In vitro activities of ceftobiprole combined with amikacin or levofloxacin against Pseudomonas aeruginosa: evidence of a synergistic effect using time-kill methodology

Ceftobiprole is an investigational intravenous broad-spectrum cephalosporin with in vitro activity against Gram-positive and Gram-negative pathogens, including meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Pseudomonas aeruginosa is a frequent nosocomial pathogen, increasingly associated with complicated skin and skin-structure infections. Combination antimicrobial therapy is recommended as empirical therapy for serious infections where P. aeruginosa is suspected. Therefore, in this study the interaction of ceftobiprole with two other antipseudomonal agents (amikacin and levofloxacin) was investigated. Time-kill studies were performed for each single agent and for the combination of ceftobiprole 4 mg/L with either amikacin or levofloxacin at 0.5×, 1× and 2× the minimum inhibitory concentration. Five clinical isolates of P. aeruginosa as well as the P. aeruginosa ATCC 27853 reference strain were tested at initial inocula of 5 × 10⁵ colony-forming units (CFU)/mL (low inoculum) or 5 × 10⁷ CFU/mL (high inoculum). Synergy was defined as a decrease of ≥2 log₁₀ CFU/mL with the combination compared with the most active single drug at 6 h and 24 h. At low inoculum with ceftobiprole as a single agent, viable counts were decreased by 1.5-2 log₁₀ at 6 h. Addition of either amikacin or levofloxacin resulted in synergistic bactericidal activity at 24 h. At high inoculum the combination of ceftobiprole with amikacin or levofloxacin demonstrated synergism in one of three and three of five strains, respectively. This study demonstrated that the combination of ceftobiprole at a clinically achievable concentration of 4 mg/L with amikacin or levofloxacin exhibited synergistic activity against P. aeruginosa. There was no evidence of antagonism for either combination.     

In vitro activities of the newly synthesised ER-2 against clinical isolates of Mycobacterium tuberculosis susceptible or resistant to antituberculosis drugs

Two hundred isolates of Mycobacterium tuberculosis were evaluated for their susceptibility to a newly synthesised quinolone derivative, ER-2, compared with ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin. ER-2 and moxifloxacin showed the greatest activity [MIC for 90% of strains tested (MIC₉₀) = 0.5 μg/mL], although levofloxacin and ciprofloxacin showed good activity with an MIC₉₀ of 1 μg/mL. More importantly, ER-2 showed excellent activity against M. tuberculosis H37Rv both in the lungs and spleen of mice, indicating the potential therapeutic value of ER-2 against M. tuberculosis.     

Clofazimine protects against Mycobacterium tuberculosis dissemination in the central nervous system following aerosol challenge in a murine model

Tuberculosis (TB) has been the scourge of the human race for many decades, claiming countless number of lives. This is further complicated by the ability of Mycobacterium tuberculosis to infect extrapulmonary sites, specifically the brain. These extrapulmonary forms of TB are difficult to treat owing to problems associated with drug delivery across the blood–brain barrier. Linezolid (LIN) and clofazimine (CFZ) are two of the more promising anti-TB drugs in recent times. In this study, BALB/c mice were aerosol-infected with M. tuberculosis H37Rv and were treated for 4 weeks with LIN [100?mg/kg body weight (BW)] or CFZ (100?mg/kg BW). Concurrently, it was investigated whether an aerosol TB infection would lead to dissemination of TB bacilli into the brain. Post-treatment brain and lung CFUs were determined together with serum, lung and brain drug concentrations. CFZ displayed a strong bactericidal effect in the lung, whilst LIN had a bacteriostatic effect. Mycobacterium tuberculosis appeared at 2 weeks post-infection in the untreated group (2.38?±?0.43 log₁₀ CFU) and more surprisingly at 3 weeks post-infection in the LIN-treated group (1.14?±?0.99 log₁₀ CFU). TB bacilli could not be detected in the brains of the CFZ-treated group. To the best of our knowledge, this is the first study showing the appearance of M. tuberculosis in the brain following a murine aerosol TB infection. This study may advocate the use of CFZ as prophylactic treatment to prevent the development of extrapulmonary TB of the central nervous system using a two-pronged approach.     

