Drug Targeting

The renin-angiotensin-aldosterone system (RAAS) is important for the development of hypertension, and several antihypertensive drugs target this system. Our aim was to determine whether specific single nucleotide polymorphisms (SNPs) in RAAS genes were related to the blood pressure (BP) lowering effect of antihypertensive treatment. Methods: Patients with mild to moderate primary hypertension and left ventricular hypertrophy were randomized in a double-blind fashion to treatment with either the angiotensin II type 1 receptor antagonist irbesartan (n ϭ 48) or the ␤ 1 -adrenergic receptor blocker atenolol (n ϭ 49) as monotherapy. A microarray-based minisequencing system was used to genotype 30 SNPs in seven genes in the RAAS. These polymorphisms were related to the antihypertensive response after 12 weeks treatment. Results: The BP reductions were similar in the atenolol and the irbesartan groups. Presence of the angiotensinogen (AGT) -6A allele or the AGT 235T allele were both associated with the most pronounced systolic BP response to atenolol treatment (P ϭ .001 when -6 AAϩAG was compared with GG and P ϭ .008 for presence of the 235T variant compared with 235 MM).

Unlike traditional biological research that focuses on a small set of components, systems biology studies the complex interactions among a large number of genes, proteins, and other elements of biological networks and systems. Host-pathogen systems biology examines the interactions between the components of two distinct organisms: a microbial or viral pathogen and its animal host. With the availability of complete genomic sequences of a variety of hosts and pathogens, together with breakthroughs in proteomics, metabolomics, and other experimental areas, the investigation of host-pathogen systems on a multitude of levels of detail has come within reach. This chapter introduces methods and approaches for the analysis of host-pathogen systems. We will particularly emphasize the role of biochemical networks for the study of complex relationships across species boundaries. Although the research area of host-pathogen systems biology spans multiple spatial and temporal scales, we will focus on the molecular and cellular aspects of pathogen-host interactions. We will cover the construction of biochemical networks, the identification of functional response sub-networks, and their comparative network analysis. These methods find application in the identification of host markers and drug targets for further drug development and therapeutic interventions. We will also provide a brief review of other modeling techniques and applications within host-pathogen systems biology, including rule-based modeling of signal transduction pathways, immune system models, and physiological top-down approaches.

Efficient and safe drug delivery has always been a challenge in medicine. The use of nanotechnology, such as the development of nanocarriers for drug delivery, has received great attention owing to the potential that nanocarriers can theoretically act as "magic bullets" and selectively target affected organs and cells while sparing normal tissues. During the last decades the formulation of surfactant vesicles, as a tool to improve drug delivery, brought an ever increasing interest among the scientists working in the area of drug delivery systems. Niosomes are self assembled vesicular nanocarriers obtained by hydration of synthetic surfactants and appropriate amounts of cholesterol or other amphiphilic molecules. Just like liposomes, niosomes can be unilamellar or multilamellar, are suitable as carriers of both hydrophilic and lipophilic drugs and are able to deliver drugs to the target site. Furthermore, niosomal vesicles, that are usually non-toxic, require less production costs and are stable over a longer period of time in different conditions, so overcoming some drawbacks of liposomes. The niosome properties are specifically dictated by size, shape, and surface chemistry which are able to modify the drug's intrinsic pharmacokinetics and eventual drug targeting to the areas of pathology. This up-to-date review deals with composition, preparation, characterization/evaluation, advantages, disadvantages and application of niosomes.

Rapid synthesis and screening of compound libraries enables the accelerated identification of novel protein ligands in order to support processes like analysis of protein interactions, drug target discovery or lead structure discovery. SPOT synthesis-a well established method for the rapid preparation of peptide arrays-has recently been extended to the field of nonpeptides. In this contribution we report on the systematic evaluation of the SPOT technique for the assembly of N-alkylglycine (peptoid) library arrays. In the course of this investigation bromoacetic acid 2,4-dinitrophenylester (1a) was identified to be the most suited agent for bromoacetylation in terms of yield and N-selectivity enabling straightforward submonomer synthesis on hydroxy-group rich cellulose membranes. The potential of this method for the rapid identification of novel nonpeptidic protein ligands was demonstrated by synthesis and screening of a library consisting of 8000 peptoids and peptomers (i.e. their hybrids with a-substituted amino acids) allowing the identification of micromolar ligands for the monoclonal antibody Tab-2. q

Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93 nmol/hr/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.

A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC-MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is ∼20 fmol. Plasma CEPM concentration is determined using LC-MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4 h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.

