[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Following a US National Academy of Sciences report in 2007 entitled "Toxicity Testing of the 21st Century: a Vision and a Strategy," significant advances within translational drug safety sciences promise to revolutionize drug discovery and development. The purpose of this review is to outline why investigative safety science is a competitive advantage for the pharmaceutical industry.
The article discusses the essential goals for modern investigative toxicologists including: cross-species target biology; molecular pathways of toxicity; and development of predictive tools, models and biomarkers that allow discovery researchers and clinicians to anticipate safety problems and plan ways to address them, earlier than ever before. Furthermore, the article emphasizes the importance of investigating unanticipated clinical safety signals through a combination of mechanistic preclinical studies and/or molecular characterization of clinical samples from affected organs.
The traditional boundaries between pharma industry teams focusing on safety/efficacy and preclinical/clinical development are rapidly disappearing in favor of translational safety science-centric organizations with a vision of bringing more effective medicines forward safely and quickly. Comparative biology and mechanistic toxicology approaches facilitate: i) identifying translational safety biomarkers; ii) identifying new drug targets/indications; and iii) mitigating off-target toxicities. These value-adding safety science contributions will change traditional toxicologists from side-effect identifiers to drug development enablers.
No preview · Article · Jul 2012 · Expert Opinion on Drug Metabolism & Toxicology
[Show abstract][Hide abstract] ABSTRACT: Kidney toxicity accounts both for the failure of many drug candidates as well as considerable patient morbidity. Whereas histopathology remains the gold standard for nephrotoxicity in animal systems, serum creatinine (SCr) and blood urea nitrogen (BUN) are the primary options for monitoring kidney dysfunction in humans. The transmembrane tubular protein kidney injury molecule-1 (Kim-1) was previously reported to be markedly induced in response to renal injury. Owing to the poor sensitivity and specificity of SCr and BUN, we used rat toxicology studies to compare the diagnostic performance of urinary Kim-1 to BUN, SCr and urinary N-acetyl-beta-D-glucosaminidase (NAG) as predictors of kidney tubular damage scored by histopathology. Kim-1 outperforms SCr, BUN and urinary NAG in multiple rat models of kidney injury. Urinary Kim-1 measurements may facilitate sensitive, specific and accurate prediction of human nephrotoxicity in preclinical drug screens. This should enable early identification and elimination of compounds that are potentially nephrotoxic.
Full-text · Article · May 2010 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and beta2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.
Full-text · Article · May 2010 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.
Full-text · Article · May 2010 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Drug-induced kidney injury is a serious and not uncommon adverse event which needs to be considered during drug development. The current standards used to monitor kidney function, such as blood urea nitrogen and serum creatinine, are late indicators of kidney injury and thus do not allow for timely intervention before loss of function. Improving the diagnosis and monitoring of kidney damage goes hand-in-hand with the identification of new biomarkers and the development of technologies that enable their sensitive and specific measurements. In order to move beyond restriction to internal company decisions, every entity that demonstrates the qualities of a biomarker must gain acceptance by health authorities if it is to be used for regulatory decision making in preclinical studies and clinical trials. This review focuses on the most promising achievements of new technologies applied to monitoring drug-induced nephrotoxicity (eg, gene expression, imaging, in vitro screening, protein assays) and on the use and implications of peripheral biomarkers such as the urinary protein biomarkers glutathione S-transferase-alpha, N-acetyl-beta-d-glucosaminidase, total protein, cystatin C, beta2-microglobulin, KIM-1, lipocalin-2 and serum cystatin C. Finally, the associated regulatory processes for use in clinics are also discussed.
No preview · Article · Feb 2008 · Current opinion in drug discovery & development
[Show abstract][Hide abstract] ABSTRACT: The aim of this study is to test the predictive power of in vivo multiorgan RNA expression profiling in identifying the biologic activity of molecules.
Animals were treated with compound A or B. At the end of the treatment period, in vivo multiorgan microarray-based gene expression data were collected. Investigators masked to the identity of the compounds analyzed the transcriptome signatures to define the molecular pathways affected by treatment and to hypothesize the biologic activity and potential therapeutic indications of the blinded compounds.