Activity of 2-(quinolin-4-yloxy)acetamides in Mycobacterium tuberculosis clinical isolates and identification of their molecular target by whole-genome sequencing

The 2-(quinolin-4-yloxy)acetamides (QOAs) have been reported to be promising molecules for tuberculosis treatment. Recent studies demonstrated their potent antimycobacterial activity, biological stability and synergism with rifampicin. The identification of the molecular target is an essential step towards the development of a novel drug candidate. Here, we report the target identification of the QOAs. We found that these compounds are active against Mycobacterium tuberculosis clinical isolates resistant to isoniazid, rifampicin, ethambutol, streptomycin and ethionamide. The initial evidence that DNA gyrase might be the target of QOAs, based on high minimum inhibitory concentration (MIC) values against ofloxacin-resistant clinical isolates and structural similarities with fluoroquinolones, was discarded by experiments performed with M. tuberculosis GyrA point mutant, DNA gyrase supercoiling inhibition assay and overexpression of DNA gyrase. We selected spontaneous mutants for our lead compound 21 and observed that these strains were also resistant to all QOA derivatives. The genomes of the spontaneous mutants were sequenced, and the results revealed a single mutation in qcrB gene (T313A), which indicates that the QOAs target the cytochrome bc₁ complex. The protein-compound interaction was further investigated by molecular docking. These findings reinforce the relevance of these compounds as promising candidates for the treatment of multidrug-resistant tuberculosis.     

Synthesis and pharmacological evaluation of a major metabolite of ameltolide, a potent anticonvulsant

The 4-aminobenzamides have provided several anticonvulsants that have been extensively investigated. Ameltolide, 4-amino-N-(2,6-dimethylphenyl)benzamide (compound 2,LY201116), is the most potent analogue studied to date. This drug is inactivated in vivo by metabolic N-acetylation and addition of a hydroxy moiety to one of the methyl substituents, resulting in compound 7,N-[4-[[[2-(hydroxymethyl)-6- methylphenyl] amino] carbonyl] phenyl] acetamide. This metabolite was prepared in five steps from a readily available starting material. Compound 7 and its nonacetylated analogue 6 were compared to ameltolide as anticonvulsants. After oral administration to mice, the MES ED50 values of ameltolide, 6, and 7 were 1.4, 10.9, and greater than 100 mg/kg, respectively, demonstrating that hydroxylation and acetylation dramatically decrease the anticonvulsant potency of ameltolide. This rank order of MES anticonvulsant potency was also seen after iv administration to mice, suggesting that these data reflect intrinsic pharmacological activities. After oral administration of 2.0 mg/kg of ameltolide to mice, parent drug, N-acetyl metabolite 3, and the hydroxy metabolite 7 were detected in plasma; the Cmax values were 572, 387, and 73 ng/mL, respectively. Compound 7 was the primary metabolite excreted in urine. These data indicate that 7 is a major metabolite of ameltolide, but does not contribute significantly to the pharmacological effects seen after administration of ameltolide to mice.     

Tiratricol,a thyroid hormone metabolite,has potent inhibitory activity against human dihydroorotate dehydrogenase

Human dihydroorotate dehydrogenase (hDHODH) is a promising drug target for many diseases including autoimmune diseases, cancer, and viral infection. To develop more novel and potent hDHODH inhibitors, we screened our in-house library of old drugs. We found that tiratricol (3,3′,5-triiodothyroacetic acid), a thyroid hormone metabolite, has potent hDHODH inhibitory activity (IC₅₀: 0.754 ± 0.126 μM), and its precursor tetrac (3,3′,5,5′-tetraiodothyroacetic acid) also shows a certain inhibitory activity against hDHODH (IC₅₀: 11.960 ± 1.453 μM). Enzyme kinetic analysis shows that tiratricol and tetrac are noncompetitive inhibitors versus CoQ₀, which is different from the positive control A771726. ThermoFMN assay, molecular docking and site-directed mutagenesis all indicate that tiratricol and tetrac interact with more key residues of hDHODH than A771726, especially some hydrophobic residues in Subsite 1. In conclusion, our experiment results indicate a potential new use for the old drug, tiratricol, and provide a novel chemical scaffold for the design of hDHODH inhibitors.     