Endogenous cysteine proteases were given much attention lately, as their role in a variety of pathophysiological disorders became evident. Amongst them cathepsins, which are thought to be implicated in mediation of osteoporosis, cancer progression, atherosclerosis, and many other conditions, are of considerable interest as drug targets. In the presented work, papain was chosen as a model cysteine protease and panning protocol was optimized for selection of papain-binding phage-displayed peptides from a commercially available combinatorial peptide library. Different selection strategies were applied in order to select high-affinity binders. Ultimately, five cyclic peptides (CNWAAGYNCGGGS-NH 2 , CWSMMGFQCGGGS-NH 2 , CWEWGGWHCGGSS-OH, CNW TLGGYKCGGGS-NH 2 (all cyclized through formation of intramolecular disulphide bond), and GNWTLGGYKGG (cyclized head-to-tail)) were synthesized and tested for inhibitory activity towards papain and human cathepsins L, B, H, and K. The peptides possess inhibitory constants in the low micromolar to mid-nanomolar range and exhibit certain selectivity for different lysosomal cysteine proteases included in this study.

The non-neuronal cholinergic system is widely expressed in human airways. Choline acetyltransferase (ChAT) and/or acetylcholine are demonstrated in more or less all epithelial surface cells (goblet cells, ciliated cells, basal cells), submucosal glands and airway smooth muscle fibres. Acetylcholine is also demonstrated in the effector cells of the immune system (lymphocytes, macrophages, mast cells). Epithelial, endothelial and immune cells express nicotinic and muscarinic receptors. Thus the cytomolecule acetylcholine can contribute to the regulation of basic cell functions via auto-/paracrine mechanisms (proliferation, differentiation, ciliary activity, secretion of water, ions and mucus, organization of the cytoskeleton, cell-cell contact). Acetylcholine also modulates immune functions (release of cytokines; proliferation, activation and inhibition of immune cells). Preliminary experimental evidence suggests that mucosal inflammation may be associated with raised acetylcholine le...

Validation of antibiotic mode of action in whole bacterial cells is a key step for antibiotic drug discovery. In this study, one potential drug target, enoyl-acyl carrier protein reductase (FabI), an essential enzyme in the fatty acid biosynthesis pathway, was used to evaluate the feasibility of using a regulated antisense RNA interference approach to determine antibiotic mode of action. Antisense isogenic strains expressing antisense RNA to fabI were created using a tetracycline-regulated vector in Staphylococcus aureus. We demonstrated that down-regulation of FabI expression by induction of fabI antisense RNA induces a conditional lethal phenotype. In contrast, partial down-regulation gives a viable cell with a significant increase in sensitivity to FabI-specific inhibitors (i.e., a sensitized phenotype). More importantly, the mode of action for novel FabI inhibitors has been confirmed using this genetic approach in whole cell assay. These results indicate that controlled antisense technology provides a robust tool for defining and tracking the mode of action of novel antibacterial agents.

Type 2 diabetes Energy metabolism Activation of 5′-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5′-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase ce:sup>/ce:sup>/Mn 2+ -dependent (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the ce:sup>/ce:sup>/Mn 2+ -dependent protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggests that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.

Phospholipase A2 is potentially an important target for structure-based rational drug design. In order to determine the involvement of phospholipase A2 in the action of non-steroidal anti-inflammatory drugs (NSAIDs), the crystal structure of the complex formed between ...

Microarray based transcription profiling is now a consolidated methodology and has widespread use in areas such as pharmacogenomics, diagnostics and drug target identification. Large-scale microarray studies are also becoming crucial to a new way of conceiving experimental biology. A main issue in microarray transcription profiling is data analysis and mining. When microarrays became a methodology of general use, considerable effort was made to produce algorithms and methods for the identification of differentially expressed genes. More recently, the focus has switched to algorithms and database development for microarray data mining. Furthermore, the evolution of microarray technology is allowing researchers to grasp the regulative nature of transcription, integrating basic expression analysis with mRNA characteristics, i.e. exon-based arrays, and with DNA characteristics, i.e. comparative genomic hybridization, single nucleotide polymorphism, tiling and promoter structure. In this article, we will review approaches used to detect differentially expressed genes and to link differential expression to specific biological functions.

