacy against P. aeruginosa due to the increased Fmoc-F permeability by AZT through the bacterial membrane. This combinatorial approach could be an effective strategy for other Fmoc-AA having a Gram-positive specific antibacterial effect for the better management of bacterial wound infections.

β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-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 β-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 [IC 50 ]). MIC assays indicate relebactam potentiates β-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-β-lactam combinations., (Copyright © 2019 Tooke et al.)

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).

CTX-M-15 type β-lactamases are a class of enzymes which hydrolyzes cefotaxime and aztreonam (a monobactam) antibiotics. The emergence of CTX-M-15 producing Enterobacteriaceae member is a major threat to public health. The objective of the study was to check the potency of aztreonam and cefotaxime in combination against β-lactamase producing strains and to monitor the mechanism behind their interaction. FICI results showed the synergistic effect of aztreonam-cefotaxime against CTX-M-15 producing strain. The expressed and purified CTX-M-15 protein was used as the source of enzyme. Kinetic studies confirmed that the catalytic efficiency of the CTX-M-15 enzyme was decreased to about 78% when it was treated with aztreonam then with cefotaxime in 5 and 10 times molar ratio, respectively, in comparison to the studies where efficiency was enhanced by 33% when cefotaxime was taken alone. Fluorescence study showed that aztreonam binding with CTX-M-15 was an endothermic and spontaneous process with Ka of the order of 10 4  M -1 . CD spectroscopic study showed conformational changes upon aztreonam/aztreonam-cefotaxime binding with CTX-M-15. The study concludes that aztreonam in combination with cefotaxime synergistically inhibits CTX-M-15 efficiency significantly. Hence the combination of a monobactam and cephalosporin can be used as the potential therapeutic candidates against β-lactamase producing CTX-M-15 strains., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Enhanced sampling of large number of collective variables (CVs) is inevitable in molecular dynamics (MD) simulations of complex chemical processes such as enzymatic reactions. Because of the computational overhead of hybrid quantum mechanical/molecular mechanical (QM/MM)-based MD simulations, especially together with density functional theory, predictions of reaction mechanism, and estimation of free-energy barriers have to be carried out within few tens of picoseconds. We show here that the recently developed temperature-accelerated sliced sampling method allows one to sample large number of CVs, thereby enabling us to obtain rapid convergence in free-energy estimates in QM/MM MD simulation of enzymatic reactions. Moreover, the method is shown to be efficient in exploring flat and broad free-energy basins that commonly occur in enzymatic reactions. We demonstrate this by studying deacylation and reverse acylation reactions of aztreonam drug catalyzed by a class-C β lactamase (CBL) bacterial enzyme. Mechanistic details and nature of kinetics of aztreonam hydrolysis by CBL are elaborated here. The results of this study point to characteristics of the aztreonam drug that are responsible for its slow hydrolysis.

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed., (Copyright © 2016 Vandavasi et al.)

