QWBA studies provide a rapid method to determine the extent to which a compound is absorbed, and where drug-related radioactivity distributes within the body. They can indicate passage across physiochemical barriers, such as across the placenta into fetuses, or distribution to the site of action, e.g., into tumors or across the blood–brain barrier to the CNS. They can help identify unsuspected routes of elimination (sweat, saliva, etc.) and provide information on the retention of radiolabeled material in tissues or organ systems. QWBA studies can provide a method for correlating the distribution and/or retention of radiolabeled material with observed or potential toxicity. The potential for autoradiography studies to evaluate unanticipated sites of distribution and/or retention is one of the core strengths of this technique. Data from QWBA studies are also required to support the administration of radiolabeled test compounds to humans.
The technique allows only for the detection of radioactivity, that may correlate to parent compound, metabolite or impurities. Whilst the technique allows visualization of the distribution of drug-related radioactivity into tissues or organs, it does not give sufficient resolution at the cellular level. Where cellular distribution within a tissue is required, a micro-autoradiography study combining autoradiography with conventional histology techniques should be utilized and, again, we have wide experience in this field.
Test-animal selection is important as it should generally correlate with the rodent species used in the toxicity testing of the compound, to support interpretation of toxicity data. Where data are to be used to support a human dosimetry calculation, it is essential to evaluate the potential for melanin binding. Melanin is principally found in the skin and the uveal tract of the eye in pigmented rats; therefore, a limited number of partially pigmented rats is also required for inclusion in the study design. Where specific distribution questions need addressing, it is possible to perform autoradiography studies in other species; however, because of the size limitations of the microtome, specimens should be less than 3 kg in mass.
The most widely utilized isotope for autoradiography studies is 14C, due to its ideal path length, decay half-life and ease at which it is radiosynthesized. