A Baeyer-Villiger oxidation specifically catalyzed by human flavin-containing monooxygenase 5.

10-((4-Hydroxypiperidin-1-yl)methyl)chromeno[4,3,2-de]phthalazin-3(2H)-one (E7016), an inhibitor of poly(ADP-ribose) polymerase, is being developed for anticancer therapy. One of the major metabolites identified in preclinical animal studies was the product of an apparent oxidation and ring opening of the 4-hydroxypiperidine. In vitro, this oxidized metabolite could not be generated by incubating E7016 with animal or human liver microsomes. Further studies revealed the formation of this unique metabolite in hepatocytes. In a NAD(P)(+)-dependent manner, this metabolite was also generated by liver S9 fractions and recombinant human flavin-containing monooxygenase (FMO) 5 that was fortified with liver cytosol fractions. In animal and human liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), or a brief heat treatment at 50°C. Based on these results, the overall metabolic pathway was believed to involve a two-step oxidation process: dehydrogenation of the secondary alcohol in liver cytosol followed by an FMO5-mediated Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps were coupled via regeneration of NAD(P)(+) and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In an NAD(P)H-dependent manner, the synthetic ketone intermediate was metabolized to the same ring-opened metabolite in animal and human liver microsomes. This metabolic reaction was also inhibited by BNPP or a brief heat treatment at 50°C. Methimazole, the substrate/inhibitor of FMO1 and FMO3, did not inhibit this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the synthetic ketone intermediate in recombinant enzymes.

Characterization of recombinant human carboxylesterases: fluorescein diacetate as a probe substrate for human carboxylesterase 2.

Human carboxylesterase (CES) 1 and CES2 are members of the serine hydrolase superfamily, and both exhibit broad substrate specificity and are involved in xenobiotic and endobiotic metabolism. Although expression of CES1 and CES2 occurs in several organs, their expression in liver and small intestine is predominantly attributed to CES1 and CES2, respectively. We successfully expressed CES1 form b (CES1-b) and form c (CES1-c) as well as CES2 in baculovirus-infected High Five insect cells. With 4-nitrophenyl acetate (4-NPA) as the probe substrate, the K(m) values of recombinant CES1-b and CES2 matched those of human liver microsomes (HLM) and human intestinal microsomes (HIM) with approximately 200 and 180 µM, respectively. Bis(4-nitrophenyl) phosphate potently inhibited 4-NPA hydrolysis by HLM, CES1-b, CES1-c, HIM, and CES2 with IC(50) values less than 1 µM. With fluorescein diacetate (FD) as the substrate, the K(m) values were similar for all enzyme systems, with the exception of CES1-b, which was slightly lower; however, the V(max) values for HIM and CES2 were 39.5 and 14.6 µmol · mg(-1) · min(-1), respectively, which were at least 50-fold higher than those of CES1-b or CES1-c. Loperamide potently inhibited HLM, HIM, and CES2 with similar IC(50) values of approximately 1 µM. Substrate specificity was compared between human tissues and recombinant enzymes. The data suggest the following: 1) FD is a probe substrate for CES2; 2) CES1-b is the predominant form in human liver; and 3) recombinant CES1-b and CES2 expressed in insect cells are functionally consistent with native carboxylesterases expressed in human liver and intestine, respectively.

Interactions between the chemotherapeutic agent eribulin mesylate (E7389) and P-glycoprotein in CF-1 abcb1a-deficient mice and Caco-2 cells.

