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Introduction: Increasing resistance to beta-lactam antibiotics is an alarming development worldwide. Fecal carriership of TEM, SHV, CTX-M and CMY was studied in a community-dwelling population of middle-aged and elderly individuals., Patients and Methods: Feces was obtained from individuals of the Rotterdam Study. Carriership of the TEM, SHV, CTX-M and CMY genes was determined using real-time polymerase chain reaction (qPCR). Possible associations were investigated between carriership of these genes and several risk factors, such as the use of antimicrobial drugs, diabetes mellitus, protein pump inhibitor (PPI) use, travelling, the composition of the gut microbiota, and intake of certain foods., Results: The most prevalent gene was TEM (53.0%), followed by SHV (18.4%), CTX-M (5.4%) and CMY (3.6%). Use of penicillins with extended spectrum was associated with TEM carriership, whereas use of macrolides and lincosamides was associated with TEM and SHV carriership. Interestingly, use of PPIs was associated with a higher prevalence of carriership of TEM, SHV and CMY (TEM: odds ratio [OR] 1.34; 95% confidence interval [CI] 1.05-1.77; SHV: OR 2.17; 95%CI 1.55-2.87; CMY: OR 2.26; 95%CI 1.23-4.11). Furthermore, associations were found between the richness and composition of the gut microbiota and TEM and SHV carriership., Conclusions: The prevalence of carriership of TEM was substantial, but the prevalence of carriership of the extended-spectrum β-lactamase gene, CTX-M and the AmpC β-lactamase gene, CMY was relatively low in this community-dwelling, population-based cohort. The composition of the microbiota might play a role in the retention of resistance genes, but future studies are necessary to further elucidate this relationship., (Copyright © 2021. Published by Elsevier Ltd.)

SYN-004 (ribaxamase) is a β-lactamase designed to be orally administered concurrently with intravenous β-lactam antibiotics, including most penicillins and cephalosporins. Ribaxamase's anticipated mechanism of action is to degrade excess β-lactam antibiotic that is excreted into the small intestine. This enzymatic inactivation of excreted antibiotic is expected to protect the gut microbiome from disruption and thus prevent undesirable side effects, including secondary infections such as Clostridium difficile infections, as well as other antibiotic-associated diarrheas. In phase 1 clinical studies, ribaxamase was well tolerated compared to a placebo group and displayed negligible systemic absorption. The two phase 2a clinical studies described here were performed to confirm the mechanism of action of ribaxamase, degradation of β-lactam antibiotics in the human intestine, and were therefore conducted in subjects with functioning ileostomies to allow serial sampling of their intestinal chyme. Ribaxamase fully degraded ceftriaxone to below the level of quantitation in the intestines of all subjects in both studies. Coadministration of oral ribaxamase with intravenous ceftriaxone was also well tolerated, and the plasma pharmacokinetics of ceftriaxone were unchanged by ribaxamase administration. Since ribaxamase is formulated as a pH-dependent, delayed-release formulation, the activity of ribaxamase in the presence of the proton pump inhibitor esomeprazole was examined in the second study; coadministration of these drugs did not adversely affect ribaxamase's ability to degrade ceftriaxone excreted into the intestine. These studies have confirmed the in vivo mechanism of action of ribaxamase, degradation of β-lactam antibiotics in the human intestine (registered at ClinicalTrials.gov under NCT02419001 and NCT02473640)., (Copyright © 2017 Kokai-Kun et al.)

