Peptide-Radionuclide Conjugates: Interpretation of FDA Approved Drugs and DMPK Strategies

Introduction

Radionuclides bearing excessive energy can emit particles or rays spontaneously and release energy to achieve the purpose of diagnosis or therapy. However, the shortcomings of radionuclides, such as low stability , uncontrollability, and off-target toxicity, limit their clinical application. Radionuclides are chelated with a chelator and then coupled with peptides through a linker to form peptide-radionuclide conjugates, ensuring the precise delivery of radionuclides to the target. Combined with the excellent tissue penetration and low immunogenicity of peptides, peptide-radionuclide conjugates can achieve the ideal drug delivery, enhancing therapeutic efficiency and reducing side effects associated with systemic toxicity. In recent years, a number of peptide-radionuclide conjugates have been approved by the FDA (Table 1), which play an increasingly important role in the diagnosis and therapy of cancer treatment.

Table 1. The peptide-radionuclide conjugates approved by FDA

The historical development and types of peptide-radionuclide conjugates

Since the 1920s, radionuclides have been used for diagnosis and therapy. ¹⁷⁷Lu-DOTATATE, the first peptide-radionuclide conjugate for therapy, was approved in 2018, unveiling the prelude to the rapid development of peptide-radionuclide conjugates. Pharmaceutical companies represented by Novartis have developed multiple pipelines targeting SSTR, PSMA, CXC chemokine receptor 4 (CXCR4), and fibroblast activation protein (FAP) and opened the era of integration of diagnosis and therapy (Figure 1).

PET: Positron Emission Tomography；

CT: Computed Tomography；

¹⁸F-FDG: ¹⁸F-Fludeoxyglucose；

RGD: (Arg-Gly-Asp) peptides；

Ra: Radium; Y: Yttrium; Ga: Gallium; Lu: Lutecium; I: Iodine。

Figure 1. Nuclear drug development timeline.

Peptide-radionuclide conjugates can be divided into diagnostic and therapeutic radionuclides based on emitted particles and usage.

Diagnosis nuclides include positron-emitting nuclides such as 18F, 68Ga, and 123I, which can be imaged by PET, and γ-emitting nuclides such as 99Tc and 67Ga, which can be scanned by single photon emission computed tomography (SPECT). The physical half-lives of diagnostic radionuclides are usually one to a dozen hours, and the short half-lives help prevent long-term exposure to radiation damage.

Therapeutic nuclides include α particles-emitting nuclides such as ²²³Ra, ²²⁵Ac, and β particles-emitting nuclides such as 90Y, 131I, 177Lu, and ⁹⁰Sr. Therapeutic nuclides can damage DNA mitochondria and cell membranes directly through radiation. They can also play a bystander effect in inducing the death of adjacent cancer cells through the communication between target cells and adjacent cells. They can also exert an abscopal effect to attack distant tumor cells outside the irradiated field¹. These three processes cause oxidative stress, stimulate the secretion of endogenous danger signal factors, and activate the immune system². The physical half-lives of therapeutic nuclides are usually longer than diagnostic nuclides, varying from one day to a dozen days, to ensure a relatively long residence time in the body for efficacy (Figure 2).

Figure 2. The peptide-radionuclide conjugates classification and corresponding radionuclides.

The selection of radionuclides depends on multiple factors, such as the penetration range, linear energy transfer (LET) of emitters, and physical half-life of particles emitted by radionuclides, combined with the size, type, density, and heterogeneity of tumors (Figure 3) ³. The characteristics and application of emitted particles are summarized in Table 2.

Figure3. Types of radioactive decay and their tissue penetration range³.



Table 2. The characteristics and application of emitted particles³

Examples of FDA-approved peptide-radionuclide conjugates

Disease background:

Prostate cancer is the second most common cancer in men worldwide. Current treatments include surgery, androgen deprivation therapy， chemotherapy, etc., but the recurrence rate after primary treatment is high (20-60%), with a poor end-stage prognosis. The 5-year survival rate is low (below 30%). Moreover, conventional imaging techniques have a low detection rate and have insufficient performance in describing disease progression⁴.

