Analysis of the molecular association of rifampicin with hydroxypropyl-²-cyclodextrin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Horseradish peroxidase-catalyzed oxidation of rifampicin: Reaction rate enhancement by co-oxidation with anti-inflammatory drugs

The tuberculostatic drug rifampicin has been described as a scavenger of reactive species. Additionally, the recent demonstration that oral therapy with a complex of rifampicin and horseradish peroxidase (HRP) was more effective than rifampicin alone, in an animal model of experimental leprosy, suggested the importance of redox reactions involving rifampicin and their relevance to the mechanism of action. Hence, we studied the oxidation of rifampicin catalyzed by HRP, since this enzyme may represent the prototype of peroxidation-mediated reactions. We found that the antibiotic is efficiently oxidized and that rifampicin-quinone is the product, in a reaction dependent on both HRP and hydrogen peroxide. The steady-state kinetic constants Km app (101±23 mmol/l), Vmax app (0.78±0.09 μmol/l·s-1) and kcat (5.1±0.6 s-1) were measured (n=4). The reaction rate was increased by the addition of co-substrates such as tetramethylbenzidine, salicylic acid, 5-aminosalicylic acid and paracetamol. This effect was explained by invoking an electron-transfer mechanism by which these drugs acted as mediators of rifampicin oxidation. We suggested that this drug interaction might be important at the inflammatory site. © 2005 Pharmaceutical Society of Japan.

Staphylococcus epidermidis biofilms control by N-acetylcysteine and rifampicin

Medical device-associated infections caused by Staphylococcus epidermidis usually involve biofilm formation and its eradication is particularly challenging. Although rifampicin has been proving to be one of the most effective antibiotics against S. epidermidis biofilms, its use as a single agent can lead to the acquisition of resistance. Therefore, we assessed the combined effect of rifampicin with N-acetylcysteine (NAC) known by its mucolytic effect, in the control of S. epidermidis biofilms. Biofilms of 2 S. epidermidis strains (9142 and 1457) were treated with 1× minimum inhibitory concentration (4 mg/mL) and 10× minimum inhibitory concentration (40 mg/mL) of NAC and 10 mg/L (peak serum) of rifampicin alone and in combination. NAC at 40 mg/L alone or in combination with rifampicin (10 mg/L) significantly reduced (4 log 10) the number of biofilm cells. Considering their different modes of action, the association of NAC with rifampicin constitutes a promising therapeutic strategy in the treatment of infections associated to S. epidermidis biofilms. © 2013 Lippincott Williams & Wilkins.

Effect of pH, mucin and bovine serum on rifampicin permeability through Caco-2 cells

Rifampicin, a poorly soluble drug, has great importance in therapeutics as it is the main drug used to treat tuberculosis. The characterization of its permeability and the factors that influence it represent an important tool for predicting its bioavailability. Caco-2 cell monolayers were used as models of the intestinal mucosa to assess the uptake and transport of rifampicin and the effects of various experimental conditions were investigated, in order to establish the influence of these variables on rifampicin permeability. Different pHs (5.8, 6.8 and 7.4) in the apical medium, the presence or absence of mucin (3.0% w/v) in the donor site and the presence or absence of bovine serum albumin (4.0% v/v) in the receptor chamber were the evaluated conditions. The quantification of rifampicin in the apical or basolateral chambers was performed by a validated HPLC-UV method. The change in the donor chamber pH showed that permeability values were greater at pH 6.8, although this increase does not result in an alteration of the qualitative classification of rifampicin, which has high permeability. Mucin and bovine serum showed no effects on the permeability of rifampicin at the concentration tested. Overall, the current study suggests that pH, artificial mucin and bovine serum proteins have no influence on rifampicin permeability. Copyright (c) 2012 John Wiley & Sons, Ltd.

Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity

Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

P-glycoprotein: a major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and humans.

The P-glycoprotein (Pgp) efflux pump can influence the hepatocellular concentration of xenobiotics that are modulators and substrates of cytochrome P4503A (CYP3A). We tested the hypothesis that Pgp is a determinant of drug-inducible expression of CYP3A. The magnitude of CYP3A induction by rifampicin was compared in the human parental colon carcinoma cell line LS 180/WT (wild type) and in two derivative clones overexpressing the human multidrug resistance gene MDR1 (also designated PGY1) because of either drug selection (LS 180/ADR) or transfection with MDRI cDNA (LS 180/MDR). In both MDR1 cDNA-overexpressing clones, rifampicin induction of CYP3A mRNA and protein was decreased and required greater rifampicin concentrations compared with parental cells. The role of Pgp in regulation of CYP3A expression in vivo was analyzed in mice carrying a targeted disruption of the mdr1a mouse gene. Oral treatment with increasing doses of rifampicin resulted in elevated drug levels in the livers of mdr1a (-/-) mice compared with mdr1a (+/+) mice at all doses. Consistent with the enhanced accumulation of rifampicin in mdr1a (-/-) mice, lower doses of rifampicin were required for induction of CYP3A proteins, and the magnitude of CYP3A induction was greater at all doses of rifampicin in mdr1a (-/-) mice compared with mdr1a (+/+) mice. We conclude that Pgp-mediated transport is a critical element influencing the CYP3A inductive response.