In vitro evaluation of permeation,toxicity and effect of praziquantel-loaded solid lipid nanoparticles against Schistosoma mansoni as a strategy to improve efficacy of the schistosomiasis treatment

Solid lipid nanoparticles (SLN) are a promising drug delivery system for oral administration of poorly-water soluble drugs because of their capacity to increase the solubility of drug molecules when loaded in their lipid matrices, with the resulting improvement of the drug bioavailability. In the present work, we have developed praziquantel (PZQ)-loaded SLN and explored the biological applications of this system for intestinal permeation of PZQ. The effect in vitro on Schistosoma mansoni culture and the cytotoxicity in HepG2 line cell were also evaluated. The results showed a significant decrease in the intestinal absorption of PZQ loaded in SLN compared to free PZQ, suggesting that the SLN matrix could act as reservoir system. In culture of S. mansoni, we observed that PZQ-loaded SLN were more effective than free PZQ, leading the death of the parasites in less time. The result was proportional to doses of PZQ (25 and 50 μg mL⁻¹) and lipid concentration. Regarding cytotoxicity, the encapsulation of PZQ into SLN decreased the toxicity in HepG2 cells in comparison to the free PZQ. From the obtained results, PZQ-loaded SLN could be a new drug delivery system for the schistosomiasis treatment especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ.     

In vitro activity of azlocillin, metronidazole and its hydroxy metabolite against anaerobes. Bacteriostatic synergism studies

The in vitro inhibitory activity of azlocillin (Securopen), metronidazole (Cloni) and its hydroxy metabolite was determined against 27 gram-negative and 13 gram-positive species of anaerobes by means of agar dilution tests. The 63 anaerobic strains were also tested against the pairs azlocillin-metronidazole and azlocillin-hydroxy metabolite by means of agar dilution tests. Gram-negative species (Bacteroides spp., Fusobacterium spp. etc.) were inhibited by 0.125-256 micrograms/ml azlocillin, 0.01-4 micrograms/ml metronidazole and 0.01-4 micrograms/ml hydroxy metabolite. With gram-positive anaerobes (Clostridium spp., Peptococcaceae etc.) the MIC ranges were 0.125-4 micrograms/ml for azlocillin, 0.03-1 micrograms/ml for metronidazole and 0.125-2 micrograms/ml for the hydroxy metabolite. A synergistic effect was observed exclusively with gram-negative anaerobes (Bacteroides fragilis, B. thetaiotaomicron, B. disiens etc.). There were few instances of antagonism, likewise with gram-negative species. The preponderant combination effect against gram-positive anaerobes was addition. In view of the broad antiaerobic spectrum of azlocillin, the present in vitro findings do not preclude combined therapy with metronidazole in cases of anaerobic-aerobic poly-bacterial infections.     

In vitro pharmacokinetic/pharmacodynamic activity of NXL103 versus clindamycin and linezolid against clinical Staphylococcus aureus and Streptococcus pyogenes isolates

NXL103 (linopristin/flopristin, 30/70) is a novel oral streptogramin combination with activity against a large variety of multidrug-resistant Gram-positive pathogens. The objective of this study was to evaluate the in vitro activity of NXL103 in comparison with oral comparators (clindamycin and linezolid). Six clinical isolates [four meticillin-resistant Staphylococcus aureus (MRSA) and two Streptococcus pyogenes] were exposed for 48 h in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model at a starting inoculum of ca. 10⁶ colony-forming units (CFU)/mL. Antimicrobial simulations included NXL103 500 mg every 12 h, linezolid 600 mg every 12 h and clindamycin 450 mg every 6 h. Bactericidal and static effects were defined as ≥3 log₁₀ and <3 log₁₀ CFU/mL kill from the starting inoculum, respectively. Experiments were performed in duplicate to ensure reproducibility, and differences between regimens were evaluated by analysis of variance (ANOVA) with Tukey's post-hoc test. NXL103 exhibited lower minimum inhibitory concentrations than comparators, with values ≤0.06 mg/L for S. pyogenes and 0.125-0.25 mg/L for MRSA isolates. In the PK/PD model, NXL103 demonstrated significantly better activity than linezolid and clindamycin (P < 0.05), achieving sustained bactericidal activity within <2 h against S. pyogenes strains and between 7.3-32 h against MRSA isolates. In contrast, linezolid only exhibited a static effect, whereas clindamycin achieved 3 log₁₀ kill at 6 h against the unique clindamycin-susceptible S. pyogenes strain evaluated. In conclusion, at therapeutic concentrations NXL103 exhibits promising activity against both MRSA and S. pyogenes strains, including clindamycin-resistant organisms. Further in vitro and in vivo experiments are warranted to explore the therapeutic benefit of NXL103 for the treatment of Gram-positive skin and soft-tissue infections.     