The brain is a delicate organ, and nature has very efficiently protected it. The brain is shielded against potentially toxic substances by the presence of two barrier systems: the blood brain barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB). Unfortunately, the same mechanisms that protect it against intrusive chemicals can also frustrate therapeutic interventions. Despite aggressive research, patients suffering from fatal and/or debilitating central nervous system (CNS) diseases, such as brain tumours, HIV encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders, far outnumber those dying of all types of systemic cancers or heart diseases. The abysmally low number of potential therapeutics reaching commercial success is primarily due to the complexity of the CNS drug development. The clinical failure of many probable candidates is often, ascribable to poor delivery methods which do not pervade the unyielding BBB. It restricts the passive diffusion of many drugs into the brain and constitutes a significant obstacle in the pharmacological treatment of central nervous system (CNS) disorders. General methods that can enhance drug delivery to the brain are, therefore, of great pharmaceutical interest. Various strategies like non-invasive methods, including drug manipulation encompassing transformation into lipophilic analogues, prodrugs, chemical drug delivery, carrier-mediated drug delivery, receptor/vector mediated drug delivery and intranasal drug delivery, which exploits the olfactory and trigeminal neuronal pathways to deliver drugs to the brain, are widely used. On the other hand the invasive methods which primarily rely on disruption of the BBB integrity by osmotic or biochemical means, or direct intracranial drug delivery by intracerebroventricular, intracerebral or intrathecal administration after creating reversible openings in the head, are recognised. Extensive review pertaining specifically, to the patents relating to drug delivery across the CNS is currently available. However, many patents e.g. US63722506, US2002183683 etc., have been mentioned in a few articles. It is the objective of this article to expansively review drug delivery systems for CNS by discussing the recent patents available.

Models for drugs exhibiting target-mediated drug disposition (TMDD) play an important role in the investigation of biological products (Mager and Jusko 2001). These models are often overparameterized and difficult to converge. A simpler quasi-equilibrium (QE) approximation of the general model has been suggested (Mager and Krzyzanski 2005), but even this simpler form can be overparameterized when, for example, drug target level is not available. This work (a) introduces quasisteady-state (QSS) and Michaelis-Menten (MM) approximations of the TMDD model, (b) derives the relationships between the parameters of the TMDD, QE, QSS and MM models, (c) investigates the parameter ranges where the simplified approximations are equivalent to the TMDD model, (d) proposes an algorithm for establishing identifiability of these models, and (e) tests this algorithm on simulated datasets. The proposed QSS approximation is more general than the QE approximation: it degenerates into the QE approximation when the internalization rate of the drug-target complex is much smaller than its dissociation rate. The proposed identifiability analysis algorithm may be applied to provide justification for use of simplified approximations, avoiding use of incorrect parameter estimates of over-parameterized TMDD models while simultaneously saving time and resources required for the pharmacokinetics analysis of drugs with TMDD. The utility of the derived approximations and of the identifiability algorithm was demonstrated on the examples of the simulated data sets. The simulation examples indicated that the QSS model may be preferable to the QE model L. Gibiansky (B)

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using ''far-UV'' circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H 131 and the phosphoryl group is then transferred to D 73 of TcrA.

Background: The toxicity of ricin resides in the ricin A-chain (RTA) and is attributed to the inhibition of protein synthesis but inflammation and oxidative stress have also been implicated. RTA can independently enter cells producing comparable tissue injury and inflammation, although at much higher concentrations than intact ricin. Treatment for exposure to ricin or RTA is supportive. Purpose: To examine the effectiveness of conventional or liposome-encapsulated N-acetylcysteine (Lipo-NAC) in ameliorating RTA-induced hepatotoxicity. Methods: Four hours after RTA administration (90 µg/kg b.wt, iv), rats were treated with conventional NAC or Lipo-NAC (25 mg/kg NAC). The hepatoprotective effects of the antioxidant formulations were assessed by measuring indexes for liver injury (alanine [ALT] and aspartate [AST] aminotransferase activities), inflammation (myeloperoxidase, tumor necrosis factor-α, chloramine levels), and oxidant response (lipid peroxidation, nitrotyrosine, glutathione levels) 24-h post-RTA exposure. Results: Administration of RTA to animals resulted in hepatotoxicity as demonstrated by elevated plasma ALT and AST levels, increases in an inflammatory response, and increases in oxidant response. Treatment of animals with the antioxidant formulations reversed the RTA-induced hepatotoxicity, being most evident following the administration of Lipo-NAC. Conclusion: NAC, administered in a liposomal form, may serve as a potentially effective pharmacological agent in the treatment of RTA-induced liver injuries.

