Cyprotex US, LLC

Cell viability assessment

In addition to multi-parametric cytotoxicity assessment using high content screening, Cyprotex offer general cell viability assessment using single endpoints such as LDH (lactate dehydrogenase), neutral red or MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide).

• LDH is a stable enzyme, present in all cell types, and is rapidly released into the cell culture medium upon damage of the plasma membrane. Neutral red measures the ability of viable cells to incorporate and bind the neutral red dye in the lysosomes. MTT is a yellow, water-soluble tetrazolium dye that is converted by mitochondrial dehydrogenases in viable cells to a water-insoluble, purple formazan.
• LDH, neutral red and MTT are well established, sensitive and reliable endpoints of cytotoxicity.
• Cyprotex offer assessment of LDH, neutral red or MTT in HepG2, NIH3T3, HaCaT cells or primary hepatocytes.

Through its acquisition of the business and assets of CeeTox in January 2014, Cyprotex can offer a range of services to evaluate skin sensitization. These include the Direct Peptide Reactivity Assay (DPRA), the KeratinoSens™ and the SenCeeTox assays.

Understand the potenial irritation to the eye of your individual ingredients / chemicals or finished products using our ocular irritation assay.

Ocular irritation is in our portfolio of in vitro topical services for the cosmetics/personal care, chemical or pharmaceutical industry. Cyprotex deliver consistent, high quality data under either GLP or non-GLP conditions.

Ocular irritation and its measurement in vitro

Cyprotex’s ocular irritation test is performed using MatTek's in vitro 3D EpiOcular™ model. Cyprotex are a MatTek-approved laboratory for performing these studies.
The EpiOcular™ tissue model consists of normal, human-derived epidermal keratinocytes that have been cultured to form a stratified, squamous epithelium which closely mimics the human corneal epithelium.
EpiOcular™ was developed by MatTek to create an in vitro (non-animal) alternative to the animal-based Draize Eye Irritation Test used in the cosmetics, personal care, household products, chemical, pharmaceutical, and biotech industries.
The EpiOcular™ eye irritation test model is currently undergoing ECVAM validation. Peer-reviewed publications have shown the EpiOcular™ eye irritation model achieves greater than 80% accuracy when compared with the Draize eye irritation test.
The EpiOcular™ model is metabolically and mitotically active and produces the same cytokines involved in irritation and inflammation in vivo.
A range of different test articles can be assessed including liquids, solids, semi-solids, pastes, gels, creams and waxes.
Cyprotex's ocular irritation test is based upon assessment of the cytotoxicity following exposure to a test chemical, typically at three time points. Cytotoxicity is expressed as a decrease in mitochondrial conversion of MTT to formazan.

Through its acquisition of the business and assets of CeeTox in January 2014, Cyprotex can offer a range of services to evaluate skin sensitization. These include the Direct Peptide Reactivity Assay (DPRA), the KeratinoSens™ and the SenCeeTox assays.

Cyprotex’s skin irritation test is performed using MatTek's in vitro 3D human skin tissue model (EpiDerm™). Cyprotex are a MatTek-approved laboratory for performing these studies.

Contact us to find out more about our dermal penetration and toxicity services.

The EpiDerm™ tissue model, which closely mimics human epidermis, was developed by MatTek to create an in vitro (non-animal) alternative to the animal-based Draize Skin Irritation Test used in the cosmetics, personal care, household products, chemical, pharmaceutical, and biotech industries.
The scientific validity of the EpiDerm™ skin irritation test model has been endorsed by the ECVAM Scientific Advisory Committee (ESAC) as an in vitro replacement for the Draize Skin Irritation Test. This led to the adoption of the OECD Test guidelines 439 which were published in 2010.
Cyprotex follows the standardized skin irritation test procedure within the OECD guidelines. Our skin irritation test results provide a robust assessment of skin irritation using a model accepted by the EU regulatory authorities.

Endocrine Disruption

Endocrine disruptors are chemicals that interfere with the hormonal system in the body which can lead to adverse effects on the developmental, reproductive, neurological and immune systems. Both natural and man-made chemicals are reported to cause endocrine disruption. These include pharmaceuticals (e.g., 17α-ethinyl estradiol), pesticides and herbicides (e.g., DDT), heavy metals (e.g., cadmium and lead), plasticisers (e.g., bisphenol A) and various other chemicals.

Endocrine disruptors are found in common everyday products such as metal food cans, plastic bottles, detergents, toys, cosmetics and pesticides. Although the link between endocrine disruptors and disease continues to be debated, epidemiological data show increases in incidence and prevalence of diseases associated with endocrine-disrupting chemicals, such as breast, prostate, and testis cancer, diabetes, obesity, and decreased fertility over the last 50 years. There is particular concern regarding low levels of environmental chemical and their effects on foetal development and on the behaviour or development of children.