Three new degradants have been identified from drug product and active pharmaceutical ingredient stability samples of aztreonam, a marketed synthetic monocyclic beta-lactam antibiotic. The degradants were detected following the implementation of a new, more selective HPLC method for the determination of impurities and degradants. The new method was developed in response to changes in the regulatory requirement for mature products. Two of the new unknown Degradants (I and II) were observed in chromatograms from stability samples of aztreonam injection. The third new Degradant (III) was observed during a stability study of the aztreonam active pharmaceutical ingredient. These degradants were structurally characterized. A small amount (ca. 1-3mg) of each degradant was isolated via preparative HPLC for structure elucidation using accurate MS, one and two-dimensional NMR spectroscopy. The small amount of each NMR sample was then reused as a standard for HPLC purity/impurity method validation. Their exact concentrations were determined using quantitative NMR which enabled the execution of the quantitative elements of the HPLC method validation. This innovative approach eliminated the need to isolate or synthesize larger quantities of markers for HPLC/UV method validation, thus saving significant time and reducing costs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We solved the crystal structure of the class C β-lactamase MOX-1 complexed with the inhibitor aztreonam at 1.9Å resolution. The main-chain oxygen of Ser315 interacts with the amide nitrogen of aztreonam. Surprisingly, compared to that in the structure of free MOX-1, this main-chain carboxyl changes its position significantly upon binding to aztreonam. This result indicates that the interaction between MOX-1 and β-lactams can be accompanied by conformational changes in the B3 β-strand main chain., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Studies performed on samples larger than 100 subjects with a documented IgE-mediated hypersensitivity to penicillins have demonstrated a cross-reactivity rate of approximately 1% between penicillins and both imipenem and meropenem, whereas a single study found a cross-reactivity rate of 6.2% with aztreonam in 16 such subjects., Objective: To assess the cross-reactivity and tolerability of aztreonam and 3 carbapenems (imipenem-cilastatin, meropenem, and ertapenem) in patients with documented IgE-mediated hypersensitivity to penicillins., Methods: A total of 212 consecutive subjects with immediate reactions to penicillins and positive results on skin tests to at least 1 penicillin reagent underwent skin tests with aztreonam and carbapenems; subjects with negative results were challenged with escalating doses of aztreonam and carbapenems., Results: All subjects displayed negative skin test results to both aztreonam and carbapenems; 211 accepted challenges and tolerated them. Challenges were not followed by full therapeutic courses., Conclusions: These data indicate the tolerability of both aztreonam and carbapenems in penicillin-allergic subjects. In those who especially require these alternative β-lactams, however, we recommend pretreatment skin tests, both because rare cases of cross-reactivity have been reported and because negative results indicate tolerability., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement.

Bacteria that cause most of the hospital-acquired infections make use of class C β-lactamase (CBL) among other enzymes to resist a wide spectrum of modern antibiotics and pose a major public health concern. Other than the general features, details of the defensive mechanism by CBL, leading to the hydrolysis of drug molecules, remain a matter of debate, in particular the identification of the general base and role of the active site residues and substrate. In an attempt to unravel the detailed molecular mechanism, we carried out extensive hybrid quantum mechanical/molecular mechanical Car-Parrinello molecular dynamics simulation of the reaction with the aid of the metadynamics technique. On this basis, we report here the mechanism of the formation of the acyl-enzyme complex from the Henry-Michaelis complex formed by β-lactam antibiotics and CBL. We considered two β-lactam antibiotics, namely, cephalothin and aztreonam, belonging to two different subfamilies. A general mechanism for the formation of a β-lactam antibiotic-CBL acyl-enzyme complex is elicited, and the individual roles of the active site residues and substrate are probed. The general base in the acylation step has been identified as Lys67, while Tyr150 aids the protonation of the β-lactam nitrogen through either the substrate carboxylate group or a water molecule.

β-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple β-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple β-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.

New Delhi metallo-β-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (k cat/K m ratios between 0.03 to 1.28 µmol⁻¹.s⁻¹), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydrophilic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.

Herein we examine the viability of physicochemical approaches based on standard computational chemistry tools to characterize the structure and energetics of flexible drug molecules with various titratable sites. We focus on the case of the monobactam antibiotic aztreonam, whose structure and physicochemical properties have been ascribed to several tautomeric forms, although it is still unclear which protonation states are responsible for its biological activity. First, we experimentally determined the pKa values for aztreonam over the pH range 0.8-7.0 using both 1H NMR and 13C NMR spectroscopy. Second, we carried out quantum chemical calculations on snapshots extracted from classical molecular dynamics simulations. Various levels of approximation were used in the energy calculations: ONIOM(HF/3-21G*:AMBER) for geometry relaxation, B3LYP/6-31+G** for electronic and electrostatic solvation energies, and molecular mechanics for attractive dispersion energy. The value of the free energy of solvation of a proton was treated as a parameter and chosen to give the best match between calculated and experimental pKa values for small molecules. Overall, this computational scheme can give satisfactory results in the pKa calculations for drug molecules.

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.

The influence of temperature and relative humidity on the stability of aztreonam in AZACTAM was investigated. Changes of the concentration of aztreonam were followed using the HPLC method with UV detection. The first-order rate constants of the reversible reaction of isomerization Z-aztreonam right harpoon over left harpoonE-aztreonam and the parallel reaction Z-aztreonam-->products were determined at RH=76.4% and T=313, 323, 333, 343 and 353 K, and at T=343 K and RH=50.9%, 60.5%, 66.5% and 76.4%. The thermodynamic parameters-energy, enthalpy and entropy of these reactions were calculated.

