Development of mouse models with restricted HLA-B∗57:01 presentation for the study of flucloxacillin-driven T-cell activation and tolerance in liver injury.

BACKGROUND: Flucloxacillin (FLX)-induced liver injury is immune-mediated and highly associated to HLA-B∗57:01 expression. Host factors leading to drug-induced liver injury are not yet well understood. OBJECTIVE: Characterize in vivo immune mechanisms determining the development of CD8+ T cells reactive to FLX in animals expressing the risk human leukocyte antigen (HLA) allotype. METHODS: HLA-B∗57:01 transgenic mice (Tg) or Tg strains with H2-KbDb knockout (Tg/KO) or H2-KbDb/PD-1 double knockout (Tg/DKO) were treated with drug and/or anti-CD4 antibody. Drug-induced liver injury was evaluated on the basis of liver enzyme and histologic changes at day 10 of treatment. FLX-reactive CD8+ T cells were characterized in vitro by release of effector molecules on drug restimulation, gene expression, and flow cytometry analysis, and functionality tested for hepatic cytotoxicity. RESULTS: CD8+ T-cell responses to FLX in Tg were dependent on both HLA and mouse major histocompatibility complex I presentation and in vivo priming. Eliminating H2-KbDb in Tg/KO to allow exclusive presentation of FLX by HLA resulted in a less robust drug-specific CD8+T-cell response unless CD4+ cells, including regulatory T cells, were depleted. Treatment of Tg/KO with anti-CD4 antibody and FLX led to subclinical liver inflammation associated with an increase in PD1+CD8+ T cells in the lymphoid organs and liver. Impaired PD-1 expression in Tg/DKO led to liver histopathologic and transcriptional alterations but without hepatic enzyme elevations. Moreover, effector lymphocytes accumulated in the liver and showed FLX-dependent hepatic cytotoxicity in vitro when tolerogenic liver cells were depleted. CONCLUSIONS: In our in vivo models, FLX primes CD8+ T cells to recognize drug presented by HLA-B∗57:01 and murine major histocompatibility complex I. HLA-B∗57:01-dependent CD8+ T-cell reaction to FLX is limited by the presence of CD4+ cells, presumably regulatory T cells, and PD-1 expression. Tolerogenic hepatic cells limit clinical disease through PD-L1 or additional unexplored mechanisms.

Characterization of kinetics of human cytochrome P450s involved in bioactivation of flucloxacillin: inhibition of CYP3A-catalysed hydroxylation by sulfaphenazole.

BACKGROUND AND PURPOSE: The aim of this study was to characterize the human cytochrome P450s (CYPs) involved in oxidative bioactivation of flucloxacillin to 5-hydroxymethyl flucloxacillin, a metabolite with high cytotoxicity towards biliary epithelial cells. EXPERIMENTAL APPROACH: The CYPs involved in hydroxylation of flucloxacillin were characterized using recombinant human CYPs, pooled liver microsomes in the presence of CYP-specific inhibitors and by correlation analysis using a panel of liver microsomes from 16 donors. KEY RESULTS: Recombinant CYPs showing the highest specific activity were CYP3A4, CYP3A7 and to lower extent CYP2C9 and CTP2C8. Michaelis-Menten enzyme kinetics were determined for pooled human liver microsomes, recombinant CYP3A4, CYP3A7 and CYP2C9. Surprisingly, sulfaphenazole appeared to be a potent inhibitor of 5'-hydroxylation of flucloxacillin by both recombinant CYP3A4 and CYP3A7. CONCLUSIONS AND IMPLICATIONS: The combined results show that the 5'-hydroxylation of flucloxacillin is primarily catalysed by CYP3A4, CYP3A7 and CYP2C9. The large variability of the hepatic expression of these enzymes could affect the formation of 5'-hydroxymethyl flucloxacillin, which may determine the differences in susceptibility to flucloxacillin-induced liver injury. Additionally, the strong inhibition in CYP3A-catalysed flucloxacillin metabolism by sulfaphenazole suggests that unanticipated drug-drug interactions could occur with coadministered drugs.

