Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product, or procedure?

It is not clear whether improvement in environmental decontamination is more efficiently achieved through changes in cleaning products, cleaning procedures, or performance of cleaning personnel. To assess the impact of cleaning performance on environmental contamination with vancomycin-resistant enterococci (VRE), we conducted a sequential trial in which a multifaceted environmental cleaning improvement intervention was introduced in a medical intensive care unit and respiratory step-down unit. The intervention included educational lectures for housekeepers and an observational programme of their activities without changes in cleaning products or written procedures. Following these interventions, the proportion of environmental sites cleaned improved from 49% to 85% (P<0.001); contamination of environmental sites declined from 21% to 8% (P<0.0001) before cleaning and from 13% to 8% (P<0.0001) after cleaning. The improved cleaning and contamination rates persisted in a washout period. In a multivariate model, cleaning thoroughness strongly influenced the degree of environmental contamination, with a 6% decline in VRE prevalence with every 10% increase in percentage of sites cleaned. These findings suggest that surface contamination with VRE is due to a failure to clean rather than to a faulty cleaning procedure or product.

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 2: N-acetamidoimidazoles.

Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.

Potent noncovalent thrombin inhibitors that utilize the unique amino acid D-dicyclohexylalanine in the P3 position. Implications on oral bioavailability and antithrombotic efficacy.

In an effort to prepare orally bioavailable analogs of our previously reported thrombin inhibitor 1, we have synthesized a series of compounds that utilize the unique amino acid D-dicyclohexylalanine as a P3 ligand. The resulting compounds are extremely potent and selective thrombin inhibitors, and the N-terminal Boc derivative 8 exhibited excellent oral bioavailability and pharmacokinetics in both rats and dogs. The des-Boc analog 6 was not orally bioavailable in rats. The high level of oral bioavailability observed with 8 appears to be a direct function of its increased lipophilicity versus other close analogs. Although increased lipophilicity may serve to increase the oral absorption of tripeptide thrombin inhibitors, it also appears to have detrimental effects on the antithrombotic properties observed with the compounds. Compound 6 performed extremely well in our in vivo antithrombotic assay, while the much more lipophilic but essentially equipotent analog 8 performed poorly. We have found that in general with this series of thrombin inhibitors as well as with other unreported series, increased lipophilicity and the associated increases in plasma protein binding have detrimental effects on 2X APTT values and subsequent performance in in vivo antithrombotic models.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Discovery and development of the novel potent orally active thrombin inhibitor N-(9-hydroxy-9-fluorenecarboxy)prolyl trans-4-aminocyclohexylmethyl amide (L-372,460): coapplication of structure-based design and rapid multiple analogue synthesis on solid support.

Early studies in these laboratories of peptidomimetic structures containing a basic P1 moiety led to the highly potent and selective thrombin inhibitors 2 (Ki = 5.0 nM) and 3 (Ki = 0.1 nM). However, neither attains significant blood levels upon oral administration to rats and dogs. With the aim of improving pharmacokinetic properties via a more diverse database, we devised a resin-based route for the synthesis of analogues of these structures in which the P3 residue is replaced with a range of lipophilic carboxylic amides. Assembly proceeds from the common P2-P1 template 7 linked via an acid-labile carbamate to a polystyrene support. Application of the methodology in a repetitive fashion afforded several interesting analogues out of a collection of some 200 compounds. Among the most potent of the group, N-(9-hydroxy-9-fluorenecarboxy)-prolyl trans-4-aminocyclohexylmethyl amide (L-372,460 8, Ki = 1.5 nM), in addition to being fully efficacious in a rat model of arterial thrombosis at an infusion rate of 10 micrograms/kg/min, exhibits oral bioavailability of 74% in dogs, and oral bioavailability of 39% in monkeys with a serum half-life of just under 4 h. On the basis of its favorable biological properties, inhibitor 8 has been subject to further evaluation as a possible treatment for thrombogenic disorders.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Inhibition of thrombin by peptides containing Lysyl-alpha-keto carbonyl derivatives.

Several H-N-Me-D-Phe-Pro-Lysyl-alpha-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by alpha-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The alpha-keto carbonyl inhibitors bound thrombin with a second order rate constant (k1 1-4 microM-1s-1) that was 10-100-fold slower than that expected for a diffusion-controlled reaction. Certain alpha-keto carbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10-19%) in rats demonstrated for three of the alpha-keto carbonyl thrombin inhibitors suggests the possibility that alpha-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.