Spectrophotometric and fluorimetric determination of aztreonam were achieved through its reaction with cerium (IV) in acidic medium. The spectrophotometric method involves the quantitation of the amount of ceric equivalent to aztreonam by measuring the absorbance at 317 nm and the corresponding first-derivative value at 284 nm for the blank solution against the reaction solution. Beer's law is obeyed over the concentration ranges of 1.5-4 and 1-4 microg ml(-1), respectively. Meanwhile, in the fluorimetric method, higher sensitivity was achieved by measuring the fluorescence intensity of the formed cerium (III) at lambda(em)=357 nm (lambda(ex)=257 nm) within a concentration range 150-350 ng ml(-1). Study of the reaction conditions and reaction stoichiometry were presented. Interference from L-arginine, which is frequently co-formulated with aztreonam, was tested. The proposed procedures were applied successfully to the determination of aztreonam in pure form and in presence of arginine both in laboratory mixtures and commercial vials. The proposed methods are sensitive, accurate and precise as compared with the official USP 24 HPLC method.

Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions. The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.

Three rapid and sensitive, colorimetric and atomic absorption spectrometric methods were developed for the determination of aztreonam. The proposed methods depend upon the reaction of cobaltthiocyanate (I) or reineckate (II) ions with the drug to form stable ion-pair complexes which extractable with chloroform. The greenish blue and pink color complexes are determined either colorimetrically at lambda(max) 625 and 525 nm for I and II reagents, respectively, or by atomic absorption spectrometry, directly using the organic extracted complex, or indirectly, using the supernatant. The three procedures are applied for the determination of aztreonam in pure and in pharmaceutical dosage forms applying the standard additions technique and the results obtained agreed well with those obtained by the official method.

Objective: To evaluate the physical compatibility and chemical stability of linezolid (Zyvox-Pharmacia) 200 mg/100 mL admixed with aztreonam (Azactam-Squibb) 2 grams and separately with piperacillin sodium (Pipracil-Lederle) 3 grams over 7 days at 4 degrees C and 23 degrees C., Design: Controlled experimental trial., Setting: Laboratory., Interventions: Test samples were prepared by adding the required amount of aztreonam or piperacillin sodium to separate bags of linezolid injection 200 mg/100 mL., Main Outcome Measures: Physical compatibility and chemical stability based on drug concentrations initially and after 1, 3, 5, and 7 days of storage at 4 degrees C and 23 degrees C., Results: All of the linezolid admixtures with aztreonam and with piperacillin sodium were clear when viewed in normal fluorescent room light and with a Tyndall beam. Measured turbidity and particulate content were low and exhibited little change throughout the study at both storage temperatures. High-performance liquid chromatography analysis found little or no loss of linezolid in any sample stored at either temperature throughout the study. Aztreonam in the linezolid admixtures was stable for 7 days, exhibiting less than 5% loss at 4 degrees C and 9% loss at 23 degrees C. Piperacillin sodium in the linezolid admixtures was stable for 7 days at 4 degrees C, exhibiting no loss, but was stable for only 3 days at 23 degrees C with losses of about 5%. Losses had increased to 9% to 12% after 5 days of storage at room temperature., Conclusion: Admixtures of linezolid 200 mg/100 mL with aztreonam 2 grams or piperacillin sodium 3 grams were physically compatible and chemically stable for at least 7 days stored at 4 degrees C and for 7 days or 3 days, respectively, at 23 degrees C.