Eribulin is a new anticancer agent currently in Phase III clinical trials for the treatment of metastatic breast cancer. In the current studies, we have investigated the effects of P-glycoprotein (P-gp) on the in vivo disposition of eribulin using CF-1 abcb1a-deficient mice, and the influence of eribulin on P-gp-mediated efflux of digoxin in Caco-2 cells. Eribulin was administered intravenously and orally in both CF-1 wild-type and CF-1 abcb1a-deficient mice. P-gp-mediated efflux of digoxin in Caco-2 cell monolayers was measured in the presence of eribulin. The plasma exposure to eribulin was higher in CF-1 abcb1a-deficient mice than that in CF-1 wild-type mice after intravenous (IV) and oral (PO) administrations. The oral bioavailability of eribulin was 62.3% in CF-1 abcb1a-deficient mice compared with 7.6% in wild-type mice. The brain penetration of eribulin in CF-1 abcb1a-deficient mice was 30-fold greater than that in wild-type mice. Eribulin decreased the efflux ratio of digoxin in a concentration-dependent manner, with the result of IC(50) greater than 10 µM. The [I]/IC(50) of eribulin was estimated to be <0.05. P-gp is likely to limit the oral absorption and brain penetration of eribulin in CF-1 wild-type mice. Eribulin inhibited the efflux of digoxin with IC(50) greater than 10 µM in Caco-2 cells. These results suggest that eribulin, given intravenously at the clinically relevant concentrations, may not alter P-gp-mediated disposition of concurrently administered drugs.

The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America.

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.

A high-performance liquid chromatography-tandem mass spectrometry method for the clinical combination study of carboplatin and anti-tumor agent eribulin mesylate (E7389) in human plasma.

An LC/MS/MS method was developed to quantify carboplatin and eribulin mesylate (E7389) in human plasma and urine. For carboplatin, sample clean-up by protein precipitation and supernatant injection into a Waters Spherisorb((R)) S5 SCX column was used. Liquid-phase extraction and reverse-phase chromatography on a Polaris C18 column were used for eribulin. Quantitation involved LC/MS/MS with positive electrospray ionization. Accuracy, precision, linearity, range, specificity, recovery and stability were also evaluated. Both compounds were stable in human plasma (>or=80 days at -80 degrees C), at room temperature (>or=4h), following three freeze-thaw cycles and in 50/50 methanol/H(2)O (<4 degrees C for >or=252 days).

Enzyme kinetics for clinically relevant CYP inhibition.

In vitro cytochrome P450 (CYP)-associated metabolic studies have been considered cost-effective for predicting the potential clinical drug-drug interactions (DDIs), one of the major attritions in drug development. The breakthroughs during the past decade in understanding the biochemistry of CYP-mediated biotransformation and molecular biology of CYP gene regulation in humans have provided the scientific bases for such endeavors in early drug development. In this review, the enzyme kinetics of CYP inhibitions is described, with the primary focus on the ones proven with clinical relevance, namely the competitive inhibition and mechanism-based inactivation (MBI). Competitive CYP inhibition, the most often detected reversible inhibition, is well understood and has been studied extensively both in vitro and in clinical setting. Recently, MBI has received increasing attention. It has been recognized that MBI could occur more often than anticipated, due in part to the redox cycling-allied enzymatic action of CYPs. As commonly as an irreversible inhibition, MBI would inactivate the target proteins, and thus would be generally considered of high potential for causing clinical DDI. Moreover, the reversible inhibitions other than the competitive, namely noncompetitive, uncompetitive and mixed, were also documented for the important drug-metabolizing CYP members, particularly CYP1A2 and CYP2C9. Finally, the unusual kinetic interactions, which did not follow the Michaelis-Menten (M-M) kinetics, were detected in vitro for the majority of drug-metabolizing CYP members, and manifested for CYP3A4. However, the clinical relevance of the interactions involving the unusual CYP kinetics has not yet been fully understood. Nonetheless, the reversibility and inhibitory potency should be considered as the major determinants of the clinical relevance, particularly in combination with the therapeutic exposure levels. With rapid expansion of knowledge and technology, the evaluation of the clinically relevant CYP-associated DDIs in vitro is not only desirable but also achievable.

Safety, pharmacokinetics, and pharmacodynamics of E5564, a lipid A antagonist, during an ascending single-dose clinical study.