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer, and the expression level is 100-1000 fold than that in normal tissues⁵. It promotes angiogenesis, shows a significant proliferative advantage, and can translocate from the apical to the luminal surface of the prostatic duct, where it becomes accessible on the outside of the cell for binding to ligands⁶. The expression of PSMA increases with tumor aggressiveness, metastatic disease, and recurrence. So, it is suitable as a molecular target for imaging and therapy. ⁶⁸Ga-PSMA-11 and ¹⁷⁷Lu-PSMA-617 binding to PSMA with high-affinity, realize precise diagnosis and treatment in prostate cancer, respectively.

2.1 Peptide-radionuclide conjugate ⁶⁸Ga-PSMA-11 for diagnosis

In December 2020, the FDA approved 68 Ga PSMA-11 as the first diagnostic reagent indicated for PET in men with PSMA-positive lesions. The drug consists of a peptide PSMA-11, which targets PSMA, a linker, a chelator, and radionuclide68Ga, and the structure is shown in Figure 4. The clinically recommended dosage is 111 MBq-259 MBq (3 mCi-7 mCi) by intravenous injection. Imaging can be achieved after 50 to 100 minutes post-dose⁷.

Figure 4. The structure of ⁶⁸Ga-PSMA-11（targeting peptide in purple, linker in blue, chelator in red, and radionuclide in black）.

The peptide PSMA-11 targets and binds to the overexpressed PSMA on prostate cancer cells, and ⁶⁸Ga emits positron, which is scanned by PET. The physical half-life of ⁶⁸ Ga is 68 minutes, followed by transformation to stable ⁶⁸ Zn. It takes only a few hours to complete imaging after administration. ⁶⁸Ga-PSMA-11 has a considerable detection rate in patients clinically evaluated for prostate cancer metastasis and recurrence (sensitivity 47%, specificity 90%, positive predictive value 61%, and negative predictive value 84% ), and the incidence of adverse effects is low (< 1%) with the most commonly reported adverse reactions being nausea, diarrhea, and dizziness.

Pharmacokinetics of ⁶⁸Ga-PSMA-11 ⁷:

• Binding to PSMA and internalization of ⁶⁸Ga-PSMA-1:

⁶⁸ Ga-PSMA-11 is bound to the membrane surface and internalized into the human prostate cancer cell line (LNCaP). The uptake of ⁶⁸ Ga-PSMA-11 into cells at 37 °C was approximately 20­35%, which was higher than that at 4 °C. The binding and internalization of ⁶⁸ Ga-PSMA-11 were completely blocked when co-incubated with 500 μM of 2-PMPA (a competitive blocker of PSMA).

• Distribution of ⁶⁸Ga-PSMA-11:

⁶⁸Ga-PSMA-11 intravenously injected was cleared from the blood, and the blood radiation activity showed bi-exponential behavior with half-lives of 6.5 minutes and 4.4 hours, respectively.

In healthy humans, ⁶⁸ Ga-PSMA-11 preferentially accumulated in the liver (15%), kidneys (7%), spleen (2%), and salivary glands (0.5%). ⁶⁸Ga-PSMA-11 was also seen in the adrenal glands and prostate. No ⁶⁸Ga-PSMA-11 uptake was found in the cerebral cortex or heart, and uptake of ⁶⁸Ga-PSMA-11 in the lungs was usually very low.

In patients with prostate cancer, lymphatic metastasis of prostate cancer in the pelvis can be observed by PET/CT 1 hour after the injection of 170 MBq of ⁶⁸Ga-PSMA-11 (red arrows in Figure 5a and Figure 5b), and the radiation intensity gradually increased and reached the peak at 4 hours, indicating targeted delivery of ⁶⁸Ga-PSMA-11 in prostate cancer lesions.

Figure 5. Sequential PET/CT of a Patient Following the Injection of 170 MBq 68Ga-PSMA-11.

• Metabolism of ⁶⁸Ga-PSMA-11:

After internalization of the ligand-bound receptor, the ligand dissociated from the receptor and was degraded by proteolytic enzymes into amino acids. ⁶⁷Ga-PSMA-11 was stable after 48 hours of incubation at 37 ℃ in human serum, and no free ⁶⁷Ga was detected⁸. Incubation of ⁶⁸Ga-PSMA-11 in the presence of excess amount of transferrin at 37℃ for 2 hours, ⁶⁸Ga was notchelated to transferrin.

• Excretion of ⁶⁸Ga-PSMA-11:

⁶⁸Ga-PSMA-11 was primarily excreted via the kidneys.

• Drug interactions of ⁶⁸Ga-PSMA-11:

The PSMA blockers like 2-PMPA can potentially inhibit the binding and cellular internalization of ⁶⁸Ga-PSMA-11 to PSMA. However, there is no clinical concern currently, given that no approved drugs currently contain PSMA blockers⁷.

2.2 Peptide-radionuclide conjugate ¹⁷⁷Lu-PSMA-617 for therapy

In March 2022, the FDA approved ¹⁷⁷ Lu-PSMA-617 as a radioligand therapy targeting PSMA. ¹⁷⁷Lu-PSMA-617 consists of a peptide PSMA-611, which targets PSMA, a linker, a chelator, and radionuclide 177Lu, and the structure is shown in Figure 6. It is the first radioligand therapy -approved by the FDA to treat patients with metastatic trend-resistant prostate cancer and has received breakthrough therapy designation. The results of the phase III clinical study showed that the combination therapy (BSC/BSoC + ¹⁷⁷Lu-PSMA-617) can prolong the radiographic progression-free survival and overall survival of patients by about 4 months compared with BSC/BSoC standard therapy⁹.

Figure 6. The structure of ¹⁷⁷Lu-PSMA-617 (targeting peptide in purple, linker in blue, chelating agent in red, and radionuclide in black).

After entering the body, ¹⁷⁷Lu-PSMA-617 binding to PSMA overexpressed on the surface of prostate cancer cells is internalized into the tumor cells via clathrin-mediated endocytosis and transported intracellularly via endosomes. The antigen PSMA is recycled to the cell membrane surface or degraded by lysosomal enzymes. ¹⁷⁷Lu is released and emits β-particles to kill cancer cells (Figure 7) ¹⁰.

Figure 7. Schematic model of ¹⁷⁷Lu-PSMA-617 internalization.

The advantage of ¹⁷⁷Lu

• The β-rays emitted by ¹⁷⁷Lu can not only exert cell killing directly, but also induce oxygen free radicals to break DNA.
• The average penetration range of β-rays emitted by ¹⁷⁷Lu is 0.28 mm in the tissue, which can play a crossfire effect to destroy the surrounding tumor cells that do not express the specific antigen. Combined with the targeting components, 177 Lu can precisely kill tumors and overcome tumor heterogeneity.
• ¹⁷⁷Lu can produce γ photon with an emission energy of 208.4 keV, which can be used for SPECT imaging, integrating diagnosis and therapy.
• ¹⁷⁷Lu, with a physical half-life of 6.7 days, is more stable and less volatile than ¹³¹I.

Pharmacokinetics of ¹⁷⁷Lu-PSMA-617:

• Distribution of ¹⁷⁷Lu-PSMA-617:

The geometric mean distribution volume of PSMA-617 was 123 L. Unlabeled PSMA-617, and non-radioactive ¹⁷⁵Lu-PSMA-617 showed moderately bound (approximately 60%-70%) with human plasma. The human blood plasma partitioning ratio were both less than 1, indicating PSMA-617 and ¹⁷⁵Lu-PSMA-617 are not distributed into human erythrocytes specifically.

In healthy humans, ¹⁷⁷Lu-PSMA-617 is mainly distributed in the gastrointestinal tract, liver, lungs, kidneys, heart wall, bone marrow, and salivary glands within 2.5 hours of intravenous administration.

After administration of one to two cycles of ¹⁷⁷Lu-PSMA-617 to patients with low, low to intermediate, intermediate to high, and high prostate tumor load, ¹⁷⁷Lu-PSMA-617 showed strong targeting to prostate cancer metastases and low accumulation in other tissues (Table 3) ¹¹.

Table 3.Organ dosimetry of 4 patients with prostate cancer during their first and second cycles of ¹⁷⁷ Lu-PSMA-617（Gy/GBq）

Note: Tumor load refers to the number of cancer cells and tumor size.

• Metabolism of ¹⁷⁷Lu-PSMA-617:

¹⁷⁵Lu-PSMA-617 and PSMA-617 were stable in the plasma from humans, rats, and minipig for up to 2 hours at 37℃. ¹⁷⁵Lu-PSMA-617 and PSMA-617 were stable in the liver and kidney S9 fractions from human, rat, and minipig for up to 1 hour at 37℃.

• Excretion of ¹⁷⁷Lu-PSMA-617:

In humans, the terminal elimination half-life of ¹⁷⁷Lu-PSMA-617 was about 41.6 hours. The total systemic clearance (CL) was 2.04 L/h, and ¹⁷⁷Lu-PSMA-617 was primarily eliminated via the kidneys as intact. Only 1%-5% of the injected radioactivity was detected in feces.

• Drug Interaction of ¹⁷⁷Lu-PSMA-617:

Interactions with metabolic enzymes: ¹⁷⁷Lu-PSMA-617 and PSMA-617 were not inhibitors for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A, and were not inducers of CYP1A2, 2B6 and 3A4. PSMA-617 was not a substrate of CYP450s.

Interactions with transporters: PSMA-617 was not a substrate of BCRP, P-gp, MATE1, MATE2-K, OAT1, OAT3 and OCT2 in vitro; PSMA-617 was not an inducer of BCRP, P-gp, MATE1, MATE2-K, OAT1, OAT3, OATP1B1, OATP1B3, OCT1 and OCT2 in vitro ⁹.

Characteristics of ideal peptide-radionuclide conjugates (Figure 8)

• Appropriate ray types and emission energy: Radionuclides used for diagnosis normally emit γ rays or positrons (β +), preferably no or less emission of β- and α-rays to reduce radiation damage. Radionuclides used for therapy normally emit α or β- rays mostly and no or less γ rays to improve the therapeutic effect. The emission energy of β- is recommended to be lower than 1 MeV, and the counterpart for α is recommended to be lower than 6 MeV.

• Appropriate physical half-lives: The ideal physical half-lives of radionuclides are normally in the range of 30 minutes to 10 days. On the premise of successful imaging, the physical half-lives of diagnostic radionuclides should be as short as possible to reduce the exposure time in vivo to protect patients from radiation. The physical half-life of therapeutic radionuclides should not be too short, generally 1 to 10 days, to ensure efficacy.

• Low toxicity: The radionuclides and their decay products should have little or no toxicity. The decay products are preferably stable nuclides.

• Stable linkers: Non-cleavable linkers are generally used to enhance stability and decrease off-target risks.

• Target specificity: Peptide-radionuclide conjugates emit radiation once administered. Therefore, high specificity of the target and low accumulation in non-specific tissues are preferred.

Figure 8. Characteristics of an ideal peptide-radionuclide conjugates.

DMPK strategies for peptide-radionuclide conjugates

Many DMPK studies could be carried out with cold counterparts that are not radioactive, such as conjugates containing stable isotopes or the decay products from radionuclides, due to the high threshold for radioisotope experiments if the nuclides are already in clinical use¹². Of course, DMPK studies could be carried out directly with radioactive conjugates if the condition is allowed.

Generally, peptides are easily degraded by proteases, so the stability of peptide-radionuclide conjugates in matrices like plasma, liver S9, kidney homogenate, and so on is essential for DMPK research. Meanwhile, the structure will be optimized based on in vivo and in vitro metabolite identification studies. Plasma protein binding, whole blood to plasma partitioning, and rodent PK with peptide or cold conjugates help to further understand the disposition of peptide-radionuclide conjugates. In addition, it is also necessary to pay attention to the interaction of drugs with metabolic enzymes and transporters.

Tissue distribution in rodents or large animals, or model animals with hot conjugates are useful for studying organ distribution and is beneficial to predict the dosage for first in human studies. Excretion study in rodents with the hot conjugate facilitates understanding of mass balance and excretion pathway.

The DMPK strategies of peptide-radionuclide conjugates for pre-clinical studies and IND-enabling studies are summarized in Table 4.



Table 4. DMPK Strategies of peptide-radionuclide conjugates

Conclusion:

The global nuclear medicine market is in a stage of rapid development and is expected to reach 10 billion dollars by 2030. The increased incidence of cancer also raises the need for nuclear medicine. With more pipelines from pharmaceutical companies combining the facilitation from regulatory agencies and governments, the global enthusiasm for research and development of peptide-radionuclide conjugates is unprecedented, which leads the diagnosis and therapy of cancer into a whole new era.

Talk to a WuXi AppTec expert today to get the support you need to achieve your drug development goals.

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Authors: Chong Chen, Qigan Cheng, Jing jin

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,600+ global clients, and have successfully supported 1,500+ IND applications.