McKim Jr. J.M.,CeeTox Inc. |
Keller III D.J.,CeeTox Inc. |
Cutaneous and Ocular Toxicology | Year: 2012
Chemical sensitization is a serious condition caused by small reactive molecules and is characterized by a delayed type hypersensitivity known as allergic contact dermatitis (ACD). Contact with these molecules via dermal exposure represent a significant concern for chemical manufacturers. Recent legislation in the EU has created the need to develop non-animal alternative methods for many routine safety studies including sensitization. Although most of the alternative research has focused on pure chemicals that possess reasonable solubility properties, it is important for any successful in vitro method to have the ability to test compounds with low aqueous solubility. This is especially true for the medical device industry where device extracts must be prepared in both polar and non-polar vehicles in order to evaluate chemical sensitization. The aim of this research was to demonstrate the functionality and applicability of the human reconstituted skin models (MatTek Epiderm ® and SkinEthic RHE) as a test system for the evaluation of chemical sensitization and its potential use for medical device testing. In addition, the development of the human 3D skin model should allow the in vitro sensitization assay to be used for finished product testing in the personal care, cosmetics, and pharmaceutical industries. This approach combines solubility, chemical reactivity, cytotoxicity, and activation of the Nrf2/ARE expression pathway to identify and categorize chemical sensitizers. Known chemical sensitizers representing extreme/strong-, moderate-, weak-, and non-sensitizing potency categories were first evaluated in the skin models at six exposure concentrations ranging from 0.1 to 2500 M for 24 h. The expression of eight Nrf2/ARE, one AhR/XRE and two Nrf1/MRE controlled gene were measured by qRT-PCR. The fold-induction at each exposure concentration was combined with reactivity and cytotoxicity data to determine the sensitization potential. The results demonstrated that both the MatTek and SkinEthic models performed in a manner consistent with data previously reported with the human keratinocyte (HaCaT) cell line. The system was tested further by evaluating chemicals known to be associated with the manufacture of medical devices. In all cases, the human skin models performed as well or better than the HaCaT cell model previously evaluated. In addition, this study identifies a clear unifying trigger that controls both the Nrf2/ARE pathway and essential biochemical events required for the development of ACD. Finally, this study has demonstrated that by utilizing human reconstructed skin models, it is possible to evaluate non-polar extracts from medical devices and low solubility finished products. © 2012 Informa Healthcare USA, Inc.
McKim Jr. J.M.,CeeTox Inc.
Cutaneous and ocular toxicology | Year: 2010
Allergic contact dermatitis (ACD) is a significant safety concern for developers of cosmetic, personal care, chemical, pharmaceutical, and medical device products. The guinea pig maximization test (GMPT) and the murine local lymph node assay (LLNA) are accepted methods for determining chemical sensitization. Recent legislative initiatives in Europe require the development of new in vitro alternatives to animal tests for chemical sensitization. The aim of this project was to develop an in vitro screening method that uses a human skin cell line (HaCaT), chemical reactivity, and gene expression profiling to identify positive and negative responses, to place chemicals into potency categories of extreme/strong (ES), moderate (M), weak (W), and nonsensitizers (N), and to provide an estimate of corresponding LLNA values. The method and processing algorithm were developed from a training set of 39 chemicals possessing a wide range of sensitization potencies. Three cationic metals, chromium (Cr), nickel (Ni), and silver (Ag), were also evaluated in this model. Chemical reactivity was determined by measuring glutathione (GSH) depletion in a cell free matrix. Three signaling pathways (Keap1/Nrf 2/ARE/EpRE, ARNT/AhR/XRE, and Nrf1/MTF/MRE) that are known to be activated by sensitizing agents were monitored by measuring the relative abundance of 11 genes whose expression is controlled by one of these 3 pathways. Final exposure concentrations were based on toxicity and solubility. A range-finding experiment was conducted with each compound to determine cytotoxicity and solubility. Six exposure concentrations (0.1 to 2,500 microM) and an exposure time of 24 hours were used in the final experiments. Glutathione depletion alone did not provide the accuracy necessary to differentiate potency categories. However, chemical reactivity combined with gene expression profiles significantly improved the in vitro predictions. A predicted toxicity index (PTI) was determined for each test chemical. A comparison of LLNA values with PTI values revealed an inverse relationship. The large variation in LLNA data for compounds in the same potency category makes direct extrapolation from PTI to LLNA difficult. To challenge the system, 58 additional compounds were submitted in a blinded manner. Compounds placed into ES and M categories were considered positive, whereas compounds classified as W or N were considered negative. Accuracy was approximately 84%, with a sensitivity of 81% and a specificity of 92%. The model correctly identified 2 of 3 cationic metals as positive. In conclusion, the method described here demonstrates a valuable in vitro method for identifying chemicals and metals that induce skin sensitization.
Lapchak P.A.,Cedars Sinai Medical Center |
McKim Jr. J.M.,CeeTox Inc
Translational Stroke Research | Year: 2011
In the present study, we used a comprehensive cellular toxicity (CeeTox) analysis panel to determine the toxicity profile for CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vin yl)-2-methoxy-phenol)], which is a hybrid molecule created by combining cyclohexyl bisphenol A, a molecule with neurotrophic activity and curcumin, a spice with neuroprotective activity. CNB-001 is a lead development compound since we have recently shown that CNB-001 has significant preclinical efficacy both in vitro and in vivo. In this study, we compared the CeeTox profile of CNB-001 with two neuroprotective molecules that have been clinically tested for efficacy: the hydrophilic free radical spin trap agent NXY-059 and the hydrophobic free radical scavenger edaravone (Radicut). CeeTox analyses using a rat hepatoma cell line (H4IIE) resulted in estimated CTox value (i. e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) of 42 μM for CNB-001 compared with >300 μM for both NXY-059 and Radicut. The CeeTox panel suggests that CNB-001 produces some adverse effects on cellular adenosine triphosphate content, membrane toxicity, glutathione content, and cell mass (or number), but only with high concentrations of the drug. After a 24-h exposure, the drug concentration that produced a half-maximal response (TC50) on the measures noted above ranges from 55 to 193 μM. Moreover, all CNB-001-induced changes in the markers were coincident with loss of cell number, prior to acute cell death as measured by membrane integrity, suggesting a cytostatic effect of CNB-001. NXY-059 and Radicut did not have acute toxic effects on H4IIE cells. We also found that CNB-001 resulted in an inhibition of ethoxyresorufin-odeethylase activity, indicating that the drug may affect cytochrome P4501A activity and that CNB-001 was metabolically unstable using a rat microsome assay system. For CNB-001, an estimated in vitro efficacy/toxicity ratio is 183-643-fold, suggesting that there is a significant therapeutic safety window for CNB-001 and that it should be further developed as a novel neuroprotective agent to treat stroke. © 2010 Springer Science+Business Media, LLC.
McKim J.M.,CeeTox Inc.
Critical Reviews in Toxicology | Year: 2014
Carrageenan (CGN) has been used as a safe food additive for several decades. Confusion over nomenclature, basic CGN chemistry, type of CGN tested, interspecies biology, and misinterpretation of both in vivo and in vitro data has resulted in the dissemination of incorrect information regarding the human safety of CGN. The issue is exacerbated when mechanistic data obtained from in vitro experiments are directly translated to human hazard and used for risk assessment. This can lead to information that is taken out of experimental context and reported as a definitive effect in humans. In recent years, the use of cell-based models has increased and their ability to provide key information regarding chemical or drug safety is well established. In many instances, these new alternative approaches have started to replace the need to use animals altogether. In vitro systems can be extremely useful for understanding subcellular targets and mechanisms of adverse effects. However, care must be exercised when extrapolating the in vitro findings to in vivo effects. Often, issues such as chemical identity and purity, relevant dose, pharmacokinetic properties, solubility, protein binding, adsorption to plastics, and the use of cell models that are biologically and mechanistically relevant are overlooked or ignored. When this occurs, in vitro findings can provide misleading information that is not causally linked to in vivo events in animals or in humans. To date, there has not been a comprehensive review of the CGN in vitro literature, which has reported a wide range of biochemical effects related to this compound. An extensive effort has been made to evaluate as much of this literature as possible. This review focuses on the in vitro observation, the unique chemistry of CGN, and potential pitfalls of in vitro models used for hazard identification. The discussion of the in vitro studies discussed this review are supported by numerous in vivo studies. This provides a unique opportunity to have both the in vitro and in vivo studies reviewed together. © 2014 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted.
McKim Jr. J.M.,CeeTox Inc.
Combinatorial Chemistry and High Throughput Screening | Year: 2010
One of the greatest challenges facing the pharmaceutical industry today is the failure of promising new drug candidates due to unanticipated adverse effects discovered during preclinical animal safety studies and clinical trials. Late stage attrition increases the time required to bring a new drug to market, inflates development costs, and represents a major source of inefficiency in the drug discovery/development process. It is generally recognized that early evaluation of new drug candidates is necessary to improve the process. Building in vitro data sets that can accurately predict adverse effects in vivo would allow compounds with high risk profiles to be deprioritized, while those that possess the requisite drug attributes and a lower risk profile are brought forward. In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity. However, when used in a prospective manner, they have not been highly predictive of in vivo toxicity. Therefore, the issue may not be how to collect in vitro toxicity data, but rather how to translate in vitro toxicity data into meaningful in vivo effects. This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation. © 2010 Bentham Science Publishers Ltd.
Ceetox Inc. | Date: 2014-01-29
The invention provides methods for analyzing and predicting the in vivo respiratory toxicity of a compound (e.g., pharmaceutical, biological, cosmetic, or chemical compounds) or composition comprising a combination of an in vitro mammalian cell model with multiple endpoint analysis, and time and concentration response curves. The methods allow the determination of a predicted in vivo respiratory toxicity value of a compound without the use of animals, with a high degree of accuracy. The methods comprise detecting any combination of cell viability markers and expression levels of genes implicated in respiratory toxicity and/or sensitization, such as pro-inflammatory response genes, combining the viability and gene expression level data with concentration response and time response data, conducting a computational analysis, and comparing test compound data to a database of known respiratory toxicants/sensitizers to predict and/or analyze the respiratory toxicity. An indication of organ specificity is provided by a toxicity index, which is determined by comparing mean 10_(50 )values in lung cells to mean 10_(50 )values in liver cells.
Ceetox Inc. | Date: 2010-06-07
The invention provides methods for analyzing and predicting the in vivo respiratory toxicity of a compound (e.g., pharmaceutical, biological, cosmetic, or chemical compounds) or composition comprising a combination of an in vitro mammalian cell model with multiple endpoint analysis, and time and concentration response curves. The methods allow the determination of a predicted in vivo respiratory toxicity value of a compound without the use of animals, with a high degree of accuracy. The methods comprise detecting any combination of cell viability markers and expression levels of genes implicated in respiratory toxicity and/or sensitization, such as pro-inflammatory response genes, combining the viability and gene expression level data with concentration response and time response data, conducting a computational analysis, and comparing test compound data to a database of known respiratory toxicants/sensitizers to predict and/or analyze the respiratory toxicity. An indication of organ specificity is provided by a toxicity index, which is determined by comparing mean IC_(50 )values in lung cells to mean IC_(50 )values in liver cells.
Ceetox Inc. | Date: 2011-02-23
The present invention provides methods of determining a level of toxicity of a given compound based on in vitro assays. The present invention provides particular methods of determining organ-specific toxicity and species-specific toxicity of a given compound based on in vitro assays. In addition, the present invention provides methods of determining a level of toxicity in normal tissue for an anti-tumor compound. The methods include providing at least one cell type and culturing the cell type in the presence of at least one concentration of the chemical compound, measuring at least one indicator of cell health at the at least one concentration of compound for the at least one cell type, and performing a concentration response analysis, from which a toxic concentration can be determined.
Ceetox Inc. | Date: 2013-01-03
Methods of evaluating an anti-tumor compound based on in vitro assays are disclosed. The methods may determine a potency/efficacy of the anti-tumor compound as well as a toxicity of the anti-tumor compound to non-cancerous cells.
CeeTox Inc. | Date: 2012-11-08
Methods of determining a level of ocular irritation and/or toxicity for a chemical compound are described. Kits for use in methods of determining a level of ocular irritation and/or toxicity for a chemical compound are also described.