E5564, a structural analog of the lipid A portion of lipopolysaccharide (LPS), is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models and is currently under clinical development as a possible therapeutic for the treatment of sepsis and septic shock. The objectives of this study were to (1) assess the safety and tolerability of E5564 following a 30-minute intravenous (i.v.) infusion, (2) evaluate the pharmacokinetic profile of E5564, and (3) measure the ability of E5564 to block LPS stimulation ex vivo in blood taken from subjects up to 8 hours after ending the infusion. Healthy male volunteers (n = 7/dose group) were randomly assigned to each of four dose levels (350, 1000, 2000, or 3500 micrograms). Within each dose group, 5 subjects received drug and 2 received placebo. E5564 or matching placebo was administered by a 30-minute infusion, and blood samples were collected at predetermined time points. All doses of E5564 were demonstrated to be safe and well tolerated. E5564 plasma concentrations were determined using a validated LC/MS/MS method. The Cmax and AUC of E5564 increased in a dose-proportional manner. E5564 pharma-cokinetics were characterized by a slow clearance (0.67-0.95 mL/h/kg), a small volume of distribution (41-54 mL/kg), and a relatively long elimination half-life (42-51 h). As measured in the ex vivo assay, E5564 inhibited LPS-induced tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 hours postinfusion. E5564 lacked LPS-like agonist activity at doses up to 3.5 mg. Taken together, we believe that E5564 is a safe, potent antagonist of LPS in blood and will likely benefit patients in the treatment of LPS-related diseases.

Pharmacokinetics of E5564, a lipopolysaccharide antagonist, in patients with impaired hepatic function.

E5564 is a structural analog of the Lipid A portion of lipopolysaccharide (LPS). E5564 has been tested in several in vitro and in vivo models and has demonstrated its effectiveness against LPS. It is intended to be an antagonist of LPS to reduce the morbidity and mortality associated with sepsis syndrome. This study assessed the pharmacokinetics (PK) of E5564 in patients with impaired hepatic function. E5564 was administered via intermittent intravenous infusion every 12 hours for six times to 24 hepatic-impaired patients (12 each to Child-Pugh Classifications A and B) and 24 matching healthy volunteers. Plasma samples were analyzed by LC/MS/MS. A one-compartment model resulted in good and comparable fits for all volunteers. Regardless of liver disease state, none of the PK parameters compared (i.e., Cmax (0-12),tmax (0-12),CL,t1/2, Vss, AUC(0-12), AUC(0-last), AUC(0-infinity), C(ss,min), C(ss,max), and C(ss,av)) exhibited any difference between these two groups. This suggested that the exposure of E5564 in volunteers was independent of hepatic function. Thus, no dose adjustment is needed in patients with hepatic impairment classified as Child-Pugh A and B.

Application of a substrate cocktail approach in the assessment of cytochrome P450 induction using cultured human hepatocytes.

Induction of the cytochrome P450 (CYP) family of enzymes by coadministered compounds can result in drug-drug interactions, as in the case of the coadministration of rifampicin with many CYP3A substrates, including midazolam. Identification of potential drug-drug interactions due to CYP induction during drug discovery is critical. We present a substrate cocktail method that was applied to assess the induction of CYP1A, CYP2B6, CYP2C9, and CYP3A using a 96-well high-throughput format. Viable cell counts were determined using a high-content screening system to normalize activities. Substrate cocktail incubations demonstrated a similar fold induction for known inducers as compared with discrete probe incubations. The system was further validated by determining the induction potency of rifampicin. The E(max) and EC(50) values in two separate lots of hepatocytes for CYP3A induction by rifampicin in a 96-well format were similar when discrete probe was compared with the probe cocktail. This system has been demonstrated to be suitable for high-throughput assessments of CYP induction.

Characterization of the pharmacokinetics of a liposomal formulation of eribulin mesylate (E7389) in mice.

