Nonclinical PK and Toxicokinetic Data Required Before First-in-Human Small-Molecule Trials Under ICH M3(R2) and S3A

Nonclinical PK and TK before first-in-human trials under ICH M3(R2) and S3A

FDA has adopted the two ICH guidelines that govern this question as final guidance for industry. M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals was issued in January 2010 ⁸⁹, with a companion Questions and Answers document in March 2013 ⁹⁰. S3A Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies dates from March 1995 ⁹³, and an S3A Q&A focused on microsampling followed in May 2018 ⁹⁴.

Read together, they set a short list of exposure-related data you need before the first human dose. Fuller ADME characterization can follow later. M3(R2) sets what pharmacokinetic (PK) information is needed and when. S3A sets how systemic exposure is measured and interpreted inside the toxicity studies that support the IND.

The minimum PK package before first-in-human dosing

M3(R2) Section III ("Toxicokinetic and Pharmacokinetic Studies") states that three data sets should generally be evaluated before human clinical trials begin ⁸⁷:

In vitro metabolism data for both the animal species and humans.
Plasma protein binding data for both the animal species and humans.
Systemic exposure (toxicokinetic) data in the species used for the repeated-dose toxicity studies.

The M3(R2) Q&A repeats that in vitro metabolism data for animals and humans should be evaluated before human trials ¹⁸². Separately, it explains what the deferrable "in vitro biochemical information" on potential drug interactions can include: standard metabolic evaluations such as CYP inhibition and PXR activation assays, studies in hepatic microsomes or hepatocytes, and potential drug-transporter interactions ¹⁸³. Those interaction assays are not part of the pre-FIH minimum; M3(R2) generally expects them before phase 3 (see below).

These data serve a practical purpose. Combined with the repeated-dose toxicity results, they let the sponsor relate animal exposures to proposed human exposures and support dose selection ⁸⁷⁸⁸.

What can be deferred until later development

Fuller PK characterization in the test species does not have to come before first-in-human (FIH) dosing. This includes absorption, distribution, metabolism and excretion (ADME) studies and in vitro biochemical data relevant to potential drug interactions. These data should be available before large numbers of subjects are exposed or subjects are treated for a long time, which generally means before phase 3 ⁸⁷. At that stage they support comparisons of human and animal metabolites and decisions about whether more testing is needed ⁸⁷¹⁴⁰.

Data element	Timing under M3(R2)
In vitro metabolism, animal and human	Before human trials ⁸⁷¹⁸²
Plasma protein binding, animal and human	Before human trials ⁸⁷
Systemic exposure (TK) in the repeated-dose toxicity species	Before human trials ⁸⁷
In vivo ADME in test species	Before large or long-duration exposure, generally before phase 3 ⁸⁷
In vitro drug-interaction data	Before large or long-duration exposure, generally before phase 3 ⁸⁷
Nonclinical characterization of human metabolites (when triggered)	To support phase 3 ¹³⁵

Human metabolites: the 10% trigger and its timing

The metabolites-in-safety-testing question is often treated as an FIH issue, but M3(R2) places dedicated metabolite toxicity work later. Nonclinical characterization of a human metabolite is warranted only when both of these conditions apply ¹³⁵:

the metabolite accounts for more than 10% of total drug-related exposure, and
its human exposure is significantly greater than the maximum exposure seen in the toxicity studies.

When those studies are warranted, they should support phase 3 trials ¹³⁵. The guidance lists exceptions. For drugs dosed below 10 mg/day, a higher fraction may be a more appropriate trigger. Some metabolites, such as most glutathione conjugates, do not warrant testing. Metabolites with an identified cause for concern are handled case by case ¹³⁵.

The Q&A makes the trigger operational ¹³⁸¹³⁹:

The 10% threshold means more than 10% of measured total exposure to the drug and its metabolites, usually based on group-mean AUC.
"Significantly greater" is not a statistical test. A difference of at least twofold in mean AUC is generally meaningful.
Toxicity characterization of a metabolite is generally considered adequate when animal exposure is at least 50% of human exposure.