This paper describes the synthesis, physico-chemical characteristics and results of selected biological tests of conjugates of antibodies or proteins with poly(HPMA) or with poly(HPMA) carriers of anti-cancer drug doxorubicin, designed for targeted cancer therapy. Two types of conjugates differing in the method of conjugation of polymer with protein were synthesized. In the first, protein is attached to the polymer via an oligopeptide sequence in the side chain of the polymer backbone and, in the second, the polymer is attached to protein via its end-chain functional group. Conjugation of an antibody with poly(HPMA) does not influence the binding activity of the antibody for cell surface antigen. The physico-chemical characteristics and biological activity of both systems depend on the detailed structure of the polymer, the type of antibody or protein moiety and the structure of the whole system.

A major challenge in the field of functional genomics is the development of computational techniques for organizing and interpreting large amounts of gene expression data. These methods will be critical for the discovery of new therapeutic drug targets. Here, we present a simple method for determining the most likely drug target candidates from temporal gene expression patterns assayed with reverse-transcription polymerase chain reaction (RT-PCR) and DNA microarrays.

Microbial biofilms have been observed as congregates and attached communities on a diverse range of microecosystems of medicinal and industrial importance. Until recently, most investigations have been performed on planktonic (floating or fluid phase) microorganisms. After realization of the biofilm existence and their recalcitrance toward conventionally adopted preventive strategies and antimicrobial agents, research has been shifted toward novel therapeutics based drug delivery and targeting approaches. With the emergence of various biofilm models and methods to assess biofilm formation and physiology, it is pivotal to discuss various novel strategies that may become the therapeutic tools and clinically adaptable strategies of the future. This review explores various novel research strategies studied to date for their potential in effective biofilm eradication.

Purpose: Recently the emphasis has been laid upon the carbohydrate mediated liposomal interactions with the target cells. Among the various carbohydrate ligands, such as glycoproteins, glycolipids, viral proteins, polysaccharides, lipo-polysaccharides and other oligosaccharides, this review deals with the polysaccharide anchored liposomal system for their potential in drug delivery, targeting and immunization. Over the years, various strategies have been developed which include coating of the liposomal surface with natural or hydrophobized polysaccharides, namely mannan, pullulan, amylopectin, dextran etc., or their palmitoyl or cholesteroyl derivatives. The polysaccharide(s) coat tends vesicular constructs physicochemically stable in bio-environments and site-specific. The aim of improving the physical and biochemical stability of liposomes and the ability to target liposomes to specific organs and cells, were the major attributes of the polysaccharide anchored liposomes. In this review the authors attempted to overview various applications of polysaccharide bearing liposomes, including lung therapeutics, targeted chemotherapy, cellular targeting, cellular or mucosal immunity and macrophage activation. Future prospects of the delivery module are also discussed. The review in general explores the concepts, options and opportunities of polysaccharide anchored liposomes with newer perspectives.

Claudin-18 isoform 2 (CLDN18.2) is one of the few members of the human claudin family of tight junction molecules with strict restriction to one cell lineage. The objective of the current study was to compare molecular structure and tissue distribution of this gastrocyte specific molecule in mammals. We show here that the CLDN18.2 protein sequence is highly conserved, in particular with regard to functionally relevant domains in mouse, rat, rabbit, dog, monkey and human and also in lizards. Moreover, promoter regions of orthologs are highly homologous, including the binding site of the transcription factor cyclic AMP-responsive element binding protein (CREB), which is known to regulate activation of human CLDN18.2. Employing RT-PCR and immunohistochemistry, we found that, analogous to the human gene, all orthologous CLDN18.2 transcripts and proteins are exclusively expressed in differentiated gastric cells. Gene structure, promoter elements and RNA expression pattern of the lung-tissue specific Claudin-18 isoform 1 (CLDN18.1) as well, are homologous across species. These findings exemplify phylogenetic conservation of lineage-specific members of a multigene family. Given that CLDN18.2 is a novel drug target candidate, our data is also relevant for drug development as it reveals all six investigated mammalian species as suitable models for testing safety of CLDN18.2 targeting regimen.

One of the key players in many thrombotic complications is von Willebrand factor (VWF), a large, multimeric glycoprotein that is present in plasma where it fulfils a crucial role in haemostasis. First, VWF recruits platelets to vascular lesions by acting as a linker molecule between the exposed collagen and free-flowing platelets in the circulation. Second, by serving as a carrier protein for the coagulation factor VIII, VWF protects this anti-haemophilic factor from rapid degradation. Quantitative or qualitative defects in VWF result in the most common bleeding disorder in man, known as von Willebrand disease, illustrating the central role of VWF in haemostasis. On the other hand, a thrombotic risk emerges when over-reactive VWF molecules can bind spontaneously to platelets. It is clear that because of its pivotal role in maintaining the fine balance between bleeding and thrombosis, VWF is an attractive but delicate drug target. This review focuses on the role of VWF in both haemostasis and thrombosis with special attention to the molecule as drug and drug target respectively.

