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How to transform your compound into in-human data in five steps

Prof. G.M. van Dam, MD, PhD

In-human drug-distribution data obtained by molecular imaging is a key read-out for determining the efficacy of a drug. If generated early in the drug development process (before Phase I), it can significantly accelerate the drug’s clinical development. One way to transform a compound into (early) in-human data is through a concept called microdosing in molecular imaging. The process of microdosing contains five essential steps: product labelling, toxicity study, systemic injection, imaging, and in vivo and ex vivo analysis.

From compound to in-human data graph

Step 1: Label your New Molecular Entity.

The first step towards generating in-human imaging data is to label the New Molecular Entity (NME) with a fluorescent dye or radionuclide. The dye causes the compound to illuminate when activated with a specified light source (e.g., Near Infrared Fluorescence – NIRF). The illuminated dye and its emitted fluorescent light can be visualized with specialized cameras to image the drug binding in-human in vivo at a later stage in the process. Common used NIRF dyes are IRDye800CW and indocyanine green (ICG).

The photons emitted by radionuclides can be visualized by specialized cameras (gamma cameras, PET and SPECT). Commonly used radionuclides for medical imaging are technicium-99m, gallium-68, fluorine-18 and zirconium-89.

Label a MNE

The (chemico-physical) properties of the label have to match the carrier (NME). For example, when you have a long circulation compound, you will need a radionuclide with a longer half-life and vice versa.

Step 2: Conduct a toxicity study if necessary.

Generally, after labelling the compound, an animal toxicity study to ensure the safety for in vivo in-human use is conducted. The need for a toxicity study depends on if the compound and dye/nuclide are considered to be ‘known’ or ‘unknown’. The former implies that the tox levels are known (to cause no harm). The latter means there is no data available yet.

If both the compound and dye are considered to be known, a toxicity test is in most cases not necessary. This means you can go straight to step 3, significantly increasing the time to in-human usage. However, if it is expected that the ‘known’ label affects the biodistribution of the ‘known’ drug, then a toxicity study is often needed. All other cases always require a toxicity study in the form of a single-dose extended animal toxicity study. In such a study you look at the effect on mice or rats with 1000-fold the clinical dose on a mg/kg basis for i.v. and mg/m2 for oral administration (EMA/FDA guidelines). If the results show that the compound and dye/nuclide are safe to use, you continue with the next step.

Do you need a toxicity test?

Step 3: Systemically inject the drug into target population / healthy volunteers.

The next step is to systemically administer microdoses of the drug into volunteers. This means that the drug is directly injected into the circulatory system, and thus, reaches the entire body. This allows a full image of the on- and off-target binding of the drug to be developed. To safeguard the process there are strict regulations for the dosing of the drug. Namely, a microdose can be a maximum of <100 ug (<30 nmol for large proteins) or should be 100x lower than the intended therapeutic dose. The moment of administration depends on the biological characteristics of the drug. For example, one-three hours before surgery for small-sized compounds, but up to 2-3 days for larger proteins like antibodies.

Study participants can either be healthy volunteers or the drug’s target population. Molecular imaging studies with patients belonging to the target population are preferred, as they actually represent the intended end-user. Namely, the disease the drug is developed for is present. Consequently, the on- and off-target data, pharmacokinetics and biodistribution is a better resemblance of the actual efficacy of the drug in vivo.

Step 4: Image the distribution of the drug in and ex vivo.

After injection of the labelled drug to the subject, the drug-label conjugate can be detected in vivo and ex vivo using dedicated fluorescent or nuclear imaging camera systems.

Fluorescent labelled drugs, can be detected by state-of-the-art specific fluorescence cameras, endoscopes and light-tight boxes, for in vivo and ex vivo fluorescence imaging of the excised or biopsied specimen after surgery or endoscopic procedures.

Radiolabelled drugs can be visualized in vivo by PET/CT – PET/MRI and SPECT/CT scanners used for clinical routine medical imaging. In vivo imaging provides true quantitative data  of the drug’s distribution of the labelled drug. When imaging in the target population is performed, usually samples of the tissue of interest can be obtained after surgery. The advantage of additional detailed ex vivo imaging, both fluorescent and nuclear, is that such systems provide higher resolution and sensitivity to detect the label in a more controlled and standardized imaging setting, providing reliable, reproducible and detailed information of the drug distribution. In particular at the cellular level when using a fluorescently labelled drug compound.

Step 5: Analyse image results and draw conclusions.

The final step towards in-human data is to analyse the imaging results of step 4. During the analysis the distribution of the drug in vivo and at the tissue level ex vivo is evaluated and established. Questions such as “Is there a high uptake in the target tissue? And a low uptake in healthy organs?” can be answered during this process, serving the next steps in drug development.

Once the data is compiled you can make reliable and well-founded go/no-go decisions on the tested compound for further clinical development. Thus, the earlier you obtain valuable and reliable in-human data of the drug compound of interest, the earlier you can make go/no-go decisions. In turn, early go/no-go decisions help avoid large investments in non-effective compound in later (more expensive) clinical stages.

The described process provides a general overview of how to transform a drug compound into in-human data. Every compound follows the same steps, however, the specifics of each step are depended on the nature of the drug. Want to know more about what your drug development plan would look like by using the unique microdosing concept for a 1st in-human small smart early phase clinical trial? Contact us at


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