For compound A, G-protein-coupled receptors and factors associated with cell growth were affected-growth hormone/insulin-like growth factor-1, glucagon/insulin axes, and general somatomedin-like activity. Deblinding showed the compound to be a somatostatin analog, SOM230, confirming the accuracy of the predicted biologic activity. For compound B, components of the inflammatory cascade potentially mediated by lipopolysaccharide, tumor necrosis factor, or proinflammatory cytokines were affected. The gene expression signatures were most consistent with an interleukin-6 family activity. Deblinding revealed that compound B was leukemia inhibitory factor.
VeloceGenomics is a strategy of coupling in vivo compound testing with genomic technologies. The process enables prediction of the mechanism of action and, coupled with other relevant data, prediction of the suitability of compounds for advancement in the drug development process.
No preview · Article · Nov 2005 · Pharmaceutical Research
[Show abstract][Hide abstract] ABSTRACT: A discovery process beginning with an in vivo screening of proteins, peptides, natural products, classical medicinal compound or other substances. The administration of compounds to the animal can be either direct or indirect, such as by the administration and expression of cDNA-containing plasmids. Since the discovery process of the invention is based on a non-preconceived hypothesis and whole organism multi-organ analysis, a compound can be selected for testing in the absence of any biological selection criteria. The resulting organism-wide pattern of the gene expression changes in the transcriptome provides an overview of the activities at the molecular and organism-wide levels. The discovery process of the invention then integrates in vivo profiling and internal and external genomic databases to elucidate the function of unknown proteins, typically within few months. The invention further relates to medical uses of fibroblast growth factor 23 (FGF-23), FGF-23 fragments, FGF-23 C-terminal polypeptides, FGF-23 homologs and/or FGF-23 variants.
[Show abstract][Hide abstract] ABSTRACT: The ascomycin macrolactam pimecrolimus is a novel inflammatory cytokine release inhibitor that so far has not been administered systemically to humans. In this phase I/II randomized double-blind, placebo-controlled, multiple rising dose proof of concept study psoriasis patients were treated with oral pimecrolimus or placebo. Gene profiling identified a common genomic profile with a downregulation of genes associated with inflammation but no changes in gene expression linked to drug-related side-effects. A steady state of pimecrolimus was reached after 5-10 d, Cmax, and area under the curve (0-24) was 54.5 ng per ml and 589.9 ng h per ml, respectively, at steady state at the highest dose. There was clear clinical efficacy in patients receiving 20 mg pimecrolimus twice daily and 30 mg twice daily with a reduction of Psoriasis Area and Severity Index by 60% and 75%, respectively. Histopatho logically and immunopathologically there was a reversion of the psoriatic phenotype towards normal. There were no notable clinical, laboratory, kidney function, or immunologic side-effects. We conclude that pimecrolimus taken orally is highly effective in a concentration-dependent manner in patients with psoriasis and on a short-term basis it is well tolerated and this is confirmed by its pharmacogenomic profile. The latter also indicates that pimecrolimus should be equally effective in other inflammatory skin diseases.
Full-text · Article · Nov 2002 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: The main goal of the present study was to investigate the response of the human skin equivalent Apligraf in vitro to the application of irritant substances and its predictivity as a screening tool for cumulative skin irritant potential in humans. Vaseline, calcipotriol, trans-retinoic acid, and sodium lauryl sulfate were applied to Apligraf in vitro for 24 h. Cell viability (lactate dehydrogenase leakage), release and mRNA expression of the proinflammatory cytokines IL-1α and IL-8, and morphological changes were assessed. The same products were applied to 30 healthy volunteers in a double-blind, randomized, vehicle-controlled within-subject study. The skin reactions after repeated 24-h applications over 3 weeks under Finn chamber patches were monitored by visual scoring and biophysical methods (trans-epidermal water loss, chromametry, and blood flow). Sodium lauryl sulfate was cytotoxic to Apligraf, and increased the release and expression of cytokines at low (0.2%, 0.4%), but not at high (0.8%, 1%) concentrations. It induced severe irritancy in vivo. Trans-retinoic acid increased the expression and release of cytokines with no detectable cytotoxicity and showed moderate irritancy in humans. Although calcipotriol did neither affect cell viability nor the production of cytokines, it induced morphological signs of irritation and was mildly irritant for healthy volunteers. Vaseline was innocuous in vivo and induced no changes in Apligraf. In conclusion, the cumulative skin irritation potential of the tested products could be predicted with Apligraf in a sensitive and specific manner, by monitoring cytotoxicity, proinflammatory cytokines, and morphological changes.
