Optimization of human absorption, metabolism, and excretion (hAME) studies, and their timing, increases the chance of drug development success, saving time and resources. This digital whitepaper provides advice and insight on how to make the right choices when planning and conducting hAME studies and how to leverage preclinical and clinical partnerships to maximize the benefits.
Why are ADME/human AME (hAME) studies required?
Understanding how drugs are absorbed, metabolized and excreted after dosing is a critical part of the drug development process and the hAME study (also referred to as human mass balance study) which is one of the most informative studies for evaluating the pharmacokinetic (PK) properties of a new drug.
The metabolite profiles of candidate drugs in humans and animals are usually generated by mass balance studies using radiolabeled drugs.
Regulatory agencies, including the FDA and the International Conference on Harmonization (ICH), require the following mass balance studies as part of the new drug approval process:1
- Preclinical phase: absorption, distribution, metabolism and elimination (ADME) study
- The main objectives of this study are to identify and quantify circulating parent drug and metabolites, evaluate tissue distribution, and to elucidate the elimination pathways and the rates of elimination in animal models
- Clinical phase: hAME study
- A radiolabeled version of the drug is used to evaluate the total fate of drug-related material in humans
- This study provides safety data on the absorption, metabolism, plus routes and rates of elimination of a drug by assessing the pharmacokinetics, mass balance, routes of excretion and metabolic pathways of the parent drug
- The key objective is to determine whether there are any human-specific (i.e., ‘unique’ metabolites) or disproportionate metabolites. A disproportionate drug metabolite is a metabolite present at higher plasma concentrations in humans than in the animals used in nonclinical studies2
It is recognized that conducting ADME studies in animals is not sufficient to identify the human excretory and circulating metabolite profiles to mitigate the risk of disproportionate metabolites in humans. Therefore, a hAME study must be performed to ensure that the metabolite profile is comparable to that observed in preclinical ADME studies and to identify any disproportionate or unique human metabolites.3
Human AME studies should, ideally, be conducted before Phase III of a clinical trial to address the safety of drug metabolites.
Although there is consensus on the need to conduct a radiolabeled hAME study to identify and manage any risks related to the safety of drug metabolites, the guidance on what constitutes an adequate study to meet this objective is not always clear.
Regular updates in regulatory requirements has improved this situation; however, uncertainty still exists, so many drug developers operate risk-based strategies to fulfil the requirements of the guidance on the safety testing of drug metabolites.
One of these strategies is deciding when to run an hAME study with some developers wanting information early to allow development on potentially problematic drugs to be halted before any major research spend, while others, with more predictable compounds, may wait until late in Phase II.
However, conducting the hAME study as early as possible, before large scale clinical trials begin, has multiple benefits: it enables efficient management of possible metabolites that need safety testing; allows more time for elucidation of major elimination pathways that can be time consuming (and often includes clinical interaction studies); and can help reduce barriers to marketing authorization.
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What are the regulatory requirements for hAME studies?
Regulations across the world are being constantly updated with the common aim of gaining pharmacokinetic data relevant to understanding the metabolism of a drug and potential drug interactions in advance of entry into Phase III. The key worldwide regulations are outlined in Table 1.
|Food and Drug Administration (FDA) Safety Testing of Drug Metabolites Guidance2||Human metabolites that can raise a safety concern are those present at greater than 10% of total drug-related exposure at steady stateGenerally, metabolites identified only in human plasma or metabolites present at higher levels in humans than in any of the animal test species should be considered for safety assessment. Such metabolites are called disproportionate drug metabolites|
|International Conference on Harmonization (ICH) M3(R2) Guidance4||Nonclinical characterization of a human metabolite(s) is warranted when that metabolite(s) is observed at exposures >10% of total drug-related exposure and at significantly greater levels in humans than the maximum exposure seen in the toxicity studiesFor drugs for which the daily administered dose is <10 mg, greater fractions of the drug related material might be more appropriate triggers for testing|
|European Medicines Agency (EMA) Drug–Drug Interaction Guidance5||It is recommended that metabolites contributing to >10% of the area under the curve (AUC) of drug related material are structurally characterized Total recovery of radioactivity in urine and feces should exceed 90% of dose and >80% of the recovered radioactivity identified Information supporting the prediction of the effects by other medicinal products on the pharmacokinetics of the investigational drug should generally be available before starting Phase III clinical trials|
Table 1. An overview of the different regulatory requirements for hAME studies.
