Dicloxacillin-warfarin drug-drug interaction-A register-based study and in vitro investigations in 3D spheroid primary human hepatocytes.

AIMS: Dicloxacillin is used to treat staphylococcal infections and we have previously shown that dicloxacillin is an inducer of cytochrome P450 enzymes (CYPs). Here, we employed a translational approach to investigate the effect of a treatment with dicloxacillin on warfarin efficacy in Danish registries. Furthermore, we assessed dicloxacillin as an inducer of CYPs in vitro. METHODS: We conducted a register-based study and analysed international normalized ratio (INR) levels in chronic warfarin users before and after short- and long-term use of dicloxacillin (n = 1023) and flucloxacillin (n = 123). Induction of CYPs were investigated in a novel liver model of 3D spheroid primary human hepatocytes at the level of mRNA, and protein and enzyme activity. RESULTS: Short- and long-term dicloxacillin treatments decreased INR levels by -0.65 (95% confidence interval [CI]: -0.57 to -0.74) and -0.76 (95% CI: -0.50 to -1.02), respectively. More than 90% of individuals experienced subtherapeutic INR levels (below 2) after long-term dicloxacillin treatment. Flucloxacillin decreased INR levels by -0.37 (95% CI: -0.14 to -0.60). In 3D spheroid primary human hepatocytes, the maximal induction of CYP3A4 mRNA, protein and enzyme activity by dicloxacillin were 4.9-, 2.9- and 2.4-fold, respectively. Dicloxacillin also induced CYP2C9 mRNA by 1.7-fold. CONCLUSION: Dicloxacillin induces CYPs and reduces the clinical efficacy of warfarin in patients. This effect is substantially exacerbated during long-term treatment with dicloxacillin. The in vitro results corroborated this drug-drug interaction and correlated to the clinical findings. Caution is warranted for warfarin patients that initiate dicloxacillin or flucloxacillin, especially for a long-term treatment of endocarditis.

Dicloxacillin induces CYP2C19, CYP2C9 and CYP3A4 in vivo and in vitro.

AIM: The aim of this study was to study potential cytochrome P450 (CYP) induction by dicloxacillin. METHODS: We performed an open-label, randomized, two-phase, five-drug clinical pharmacokinetic cocktail crossover study in 12 healthy men with and without pretreatment with 1 g dicloxacillin three times daily for 10 days. Plasma and urine were collected over 24 h and the concentration of all five drugs and their primary metabolites was determined using a liquid chromatography coupled to triple quadrupole mass spectrometry method. Cryopreserved primary human hepatocytes were exposed to dicloxacillin for 48 h and changes in gene expression and the activity of CYP3A4, CYP2C9, CYP2B6 and CYP1A2 were investigated. The activation of nuclear receptors by dicloxacillin was assessed using luciferase assays. RESULTS: A total of 10 days of treatment with dicloxacillin resulted in a clinically and statistically significant reduction in the area under the plasma concentration-time curve from 0 to 24 h for omeprazole (CYP2C19) {geometric mean ratio [GMR] [95% confidence interval (CI)]: 0.33 [0.24, 0.45]}, tolbutamide (CYP2C9) [GMR (95% CI): 0.73 (0.65, 0.81)] and midazolam (CYP3A4) [GMR (95% CI): 0.54 (0.41, 0.72)]. Additionally, other relevant pharmacokinetic parameters were affected, indicating the induction of CYP2C- and CYP3A4-mediated metabolism by dicloxacillin. Investigations in primary hepatocytes showed a statistically significant dose-dependent increase in CYP expression and activity by dicloxacillin, caused by activation of the pregnane X receptor. CONCLUSIONS: Dicloxacillin is an inducer of CYP2C- and CYP3A-mediated drug metabolism, and we recommend caution when prescribing dicloxacillin to users of drugs with a narrow therapeutic window.

Antibiotic selection of Escherichia coli sequence type 131 in a mouse intestinal colonization model.