A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants.

The 5'-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5'-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5'-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5'-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5'-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

Pharmacokinetics of flucloxacillin and its metabolites in patients with renal failure: Impact on liver toxicity
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OBJECTIVE: The antimicrobial agent flucloxacillin is a potential cause of drug-induced liver disease, but the underlying mechanisms for toxicity have not been fully elucidated. As in-vitro and in-vivo findings suggest that biotransformation products contribute to hepatotoxicity, the purpose of this study was to characterize formation and accumulation of its metabolites in patients with renal failure. METHODS: Twelve intensive care patients undergoing continuous venovenous hemofiltration received 4.0 g flucloxacillin as single and repeated infusion. Blood and dialysate samples were collected and analyzed for flucloxacillin and its metabolites by HPLC. RESULTS: The overall amounts of the flucloxacillin metabolites 5'-hydroxymethylflucloxacillin (5-OH-FX), 5'-hydroxymethylflucloxacillin-penicilloic acid (5-OH-PA), and flucloxacillin-penicilloic acid (FX-PA) produced varied considerably between patients, and accounted for 3.62 - 35.9% of total flucloxacillin concentration (flucloxacillin + metabolites) in the plasma. Clearance rates and sieving coefficients for 5-OH-FX and FX-PA were comparable to that of the parent drug, although removal of 5-OH-PA was decreased. Using an isolated perfused rat liver model we demonstrated that 5-OH-FX reached concentrations in the bile (240.5 ± 84.2 nmoles/mL) that were sufficient to exert cytotoxic effects, unlike either of the two penicilloic acids. CONCLUSIONS: Based on data from perfused rat livers, high biliary concentrations of 5-OH-FX might also be observed in our patients explaining why LDH, bilirubin, and alkaline phosphatase were elevated in up to 8/12 patients after repeated infusion of flucloxacillin. Liver toxicity of flucloxacillin might therefore be observed in patients with renal impairment after continuously elevated 5-OH-FX levels.
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Human leukocyte antigen (HLA)-B*57:01-restricted activation of drug-specific T cells provides the immunological basis for flucloxacillin-induced liver injury.

UNLABELLED: The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin-responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA-B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T-cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon-gamma (IFN-γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time-dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing-dependent and restricted by HLA-B*57:01 and the closely related HLA-B*58:01. Clones displayed additional reactivity against ß-lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. CONCLUSION: This work defines the immune basis for flucloxacillin-induced liver injury and links the genetic association to the iatrogenic disease.

Protein binding of flucloxacillin in neonates.

The isoxazolyl penicillins, including flucloxacillin, have the highest levels of plasma protein binding among the semisynthetic penicillins. Because only the free fraction of the penicillin is pharmacologically active, it would be useful to measure both protein-bound and free flucloxacillin to determine its protein binding. Until now, flucloxacillin protein binding in newborn infants has been investigated in only two studies with relatively small populations. In the present study, flucloxacillin protein binding was investigated in 56 (preterm) infants aged 3 to 87 days (gestational age, 25-41 weeks). Surplus plasma samples from routine gentamicin assays of each infant were collected and combined to obtain a sufficiently large sample for analysis. Free flucloxacillin was separated from protein-bound flucloxacillin using ultrafiltration. Reversed-phase high-performance liquid chromatography with ultraviolet detection was used to measure free flucloxacillin concentrations in ultrafiltrate and total flucloxacillin concentrations in pooled plasma. Flucloxacillin protein binding was 74.5% +/- 13.1% (mean +/- standard deviation) with a high variability among the infants (34.3% to 89.7%). High Pearson correlations were found between protein binding and the covariates-plasma albumin concentration (r = 0.804, P < 0.001, n = 18) and plasma creatinine concentration (r = -0.601, P < 0.001, n = 45). Statistically significant but less striking correlations were found between protein binding and gestational age, postconceptional age, body weight, and triglyceride concentration. Because of the high variability of protein binding among infants, it is difficult to devise a flucloxacillin dosage regimen effective for all infants. Individualized dosing, based on free flucloxacillin concentrations, might help to optimize treatment of late-onset neonatal sepsis, but practical obstacles will probably prevent analysis of free flucloxacillin concentrations in newborn infants on a routine basis.