Updated Food and Drug Administration (FDA) guidelines “encourage identifying any differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug development process. Discovering such differences in drug metabolites late in drug development can cause development delays.”2
The FDA, International Conference on Harmonization (ICH) M3(R2) Guidance, and EMA agree that a metabolite constituting >10% of the total drug-related exposure in human plasma requires evaluation (Table 1).2,4
In addition, there should be no metabolites in humans that were not observed in preclinical studies. Further toxicological testing on metabolites that have higher exposure in humans than in preclinical species may be required.
Key steps in conducting a hAME study
Human AME studies require substantial resources, investment and a multidisciplinary multi-site collaboration; therefore, these studies require thorough planning to ensure that you achieve all the objectives and to save time and resources.
The optimized timeline we work to when planning the nonclinical and clinical phases of an hAME study are shown in Figure 1. A comprehensive planning process comprising integrated preclinical and clinical stages takes about 22 months.
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The rate limiting step is often the radiosynthesis stage, which takes about 10 to 11 months from initiation to the GMP (good manufacturing practice) radiolabeled drug being available for the clinic. The availability of the radiolabeled drug and its radiochemical stability determines when you can start dosing in the hAME study.
The radiosynthesis stage is usually conducted in two parts: first, production of the nonGMP material, which is used in preclinical studies, and second, the GMP synthesis that produces the material for human use.
As a minimum, rodent tissue distribution investigation (typically by quantitative whole-body autoradiography [QWBA]) and a rodent excretion balance study are preclinical ADME studies required to support the radiolabeled hAME study.
The dosimetry data from the QWBA studies define the clinical radiochemical dose and, therefore, the specific activity of the material needed for the technical batch and GMP synthesis. The number of GMP synthesis stages is determined on a case-by-case basis and is dependent on three factors: the radiochemical dose, the isotopic dilution, and the formulation to be administered.
Combining radiosynthesis of ADME radiolabeled drug and GMP hAME radiolabeled drug as one project within one radiosynthesis laboratory, can significantly reduce timelines and costs. If the drug development strategy includes conducting the hAME earlier in the clinical phases, then creating additional stock of the key intermediates for the radiosynthesis during the nonGMP manufacture of the ADME radiolabeled drug has only a small incremental additional cost, but can pay back dividends in saved time and cost for the clinical GMP manufacture, often cutting many months from the timeline.
The major endpoint of the hAME trial is to identify and quantify the metabolites present in the plasma, urine and feces samples. By establishing the identification and quantitation of the metabolites in humans early on, drug development scientists are better informed in decision making and and are able to de-risk a project. This often takes place during and following database lock for the primary endpoints of the clinical phase, i.e., following radioanalysis and PK determinations.
A phase report is often appended to the final clinical report when the metabolite investigations are complete. Critical to the success of the human metabolite investigations is building upon the experiences with the nonclinical samples generated during the rat or nonrodent ADME studies. The benefit of having the same teams overseeing the extraction, concentration and analysis of the precious clinical samples as those that had conducted the nonclinical analyses should not be overlooked.
Analytical strategies for conducting an hAME study
Accurate sample collection and processing is critical for hAME study success. Urine and fecal samples for radioactivity should be collected at least daily for the entire duration of the study until the discharge criteria are met. Blood samples are required for analysis of whole blood and plasma radioactivity as well as for bioanalysis of parent and known metabolites.
Sample collection should follow a typical PK plasma profile, and for the radioanalysis should also continue until the discharge criteria are met. Collection of expired air for radioanalysis is generally not required unless the radiolabel is in a labile position, or the molecule (or its drug class) are known to have a route of excretion via expired air.