Background: SYN-004 is an orally administered β-lactamase enzyme, designed to be given concurrently with certain intravenous β-lactam antibiotics like cephalosporins. SYN-004 is intended to degrade residual antibiotics excreted into the intestine as a result of hepatobiliary excretion and to prevent the disruption of the gut microbiome by these excess antibiotics. Preserving the gut microbiome is expected to prevent secondary infections by pathogens like Clostridium difficile and protect against other antibiotic-associated diarrheas., Methods: Two, randomized, double blind, placebo-controlled Phase 1 clinical studies were conducted in normal healthy adult volunteers to assess the tolerability and systemic absorption of single and multiple doses of SYN-004. A single-ascending dose study investigated single oral doses of 75-750 mg SYN-004 and was conducted in 40 subjects (five cohorts of six active and two placebo subjects). A multiple-ascending dose study investigated doses of 75-300 mg SYN-004, administered every 6 h for 7 days and was conducted in 24 subjects (three cohorts of six active and two placebo subjects). The safety and tolerability of SYN-004 was assessed and serial plasma and serum samples were collected to assess the pharmacokinetics and potential immunogenicity of SYN-004., Results: Minimal and mild adverse events were reported in ~30 % of the subjects who received active drug and placebo and no antidrug antibodies were detected in any subject. Analysis of serial plasma samples demonstrated negligible systemic bioavailability of SYN-004 with most plasma concentrations being below the lower limit of quantitation (0.8 ng/mL) for the assay. SYN-004 was well tolerated in the 48 subjects who received active drug, and adverse events in those subjects were comparable to the 16 subjects who received placebo, up to the maximum doses administered in each study., Conclusion: SYN-004 was well tolerated up to a single oral dose of 750 mg and multiple doses of 300 mg every 6 h for 7 days. The pharmacokinetic results support that SYN-004 remained localized in the intestine.

SYN-004 is a first in class, recombinant β-lactamase that degrades β-lactam antibiotics and has been formulated to be administered orally to patients receiving intravenous β-lactam antibiotics including cephalosporins. SYN-004 is intended to degrade unmetabolized antibiotics excreted into the intestines and thus has the potential to protect the gut microbiome from disruption by these antibiotics. Protection of the gut microbiome is expected to protect against opportunistic enteric infections such as Clostridium difficile infection as well as antibiotic-associated diarrhea. In order to demonstrate that oral SYN-004 is safe for human clinical trials, 2 Good Laboratory Practice-compliant toxicity studies were conducted in Beagle dogs. In both studies, SYN-004 was administered orally 3 times per day up to the maximum tolerated dose of the formulation. In the first study, doses of SYN-004 administered over 28 days were safe and well tolerated in dogs with the no-observed-adverse-effect level at the high dose of 57 mg/kg/day. Systemic absorption of SYN-004 was minimal and sporadic and showed no accumulation during the study. In the second study, doses up to 57 mg/kg/day were administered to dogs in combination with an intravenous dose of ceftriaxone (300 mg/kg) given once per day for 14 days. Coadministration of oral SYN-004 with intravenous ceftriaxone was safe and well tolerated, with SYN-004 having no noticeable effect on the plasma pharmacokinetics of ceftriaxone. These preclinical studies demonstrate that SYN-004 is well tolerated and, when coadministered with ceftriaxone, does not interfere with its systemic pharmacokinetics. These data supported advancing SYN-004 into human clinical trials., (© The Author(s) 2015.)

For the determination of in vivo beta-lactamase activity, a high-performance liquid chromatographic (HPLC) method was established, and the pharmacokinetics of beta-lactamase after intravenous administration to the rats was analyzed using this standardized HPLC method. The plasma samples containing beta-lactamase were reacted with ampicillin (substrate) and further processed to make them fluorescent. The fluorescent compound of interest was separated using HPLC at room temperature using the excitation and the emission wavelengths of 410 nm and 475 nm, respectively. For the pharmacokinetic studies, 252 mU of beta-lactamase solution was administered to the rats through the tail vein injection (n = 6). The blood samples were withdrawn from the tail vein at different time points and analyzed by HPLC for beta-lactamase activity. For the HPLC method of beta-lactamase in plasma samples, the peak area showed a good correlation within the concentration ranges of 0.126-12.6 mU/mL (10-1000 ng/mL). The coefficients of variations were within 0.56-6.24, and the percentage recovery were within 102-107. After the intravenous injection, plasma concentration at the time zero (C(p0)) was 11.47 +/- 0.48 mU/mL, and no beta-lactamase was detected 24 h after the injection. The volume of distribution (V(d)) was 22 mL. An elimination half-life (t(1/2)) of 4.12 +/- 0.5 h and AUC of 79.4 +/- 12.9 mU.hr/mL were also calculated. The HPLC fluorimetric method was a very sensitive and reproducible method for the detection of beta-lactamase in plasma. The disposition of beta-lactamase after intravenous administration followed one-compartment and first-order kinetics.