Summary Genomics, particularly high-throughput sequencing medicine is that disease could be treated according to genetic and specific individual markers, selecting and characterization of expressed human genes, has created new opportunities for drug discovery. medications and dosages that are optimized for indi- vidual patients. The possibility of defining patient Knowledge of all the human genes and their func- tions may allow

It is estimated that over 10% of the adult population in developed countries have some degree of chronic kidney disease (CKD). CKD is a progressive and irreversible deterioration of the renal excretory function that results in implementation of renal replacement therapy in the form of dialysis or renal transplant, which may also lead to death. CKD poses a growing problem to society as the incidence of the disease increases at an annual rate of 8%, and consumes up to 2% of the global health expenditure. CKD is caused by a variety of factors including diabetes, hypertension, infection, reduced blood supply to the kidneys, obstruction of the urinary tract and genetic alterations. The nephropathies associated with some of these conditions have been modeled in animals, this being crucial to understanding their pathophysiological mechanism and assessing prospective treatments at the preclinical level. This article reviews and updates the pathophysiological knowledge acquired primarily from experimental models and human studies of CKD. It also highlights the common mechanism(s) underlying the most relevant chronic nephropathies which lead to the appearance of a progressive, common renal phenotype regardless of aetiology. Based on this knowledge, a therapeutic horizon for the treatment of CKD is described. Present therapy primarily based upon renin-angiotensin inhibition, future diagnostics and therapeutic perspectives based upon anti-inflammatory, anti-fibrotic and hemodynamic approaches, new drugs targeting specific signaling pathways, and advances in gene and cell therapies, are all elaborated.

There were 13,176 roadside drug tests performed in the first year of the random drug-testing program conducted in the state of Victoria. Drugs targeted in the testing were methamphetamines and Delta(9)-tetrahydrocannabinol (THC). On-site screening was conducted by the police using DrugWipe, while the driver was still in the vehicle and if positive, a second test on collected oral fluid, using the Rapiscan, was performed in a specially outfitted "drug bus" located adjacent to the testing area. Oral fluid on presumptive positive cases was sent to the laboratory for confirmation with limits of quantification of 5, 5, and 2 ng/mL for methamphetamine (MA), methylenedioxy-methamphetamine (MDMA), and THC, respectively. Recovery experiments conducted in the laboratory showed quantitative recovery of analytes from the collector. When oral fluid could not be collected, blood was taken from the driver and sent to the laboratory for confirmation. These roadside tests gave 313 positive...

Dosimetry, safety and the efficacy of drugs in the lungs are critical factors in the development of inhaled medicines. This article considers the challenges in each of these areas with reference to current industry practices for developing inhaled products, and suggests collaborative scientific approaches to address these challenges. The portfolio of molecules requiring delivery by inhalation has expanded rapidly to include novel drugs for lung disease, combination therapies, biopharmaceuticals and candidates for systemic delivery via the lung. For these drugs to be developed as inhaled medicines, a better understanding of their fate in the lungs and how this might be modified is required. Harmonised approaches based on 'best practice' are advocated for dosimetry and safety studies; this would provide coherent data to help product developers and regulatory agencies differentiate new inhaled drug products. To date, there are limited reports describing full temporal relationships between pharmacokinetic (PK) and pharmacodynamic (PD) measurements. A better understanding of pulmonary PK and PK/PD relationships would help mitigate the risk of not engaging successfully or persistently with the drug target as well as identifying the potential for drug accumulation in the lung or excessive systemic exposure. Recommendations are made for (i) better industry-academiaregulatory co-operation, (ii) sharing of pre-competitive data, and (iii) open innovation through collaborative research in key topics such as lung deposition, drug solubility and dissolution in lung fluid, adaptive responses in safety studies, biomarker development and validation, the role of transporters in pulmonary drug disposition, target localisation within the lung and the determinants of local efficacy following inhaled drug administration.

Jump dilution analysis is commonly used to evaluate the reversibility of inhibition and to quantify the residence time of the inhibitor-enzyme complex. During hit and lead characterization, one sometimes observes apparently linear progress curves after jump dilution that display activity recoveries that are intermediate between those expected for fully reversible and irreversible inhibition. Computer simulations of progress curves after jump dilution indicate that seemingly linear progress curves can result when dealing with tight-binding inhibitors if substoichiometric concentrations of inhibitor are preincubated with enzyme. In this situation, the activity recovered is comparable to that expected for instantaneously reversible inhibitors. In addition, simulations demonstrate that intermediate values of activity recovery may be observed for compounds with modestly slow dissociation rates (i.e., residence times >0 min but 620 min) when the attending curvature of the data is not accounted for. The observation of intermediate values of recovery can, thus, serve as an indication of either modest residence time or a contaminating inactivator within an inhibitor sample, in either case prompting greater scrutiny of the test compound.