Eribulin mesylate (E7389), a tubulin and microtubule inhibitor, has been approved to treat metastatic breast cancer in certain patient populations. A liposomal formulation of E7389, E7389-LF, aims to increase the therapeutic profile of E7389. As determining the free drug concentration is crucial for the assessment of efficacy and toxicity of liposomal drug, in this study, an ultracentrifugation method coupled with LC-MS/MS was developed to separate the free E7389 from liposomal and protein bound E7389. The pharmacokinetics of the free E7389 after dosing either E7389 or E7389-LF was characterized. The concentration ratio of E7389 in ultracentrifuged mice plasma (UCM) vs E7389 in plasma after a 2mg/kg i.v. of E7389 ranged from 54.19% to 65.41%, which was similar to the free fraction in the mouse plasma. The respective concentration ratio of E7389 in UCM vs E7389 in plasma after a 2mg/kg i.v. of E7389-LF ranged from 0.07% to 0.59%, and the exposure, expressed as AUC, of UCM/plasma ratio was determined to be 0.2%. Pharmacokinetic modeling was performed to estimate the release kinetics of E7389 from E7389-LF, and the release was best described by a first order rate constant k(rel) 0.078 h(-1). Sensitivity analysis demonstrated that further decrease the release rate constant by adjusting liposome formulation would lead to decreased C(max) and much longer half-life of UCM E7389, which might result in better efficacy and lower toxicity.

Pharmacokinetic characterization of a natural product-inspired novel MEK1 inhibitor E6201 in preclinical species.

PURPOSE: E6201 is a natural product-inspired novel inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEK1) and other kinases and is currently under development as an anticancer (parenteral administration) and antipsoriasis agent (topical application). In vitro and in vivo preclinical studies were performed to characterize the pharmacokinetics of E6201. Allometric scaling was applied to predict human pharmacokinetics of E6201. METHODS: In vitro metabolism studies for CYP induction and CYP inhibition were conducted using human hepatocytes and microsomes, respectively. Metabolic stability using microsomes and protein-binding studies using pooled plasma were performed for mice, rats, dogs, and human. Pharmacokinetics of E6201 and its isomeric metabolite, ER-813010, in mice, rats, and dogs was determined following single IV administration of E6201 at three dose levels. Bioanalysis was performed using LC/MS/MS. Pharmacokinetic parameters were determined using non-compartmental analysis, and allometric scaling with a two-compartment model was used to predict E6201 pharmacokinetics in humans. RESULTS: E6201 showed high plasma protein binding (>95%), and metabolic stability half-life ranged from 36 to 89 min across species. In vitro CYP inhibition (CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A) and CYP induction (CYP1A, 3A, 2C9, and 2C19) suggested no inhibitory or induction effect on the tested human CYPs up to 10 µM of E6201. Pharmacokinetics of E6201 in mice, rats, and dogs was characterized by mean clearance ranging from 3.45 to 10.92 L/h/kg, distribution volume ranging from 0.63 to 13.09 L/kg, and elimination half-life ranging from 0.4 to 1.6 h. ER-813010 was detected in all species with metabolite to parent exposure ratio (AUC(R)) ranging from 3.1 to 33.4% and exhibited fast elimination (<3 h). The allometry predicted high clearance and large volume of distribution of E6201 in humans and was in general in good agreement with the observed first human subject pharmacokinetics. CONCLUSIONS: E6201 exhibited high clearance, high to moderate distribution, and fast elimination in preclinical species. In vitro results suggested that E6201 has low risk of drug-drug interactions due to CYP inhibition and induction in humans. In the first-in-man study, E6201 exhibited high clearance, which was well predicted by allometric scaling.

Safety, pharmacokinetics and pharmacodynamics of four-hour intravenous infusions of eritoran in healthy Japanese and Caucasian men.