Objectives: It is unclear to what extent pharmacokinetic data from bone outside the facial area can be transferred to the maxillofacial area. The aim of this study was to evaluate the penetration characteristics of piperacillin-tazobactam into human jaw and hip bone as a model for different bone characteristics., Materials and Methods: The drug was administered at the start of surgery in a single 15-min intravenous infusion dose (4g piperacillin & 0.5g tazobactam i.v.). Plasma and bone samples of ten patients were analyzed., Results: Mean concentration of piperacillin in plasma was 309microg/ml at 0.5h declining at 4h to 14microg/ml. The respective values for tazobactam were 34microg/ml declining to 2.8microg/ml. The piperacillin-tazobactam ratio dropped during the study interval from 0.5h: 9.2%+/-0.8 to 2h: 7.2%+/-1.1 and 4h: 4.9%+/-0.7. Mean bone concentrations of piperacillin and tazobactam were 9.0+/-11.6microg/g and 1.2+/-1.3microg/g, respectively. Mean penetration ratios for all bone samples were 15% (+/-17) for piperacillin and 13% (+/-14) for tazobactam without a difference between bone of different origin., Discussion: Piperacillin-tazobactam levels in jaw bone tissue after a single dose are sufficient to assure antibacterial activity of the combination and are above the minimal inhibitory concentrations of the most relevant pathogens in head and neck surgery. Our data suggests the use of piperacillin-tazobactam as an alternative for the therapy of severe infections of the head and neck.

To develop a self-nanoemulsifying drug delivery system (SNEDDS) for protein drugs, and particularly, to test the in vitro transport of beta-lactamase (BLM) by SNEDDS across the cell monolayer. Fluorescently labeled BLM (FITC-BLM), a model protein, formulated into 16 SNEDDS preparations through a solid dispersion technique were studied for transport across MDCK monolayer. All the SNEDDS nanoemulsions resulted in higher transport rate than the free solution. The transport rate by SNEDDS depends on the SNEDDS composition. SNEDDS NE-12-7 (oil: Lauroglycol FCC, surfactant: Cremophor EL and a cosurfactant: Transcutol HP) at the ratio of 5:4:3, rendered the highest transportation rate, 33% as compared to negligible transport by the free solution. FITC-BLM solution mixed with the surfactant and the cosurfactant of SNEDDS NE-12-7 or with blank SNEDDS NE-12-7 increased the transport only by 3.3 and 1.5 folds, respectively, compared to free solution alone. It was found that the monolayer integrity was not compromised in the presence of SNEDDS NE-12-7 or its surfactant/cosurfactant. The SNEDDS significantly increased the transport of FITC-BLM across MDCK monolayer in vitro. SNEDDS may be a potential effective delivery system for non-invasive protein drug delivery.

To use self-nanoemulsifying drug delivery system (SNEDDS) to deliver hydrophilic proteins orally. beta-Lactamase (BLM), a 29 kDa protein was used as a model protein, and formulated into the oil phase of a SNEDDS through solid dispersion technique. The oral absorption of BLM in rats when delivered by such a SNEDDS was investigated. Oral delivery of 4500 mU/kg of BLM in SNEDDS nanoemulsion resulted in the relative bioavailability of 6.34%, C(max) of 1.9 mU/ml and mean residence time of 12.12h which was 1.5-, 2.7- and 1.3-fold higher than that by free solution, respectively. Delivery of BLM in the aqueous phase of the nanoemulsion resulted in a PK profile similar to that by the free solution. BLM when loaded in oil phase of SNEDDS, can significantly enhance the oral bioavailability of BLM. SNEDDS has a great potential for oral protein delivery.

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae beta-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody-enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumor approach that uses a combination of a polymeric prodrug and polymer-enzyme conjugate to generate a cytotoxic drug rapidly and selectively at the tumor site. Previously we have shown that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound cathepsin B can release doxorubicin intratumorally from an HPMA copolymer conjugate PK1. Here we describe for the first time the synthesis and biological characterization of a PDEPT model combination that uses an HPMA-copolymer-methacryloyl-glycine-glycine-cephalosporin-doxorubicin (HPMA-co-MA-GG-C-Dox) as the macromolecular prodrug and an HPMA copolymer conjugate containing the nonmammalian enzyme beta-lactamase (HPMA-co-MA-GG-beta-L) as the activating component. HPMA-co-MA-GG-C-Dox had a molecular weight of approximately 31 600 Da and a C-Dox content of 5.85 wt %. Whereas free beta-L has a molecular weight of 45 kDa, the HPMA-co-MA-GG-beta-L conjugate had a molecular weight in the range of 75-150 kDa, and following purification no free enzyme was detectable. Against the cephalosporin C or HPMA-co-MA-GG-C-Dox substrates, the HPMA-co-MA-GG-beta-L conjugate retained 70% and 80% of its activity, respectively. In vivo (125)I-labeled HPMA-co-MA-GG-beta-L showed prolonged plasma concentration and greater tumor targeting than (125)I-labeled beta-L due to the enhanced permeability and retention (EPR) effect. Moreover, administration of HPMA-co-MA-GG-C-Dox iv to mice bearing sc B16F10 melanoma followed after 5 h by HPMA-co-MA-GG-beta-L led to release of free Dox. The PDEPT combination caused a significant decrease in tumor growth (T/C = 132%) whereas neither free Dox nor HPMA-co-MA-GG-C-Dox alone displayed activity. The PDEPT combination displayed no toxicity at the doses used, so further evaluation of this approach to establish the maximum tolerated dose (MTD) is recommended.

Considerable research has been aimed at improving the efficacy of chemotherapeutic agents for cancer therapy. A promising two-step approach that is designed to minimize systemic drug toxicity while maximizing activity in tumors employs monoclonal antibody-enzyme conjugates for the activation of anti-cancer prodrugs. A mathematical model based on the biology of human 3677 melanoma xenografts in nude mice is presented, analyzed, and numerically simulated to study the biodistribution, pharmacokinetics, and intratumoral localization properties of L49-beta-lactamase fusion proteins in solid tumor masses. The model predictions were compared with published experimental data and an excellent correlation was found to exist. Analytic expressions for the total concentration of conjugate in the tumor, the time at which the concentration is maximal, and the half life of conjugate in the tissue were derived. From these results, key parameters were isolated; and the effects of the tumor vasculature, binding kinetics, and administration schedule were investigated. The antibody-antigen dissociation ratio, the conjugate permeability, and the inter-capillary half distance within the tumor mass were found to strongly influence localization and retention in the tumor. The model was used to examine various dosing strategies in an attempt to determine which regimen would provide the best biodistribution results. The results of administering a uniform dose of conjugate via bolus injection, multiple injections, and continuous infusion were compared. The model predicts that when saturation of binding sites does not occur, dosing strategy has little effect on the amount of conjugate that localizes in the tumor.

Objective: Kinetics of piperacillin (pip), in combination with the beta-lactamase inhibitor tazobactam (taz) have been studied in volunteers and patients in relatively stable conditions. The fixed drug preparation appeared to have ideal pharmacokinetic properties if renal function was normal or slightly impaired, but no data are available for critically ill patients in anuric renal failure. This study should provide such data. PATIENTS, DESIGN: We studied the pharmacokinetics in nine patients with multiple organ failure, including anuric renal failure, treated with continuous veno-venous hemofiltration (CVVH). Patients received a standard schedule of 4 g pip and 0.5 g taz administered over 0.5 h intravenously, 8 hourly. During 2 consecutive days, the serum levels of both compounds were determined, and total clearance (CIT) was calculated from serum concentrations., Results: All nine patients completed day 1, and 8 completed day 2 of the protocol. On day 1, single-dose kinetics showed considerable spread, but pip/taz serum levels followed the pattern as expected, with a pip/taz concentration ratio of 20:1. On day 2, however, taz serum concentrations showed a relative increase as compared to pip, resulting in a change in the serum pip/taz concentration ratio to 10:1 on day 2. The CIT of pip was 2.52 +/- 1.38 l/h (t 1/2: 5.9 +/- 2.9 h), and CIT of taz 4.44 +/- 2.28 l/h (t 1/2: 8.1 +/- 3.7 h). The CIT and t 1/2 of pip and taz correlated highly significantly with clearance by CVVH. Despite a higher CIT, taz has a longer half-life, because of a higher volume of distribution., Conclusion: In CVVH dependent patients, pip/taz fixed drug preparations can be used initially, but the pip dosage should be increased relative to that of taz (or interval-adjusted) to prevent cumulation of taz, as compared to the active antimicrobial agent pip.

A cephalosporin derivative of doxorubicin (C-Dox) was evaluated as a prodrug for activation by mAb conjugates of the beta-lactamase from Enterobacter cloacae P99 (beta L; EC 3.5.2.6). The conjugates consisted of beta L and the F(ab') fragments of either of the mAbs L6, P1.17, or 96.5. L6 binds to antigens on a variety of carcinomas, including the two lung adenocarcinoma cell lines H2981 and H2987 used in this study. 96.5 binds to the melanoma-associated antigen p97, and P1.17 was used for the control conjugate. C-Dox was found to be less cytotoxic to three different tumor cell lines in vitro compared to the parent drug doxorubicin (Dox). Immunospecific activation took place when the cells were pretreated with beta L conjugates that could bind to antigens on the tumor cells. In vivo toxicity and pharmacokinetics studies in athymic female nu/nu mice revealed that C-Dox was at least 7-fold less toxic than Dox (on a molar basis), despite the fact that a > or = 320-fold greater area-under-the-curve (blood concentration versus time) of C-Dox compared to Dox was obtained 0-2 h after administration of the two agents. Pharmacokinetic studies at maximum tolerated doses in mice bearing xenografts of either H2981 or H2987 revealed that the intratumoral levels of Dox after treatment with L6-beta L in combination with C-Dox were higher than were obtained by either systemic treatment with Dox or a combination of P1.17-beta L and C-Dox. This finding suggested that the conversion of C-Dox to Dox was tumor specific and dependent on the presence of the targeted antigen. Furthermore, the best antitumor activity against both H2981 and H2987 tumors was obtained by treatment with L6-beta L and C-Dox compared to P1.17-beta L and C-Dox or Dox alone. Thus, higher levels of Dox corresponded to greater therapeutic effects in both of the tumor models studied.