Chemicals can disrupt the endocrine system via a variety of mechanisms including:

• Mimicking or enhancing the action of endogenous hormone.
• Blocking a hormone receptor, thereby preventing the action of an endogenous hormone.
• Affecting the synthesis, transport, metabolism or excretion of an endogenous hormone.
• Endocrine disruptors most commonly affect the sex hormones (e.g., estrogen and androgen), the hypothalamic-pituary-adrenal axis, and thyroid hormone, as well as the hormones involved in the feedback regulation of these hormones (e.g., gonadotrophin releasing hormone, corticotropin).

Through its acquisition of the business and assets of CeeTox in January 2014, Cyprotex can now offer a range of services to evaluate endocrine disruption which include standard screening assays as well as services compliant with the US EPA Endocrine Disruption Screening Program (EDSP).

Our in vitro ADME and DMPK services include in vitro metabolism, in vitro permeability and transporters, solubility and physicochemical properties, in vitro protein binding and PK and bioanalysis

• Highly reproducible, accurate data - validated and used by over 900 Clients from the pharmaceutical, biotechnology, agrochemical, tobacco, cosmetics, health care companies and academic and government organisations.
• Delivery of data for core in vitro ADME screening and physicochemical assays is within 10 working days to fit in with the make-test timelines in drug discovery. A number of different reporting options are available.
• Highly cost effective due to our emphasis on high throughput engineering for key in vitro screening assays.
• Attention to good quality customer care, with highly trained Principal Scientists on hand to explain results and suggest the most appropriate experimental strategy.
• Flexibility - studies can be tailored to our customers’ specific requirements.
• Regulations - our ADME services maintain and comply with regulatory guidelines providing constant confidence in the data.
• Our facility has passed evaluations by a range of different organisations and companies from a variety of differing industries, including government agencies: All now routinely use our services to provide support to their programs.

Metabolite Profiling and Identification using High Resolution Accurate Mass Spectrometry

Understand the drug metabolism of your compound by identifying which metabolites are formed during in vitro or in vivo studies. Metabolite profiling and identification is included in Cyprotex's portfolio of ADME services. Cyprotex delivers consistent, high quality data in line with regulatory guidelines, and can adapt protocols based on specific customer requirements.

Profiling and identification of metabolites:

• Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data.
• Cyprotex’s metabolite profiling and identification service uses the Xevo G2-S QTof to determine high resolution accurate mass of any metabolites – leading to increased sensitivity and enhanced structural characterization.
• Utilization of Waters’ proprietary MS technology* allows for MS and 'MS/MS' to be collected in parallel from a single injection without compromising on sensitivity or selectivity.
• Service can be combined with the LabLogic Beta-Ram for radiochemical detection and quantification of metabolites.
• Critical information on the formation of metabolites including, where appropriate, both Phase I and Phase II metabolism, and comparison of drug metabolism routes in different species is provided.
• Metabolites can be investigated in a number of different matrices including in vitro microsomal incubations, hepatocyte incubations, expressed enzyme incubations as well as in vivo samples.
• Cyprotex offers a range of metabolite profiling services depending upon the level of detail and interpretation required.

The parallel artificial membrane permeability assay is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.
The Parallel Artificial Membrane Permeability Assay (PAMPA) is used as an in vitro model of passive, transcellular permeation.

• PAMPA avoids the complexities of active transport, allowing test compounds to be ranked based on a simple permeability property alone.
• The ability of this assay to evaluate permeability over a large pH range is valuable for an early understanding how new oral compounds might be absorbed across the entire gastrointestinal tract.

• Establishes the risk of QT interval prolongation and potentially fatal ventricular tachyarrhythmia (TdP).
• Employs the Ionworks™ HT System (Molecular Devices) as an automated patch clamp electrophysiology measurement.
• Delivers high quality, accurate and sensitive data which is comparable with the traditional single cell patch clamp method.

Understand the potential drug-drug interaction liabilities of your compounds by using our cytochrome P450 (CYP450) reversible inhibition assay for a range of isoforms.

The cytochrome P450 inhibition assay is one of our portfolio of in vitro ADME screening services. Cyprotex delivers consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Inhibition of cytochrome P450 (CYP450) enzymes

• Cytochrome P450 are a family of enzymes which play a major role in the metabolism of drugs.
• Assessment of the potential of a compound to inhibit a specific cytochrome P450 enzyme is important as co-administration of compounds may result in one or both inhibiting the other’s metabolism. This may affect plasma levels in vivo and potentially lead to adverse drug reactions or toxicity.
• In vitro cytochrome P450 inhibition data are useful in designing strategies for investigating clinical DDI Studies.
• Cyprotex's Cytochrome P450 Inhibition assays use industry accepted probe substrates and human liver microsomes.
• In Cyprotex's Cytochrome P450 Inhibition assay, a decrease in the formation of the metabolites compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).