No preview · Article · May 2000 · Toxicology and Applied Pharmacology
[Show abstract][Hide abstract] ABSTRACT: For the development of mechanistic assays in immunotoxicology, the phenotype, cytokine production, and stimulatory function of dendritic cells (DCs) were assessed after incubation with the chemical haptens aminophenol, chlorpromazine hydrochloride, dinitrochlorobenzene (DNCB), and with the DNCB-corresponding tolerogen DCNB, the metal allergen nickel sulfate, the irritants sodium dodecyl sulfate and benzoic acid, as well as with staphylococcal enterotoxin B (SEB) and lipopolysaccharide (LPS). DCs were differentiated from human monocytes by in vitro exposure to GM-CSF and interleukin-4 (IL-4) for 7 days. Flow cytometric data revealed that only representative haptens increased the surface expression of HLA-DR, CD86, CD40, and of CD54 on DCs when compared to irritants or to the tolerogen. This event was associated with an increased ability of DCs to stimulate T cell proliferation. Moreover, after incubation with the haptens, but not with the irritants or the tolerogen, a higher production of TNF-alpha by DCs was observed. Under our experimental conditions, no release of IL-1beta, IL-10, or IL-12 was detected. Compared to the activation elicited by haptens, SEB strongly up-regulated HLA-DR and costimulatory molecule expression. In agreement with this effect, there was a marked release of TNF-alpha and a slight production of IL-12. IL-1beta and IL-10 were not detected in the culture medium. Finally, SEB-pulsed DCs showed a strong T-cell-stimulating activity. These data underline the activating potential of haptens versus irritants or a tolerogen on DC functions. The different levels of DC activation by haptens and SEB suggested that distinct cellular events were involved.
Preview · Article · Jan 2000 · Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: The immunosuppressive drug, cyclosporin A (CsA), which is successfully used to prevent rejection in organ transplantation, induces renal side-effects as shown by a decrease in glomerular filtration rate and ultrafiltration coefficient regulated by the tone of mesangial cells.The aim of the present study was to investigate the effect of CsA on isolated glomeruli and mesangial cells, which constitute appropriate in vitro models for renal vasoreactivity studies. The roles of different intracellular and extracellular mediators such as calcium, endothelin-1 (ET-1), prostaglandins (TXA(2 )and PGI(2)) and reactive oxygen intermediates (ROIs) were analysed. CsA caused a concentration- and time-dependent decrease in the planar cross-sectional areas of isolated glomeruli and mesangial cells as determined by image analysis. Intracytosolic free calcium concentration determined by fluorimetric analysis was significantly increased after 30 min CsA (10 microM) incubation. In the contraction experiment, the calcium antagonist verapamil inhibited the CsA response. ET-1, TXB(2) and keto-PGF(1alpha) were determined directly, however no changes were found statistically significantly different from respective controls. In contrast to these results, the ET-1 specific antibody was able to reduce CsA-mediated cell contraction. In the presence of a prostacyclin agonist iloprost, CsA-induced contraction was also modified. The role of ROIs using a 2'7'-dichlorofluorescein diacetate (DCFdAc) fluorimetric method was directly determined by observing, with 10 microM CsA, a significant production of hydrogen peroxide (H(2)O(2)), which was able alone to induce mesangial cell contraction. Coincubation with the antioxidants led to a significant inhibition of mesangial cell contraction. These results suggest that CsA caused an imbalance in the normal level of all investigated vasoconstrictive and vasodilator mediators, which shifted towards the advantage of vasoconstrictive action.