Rifampicin and sodium fusidate reduces the frequency of methicillin-resistant Staphylococcus aureus (MRSA) isolation in adults with cystic fibrosis and chronic MRSA infection

Nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) to patients with cystic fibrosis (CF) frequently results in chronic respiratory tract carriage. This is an increasing problem, adds to the burden of glycopeptide antibiotic use in hospitals, and represents a relative contraindication to lung transplantation. The aim of this study was to determine whether it is possible to eradicate MRSA with prolonged oral combination antibiotics, and whether this treatment is associated with improved clinical status. Adult CF patients (six mate, one female) with chronic MRSA infection were treated for six months with rifampicin and sodium fusidate. Outcome data were examined for six months before treatment, on treatment and after treatment. The patients had a mean age of 29.3 (standard deviation = 6.3) years and FEV1 of 36.1% (standard deviation = 12.7) predicted. The mean duration of MRSA isolation was 31 months. MRSA isolates identified in these patients was of the same lineage as the known endemic strain at the hospital when assessed by pulsed-field get electrophoresis. Five of the seven had no evidence of MRSA during and for at [east six months after rifampicin and sodium fusidate. The proportion of sputum samples positive for MRSA was lower during the six months of treatment (0.13) and after treatment (0.19) compared with before treatment (0.85) (P < 0.0001). There was a reduction in the number of days of intravenous antibiotics per six months with 20.3 +/- 17.6 on treatment compared with 50.7 before treatment and 33.0 after treatment (P = 0.02). There was no change in lung function. Gastrointestinal side effects occurred in three, but led to therapy cessation in only one patient. Despite the use of antibiotics with anti-staphylococcal activity for treatment of respiratory exacerbation, MRSA infection persists. MRSA can be eradicated from the sputum of patients with CF and chronic MRSA carriage by using rifampicin and sodium fusidate for six months. This finding was associated with a significant reduction in the duration of intravenous antibiotic treatment during therapy. (C) 2003 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Cost effectiveness of antimicrobial catheters in the intensive care unit : Addressing uncertainty in the decision

Introduction: Some types of antimicrobial-coated central venous catheters (A-CVC) have been shown to be cost-effective in preventing catheter-related bloodstream infection (CR-BSI). However, not all types have been evaluated, and there are concerns over the quality and usefulness of these earlier studies. There is uncertainty amongst clinicians over which, if any, antimicrobial-coated central venous catheters to use. We re-evaluated the cost-effectiveness of all commercially available antimicrobialcoated central venous catheters for prevention of catheter-related bloodstream infection in adult intensive care unit (ICU) patients. Methods: We used a Markov decision model to compare the cost-effectiveness of antimicrobial-coated central venous catheters relative to uncoated catheters. Four catheter types were evaluated; minocycline and rifampicin (MR)-coated catheters; silver, platinum and carbon (SPC)-impregnated catheters; and two chlorhexidine and silver sulfadiazine-coated catheters, one coated on the external surface (CH/SSD (ext)) and the other coated on both surfaces (CH/SSD (int/ext)). The incremental cost per qualityadjusted life-year gained and the expected net monetary benefits were estimated for each. Uncertainty arising from data estimates, data quality and heterogeneity was explored in sensitivity analyses. Results: The baseline analysis, with no consideration of uncertainty, indicated all four types of antimicrobial-coated central venous catheters were cost-saving relative to uncoated catheters. Minocycline and rifampicin-coated catheters prevented 15 infections per 1,000 catheters and generated the greatest health benefits, 1.6 quality-adjusted life-years, and cost-savings, AUD $130,289. After considering uncertainty in the current evidence, the minocycline and rifampicin-coated catheters returned the highest incremental monetary net benefits of $948 per catheter; but there was a 62% probability of error in this conclusion. Although the minocycline and rifampicin-coated catheters had the highest monetary net benefits across multiple scenarios, the decision was always associated with high uncertainty. Conclusions: Current evidence suggests that the cost-effectiveness of using antimicrobial-coated central venous catheters within the ICU is highly uncertain. Policies to prevent catheter-related bloodstream infection amongst ICU patients should consider the cost-effectiveness of competing interventions in the light of this uncertainty. Decision makers would do well to consider the current gaps in knowledge and the complexity of producing good quality evidence in this area.

