Ceftazidime/Avibactam and Other Commonly Used Antibiotics Activity Against Enterobacterales and Pseudomonas aeruginosa Isolated in Poland in 2015-2019.

Purpose: Infections caused by resistant Gram-negative bacteria are becoming increasingly common and now pose a serious public health threat worldwide, because they are difficult to treat due to few treatment options and they are associated with high morbidity and mortality. The combination of ceftazidime with the beta-lactamase inhibitor avibactam - seems to be the right choice in this situation. The aim of the study was to evaluate the activity of ceftazidime/avibactam and other commonly used antibiotics against Enterobacterales and Pseudomonas aeruginosa strains isolated within last years in Poland. Patients and Methods: This study analyzed the antibiotic susceptibility of 1607 Enterobacterales isolates and 543 nonfermenting P. aeruginosa strains collected between 2015 and 2019 in 4 medical laboratories participating in the ATLAS (Antimicrobial Testing Leadership And Surveillance) program in Poland. Unduplicated clinically significant Enterobacterales and P. aeruginosa strains were collected from patients with respiratory, skin and musculoskeletal, genitourinary, abdominal, bloodstream or other infections (ear, eye). Results: The ceftazidime/avibactam combination demonstrates the highest activity against Enterobacterales (98.9%), in both adults and children, including strains presenting MDR (multidrug-resistant) (97.5%) and ESBL (extended spectrum ß-lactamase) (96.3%) phenotypes. The activity of ceftazidime/avibactam increased to 100% when only MBL (metallo-ß-lactamase)-negative subset of Enterobacterales was considered. This combination also achieved the second highest activity result (89.3%) after colistin in P. aeruginosa, including isolates of MDR (65.9%) and carbapenem-resistant (CR) phenotypes (54.8%). When MBL-positive isolates were excluded, susceptibility rate of P. aeruginosa increased to 94.7%. It is worth to note that susceptibility of the examined P. aeruginosa strains to ceftazidime/avibactam was very high in children (93.3%), especially in a pediatric intensive care unit (94.2%). Conclusion: Enterobacterales and P. aeruginosa included in this analysis presented high susceptibility rates to ceftazidime/avibactam. Ceftazidime/avibactam showed the highest activity against Enterobacterales strains among all antibiotics studied, both for the total population as well as for MDR phenotype and ESBL phenotype. Ceftazidime/avibactam also achieved the second highest activity result against P. aeruginosa strains (including MDR and CR phenotypes). These results are much higher when excluding MBL-positive isolates that exhibit intrinsic resistance to ceftazidime/avibactam.

Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

Novel recombinant insulin analogue with flexible C-terminus in B chain. NMR structure of biosynthetic engineered A22G-B31K-B32R human insulin monomer in water/acetonitrile solution.

A tertiary structure of recombinant A22(G)-B31(K)-B32(R)-human insulin monomer (insulin GKR) has been characterized by (1)H, (13)C NMR at natural isotopic abundance using NOESY, TOCSY, (1)H/(13)C-GHSQC, and (1)H/(13)C-GHSQC-TOCSY spectra. Translational diffusion studies indicate the monomer structure in water/acetonitrile (65/35vol.%). CSI analysis confirms existence of secondary structure motifs present in human insulin standard (HIS). Both techniques allow to establish that in this solvent recombinant insulin GKR exists as a monomer. Starting from structures calculated by the program CYANA, two different refinement protocols used molecular dynamics simulated annealing with the program AMBER; in vacuum (AMBER_VC), and including a generalized Born solvent model (AMBER_GB). From these calculations an ensemble of 20 structures of lowest energy was chosen which represents the tertiary structure of studied insulin. Here we present novel insulin with added A22(G) amino acid which interacts with ß-turn environment resulting in high flexibility of B chain C-terminus.

Usefulness of the parent compound determination in bioequivalence evaluation of clopidogrel generic products.

OBJECTIVE: The aim of the presented comparative study was to evaluate the bioavailability of clopidogrel (CAS 113665-84-2) formulations containing clopidogrel bisulfate (CAS 135046-48-9, CBS) 75 mg based on the parent compound (CBS) and its metabolite SR 26334 - clopidogrel carboxylic acid (CAS 144457-28-3, CCA) determination. METHODS: This paper presents the results of a comparative, randomized, two-way cross-over study on 48 healthy male volunteers assessing the bioequivalence of two products of clopidogrel 75 mg in form of film-coated tablets. In each of the two periods, separated by a 7-day washout period, a single dose of 150 mg (2 x 75 mg) of test and reference preparations was administered under fasting condition. Nineteen blood samples for determination of CBS and CCA were collected up to 48 h post dose. The CBS and CCA concentrations were quantified by a selective ultra performance liquid chromatographic-tandem mass spectrometric (UPLC-MS/MS) method. Pharmacokinetic parameters such as AUCinf, AUCt, Cmax, tmax, t1/2 were estimated using a non-compartmental model. Bioequivalence evaluation and calculation of CI were performed for clopidogrel and its metabolite by two one-sided t-test procedures by Schuirmann. RESULTS: In case of CCA the values of pharmacokinetic parameters were similar for the two products (test vs reference): AUCinf: 15773 vs. 15691 ng x h/mL, AUCt: 15,462 vs. 15,315 ng x h/mL, Cmax: 4919 vs. 4699 ng/mL, tmax: 0.84 vs. 0.93 h, t1/2: 7.92 vs. 8.41 h. Points of estimation of the ratios test/reference were near to 100% and CI in ranges 80-125% were fulfilled for all tested parameters. Pharmacokinetic parameters values of CBS were: AUCinf: 1.96 vs. 1.84 ng x h/mL (test vs reference), AUCt: 1.91 vs. 1.81 ng x h/mL, Cmax, 1.44 vs. 1.52 ng/mL, tmax: 0.90 vs. 0.99 h, t1/2: 0.74 vs. 0.57 h. The parametric 90%-confidence interval (CI) was in the range of 80-125% for AUCt ratio and AUCinf ratio. The CI range of Cmax fulfilled the widened range of 75-133% (according to the study protocol). Unfortunately, the very high variability of pharmacokinetic parameters (over 50%) contributed to low power of the test. CONCLUSIONS: Measurement of CBS concentrations should not be a reliable one for the bioequivalence assessment, due to very low concentrations, very small and variable values of AUC and high intra-subject variability. Thus, bioequivalence evaluation should be based on CCA determination. In the presented study evaluation based on CCA unequivocally and with the proper power confirmed the bioequivalence between the investigated clopidogrel products.

NMR structure of biosynthetic engineered human insulin monomer B31(Lys)-B32(Arg) in water/acetonitrile solution. Comparison with the solution structure of native human insulin monomer.

A solution NMR-derived structure of a new long -acting, B31(Lys)-B32(Arg) (LysArg), engineered human insulin monomer, in H(2)O/CD(3)CN, 65/35 vol %, pH 3.6, is presented and compared with the available X-ray structure of a monomer that forms part of a hexamer (Smith, et al., Acta Crystallogr D 2003, 59, 474) and with NMR structure of human insulin in the same solvent (Bocian, et al., J Biomol NMR 2008, 40, 55-64). Detailed analysis using PFGSE NMR (Pulsed Field Gradient Spin Echo NMR) in dilution experiments and CSI analysis prove that the structure is monomeric in the concentration range 0.1-3 mM. The presence of long-range interstrand NOEs in a studied structure, relevant to the distances found in the crystal structure of the monomer, provides the evidence for conservation of the tertiary structure. Therefore the results suggest that this solvent system is a suitable medium for studying the native conformation of the protein, especially in situations (as found for insulins) in which extensive aggregation renders structure elucidations in water difficult or impossible. Starting from the structures calculated by the program CYANA, two different molecular dynamics (MD) simulated annealing refinement protocols were applied, either using the program AMBER in vacuum (AMBER_VC), or including a generalized Born solvent model (AMBER_GB). Here we present another independent evidence to the one presented recently by us (Bocian et al., J Biomol NMR 2008, 40, 55-64), that in water/acetonitrile solvent detailed structural and dynamic information can be obtained for important proteins that are naturally present as oligomers under native conditions.