Development and Validation of a PET/SPECT Radiopharmaceutical in Oncology

As per the World Health Organization definition, “Radiopharmaceuticals are unique medicinal formulations containing radioisotopes which are used in major clinical areas for diagnosis and/or therapy”. The most widely clinically used radiopharmaceutical, [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG), employed for the diagnosis and staging of cancer by Positron Emission Tomography (PET), accounts for the vast majority of the over 2 million PET-scans reported in 2020 in the USA. In the last two decades, a plethora of new radiopharmaceuticals have been reported in the preclinical literature, with some that were translated to early clinical trials and a small but significant number that were approved by the FDA for clinical use. The latest have been prostate-specific membrane antigen (PSMA) PET agents for prostate cancer, with ⁶⁸ Ga-PSMA 11 granted approval to UCLA and UCSF on December 1, 2020 [1] and ¹⁸F-DCFPyL (Pylarify) approved in late May 2021 [2].

One of the challenges in maintaining consistency when reporting data about production and preclinical validation of new radiopharmaceuticals is the lack of guidelines or standards that need to be met in order to provide the community with uniform and reliable data. The present white paper will address some of these issues and lay down a list of key parameters, albeit not exhaustive, in an effort by the WMIS Education Committee to standardize reports on the development of new radiopharmaceuticals.

Radiopharmaceuticals are diagnostic or therapeutic tools and are produced in a highly regulated environment, following protocols that are set with high standards of safety. As with many other drugs, the final deliverable must have a high degree of purity, but, unlike standard pharmaceuticals that can be produced in lots and stored, radiopharmaceuticals, especially the ones labeled with the short-lived isotopes, must be produced “fresh” before use. This feature lays down a series of needs that must be addressed during the development of a new radiopharmaceutical.

A new radiopharmaceutical must be produced using a reliable and reproducible synthetic method [3] and the consensus nomenclature rules for radiopharmaceutical chemistry must be obeyed [4‐6].

For ¹¹C- and ideally for ¹⁸F-tracers, it is preferable for the synthesis to be automated [7]. Many laboratories prefer to perform hands-on ¹⁸F-radiochemistry on novel radiotracers and invest in automation only when the biological value of the compound is proven. However, this must be balanced with the fact that manual ¹⁸F-radiochemistry typically adds unnecessary time to the development of an ¹⁸F-tracer because the corresponding automated synthesis can require a re-design and re-development of the whole synthetic process, steps that can be avoided if the radiosynthesis is conceived directly in automation. In addition, synthetic modules often have a built-in system for purification and reformulation of the radiopharmaceutical which allow for optimization of the entire procedure from end-of-bombardment to final formulation. Morever, an automated synthesis minimizes risks of human error, adding to the reliability of the process and often allows production of higher doses, enabling more complex biological studies. With radiometals, where the labeling procedure is usually straightforward incubation method, hands-on syntheses are still very common, although small synthetic modules are available for preclinical development [8, 9] and routinely used for human studies [10‐12]. High activity yields (non-decay corrected) are of course desirable, but for ¹¹C- and ¹⁸F-chemistry, a 3–4% activity yield from end-of-bombardment (EOB) to formulation is acceptable for initial validation. Decay corrected radiochemical yields can be a useful indication of the efficiency of the labeling method, and are commonly used in radiochemistry methodology development. They, however, have less meaning in radiotracer development, and, when reported, should be alongside activity yields. The length of the synthetic process, from end-of-bombardment to formulation, and a confirmation of the % purity is also paramount prior to utilization in biological systems.

Any new reported radiotracer must be radiochemically and chemically characterized. Identity must be confirmed by high performance liquid chromatography (HPLC) via co-elution with the non-radioactive reference compound, with one, and preferably two different mobile phases. Since radio-HPLCs are usually equipped with a γ- and a UV detector, a UV-trace is usually presented as proof of identity. If the final compound is not UV-active throughout a multistep synthesis, the identity of the UV-active intermediate (e.g. the protected analogue of the final compound) must be reported [13]. Other types of detectors, such as fluorescence and refractive index detectors, are equally valid. Mass spectrometry (MS) via a HPLC–MS is also an acceptable alternative for the detection of small amounts of carrier observed in most radiotracer preparations, even at high molar activities [14]. The UV trace of the final compound should be reported as proof of chemical purity and ideally will show only background noise. Although an accurate analysis of chemical non-radioactive (solid) contaminants is not required for preclinical validation, quantification of UV-contaminants can be reported, under the assumption that all peaks have an identical extinction coefficient to the product of interest. If a major peak is shown in the UV-chromatogram, identification of the peak is desirable even in a preclinical setting. However, in the case where the tracer is used in human studies, the peak(s) absolutely must be identified. Assessment of the radiochemical purity via radio-HPLC as the ratio between the desired compound and the sum of all other radioactive peaks should be at least 95%.

Radiochemical yields and purities for proteins (e.g. antibodies) are most often determined using radio-instant thin layer chromatography (radio-iTLC), which identifies free isotope from the radiolabeled protein. The high pressure of HPLC systems, along with the use of organic solvents, can pejoratively affect the tertiary structure of antibodies. Certain conventional HPLC systems can be outfitted with size exclusion columns and aqueous solvents and employed in the quality control or separation of radiolabeled proteins. Alternatively, dedicated fast protein liquid chromatography (FPLC) systems are very convenient for quality control and separation of antibody aggregates or dimers that result from long term storage or high temperature incubations. In the absence of an automated liquid chromatography system, purification can also be performed using a size-exclusion column or a centrifugal spin column filter with molecular weight cut-offs ranging from 10 to 50 kDa with centrifugal speeds typically at ~ 3000–4000 rpm (400–1000 × g with a rotor radius of 4–6 cm). Unfortunately, there is no existing method to separate unlabeled from labeled protein.