Setting hospital infection control policy : a decision-making framework incorporating health economics and healthcare epidemiology

Background: Reducing rates of healthcare acquired infection has been identified by the Australian Commission on Safety and Quality in Health Care as a national priority. One of the goals is the prevention of central venous catheter-related bloodstream infection (CR-BSI). At least 3,500 cases of CR-BSI occur annually in Australian hospitals, resulting in unnecessary deaths and costs to the healthcare system between $25.7 and $95.3 million. Two approaches to preventing these infections have been proposed: use of antimicrobial catheters (A-CVCs); or a catheter care and management ‘bundle’. Given finite healthcare budgets, decisions about the optimal infection control policy require consideration of the effectiveness and value for money of each approach. Objectives: The aim of this research is to use a rational economic framework to inform efficient infection control policy relating to the prevention of CR-BSI in the intensive care unit. It addresses three questions relating to decision-making in this area: 1. Is additional investment in activities aimed at preventing CR-BSI an efficient use of healthcare resources? 2. What is the optimal infection control strategy from amongst the two major approaches that have been proposed to prevent CR-BSI? 3. What uncertainty is there in this decision and can a research agenda to improve decision-making in this area be identified? Methods: A decision analytic model-based economic evaluation was undertaken to identify an efficient approach to preventing CR-BSI in Queensland Health intensive care units. A Markov model was developed in conjunction with a panel of clinical experts which described the epidemiology and prognosis of CR-BSI. The model was parameterised using data systematically identified from the published literature and extracted from routine databases. The quality of data used in the model and its validity to clinical experts and sensitivity to modelling assumptions was assessed. Two separate economic evaluations were conducted. The first evaluation compared all commercially available A-CVCs alongside uncoated catheters to identify which was cost-effective for routine use. The uncertainty in this decision was estimated along with the value of collecting further information to inform the decision. The second evaluation compared the use of A-CVCs to a catheter care bundle. We were unable to estimate the cost of the bundle because it is unclear what the full resource requirements are for its implementation, and what the value of these would be in an Australian context. As such we undertook a threshold analysis to identify the cost and effectiveness thresholds at which a hypothetical bundle would dominate the use of A-CVCs under various clinical scenarios. Results: In the first evaluation of A-CVCs, the findings from the baseline analysis, in which uncertainty is not considered, show that the use of any of the four A-CVCs will result in health gains accompanied by cost-savings. The MR catheters dominate the baseline analysis generating 1.64 QALYs and cost-savings of $130,289 per 1.000 catheters. With uncertainty, and based on current information, the MR catheters remain the optimal decision and return the highest average net monetary benefits ($948 per catheter) relative to all other catheter types. This conclusion was robust to all scenarios tested, however, the probability of error in this conclusion is high, 62% in the baseline scenario. Using a value of $40,000 per QALY, the expected value of perfect information associated with this decision is $7.3 million. An analysis of the expected value of perfect information for individual parameters suggests that it may be worthwhile for future research to focus on providing better estimates of the mortality attributable to CR-BSI and the effectiveness of both SPC and CH/SSD (int/ext) catheters. In the second evaluation of the catheter care bundle relative to A-CVCs, the results which do not consider uncertainty indicate that a bundle must achieve a relative risk of CR-BSI of at least 0.45 to be cost-effective relative to MR catheters. If the bundle can reduce rates of infection from 2.5% to effectively zero, it is cost-effective relative to MR catheters if national implementation costs are less than $2.6 million ($56,610 per ICU). If the bundle can achieve a relative risk of 0.34 (comparable to that reported in the literature) it is cost-effective, relative to MR catheters, if costs over an 18 month period are below $613,795 nationally ($13,343 per ICU). Once uncertainty in the decision is considered, the cost threshold for the bundle increases to $2.2 million. Therefore, if each of the 46 Level III ICUs could implement an 18 month catheter care bundle for less than $47,826 each, this approach would be cost effective relative to A-CVCs. However, the uncertainty is substantial and the probability of error in concluding that the bundle is the cost-effective approach at a cost of $2.2 million is 89%. Conclusions: This work highlights that infection control to prevent CR-BSI is an efficient use of healthcare resources in the Australian context. If there is no further investment in infection control, an opportunity cost is incurred, which is the potential for a more efficient healthcare system. Minocycline/rifampicin catheters are the optimal choice of antimicrobial catheter for routine use in Australian Level III ICUs, however, if a catheter care bundle implemented in Australia was as effective as those used in the large studies in the United States it would be preferred over the catheters if it was able to be implemented for less than $47,826 per Level III ICU. Uncertainty is very high in this decision and arises from multiple sources. There are likely greater costs to this uncertainty for A-CVCs, which may carry hidden costs, than there are for a catheter care bundle, which is more likely to provide indirect benefits to clinical practice and patient safety. Research into the mortality attributable to CR-BSI, the effectiveness of SPC and CH/SSD (int/ext) catheters and the cost and effectiveness of a catheter care bundle in Australia should be prioritised to reduce uncertainty in this decision. This thesis provides the economic evidence to inform one area of infection control, but there are many other infection control decisions for which information about the cost-effectiveness of competing interventions does not exist. This work highlights some of the challenges and benefits to generating and using economic evidence for infection control decision-making and provides support for commissioning more research into the cost-effectiveness of infection control.