In vitro effects of essential oils fromOstericum koreanum against antibiotic-resistantSalmonella spp.

The essential oil fraction of Ostericum koreanum was analyzed by GC-MS. Inhibiting activities of this oil and its main components were tested by the broth dilution assay and disk diffusion test against one antibiotic-susceptible and two resistant strains of Salmonella enteritidis and S. typhimurium, respectively. The GC-MS analysis revealed thirty-four compounds; the main components were alpha-pinene (41.12%), rho-cresol (17.99%) and 4-methylacetophenone (7.90%). The essential oil of O. koreanum and its main components were significantly effective against the tested antibiotic-susceptible strains as well as against the resistant strains of the two Salmonella species, with MICs (minimum inhibitory concentrations) ranging from 2 mg/mL to 16 mg/mL. The anti-Salmonella effects of the oils were dose-dependent on Müller-Hinton agar plates in this experiment. Additionally, checkerboard titer test results demonstrated significant combined effects of streptomycin and O. koreanum oil or cresol, one of the main components of this oil, against the two streptomycin resistant strains of S. typhimurium, with FICIs ranging from 0.12 to 0.37.     

In vitro evaluation of the inhibition and induction potential of olaparib,a potent poly(ADP-ribose) polymerase inhibitor,on cytochrome P450

1.?In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes.

2.?Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100?μM. However, olaparib (17–500?μM) inhibited CYP3A4/5 with an IC₅₀ of 119?μM. In time-dependent CYP inhibition assays, olaparib (10?μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2–200?μM) functioned as a time-dependent inhibitor of CYP3A4/5 (K_I, 72.2?μM and K_inact, 0.0675?min^?1). Assessment of the CYP induction potential of olaparib (0.061–44?μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed.

3.?Clinically significant drug–drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.     

In vitro evaluation of the antiviral activity of SP-303, an euphorbiaceae shrub extract,against a panel of respiratory viruses

The antiviral dn cytotoxic activity of SP-303, a naturally occurring polyphenolic polymer (average M.W. = 2,100 daltons) isolated from a Euphorbiaceae shrub, was evaluated in tissue culture. The compound exhibited selective antiviral activity (ratio IC₅₀/EC₅₀ ≥10) against parainfluenza virus type 1, respiratory syncytial virus, influenza A viruses, and influenza B viruses; marginal selective antiviral activity (IC₅₀/EC₅₀ ratio >2 to <10) against parinfluenza virus type 3; and no selective antiviral activity (IC₅₀/EC₅₀ ratio ≤1) against measles virus or adenovirus type 5. Hemagglutination and other studies suggested that SP-303 may at least partially inactivate virus by direct interaction with virus or host cell lipid membranes. © Wiley-Liss, Inc.     

哌拉西林／舒巴坦对临床分离菌的体外抗菌作用

目的 对哌拉西林／舒巴坦进行体外抗菌活性研究。方法 采用琼脂二倍稀释法测定最低抑菌浓度,采用肉汤稀释法测定最低杀菌浓度,并对其影响因素进行测定。结果 本品对肠杆菌科细菌、非发酵菌、葡萄球菌、粪肠球菌抗菌活性均低于亚胺培南,对不产酶流感嗜血杆菌和粘膜莫拉氏菌抗菌活性高于亚胺培南2－16倍。对肺炎链球菌和嗜麦芽黄单胞菌的抗菌活性同本次所研究的所有对照药。对临床常见5种致病菌（铜绿假单胞菌、大肠埃希氏菌、肺炎克雷伯氏菌、金葡球菌和粪肠球菌）的MIC和MBC非常接近,差异在0－2之间,表明本品为一杀菌剂。     

In vitro metabolite identification of ML3403, a 4-pyridinylimidazole-type p38 MAP kinase inhibitor by LC-Qq-TOF-MS and LC-SPE-cryo-NMR/MS

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4-[5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600?MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.     

In vitro metabolite identification of ML3403, a 4-pyridinylimidazole-type p38 MAP kinase inhibitor by LC-Qq-TOF-MS and LC-SPE-cryo-NMR/MS

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4- [5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl -(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600 MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.     

In vitro antibacterial activity of a new cephalosporin, FR295389, against IMP-type metallo-beta-lactamase-producers

FR295389 is a novel dihydroimidazopyrazolium cephalosporin. Although all previously reported cephalosporins had been ineffective against metallo-beta-lactamase (MBL)-producers, FR295389 showed moderate activity against these strains. The MIC values of FR295389 at which 50% and 90% of 21 clinical isolates of IMP-type MBL-producing Pseudomonas aeruginosa were inhibited were 8.0 and 32 mug/ml, respectively. The kinetic study of IMP-1 MBL showed that the K(m) and the k(cat) values against FR295389 were over 20-fold-higher and 12-fold-lower than those against ceftazidime, respectively, suggesting that FR295389 is a poor substrate for IMP-type MBL. This is the first report of a cephalosporin with activity against IMP-type MBL-producers.     

In vitro characterization,ADME analysis,and histological and toxicological evaluation of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains

BackgroundCandida species are one of the most common causes of nosocomial bloodstream infections among the opportunistic fungi. Extensive use of antifungal agents, most of which were launched on the market more than 20 years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. We recently described a novel class of antifungal macrocyclic compounds with an amidinourea moiety that is highly active against azole-resistant Candida strains.ObjectiveA compound from this family, BM1, was investigated in terms of in vitro activity against various Candida species, including C. auris isolates, interaction with the ABC transporter, CDR6, and in vivo distribution and safety.MethodsIn vitro assays (CYP inhibition, microsomal stability, permeability, spot assays) were used to collect chemical and biological data; animal models (rat) paired with LC-MS analysis were utilised to evaluate in vivo toxicology, pharmacokinetics, and distribution.ResultsThe current research shows BM1 has a low in vivo toxicity profile, affinity for the renal system in rats, and good absorption, distribution, metabolism, and excretion (ADME). BM1 also has potent activity against azole-resistant fungal strains, including C. auris isolates and CDR6-overexpressing strains.ConclusionsThe results confirmed low minimum inhibitory concentrations (MICs) against several Candida species, including preliminary data vs. C. auris. BM1 has good ADME and biochemical characteristics, is suitable and safe for daily administration and is particularly indicated for renal infections. These data indicate BM1 and its derivatives form a novel, promising antifungal class.     

In vitro and in vivo safety assessment of edible blue-green algae, Nostoc commune var. sphaeroides Kützing and Spirulina plantensis

Blue-green algae (BGA) have been consumed as food and herbal medicine for centuries. However, safety for their consumption has not been well investigated. This study was undertaken to evaluate in vitro and in vivo toxicity of cultivated Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP). Neither NO nor SP contained detectable levels of microcystin (MC)-LA, MC-RR, MC-LW and MC-LR by LC/MS/MS. Cell viability remained ∼70-80% when HepG2 cells were incubated with 0-500 μg/ml of hexane, chloroform, methanol and water-extractable fractions of NO and SP. Four-week-old male and female C57BL/6J mice were fed an AIN-93G/M diet supplemented with 0%, 2.5% or 5% of NO and SP (wt/wt) for 6 months. For both genders, BGA-rich diets did not induce noticeable abnormality in weight gain and plasma alanine aminotransferase (ALT) and aspartate aminotransferase concentrations except a significant increase in plasma ALT levels by 2.5% NO supplementation in male mice at 6 month. Histopathological analysis of livers, however, indicated that BGA did not cause significant liver damage compared with controls. In conclusion, our results suggest that NO and SP are free of MC and the long-term dietary supplementation of up to 5% of the BGA may be consumed without evident toxic side-effects.