The ability of different antibiotics to select for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli remains a topic of discussion. In a mouse intestinal colonization model, we evaluated the selective abilities of nine common antimicrobials (cefotaxime, cefuroxime, dicloxacillin, clindamycin, penicillin, ampicillin, meropenem, ciprofloxacin, and amdinocillin) against a CTX-M-15-producing E. coli sequence type 131 (ST131) isolate with a fluoroquinolone resistance phenotype. Mice (8 per group) were orogastrically administered 0.25 ml saline with 10(8) CFU/ml E. coli ST131. On that same day, antibiotic treatment was initiated and given subcutaneously once a day for three consecutive days. CFU of E. coli ST131, Bacteroides, and Gram-positive aerobic bacteria in fecal samples were studied, with intervals, until day 8. Bacteroides was used as an indicator organism for impact on the Gram-negative anaerobic population. For three antibiotics, prolonged colonization was investigated with additional fecal CFU counts determined on days 10 and 14 (cefotaxime, dicloxacillin, and clindamycin). Three antibiotics (cefotaxime, dicloxacillin, and clindamycin) promoted overgrowth of E. coli ST131 (P < 0.05). Of these, only clindamycin suppressed Bacteroides, while the remaining two antibiotics had no negative impact on Bacteroides or Gram-positive organisms. Only clindamycin treatment resulted in prolonged colonization. The remaining six antibiotics, including ciprofloxacin, did not promote overgrowth of E. coli ST131 (P > 0.95), nor did they suppress Bacteroides or Gram-positive organisms. The results showed that antimicrobials both with and without an impact on Gram-negative anaerobes can select for ESBL-producing E. coli, indicating that not only Gram-negative anaerobes have a role in upholding colonization resistance. Other, so-far-unknown bacterial populations must be of importance for preventing colonization by incoming E. coli.

Relative efficacy of cefuroxime versus dicloxacillin as definitive antimicrobial therapy in methicillin-susceptible Staphylococcus aureus bacteraemia: a propensity-score adjusted retrospective cohort study.

OBJECTIVES: The objective of the present study was to compare the efficacy of cefuroxime with that of dicloxacillin as definitive antimicrobial therapy in methicillin-susceptible Staphylococcus aureus bacteraemia (MS-SAB) using a Danish bacteraemia database, information on the indication for antimicrobial therapy, multivariate adjustment and propensity score (PS) matching. METHODS: This was a retrospective cohort study. MS-SAB cases from 1 January 2006 to 31 December 2008 were included from a total of seven hospitals in the greater Copenhagen area and seven hospitals in the North Denmark Region. Information including demographics, antimicrobial therapy and clinical condition was obtained. The physician's note detailing the indication for starting empirical antimicrobial therapy was given special attention. Hazard ratios (HRs) and 95% CIs for 30 day and 90 day mortality were calculated using PS-adjusted Cox proportional hazards regression analyses. In addition, PS matching was performed. RESULTS: A total of 691 patients with MS-SAB received either dicloxacillin (n = 368) or cefuroxime (n = 323) as definitive antimicrobial therapy. Twenty-eight different indications for empirical antimicrobial therapy were identified and grouped into eight categories. There was no statistically significant difference in 30 day mortality between the two groups (HR 1.02, 95% CI 0.68-1.52). Definitive antimicrobial therapy with cefuroxime was associated with increased 90 day mortality in a PS-adjusted multivariate analysis (HR 1.43, 95% CI 1.03-1.98) and in the PS matching (OR 1.65, 95% CI 1.06-2.56). Antimicrobial therapy for an indication of 'severe infection' was independently associated with 90 day mortality (HR 1.97, 95% CI 1.19-3.28). CONCLUSIONS: Definitive antimicrobial therapy with cefuroxime was associated with significantly higher 90 day mortality than was dicloxacillin therapy in patients with MS-SAB.

Hollow-fiber pharmacodynamic studies and mathematical modeling to predict the efficacy of amoxicillin for anthrax postexposure prophylaxis in pregnant women and children.

