Pipeline Impact of Radiolabeled Compounds in Drug Discovery and Development

Abstract

The long-lived radionuclides tritium and carbon-14 have been used for many years in pharmaceutical research and development for making key efficacy and toxicological decisions. Early discovery utilizes radiolabels for compound selection through radioligand binding assays and autoradiography. In preclinical safety evaluation, the use of labeled compounds for adsorption, distribution, metabolism, and excretion studies is often preferred for the added detection sensitivity. As the drug substance proceeds to the clinic, human metabolism studies are reliant on the use of labeled materials to fulfill required regulatory applications.

The use of long-lived radioactive compounds in drug discovery and development can provide highly valuable data due to their ultrahigh sensitivity and ease of detection.¹ Diverse radiolabel probes have been used in early discovery, lead optimization, and candidate selection² across multiple therapeutic areas throughout the pharmaceutical industry, as is well documented.³ At AbbVie, compounds labeled with tritium (³H) are widely used in early discovery for receptor binding, in vitro hepatocellular activity, and autoradiography (ARG). As the drug substances advance to the preclinical stage, the identification and quantification of metabolites across species is necessary. This is typically accomplished using radiolabeled compounds in quantitative whole-body autoradiography (QWBA)⁴ and in vivo adsorption, distribution, metabolism, and excretion (ADME) studies.⁵ Advancement to late-stage clinical trials often requires human radiolabeled studies to properly identify any unique circulating metabolites.⁶

The choice of ³H versus ¹⁴C label is often dictated by the stage of a compound in development and the purpose of the study being conducted. The higher specific activity of tritium (∼29 Ci/mmol) compared to ¹⁴C (∼62 mCi/mmol) makes it ideal for studies in which the molar concentration is very low, such as ARG or receptor occupancy screens. Tritium is also the preferred isotope for early ADME studies due to the relatively low cost (often ∼100-fold less expensive than ¹⁴C per synthesis) and synthetic ease.⁷ Another advantage is that tritium can often be incorporated by late-stage synthesis through metal-mediated hydrogen/tritium exchange reactions.⁸ However, tritium labels are more susceptible to hydrolysis and exchange,⁹ especially during in vivo experiments, which can hamper mass balance and excretion studies.¹⁰ For these reasons, ¹⁴C is the preferred isotope for late-stage developmental studies in animals and humans and for studies that can take several months to complete, such as environmental fate.⁹

Radioligand binding assays can often be used to study receptor dynamics and pharmacokinetic and pharmacodynamic characteristics, in addition to identifying novel chemical structures to define ligand activity/selectivity in normal and diseased tissues.¹¹ High specific activity radiolabeled compounds with high affinity and low non-specific binding are used for IC₅₀ measurements and kinetic experiments to help quantify terms such as receptor occupancy (B_max) and equilibrium dissociation constant (K_D).¹² An example from AbbVie was the use of radioligand binding for the discovery of a potent selective antagonist of P2X7 receptors and its utility for screening of possible new molecular entities (NMEs).¹³ The P2X7 receptors belong to the ATP family, localized in peripheral macrophages and glial cells of the central nervous system. Activation of P2X7 receptors enhances the calcium concentration, releasing the cytokine IL-1β, causing tissue inflammation/damage. We developed a structurally novel radioligand, [³H]A-804598, which was prepared in three steps as shown in Scheme 1.

Scheme 1. Preparation of Structurally Novel Radioligand [³H]A-804598.

With the high affinity for the receptor in rat (IC₅₀ = 10 nm), mouse (IC₅₀ = 9 nm) and human (IC₅₀ = 11 nm), [³H]A-804598 enabled additional pharmacological studies to elucidate the functional role of P2X7 receptors in rodents to aid in the advancement of NMEs.

Interleukin (IL-23) is indicated in several immunological diseases, such as psoriasis and arthritis.¹⁴ Despite a huge advancement in this field, the pharmacokinetics of IL-23 and other cytokines are not well understood, primarily due to their ultra-low endogenous levels.^15,16 At AbbVie, we synthesized radiolabeled human recombinant IL-23 by reductive methylation on the lysine residues using [¹⁴C]formaldehyde (Scheme 2). The average incorporation of [¹⁴C] labels was 23 methyl groups, for a specific activity of 1249 mCi/mmol. The radiolabeled IL-23 was intravenously dosed to cynomolgus monkeys, and their serum samples were analyzed for the disappearance of [¹⁴C]IL-23 by accelerated mass spectrometry (AMS).¹⁷ The measured pharmacokinetic parameters are shown in Table 1. This data enabled the development of a model to predict the suppression of free IL-23 by therapeutic antibodies.

Scheme 2. Synthesis of Radiolabeled Human Recombinant IL-23.