Samples for metabolite profiling are collected using a refined PK profile to ensure the Tmax and a number of samples around Tmax are collected and continue for the entire study period to ensure that any late emerging or persistent metabolites can be fully defined.
Careful consideration should be given to the sample collection times to ensure that the maximum limit for the total volume of blood (500 mL per person) is not exceeded.
Quantification and identification of metabolites
The expectation is that you identify all the metabolites that may contribute to the overall exposure in humans. Typically, this includes those below the 10% indicated by regulatory guidelines, as low as metabolites present at 1-5% of the total drug-related exposure circulating systemically, and similarly 1% in urine and feces too.
Data is usually presented as percent total radioactivity, but can also be presented as percent parent contribution. Metabolite investigations are relatively resource intensive, so several strategies can be used to reduce the number of samples analyzed while maximizing per subject data.
For example, sample pooling may be appropriate for plasma, urine or feces. Typically, a plasma pool will be prepared within a subject to generate an area under the curve (AUC) pool representative of the plasma exposure. Equally, pools may be prepared across subjects at selected time points to generate pools for further analysis.
Assessing the radioactive dose for metabolite profiling/ID
To select the dose to be administered to humans and to determine whether the dose can be justified to the Administration of Radioactive Substance Advisory Committee (ARSAC), the following must the illustrated:
- The potential concentration of circulating metabolites
- The limits of detection that is likely to be achieved in hAME trials
This is illustrated using dosimetry data along with earlier data, such as from single or multiple ascending dose (SAD/MAD) studies and preclinical ADME data.
The data obtained from the dosimetry studies, which provides a linear scale for the amount of radioactivity, sets what is a reasonable radioactive dose level.
This will indicate whether an increase in radioactive dose would increase the sensitivity for detection of the radiolabeled metabolites. Most studies fall within the range of 0.1–1 mSv [International Commission on Radiological Protection (ICRP) Category IIa guidance]; however, up to 2 mSv can be justified.
What are the radiosynthesis challenges?
Selecting the optimum radiolabel position to ensure full tracking of all metabolites
The position of the radiolabel is vital because all metabolites must be tracked and quantified. Ideally, the radiolabel is incorporated in a metabolically stable position that allows most of the metabolites circulating in the plasma, urine and other matrices to be followed.
The decision on where to position the radiolabel is often a trade-off between incorporating the radiolabel towards the end of the synthesis (to reduce costs) and having the radiolabel in the core of the molecule, i.e., in a more metabolically stable position (to give better data)
Calculating the level of radioactive dose for sufficient quantification
The level of radioactive dose administered to humans is critical in determining the scientific outcomes of an hAME study. If the dose is too low, the radiolabeled test material and/or its radiolabeled metabolites may not be detectable.
If there is insufficient radiolabeled dose to give suitable response for radiodetection, you may have to consider accelerator mass spectrometry (AMS) as an alternative. However, this technique is very expensive, and any processing occurs in ‘cold’ lab.
Setting a dose higher than necessary may put the subject at increased risk of cancer and doesn’t comply with ALARA (As Low As Reasonably Acceptable) guidance. These risks can be significantly reduced if correct calculations are conducted upfront, by experts, based on preclinical profiling data and human pharmacokinetic data.
What are the pharmacy and clinical conduct challenges?
A ‘fit-for-purpose’ formulation needs to be developed in parallel to hAME studies. The formulation must be tested for stability and must be compatible with any device/equipment used for dosing.
The route of administration should be considered early on because it affects release testing requirements of the active pharmaceutical ingredient (API) and the amount of material required for analysis. Also, depending on the specific activity, can add considerable costs to the synthesis. In addition, the correct radioactive dose quantity must be established so that this does not exceed that approved by the regulatory committee.
Critical to the success of an hAME study, is having an onsite GMP pharmacy that is licensed to handle radiolabeled material. The site for manufacture depends on the preference of the client and on the stability of the radiolabeled material.
