Showing papers in "Current Topics in Medicinal Chemistry in 2001"

Toxic Metals and Oxidative Stress Part I: Mechanisms Involved in Me-tal induced Oxidative Damage

Abstract: Toxic metals (lead, cadmium, mercury and arsenic) are widely found in our environment. Humans are exposed to these metals from numerous sources, including contaminated air, water, soil and food. Recent studies indicate that transition metals act as catalysts in the oxidative reactions of biological macromolecules therefore the toxicities associated with these metals might be due to oxidative tissue damage. Redox-active metals, such as iron, copper and chromium, undergo redox cycling whereas redox-inactive metals, such as lead, cadmium, mercury and others deplete cells major antioxidants, particularly thiol-containing antioxidants and enzymes. Either redox-active or redox-inactive metals may cause an increase in production of reactive oxygen species (ROS) such as hydroxyl radical (HO.), superoxide radical (O2.-) or hydrogen peroxide (H2O2). Enhanced generation of ROS can overwhelm cells intrinsic antioxidant defenses, and result in a condition known as “oxidative stress”. Cells under oxidative stress display various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA. Consequently, it is suggested that metal-induced oxidative stress in cells can be partially responsible for the toxic effects of heavy metals. Several studies are underway to determine the effect of antioxidant supplementation following heavy metal exposure. Data suggest that antioxidants may play an important role in abating some hazards of heavy metals. In order to prove the importance of using antioxidants in heavy metal poisoning, pertinent biochemical mechanisms for metal-induced oxidative stress should be reviewed.

Physicochemical Profiling (Solubility, Permeability and Charge State)

Abstract: About 30% of drug candidate molecules are rejected due to pharmacokinetic-related failures. When poor pharmaceutical properties are discovered in development, the costs of bringing a potent but poorly absorbable molecule to a product stage by "formulation" can become very high. Fast and reliable in vitro prediction strategies are needed to filter out problematic molecules at the earliest stages of discovery. This review will consider recent developments in physicochemical profiling used to identify candidate molecules with physical properties related to good oral absorption. Poor solubility and poor permeability account for many PK failures. FDA's Biopharmaceutics Classification System (BCS) is an attempt to rationalize the critical components related to oral absorption. The core idea in the BCS is an in vitro transport model, centrally embracing permeability and solubility, with qualifications related to pH and dissolution. The objective of the BCS is to predict in vivo performance of drug products from in vitro measurements of permeability and solubility. In principle, the framework of the BCS could serve the interests of the earliest stages of discovery research. The BCS can be rationalized by considering Fick's first law, applied to membranes. When molecules are introduced on one side of a lipid membrane barrier (e.g., epithelial cell wall) and no such molecules are on the other side, passive diffusion will drive the molecules across the membrane. When certain simplifying assumptions are made, the flux equation in Fick's law reduces simply to a product of permeability and solubility. Many other measurable properties are closely related to permeability and solubility. Permeability (Pe) is a kinetic parameter related to lipophilicity (as indicated by the partition and distribution coefficients, log P and log D). Retention (R) of lipophilic molecules by the membrane (which is related to lipophilicity and may predict PK volumes of distribution) influences the characterization of permeability. Furthermore, strong drug interactions with serum proteins can influence permeability. The unstirred water layer on both sides of the membrane barrier can impose limits on permeability. Solubility (S) is a thermodynamic parameter, and is closely related to dissolution, a kinetic parameter. The unstirred water layer on the surfaces of suspended solids imposes limits on dissolution. Bile acids effect both solubility and dissolution, by a micellization effect. For ionizable molecules, pH plays a crucial role. The charge state that a molecule exhibits at a particular pH is characterized by the ionization constant (pKa) of the molecule. Buffers effect pH gradients in the unstirred water layers, which can dramatically affect both permeability and dissolution of ionizable molecules. In this review, we will focus on the emerging instrumental methods for the measurement of the physicochemical parameters Pe, S, pKa, R, log P, and log D (and their pH-profiles). These physicochemical profiles can be valuable tools for the medicinal chemists, aiding in the prediction of in vivo oral absorption.