Inosine-5′-monophosphate dehydrogenase (IMPDH) is an attractive drug target for the control of parasitic infections. The enzyme catalyzes the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), the committed step in de noVo guanosine monophosphate (GMP) biosynthesis. We have determined the crystal structures of IMPDH from the protozoan parasite Tritrichomonas foetus in the apo form at 2.3 Å resolution and the enzyme-XMP complex at 2.6 Å resolution. Each monomer of this tetrameric enzyme is comprised of two domains, the largest of which includes an eight-stranded parallel /R-barrel that contains the enzyme active site at the C termini of the barrel -strands. A second domain, comprised of residues 102-220, is disordered in the crystal. IMPDH is expected to be active as a tetramer, since the active site cavity is formed by strands from adjacent subunits. An intrasubunit disulfide bond, seen in the crystal structure, may stabilize the protein in a less active form, as high concentrations of reducing agent have been shown to increase enzyme activity. Disorder at the active site suggests that a high degree of flexibility may be inherent in the catalytic function of IMPDH. Unlike IMPDH from other species, the T. foetus enzyme has a single arginine that is largely responsible for coordinating the substrate phosphate in the active site. This structural uniqueness may facilitate structurebased identification and design of compounds that specifically inhibit the parasite enzyme.

Tumor vascular permeability factor (VPF) was initially identified by Senger et al. in the laboratory of Dvorak [1] and was later confirmed as identical to VEGF [2, 3, 4 and 5]. Although more attention has been focused on angiogenesis, the other side of biological activity of VEGF, the ...

A B S T R A C T Peptide and protein (P/P) drugs have been identified as showing great promises for the treatment of various neurodegenerative diseases. A major challenge in this regard, however, is the delivery of P/P drugs over the blood-brain barrier (BBB). Intense research over the last 25 years has enabled a better understanding of the cellular and molecular transport mechanisms at the BBB, and several strategies for enhanced P/P drug delivery over the BBB have been developed and tested in preclinical and clinicalexperimental research. Among them, technology-based approaches (comprising functionalized nanocarriers and liposomes) and pharmacological strategies (such as the use of carrier systems and chimeric peptide technology) appear to be the most promising ones. This review combines a comprehensive overview on the current understanding of the transport mechanisms at the BBB with promising selected strategies published so far that can be applied to facilitate enhanced P/P drug delivery over the BBB. ß

DNA clones for the b-class carbonic anhydrase (CA, EC 4.2.1.1) of Helicobactor pylori (hpbCA) were obtained. A recombinant hpbCA protein lacking the N-terminal 15-amino acid residues was produced and purified, representing a catalytically efficient CA. hpbCA was strongly inhibited (K I s in the range of 24-45 nM) by many sulfonamides/sulfamates, among which acetazolamide, ethoxzolamide, topiramate, and sulpiride, all clinically used drugs. The dual inhibition of a-and/or b-class CAs of H. pylori might represent a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This is also the first study showing that a bacterial b-CA can be a drug target.

The brain is a delicate organ, and nature has very efficiently protected it. The brain is shielded against potentially toxic substances by the presence of two barrier systems: the blood brain barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB). Unfortunately, the same mechanisms that protect it against intrusive chemicals can also frustrate therapeutic interventions. Despite aggressive research, patients suffering from fatal and/or debilitating central nervous system (CNS) diseases, such as brain tumours, HIV encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders, far outnumber those dying of all types of systemic cancers or heart diseases. The abysmally low number of potential therapeutics reaching commercial success is primarily due to the complexity of the CNS drug development. The clinical failure of many probable candidates is often, ascribable to poor delivery methods which do not pervade the unyielding BBB. It restricts the passive diffusion of many drugs into the brain and constitutes a significant obstacle in the pharmacological treatment of central nervous system (CNS) disorders. General methods that can enhance drug delivery to the brain are, therefore, of great pharmaceutical interest. Various strategies like non-invasive methods, including drug manipulation encompassing transformation into lipophilic analogues, prodrugs, chemical drug delivery, carrier-mediated drug delivery, receptor/vector mediated drug delivery and intranasal drug delivery, which exploits the olfactory and trigeminal neuronal pathways to deliver drugs to the brain, are widely used. On the other hand the invasive methods which primarily rely on disruption of the BBB integrity by osmotic or biochemical means, or direct intracranial drug delivery by intracerebroventricular, intracerebral or intrathecal administration after creating reversible openings in the head, are recognised. Extensive review pertaining specifically, to the patents relating to drug delivery across the CNS is currently available. However, many patents e.g. US63722506, US2002183683 etc., have been mentioned in a few articles. It is the objective of this article to expansively review drug delivery systems for CNS by discussing the recent patents available.