No preview · Article · Sep 1999 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: Administration of antiasthmatic drugs in the form of inhalation particles may alter the cytokine network in the airways, independently of their pharmacological actions. Changes induced by drugs not well tolerated may potentially contribute to the immunopathology of the disease, a strongly undesirable effect. In this study, cell viability assays and characterization of the cellular profile of cytokines and chemokines were performed in order to investigate the response of human bronchoalveolar macrophages and bronchial epithelial cells in culture to inhalation particles of the cyclosporine derivative IMM 125. Interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-8 were assayed by enzyme-linked immunosorbent assay (ELISA) in the supernatants of bronchoalveolar macrophages, and RANTES, granulocyte--macrophage colony-stimulating factor (GM-CSF), and IL-8 in those of bronchial epithelial cells. Cells were studied both under basal and stimulated conditions (lipopolysaccharide and TNFalpha were used for activating macrophages and epithelial cells, respectively). The immunosuppressant FK 506 and the glucocorticoid Budesonide served as comparison. IMM 125 did not affect cell viability (except at high concentrations and long time periods). Moreover, IMM 125 did not induce an increase in the secretion of any of the cytokines and chemokines measured with respect to nontreated cells, except for a slight increase in IL-8, an effect that was also observed for FK 506, Budesonide, and inert latex particles, and was therefore regarded as nonspecific. Furthermore, IMM 125 significantly decreased the secretion of TNFalpha, IL-1beta by macrophages, and GM-CSF by epithelial cells, suggesting an antiinflammatory potential. In conclusion, the present in vitro results point to a good tolerance of human airways to IMM 125 inhalation particles.
No preview · Article · Sep 1999 · Inhalation Toxicology
[Show abstract][Hide abstract] ABSTRACT: An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1alpha and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs.
No preview · Article · May 1999 · Cell Biology and Toxicology
[Show abstract][Hide abstract] ABSTRACT: In order to develop an in vitro predictive assay for the detection of contact sensitizers, we investigated the possible modulation of the expression of cell-surface molecules in the early phases of treatment of murine epidermal cells (EC) with known contact sensitizers. After in vitro treatment of Balb/c EC with the strong contact sensitizer, TNBS, Langerhans cells (LCs) demonstrated a rapid up-regulation of CD45, CD40, CD32/16 (Fc gamma RII/III) and CD23 (Fc epsilon RII) molecules. CD45 and CD40 were also rapidly up-regulated on the dendritic epidermal T cells. Interestingly, after treatment with this severe sensitizer, a marked induction of CD40 expression was found on a CD45 negative population, most probably keratinocytes. In contrast to these cell-surface molecules, I-Ad/I-Ed and CD90.2 expression were unchanged. No change was observed on the expression of CD45 and CD40 after treatment with a mild or a weak contact sensitizer, citral and citronellal respectively. In contrast, like TNBS, they up-regulated the expression of CD32/16 and CD23 on LCs. The irritant sodium dodecyl sulfate had no effect on all these cell-surface molecules. Our results indicated that in vitro, chemicals with allergic potential induced early specific phenotype changes that may represent an early-activated state of the cells. This state may be responsible for initiating the afferent phase of contact sensitivity in vivo. Based on these findings, it might be possible to develop an in vitro assay to reduce the number of experimental animals for a fast screening of contact sensitizers and for discriminating between mild contact sensitizers and irritants.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to compare two types of cyclosporin (Cs) particles, SDZ OXL 400 and SDZ IMM 125, the latter being more hydrophilic, to understand their uptake by airway macrophages. Alveolar macrophages (AM), harvested by bronchoalveolar lavage (BAL) of hamster lungs, were cultured with two different doses (0.1 mg and 0.5 mg) for 1 h, 6 h, and 24 h. Control incubations without Cs particles or with latex particles were carried out simultaneously. Cell viability, cell activation (i.e., respiratory burst, interleukin-6 (IL-6) synthesis) and mean volume of particles phagocytosed per macrophage were measured. Both types of Cs particles did not modify the AM viability, and failed to induce IL-6 synthesis during phagocytosis but slightly decreased the cell oxidative respiratory burst. The comparison between SDZ OXL 400 and SDZ IMM 125 showed that for the lower dose the mean volume of both Cs types phagocytosed was similar at 1 h and 6 h. At 24 h an increase of the mean volume phagocytosed was seen for SDZ IMM 125 but not for SDZ OXL 400. For the higher dose the mean volume of SDZ IMM 125 phagocytosed was higher than SDZ OXL 400 at 1 h and 6 h and comparable for both types at 24 h. SDZ IMM 125 particles were phagocytosed more rapidly than SDZ OXL 400. The mean volume of phagocytosed latex particles increased with time and dose and was higher than for both Cs particle types. In conclusion, AM were seen to phagocytose particles of different physical properties (i.e., form, size, and shape), chemical properties (i.e., inert or peptidic) and degrees of hydrophilicity in a different manner.
No preview · Article · Jan 1999 · Cell Biology and Toxicology