Toxicology (Tox) Studies

In-vivo toxicology studies are required before FIH (First In Human) dosing and are usually performed under GLP conditions using detailed protocols. A general scheme used in the industry for preclinical studies of lead compounds before the commencement of clinical trials is as follows: Acute (single-dose) toxicity dose ranging study, maximum tolerated dose (MTD) study, Repeat-dose toxicity – 2 to 14 weeks (daily dosing, required by regulators in two species, one rodent and one non-rodent), carcinogenicity – up to 2 years of dosing in rodents for chronic treatments, genotoxicity- in vivo mouse micronucleus test, mutagenicity – in vitro AMES test, cardiovascular risk by hERG inhibition, chromosome aberration, and in-vivo respiratory and cardiovascular safety pharmacology studies, as applicable. The majority of these GLP toxicity studies follow regulatory guidance with quality oversight and are performed as toxicology services by CROs.

Toxicokinetic (TK) study is simply a pharmacokinetic study of the drug when administered at quantities higher than the therapeutic dose to evaluate potentially toxic levels of the drug. Thus, Toxicokinetics is the appropriate term for studying the systemic kinetics of all substances at exposure levels ranging from therapy to toxicity. During a GLP toxicity study by FDA standards, the body reaches the maximum tolerated dose (MTD), the dose level yielding the drug’s exposure to the point of toxicity, or the maximum feasible dose (MFD), the highest dose level that can be administered based on physicochemical limitations. Understanding the dosage and systemic exposure of the drug during toxicology studies is essential as this aspect defines the difference in medicine being therapeutic vs. toxic. This difference explains the drug’s therapeutic index, representing the safety margins between therapy (minimum efficacious dose) and toxicity (maximum tolerated dose or maximum feasible dose). The same drug given in a small quantity may start healing at the site of action. But, when administered at a high dose, it can even be fatal for the human body. The necessity for toxicokinetic analysis lies in the study’s ability to provide insights on molecules’ systemic exposure related to adverse effects. This aspect is an incredibly important consideration for the cardiovascular system, the central nervous system, and respiratory assessments. Furthermore, acute single dose toxicity studies carried under this analysis assist in predicting the duration and rate of exposure at the time of one dosing interval and whether systemic accumulation occurs following multiple drug administrations in repeated dose toxicity studies. This benchmark information provides the right dose levels for later stages of the compound development. Overall, the data obtained from toxicokinetic studies assess the toxic responses of various drug compounds. This study’s result becomes the basis for knowing the right starting dose of a new drug that is safe for the human body. Moreover, the analysis may provide insights into how certain medications can diversely affect people of different ages, genders, and ethnicities.

Toxicokinetic analysis is typically performed for acute and repeated dose toxicity studies. It is essentially the process that helps us understand how a substance enters the body and what happens to it inside the body. The term “disposition” is often used in the place of Toxicokinetics to describe the body’s exposure to the drug or toxicant over time. Many factors impact Toxicokinetic studies, but the four critical descriptive underlying processes of disposition are absorption, distribution, metabolism, and elimination. TK analysis mathematically describes these dispositional principles by non-compartmental or compartmental analyses. These stages or processes help learn how the body absorbs, metabolizes, and clears the administered drugs and correlates to clinical toxicological analysis of exposure margins and adverse events for safe clinical administration.

Absorption
Absorption, the first dispositional element in toxicokinetic studies, is the process of a substance entering systemic blood circulation. Depending on the route of administration, drugs may need to overcome some barriers to reach circulation. For example, following oral dosing, a drug must be soluble in the gastrointestinal space and capable of passive or active diffusion across the gut wall. Medicines absorbed through the gastrointestinal tract then reach the portal vein, where first pass metabolism occurs before systemic absorption. In PK and TK studies, the absorption process may be bypassed by administering an intravenous (IV) dose directly into the systemic circulation. Comparison of exposures following extravascular doses (i.e., oral, subcutaneous, dermal, inhaled, etc.) to exposures following IV doses during PK/TK analysis yields bioavailability.

Distribution
Distribution describes the reversible transfer of drugs within the body from one location to another. The distribution of a drug is affected by multiple factors, including lipid-solubility, concentration in plasma and various tissues, and binding to plasma proteins, transport proteins, and tissues. The volume of distribution calculated during PK or TK analysis describes a drug’s ability to enter cells and tissues from systemic circulation.

Metabolism
Metabolism signifies a process by which a drug is converted to other chemical entities (metabolites). Metabolism happens primarily in the liver but can occur throughout the body. At the time of metabolism, the xenobiotic chemicals administered in the body are altered with non-enzymatic or enzymatic reactions. The aim is to convert lipophilic compounds, which are poorly excreted, to more polar entities that can be readily discharged from the body. Understanding drug metabolism rates and sites allow researchers to predict safe exposure levels and potentials for drug-drug or drug-food interactions.

Elimination
Drug chemicals are excreted from the system either as metabolites or in their unchanged form. While the kidney plays an essential role in excreting water-soluble toxins from the body, excretion of other compounds needs the hepatic and biliary systems, etc. During toxicokinetic studies, parameters such as apparent clearance and elimination half-life are calculated to describe compound metabolism and excretion.