Background: Taenia pisiformis is one of the most common intestinal tapeworms and can cause infections in canines. Adult T. pisiformis (canines as definitive hosts) and Cysticercus pisiformis (rabbits as intermediate hosts) cause significant health problems to the host and considerable socio-economic losses as a consequence. No complete genomic data regarding T. pisiformis are currently available in public databases. RNA-seq provides an effective approach to analyze the eukaryotic transcriptome to generate large functional gene datasets that can be used for further studies.

Abstract—The first step from the scientific data to drugs, it is the identification of drug active substance candidates called "drug discovery". As technology in the medical world evolves, the use of new methods has enabled some questions to be answered. Biology has emerged from being a science field that is currently being studied only in the laboratory and has become a scientific discipline working with information technology. Bioinformatics is the process of storing and processing biological data using computers and software to respond to these biological issues. In this context, drug discovery has accelerated in the process of organizing, directing and interpreting results from biological information. In this study, it is mentioned which phases are passed through bioinformatics during the drug discovery process and which methods are used at these stages. The first step in the drug discovery process is to set an appropriate target. This can be a disease state, a molecule or protein associated with a gene. Within the context of bioinformatics information, drugs can be designed using biological data with various algorithms.

Özet—Bilimsel verilerden ilaçlara uzanan aşamalardan ilki ("drug dıscovery") yani "ilaç keşfi" olarak adlandırılan ilaç etkin madde adaylarının saptanmasıdır. Tıp dünyasındaki teknoloji geliştikçe yeni methotların kullanılması bazı soruların açıklığa çıkabilmesini sağlamıştır. Biyoloji günümüzde sadece laboratuvarda çalışılan bir bilim alanı olmaktan çıkmış ve bilgi teknolojisiyle iç içe çalışan bir bilim dalı haline gelmiştir. Biyoinformatik de bu biyolojik meselelere cevap verebilmek için bilgisayarlar ve yazılımlarının kullanılarak biyolojik verilerin depolanması ve işlenmesi süreçleridir. Bu kapsamda biyolojik bilgilerden veriyi organize etmede, yönetmede ve yorumlama sonuçlarında ilaç keşfi sürecide hız kazanmıştır. Bu çalışmada ilaç keşfi sürecinde biyoinformatik kullanılarak hangi aşamalardan geçildiğini ve bu aşamalarda hangi yöntemlerin kullanıldığına değinilmiştir. İlaç keşif işleminde ilk adım olarak, uygun bir hedef belirlemektir. Bu bir hastalık durumu, bir gen ile ilişkili bir molekül veya protein olabilir. Biyoinformatik bilgiler kapsamında da çeşitli algoritmalar ile biyolojik veriler kullanılarak ilaçlar tasarlanabilmektedir. Anahtar Kelimeler—biyoinformatik, ilaç keşfi, genomik, hedef tanımlama

The botanical collections of early explorers and the later ethnobotany have played important roles in the development of new drugs for many centuries. In the middle of the last century interest in this approach had declined dramatically, but has risen again during its last decade, and new foci have developed. The systematic evaluation of indigenous pharmacopoeias in order to contribute to improved health care in marginalized regions has been placed on the agenda of international and national organizations and of NGOs. In this paper the results of various projects on Mexican Indian ethnobotany and some of the subsequent pharmacological and phytochemical studies are summarized. Medicinal plants are an important element of indigenous medical systems in Mexico. This study uses the medicinal plants in four indigenous groups of Mexican Indians-Maya, Nahua, Zapotec and Mixe-as an example. The relative importance of a medicinal plant within a culture is documented using a quantitative method and the data are compared intra-and interculturally. While the species used by the indigenous groups vary, the data indicate that there exist well-defined criteria specific for each culture, which lead to the selection of a plant as a medicine. For example, a large number of species are used for gastrointestinal illnesses by two or more of the indigenous groups. At least in this case, the multiple transfers of species and their uses within Mexico seems to be an important reason for the widespread use of a species.

