IONTOX is a biotechnology company founded in 2014 dedicated to providing expertise to the area of in vitro toxicology, with a mission to build improved methods for predicting human adverse effects from chemical exposure.
From laboratories located in Kalamazoo, Michigan, IONTOX serves a global client base in the pharmaceutical, cosmetic, chemical, tobacco, and food additive industries. Through, consulting, product development, and laboratory services the company contributes to the development, application, and interpretation of alternative testing methods. IONTOX aims to advance current alternative toxicology methods through continuous efforts in the research and development of new in vitro technology. Through the company’s contract research laboratories IONTOX offers Advanced in vitro solutions that promise to increase the reliability of in vitro toxicology.
Predict the intestinal permeability and oral absorption of your test compound using our Caco-2 permeability assay.
Caco-2 cells are cultured on transwell membranes (0.4 um) for approximately 3 weeks in order to establish a fully differentiated monolayer. The test compound and controls are applied to the apical surface and the rate of transport across the barrier over time is measured. Control compounds for low (Ranitidine) and high (pindolol) permeability are always included in order to monitor assay performance.
These data are then used to determine the apparent permeability coefficient (Papp; units are cm/s), which is calculated using the following equation: Papp = (dQ/dt) (1/(AC0))
Additional assessments of the test compound can be made as well. These include the efflux ratio (measured by assessing the basal to apical Papp as well as the standard apical to basal Papp). This information can also determine whether the test compound is actively transported by P-glycoprotein (P-gp).
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Predict the metabolic stability (half-life and clearance) of your test compound or xenobiotic using our Metabolic Stability assay.
In the human body, the liver plays a major role in the metabolism and clearance of drugs absorbed through the gut. Therefore, in vitro metabolic stability is an important early ADME test for predicting in vivo half-life and clearance of a test compound. Our Metabolic Stability Assay uses microsomes (various species available) and human primary hepatocytes in a sandwich culture. While microsomes contain Phase I enzymes, primary human hepatocytes in sandwich culture contain both the Phase I and the Phase II enzymes. The microsomes and/or primary human hepatocytes in sandwich culture are exposed to the test compound for a number of exposure times and the samples are then assessed by LC-MS/MS to determine intrinsic clearance of a compound.
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Predict the potential drug-drug interactions of your test compound or xenobiotic using our Cytochrome P450 (CYP) Induction assay.
Cultured primary hepatocytes from three donors are exposed to the controls and the test compound for 72 hours. The test compound should be assessed at several concentrations surrounding the expected human plasma drug concentrations. After the exposure, the mRNA is isolated and CYP1A2, CYP2B6 and CYP3A4 gene expression levels are analyzed by real time RT-PCR (qRT-PCR). The data are normalized to housekeeping genes and presented as fold induction vs negative (vehicle) control. IONTOX can also evaluate changes in CYP activity toward specific substrates. This information provides an added level of confidence.
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Predict the potential drug-drug interactions of your test compound or xenobiotic using our Cytochrome P450 (CYP) Inhibition assay.
In the human body, Cytochrome P450 (CYP) enzymes play a major role in the metabolism of drugs and therefore, CYPs are primary targets in the assessment of drug-drug interactions. Inhibition of CYPs can lead to altered metabolic capacity resulting in the inhibition of the metabolism of one, or both, drugs. This can lead to potentially toxic accumulation of one, or both, drugs. Therefore, it is crucial to assess the CYP inhibition potential of a test compound. Our CYP Inhibition assays uses industry accepted substrates and human liver microsomes expressing the seven main cytochrome P450 isoforms (CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4; other isoforms also available) to assess the decrease in metabolite formation. This assay is performed in accordance with the FDA guidelines on Drug Interaction Studies.
CYP Inhibition assay overview
Recombinant human microsomes containing the seven main CYPs (and other isoforms of your choosing if needed) are incubated with the industry accepted substrates and the controls/test compound. The test compound is assessed at several concentrations. The metabolite for each CYP is measured to determine the % inhibition and calculate an IC50. Test compounds can be classified into three categories:
Strong Inhibitor: IC50 < 1µM
Moderate Inhibitor: IC50 between 1 and 10 µM
Weak/Non-Inhibitor: IC50 > 10 µM
Predict the plasma protein binding potential of your test compound or xenobiotic using our Plasma Protein Binding assay.
In humans, ingested drugs are first absorbed through the gut and then enter the bloodstream, where they interact with blood plasma proteins. The extent of the plasma protein binding influences the drugs distribution and availability. For example, a compound that binds with high affinity to blood plasma proteins means there will be less free compound available for metabolism. This can reduce the therapeutic effects at a particular dose, and can affect clearance of the compound. IonTox uses solid phase microextraction (SPM) to assess plasma protein binding. The test compound is incubated with human (or rat) blood plasma and a buffered solution (PBS). After incubation, the presence of the test compound is analyzed by LC-MS/MS.