Immunochemical detection of flucloxacillin adduct formation in livers of treated rats.

Flucloxacillin is a semi-synthetic penicillin widely used in the prophylaxis and treatment of staphylococcal infections. Severe liver reactions, characterised by delayed cholestatic hepatitis and a prolonged course of recovery, are associated with flucloxacillin therapy. Clinical findings are suggestive of an immune mediated reaction but there exists little supporting experimental evidence. The formation of drug modified hepatic protein adducts has been proposed to play an important role in the hepatotoxicity of many drugs. The aim of the present study was to investigate whether flucloxacillin treatment results in adduct formation in vivo. Flucloxacillin was conjugated to rabbit serum albumin by formation of a penicilloyl determinant and used as an immunogen to raise a polyclonal antiserum specific for flucloxacillin-modified proteins. Antibody specificity was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with free drug. The antiserum was used in combination with western blotting to detect adduct formation in the livers of flucloxacillin treated rats. Western blot analysis of rat liver subcellular fractions revealed the formation of six flucloxacillin adducts in various subcellular fractions. These studies demonstrate for the first time that treatment with flucloxacillin results in the formation of hepatic protein adducts.

Indirect cytotoxicity of flucloxacillin toward human biliary epithelium via metabolite formation in hepatocytes.

Flucloxacillin, an isoxazolyl-penicillin, causes cholestasis and biliary epithelium injury. The aim of the study was to determine whether flucloxacillin, either directly or through metabolite formation, may induce cytotoxicity in hepatic or biliary cells. Cytotoxicity was assessed by lactate dehydrogenase release in primary cultures of human hepatocytes and of gallbladder-derived biliary epithelial cells (BEC). Metabolite production in microsome and cell preparations was analyzed by chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. While flucloxacillin induced no direct cytotoxicity in any of the hepatocyte (n = 12) and BEC (n = 19) preparations, the conditioned media from cultured hepatocytes preincubated with flucloxacillin (50-500 mg/L) triggered a significant increase in lactate dehydrogenase release over controls in approximately 50% of BEC preparations (7/12), and this effect depended upon flucloxacillin concentration. Remaining BEC preparations exhibited no toxic response. Cytotoxicity in BEC preparations (9/13) was also induced by the supernatants of human liver microsomes and of recombinant human cytochrome P450 (CYP)3A4 preincubated with flucloxacillin (500 mg/L). Supernatants from both liver microsome and CYP3A4 preparations contained one major metabolite which was identified as 5'-hydroxymethylflucloxacillin. The production of this metabolite was inhibited following CYP3A4 inhibition by troleandomycin in human liver microsomes, and markedly enhanced following CYP3A induction by dexamethasone in rat liver microsomes. As opposed to BEC, cultured hepatocytes displayed significant CYP3A activity and produced low amounts of this metabolite. The purified metabolite (0.01-5 mg/L) exerted toxic effects in BEC but not in hepatocytes. In conclusion, hepatocytes mainly via CYP3A4 activity, generate flucloxacillin metabolite(s) including 5'-hydroxymethylflucloxacillin that may induce cytotoxicity in susceptible BEC. These metabolic events may contribute to the pathogenesis of drug-induced cholangiopathies.

Pharmacokinetics and bioavailability of flucloxacillin in elderly hospitalized patients.