Oxytosis: A novel form of programmed cell death.

Abstract: Extensive nerve cell death occurs during the development of the central nervous system as well as in episodes of trauma and in neurodegenerative disease. The mechanistic details of how these cells die are poorly understood. Here we describe a unique oxidative stress-induced programmed cell death pathway called oxytosis, and outline pharmacological approaches which interfere with its execution. Oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake through the cystine/glutamate antiporter leading to a depletion of glutathione, is used as an example of oxytosis. It is shown that there is a sequential requirement for de novo macromolecular synthesis, lipoxygenase activation, reactive oxygen species production, and the opening of cGMP-gated channels which allow the influx of extracellular calcium. The translation initiation factor elF2alpha plays a central role in this pathway by regulating the levels of glutathione. Finally, examples are given in which the reduction in glutathione, the production of reactive oxygen species, and calcium influx can be experimentally manipulated to prevent cell death. Data are reviewed which suggest that oxytosis may be involved in nerve cell death associated with nervous system trauma and disease.

Role of flavonoids in oxidative stress.

Abstract: Flavonoids are a group of naturally occuring compounds which are widely distributed in nature. Epidemiological evidence suggests an inverse relationship between dietary intake of flavonoids and cardiovascular risk. The biological activities of flavonoids are related to their antioxidative effects. But a number of studies have found both anti and prooxidant effects for many of these compounds. This review article presents the synthetic pathways of flavonoids and discusses the structure-activity relationships between, xanthine oxidase inhibitive activities and their chemical structures, between the antioxidant and prooxidant activities and the chemical structure. Then we will show the antioxidant properties of new flavonoids in a few models. In these compounds one or two di-tert-butylhydroxyphenyl (DBHP) groups replace the catechol moiety at the position 2 of the benzopyrane heterocycle. New structures are compared with quercetin and BHT in an LDL-oxidation system, in protecting cultured bovine aortic endothelial cells against mO-LDL cytotoxicity and on myocardial functional recovery during reperfusion after 30 min global ischemia in isolated rat hearts.

From cocaine to ropivacaine: the history of local anesthetic drugs.

Abstract: In 1850, about three centuries after the conquest of Peru by Pizzaro, the Austrian von Scherzer brought a sufficient quantum of coca leaves to Europe to permit the isolation of cocaine. As suggested by his friend Sigmund Freud, descriptions of the properties of the coca prompted the Austrian Koller to perform in 1884 the first clinical operation under local anesthesia, by administration of cocaine on the eye. The use of cocaine for local and regional anesthesia rapidly spread throughout Europe and America. The toxic effects of cocaine were soon identified resulting in many deaths among both patients and addicted medical staff. Local anesthesia was in a profound crisis until the development of modern organic chemistry which led to the synthesis of pure cocaine in 1891. New amino ester local anesthetics were synthesized between 1891 and 1930, such as tropocaine, eucaine, holocaine, orthoform, benzocaine, and tetracaine. In addition, amino amide local anesthetics were prepared between 1898 and 1972 including nirvaquine, procaine, chloroprocaine, cinchocaine, lidocaine, mepivacaine, prilocaine, efocaine, bupivacaine, etidocaine, and articaine. All of these drugs were ostensibly less toxic than cocaine, but they had differing amounts of central nervous system (CNS) and cardiovascular (CV) toxicity. Bupivacaine is of special interest because of its long duration of action and history of clinical application. Synthesized in 1957, the introduction of bupivacaine on the market in 1965 paralleled the progressive and cumulative reports of CNS and CV toxicity, leading to the restriction of its use and the identification of a special therapy-resistant CV toxicity. Numerous experimental studies were conducted to identify the fine cellular mechanism of this toxicity, which refines our understanding of the action of local anesthetics. The identification of optically active isomers of the mepivacaine family led to the selection of ropivacaine, a pure S-(-) enantiomer, whose toxicology was selectively and extensively studied before its introduction on the market in 1996. During the rapid and extensive use of ropivacaine in the clinic, unwanted side-effects have been found to be very limited.

Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance.

Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy. Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date. These include MexA-Mexs-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains. Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown. These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor [OMF]) and a periplasmic membrane fusion protein (MFP) that links the other two. In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing. While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P. aeruginosa makes them reasonable targets for therapeutic intervention.

Strategies for Absorption Screening in Drug Discovery and Development

Abstract: This review gives an overview of the current approaches to evaluate drug absorption potential in the different phases of drug discovery and development. Methods discussed include in silico models, artificial membranes as absorption models, in vitro models such as the Ussing chamber and Caco-2 monolayers, in situ rat intestinal perfusion and in vivo absorption studies. In silico models such as iDEA can help optimizing chemical synthesis since the fraction absorbed (Fa) can be predicted based on structural characteristics only. A more accurate prediction of Fa can be obtained by feeding the iDEA model with Caco-2 permeability data and solubility data at various pH's. Permeability experiments with artificial membranes such as the filter-IAM technology are high-throughput and offer the possibility to group compounds according to a low and a high permeability. Highly permeable compounds, however, need to be further evaluated in Caco-2 cells, since artificial membranes lack active transport systems and efflux mechanisms such as P-glycoprotein (PgP). Caco-2 and other "intestinal-like" cell lines (MDCK, TC-7, HT29-MTX, 2/4/A1) permit to perform mechanistic studies and identify drug-drug interactions at the level of PgP. The everted sac and Ussing chamber techniques are more advanced models in the sense that they can provide additional information with respect to intestinal metabolism. In situ rat intestinal perfusion is a reliable technique to investigate drug absorption potential in combination with intestinal metabolism, however, it is time consuming, and therefore not suited for screening purposes. Finally, in vivo absorption in animals can be estimated from bioavailability studies (ratio of the plasma AUC after oral and i.v. administration). The role of the liver in affecting bioavailability can be evaluated by portal vein sampling experiments in dogs.

Assessing the absorption of new pharmaceuticals.

Abstract: The advent of more efficient methods to synthesize and screen new chemical compounds is increasing the number of chemical leads identified in the drug discovery phase. Compounds with good biological activity may fail to become drugs due to insufficient oral absorption. Selection of drug development candidates with adequate absorption characteristics should increase the probability of success in the development phase. To assess the absorption potential of new chemical entities numerous in vitro and in vivo model systems have been used. Many laboratories rely on cell culture models of intestinal permeability such as, Caco-2, HT-29 and MDCK. To attempt to increase the throughput of permeability measurements, several physicochemical methods such as, immobilized artificial membrane (IAM) columns and parallel artificial membrane permeation assay (PAMPA) have been used. More recently, much attention has been given to the development of computational methods to predict drug absorption. However, it is clear that no single method will sufficient for studying drug absorption, but most likely a combination of systems will be needed. Higher throughput, less reliable methods could be used to discover 'loser' compounds, whereas lower throughput, more accurate methods could be used to optimize the absorption properties of lead compounds. Finally, accurate methods are needed to understand absorption mechanisms (efflux-limited absorption, carrier-mediated, intestinal metabolism) that may limit intestinal drug absorption. This information could be extremely valuable to medicinal chemists in the selection of favorable chemo-types. This review describes different techniques used for evaluating drug absorption and indicates their advantages and disadvantages.

Metabolism Profiling, and Cytochrome P450 Inhibition & Induction in Drug Discovery

Abstract: To reduce the high attrition rates of NCEs in preclinical and clinical development uncovering pharmacokinetics, toxicokinetics, drug metabolism, and drug-drug interactions early in drug discovery would be highly valuable. There have been many in vitro screens developed for these areas that have higher sample throughput, which is consistent with the iterative cycle of a typical drug discovery research project. We have presented the present status and given detailed descriptions of biotransformation, metabolic stability assays, identification of drug metabolizing P450 enzymes, prediction of pharmacokinetic parameters from in vitro metabolism data, structure elucidation of metabolites, CYP450 inhibition assays and CYP450 induction assays from a drug discovery perspective. Strategies for the proper sequencing of primary and secondary assays employedfor drug metabolism and CYP450 inhibition & induction is discussed.

Production of reactive oxygen species from aggregating proteins implicated in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases.

