P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.

Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes as thrombin inhibitors.

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.

Characterization of the cell surface glycolipid from Spirochaeta aurantia.

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

Risk of hand or glove contamination after contact with patients colonized with vancomycin-resistant enterococcus or the colonized patients' environment.

OBJECTIVE: To estimate the level of hand or glove contamination with vancomycin-resistant enterococci (VRE) among healthcare workers (HCWs) who touch a patient colonized with VRE and/or the colonized patient's environment during routine care. DESIGN: Structured observational study. SETTING: Medical intensive care unit of a 700-bed, tertiary-care teaching hospital. PARTICIPANTS: VRE-colonized patients and their caregivers. METHODS: We obtained samples from sites on the intact skin of 22 patients colonized with VRE and samples from sites in the patients' rooms, before and after routine care, during 27 monitoring episodes. A total of 98 unique HCWs were observed during 131 HCW observations. Observers recorded the sites touched by HCWs. Culture samples were obtained from HCWs' hands and gloves before and after care. RESULTS: VRE were isolated from a mean (+/-SD) of 55% +/- 24% of patient sites (n=256) and 17% +/- 12% of environmental sites (n=1,572). Most HCWs (131 [56%]) touched both the patient and the patient's environment; no HCW touched only the patient. Of 103 HCWs whose hand samples were negative for VRE when they entered the room, 52% contaminated their hands or gloves after touching the environment, and 70% contaminated their hands or gloves after touching the patient and the environment (P=.101). In a univariate logistic regression model, the risk of hand or glove contamination was associated with the number of contacts made (odds ratio, 1.1 [95% confidence interval, 1.01-1.19). In a multivariate model, the effect of the number of contacts could not be distinguished from the effect of type of contact (ie, touching the environment alone or touching both the patient and the environment). Overall, 37% of HCWs who did not wear gloves contaminated their hands, and 5% of HCWs who wore gloves did so (an 86% difference). CONCLUSION: HCWs were nearly as likely to have contaminated their hands or gloves after touching the environment in a room occupied by a patient colonized by VRE as after touching the colonized patient and the patient's environment. Gloves were highly protective with respect to hand contamination.

Identification and characterization of 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate, an orally bioavailable, brain penetrant NR2B selective N-methyl-D-aspartate receptor antagonist.

The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease.

Reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures.

BACKGROUND: The role of environmental contamination in nosocomial cross-transmission of antibiotic-resistant bacteria has been unresolved. Using vancomycin-resistant enterococci (VRE) as a marker organism, we investigated the effects of improved environmental cleaning with and without promotion of hand hygiene adherence on the spread of VRE in a medical intensive care unit. METHODS: The study comprised a baseline period (period 1), a period of educational intervention to improve environmental cleaning (period 2), a "washout" period without any specific intervention (period 3), and a period of multimodal hand hygiene intervention (period 4). We performed cultures for VRE of rectal swab samples obtained from patients at admission to the intensive care unit and daily thereafter, and we performed cultures of environmental samples and samples from the hands of health care workers twice weekly. We measured patient clinical and demographic variables and monitored intervention adherence frequently. RESULTS: Our study included 748 admissions to the intensive care unit over a 9-month period. VRE acquisition rates were 33.47 cases per 1000 patient-days at risk for period 1 and 16.84, 12.09, and 10.40 cases per 1000 patient-days at risk for periods 2, 3, and 4, respectively. The mean (+/-SD) weekly rate of environmental sites cleaned increased from 0.48+/-0.08 at baseline to 0.87+/-0.08 in period 2; similarly high cleaning rates persisted in periods 3 and 4. Mean (+/-SD) weekly hand hygiene adherence rate was 0.40+/-0.01 at baseline and increased to 0.57+/-0.11 in period 2, without a specific intervention to improve adherence, but decreased to 0.29+/-0.26 in period 3 and 0.43+/-0.1 in period 4. Mean proportions of positive results of cultures of environmental and hand samples decreased in period 2 and remained low thereafter. In a Cox proportional hazards model, the hazard ratio for acquiring VRE during periods 2-4 was 0.36 (95% confidence interval, 0.19-0.68); the only determinant explaining the difference in VRE acquisition was admission to the intensive care unit during period 1. CONCLUSIONS: Decreasing environmental contamination may help to control the spread of some antibiotic-resistant bacteria in hospitals.

Transfer of vancomycin-resistant enterococci via health care worker hands.

BACKGROUND: The roles of the contaminated hospital environment and of patient skin carriage in the spread of vancomycin-resistant enterococci (VRE) are uncertain. Transfer of VRE via health care worker (HCW) hands is assumed but unproved. We sought to determine the frequency of VRE transmission from sites in the environment or on patients' intact skin to clean environmental or skin sites via contaminated hands of HCWs during routine care. METHODS: We cultured sites on the intact skin of 22 patients colonized by VRE, as well as sites in the patients' rooms, before and after routine care by 98 HCWs. Observers recorded sites touched by HCWs. Cultures were obtained from HCW hands and/or gloves before and after care. All isolates underwent pulsed-field gel electrophoresis. We defined a transfer to have occurred when a culture-negative site became positive with a VRE pulsotype after being touched by an HCW who had the same pulsotype on his or her hands or gloves and who had previously touched a colonized or contaminated site. RESULTS: Health care workers touched 151 negative sites after touching a site that was positive for VRE. Sixteen negative sites (10.6%) became positive after contact. The percentage of times that contact with a site led to a transfer was highest for antecubital fossae and blood pressure cuffs. CONCLUSIONS: Vancomycin-resistant enterococci were transferred from contaminated sites in the environment or on patients' intact skin to clean sites via HCW hands or gloves in 10.6% of opportunities. Controlling VRE by decontaminating the environment and patients' intact skin may be an important adjunctive infection control measure.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Differential effects of sodium nitroprusside and hydralazine in a rat model of topical FeCl3-induced carotid artery thrombosis.

INTRODUCTION: In the rat model of topical ferric chloride-induced carotid artery thrombosis, a transient blood flow velocity (VEL) increase is observed immediately following ferric chloride application. The immediacy of the response suggested vasoconstriction, as thrombotic narrowing of the vessel lumen was hypothesized to be too slow to account for the rapidity of the response. METHODS: To explore this phenomenon, the effects of two mechanistically distinct vasodilators, sodium nitroprusside (SNP) and hydralazine (HYD), on velocity increase, ex vivo platelet aggregation and thrombosis, were assessed in the rat ferric chloride-induced thrombosis model. RESULTS: Sodium nitroprusside (10, 30 and 50 microg/kg/min i.v.) and hydralazine (0.1, 0.3 and 1.0 mg/kg/min i.v.) reduced the mean arterial pressure with the higher dose regimens eliciting equivalent hypotensive effects. Both sodium nitroprusside and hydralazine blunted the initial velocity increase, but only sodium nitroprusside significantly reduced the incidence of thrombotic occlusion. No differences in ex vivo platelet aggregation responses to adenosine diphosphate (ADP), collagen (COLL) and arachidonic acid (AA) were observed between the sodium nitroprusside and hydralazine treatment groups. However, platelet aggregation response to thrombin was significantly reduced in the 50 microg/kg/min i.v. sodium nitroprusside compared to the 1.0 mg/kg/min i.v. hydralazine and vehicle groups. CONCLUSIONS: Inhibition of the initial velocity increase by two mechanistically distinct vasodilators, and the dissociation between this velocity change and antithrombotic efficacy, support the hypothesis that the early velocity increase results from a change in vascular tone rather than due to enhanced platelet activation and thrombus formation. Inhibition of thrombin-induced platelet activation may contribute to the antithrombotic actions of sodium nitroprusside in this preparation.

The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components.

As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9-12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.

Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy.