The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first 'tagged' by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed. [Nandi D, Tahiliani P, Kumar A and Chandu D 2006 The ubiquitin-proteasome system; J. Biosci. 31 137-155] http://www.ias.ac.in/jbiosci Abbreviations used: AAA-ATPase, ATPases associated with various cellular activities-ATPase; BrAAP, branched chain amino acids pre-ß ß 1 ß 2 ß 2 ß 5 ß 5 ß 1i ß 1i ß 2i ß 2i ß 5i ß 5i

There is an urgent need to develop new drugs against eukaryotic parasitic protozoa such as Plasmodium, Trypanosoma and Leishmania, which cause the diseases malaria, trypanosomiasis and the leishmaniases respectively. The biology of these organisms has many unusual facets that might be exploited for drug design, and the recent availability of parasite genome sequence data has facilitated the search for novel drug targets. Here we review current understanding of the cell cycle in these parasites and show that important structural and functional differences exist between parasite and mammalian cell cycle control machineries and signal transduction pathways, which might be utilised for rational drug design. Potential targets include protein kinases from the cyclin-dependent kinase, cAMP-dependent kinase and mitogen activated protein kinase families.

Although the cathecholamine systems have long been the focus of drug therapy in anxiety and depression, the development of novel drugs specifically aimed at new targets within these traditional neurotransmitter systems and at targets outside of these systems is now propelling the field of drug development in anxiety. A greater understanding of regional brain networks implicated in stress, anxiety, and anxious behaviors has provided localized targets for anxiolytics. Within the serotonin and norepinephrine systems, increased understanding of postsynaptic receptor regulation with chronic treatment and cross-system effects of drug therapy have been critical in furthering our understanding of effective pharmacological interventions. Receptors within the glutamate, ␥-aminobutyric acid, and neuropeptide systems provide a rich diversity of drug targets, both in localization and function. While acknowledging significant clinical and biological differences between the various anxiety disorders, an important aspect of modern neurobiological research is to look for similarities among these disorders, given that they are highly comorbid with each other and often respond to the same spectrum of treatments. Here we review current views on both traditional and new molecular targets in the treatment of anxiety, realizing that the ultimate challenge in effective anxiolytic drug development may be achieving specificity in brain regions important in generating and sustaining anxiety.

Drug delivery systems present an opportunity to potentiate the therapeutic effect of antileishmanial drugs. Colloidal carriers are rapidly cleared by the phagocytic cells of the reticuloendothelial system (RES), rendering them ideal vehicles for passive targeting of antileishmanials. This paper describes the development of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the antileishmanial saponin β-aescin. NPs were prepared using the combined emulsification solvent evaporation/salting-out technique. Confocal microscopy was used to visualise the internalisation and intracellular trafficking of fluorescein-and nile red-labelled PLGA NPs in J774A.1 macrophages infected with GFP-transfected Leishmania donovani. The in vitro activity of aescin and aescin-loaded NPs on L. infantum was determined in the axenic model as well as in the ex vivo model. The developed PLGA NPs were monodispersed with Z ave <300 nm, exhibited negative zeta potentials and had relatively high drug loadings ranging from 5.80 to 8.68% w/w PLGA. The fluorescent NPs were internalised by the macrophages and trafficked towards the lysosomes after 2 h in vitro incubation. Co-localisation of the NPs and the parasite was not shown. A two-fold increase in activity was observed in the ex vivo macrophage model by encapsulating β-aescin in PLGA NPs (IC 50 , 0.48-0.76 µg/mL vs. 1.55 ± 0.32 µg/mL for the free drug).

Ethnopharmacological relevance: The study was done to establish medicinal plants used in the treatment of various diseases by the people in the Northern sector of Kibale National Park in western Uganda. It was also aimed at establishing the plant parts used and the mode of preparation of remedies. These plants create a basis for phytochemical evaluation which can lead to the discovery of biologically active compounds that can be used as starting materials in the development of new drugs targeting selected diseases such as malaria. Materials and methods: The required information was obtained using open interviews, semi-structured questionnaires, focus group discussions and transect walks. Results: Different medicinal plants (131 species) distributed over 55 families were observed to be used by the local communities around the Northern sector of Kibale National Park. The plants as reported in this paper are used to treat 43 physical illnesses/diseases. The most used parts of the plants are the leaves. Water is the main medium used for the preparation of the remedies which are mostly administered orally. Conclusion: The people in the study area have a rich heritage of traditional plants that are used in the health care system to treat diseases. These medicinal plants have contributed significantly to several disease therapies. The most common diseases treated are malaria and cough, which are mostly treated by Vernonia amygdalina Del. and Albizia coriaria Welw. respectively. The main sources of medicinal plants include bush land, home gardens, grasslands, and the forest.