Abstract: The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), motor neurone disease and the 'prion' dementias. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the beta-amyloid (Abeta), which accumulates in the brain in AD, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown recently that solutions of Abeta liberate readily detectable amounts of hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). We have also obtained similar results with alpha-synuclein, which accumulates in Lewy bodies in PD. Our data suggest that hydrogen peroxide accumulates during Abeta or alpha-synuclein incubation and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, we now support the idea that one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD, PD, and possibly some other protein conformational diseases, could be the direct production of ROS during formation of the abnormal protein aggregates. This hypothesis suggests a novel approach to the therapy of this group of diseases.

Structural biology of bacterial iron uptake systems.

Abstract: Numerous bacterial proteins are involved in microbial iron uptake and transport and considerable variation has been found in the uptake schemes used by different bacterial species. However, whether extracting iron from host proteins such as transferrin, lactoferrin or hemoglobin or importing low molecular weight iron-chelating compounds such as heme, citrate or siderophores, Gram-negative pathogenic bacteria typically employ a specific outer membrane receptor, a periplasmic binding protein and two inner membrane associated proteins: a transporter coupled with an ATP-hydrolyzing protein. Often, studies have shown that proteins with similar function but little amino acid sequence homology are structurally related. Elucidation of the structures of the Escherichia coli outer membrane siderophore transport proteins FepA and FhuA have provided the first insights into the conformational changes required for ligand transport through the bacterial outer membrane. The variations between the structures of the prototypical periplasmic ferric binding protein FbpA from Neisseria and Haemophilus influenzae and the unusual E. coli periplasmic siderophore binding protein FhuD reveal that the different periplasmic ligand binding proteins exercise distinct mechanisms for ligand binding and release. The structure of the hemophore HasA from Serratia marcescens shows how heme may be extracted and utilized by the bacteria. Other biochemical evidence also shows that the proteins that provide energy for iron transport at the outer membrane, such as the TonB-ExbB-ExbD system, are structurally very similar across bacterial species. Likewise, the iron-sensitive gene regulatory protein Fur is found in most bacteria. To date, no structural information is available for Fur, but the structure for the related protein DxtR has been determined. Together, these three-dimensional structures complement our knowledge of iron transport systems from other pathogenic bacteria, including Pseudomonas aeruginosa, which has a number of homologous iron uptake proteins. More importantly, the current structures for iron transport proteins provide rational starting points for design of novel antimicrobial agents.

The new pre-preclinical paradigm: compound optimization in early and late phase drug discovery.

Abstract: The attrition rates of new chemical entities (NCEs) in preclinical and clinical development are staggeringly high. NCEs are abandoned due to insufficient efficacy, safety issues, and economic reasons. Uncovering drug defects that produce these failures as early as possible in drug discovery would be highly effective in lowing the cost and time of developing therapeutically useful drugs. Unfortunately, there is no single factor that can account for these NCE failures in preclinical and clinical development since factors, such as solubility, pKa, absorption, metabolism, formulation, pharmacokinetics, toxicity and efficacy, to name a few, are all interrelated. In addition, there are many problems in scaling-up drug candidates from the laboratory bench scale to the pilot plant scale. To address the problem of attrition rates of NCEs in preclinical and clinical development and drug scale-up issues, pharmaceutical companies need to reorganize their preclinical departments from a traditional linear approach to a parallel approach. In this review, a strategy is put forth to integrate certain aspects of drug metabolism/pharmacokinetics, toxicology functions and process chemistry into drug discovery. Compound optimization in early and late phase drug discovery occurs by relating factors such as physicochemical properties, in vitro absorption, in vitro metabolism, in vivo pharmacokinetics and drug scale-up issues to efficacy optimization. This pre-preclinical paradigm will improve the success rate of drug candidates entering development.

Coagulation factor Xa inhibition: biological background and rationale.

Abstract: Ischemic heart disease and cerebrovascular disease are the leading causes of death in the world. Surprisingly, these diseases are treated by relatively antiquated drugs. However, due to our improved understanding of the underlying pathology of these diseases, and a number of technological advances in tools for drug discovery and chemical optimization, an exciting new wave of antithrombotic compounds is beginning to emerge in clinical trials. These agents, referred to as direct coagulation factor Xa inhibitors, appear to provide an enhanced risk-benefit margin compared to conventional therapy. Preclinical and early clinical data gathered over the past few years suggests that direct fXa inhibitors will provide the necessary advancements in efficacy, safety, and ease of use required to displace conventional therapy. Whether or not these agents will succeed will be determined as this class of agents advances through clinical trials in the near future. This review describes some of the key studies that sparked an interest in fXa as a therapeutic target, highlighting the findings that provided important rationale for continuing the development of potent and selective direct fXa inhibitors.

Siderotyping A Powerful Tool for the Characterization of Pyoverdines

Abstract: Tools for the identification of bacteria are of great importance especially for taxonomical and medical purposes. In the case of fluorescent pseudomonads a quick and unambiguous identification is possible by methods that are referred to as siderotyping. All of them are based upon the characterization of the bacterial siderophores or the receptors expressed for the uptake of these compounds. Different microbiological and bioanalytical tests that are accurate, rapid and easy to use will be described.

Siderophore-antibiotic conjugates used as trojan horses against Pseudomonas aeruginosa.

Abstract: Pseudomonas aeruginosa is a dangerous opportunistic bacterium responsible for frequently lethal hospital (nosocomial) infections. It endangers especially severely injured patients suffering from large wounds or severe burns, as well as persons whose immune system is weakened. An extremely critical situation exists for patients suffering from mucoviscidosis (cystic fibrosis), when P. aeruginosa infects the bronchial tubes. P. aeruginosa is resistant against many disinfecting agents and, more important, an increasing number of strains especially from hospital isolates have become highly resistant against most antibiotics. The low permeability of the outer membrane and an active export mechanism for low molecular weight substances are the main reasons for the resistance. In addition, beta-lactamase activity affects treatment with beta-lactam antibiotics. An approach to overcome the problem of resistance lies in the synthesis of antibiotics conjugated with compounds active as siderophores. In this way the transport ways for iron complexes into the cell can be used ("Trojan Horse strategy"), and the presence of large substituents reduces the export and the beta-lactamase activity. The results obtained with natural (pyoverdins) and synthetic (mainly catecholate) siderophores will be reviewed.

Oxidative stress in neurodegeneration: mechanisms and therapeutic perspectives.

Abstract: Neural tissue is especially sensitive to oxidative stress, which is considered a prominent factor in both acute and chronic neurodegenerative diseases and traumatic brain insults. On this basis, therapeutical strategies centered on antioxidants and on drugs able to scavenge excess free radicals and to re-establish the redox equilibrium, have been proposed for treatment of several brain pathologies. The present paper shortly summarizes the main sources of free radical production in the brain and reviews some of the recent data on mechanisms of cellular transduction through which free radicals are believed to damage cells and, eventually, to bring them to death. Some of the most promising therapeutical perspectives for treatment of oxidative stress in neurodegeneration, are then considered. Their choice is the result of a selection, that is unavoidably due to the enormous amount of the literature data, based on personal evaluation as well as on the personal experimental experience of the author. Four main categories of possible therapeuticals are considered: inhibitors of antioxidant enzymes, endogenous antioxidants and their precursors, vitamins and related compounds, other natural antioxidants from fruits and vegetables. Some theoretical and practical issues relevant to the adoption of antioxidant therapies for neurodegeneration are highlited, with particular reference to the fact that a basal production of free radicals must be maintained in the brain due to the host of essential cellular functions subserved by them. In this connection, it seems advisable that future antioxidant strategies for neurodegeneration are based on mixtures of agents able to modulate multiple mechanisms of free radical production and scavenging, without dangerously hampering essential physiological defense based on free radical cellular signaling.