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy3-(p- nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

Pharmacokinetic interactions of pioglitazone

Pioglitazone is a thiazolidinedione compound used in the treatment of type 2 diabetes. It has been reported to be metabolised by multiple cytochrome P450 (CYP) enzymes, including CYP2C8, CYP2C9 and CYP3A4 in vitro. The aims of this work were to identify the CYP enzymes mainly responsible for the elimination of pioglitazone in order to evaluate its potential for in vivo drug interactions, and to investigate the effects of CYP2C8- and CYP3A4-inhibiting drugs (gemfibrozil, montelukast, zafirlukast and itraconazole) on the pharmacokinetics of pioglitazone in healthy volunteers. In addition, the effect of induction of CYP enzymes on the pharmacokinetics of pioglitazone in healthy volunteers was investigated, with rifampicin as a model inducer. Finally, the effect of pioglitazone on CYP2C8 and CYP3A enzyme activity was examined in healthy volunteers using repaglinide as a model substrate. Study I was conducted in vitro using pooled human liver microsomes (HLM) and human recombinant CYP isoforms. Studies II to V were randomised, placebo-controlled cross-over studies with 2-4 phases each. A total of 10-12 healthy volunteers participated in each study. Pretreatment with clinically relevant doses with the inhibitor or inducer was followed by a single dose of pioglitazone or repaglinide, whereafter blood and urine samples were collected for the determination of drug concentrations. In vitro, the elimination of pioglitazone (1 µM) by HLM was markedly inhibited, in particular by CYP2C8 inhibitors, but also by CYP3A4 inhibitors. Of the recombinant CYP isoforms, CYP2C8 metabolised pioglitazone markedly, and CYP3A4 also had a significant effect. All of the tested CYP2C8 inhibitors (montelukast, zafirlukast, trimethoprim and gemfibrozil) concentration-dependently inhibited pioglitazone metabolism in HLM. In humans, gemfibrozil raised the area under the plasma concentration-time curve (AUC) of pioglitazone 3.2-fold (P < 0.001) and prolonged its elimination half-life (t½) from 8.3 to 22.7 hours (P < 0.001), but had no significant effect on its peak concentration (Cmax) compared with placebo. Gemfibrozil also increased the excretion of pioglitazone into urine and reduced the ratios of the active metabolites M-IV and M-III to pioglitazone in plasma and urine. Itraconazole had no significant effect on the pharmacokinetics of pioglitazone and did not alter the effect of gemfibrozil on pioglitazone pharmacokinetics. Rifampicin decreased the AUC of pioglitazone by 54% (P < 0.001) and shortened its dominant t½ from 4.9 to 2.3 hours (P < 0.001). No significant effect on Cmax was observed. Rifampicin also decreased the AUC of the metabolites M-IV and M-III, shortened their t½ and increased the ratios of the metabolite to pioglitazone in plasma and urine. Montelukast and zafirlukast did not affect the pharmacokinetics of pioglitazone. The pharmacokinetics of repaglinide remained unaffected by pioglitazone. These studies demonstrate the principal role of CYP2C8 in the metabolism of pioglitazone in humans. Gemfibrozil, an inhibitor of CYP2C8, increases and rifampicin, an inducer of CYP2C8 and other CYP enzymes, decreases the plasma concentrations of pioglitazone, which can necessitate blood glucose monitoring and adjustment of pioglitazone dosage. Montelukast and zafirlukast had no effects on the pharmacokinetics of pioglitazone, indicating that their inhibitory effect on CYP2C8 is negligible in vivo. Pioglitazone did not increase the plasma concentrations of repaglinide, indicating that its inhibitory effect on CYP2C8 and CYP3A4 is very weak in vivo.

A distinct physiological role of MutY in mutation prevention in mycobacteria

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-B-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG: A, 8-oxoG: G or 8-oxoG: T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria.