ADME Studies of 37 FDA Approved Peptides: Strategies and Radiolabeling Considerations

Since the clinical introduction of insulin dating back to 1922, therapeutic peptides have undergone over a century of development. Typically comprising 10 to 40 amino acids, peptides exhibit a molecular weight (MW) of less than 10 kDa. Positioned between small molecules and biological macromolecules in terms of MW, therapeutic peptides stand out due to their high specificity and potency, low dosage, and low toxicity. In recent years, with the development of many blockbuster drugs such as dulaglutide, semaglutide, and tirzepatide, therapeutic peptides have become one of the most emerging fields in drug research and development globally. By November 2023, around 90 therapeutic peptides have been marketed worldwide. Notably, these encompass diverse targets including GLP-1R, Insulin, HER 2, and others, with GLP-1R emerging as a prominent focus.

In this article, we will focus on an industry white paper about ADME research of therapeutic peptides published in 2023: Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations ^[1]. This white paper explores the current ADME research landscape, research strategy of therapeutic peptides in vivo and shares the key considerations of radiolabeling technology for the pharmacokinetic study of therapeutic peptides.

What Is the Current Landscape of Radiolabeled ADME Studies for Therapeutic Peptides

Therapeutic peptides can be classified into three types based on structures: TPepA, linear peptides composed of only natural amino acids; TPepB, linear peptides containing unnatural amino acids and/or organic linkers with lipophilic side chains, and TPepC, cyclic peptides.

The white paper summarizes 31 therapeutic peptides approved by the FDA from 2011 to 2022, and we supplemented 6 new FDA approved peptides authorized in 2023, which are trofinetide, rezafungin, paxlovid, flotufolastat F 18, motixafortide and zilucoplan (listed by approval order in Figure 1). These 37 FDA approved peptides include 8 TPepA (natural amino acid linear peptides), 19 TPepB (unnatural amino acid linear peptides), and 10 TPepC (cyclic peptides).

Figure 1. Summary of therapeutic peptides approved by the FDA from 2011 to 2023

The in vitro metabolism studies of 37 therapeutic peptides approved by the FDA from 2011 to 2023 are summarized in Table 1, with additional details provided in End-of-Text Table 4. The metabolism of the three types of therapeutic peptides was studied in vitro in a variety of biological materials, whereas the liver-associated matrices were commonly used to study TPepB and TPepC. Some of these drugs, such as difelikefalin and odevixibat, have been studied in vitro with a variety of biological substrates using ¹⁴C and/or ³H.

Table 1. Summary of in vitro metabolism studies of therapeutic peptides approved by the FDA from 2011 to 2023

Note: NS, no studies mentioned in submission document(s).

Figures in parentheses indicate the number of peptides utilized for in vitro metabolism studies in this matrix type among 37 therapeutic peptides approved by the FDA.

Seventy-three percent (mainly TPepB and TPepC) of these 37 therapeutic peptides have undergone radiolabeled ADME studies in animals and/or humans (summarized in Table 2) (metabolism and excretion studies summarized in End-of-Text Table 4). Among these approved therapeutic peptides, the structures of nine drugs (boceprevir, telaprevir, ombitasvir, paritaprevir, grazoprevir, voxilaprevir, difelikefalin, odevixibat, and trofinetide) are similar to small molecules (MW less than 1,000), and all contain unnatural amino acids. Human radiolabeled ADME studies were conducted for all of them, elucidating the pattern of metabolism and elimination of therapeutic peptides in humans.

Table 2. Summary of in vivo radiolabeled metabolism and excretion studies of therapeutic peptides approved by the FDA from 2011 to 2023

Note: NS, no studies mentioned in submission document(s).

Figures in parentheses indicate the number of peptides utilized for in vivo radiolabeled studies among 37 therapeutic peptides approved by the FDA.

How to Conduct In Vivo ADME Studies for Therapeutic Peptides

There is no regulatory guidance on conducting metabolism and excretion studies for therapeutic peptides. Based on industry status and regulatory requirements, the white paper provides a decision tree delineating the necessity of radiolabeled ADME experiments to support the marketing of therapeutic peptides. (Figure 2).

For a peptide containing only natural amino acids, in vitro metabolite identification (MetID) and in vivo metabolism/excretion studies are not mandated, following ICH-S6(R1) ^[2] for regulatory filing. However, these studies are required when metabolic insights are critical to understanding the pharmacokinetic/pharmacodynamic (PK/PD) relationship.

In contrast, for peptides containing unnatural amino acids and/or organic linker moieties (TPepB and TPepC), there are three distinct scenarios meriting considerations:

• If the unnatural amino acids or the organic linker moiety have been used in other marketed therapeutic peptides and shown safety in humans, it may not be necessary to conduct radiolabeled ADME studies. Nonetheless, metabolism studies are still recommended to evaluate major circulating and potential active metabolites if needed.

• If the therapeutic peptides contain novel unnatural amino acids and/or a novel organic linker moiety that has not been incorporated in any marketed drugs, radiolabeled ADME studies are generally recommended to characterize its metabolism and elimination pathways. It is worth noting that if a therapeutic peptide contains multiple (≥ 3) novel unnatural amino acids and/or organic linker moieties, it is not obligatory to label all those novel moieties.