The Cardiotoxicity of Local Anesthetics: The Place of Ropivacaine

Abstract: Central and regional block procedures have a well-defined role as safe and effective methods in modern anesthesia and analgesia with long-acting local anesthetics. Recent studies have shown that the incidence of intoxication by these drugs is a rare but catastrophic event. As classic neuronal sodium channel inhibitors, local anesthetics block peripheral fast voltage-gated sodium channels on neuronal axons, and these drugs have a particularly high level of activity in the CNS and the cardiovascular system. CNS-toxicity follows a two-stage process, whereby at lower concentrations inhibitory neurons are blocked first resulting in generalized convulsions, and at higher concentrations a global CNS depression can be seen. Although seizures are an impressive clinical syndrome, they can often be treated safely without permanent damage. More important is the cardiotoxicity of these drugs, which can be divided into indirect cerebrally mediated and a direct myocardial component. Like CNS-toxicity in general, indirect cardiotoxicity demonstrates an initial stimulating effect, followed by a depressive component at higher concentrations. Direct myocardial actions are comprised of negative chronotropic, dromotropic and inotropic effects. For dromotropy, stereoselectivity was found. The S-(-)-isomers of the longacting local anesthetics were less delayed compared to racemic mixtures and the R-(+)-enantiomers. For inotropy, no stereospecific depression of this parameter was noted between isomers of ropivacaine or bupivacaine, but bupivacaine produced a significantly greater depression of LV pressure than ropivacaine, mepivacaine, or lidocaine. Pharmacokinetic differences in lipophilicity of local anesthetics correlate well with the depression mitochondrial ATP-synthesis in fast metabolizing cells. Intracellular ATP-level may be involved in contractility and resuscitation of cardiomyocytes, as be proven by in-vitro and in-vivo data. Therefore the use of pure optical S-(-)-isomers of local anesthetics may help to reduce these rare but catastrophic events. Presently, ropivacaine appears to be the safest long-acting local anesthetic.

Integration of computational analysis as a sentinel tool in toxicological assessments.

Abstract: Computational toxicity modeling can have significant impact in the drug discovery process, especially when utilized as a sentinel filter for common drug safety liabilities, such as mutagenicity, carcinogenicity and teratogenicity. This review will focus on the strengths and limitations of the current computational models for predicting these drug safety liabilities, and the various strategies for incorporating these predictive models into the drug discovery process.

The use of 3D structural data in the design of specific factor Xa inhibitors

Abstract: Factor Xa (fXa) is a serine protease that plays a critical role in the blood coagulation process and qualifies as an attractive target for finding new antithrombotics. In the case of fXa several structure based drug design strategies have been followed because of the difficulty in growing fXa co-crystals routinely. This has led to the use of surrogate proteins such as trypsin. Factor Xa inhibitors for which the binding mode has been determined experimentally or modeled are described in this review. The inhibitors are divided into three fragments: a P1 group, a central scaffold and a P4 group. In this review, interactions in each sub-site of fXa with various inhibitor fragments have been examined at the molecular level and were shown to bind, in most cases, independently of the rest of the molecule. Knowledge of the 3D structure of the binding mode of ligands to target proteins has been successfully applied in designing fXa inhibitors with enhanced specificity, affinity and has provided hints to modulate the physico-chemical properties of the small molecule ligand.

Siderophores of the human pathogenic fluorescent pseudomonads.

Abstract: Bacteria need a sufficient supply of iron in ionic form for their metabolism. When living in an environment where this is not possible (as in the soil due to the presence of highly unsoluble ferric oxide hydrates, or in living organisms where iron is bound to peptidic chelators) Fe3+ complexing compounds, called siderophores, are produced. The siderophores of Pseudomonas aeruginosa, a dangerous opportunistic human pathogen, and of related potentially pathogenic species will be presented.

Brain oxidative stress--analytical chemistry and thermodynamics of glutathione and NADPH.

Abstract: Oxidative stress occurs in the brain due to stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, trauma, aging and other conditions. Analysis of the effects of oxidative stress can involve quantitation of brain GSH, GSSG, NADPH and NADP. Reliable and rapid assays have been developed for these compounds and will be presented in detail. The assays have been used to analyze the effects of brain oxidative stress. Thermodynamic calculations can be performed to find the observed electrochemical potentials of the GSSG/GSH and the NADP/NADPH couples during oxidative stress. The biochemical consequences of these thermodynamic changes in the cell will be discussed as well as the defense mechanisms available to the cell to recover from oxidative stress.