Poor recovery of radioactivity can have multiple causes
Non-compliance of subjects (e.g., spilling the sample or forgetting to collect the sample) is an example of how human behavior can result in low recovery of radioactivity.
Another consideration is that the selection of a suitable solvent for feces samples impacts the homogeneity, and this will also impact the recovery of radioactivity.
Ensuring appropriate informed consent
Informed consent must cover the both the radioanalysis and metabolite profiling of samples.The maximum duration of sample retention should be defined in the informed consent form and reflect the time needed to conduct this work. Once completed the samples may be disposed of in accordance with the Human Tissue Act (HTA).
Examples of the differences in front-end approval for hAME studies from two global sites
The regulations to the approval of hAME studies varies around the world. Figure 2 shows examples from the UK and US.
In the UK, as with all clinical trials, Clinical Trial Authorization (CTA) submission needs to be made to the Medicine and Healthcare Products Regulatory Agency (MHRA) for a Clinical Trial of an Investigational Medicinal Product (CTIMP). The main difference for submission for an hAME study compared with other clinical studies is that an Investigational Medicinal Product Dossier (IMPD) for the radiolabeled material needs to be submitted, including the necessary supporting data for radiolabeled API and non-radiolabeled API.
The MHRA review can take approximately 3–6 weeks, depending on if comments are raised by the MHRA after their initial review. The Ethics Committee (EC) review can be conducted in parallel to the MHRA review, as can submission to the ARSAC. Both the EC and ARSAC submissions are made via the Integrated Research Application System (IRAS) system. The role of ARSAC is to check that the radiation exposure of trial subjects is within acceptable limits. In addition to the documentation that the EC receive, the ARSAC review also includes a form containing the following:
- An overview of the research
- The purpose of the research
- Details of the radioactive material (extracted from the dosimetry report)
- A radiation risk assessment
The ARSAC review can take up to 8 weeks, depending on queries raised. The ARSAC approval is only required for dosing, therefore screening can be initiated ahead of ARSAC approval, taking this off the critical path for the hAME study. Recruitment of participants can take place in parallel to all three submissions (the CTA, EC and ARSAC submissions) so that screening can take commence once the MHRA and EC approvals are received.
Briefly, in the US, the process is governed by the Institutional Review Board (IRB) under FDA regulations. After IRB review, which can take up to 1 week, recruitment can begin up to 4 weeks prior to screening depending the investigational new drug (IND) status of the test compound (Figure 2). Following this, a 28-day screening period takes place before dosing.
How to overcome the key challenges of hAME studies
As you have seen, there are multiple challenges that can impact the success of an hAME study. One overarching way of addressing these is by using an integrated early drug development platform.
Use an integrated preclinical to clinical approach
The optimal approach for a clinical development program is an integrated early drug development platform. Having the resources, expertise and necessary infrastructure available to design and execute regulatory strategies and early clinical development plans is an effective way of mitigating risk. An integrated development platform involves a collaborative and coordinated effort between sponsor and contract research organizations (CROs).
An integrated approach allows you to save time on approvals, helps you to understand the molecule as completely as possible, ensures better planning for optimized analysis and provides consistent results. Various components span the planning of an hAME study, from the radiosynthesis to nonclinical studies; clinical conduct, analytical involvement, and generation of data that addresses both regulatory and safety questions.
The hAME studies combine the technical areas of radiosynthesis, chemistry, manufacturing, and control (CMC), preclinical ADME with metabolite ID (MetID) dosimetry calculations, clinical pharmacology and pharmacy.
Design preclinical studies that support the clinical studies
To ensure that your preclinical data supports the clinical studies, you need to ensure alignment between these studies on the following factors:
- Radioactive isotope – different isotopes have different properties, therefore different risks to tissues in terms of radioactivity
- Route of administration (or with additional justification and extrapolation if a different route is used)
- Sufficient sampling times to provide reliable AUC data, including Cmax and elimination phases
In addition, the tissue distribution study should cover an appropriate time range, so that the absorption, exposure and elimination can be evaluated to enable an accurate dosimetry calculation to be performed.