• Radiolabeled metabolism and excretion studies may not be performed if sufficient in vitro and/or in vivo ADME data or literature knowledge is provided to demonstrate the safety of specific unnatural amino acids or organic linker moieties.

Figure 2. Industry recommendations for ADME studies of therapeutic peptides. Adapted from reference ^[1]

How to Select Radioisotopes and Labeling Sites for Radiolabeled Peptides in In Vivo ADME Studies

Radioactive tracer labeling is a widely used technique to study the in vivo ADME properties of small-molecule drugs, as well as drugs such as peptides, oligonucleotides, and ADCs. Similar to small molecules, peptides are commonly labeled with either ¹⁴C or ³H ^[3-6], with occasional application of other radioisotopes (e.g. ¹⁸F or ¹²⁵I). Among the 37 peptide drugs approved by the FDA between 2011 and 2023, 17 drugs were labeled with ¹⁴C for in vivo ADME studies, 4 drugs were labeled with ³H, and 4 drugs were developed with the initial application of ³H labeling; before transitioning to ¹⁴C-labeled compounds, 1 drug was labeled with ¹²⁵I, and 1 drug (diagnostic drug) was labeled with ¹⁸F, and other drugs were linear peptides containing only natural amino acids (TPepA) or had sufficient safety support data, meaning no radiolabeled ADME studies were conducted.

¹⁴C-labeling is a more prevalent choice than other radioisotopes ^[7]: ³H can be lost from the molecule due to hydrogen-tritium exchange ^[8], thus affecting the accuracy of the ADME study; ¹²⁵I-labeling may alter the structure and composition of the molecule and ADME properties, particularly for small peptides.

Although ¹⁴C is a commonly used radioisotope, it has limitations compared to ³H labeling. The specific activity of ¹⁴C is much lower than that of ³H ^[7] , which can make detection of the ¹⁴C labeling difficult for low-dose drugs. The specific activity could be increased by introducing multiple ¹⁴C atoms to the same molecule. However, analytical challenges with ¹⁴C labeling persist for low-dose peptides with a large MW. The traditional radioactivity detection methods may lack the requisite sensitivity. To overcome this limitation, accelerator mass spectrometry can be used to analyze low radioactivity concentrations of ¹⁴C samples ^[9].

The selection of a radiolabeling site is critical and warrants careful deliberation. The radioisotope should be introduced in a position to monitor significant biotransformation pathways or to track the long-lived circulating metabolites. Prior to radiolabeled ADME studies, a qualitative metabolic profile for the therapeutic peptides from in vitro and in vivo nonradiolabeled metabolism studies can assist in determining the optimal labeling site ^[10].

Additionally, consideration must be given to the potential reincorporation of radiolabeled natural amino acids into the proteome of test species, which is unethical in the case of humans, and may potentially affect the total recovery of radioactivity in the excreta ^[2]. As a rule of thumb, xenobiotic moieties like novel unnatural amino acids and organic linkers with lipophilic side chains are preferred labeling positions over the natural amino acids within a peptide. In cases where multiple parts of a therapeutic peptide need to be monitored, efforts may be made to label a therapeutic peptide at two or more distinct positions.

Case Study: Radioactive Tracer Technique in the In Vivo ADME Study of Therapeutic Peptides

Figure 3. [³H]-Semaglutide Structure

Semaglutide is a GLP-1 receptor agonist developed by Novo Nordisk for the treatment of type 2 diabetes. It has a MW of 4113.6 and uses radioisotope ³H. As shown in Figure 3, the labels are located at the α and β positions of the carbonyl group in the amide bond of the octadecanedioic acid group of the side chain of the semaglutide (two labeling positions in total) ^[11]. The investigators conducted separate radiolabeled ADME studies of ³H-labeled semaglutide in rats, monkeys, and humans using a liquid scintillation counter (LSC) to determine total radioactivity. The results showed that the recovery of total radioactivity in rats was 94%, much higher than 75% in humans and 58% in monkeys, as detailed in Table 3 ^[11]. The total radioactivity of rat carcasses accounted for 22%, which primarily explains why the total recovery in rats was higher than that in humans and monkeys. Lower recovery of total radioactivity in humans and monkeys presumably may be due to in vivo hydrogen-tritium exchange resulting in the loss of ³H from the molecule. It may also result from the hydrolysis of ³H-semaglutide to octadecanedioic acid, which enters into the body’s circulation. To improve the recovery of total radioactivity in human mass balance and reduce the effect of hydrogen-tritium exchange, a ¹⁴C-labeled compound for therapeutic peptides ADME studies is recommended, but there is still a need to comprehensively consider the administered dosage of therapeutic peptides, MW, and ¹⁴C-labeled radioactivity.

Table 3. Summary of radioactivity excretion recovery following subcutaneous administration of [³H]-semaglutide ^[11]

Conclusion

In conclusion, radiolabeling technology provides a powerful tool for in vivo ADME studies of therapeutic peptides. In applications, it is necessary to select the appropriate isotope and labeling position based on the specific situation to ensure the accuracy and reliability of the study results. By leveraging techniques for radiolabeled peptides, invaluable ADME insights can be obtained, thereby advancing peptide drug development and therapeutic optimization.

Table 4. Summary of Metabolism and Excretion Studies of Therapeutic Peptides Approved by the FDA from 2011 to 2023^[12]

Authors: Jia Xue, Peng Li, Lian Guo, Weiqun Cao, Lingling Zhang

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