The design and synthesis of noncovalent factor Xa inhibitors.

Abstract: Thrombosis is a major cause of mortality in the industrialized world. Therefore, the control of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (fXa), the enzyme directly responsible for thrombin generation. In this review we describe our approaches in the design and synthesis of small molecule, noncovalent fXa inhibitors. Rational drug design and selective screening of our GPIIb/IIIa library afforded several lead compounds for our fXa program. Following-up the leads in the isoxazoline series led to potent fXa inhibitors such as SF303 and SK509 with only one basic group. The isoxazole series was then designed to remove the chiral center in the isoxazoline ring, and this effort led to SA862 which has subnanomolar fXa affinity. Optimizing the core structure generated a series of novel five-membered ring heterocycles substituted with benzamidine, which are potent fXa inhibitors. Further optimization in the pyrazole series resulted in the discovery of fXa inhibitors such as SN429 with picomolar fXa affinity. Efforts to improve the oral bioavailability by lowering the basicity of these compounds, while simultaneously maintaining potency against fXa, culminated in the discovery of DPC 423. DPC 423 was selected for clinical evaluation as a potent and orally bioavailable fXa inhibitor.

The Design of Competitive, Small-molecule Inhibitors of Coagulation Factor Xa

Abstract: The last five years have seen an explosion of research into inhibitors of Factor Xa as potential antithrombotic agents. Aventis Pharma was a participant in this effort and its two founder companies have substantially contributed to the discovery of new inhibitors over the years. This review traces the systematic development of the former Rhone-Poulenc Rorer factor Xa program from conception to the realization of potent, orally bioavailable inhibitors with exquisite selectivity against other serine proteases. The work on beta-aminoesters described in Part 1 culminates in the development of FXV673 (Ki = 0.5 nM), an effective anticoagulant for acute indications. Part 2.2 details the de novo design of the pyrrolidinone series of inhibitors (RPR120844), within which a group of efficacious i.v. agents were identified (e.g. RPR130737, Ki = 2 nM). The first active and bioavailable benzamidine isostere i.e. the 1-aminoisoquinoline (RPR208815, Ki = 22 nM) was discovered on the pyrrolidinone scaffold (Part 2.3). Ultimately a variety of benzamidine mimics were explored and incorporated into the ketopiperazine series; the 6-substituted aminoquinazolines were found to be the most potent (Part 3). The azaindole, as represented by RPR200443 (Ki = 4 nM), stands out as imparting favorable pharmacokinetic properties to the sulfonamido-ketopiperazines.

The Chemistry of Transition Metals in Relation to Their Potential Role in Neurodegenerative Processes

Abstract: Cells rely on several transition metals to regulate a wide range of metabolic and signaling functions. The diversity and efficiency of their physiological functions are derived from atomic properties that are specific to transition metals, most notably an incomplete inner valence subshell. These properties impart upon these elements the ability to fluctuate among a variety of positively charged ionic forms, and a chemical flexibility that allows them to impose conformational changes upon the proteins to which they bind. By this means, transition metals can serve as the catalytic centers of enzymes for redox reactions including molecular oxygen and endogenous peroxides. This review addresses the consequences of the aberrant translocation of the redox-capable essential transition elements, iron, copper, and manganese, upon the brain with an emphasis on uncontrolled and deleterious oxidative events. The potential of metal-protein interactions in facilitating such events, and their association with the physiologically redox-inert metals zinc and aluminum, are related to their postulated contribution to the pathology of neurodegeneration.

Nuclear magnetic resonance as a tool in drug discovery, metabolism and disposition.

Abstract: Nuclear magnetic resonance techniques have become critically important in the design of new pharmaceuticals, the characterization of drug-receptor interactions and metabolite identification. Advances in solvent suppression, coherent and incoherent magnetization transfer pathway selection, isotope editing and filtering, and diffusion filtering have made it possible to examine the interactions between small molecules and proteins or nucleic acids in great detail. Multiple schemes for high-throughput lead compound identification, metabolite screening and drug disposition have been proposed and reduced to practice. In particular, the coupling of NMR with other analytical methods, especially HPLC, combine the structural and dynamic detail available from NMR methods with the resolution and sensitivity of other analytical techniques.