beta-Lactamase from Enterobacter cloacae (beta L) was conjugated to the Fab'2 fragment of the monoclonal antibody L6 through a thioether linkage. Although L6-Fab'2-beta L was capable of activating the antitumor prodrug, 7-(phenylacetamido)cephalosporin mustard, it was impaired in its ability to bind to antigens on the H2981 human lung adenocarcinoma cell line. As a result, studies were undertaken to prepare conjugates with preserved binding activities. L6-Fab'-beta L and a dimeric conjugate consisting of two individual L6-Fab' units linked to a single beta L molecule (dimeric L6-beta L) were prepared by linking L6-Fab'-SH to maleimide-substituted beta L. Analysis of these conjugates by SDS-PAGE indicated that the linkage involved heavy-chain thiol groups on L6 that are most likely in the hinge region and are therefore removed from the antigen binding site of the antibody. Cell binding studies revealed that the monovalent conjugate L6-Fab'-beta L bound as well as L6-Fab'. Dimeric L6-beta L displayed slightly less binding than L6-Fab'2, but bound substantially better than L6-Fab'2-beta L. Lower concentrations of dimeric L6-beta L compared to L6-Fab'2-beta L were required to convert the prodrug 7-(phenylacetamido)-cephalosporin mustard into the cytotoxic drug phenylenediamine mustard. Localization studies were performed in nude mice with H2981 subcutaneous tumor xenografts. At 96 h post conjugate treatment, there was no significant difference in tumor concentration between L6-Fab'2-beta L and dimeric L6-beta L.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro activity of FK-037, 5-amino-2-[[(6R, 7R)-7-[[(Z)-2-(2-amino-4-thiazolyl)-2- methoxyimino) acetyl] amino]-2-carboxy-8-oxo-5-thia-1- azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-1-(2-hydroxyethyl)-1H-pyrazoli um hydroxide, inner salt, sulfate (1:1), a new parenteral cephem, was compared with those of cefepime, ceftazidime, imipenem, and ciprofloxacin. FK-037 inhibited methicillin-susceptible staphylocci at < or = 4 micrograms/ml. Of 98 isolates of homogenous methicillin-resistant Staphylococcus aureus, 55 (56.1%) were inhibited by 8 micrograms of FK-037 per ml, compared to 3.1% for cefepime. Imipenem was the most active beta-lactam tested against staphylococci. The MIC of FK-037 for 90% of the strains tested (MIC90) was 0.06 micrograms/ml for hemolytic streptococci, Streptococcus pneumoniae, viridans group streptococci, and Streptococcus bovis. The MIC90 for many of the members of the family Enterobacteriaceae was 1 microgram/ml, similar to that of cefepime and lower than those of ceftazidime and imipenem. The MIC90 for Klebsiella pneumoniae and Enterobacter cloacae was 8 micrograms/ml, similar to that for cefepime, but all isolates were inhibited by 2 micrograms of imipenem per ml. K. pneumoniae isolates with cefotaxime and ceftazidime MICs of > 32 micrograms/ml with Bush type 2b' beta-lactamases were inhibited by 4 micrograms of FK-037 per ml. E. cloacae, Citrobacter freundii, and S. aureus stably resistant to FK-037 could be selected by repeated transfer in the presence of FK-037. The FK-037 MIC90 for Pseudomonas aeruginosa was 4 microgram/ml, compared to 32 microgram/ml for cefepime and ceftazidime and 8 microgram/ml for imipenem. Xanthomonas maltophilia, Pseudomonas cepacia, Acinetobacter anitratus, and Bacteroides species were resistant to FK-037 (MIC, more than or equal 32 microgram/ml). MBCs were identical to or within twofold of the MICs except for a 32-fold greater MBC for P. aeruginosa. Inoculum size and acid environment did not lower the activity of FK-037. FK-037 was not appreciably hydrolyzed by Bush group 1, 2a, 2b, and 2e beta-lactamases but was hydrolyzed by 2b' and 2d enzymes at rates comparable to that of ceftazidime. Nonetheless, FK-037 inhibited bacteria possessing TEM-3, -5, and -7 and SHV -5 at less than or equal 8 microgram/ml. Overall, FK-037 has lower MICs against staphylococci and P. aeruginosa than the currently available iminomethoxy aminothiazolyl cephalosporins and has activity against members of the family Enterobacteriaceae comparable to that of cefepime.

Brief exposure of Pseudomonas aeruginosa to a temperature of 10 degrees C or lower caused a significant leakage of the periplasmic beta-lactamase into the medium. The extent of leakage increased as the incubation temperature was lowered to 4 degrees C and reached a maximum at 0 degrees C. Cells grown in the presence of beta-lactamase inducers were unsuitable for the permeability assay. It was found that the diffusion rates of beta-lactams through the outer membrane of P. aeruginosa were much lower than those previously reported, as assayed under refined conditions. The diffusion rates of beta-lactams in one of the mutants tested were an order of magnitude lower than those of the other strains, despite the fact that the outer membrane protein profile of the strain appeared to be indistinguishable from those of the others. These results suggest that beta-lactam antibiotics diffuse through the outer membrane of P. aeruginosa, at least partly, through a non-porin pathway.