Amoxicillin is considered an option for postexposure prophylaxis of Bacillus anthracis in pregnant and postpartum women who are breastfeeding and in children because of the potential toxicities of ciprofloxacin and doxycycline to the fetus and child. The amoxicillin regimen that effectively kills B. anthracis and prevents resistance is unknown. Fourteen-day dose range and dose fractionation studies were conducted in in vitro pharmacodynamic models to identify the exposure intensity and pharmacodynamic index of amoxicillin that are linked with optimized killing of B. anthracis and resistance prevention. Studies with dicloxacillin, a drug resistant to B. anthracis beta-lactamase, evaluated the role of beta-lactamase production in the pharmacodynamic indices for B. anthracis killing and resistance prevention. Dose fractionation studies showed that trough/MIC and not time above MIC was the index for amoxicillin that was linked to successful outcome through resistance prevention. Failure of amoxicillin regimens was due to inducible or stable high level expression of beta-lactamases. Studies with dicloxacillin demonstrated that a time above MIC of ≥94% was linked with treatment success when B. anthracis beta-lactamase activity was negated. Recursive partitioning analysis showed that amoxicillin regimens that produced peak concentrations of <10.99 µg/ml and troughs of >1.75 µg/ml provided a 100% success rate. Other amoxicillin peak and trough values produced success rates of 28 to 67%. For postpartum and pregnant women and children, Monte Carlo simulations predicted success rates for amoxicillin at 1 g every 8 h (q8h) of 53, 33, and 44% (30 mg/kg q8h), respectively. We conclude that amoxicillin is suboptimal for postexposure prophylaxis of B. anthracis in pregnant and postpartum women and in children.

Activity of oxacillin versus that of vancomycin against oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates evaluated by population analyses, time-kill assays, and a murine thigh infection model.

We compared the activity of dicloxacillin with that of vancomycin against 15 oxacillin-susceptible, methicillin-resistant Staphylococcus aureus (OS-MRSA) clinical isolates. By population analyses, we found that 6 OS-MRSA isolates were able to grow in the presence of up to 8 µg/ml dicloxacillin and 9 isolates were able to grow in 12 to >32 µg/ml dicloxacillin; all isolates grew in up to 2 µg/ml vancomycin. Both drugs exhibited similar bactericidal activities. In experimental infections, the therapeutic efficacy of dicloxacillin was significant (P < 0.05 versus untreated controls) in 10 OS-MRSA isolates and vancomycin was effective (P < 0.05) against 12 isolates; dicloxacillin had an efficacy that was comparable to that of vancomycin (P > 0.05) in 8 isolates. The favorable response to dicloxacillin treatment might suggest that antistaphylococcal penicillins could be used against OS-MRSA infections.

Risk factors for Clostridium difficile toxin-positive diarrhea: a population-based prospective case-control study.

Increased incidence and severity of Clostridium difficile infections (CDIs) is of major concern. However, by minimizing known risk factors, the incidence can be decreased. The aim of this investigation was to calculate the incidence and assess risk factors for CDI in our population. A 1-year prospective population-based nationwide study in Iceland of CDIs was carried out. For risk factor evaluation, each case was matched with two age- and sex-matched controls that tested negative for C. difficile toxin. A total of 128 CDIs were identified. The crude incidence was 54 cases annually per 100,000 population >18 years of age. Incidence increased exponentially with older age (319 per 100,000 population >86 years of age). Community-acquired origin was 27 %. Independent risk factors included: dicloxacillin (odds ratio [OR]: 7.55, 95 % confidence interval [CI]: 1.89-30.1), clindamycin (OR: 6.09, 95 % CI: 2.23-16.61), ceftriaxone (OR: 4.28, 95 % CI: 1.59-11.49), living in a retirement home (OR: 3.9, 95 % CI: 1.69-9.16), recent hospital stay (OR: 2.3, 95 % CI: 1.37-3.87). Proton pump inhibitors (PPIs) were used by 60/111 (54 %) versus 91/222 (41 %) (p = 0.026) and ciprofloxacin 19/111 (17 %) versus 19/222 (9 %) (p = 0.027) for cases and controls, respectively. In all, 75 % of primary CDIs treated with metronidazole recovered from one course of treatment. CDI was mostly found among elderly patients. The most commonly identified risk factors were broad-spectrum antibiotics and recent contact with health care institutions. PPI use was significantly more prevalent among CDI patients.

Intra- and extracellular activities of dicloxacillin and linezolid against a clinical Staphylococcus aureus strain with a small-colony-variant phenotype in an in vitro model of THP-1 macrophages and an in vivo mouse peritonitis model.

The small-colony-variant (SCV) phenotype of Staphylococcus aureus has been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT) S. aureus strain and its counterpart with an SCV phenotype both in vitro and in vivo, using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in S. aureus pathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log(10) in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present.

The alpha-synuclein 5'untranslated region targeted translation blockers: anti-alpha synuclein efficacy of cardiac glycosides and Posiphen.

Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson's disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5'untranslated region (5'UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5'UTR, whereas, by contrast, the H-ferritin 5'UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5'UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5'UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 µM IC(50)) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

Intra- and extracellular activities of dicloxacillin against Staphylococcus aureus in vivo and in vitro.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT(MIC)]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT(MIC) is the index that is the most predictive of the outcome of infection both intra- and extracellularly.

Ischemic Stroke and Systemic Embolism in Warfarin Users With Atrial Fibrillation or Heart Valve Replacement Exposed to Dicloxacillin or Flucloxacillin.

The antibiotics dicloxacillin and flucloxacillin induce cytochrome P450-dependent metabolism of warfarin. We explored the influence of these drug-drug interactions on the clinical effectiveness of warfarin therapy due to atrial fibrillation or heart valve replacement. Using the population-based Danish registers, we performed a propensity-score matched cohort study including around 50,000 episodes of dicloxacillin/flucloxacillin matched to phenoxymethylpenicillin and to no antibiotic, respectively. We estimated hazard ratios (HRs) with 95% confidence intervals (CIs) by comparing 21-day (days 7-28) risks of ischemic stroke/systemic embolism (SE) following initiation of each exposure. When compared with phenoxymethylpenicillin, dicloxacillin/flucloxacillin was associated with an HR of ischemic stroke/SE of 2.09 (95% CI 1.51-2.90; strongest for dicloxacillin (HR 2.17; 95% CI 1.56-3.02)). Use of an untreated comparator strengthened the association (HR 2.84; 95% CI 1.97-4.09). Dicloxacillin should be used with caution in patients receiving warfarin. This may also apply to flucloxacillin; however, more data on the risks associated with flucloxacillin exposure during warfarin therapy are needed.

Intracellular activity of antibiotics against Staphylococcus aureus in a mouse peritonitis model.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log(10) number of CFU/ml (Delta log(10) CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Delta log(10) CFU/ml) > cefuroxime (3.56 Delta log(10) CFU/ml) > rifampin (1.86 Delta log(10) CFU/ml) > gentamicin (0.61 Delta log(10) CFU/ml) > azithromycin (0.21 Delta log(10) CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.

Effects of Dicloxacillin on Warfarin Dose in Patients With a Left Ventricular Assist Device.

Patients with a durable, continuous flow left ventricular assist device (CF-LVAD) require anticoagulation with warfarin to prevent thromboembolic events. Driveline infections (DLIs) are a common CF-LVAD complication. A common pathogen implicated in DLI is oxacillin-sensitive Staphylococcus aureus (OSSA), which is effectively treated by oral dicloxacillin. Previous published experiences have observed a significant drug interaction between dicloxacillin and warfarin resulting in decreased international normalized ratio (INR) and increased warfarin dosing requirements. We sought to analyze the effect of dicloxacillin on INR and warfarin dose when used for DLI in our CF-LVAD program. Five of 106 patients having received an CF-LVAD at our institution met the inclusion criteria for this case series. These patients required a mean 51.8% (standard deviation of 29.8%) weekly warfarin dose increase to restore INR to the therapeutic range after the addition of dicloxacillin. Three of the five patients subsequently had their dicloxacillin discontinued, with a mean decrease in weekly warfarin dose of 30.6% (standard deviation of 19.1%). In our experience, when coalesced with prior published reports, an empiric warfarin dose increase of 25% to 33% is reasonable upon initiation of dicloxacillin and an empiric warfarin dose reduction of 10% to 15% is recommended upon discontinuation of dicloxacillin. Close INR follow-up is warranted during and after dicloxacillin treatment.

In vitro and in vivo evaluations of oxacillin efficiency against mecA-positive oxacillin-susceptible Staphylococcus aureus.

Community-type Staphylococcus aureus strains that are positive for mecA and PBP2a but appear phenotypically susceptible to oxacillin are increasingly reported worldwide. Four S. aureus clinical isolates carrying the mecA gene with oxacillin MICs of <2 microg/ml were tested for oxacillin efficiency by population analyses and experimental thigh infections. These isolates harbored staphylococcal cassette chromosome mec type IV and belonged to two genotypes. Two of the four isolates were found by population analysis to be truly oxacillin susceptible. All four isolates exhibited significant reductions in the numbers of colonies grown after dicloxacillin treatment of experimental thigh infections, as also did a mecA-negative S. aureus control strain. These observations indicate that some of the phenotypically oxacillin susceptible mecA-positive Staphylococcus aureus isolates may be at least partially responsive to oxacillin.