DNA Damage and Repair in the Brain After Cerebral Ischemia

Abstract: In experimental models of brain injury of the ischemia-reperfusion type, there is a period of time in which the formation of oxidative damage exceeds its repair. Simultaneously, the expression of immediate early genes is induced to activate the expression of late effector genes. Drugs that reduce the need to repair during this transient period of time also attenuate neuronal death after brain injury. An example discussed in this review is the activator protein-1 (AP-1), the product of the c-fos gene and other immediate early genes. What is the effect of a delayed expression of these genes in relationship to the process of cell death? This short period presents a window of opportunity to study the effects of oxidative damage on gene expression in the brain and specific deficiencies in gene repair that have been associated with particular neurological disorders.

Nerve injury associated with regional anesthesia.

Abstract: Neural damage is a possible consequence of general anesthesia, central nervous system blockade, and regional anesthesia. Dainage may be caused by ischaemic and mechanical or chemical factors, which may occur either alone or in combination. Neural damage may be secondary to prolonged and severe arterial hypotension compromising blood supply to the cord, a spinal haematoma whose main etiological factor is a coagulation abnormality, an intraneural injection, and peripheral neuropathy related to perioperative positioning. Mechanical trauma by the needle bevel is an important factor contributing to neuropathy. Neurological complications may also result from a direct neurotoxic effect of local anesthetic agents which is concentration and dose-dependent. A better understanding of these mechanisms will provide a reliable basis for the development of improved pharmaceutical therapy.

Clinical application of ropivacaine for the lower extremity.

Abstract: Ropivacaine is a new amide local anaesthetic, which is the first commercially available in its category as a pure S-(-) enantiomer. In most recent studies, ropivacaine exhibited a very close pharmacodynamic profile to equipotent doses of bupivacaine. Concentrations of 0.5%, 0.75% and 1% (5, 7.5 and 10 mg/mL, respectively) ropivacaine are used for intraoperative anaesthesia, while the concentration of 0.2% (2 mg/mL) is preferred for postoperative analgesia, either alone or in combination with opioids and/or clonidine. Ropivacaine is responsible for excellent postoperative analgesia following epidural and peripheral perineural injections, using single-shot injections and continuous infusions. Differential sensory/motor block is only apparent at low concentrations (0.2% and less). A significant amount of recent literature focuses on its use for peripheral blocks of the lower limbs, i.e. sciatic and femoral nerve blocks. The primary benefit of ropivacaine is its lower toxicity, mainly lower cardiotoxicity, following accidental intravascular injection. This higher therapeutic index leads to an improved safety profile as compared with potent local anaesthetics such as racemic bupivacaine. For that reason, ropivacaine is a good choice for both intraoperative and postoperative regional anaesthesia and analgesia.

Alpha2-Adrenergic Receptor Agonists as Analgesics

Abstract: Alpha2-adrenergic receptor agonists are analgesic agents, and the alpha2-adrenergic agonist clonidine has been used in clinical studies for regional analgesia after intrathecal administration. We review here recent developments concerning the structure activity relationships of a new class of potent alpha2-adrenergic agonists and their use as analgesic agents. The effect of structure upon cardiovascular side-effects is also monitored, such as the prolongation of the QT portion of the cardiac action potential.

Assessing the potential toxicity of new pharmaceuticals.

Abstract: Optimizing chemical structures to create potentially safe drugs during discovery and early development relies on a combination of predictive algorithms, screening, formal toxicology studies, and early clinical trials. Early in the process three critical questions emerge that must be answered by a detailed "profiling" approach. These questions are: 1) is there a correlation between the chemical structure and potential toxicity that can be used to optimize structures of lead compounds, 2) can specific markers of potential toxicity can be identified early and used as mechanistic decision-making screens, and 3) will exposures (plasma levels) in animal studies correlate with exposures encountered in the clinic thereby providing "coverage" for safety? Depending on the therapeutic class of compounds being considered and the level of knowledge available, feedback loops of information can be established to guide the development process.