Cyprotex's Caco-2 Permeability assay uses an established method for predicting the in vivo absorption of drugs across the gut wall by measuring the rate of transport of a compound across the Caco-2 cell line.

• The Caco-2 cell line is derived from a human colon carcinoma. The cells have characteristics that resemble intestinal epithelial cells such as the formation of a polarized monolayer, well-defined brush border on the apical surface and intercellular junctions.
• Assessing transport in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer enables an efflux ratio to be determined which provides an indicator as to whether a compound undergoes active efflux.
• The P-glycoprotein (P-gp) inhibitor, verapamil, can be included to identify whether active transport is mediated by P-gp.

Understand the potential drug-drug interaction liabilities of your compounds by using our cytochrome P450 (CYP450) induction assay.

Cytochrome P450 induction is one of Cyprotex's in vitro experimental ADME services. Cyprotex delivers consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Induction of cytochrome P450 enzymes is associated with an increased prevalence of clinical drug-drug interactions.

• Cyprotex's Cytochrome P450 Induction assay identifies the potential of test compounds to induce CYP1A2, CYP2B6 or CYP3A4 in fresh cultured human hepatocytes by evaluating catalytic activity and mRNA levels as recommended in a recent PhRMA perspective (Chu et al., 2009 Drug Metab Dispos 37; 1339-1354).
• Test drug concentrations should be based on the expected human plasma drug concentrations. At least three concentrations spanning the therapeutic range should be studied, including at least one concentration that is an order of magnitude greater than the average expected plasma drug concentration. If this information is not available, concentrations ranging over at least two orders of magnitude should be studied.
• Cyprotex's Cytochrome P450 Induction assay delivers data elucidating the extent of induction relative to positive and negative controls.
• The clinical consequences of induction may be therapeutic failure caused by a decreased systemic exposure of the drug itself or a co-administered therapy, or toxicity as a result of increased bioactivation.

PXR and AhR Nuclear Receptor Activation

• The primary mechanism of cytochrome P450 induction is via increased gene transcription which typically occurs through nuclear receptor activation.
• The most common nuclear receptors involved in the induction of drug metabolizing enzymes include the pregnane X receptor (PXR), the aryl hydrocarbon receptor (AhR), and the constitutive androstane receptor (CAR) which are known to regulate CYP3A4, CYP1A2 and CYP2B6, respectively.
• An industry survey of current practices and recommendations (Chu et al., (2009) Drug Metab Dispos 37; 1339) indicates 64% of survey respondents routinely use nuclear receptor transactivation assays to assess the potential of test compounds to cause enzyme induction.
• Cyprotex can evaluate PXR and AhR nuclear receptor activation utilizing stably-transfected human hepatoma cell lines (DPX2™ for PXR and 1A2-DRE™ for AhR) and a luciferase reporter gene assay.

Monoamine oxidase (MAO) inhibition assay

Understand if your compound is an inhibitor of MAO-A or MAO-B enzyme. Monoamine oxidase (MAO) inhibition is a non-CYP mediated metabolism assay within our portfolio of in vitro ADME screening services. Cyprotex delivers consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Determining potential inhibition of MAO enzymes

• Monoamine oxidases (MAO) are membrane-associated enzymes located specifically to the outer mitochondrial membrane. They are the major enzymes participating in the catabolism of monoamine neurotransmitters and related exogenous amines.
• Two isoforms of MAO exist, MAO-A and MAO-B, which differ in their substrate specificity, inhibitor selectivity and tissue distribution.
• Selective MAO-A inhibitors are useful in the therapy of depression and anxiety whereas MAO-B inhibitors are often used in the treatment of Parkinson’s and Alzheimer’s diseases
• Cyprotex’s MAO inhibition assay identifies if your compound is an inhibitor for either MAO-A or MAO-B.

High Content Toxicology: Cytotoxicity Screening Panel

Drug toxicity is often a combination of multiple mechanisms. A single experimental approach is unlikely to capture the complexity involved in cellular toxicity.