For animal-to-human metabolite comparisons, the Q&A generally compares animal exposure at the MTD with maximum human exposure at the therapeutic dose, provided the relevant toxicity is monitorable and does not pose unacceptable risk. Otherwise the NOAEL for that toxicity is the comparator ¹⁸⁷¹⁸⁸.

In practice, the in vitro metabolism data you have before FIH are what let you anticipate this issue early. The in vivo exposure comparison and any dedicated studies come later.

Toxicokinetics in the IND-enabling repeated-dose studies

Species and duration

To support FIH and early clinical trials, M3(R2) generally recommends repeated-dose toxicity studies in two mammalian species, one rodent and one nonrodent ⁸⁵⁸⁶:

A minimum of 2 weeks in each species generally supports clinical dosing for up to 2 weeks.
For trials longer than 2 weeks and up to 6 months, the toxicity studies should generally last at least as long as the clinical trial.
For trials longer than 6 months, the minimums are 6 months in rodents and 9 months in nonrodents, subject to the guidance's exceptions.

The systemic exposure data that M3(R2) requires before FIH come from these studies ⁸⁷⁸⁸.

What S3A expects TK to accomplish

The primary aim of toxicokinetics under S3A is to describe systemic exposure in animals in relation to dose and the time course of the study. TK also helps relate exposure to toxicological findings and their relevance to clinical safety, supports the choice of species and treatment regimen, and informs the design of later studies ⁵³.

Exposure parameters

The commonly used exposure measures are plasma, serum or whole-blood AUC, Cmax and the concentration at a specified time, C(time). The right choice depends on the compound, and for some compounds exposure is better expressed as unbound concentration ⁵⁴. S3A distinguishes two levels of sampling ⁵⁵:

Monitoring: a small number of samples per dosing interval (for example, 1 to 3) to estimate C(time) or Cmax.
Profiling: more samples (for example, 4 to 8) to estimate Cmax and/or C(time) and AUC.

Sampling design and animals

Take enough time points to estimate exposure. Justify them from earlier toxicity, pilot or dose-ranging studies, or from kinetic data that can reasonably be extrapolated. Sampling should not disrupt the study or cause undue physiological stress ⁵⁶.
Use the minimum number of animals that still gives adequate TK data, across an appropriate number of animals and dose groups. Samples can come from all main-study animals, from representative main-study animals, or from satellite groups. Main-study sampling is normally feasible in larger animals, while small rodents may need satellites ⁵⁷.
Satellite animals are treated and housed like main-study animals but are used mainly for TK ⁵⁸.
When both sexes are in the main study, both are normally assessed unless there is a justification for leaving one out ⁵⁷.

Timing within the first repeated-dose study

In the first repeated-dose study, include exposure monitoring or profiling at appropriate doses at the start and toward the end of treatment. Later TK plans depend on those results and on any changes to the regimen. They can be extended, reduced or modified to resolve problems in interpretation ⁶². Single-dose kinetic data can help choose a formulation, predict the rate and duration of exposure within a dosing interval, and select doses for later studies ⁶¹.

Analytes, metabolites and GLP

Measure the parent compound, metabolites or both, depending on the compound ⁵⁴⁵⁹. S3A highlights three situations where metabolite measurement is especially important ⁵⁹¹⁹³¹⁹⁴:

the compound is a prodrug and a metabolite is the main active entity;
active metabolites may contribute significantly to tissue or organ responses; or
extensive metabolism means a major metabolite is the only practical measure of exposure.

S3A also notes that documenting exposure to human metabolites in the toxicity studies may be important. Measuring metabolites in plasma cannot account for reactive intermediate metabolites ¹⁹³¹⁹⁴.

When the toxicity study is conducted under GLP, the concomitant TK must also conform to GLP. Some retrospectively designed TK studies needed for the safety evaluation should conform as well ⁶⁰.

Microsampling (S3A Q&A, 2018)

The S3A Q&A defines microsampling as collecting a very small blood sample, typically 50 µL or less, to measure drug and/or metabolite levels and calculate TK parameters. Blood, plasma or serum can be analyzed in liquid or dried form, in both rodents and nonrodents ⁶⁷.