TLRs constitute an essential family of pattern recognition molecules that, through direct recognition of conserved microbial components, initiate inflammatory responses following infection. In this role, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. In this study, we report that I602S, a common single nucleotide polymorphism within TLR1, is associated with aberrant trafficking of the receptor to the cell surface and diminished responses of blood monocytes to bacterial agonists. When expressed in heterologous systems, the TLR1 602S variant, but not the TLR1 602I variant, exhibits the expected deficiencies in trafficking and responsiveness. Among white Europeans, the 602S allele represents the most common single nucleotide polymorphism affecting TLR function identified to date. Surprisingly, the 602S allele is associated with a decreased incidence of leprosy, suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion.

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus.

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPS-binding protein was more important for LPS- than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligand-binding receptors, which may differentially influence innate immune responses.

Domain exchange between human toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination.

Among the 10 human Toll-like receptors (TLRs), TLR2 appears to be unique in its requirement for cooperation with other TLRs, namely TLR1 and TLR6, to mediate cell signaling. Through reconstitution experiments, we have defined more precisely the function of these human TLRs. Human colonic epithelial cells cotransfected with TLR1 and -2 preferentially respond to a synthetic tripalmitoylated bacterial lipopeptide analogue (Pam(3)CSK(4)). However, examination of a wide variety of lipopeptide derivatives indicates that recognition by human TLR1 and -2 does not strictly correlate with the number or position of the acyl chains on the modified cysteine residue. Conversely, human TLR2 and -6 exclusively respond to lipopeptides possessing a diacylglycerol group. Most surprisingly, we have found that an R stereoisomer of diacylated macrophage-activating lipopeptide 2 (MALP-2) exclusively activates epithelial cells through TLR6 and -2 but not through TLR1 and -2. These results suggest that the chirality of the central carbon of the diacylglycerol group of these agonists is a structural determinant for human TLR recognition. Examination of chimeric receptors, generated by domain exchange between TLR1 and -6, has revealed that leucine-rich repeats 9-12 of the extracellular domain enable these receptors to discriminate between structurally similar lipopeptides. However, additional chimeric constructs reveal that this region alone is not sufficient to generate receptors that can functionally cooperate with TLR2. Our results support the idea that TLR1 and TLR6 diverged during evolution to differentially recognize natural lipoprotein structures and that this function has been conserved with respect to the human receptors.

Toll-like receptor 2 mediates cellular activation by the B subunits of type II heat-labile enterotoxins.

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1beta (IL-1beta), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-kappaB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities.

The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGLB.

In an attempt to isolate lipopolysaccharide from Spirochaeta aurantia, Darveau-Hancock extraction of the cell mass was performed. While no lipopolysaccharide was found, two carbohydrate-containing compounds were detected. They were resolved by size-exclusion chromatography into high molecular mass (LGLA) and low molecular mass (LGLB) fractions. Here we present the results of the analysis of the glycolipid LGLB. Deacylation of LGLB with hydrazine and separation of the products by using anion-exchange chromatography gave two major products. Their structure was determined by using chemical methods, NMR and mass spectrometry. All monosaccharides had the D-configuration, and aspartic acid had the L-configuration. Intact LGLB contained two fatty groups at O-2 and O-3 of the glycerol residue. Nonhydroxylated C14 to C18 fatty acids were identified, which were predominantly unsaturated or branched. LGLB was able to gel Limulus amebocyte lysate, albeit at a lower level than that observed for Escherichia coli O113 lipopolysaccharide. However, even large amounts of LGLB were unable to stimulate any Toll-like receptor (TLR) examined, including TLR4 and TLR2, previously shown to be sensitive to lipopolysaccharide and glycolipids from diverse bacterial origins, including other spirochetes.

3-amino-4-sulfonylpyridinone acetamide and related pyridothiadiazine thrombin inhibitors.

We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-substituent, and forms a hydrogen bond to the insertion loop tyrosine OH. We also describe a related series of fused bicyclic dihydrothiadiazinedioxide derivatives, of which one had improved pharmacokinetics in dogs after oral dosing.

Low molecular weight thrombin inhibitors with excellent potency, metabolic stability, and oral bioavailability.

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.

Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.