Eritoran, a synthetic analogue of lipid A, has been shown to bind to TLR4/MD-2 complex and thereby block the interaction of endotoxins with TLR4. We report here the results of a study conducted to assess the single-dose safety and tolerability, as well as the pharmacokinetics and pharmacodynamics, of eritoran infusion in Japanese and Caucasian healthy adult men. Sixty-four men (aged 20-45 years; body mass index 18-30 kg/m(2)) were randomized into four groups: 4-mg total dose (six Japanese and six Caucasian men); 12-mg total dose (12 Japanese and 12 Caucasian men); 28-mg total dose (six Japanese and six Caucasian men); and placebo (eight Japanese and eight Caucasian men). Eritoran in single doses up to 28 mg over 4 h was well tolerated, with no apparent ethnic differences noted. Plasma concentrations were slightly higher in Japanese versus Caucasian men; these differences were not significant after adjustment for differences in body mass (clearance: approximately 1.2 ml/h/kg; volume of distribution at steady state: approximately 0.07 l/kg). The ex vivo endotoxin inhibitory activity of eritoran was similar in Japanese and Caucasian men. The data do not indicate any need for clinical dose adjustment for possible ethnic-based differences in drug distribution or metabolism.

Investigation of the metabolism of rufinamide and its interaction with valproate.

Rufinamide was evaluated in vitro to determine which enzyme(s) are responsible for rufinamide hydrolysis and whether valproate, one of its metabolites (valproyl-CoA), and/or the rufinamide hydrolysis product (CGP 47292) could inhibit hydrolysis. Rufinamide hydrolysis was mediated primarily by human carboxylesterase (hCE) 1 and was nonsaturable up to 500 µM. Two-thirds of rufinamide hydrolysis was estimated to occur in human microsomes and one-third in cytosol. Valproate was a selective inhibitor for hCE1 compared to hCE2 and inhibition had a greater impact on rufinamide hydrolysis in microsomes than in cytosol. Valproyl-CoA caused similar inhibition of rufinamide hydrolysis in both microsomes and cytosol. Carboxylesterases were not significantly inhibited by CGP 47292. Inhibition of in vitro rufinamide hydrolysis by valproate could offer an explanation for the observed in vivo drug-drug interaction between the two antiepileptic drugs.

Phase I study of eribulin mesylate administered once every 21 days in patients with advanced solid tumors.

PURPOSE: To evaluate the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and pharmacokinetics of eribulin mesylate (E7389), a halichondrin B analogue, administered every 21 days in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Eribulin mesylate was given as a 1-hour infusion every 21 days at doses of 0.25, 0.5, 1, 2, 2.8, and 4 mg/m(2). The MTD was identified using an accelerated titration design. The pharmacokinetics of eribulin were evaluated in the plasma and urine with the first dose. RESULTS: Twenty-one patients were enrolled. At 4 mg/m(2), three patients experienced a DLT of febrile neutropenia on day 7. The dose level was reduced to 2.8 mg/m(2) where two of three patients experienced dose-limiting febrile neutropenia. Six additional patients were enrolled at 2 mg/m(2) (seven patients in total received this dose) and one of these patients experienced a neutropenic DLT. The MTD of eribulin mesylate was therefore 2 mg/m(2). Nonhematologic toxicities included alopecia, fatigue, anorexia, and nausea. Pharmacokinetic analysis showed linear kinetics for eribulin over the dose range studied and a terminal half-life of 2 days. The plasma-concentration-time profile exhibited a rapid distribution phase followed by a slow elimination phase. Drug clearance was nonrenal. One patient with non-small cell lung cancer achieved an unconfirmed partial response and 12 patients had stable disease. CONCLUSIONS: Eribulin mesylate administered as a 1-hour infusion every 21 days has a manageable toxicity profile at 2 mg/m(2), with further dose escalation limited by neutropenia.

Preferential inducibility of CYP1A1 and CYP1A2 by TCDD: differential regulation in primary human hepatocytes versus transformed human cells.

