Biomedical imaging in the safety evaluation of new drugs

Toxicology accounts for approximately one-third of attrition in new drug development and is a major concern in the pharmaceutical industry. This paper reviews the role of biomedical imaging in the safety evaluation of new candidate drugs. Ex vivo high-resolution three-dimensional imaging of specimens can provide a quick overview of the specimens. Volumetric measurements of tissue structures and lesions can be made with higher precision and reproducibility than histology approaches. As opposed to histology, in vivo animal imaging permits longitudinal studies of the same animals over an extended period of time, with individual animals serving as their own control. Therefore, the number of animals required for a study can be significantly reduced and the intra-subject variability is minimized. Repeated in vivo imaging allows monitoring of the occurrence and progression, or regression, of various structural and functional abnormalities. Compared with other biological assays, imaging can provide anatomically specific information about tissue abnormality. Imaging offers the opportunity to carry forward the same methodology in animal experiments into human studies and has an important role in clinical trials when other safety biomarkers for early toxicities are not available.     

Developmental toxicology: new directions workshop: refining testing strategies and study designs

In April 2009, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute's (HESI) Developmental and Reproductive Toxicology Technical Committee held a two-day workshop entitled "Developmental Toxicology-New Directions." The third session of the workshop focused on ways to refine animal studies to improve relevance and predictivity for human risk. The session included five presentations on: (1) considerations for refining developmental toxicology testing and data interpretation; (2) comparative embryology and considerations in study design and interpretation; (3) pharmacokinetic considerations in study design; (4) utility of genetically modified models for understanding mode-of-action; and (5) special considerations in reproductive testing for biologics. The presentations were followed by discussion by the presenters and attendees. Much of the discussion focused on aspects of refining current animal testing strategies, including use of toxicokinetic data, dose selection, tiered/triggered testing strategies, species selection, and use of alternative animal models. Another major area of discussion was use of non-animal-based testing paradigms, including how to define a "signal" or adverse effect, translating in vitro exposures to whole animal and human exposures, validation strategies, the need to bridge the existing gap between classical toxicology testing and risk assessment, and development of new technologies. Although there was general agreement among participants that the current testing strategy is effective, there was also consensus that traditional methods are resource-intensive and improved effectiveness of developmental toxicity testing to assess risks to human health is possible. This article provides a summary of the session's presentations and discussion and describes some key areas that warrant further consideration.     

The autocatalytic step is an integral part of the hydrogenase cycle

We earlier proved the involvement of an autocatalytic step in the oxidation of H₂ by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H₂, we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme–substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.     

Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one

Recent research has provided new insights on the epidemiology, pathology and immunology of canine leishmaniosis (CanL) and its genetic basis. The prevalence of infection in endemic areas is considerably higher than that of apparent clinical illness. In addition, infection spreads rapidly among dogs in the presence of optimal conditions for transmission. Infection involves a variety of granulomatous and harmful immune-mediated responses, and susceptibility to the disease is influenced by a complex genetic basis. These concepts will be instrumental for devising control programs. This review, the first in a series of two articles on CanL, presents an updated view on progress in elucidating the epidemiology and pathogenesis of this challenging disease, and the second part focuses on advances in diagnosis, treatment and prevention.     

Developmental biology: an array of new possibilities

Microarrays offer biologists comprehensive and powerful tools to analyze the involvement of genes in developmental processes at an unprecedented scale. Microarrays that employ defined sequences will permit us to elucidate genetic relationships and responses, while those that employ undefined DNA sequences (ESTs, cDNA, or genomic libraries) will help us to discover new genes, relate them to documented gene networks, and examine the way in which genes (and the process that they themselves control) are regulated. With access to broad new avenues of research come strategic and logistical headaches, most of which are embodied in the reams of data that are created over the course of an experiment. The solutions to these problems have provided interesting computational tools, which will allow us to compile huge data sets and to construct a genome-wide view of development. We are on the threshold of a new vista of possibilities where we might consider in comprehensive and yet specific detail, for example, the degree to which diverse organisms utilize similar genetic networks to achieve similar ends.     

