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In-vitro Toxicity Testing in Drug Development

In-vitro Toxicity Testing in Drug Development

Before submitting an IND application, the concerned drug must go through a comprehensive series of in-vitro and in-vivo toxicity testing to ensure maximum safety in clinical trials. Considering the ethical issues and the cost of in-vivo animal tests, the pharmaceutical industry now relies more on in-vitro methods for toxicity testing in the drug development phase.

Here, we answer the common questions regarding the in-vitro toxicity testing in drug development.

What is the right time to screen for toxicity in drug discovery and development project?

Screening for toxicity should start as early as possible to reveal inappropriate drugs early. This helps to eliminate such drugs from the drug development process timely and saves resources.

What are the most common types of toxicity observed in drug development?

Hepatotoxicity (toxic effects on the liver) and cardiotoxicity (toxic effects on the heart) are the most severe problems encountered in drug development and the major reason for drug withdrawals. In addition, possible mutagenesis (genotoxicity) caused by the concerned drug candidates is also observed frequently.

What types of assays are used for liver toxicity testing?

Liver toxicity or Drug-induced liver injury (DILI) can occur through several mechanisms like direct cell injury, mitochondrial injury, etc. To study direct cell toxicity, you can use membrane integrity assay (LDH release). The viability of the cells can be assessed by measuring cell metabolic activity using MTT assay or by measuring the ATP levels of the cells.

Mitochondrial toxicity can be studied by replacing the cell’s glucose with galactose in the incubation medium to augment toxicity.

How do you screen for cardiotoxicity?

The severe cardiotoxic effect is mainly caused by the blockage of a potassium ion channel, hERG (human Ether-à-go-go-related gene) which prolongs the QT interval. Thus, cardiotoxic effects can be screened by testing the hERG blockage. They use HTS 384-plate fluorescence polarisation assay for this purpose as it is cost-effective and gives quicker results than the traditional patch-clamp assay. However, search for more comprehensive cardiotoxicity screening assays is under development.

Why should you screen for genotoxicity?

Genotoxic compounds can cause mutations in DNA and predispose to cancer and reproductive problems. Therefore, it’s crucial to screen for genotoxicity. As per the regulatory guidelines, the standard tests for genotoxicity screening include bacterial reverse mutation test (AMES) and a mammalian genotoxicity assay.

AMES test reveals whether the compound will cause any direct mutations to the DNA. AMES screening is performed with two tester strains of Salmonella; TA 98 and TA 100 (mini AMES).

Mammalian genotoxicity assay reveals if the compound causes abnormalities in chromosome distribution (aneugenity) or chromosome breaks (clastogenity) during cell division. It is performed using actively dividing mammalian cells.

What are the other toxicity tests to consider in drug development?

Based on the properties of the investigational drug, other screening assays to consider are reactive metabolite formation and time-dependent CYP inhibition or transporter inhibition. 3D cell models are useful o screen for metabolism-dependent toxicity or delayed toxicity as they allow long-term incubation with the metabolically active cells.

What are the requirements of the authorities for toxicity studies?

The safety of a new drug candidate is very important. Therefore, toxicity testing is strictly controlled and has to be performed in animals and in GLP (Good Laboratory Practice) conditions to select a safe starting dose for the clinical trial in humans. Non-GLP screening assays are not accepted any more. In addition, continuous development of more predictive toxicity screening assays is required to reduce unnecessary, laborious, and expensive animal studies.

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