Imipenem and meropenem, members of the carbapenem class of beta-lactam antibiotics, are among the most broadly active antibiotics available for systemic use in humans. They are active against streptococci, methicillin-sensitive staphylococci, Neisseria, Haemophilus, anaerobes, and the common aerobic gram-negative nosocomial pathogens including Pseudomonas. Resistance to imipenem and meropenem may emerge during treatment of P. aeruginosa infections, as has occurred with other beta-lactam agents; Stenotrophomonas maltophilia is typically resistant to both imipenem and meropenem. Like the penicillins, the carbapenems have inhibitory activity against enterococci. In general, the in vitro activity of imipenem against aerobic gram-positive cocci is somewhat greater than that of meropenem, whereas the in vitro activity of meropenem against aerobic gram-negative bacilli is somewhat greater than that of imipenem. Daily dosages may range from 0.5 to 1 g every 6 to 8 hours in patients with normal renal function; the daily dose of meropenem, however, can be safely increased to 6 g. Infusion-related nausea and vomiting, as well as seizures, which have been the main toxic effects of imipenem, occur no more frequently during treatment with meropenem than during treatment with other beta-lactam antibiotics. The carbapenems should be considered for treatment of mixed bacterial infections and aerobic gram-negative bacteria that are not susceptible to other beta-lactam agents. Indiscriminate use of these drugs will promote resistance to them. Aztreonam, the first marketed monobactam, has activity against most aerobic gram-negative bacilli including P. aeruginosa. The drug is not nephrotoxic, is weakly immunogenic, and has not been associated with disorders of coagulation. Aztreonam may be administered intramuscularly or intravenously; the primary route of elimination is urinary excretion. In patients with normal renal function, the recommended dosing interval is every 8 hours. Patients with renal impairment require dosage adjustment. Aztreonam is used primarily as an alternative to aminoglycosides and for the treatment of aerobic gram-negative infections. It is often used in combination therapy for mixed aerobic and anaerobic infections. Approved indications for its use include infections of the urinary tract or lower respiratory tract, intra-abdominal and gynecologic infections, septicemia, and cutaneous infections caused by susceptible organisms. Concurrent initial therapy with other antimicrobial agents is recommended before the causative organism has been determined in patients who are seriously ill or at risk for gram-positive or anaerobic infection.

A series of cephalosporins, 2-isocephems, and 2-oxaisocephems with C-3' catechol-containing (pyridinium-4-thio)methyl groups and 2-isocephems with C-7 catechol related aromatics have been prepared and evaluated for antimicrobial activity. It turns out that these compounds have highly potent activity against Gram-negative bacteria, especially resistant pathogens such as Pseudomonas aeruginosa. The most active compound of the series was (6S,7S)-7-[2-(2-aminothiazol-4-yl)-2-[(Z)-[(1,5-dihydroxy-4-pyr idon-2-yl) methoxy]imino]acetamido]-3-[[[(4-methyl-5-carboxymethyl)thiazol-2- yl] thio]methyl]-8-oxo-1-aza-4-thiabicyclo [4.2.0] oct-2-ene-2-carboxylic acid which exhibited potent in vitro activity against clinically isolated P, aeruginosa and Acinetobacter baumanii which is also resistant to many anti-infectives, and good in vivo efficacy against clinically isolated P aeruginosa.

Amorphos injectable form of aztreonam (BIOKTAM) was prepared. It was shown that after intramuscular or intravenous administration there are not any considerable differences in the bioavailability of aztreonam from BIOKTAM and of the drug from AZACTAM.

Parameters affecting the separation of amino acids on different RP-HPLC columns were studied. Six amino acids were separated on Zorbax TMS, Zorbax CN, Zorbax ODS and Zorbax C8, using 1.8 x 10(-3) M copper sulphate at pH 4.1 as aqueous mobile phase. The best separation was shown by Zorbax TMS followed by Zorbax CN. The column performance was maintained by serial washing after 6 h continuous work with aqueous 10(-4) M disodium edetate, 10(-4) N sulphuric acid, water and gradient elution with aqueous methanol. The separation mechanism was interpreted. The method was applied for separation and quantification of aztreonam and L-arginine in Azactam vials.