The pharmacokinetics and oral bioavailability of flucloxacillin were studied in five female and two male patients (age 68-87 yr) who had been hospitalized for orthopedic surgeries. A single dose of intravenous or oral flucloxacillin sodium (500 mg) was administered in random order on different occasions separated by at least 2 days. Blood and urine samples were taken up to 24 hours after drug administration and levels of flucloxacillin and 5-hydroxymethylflucloxacillin (5-HMF), a major metabolite, were measured by high-performance liquid chromatography. Flucloxacillin elimination, but not oral absorption, was reduced in the elderly, compared with data from young healthy subjects reported elsewhere. Total clearance, renal clearance, and volume of distribution were 0.083 +/- 0.013 L/kg/hr, 0.038 +/- 0.01 L/kg/hr, and 0.184 +/- 0.034 L/kg, respectively. Regression of flucloxacillin renal clearance (Clr) on estimated creatine clearance (CLcr) gave the relationship: Clr = 0.755 (CLcr) + 10.6 (r = 0.91; P = 0.004). Terminal half-lives for flucloxacillin and 5-HMF were 2.21 +/- 0.51 hr and 3.0 +/- 0.75 hr, respectively after intravenous administration. Flucloxacillin was absorbed rapidly after oral administration with a mean absorption time of 0.95 +/- 0.34 hr, and time to reach peak concentration of 1.20 +/- 0.29 hr. The absolute bioavailability of flucloxacillin from capsules was 54.4 +/- 18.8%.

Concentrations of flucloxacillin in heart valves and subcutaneous and muscle tissues of patients undergoing open-heart surgery.

Thirty-seven patients were given a single, 2-g intravenous bolus injection of flucloxacillin prior to open-heart surgery. Within 12 h, flucloxacillin concentrations in serum and heart valves declined from 125.2 to 4.4 micrograms/ml and from 16.5 to 3.7 micrograms/g, respectively. Concentrations in subcutaneous tissue and muscle were almost identical, declining from 14.7 or 14.2 micrograms/g to undetectable levels after 8 to 10 h.

Pharmacokinetics of free and total flucloxacillin in newborn infants.

Flucloxacillin 50 mg/kg b.w. was administered intravenously (in combination with ampicillin/gentamicin) and orally (with amoxicillin) to 9 newborn infants (gestational age 33-41 weeks) to treat bacterial infections. The concentrations of flucloxacillin in plasma and urine after i.v. injection were analysed according to an open two-compartment model, and the plasma protein binding of flucloxacillin and its distribution to blood cells and plasma water in whole blood were determined. Considerable differences were found from values reported in adults. The terminal half-life averaged 4 h 38 min and was significantly correlated with gestational age. Plasma clearance was low (0.744 ml X min-1 X kg-1), due to the small renal clearance (0.182 ml X min-1 X kg-1), whilst non-renal clearance (0.563 ml X min-1 X kg-1) was approximately the same as in adults. The mean apparent volume of distribution of total drug (Vz) was 0.280 l/kg. The corresponding volume of distribution of unbound drug (Vu1 + Vu2) was 1.74 l/kg, which indicates considerable extravascular drug binding. The plasma protein binding of flucloxacillin (mean 86.3%) was significantly correlated with gestational age and the bilirubin/albumin concentration ratio. Bioavailability after oral administration, when corrected for changes in terminal half-life, was 47.7%, which is only slightly lower than that reported in adults. Since the plasma concentrations after both i.v. and oral administration were well above the MIC-values generally reported for Staphylococcus aureus, and since few side-effects were observed, intravenous injection or, in selected cases, orl administration of flucloxacillin appears to be a reliable therapy for the treatment of infections due to sensitive strains of S. aureus in premature newborn infants.

Extravascular penetration of highly protein-bound flucloxacillin.