Plasma Protein Binding assay overview
First, the plasma (human or rat) and PBS are equilibrated with test compound for 3 hours at 37°C. The SPME filters are then added to the plasma and buffer samples and agitated for 30 minutes. The SPME filters are then incubated in samples containing internal standards for 10 minutes. LC-MS/MS is then used to assess the concentration of test compound on the SPME filters. The percent bound (%B) is calculated as follows:
% Bound = 100((C1 – C2)\C1) where C1 is the concentration of the test compound in PBS while C2 is the concentration of the test compound in the plasma sample.
IONTOX’s Plasma Protein Binding Assay can be combined with our Dynamic Multiple Organ Plate to provide significantly more information on the ADME properties of your test compound(s), The simulated blood system in the Hu-DMOP can be modified to contain plasma proteins and this allows the you to monitor the impact of plasma proteins on distribution.
Protein Binding and Blood To Plasma Ratio Cell Partitioning Assay
Understand how test molecule interacts with whole blood and plasma proteins.
Knowing a drug’s potential for partitioning is critical in situations where the test drug has a high LogP (lipophilic). In this situation a significant portion of the drug may be taken up and held by red blood cells (RBCs). For this reason, more informative data describing drug distribution in blood can be obtained by determining both the blood partitioning ratios and direct protein binding.
When the combined knowledge of protein binding and blood partitioning is understood investigators can make a better determination about whether they should collect whole blood, plasma, or serum for assaying pharmacokinetic behavior of the drug if animal or clinical ADME studies.
Protein Binding assay overview
Equilibrium dialysis or an equivalent is used to separate bound drug (drug-protein) from unbound (free) drug. Whenever possible, Teflon coated compartments are used to reduce nonspecific adsorption. The microdialyzer is agitated during incubation to optimize diffusion kinetics. Plasma pH is adjusted to 7.4 and drug is added and mixed. After the binding period, an aliquot of the plasma-drug mix is placed in the dialysis system and dialyzed against phosphate buffered saline at 37oC with agitation for 2-3 hr. Following the incubation periodsamples are transferred to Teflon cups for analysis by LC/MS/MS (Lin et al., 2005).
Distribution of drug in whole blood overview
To determine the potential for a drug or chemical to partition into red blood cells (RBC partitioning) a series of drug concentrations (e.g. 20, 10, 5, 2.5 µg/mL) are prepared by making dilutions into 6 mL of whole blood. The packed cell volume (PVC) of the test blood is determined. Once the blood-drug dilutions are made the samples are incubated for 24 hours at 37°C. After incubation, plasma and RBCs are separated by centrifugation of whole blood samples at 1200 rpm for 10 minutes. Aliquots of whole blood (after incubation), plasma, RBC pack and standard dilutions are analyzed by LC/MS/MS to determine the amount of drug present in each fraction. The concentration of drug is estimated based on calibration curves. Final results represent the average from triplicate experiments (Deshmukh et al., 2009).
For over 50 years, pharmacokinetics (PK) has been described using the 4 letter acronym ADME, or absorption, distribution, metabolism, and excretion. As the cost of bringing a drug to market increases and the potential for marketed drug withdrawals due to unforeseen ADME issues climb, it is crucial to fully characterize your drug candidates as early as possible. ADME/PK screening in the hit-to-lead and lead optimization phases can help reduce failure rates later on by offering accurate information on ADME, potential drug-drug interactions (DDIs) and potential unforeseen toxicities.
In vitro ADME-PK services
Plasma Protein Binding
Evaluation of Transporter Interactions
Determining cytotoxicity (being toxic to cells) is a critical component of identifying compound viability early in the preclinical phase of drug development or new product discovery. Cytotoxicity Screens (measuring cell death or cell viability) are used to give researchers key insight into potential adverse effects due to cell death prior to entering more costly in vivo studies.
IONTOX's cytotoxicity screens range from predictive multi-endpoint models to standard cell viability screens (ATP) to rank order compounds or mitochondrial toxicity assessments for companies wishing to understand how their compound effects mitochondrial health.
Cytotoxicity based assays
A single organ is used to identify mechanisms of toxicity, screen a large number of compounds for efficacy or toxicity relative to reference compounds and then use these data to rank order compounds.
Single organ testing is used to identify adverse outcome pathways and targets of toxicity critical to understanding overall chemical hazard and for performing risk assessment.
Many of the in vitro methods used industry wide were pioneered by IONTOX founder Dr. James Mckim 15 years ago. IONTOX continues this trend of innovation by, finding and developing the latest in vitro toxicity testing and putting them to work for your business.
Drug Drug Interaction (DDI)
General Renal Toxicity
General Lung Toxicity
ET 50 (Potency)
Predict the potential of your test compound or xenobiotic to cause ocular irritation using our Eye Irritation Test (EIT).
This test is performed according to the Organization for Economic Co-operation and Development (OECD) test guideline number 492 and allows for proper labeling of the test compound or xenobiotic according to United Nations Globally Harmonized System (UN GHS) Classification and Labelling of Chemicals.
Historically, ocular irritation was performed on rabbits using the Draize Eye Test. However, three-dimensional human corneal tissue equivalents have been developed and validated as replacements for the Draize test. These 3D tissues are nonkeratinized, corneal epithelia grown from normal human keratinocytes and are extremely similar to native human cornea, both metabolically and morphologically. In addition, the apical surface is direct contact with the air (tissue is fed basally) allowing for testing of almost all types materials, such as gels, creams liquids, and powders, regardless of aqueous solubility.
In this assay, MatTek EpiOcular™ tissues are exposed on the apical surface to neat (undiluted) test chemical for either 30 minutes (liquid test chemicals) or 6 hours (solid test chemicals) and the viability of the tissue is then determined via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This data is then normalized to the negative control and ocular irritation is determined based on the viability using the following table:
|Mean Tissue Viability||In VivoPrediction|
|≤ 60%||Irritant (Category 1/2)|
|> 60%||Non-Irritant (No Category)|
Predict the potential of your test compound or xenobiotic to cause dermal irritation using our Skin Irritation Test (SIT).
This test is performed according to the Organization for Economic Co-operation and Development (OECD) test guideline number 439 and allows for proper labelling of the test compound or xenobiotic according to United Nations Globally Harmonized System (UN GHS) Classification and Labelling of Chemicals.
Historically, dermal irritation was performed on rabbits using the Draize Skin Test. However, three dimensional human epidermis tissue equivalents have been developed and validated as replacements for the Draize skin test. These 3D tissues are dermal epithelia grown from normal human keratinocytes and are extremely similar to native human epidermis, both metabolically and morphologically, including the stratum corneum. In addition, the apical surface is direct contact with the air (tissue is fed basally) allowing for testing of almost all types materials, such as gels, creams liquids and powders, regardless of aqueous solubility.
In this assay, MatTek EpiDerm™ tissues are exposed on the apical surface to neat (undiluted) test chemical for 30 minutes and the viability of the tissue is then determined via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This data is then normalized to the negative control and ocular irritation is determined based on the viability using the following table:
|Mean Tissue Viability||In VivoPrediction|
|≤ 50%||Irritant (H315)|
|> 50%||Non-Irritant (No Category)|
Additionally, the protocol can be adapted in order to assess irritation potency (mild, moderate, severe etc.) of test chemicals by determining the ET50 over a 24 hour exposure period.
A primary focus for endocrine screens has been on the Hypothalamic-Pituitary-Gonadal axis. General screening assays to evaluate a chemical or environmental samples (water or soil) potential for estrogenic or androgenic activities is a first tier approach.
Estrogen (ER) and Androgen (AR) receptor transactivation assays that test for chemical ER or AR agonists are designed to identify estrogenic or androgenic functional activity. This means that if a positive response is measured in the system, the response is due to chemical binding to the ER or AR receptor and subsequent binding of the chemical-receptor complex to the respective DNA response elements (ERE or ARE regions) to induce the expression of a reporter gene, such as luciferase. Chemical antagonism can also be assessed by evaluating the test chemicals ability to activate the signaling pathway in the presence of the endogenous ligand (e.g. estrogen) at a single concentration. A diminished response in luciferase expression when compared to estrogen alone would indicate antagonism.
Extraction of Environmental Water Samples:
In order to improve the detection of ER or AR active chemicals in large water sources it is sometimes necessary to concentrate the samples by solid phase extraction with OASIS HLB columns. The efficiency and reproducibility of this process can be tracked by including water samples spiked with the analyte of interest and then quantifying the recovery of the analyte following the extraction procedure by LC/MS/MS.
Estrogen and Androgen Receptor Binding:
Understanding direct receptor binding can provide important information on binding affinity relative to endogenous molecules and provide information on IC50 for test material binding. These assays can be done using either human or rat estrogen or androgen receptor. The receptors are incubated with endogenous ligand that has been radiolabeled. The test material is titrated into the system and the displacement of the endogenous ligand recorded.
The control and tight regulation of circulating thyroid hormones T3/T4 is important for normal metabolism, growth, development, and reproduction. Endocrine disruptive chemicals can interfere with thyroid physiology by altering TRH release from the hypothalamus, or TSH release from the pituitary, or by interfering with thyroid hormone binding to the thyroid receptor. Polychlorinated biphenyls (PCBs) directly block or competitively inhibit thyroid hormone binding to its receptor.
Thyroid Receptor and Functionality Assay:
IONTOX can evaluate drugs or chemicals for interaction with thyroid receptor and functionality of chemical binding using a thyroid transactivation assay.
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