The stability of the monocyclic beta-lactam antibiotic aztreonam in portable pump reservoirs was studied during storage at temperatures of -20 degrees C and +5 degrees C and during drug delivery at 37 degrees C. Three 100-ml drug reservoirs and three glass containers containing 60 mg/ml aztreonam were stored at -20 degrees C and 2-ml samples were analysed in the freshly prepared solution and after thawing at days 7, 21, 28, 70 and after 6 months of storage. A separate triplicate batch of 100-ml prefilled drug reservoirs and glass containers containing a similar aztreonam concentration (60 mg/ml) were refrigerated and tested immediately after preparation and daily for 8 days and after 70 days. Solutions of aztreonam in duplicate freshly prepared reservoirs were tested for stability when the solution was pumped at 37 degrees C over a 24-h period. All solutions were inspected for visual changes and tested for pH. Drug concentration was analysed by high-performance liquid chromatography. No colour changes or pH differences were observed in any of the solutions in the reservoirs of containers. No statistically significant decrease in aztreonam concentration could be detected after 6 months of storage at -20 degrees C. Aztreonam was stable at 5 degrees C for at least 8 days. A 24-h pumping period at 37 degrees C showed a 3.6% decrease in aztreonam concentration. Aztreonam at a concentration of 60 mg/ml in a pump reservoir is sufficiently stable to be used in home intravenous antibiotic treatment programmes.

The physical compatibility and chemical stability of aztreonam and vancomycin hydrochloride when combined at clinically used high and low concentrations were studied. Admixtures consisting of aztreonam 4 mg/mL and vancomycin 1 mg/mL (as the hydrochloride salt) in 5% dextrose injection, aztreonam 4 mg/mL and vancomycin 1 mg/mL in 0.9% sodium chloride injection, aztreonam 40 mg/mL and vancomycin 10 mg/mL in 5% dextrose injection, and aztreonam 40 mg/mL and vancomycin 10 mg/mL in 0.9% sodium chloride injection were prepared in triplicate in polyvinyl chloride containers. Three containers of each type of admixture were stored at 4, 23, and 32 degrees C. Samples were removed immediately and at various time points over 31 days. Compatibility was assessed by visual examination, with a turbidimeter, and with a particle sizer-counter. Stability was determined by stability-indicating high-performance liquid chromatography (HPLC). All the admixtures initially appeared clear to the unaided eye after the disappearance of a transient white swirl in the high-concentration admixtures (aztreonam 40 mg/mL and vancomycin 10 mg/mL). However, the high-concentration admixtures immediately developed unacceptable levels of a microcrystalline precipitate when viewed with a high-intensity fiber-optic light source. Easily visible gross turbidity and precipitation formed after various periods but often within 24 hours. HPLC showed aztreonam 4 mg/mL and vancomycin 1 mg/mL in 5% dextrose injection to be a stable combination for 7 days at 32 degrees C, 14 days at 23 degrees C, and 31 days at 4 degrees C. In 0.9% sodium chloride injection, the drugs in the low-concentration admixtures were stable for 7 days at 32 degrees C and for 31 days at 4 and 23 degrees C. Stability of the combination in the high-concentration admixtures was maintained for 3 days at 23 and 32 degrees C and for 14 days at 4 degrees C. Aztreonam and vancomycin hydrochloride were considerably less compatible and stable in the high-concentration admixtures than in the low-concentration ones.

The compatibility of aztreonam injection with selected other drugs during simulated Y-site injection was evaluated. A 5-mL sample of aztreonam 40 mg/mL in 5% dextrose injection was combined with 5 mL of each of 100 other drugs including antineoplastics, anti-infectives, and supportive care drugs in 5% dextrose injection or 0.9% sodium chloride injection. The combinations were stored under fluorescent light at 23 degrees C. The combinations were evaluated initially and at one and four hours after preparation by examination with the unaided eye and with a high-intensity light beam, turbidity measurement, and particle sizing and counting. Most of the test drugs were compatible with aztreonam 40 mg/mL for the observation period. However, aztreonam combinations with 12 drugs exhibited incompatibilities including turbidity, particulate formation, precipitation, and color change. These drugs were acyclovir sodium, amphotericin B, chlorpromazine hydrochloride, daunorubicin hydrochloride, ganciclovir sodium, lorazepam, metronidazole, miconazole, mitomycin, mitoxantrone hydrochloride, prochlorperazine edisylate, and streptozocin. Aztreonam 40 mg/mL in 5% dextrose injection was compatible with most of the drugs tested for up to four hours at 23 degrees C. However, 12 drugs exhibited incompatibility with aztreonam.