The pharmacokinetics of intravenously administered flucloxacillin (2.0 g to five volunteers) are described. The passage of flucloxacillin to peripheral lymph and suction skin blisters was monitored. This drug was selected because the high serum protein binding of 96% offered a particularly good opportunity for the study of the impact on tissue penetration. Flucloxacillin was assayed by high-pressure liquid chromatography, and pharmacokinetics were assayed by computerized curve fitting to accepted models. Penetration of flucloxacillin to extravascular foci was rapid. After 30 min the drug concentrations were 0.5 +/- 0.3 microgram/ml in lymph and 0.9 +/- 0.7 microgram/ml in blister fluid. The peak concentration was 11.7 +/- 5.6 micrograms/ml in lymph and 4.6 +/- 1.4 micrograms/ml in blister fluid. Concentrations in urine were above 111 +/- 50 micrograms/ml throughout the 8-h monitoring period, and urinary recovery was 60.4%. The half-life was 2.1 +/- 0.9 h in serum, 1.4 +/- 0.6 h in lymph, and 11.0 +/- 4.1 h in blister fluid. The differences in half-life were significant (P less than 0.05) between serum and blister fluid but not between lymph and serum. Penetration, as represented by the mean ratios of areas under the curve, was 19.7 +/- 8.1% to lymph and 38.2 +/- 11.7% to blister fluid. The flucloxacillin distribution volume during the phase of elimination was 36.4 +/- 16.0 liters and the total body clearance was 12.9 +/- 5.5 liters. Flucloxacillin showed good tissue penetration, considering its very high serum protein binding. High flucloxacillin levels in lymph and blister fluid were explained in part by drug affinity to extravascular albumin. The major impacts of high protein binding are (i) slightly slower passage into extravascular sites, (ii) slightly later peak concentration, and (iii) levels in extravascular fluid that are persistently below those in serum.

Antibiotic prophylaxis in pacemaker surgery: a prospective double blind trial with systemic administration of antibiotic versus placebo at implantation of cardiac pacemakers.

In a double blind clinical trial, 106 consecutive patients scheduled for pacemaker implantation were randomly assigned either to a systemic prophylaxis group (SPG) (to be given flucloxacillin) or to a control group who would be given a placebo (CPG). The SPG group received 2 g IV flucloxacillin 1 hour before the operation, then 1 g perorally every 8 hours for the next five days. In the CPG group, placebo infusions and tablets were given at the same schedule. There were a total of 106 patients (SPG 52, CPG 54) who met the criteria of the study. Of these, 102 patients (SPG 50, CPG 52) completed a follow-up of 7-35 months. Infection of the pacemaker system was not diagnosed in any patient in either group. Tissue fluid was drawn 24 hours postoperatively from the pacemaker pocket for culture and for determination of pocket antibiotic concentration. The mean flucloxacillin concentration of pocket fluid from 23 patients in the SPG was 7.5 micrograms/ml. The bacteriological cultures were positive in 9/32 patients in the SPG group and in 10/34 patients in the CPG group. This study suggests that antibiotic prophylaxis need not routinely be given at implantation of permanent pacemaker systems.

Oral absorption of flucloxacillin in infants and young children.

The bioavailability of flucloxacillin (Heracillin) following oral administration was determined in infants and children. After a dose of 12.5 mg/kg, peak concentrations were achieved at 60 min. which declined rapidly and were low in older children only 4 hours afterwards. Infants below 6 months of age showed a better absorption when given the mixture than older children. Older children achieved higher plasma concentrations when given tablets than when given equal doses of the mixture. There was no difference in the concentration when the dose was given to the subject when fasting, or with breakfast.

Predisposing factors, bacteriology and antibiotic therapy in 35 cases of septic bursitis.

In a retrospective study the etiology was verified in 35/52 patients with suspected septic prepatellar or olecranon bursitis. Staphylococcus aureus was the most common pathogen and 86% of them were penicillinase-producing. Other strains were streptococci. Mean age was 47 (18-83) years and males predominated (91%). Predisposing factors were seen in most cases. Preceding trauma was found in 27 and/or associated diseases (e.g. diabetes mellitus) in 11 patients. Hyperglycemia was found in 38% of the patients. Treatment with antimicrobial agents, mostly penicillins, and bursal drainage were successful in 32 cases. Two patients had local spread of the infection and 1 patient chronic bursitis.