• High Content Screening uses automated fluorescence imaging to simultaneously analyze multi-parametric indicators of cellular toxicity. It can detect general cell death and/or mechanisms of cell death within the same cell population within the same well, and it can cover a wide spectrum of cytopathological changes.
• Cyprotex have the most advanced High Content Screening equipment available, including four Thermo Scientific Cellomics ArrayScan VTI’s and a Thermo Scientific Cellomics ToxInsight.
• Cyprotex offer a cell based cytotoxicity panel to evaluate key toxicity markers including cell number, nuclear condensation, total nuclear intensity, cell permeability, mitochondrial membrane potential and cytochrome c release.

Plasma Protein Binding Assay

Understand the distribution potential of your compound using our plasma protein binding assay.

Plasma protein binding is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Determination of fraction unbound in plasma using equilibrium dialysis:

• The extent of binding to plasma influences the way in which a drug distributes into tissues in the body.
  Extensive plasma protein binding also limits the amount of free compound available to access sites of action in the cell, and metabolism and elimination may be slower.
• Equilibrium dialysis is the most widely accepted method for assessing plasma protein binding as non specific binding effects are minimized compared with other methods such as ultrafiltration.
• Cyprotex's Plasma Protein Binding assay is performed using an equilibrium dialysis method and delivers a value of fraction of compound unbound to proteins (fu).
• There is a choice of three methods for assessing plasma protein binding using three different percentages of plasma to provide flexibility depending on budget and compound characteristics.

Turbidimetric Solubility Assay

Understand if poor solubility is likely to be an issue in the development of your compound.

Turbidimetric solubility is one of Cyprotex's in vitro ADME screening services. Cyprotex delivers consistent, high quality data with cost-efficiency that comes from a highly automated approach.

The role of turbidimetric solubility measurement in early discovery:

• Poor solubility can limit the quality of the data generated in other in vitro assays. Therefore, it is essential to evaluate solubility at an early stage in drug discovery.
• The solubility of a compound is an important factor in determining its absorption from the gastrointestinal tract and ultimately its oral bioavailability.
• Compounds with poor solubility can pose a development challenge and result in prolonged development time frames and increased cost.
• Turbidimetric solubility is now an accepted early stage screen in drug discovery. Cyprotex's Turbidimetric Solubility assay investigates the kinetic solubility of compounds by diluting a test compound solution prepared in DMSO into aqueous buffer. Turbidimetry is used as the end-point by measuring absorbance at 620 nm.
• Turbidimetric solubility allows a rapid determination of solubility using small amounts of compound.

Plasma stability assay

Use our plasma stability assay to measure the degradation of your compounds in plasma.

The plasma stability assay is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Determination of stability in plasma

• Determination of the stability of new chemical entities in plasma is important as compounds (with the exception of pro-drugs) which rapidly degrade in plasma generally show poor in vivo efficacy.
• Instability in plasma can result in misleading in vitro data which can be difficult to interpret (e.g., plasma protein binding data). Storing and analyzing clinical samples from in vivo pharmacokinetic studies may also prove challenging.
• Compounds with the following functional groups tend to be more susceptible to hydrolysis in plasma: esters, amides, lactones, lactams, carbamides, sulfonamides, and peptic mimetics.
• Compounds may exhibit interspecies differences in their stability in plasma.
• Plasma stability is very useful for screening of prodrugs and antedrugs, where rapid conversion in plasma is desirable.

• The Ames test assesses the mutagenic potential of a compound. Ames testing uses strains of the bacterium Salmonella typhimurium which carry a defective (mutant) gene that renders them unable to synthesize the amino acid histidine. The Ames test investigates the potential of the test compound to result in a back mutation that causes the gene to regain its function and grow in a histidine-free medium.
• Mutagenic potential can be investigated in the Ames test in the presence or absence of a metabolizing system (e.g., Aroclor 1254-induced rat liver S9 fraction) to identify pro-mutagens as well as directly acting mutagens.
  TA98 (frameshift mutation) and TA100 (base-pair substitution) are two common strains of Salmonella typhimurium strains assessed in Ames testing. Both strains have:
  • rfa mutations, a defective lipopolysaccharide layer that makes bacteria more permeable to larger molecules
  • uvrB mutations, which eliminate excision repair of DNA damage
  • the pKM101 plasmid, which increases error-prone repair of DNA damage

Our biosciences division focuses on efficacy screening using cell based assays. We specialize in 2D and 3D cell-based approaches and have expertise in a wide range of techniques to cover phenotypic-based screening, target-based screening, and exploratory biology and mechanistic studies.

Our biosciences division focuses on efficacy screening using cell based assays. We specialize in 2D and 3D cell-based approaches and have expertise in a wide range of techniques to cover phenotypic-based screening, target-based screening, and exploratory biology and mechanistic studies.

Electrophysiology Services

Product Testing Services

Drug Permeability Studies Services