Use of Dicloxacillin and Risk of Pregnancy among Users of Oral Contraceptives.

The antibiotic dicloxacillin has been shown to induce drug-metabolizing CYP enzymes to a clinically relevant extent. In this study, we investigated whether the use of dicloxacillin confers an increased risk of unwanted pregnancy among oral contraceptive users. The study population comprised Danish women falling pregnant (1997-2015) during oral contraceptive use, defined as having filled a prescription for an oral contraceptive within 120 days both before and after the estimated date of conception. Data were analysed using a case-crossover approach. For each woman, we assessed the use of dicloxacillin preceding the date of conception and during 10 previous control periods and estimated the odds ratio for such unintended pregnancies associated with the use of dicloxacillin. Among 364 women using dicloxacillin prior to conception, 40 (11%) were exposed to dicloxacillin at the time of conception, yielding an odds ratio (OR) associating use of dicloxacillin to unintended pregnancy of 1.18 (95% CI 0.84-1.65). Supplementary and sensitivity analyses generally returned similar estimates, except for a slightly increased risk among users of progestogen-only oral contraceptives (OR 1.83, 95% CI 0.63-5.34). Analysis of other antibiotics as negative controls yielded results close to unity (ORs ranging from 0.83 to 1.13). In conclusion, our study found no evidence for an increased risk of oral contraceptive failure when using dicloxacillin. However, acknowledging study limitations, we suggest the use of supplementary barrier methods during treatment with dicloxacillin, until our findings are confirmed in further studies.

Synthesis and in vitro Antioxidant Activity Study of Some Novel Substituted Piperazinyl Flavone Compounds.

BACKGROUND: A new series of 13 piperazinyl flavone derivatives has been synthesized and examined for their in vitro antiradical and antioxidant activities in response to the pharmacy industry's increasing demand for new non-toxic anti-inflammatory and anticancer drugs. METHOD: Their antioxidant activity was evaluated by the reactive oxygen species (ROS) scavenging assays, 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH•) and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS+•) scavenging assays, and the ferric reducing antioxidant potency (TAC) method, and was compared to known positive controls, herbal infusions, and penicillins. Chemiluminescence, spectrophotometry, electron spin resonance (ESR) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as the spin trap were the measurement techniques. RESULT: It was seen that synthesized compounds have a wide spectrum of antioxidant property. Some of the test compounds proved to be extremely efficient scavengers of H2O2 exhibiting, in some cases, EC50 of about 2 µM. The values of antioxidant status (TAS) were in the range of 49 ± 3.9 to 1283 ± 51.3 µM TE/g (TE = Trolox equivalent) and were lower than that of butylated hydroxytoluene (BHT) (1304 ± 43.2 µM TE/g) and green tea (1356 ± 40.0 µM TE/g), but for several synthesized compounds, they were higher than chamomille infusion and penicillins. Ferric reducing antioxidant powers (TAC) for the piperazinyl flavone derivatives were in the range 7 ± 0.5 to 104 ± 0.6 µM TE/g and were weaker than that of BHT (217 ± 5.3 µM TR/g ). CONCLUSION: Carboxylic or hydroxamic acid substituted piperazinyl flavones are potentially active as antioxidants, thus may be suggested as pharmacologically interesting ones.

Molecular mechanisms of thioridazine resistance in Staphylococcus aureus.

Staphylococcus aureus has developed resistance towards the most commonly used anti-staphylococcal antibiotics. Therefore, there is an urgent need to find new treatment opportunities. A new approach relies on the use of helper compounds, which are able to potentiate the effect of antibiotics. A well-studied helper compound is thioridazine, which potentiates the effect of the ß-lactam antibiotic dicloxacillin against Methicillin-resistant Staphylococcus aureus (MRSA). In order to identify thioridazine's mechanism of action and how it potentiates the effect of dicloxacillin, we generated thioridazine resistant strains of MRSA USA300 by serial passage experiments. Selected strains were whole-genome sequenced to find mutations causing thioridazine resistance. Genes observed to be mutated were attempted deleted in MRSA USA300. The cls gene encoding a cardiolipin synthase important for synthesis of the membrane lipid cardiolipin was found to be mutated in thioridazine resistant strains. Deletion of this gene resulted in a two-fold increased Minimum inhibitory concentrations (MIC) value for thioridazine compared to the wild type and decreased susceptibility similar to the thioridazine resistant strains. Since cardiolipin likely plays a role in resistance towards thioridazine, it might also be important for the mechanism of action behind the potentiating effect of thioridazine. TDZ is known to intercalate into the membrane and we show here that TDZ can depolarize the plasma membrane. However, our results indicate that the membrane potential reducing effect of TDZ is independent of the resistance mechanism.