DNA structure in which an adenine-cytosine mismatch pair forms an integral part of the double helix

Extensive studies using one- and two-dimensional 1H NMR at 500 MHz revealed that the oligonucleotide d(CGCCGCAGC) in solution at 5 degrees C forms a double helix under conditions of high salt (500 mM in NaCl, 1 mM sodium phosphate), low pH (pH 4.5), and high DNA concentration (4 mM in duplex). The presence of very strong nuclear Overhauser effects (NOEs) from base H8/H6 to sugar H2',H2" and the absence of NOE from base H8/H6 to sugar H3' suggested that the oligomer under these solution conditions forms a right-handed B-DNA double helix. The following lines of experimental evidence were used to conclude that C4 and A7 form an integral part of the duplex: (i) the presence of a NOESY cross-peak involving H8 of A7 and H8 of G8, (ii) the presence of a two-dimensional NOE (NOESY) cross-peak between H6 of C3 and H6 of C4, (iii) base protons belonging to C4 and A7 forming a part of the H8/H6---H1' cross-connectivity route, and (iv) the pattern of H8/H6---H2',H2" NOESY cross-connectivity based upon a B-DNA model requiring that both C4 and A7 form an integral part of the duplex. The possibility of an A-C pair involving H bonds was also examined. Two possible structural models of the duplex at pH 4.5 are proposed: in one model A-C pairing involves two H bonds, and in the other A-C pairing involves a single H bond.     

Preliminary safety evaluation of a new Bacteroides xylanisolvens isolate

Besides conferring some health benefit to the host, a bacterial strain must present an unambiguous safety status to be considered a probiotic. We here present the preliminary safety evaluation of a new Bacteroides xylanisolvens strain (DSM 23964) isolated from human feces. First results suggest that it may be able to provide probiotic health benefits. Its identity was confirmed by biochemical analysis, by sequencing of its 16S rRNA genes, and by DNA-DNA hybridization. Virulence determinants known to occur in the genus Bacteroides, such the bft enterotoxin and other enzymatic activities involved in the degradation of the extracellular matrix and the capsular polysaccharide PS A, were absent in this strain. The investigation of the antibiotic susceptibility indicated that strain DSM 23964 was sensitive to metronidazole, meropenem agents, and clindamycin. Resistance to penicillin and ampicillin was identified to be conferred by the β-lactamase cepA gene and could therefore be restored by adding β-lactamase inhibitors. The localization of the cepA gene in the genome of strain DSM 23964 and the absence of detectable plasmids further suggest that a transfer of β-lactamase activity or the acquisition of other antibiotic resistances are highly improbable. Taken together, the presented data indicate that the strain B. xylanisolvens DSM 23964 has no virulence potential. Since it also resists the action of gastric enzymes and low-pH conditions, this strain is an interesting candidate for further investigation of its safety and potential health-promoting properties.     

Developmental expression of syndecan, an integral membrane proteoglycan, correlates with cell differentiation

Syndecan is an integral membrane proteoglycan that behaves as a matrix receptor by binding cells to interstitial matrix and associating intracellularly with the actin cytoskeleton. Using immunohistology, we have now localized this proteoglycan during the morphogenesis of various derivatives of the surface ectoderm in mouse embryos. Syndecan is expressed on ectodermal epithelia, but is selectively lost from the cells that differentiate into the localized placodes that initiate lens, nasal, otic and vibrissal development. The loss is transient on presumptive ear, nasal and vibrissal epithelia; the derivatives of the differentiating ectodermal cells that have lost syndecan subsequently re-express syndecan. In contrast, syndecan is initially absent from the mesenchyme underlying the surface ectoderm, and is transiently expressed when the surface ectoderm loses syndecan. These results demonstrate that expression of syndecan is developmentally regulated in a distinct spatiotemporal pattern. On epithelia, syndecan is lost at a time and, location that correlates with epithelial cell differentiation and, on mesenchyme, syndecan is acquired when the cells aggregate in proximity to the epithelium. This pattern of change with morphogenetic events is unique and not duplicated by other matrix molecules or adhesion receptors.     

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.     

Ameltolide. I: Developmental toxicology studies of a novel anticonvulsant

Ameltolide, a novel anticonvulsant agent, has been shown in animal models to be effective in controlling seizures. The developmental toxicity of ameltolide was evaluated in two species. Naturally mated rats and rabbits were dosed once daily by gavage on gestation days (GD) 6-17 and 6-18, respectively. Rats were given doses of 0, 10, 25, or 50 mg/kg; rabbits were given 0, 25, 50, or 100 mg/kg. Laparotomy was performed on rats on GD 20 and on rabbits on GD 28. In rats, maternal toxicity was indicated at the 25- and 50-mg/kg dose levels by depressed body weight gain. Fetal body weight was depressed at the 50-mg/kg dose level. Fetal viability and morphology were not affected. The no-observed effect levels (NOEL) for adult and developmental toxicity in the rat were 10 and 25 mg/kg, respectively. In rabbits, maternal toxicity was indicated by a net loss in body weight at the 50- and 100-mg/kg dose levels. Fetal viability and body weight were depressed at the 100 mg/kg dose level. Shortened digits occurred on the right forepaw of one fetus at the 50-mg/kg dose level (in conjunction with severe maternal toxicity) and on the hindpaws of two fetuses from separate litters at the 100-mg/kg dose level. Incomplete ossification of the phalanges occurred on the forepaws of nine fetuses from four litters at the 100-mg/kg dose level. Ameltolide was weakly teratogenic in the rabbit. The NOEL for adult and developmental toxicity in the rabbit was 25 mg/kg.     

Developmental canalization with no part of stabilizing selection

Referring the developmental canalization to stabilizing selection may be a bias that results from the ignorance of developmental mechanisms. Considering the morphological evolution of one-cell trichomes in Draba plants makes it clear that the transition from continuous variation in morphological traits to developmental creods occurs in the evolution of remote lineages of the genus irrespective of contribution to the net fitness. Morphological diversification of trichome branching is not under selection control, being a physical consequence of the trichome cell volume growth equilibrated by complication of the cell surface shape. At the start of evolution, the trichome development refers not to an individual trichome, but rather to repetitive trichome modules (branches), whose spatiotemporal order is arbitrary, except that some variants of branching depend on events that occur at earlier developmental stages more than others. Under selection fluctuating at random, or with no selection at all, fixing of these variants leads to the formation of trichome ontogeny, in which earlier developmental stages correspond to later stages of developmental evolution.     

Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes.

The highly purified prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes had two still unresolved enzyme activities; the oxygenative cyclization of 8,11,14-eicosatrienoic acid to produce prostaglandin G1 and the conversion of the 15-hydro-peroxide of prostaglandin G1 to a 15-hydroxyl group, producing prostaglandin H1. The latter enzymatic reaction required heme and was stimulated by a variety of compounds, including tryptophan, epinephrine, and guaiacol, but not by glutathione. A peroxidatic dehydrogenation was demonstrated with epinephrine or guaiacol in the presence of various hydroperoxides, including hydrogen peroxide and prostaglandin G1. Higher activity and affinity were observed with the 15-hydroperoxide of eicosapolyenoic acid, especially those with the prostaglandin structure. Both the dehydrogenation of epinephrine or guaiacol and the 15-hydroperoxide reduction of prostaglandin G1 were demonstrated in nearly stoichiometric quantities. With tryptophan, however, such a stoichiometric transformation was not observed. The peroxidase activity as followed with guaiacol and hydrogen peroxide and the tryptophan-stimulated conversion of prostaglandin G1 to H1 were not dissociable as examined by isoelectric focusing, heat treatment, pH profile, and heme specificity. The results suggest that the peroxidase with a broad substrate specificity is an integral part of prostaglandin endoperoxide synthetase which is responsible for the conversion of prostaglandin G1 to H1.     

A new class of prolylcarboxypeptidase inhibitors, part 1: discovery and evaluation

A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.