Objective: To assess the impact of degradation of aztreonam, a beta-lactam antibiotic, during HPLC analysis on pharmacokinetic parameter estimates., Methods: The baseline (B) serum concentration-time data from a published pharmacokinetic study of aztreonam were degraded using first-order equations and a degradation rate constant (0.014 h-1) determined from a preliminary degradation study. Samples were mathematically degraded for autosampler run times of 8-13 h (D1) to approximate a normal work day and for autosampler run times of 16-17 h (D2) and compared with B data. It was assumed that B data were nondegraded and that changes in chromatography were the result of degradation of azetreonam and not to any changes in chromatographic conditions. A two-compartment model was used to fit the data and pharmacokinetic parameters were calculated using standard equations. Statistical significance between all pharmacokinetic parameters for B and D1 and B and D2 was determined using the paired, two-tailed Student's t-test., Results: Increased variability was noted for all pharmacokinetic parameters for D1 and D2 compared with B. Statistically significant differences were found for clearance (B < D1, p = 0.0095 and B < D2, p = 0.0194), steady-state volume of distribution (B < D2, p = 0.0392), and area under the serum concentration-time curve (B > D1, p = 0.0497)., Conclusions: Aztreonam degradation resulted in increased variability in pharmacokinetic parameter estimates. Investigators should be cognizant of this and preliminary studies should be performed to characterize degradation for the length of the expected autosampler run.

The stability of aztreonam, ampicillin sodium, and sulbactam sodium admixed in 0.9% sodium chloride injection and stored at room temperature and under refrigeration was studied. Each of the following admixtures was prepared in 0.9% sodium chloride injection: (1) aztreonam 10 mg/mL; (2) ampicillin 20 mg/mL (as the sodium salt) and sulbactam 10 mg/mL (as the sodium salt); and (3) aztreonam 10 mg/mL, ampicillin 20 mg/mL, and sulbactam 10 mg/mL. Three minibags of each admixture were stored at room temperature and three were refrigerated. Every 12 hours, up to 96 hours, the admixtures were visually inspected and 5-mL samples were withdrawn for high-performance liquid chromatography and pH testing. No color change or precipitation was observed in any sample. In admixtures containing ampicillin, ampicillin was the first or only drug to lose more than 10% of initial concentration. In the ampicillin-sulbactam admixture, ampicillin was stable for 32 hours at room temperature and 68 hours refrigerated. In the aztreonam-ampicillin-sulbactam admixture, ampicillin was stable for 30 hours at room temperature and 94 hours refrigerated. Aztreonam 10 mg/mL, ampicillin 20 mg/mL (as the sodium salt), and sulbactam 10 mg/mL (as the sodium salt) in 0.9% sodium chloride injection were stable in combination for up to 30 hours at room temperature and 94 hours under refrigeration.

A 72-year-old woman presented with generalized urticaria after receiving an injection of aztreonam. After successive in vitro and in vivo studies, allergy to aztreonam with a good tolerance of the other beta-lactams was diagnosed.

The catalytic effect of various buffer systems (citrates, acetates, phosphates, borates and carbonates) on the degradation of aztreonam and nocardicin A in aqueous solution was studied at 35 degrees C and a constant ionic strength of 0.5 mol.dm-3 over a pH range of 3.50 to 10.50. The observed degradation rates, obtained by measuring the remaining intact antibiotic, were shown to follow pseudo-first-order kinetics with regard to antibiotic concentrations and to be influenced by general acid and general base catalysis. The changes in the concentration of intact beta-lactam antibiotic in the solutions were established by reverse-phase HPLC with UV-detection. In general the buffer systems employed in the kinetic studies showed a very weak catalytic effect on the degradation of aztreonam and nocardicin A. The pH-rate profiles for these antibiotics showed degradation minimums at pH 5.38 and 6.13, respectively. Aztreonam is slightly more reactive with hydrogen ions than nocardicin A and is much more reactive with hydroxide ions. In comparison with other beta-lactamic antibiotics, aztreonam and nocardicin A are much more stable in aqueous solution, except for aztreonam in a base solution, which is just as unstable as penicillins and cephalosporins. The Arrhenius activation energies were determined for aztreonam and nocardicin A at pH's 4.23, 6.59 and 8.60.