In this study, all critical tissues that are covered in dosimetry guidelines must be assessed over an appropriate period where radioactivity is quantified.
Ensure that the radioactive dose is as low as reasonably possible to achieve study objectives
The radioactive dose must be sufficient to allow complete quantification and characterization of metabolites, but within the limits to be safe for humans in clinical trials. Various analytical strategies can be used to optimize recovery in a clinical hAME study.
For example, the data obtained from both dosimetry studies with earlier preclinical ADME data must be analyzed thoroughly to allow you to establish the likely concentrations of radiolabeled metabolites that you will obtain in human samples.
In addition, using sample preparation and concentration techniques, such as sample pooling, allows you to work up samples to lower the limited detection where possible.
Well-designed and robust ADME and hAME studies are vital to demonstrating confidence in the safety and efficacy of a potential drug. Knowledge of metabolite profiles of a drug candidate in preclinical species and humans is required by regulatory agencies for drug approval.
Conducting these studies early in development can have multiple benefits, such as enabling efficient management of metabolites in terms of safety. Discovering unique or disproportionate drug metabolites late in drug development can cause delays.
Early investment and thorough planning of ADME and hAME studies can mitigate these problems. These studies require the synthesis of radioactive material which is costly, time consuming and, in many cases, technically difficult.
Success in conducting hAME studies in a clinical development program requires an integrated early drug development platform involving a collaborative and coordinated effort between different teams.
Covance and Arcinova
Covance have a long-term relationship with Arcinova to deliver fully integrated drug development packages to the global biotech and pharmaceutical industries. By offering Arcinova services along with our own extensive range of drug development capabilities, an end-to-end service can be provided in a streamlined and optimized process.
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References and Links
- Coppola P, Andersson A, Cole S. The Importance of the human mass balance study in regulatory submissions. CPT Pharmacometrics Syst Pharmacol 2019;8(11):792–804.
- Food and Drug Administration. Safety testing of drug metabolites: Guidance for industry. Available at: https://www.fda.gov/media/72279/download. Accessed September 2020
- Covance. Human AME. Available at: https://www.covance.com/services/analytical-services/drug-metabolism-and-pharmacokinetic-services/metabolite-identification/human-ame.html. Assessed Sept 2020.
- International Council for Harmonization. M3 (R2) Non‐clinical safety studies for the conduct of human clinical trials for pharmaceuticals. EMA/CPMP/ICH/286/1995 https://www.ema.europa.eu/en/documents/scientific-guideline/ich-guideline-m3r2-non-clinical-safety-studies-conduct-human-clinical-trials-marketing-authorisation_en.pdf. Accessed Sept 2020.
- European Medicines Agency. Drug interaction guideline on the investigation of drug interactions. CPMP/EWP/560/95/Rev. 1 Corr. 2** Available at: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-investigation-drug-interactions-revision-1_en.pdf. Accessed Sept 2020.
Abbreviations and Acronyms
- hAME: human absorption, metabolism, and excretion
- PK: pharmacokinetic
- ADME: absorption, distribution, metabolism and elimination
- QWBA: quantitative whole-body autoradiography
- FDA: Food and Drug Administration
- ICH: International Council for Harmonization
- GMP: Good manufacturing practices
- ARSAC: Administration of Radioactive Substance Advisory Committee
- SAD: single ascending dose
- MAD: multiple ascending dose
- AMS: accelerator mass spectrometry
- ALARA: As Low As Reasonably Acceptable
- API: active pharmaceutical ingredient
- CTA: Clinical Trial Authorization
- MHRA: Medicine and Healthcare Products Regulatory Agency
- CTIMP: Clinical Trial of an Investigational Medicinal Product
- EC: Ethics Committee
- IRAS: Integrated Research Application system
- IRB: Institutional Review Board
- IND: investigational new drug
- CRO: contract research organization
- CMC: chemistry, manufacturing, and control
- AUC: area under the curve