Systemic thioridazine in combination with dicloxacillin against early aortic graft infections caused by Staphylococcus aureus in a porcine model: In vivo results do not reproduce the in vitro synergistic activity.

INTRODUCTION: Conservative treatment solutions against aortic prosthetic vascular graft infection (APVGI) for inoperable patients are limited. The combination of antibiotics with antibacterial helper compounds, such as the neuroleptic drug thioridazine (TDZ), should be explored. AIM: To investigate the efficacy of conservative systemic treatment with dicloxacillin (DCX) in combination with TDZ (DCX+TDZ), compared to DCX alone, against early APVGI caused by methicillin-sensitive Staphylococcus aureus (MSSA) in a porcine model. METHODS: The synergism of DCX+TDZ against MSSA was initially assessed in vitro by viability assay. Thereafter, thirty-two pigs had polyester grafts implanted in the infrarenal aorta, followed by inoculation with 106 CFU of MSSA, and were randomly administered oral systemic treatment with either 1) DCX or 2) DCX+TDZ. Treatment was initiated one week postoperatively and continued for a further 21 days. Weight, temperature, and blood samples were collected at predefined intervals. By termination, bacterial quantities from the graft surface, graft material, and perigraft tissue were obtained. RESULTS: Despite in vitro synergism, the porcine experiment revealed no statistical differences for bacteriological endpoints between the two treatment groups, and none of the treatments eradicated the APVGI. Accordingly, the mixed model analyses of weight, temperature, and blood samples revealed no statistical differences. CONCLUSION: Conservative systemic treatment with DCX+TDZ did not reproduce in vitro results against APVGI caused by MSSA in this porcine model. However, unexpected severe adverse effects related to the planned dose of TDZ required a considerable reduction to the administered dose of TDZ, which may have compromised the results.

In vitro activity effects of combinations of cephalothin, dicloxacillin, imipenem, vancomycin and amikacin against methicillin-resistant Staphylococcus spp. strains.

BACKGROUND: combinations of drugs has been proposed as an alternative for oxacillin-resistant staphylococci infections, however, limited information about in vitro combinations are available for multi-resistant strains. The objective of this study was to describe the interaction of beta-lactams in combination with vancomycin or amikacin against 26 oxacillin and amikacin-resistant nosocomial Staphylococcus spp. isolates. METHODS: activity of dicloxacillin plus amikacin, cephalothin plus amikacin, cephalothin plus vancomycin, imipenem plus vancomycin and vancomycin plus amikacin was evaluated by checkerboard synergy tests and the fractional inhibitory concentration index (FIC) was calculated. RESULTS: dicloxacillin plus amikacin, and cephalothin plus amikacin were synergistic or partially synergistic in 84.6% and 100% respectively. For nearly half of the isolates the mean concentrations of dicloxacillin, cephalothin and amikacin at which FIC indexes were calculated were achievable therapeutically. Vancomycin plus amikacin had synergistic effect only against two isolates, and partially synergistic in 38.6%. For the combinations vancomycin plus cephalothin and vancomycin plus imipenem the effect was additive in 76.9% and 80.7% respectively. CONCLUSION: in this study the checkerboard analysis showed that amikacin in combination with cephalothin or dicloxacillin was synergistic against most of the resistant strains of S. aureus and coagulase-negative Staphylococcus. Vancomycin in combination with a beta-lactam (cephalothin or imipenem) showed additivity. An indifferent effect predominated for the combination vancomycin plus amikacin. Even though a synergistic effect is expected when using a beta-lactam plus amikacin combination, it is possible that the effect cannot be clinically achievable. Careful selection of antimicrobial combinations and initial MICs are mandatory for future evaluations.