The stability of ranitidine hydrochloride after being mixed with commonly used i.v. beta-lactam antibiotics and administered by simulated Y-site injection was studied. Solutions of ranitidine 1 mg/mL (as the hydrochloride salt), aztreonam 16.7 mg/mL, ceftazidime 20 mg/mL (with sodium carbonate), and piperacillin 30 mg/mL (as the sodium salt) were prepared by reconstitution in i.v. mini-bags. To simulate Y-site injection, 2 mL of ranitidine hydrochloride was mixed with 2 mL of each antibiotic in glass test tubes. These admixtures were prepared in triplicate and stored at room temperature under fluorescent light. Concentrations of each drug in each admixture were determined by stability-indicating high-performance liquid chromatography immediately and after one, two, and four hours. Aztreonam, ceftazidime, and piperacillin each retained more than 95% of the original concentration for at least four hours when mixed 1:1 with ranitidine. Ranitidine retained more than 90% of its original concentration for at least four hours when combined with each of the other drugs. Ranitidine 1 mg/mL (as the hydrochloride salt) and aztreonam 16.7 mg/mL, ceftazidime 20 mg/mL (with sodium carbonate), or piperacillin 30 mg/mL (as the sodium salt) were stable for at least four hours during simulated Y-site administration.

A photodegradation study of aztreonam solutions exposed to UV irradiation showed that the major product of degradation was the anti-isomer together with some unidentified products. This result was similar to that obtained with cefotaxime and it is postulated that this would be generally true of compounds containing an alkoxyimino group.

Aztreonam is a synthetic monobactam antibiotic that has excellent activity against aerobic gram-negative bacilli. It can be used as a single agent for the treatment of upper urinary tract infections caused by organisms resistant to the cephalosporins and ampicillin. It also can be administered in combination with a drug such as clindamycin for treatment of pelvic inflammatory disease or postoperative pelvic infections.

The reaction of perchloric hydroxylamine was adopted for determination of new beta-lactam antibiotics within monobactam, carbapenem and oxazepam groups. Complexes formed in colour reaction are stable over 2 hours in the case of aztreonam and imipenem, and over 1 hour for moxalactam, statistical analysis proved that both hydroxylamine-perchloric and spectrophotometric methods can be used alternatively.

Three cell lines producing monoclonal antibodies, Az-1 (IgG1), Az-2 (IgG1) and Az-3 (IgM) against aztreonam were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams, which were conjugated with human serum albumin (HSA), were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. In ELISA, Az-1 and Az-2 reacted only with aztreonam and ceftazidime, which have the same acyl side chain. Furthermore, Az-2 showed a strong cross-reaction with carumonam. In the ELISA inhibition test, Az-1 and Az-2 were inhibited from binding to aztreonam-HSA by aztreonam, ceftazidime, aztreonam hydrolysate, aztreonam-epsilon-amino-n-caproic acid (EACA) and ceftazidime-EACA. Az-2 was also inhibited with carumonam. From the above results, it seems that Az-1 can recognize only the degraded structure of monobactam nucleus, and Az-2 can recognize the degraded nucleus moiety and the acyl side chain. On the other hand, Az-3 displayed broad cross-reaction to various beta-lactams in ELISA. Furthermore, the MAb showed no inhibitory reaction with various beta-lactams except aztreonam- and ceftazidime-EACA conjugates in the ELISA inhibition test, suggesting that Az-3 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. The above results indicate that antibodies can recognize at least three epitopes of the degraded product(s) of aztreonam nucleus, acyl side chain and NAD in aztreonam-protein conjugate.

The mechanism of possible incompatibility between commercially available metronidazole parenteral solutions and the injectable aztreonam leading to the development of pink color in their intravenous admixtures was studied. It was demonstrated that nitrite ions may be produced in metronidazole solutions at the time of preparation or during storage by the effects of temperature and light. Under acidic pH conditions of admixtures the aminothiazole moiety of aztreonam was diazotized by the nitrite ion contributed by metronidazole solutions. The diazotized molecule, in turn, reacted with another aztreonam molecule by diazo-coupling. The resultant pink-colored product was isolated by chromatography and its structure was determined by mass spectral and NMR analyses. Other beta-lactam antibiotics containing the 2-aminothiazole moiety also react in acidic media in a similar manner.