The toxicokinetic process mathematically models the four dispositional factors that act on a compound in a biological system referred to as ADME. They include uptake of the drug into the systemic circulation, or absorption (A), distribution (D) from the bloodstream to cells and tissues within the body, conversion to new entities by biotransformation or metabolism (M), and excretion (E) of the compound and its biotransformation products. Toxicity is often associated with the conversion of the drug compound to more reactive metabolites. We perform toxicokinetic analysis by assessing systemic exposures via bioanalysis of active pharmaceutical ingredients and metabolites, followed by toxicokinetic analysis using the in-vivo time vs. concentration results. Toxicology studies allow determination of i) No observed effect level (NOEL) – the highest dose or exposure that produces no effect, ii) No observed adverse effect level (NOAEL) – the highest dose or exposure that has manageable toxicity, and iii) Therapeutic index (margin of safety) – the range encompassing minimum efficacious dosage to the maximum tolerated dose.

Toxicology CROs offer predictive safety assessments by in-silico and in-vitro methods. In-silico methods allow predictions of potential mutagenicity and genotoxicity based on the compound’s structure and known historical data. In-vitro toxicity assays allow testing with many discovery compounds to predict specific toxicity mechanisms and reduce animal usage. In-vitro toxicity assays test for potential drug-drug interactions (DDI), mutagenicity, cytotoxicity, or organ toxicity using toxicology services including microsome, hepatocyte, hERG, and AMES assays. That said, in-vivo toxicology studies are still critical because they are the ultimate indicator of toxicity. In-vivo toxicity studies include satellite toxicity and maximum tolerated dose (MTD) study assessments, acute single-dose toxicity studies, repeat-dose GLP toxicology studies, genetic and prenatal toxicology, carcinogenic studies, safety pharmacology studies, in-vivo erythrocyte micronucleus tests, and other systems of biology techniques including metabonomics, proteomics, and transcriptomics.

Nonclinical toxicology and safety pharmacology tests assess a drug’s potential for adverse and undesired effects in animal models before initial testing in humans. The terms nonclinical and preclinical are often used interchangeably since nonclinical tests are evaluated before the inception of clinical trials on novel drug entities.

We must obtain nonclinical GLP toxicology data for all lead candidates before filing the Investigational new drug application (IND). In-vitro toxicology studies in drug development test mechanisms such as metabolic reactivity, inhibition, induction, mutagenicity, and cytotoxicity. Study results from these assays alert scientists to potential problems or limitations. We perform in-vivo acute and chronic toxicology studies in drug development to optimize and characterize lead discovery candidates. Furthermore, novel techniques including toxicometabonomics, toxicoproteomics, and toxicogenomics can provide higher throughput approaches to identify in-vivo toxic responses soon after dosing or aid in selecting specific endogenous biomarkers for both clinical toxicological analysis and preclinical detection of potential toxicities.

Before conducting animal testing, toxicity study sponsors must get approval for toxicological studies by the testing organization’s Institutional Animal Care and Use Committee (IACUC). Single-dose or Acute toxicity studies monitor the effect of a single dose on an animal species. In acute toxicological study testing, we administer the investigational product at different doses and observe the results for ~14 days following a single dose. We perform non-GLP repeated dose toxicity studies in rodents (mice or rats) to identify maximum tolerated dose, target tissues, PK, and safety pharmacology profiles during late optimization and before the compound’s preclinical development and nonclinical toxicology assessment. If the test agent has reached a steady-state, and there is a reasonable or no accumulation of test article and no observed acute toxicity at therapeutic levels, then we follow-up with repeat-dose GLP toxicity studies. In repeated dose toxicity studies, the test drug is administered daily for a minimum of 7, 14, 21, or 28 days under GLP conditions. We can also perform subchronic toxicity studies in rodent and non-rodent models with daily dosing for 90 days or more.

TK analysis is the subdivision of PK to test the relationship between systemic drug exposure and its toxicity in various species. Pharmacokinetic studies determine the systemic exposure of drug administration to living organisms over time after dosing. Toxicokinetic studies are generally carried out with higher doses as compared to pharmacokinetic studies. The objective of toxicokinetic studies is to quantify a compound’s pharmacokinetics in toxicological study species at toxicological doses. TK exposures may increase non-linearly with dosage due to absorption differences, metabolic saturation, or metabolic alterations resulting from the test article’s chronic usage. TK analyses are often used additionally to investigate drug accumulation with repeated dosing, gender differences, and quantitation of known drug metabolites or pharmacodynamic or toxicodynamic biomarkers.

Pharmacokinetics (PK) encompasses the analysis and description of a drug’s disposition in the body, with a mathematical description of all dispositional processes (ADME – Absorption, Distribution, Metabolism, and Elimination). Toxicokinetic (TK) analysis applies PK principles to the disposition of toxicants and their metabolites related to the time course of toxic or adverse events. PK or TK parameters are determined for the majority of studies using non-compartmental analysis (NCA).

CDER’s SEND (Standard for Exchange of Nonclinical Data) allows for data transfer to the FDA and other national regulatory agencies in a harmonized format allowing for more efficient and standardized regulatory review readable by Janus Nonclinical software. SEND mirrors the Study Data Tabulation Model (SDTM) used for clinical trial results. At present, tox studies fitting in the electronic Common Technical Document format (eCTD) modules 4.2.1.3 (Safety Pharmacology), 4.2.3.1 (Single Dose Toxicity), 4.2.3.2 (Repeat-Dose Toxicity), and 4.2.3.4 (Carcinogenicity) with study start dates on or after March 15, 2019 for NDA/BLA submissions and on or after March 15, 2020 for commercial IND submissions require data submission following the SENDIG 3.1 (SEND Implementation Guide version 3.1). New data standards will be needed for any studies started 24 months after the publication of a Federal Register Notice (FRN) announcing FDA support of the standard and placement on the FDA Data Standards Catalog. The FDA Office of Computational Science (OCS) currently offers a KickStart Program to assist applicants and reviewers in the SEND standard and data transfers to Janus Nonclinical.