Cytochrome P4501A1 (CYP1A1) induction, a marker of aryl hydrocarbon (Ah) receptor activation, has been associated with carcinogenicity of the environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Consistently, we show that TCDD treatment led to induction of CYP1A1 in responsive human cancer cell lines including HepG2, LS174T, and MCF-7, as determined by Western blotting and CYP1A form-selective R-warfarin 6- and 8-hydroxylation. TCDD, however, preferably induced CYP1A2, not CYP1A1, in primary human hepatocytes. Such CYP1A form-preferred induction at the protein level was apparently uncorrelated with non-preferred mRNA induction in any cells studied. Moreover, while both genes were up-regulated by TCDD in primary hepatocytes and HepG2 cells, the induction of CYP1A1 and CYP1A2 at the mRNA level was distinguishable, indicated by the marked differences in activation kinetics and the response to the protein synthesis inhibitors, anisomycin and cycloheximide. Furthermore, formation of total benzo(a)pyrene (BaP)-DNA adducts was not altered following BaP exposure in TCDD-treated primary hepatocytes, whereas significantly elevated, in a CYP1A1-dependent manner, in the treated HepG2 cells. Taken together, our findings, demonstrating the complexities of TCDD-associated human Ah receptor function and differential regulations of CYP 1A enzymes, suggest clearly the need for caution when extrapolating data obtained in cell-based models.

In vitro interactions between a potential muscle relaxant E2101 and human cytochromes P450.

E2101 or N-methyl-[1-[1-(2-fluorophenethyl)piperidine-4-yl]-1H-indol-6-yl] acetamide, an antagonist of 5-hydroxytryptamine receptor subtypes 1A and 2, is currently under development for the potential treatment of skeletal muscle associated spasticity. Here we characterized the in vitro metabolism of E2101 using human liver enzymes including human liver microsomal preparations, human liver S9 fractions, and individual forms of recombinant cytochromes P450 (P450s). Our results showed that E2101 was metabolized by P450s to form monohydroxylated (M1 and M2), dihydroxylated (M3), and N-dealkylated metabolites (M4). The structures of these major microsomal metabolites were proposed based on LC/MS/MS analyses. All four metabolites, M1-M4, were formed by CYP3A4. Metabolites, M1, M2, and M4, were also formed by CYP2C19 and M2 and M3 by CYP2D6. The potential P450 inhibition and induction of E2101 were also evaluated. E2101 was determined to be a competitive inhibitor of CYP2C19 and CYP2D6 with K(i) of 15 and 48 microM, respectively, as determined by both Dixon plots and simultaneously nonlinear regression analyses. Induction of major P450 expression was not detected immunochemically after 72-h exposure to 10 or 50 microM E2101 in primary hepatocyte cultures obtained from three subjects. Taken together, E2101 is expected to metabolically interact with major human P450 enzymes including CYP2C19, CYP2D6, and CYP3A4, and a low risk of drug-drug interaction would be anticipated in clinical studies.

Extended in vivo pharmacodynamic activity of E5564 in normal volunteers with experimental endotoxemia [corrected].

E5564 (alpha-D-glucopyranose) is a synthetic antagonist of bacterial endotoxin that has been shown to completely block human endotoxin response. Low doses of E5564 (0.35-3.5 mg) have a long pharmacokinetic half-life, but a surprisingly short ex vivo and in vivo pharmacodynamic half-life (generally less than several hours). To determine whether extended antagonistic activity can be achieved in vivo, this study assesses the pharmacodynamic activity of 4- and 72-h infusions of E5564 into normal volunteers. Administration of 3.5 mg of E5564/h x 72 h completely blocked effects of endotoxin challenge at the end of dosing (72 h), and at 48 and 72 h postdosing. Similarly, a 4-h infusion of E5564, 3 mg/h completely blocked endotoxin administered 8 h postdosing. A lower dose of E5564, 0.5 mg/h x 4 h, ameliorated but did not block most effects of endotoxin 8 h postdosing (p <0.05). Finally, the effect of varying plasma lipoprotein content on E5564 activity was studied in subjects having high or low cholesterol levels (>180 or <140 mg/dl) after 72-h infusion of 252 mg of E5564. No differences were observed. These results demonstrate that E5564 blocks the effects of endotoxin in a human model of clinical sepsis and indicate its potential in the treatment and/or prevention of clinical sepsis.