The molar activity (MA) or specific activity (SA), expressed in SI units as either MBq/μmol to GBq/μmol or MBq/ μg to GBq/μg respectively, should be reported as a range. Although higher MAs often translate into superior performance of the radiopharmaceutical, it is difficult to predefine what range of MA is acceptable or not, as it typically depends on the biological systems that are targeted. Some systems are virtually unsaturable (e.g. hypoxia), so high MAs/SAs are not a requirement for good quality images. In receptor imaging, when the target protein is present in low concentrations and is easily saturated, radiolabeled antibodies typically provide good quality images with a SA in the order of MBq/mg, which is relatively low compared to small molecule agents. This stems from the antibodies’ high affinity for their targets, slower clearance from circulation and internalization potential, with consequent recycling of the target. Small molecules, on the other hand, need to be produced in very high MA, because they often have lower affinity, may compete with high concentrations of the physiological substrate of their target, and exhibit rapid clearance. Typically, a SA of 8–10 GBq/μg and a MA higher than 20–30 GBq/μmol for small molecules are considered acceptable. For radiolabeled proteins, a SA of > 74 MBq/mg is typical with as high as 740 MBq/mg reported. Lower SA values maybe be acceptable; however, this comes with the caveat that SA affects the direct quantification of receptor/antigen sites. As an example, antibodies with lower SAs and therefore more non-radiolabeled molecules, can saturate binding sites. This consequently leads to a decrease in signal. While higher established SAs are preferred, if the antigen of interest is shed in the blood, the tracer is likely sequestered by the circulating antigen. Another factor to consider is the “antigen sink”, wherein the antigen of interest (e.g. PD-L1) is found in non-tumor targets (e.g. bone marrow, spleen, liver, kidneys). In this case, lowering the SA/MA is beneficial as non-labeled proteins or carrier-added tracers will suppress non-specific tracer binding in antigen-expressing normal tissues while increasing uptake within the tumor [15‐17]. Thus, a fine balance between SA and receptor/antigen density both in the organ/tumor of interest and the non-target tissues is needed for optimum imaging, detection and target monitoring. For both proteins and small molecules, high MA/SA yields a radiotracer that can be administered as a “microdose” as defined by the FDA (https://​www.​fda.​gov/​media/​107641/​download) and limits any concern about pharmacology effects of the radiotracer. This means that the injected mass is virtually non-existent, and the molecule itself only acts as a vector for the radioactivity and exerts no pharmaceutical effect nor elicit unwanted toxicity.

Site-specific conjugation of the chelator or prosthetic groups is paramount to the radiopharmaceutical’s design considerations. The moiety should be attached in regions where it does not hinder nor affect binding of the molecule to the receptor’s epitope or docking site. Another key component to characterize radiolabeled proteins is to determine the number of radiolabeling moieties (e.g. chelates), which is especially pertinent to radiometal-labeled antibodies/proteins. Determination of the number of chelators historically has been done through radiometric isotopic dilution assays [18]. Currently, the ratio of chelator:protein is characterized by mass spectrometry of the non-radiolabeled chelator-protein conjugate, which determines the number of attached molecules based on changes in mass between the parent and modified molecule [19].

Shelf stability of the final formulated compound (in saline or PBS with 10% ethanol or less) must be monitored via radio-HPLC with samples analyzed at different times, at least up to 4 h, at room temperature. Stability tests at 4 °C is also recommended. If radiolysis is observed, stabilizers,such as ascorbic or gentisic acid, can be added to the final formulation to attenuate this effect [20]. In vitro plasma stability, is an important information to add to the chemical characterization of the radiotracer and provides insight of its stability in vivo. This is obtained by incubation of the radiotracer with human and/or mouse plasma at 5, 30, and 60 min for short-lived radioisotopes, and at 24 h to as long as 144 h for longer-lived isotopes, depending on the isotope’s half-life.

Although the concept of “biological validation” is potentially as broad as one can possibly imagine, a few minimal key but non-trivial experiments are required to demonstrate that the radiopharmaceutical visualizes the biological process or pathway for which it has been designed. The main goal of these biological validation experiments is to ensure that the radiopharmaceutical specifically interacts with the biological target and is not just retained in a non-specific fashion. In vivo stability, and in vivo biodistribution are also a few of the basic requirements for tracer characterization.

If a preclinical validation is successful, an imaging agent can become a clinical candidate and the process of clinical translation can begin. It is beyond the scope of this white paper to provide a thorough outline of all the steps for successfully translating a novel radiotracer into first-in-human studies, which has been reviewed elsewhere [46]. Below is a brief overview on the general steps towards translation.

It is extremely important for the investigator to understand the process for bringing a candidate radiotracer from bench-to-bedside at their institution. This usually includes the development of the radiopharmaceutical in GMP conditions, familiarity with institutional rules for intellectual property coverage, execution of Material Transfer Agreements (if needed), Institutional Review Board (IRB) approval, outsourcing of the synthesis, finding the right funding to cover the costs to obtain Phase 0/1 investigational new drug (IND) approval and last but not the least, clinical investigators interested in leading a potential clinical trial. A treating physician, on board from an early stage, will help to define preclinical pilot experiments that will be important for the writing of the first clinical protocol. A biostatistician is also key to identifying the sample size or number of patients to recruit.

Although translation to human is usually the goal, radiopharmaceuticals can be developed for a variety of reasons that span from probing molecular mechanisms in mouse models, to veterinarian application. Regardless of the final objective, a strong synthetic methodology and a very careful, rigorous, mechanistic-based preclinical validation is instrumental for the success of any new compound and its adoption by the broader scientific community.