Alterations in peripheral blood lymphocyte cytokine expression in obesity.

Obesity is characterized by alterations in immune and inflammatory function. In order to evaluate the potential role of cytokine expression by peripheral blood mononuclear cells (PBMC) in obesity-associated inflammation, we studied serum protein levels and mRNA levels in PBMC of interleukin (IL)-6, IL-1beta, tumour necrosis factor (TNF)-alpha and IL-1Ra in nine lean and 10 obese subjects. Serum IL-1beta was undetectable, IL-1Ra serum levels were elevated, serum levels of TNF-alpha were decreased and serum levels of IL-6 were similar in obese subjects compared to lean subjects, while transcript levels of IL-6, IL-1beta and TNF-alpha, but not IL-1Ra, were decreased in PBMC from obese subjects. PBMC from obese subjects did, however, up-regulate cytokine expression in response to leptin. Thus, obesity-associated changes in IL-1Ra serum levels and IL-6 mRNA levels were not correlated with changes in cognate mRNA and serum levels, respectively, while TNF-alpha serum levels and PBMC mRNA levels were both decreased in obese patients. While immune alterations in obesity are manifest in peripheral blood lymphocytes, the general lack of correlation between altered serum levels and altered PBMC gene expression suggests that PBMC may not be the source of aberrant serum cytokine levels in obesity.

Nonpeptide glycoprotein IIb/IIIa inhibitors. 8. Antiplatelet activity and oral antithrombotic efficacy of L-734,217.

The antiplatelet activity of L-734,217, a nonpeptide platelet GPIIb/IIIa antagonist, was evaluated in the rat, guinea pig and dog. IC50 for inhibition of in vitro platelet aggregation for these species (agonists: adenosine diphosphate, collagen) were rat, 838,000 and > 1,100,000 nM; guinea pig, 124 and 156 nM; dog, 42 and 50 nM. In an in vivo rat/in vitro dog platelet aggregation assay, effective antiaggregatory plasma concentrations of L-734,217 were achieved after 8.0 to 16.0 mg/kg p.o. vs. 0.3 to 1.0 mg/kg i.v. to rats. Delays in platelet-dependent hemostatic plug formation in severed mesenteric arteries were observed after 2.0 to 5.0 mg/kg p.o. vs. 0.1 to 0.2 mg/kg i.v. to guinea pigs. Dose-dependent inhibitions of ex vivo platelet aggregation after 0.3 to 3.0 mg/kg p.o. and 0.03 to 0.3 mg/kg i.v. L-734,217 to conscious dogs yielded estimates of 8 to 16% oral bioavailability. The antiplatelet activity of 3.0 mg/kg p.o. L-734,217 in dogs was unaffected by dosage form or food. In a conscious dog model of left circumflex coronary artery electrolytic lesion, 3.0 mg/kg p.o. L-734,217 q4 to 8 hr reduced thrombus mass, prevented occlusive coronary artery thrombosis and reduced or prevented myocardial infarction and ventricular ectopy. In anesthetized dogs, a dissociation between inhibition of ex vivo platelet aggregation and template bleeding time prolongation was observed with i.v. L-734,217. The results of the coadministration of heparin, aspirin and L-734,217 to anesthetized dogs suggested a synergistic effect on template bleeding time with no effect on plasma L-734,217 concentrations. These findings indicate L-734,217 to be an important lead structure for the development of therapeutically useful oral antiplatelet agents.

Bicyclic pyridones as potent, efficacious and orally bioavailable thrombin inhibitors.

A new class of conformationally constrained thrombin inhibitors is described. These compounds contain a unique bicyclic pyridone scaffold which serves as a P3P2 dipeptide surrogate. The synthesis and antithrombotic activity of these inhibitors is reported.

C6 modification of the pyridinone core of thrombin inhibitor L-374,087 as a means of enhancing its oral absorption.

1 (L-374,087) is a potent, selective, efficacious, and orally bioavailable thrombin inhibitor that contains a core 3-amino-2-pyridinone moiety. Replacement of the C6 pyridinone methyl group of 1 by a propyl group gave 5 (L-375,052), which retained all the excellent properties of 1, and also yielded higher plasma levels after oral dosing in dogs and rats.

L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor.

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.