Pacemaker infections. A clinical study with special reference to prophylactic use of some isoxazolyl penicillins.

Infection is a major complication of pacemaker treatment. Antibiotic prophylaxis has been used in association with pacemaker surgery with conflicting results, and conclusive prospective trials are lacking. This investigation indicated that systemic antibiotic prophylaxis was of benefit when infections occurred frequently. The effect of local antibiotic prophylaxis was comparable with that of systemic prophylaxis at generator replacements. No serious adverse effects of the prophylaxis were noted. However, with modern surgical methods and hygienic principles, antibiotic prophylaxis did not seem to be necessary at implantation of new cardiac pacemakers. Once infection had developed it was difficult to eradicate and serious complications sometimes occurred. Most infections commenced in the pacemaker pocket. A few cases were cured by antibiotic treatment alone but, particularly if the infection spread along the electrode, surgery was strongly needed and in the presence of endocarditis and/or septicemia all foreign material should be removed if possible. The most common causal microorganisms of pacemaker infections were Staphylococcus aureus and Staphylococcus epidermidis. Routinely performed pre-, per- and postoperative cultures were of no prognostic value. Persistent use of antibiotics could select for methicillin-resistant coagulase-negative staphylococci, therefore bacteriological monitoring of wound infections was considered important. The dosage schedules used for cloxacillin and flucloxacillin gave satisfactory serum concentrations peroperatively. Local treatment with cloxacillin in the pacemaker pocket peroperatively gave adequate concentrations in tissue fluid from the pocket 24 h after the operation, as did systemic administration of flucloxacillin. The pharmacokinetics of flucloxacillin in these elderly patients differed in some respects from that found in healthy volunteers. Plasma elimination half-life was almost twice as long. Despite the high degree of plasma protein binding, flucloxacillin appeared to pass rapidly and efficiently to extravascular compartments, such as a pacemaker pocket.

Pharmacokinetics and distribution of flucloxacillin in pacemaker patients.

The pharmacokinetics of flucloxacillin in plasma and tissue fluid after i.v. infusion of 1 g was analyzed according to an open two-compartment model in 19 patients with bradyarrhythmias (mean age 70.8 years) admitted for implantation or replacement of a permanent pacemaker system. After the first infusion of flucloxacillin (5 min), the distribution phase was rapid (t 1/2 alpha = 0.13 h). The plasma half-life of elimination (t 1/2 beta) was 1.51 h, which is almost twice as long as reported in healthy volunteers. Total plasma clearance (93.1 ml/min) was also lower than is usually found in healthy individuals, due to low renal clearance of flucloxacillin (60.2 ml/min). The total apparent volume of distribution during the beta-phase (Vdarea) was 0.172 l/kg and distribution in the central compartment (Vc) 0.064 1/kg. In each patient plasma protein binding and drug distribution to plasma water, proteins and blood cells in whole blood were determined. Binding in plasma to proteins was 91.0% and distribution to blood cells in whole blood 13.8%. The mean distribution volume of free flucloxacillin during the beta-phase (Vd beta free) was 2.18 1/kg, which exceeds total body water, suggesting possible intracellular distribution and substantial tissue binding. Plasma concentrations of flucloxacillin after the fourth dose (1 g t.i.d.) were very similar to those obtained after the first infusion and those predicted from the single dose kinetics. The concentration of flucloxacillin in fluid from the pacemaker pockets in 5 patients averaged 12.1 micrograms/ml and 9.5 micrograms/ml at 1 and 5 h, respectively, which was more than ten times the MIC-values for Staphylococcus aureus and S. epidermidis.(ABSTRACT TRUNCATED AT 250 WORDS)