Table 1. Measured Pharmacokinetic Parameters for Serum Samples with Radiolabeled IL-23.

subject	T1/2 (h)	Cmax (pg/mL)	AUC (pg h/mL)	CL (L/h)	Vss (L)
1	4.3	18.1	42.8	0.374	1.36
2	5.6	20.5	47.8	0.334	1.7
3	4.6	24.4	46.1	0.347	1.22
mean	4.8	21.0	45.5	0.352	1.43
SD	0.7	3.2	2.6	0.020	0.25

ARG is a powerful method for understanding the localized binding in vivo (after administration to an animal) and in vitro (on specific tissues) of a variety of compounds. In addition, ARG can provide information for dosimetry calculations required for adsorption, metabolism, and excretion in human (hAME) studies. For the early development of positron emission tomography (PET) tracers, ARG is often used with high specific activity tritium labeling to assess binding affinity and specificity for the area of concern.¹⁸ Tritium-labeled compounds with a half-life of >12 years can be used for a longer timespan than short-lived positron-emitting isotopes, such as ¹⁸F (half-life of 110 min), and the low energy of the beta radiation from tritium requires minimal engineering controls for safe handling. Similarly, quantitative whole-body autoradiography (QWBA or in vivo ARG) can give insight into the distribution. After an intravenous or oral dose, the single animal, typically a rodent, is euthanized at a specified time point. The body is then cryogenically frozen and thin cross-section slices are evaluated by autoradioluminography techniques to quantify the radioactivity present (³H or ¹⁴C). This not only gives a snapshot of the whole-body distribution but can also alert the toxicologist to any potential safety concerns.¹⁹ Rodent QWBA is routinely used as the defining safety study before any clinical studies, such as human metabolism and/or absolute bioavailability, that are often necessary for drug development before entering Phase III trials.

ADME studies are critical in both early discovery and development phases for NMEs. Recent advances in LC/MS and MS/MS technologies have increased their sensitivity, and they are often the first line in detecting parent and metabolites from in vitro studies and preclinical in vivo animal experiments.²⁰ However, these methods can still suffer from limitations, depending on the chemical structure and the quantities of metabolite formed. The use of radioactive labeled compounds, primarily those with ³H and ¹⁴C, is still considered the most quantitative method for ADME studies and is often required by regulatory authorities in drug development.²¹

Metabolism is the predominate mode of clearance for a majority of small-molecule NMEs.²² Of the drugs that were primarily cleared by metabolism, the cytochrome P450 enzymes were heavily involved, which can often indicate drug–drug interactions.²³ Therefore, the importance of understanding this risk early in the drug discovery timeline can be essential to differentiating compounds before advancement into development. Comprehensive profiling using radiolabeled NMEs in human liver systems, such as hepatocytes, can quantitatively capture the major metabolic pathways.

As drug substances advance further in the clinic, regulatory agencies have several requirements for the characterization of metabolites and elimination pathways.²⁴ Typically, by this stage, several studies have been performed in the appropriate animal models. However, humans can often have distinct metabolism pathways, and a better understanding of this before a large-scale clinical trial is needed. At AbbVie, we have conducted several hAME studies in the past decade; one such example is venetoclax. Venetoclax is a well-known B-cell lymphoma-2 (BCl-2) inhibitor and is approved for the treatment of chronic lymphocytic leukemia (CLL). Radiolabeled [¹⁴C]venetoclax was prepared in five steps via a synthetic route developed at AbbVie²⁵ (Scheme 3) for a single-dose study involving oral administration to four human volunteers at a dose of 100 μCi/200 mg. Parent [¹⁴C]venetoclax plus a major metabolite M27 were found to be the major components present in circulation.²¹ The M27 metabolite, at 12% of the total drug-related material, was disproportionate in humans, caused by the enzymatic oxidation on the dimethylcyclohexyl ring followed by cyclization, primarily formed by cytochrome P450 (CYP3A4) (Scheme 4).

Scheme 3. Preparation of Radiolabeled [¹⁴C]Venetoclax.

Scheme 4. Preparation of M27 Metabolite.

Metabolite M27 was found not to have clinically relevant on- or off-target pharmacologic activities.

Radiolabeled compounds play a vital role in drug discovery and development. In the lead optimization process, radioligand binding can assist in the characterization of dynamics, pharmacokinetics, and pharmacodynamics at the receptor site and facilitate the identification of NMEs. Likewise, ARG studies offer an efficient way to quantify these compounds in complex matrices. Selecting a compound candidate for development also requires having a strong understanding of the metabolism. Radiolabeling studies are considered the most quantitative methods for both in vitro and in vivo ADME studies. Finally, radiolabeled human metabolism studies are required for the clinical development of compounds.

The authors declare the following competing financial interest(s): All authors are employees of AbbVie and may own stock. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Special Issue

Published as part of the ACS Medicinal Chemistry Letters virtual special issue “New Enabling Drug Discovery Technologies - Recent Progress”.