Advantages. Smaller draws reduce animal pain and distress. In rodent studies, microsampling can remove the need for a TK satellite group, or reduce its size, when TK is done in main-study animals. The main scientific advantage is that safety findings can be related directly to exposure in the same animal ⁶⁸.
Applicability. The approach generally applies to most pharmaceuticals and to single-dose, repeat-dose, carcinogenicity, juvenile and reproductive toxicology studies. Its use depends on whether the assay is sensitive enough for the small sample volume ⁶⁹.
Bioanalytical expectations. The method should be qualified, or validated for GLP studies. It should be assessed for quantitation limits, accuracy, precision, matrix effects and analyte stability through sampling, storage and processing. If other studies used conventional sampling, you may need to show that the exposure measurements are comparable, especially when sample handling differs (for example, dried versus liquid samples) ⁷⁰⁷¹.
Main-study animals. The sampling plan must account for possible effects of blood collection on the animals and on the toxicity data. Relevant findings should be compared with controls sampled the same way. If frequent sampling could worsen drug-related hematological changes, TK satellites may still be warranted. Scientifically justified sparse sampling is another option ⁷².

Using exposure data to set toxicity-study doses

High-dose selection

M3(R2) does not require reaching the MTD in every study. The high dose can instead be limited by any of the following ¹⁷⁴:

saturation of systemic exposure;
the maximum feasible dose (MFD); or
a 50-fold exposure margin.

The 50-fold margin is generally based on group-mean AUC of the parent drug (or of the pharmacologically active molecule, for a prodrug) relative to clinical systemic exposure. It is an acceptable maximum for acute and repeated-dose toxicity studies in any species ¹⁸.

The Q&A specifies the comparison. Compare the mean AUC at the highest animal dose with the anticipated therapeutic human exposure at the highest dose proposed for phase 2/3, not necessarily the highest exposure reached in phase 1. In special cases, a Cmax-based limit may be appropriate, for example where seizures are suspected. Smaller margins can be appropriate for phase 1 ¹⁹. For US phase 3 support when the 50-fold limit is used, dose-limiting toxicity should generally be identified in at least one species. Otherwise M3(R2) describes an additional study up to the lowest of 1000 mg/kg/day, the MFD or the MTD, subject to its exception for shorter studies ²⁰.

Interpreting exposure

S3A gives these principles for interpreting exposure data:

Use exposure data to relate toxicity findings to dose and to judge their relevance to clinical safety ¹⁹⁷.
Consider species differences in protein binding, tissue uptake, receptor properties and metabolic profile ¹¹⁰¹⁹⁷.
Low plasma concentrations can coexist with high concentrations in particular tissues ¹⁹⁸.
Nonlinear kinetics need careful interpretation, but they do not necessarily limit doses or invalidate findings ¹¹¹.
High statistical precision is not normally necessary, given kinetic variability and small group sizes. Report means or medians with variability, and give a rationale for any data transformation ¹⁹⁶.
The TK report should give a full account of the data, evaluate what the results mean for interpreting the toxicity findings, report or reference the analytical method, and explain the choice of matrix and analyte ¹⁹⁹.

Linking nonclinical exposure to the FIH dose and clinical escalation

M3(R2) says the first human dose should be estimated from all relevant nonclinical evidence: pharmacological dose-response, the pharmacological and toxicological profile, and pharmacokinetics. The NOAEL in the most appropriate species is generally the most important safety input ⁸⁸¹⁵⁵.

There is no single universal animal-to-human exposure ratio for the maximum clinical dose. It depends on the trial design and the nonclinical package supporting it ¹⁵⁶. For standard phase 1 trials supported by the generally recommended package, the Q&A allows the maximum dose to be based on a standard risk assessment, including whether findings are reversible and monitorable and what adverse effects are seen clinically ¹⁶⁰.

The Q&A also addresses exposure-based escalation when the toxicity studies showed no toxicity at the top dose:

If the nonclinical top dose was a limit dose or MFD with no toxicity, the clinical dose could conservatively be escalated to a plasma AUC of one-half the exposure in the species with the lowest exposure at that dose ¹⁸⁵.
Further careful escalation may be justified on risk-benefit grounds if humans show no adverse effects at that exposure ¹⁸⁵.
Where adverse nonclinical findings are present but not dose-limiting under the 50-fold approach, escalation can be based on the NOAEL and standard risk assessment rather than being capped by the 50-fold margin ¹⁸⁶.

Exploratory (microdose and limited-exposure) FIH approaches

M3(R2) allows reduced nonclinical packages for exploratory clinical trials. In these approaches, toxicokinetics and AUC-based limits tie the clinical dose directly to animal exposure ²¹:

Approaches 1 and 2 (microdose). Approach 1 limits the total clinical dose to ≤100 µg and to ≤1/100 of both the NOAEL and the pharmacologically active dose. Approach 2 allows up to five administrations of ≤100 µg each (≤500 µg cumulative) with a washout between doses ²². The supporting animal study includes TK, and its maximum dose may be limited to 1000 times the clinical dose (mg/kg basis for intravenous, mg/m² for oral administration) ²². The Q&A says TK is there to show that systemic exposure occurred. It is not needed when the toxicity study uses the intravenous route ¹⁸⁴.
Approach 3 (single subtherapeutic or therapeutic dose). Extended single-dose toxicity studies in a rodent and a nonrodent by the clinical route should include TK, hematology, clinical chemistry, necropsy and histopathology. The maximum clinical exposure may reach ½ the NOAEL exposure in the more sensitive species if relevant toxicity is expected to be monitorable and reversible in humans ¹⁵⁷.
Approach 4 (multiple doses). Two-week repeated-dose studies in a rodent and a nonrodent, with animal doses chosen as exposure multiples of the anticipated AUC at the maximum clinical dose ¹⁶⁶. The clinical AUC limit depends on whether toxicity appears in neither, one or both species ¹⁵⁸.
Approach 5 (multiple doses). A standard 2-week rodent study plus a confirmatory or escalating-dose nonrodent study that assesses the anticipated rodent NOAEL exposure. The starting dose should give predicted exposure no greater than 1/50 of the NOAEL in the more sensitive species (mg/m² basis). Maximum human exposure should not exceed the lower of the nonrodent NOAEL AUC or ½ the rodent NOAEL AUC ¹⁵⁹¹⁶⁷.

TK beyond the IND package

S3A notes that toxicokinetic information can also support genotoxicity, carcinogenicity and reproductive toxicity studies ¹⁰⁹:

Genotoxicity. When an in vivo study is negative, it may be appropriate to show systemic exposure in the species used or to characterize exposure in the indicator tissue ¹⁴¹.
Carcinogenicity. S3A recommends TK monitoring or profiling in the preliminary dose-ranging studies, with particular attention to new species or strains, new routes and dietary dosing ¹⁶²¹⁶³. In the main study, monitoring on a few occasions should confirm that exposure stays consistent with earlier profiles. Continuing that monitoring beyond six months is not considered essential ¹⁶⁴¹⁶⁵.
New clinical routes. If a new clinical route is proposed, compare human AUC and Cmax across routes to see whether the safety margin shrinks. If exposure is not substantially greater or different, additional nonclinical work may focus on local toxicity ¹⁹⁵.

Practical takeaways for IND planning

Before FIH, you need three things: in vitro metabolism and plasma protein binding data for animals and humans, and TK from the GLP repeated-dose studies in the rodent and nonrodent species ⁸⁷.
Build TK into the pivotal repeated-dose studies, with sampling at the start and end of dosing and enough time points to estimate AUC and Cmax for the relevant analytes ⁵⁵⁶².
Plan for metabolite questions early using the in vitro data, but expect dedicated metabolite toxicity work only when the M3(R2) triggers are met, and then to support phase 3 ¹³⁵¹³⁸.
Use the toxicity-study exposures to anchor high-dose selection ¹⁸, to justify the starting dose ⁸⁸ and to plan clinical escalation ¹⁸⁵. In exploratory designs, the AUC- and NOAEL-based limits are written into the approach itself ¹⁵⁷¹⁵⁹.
Consider microsampling to get TK from main-study animals, provided the assay is qualified or validated and the effects of blood collection are controlled for ⁷⁰⁷².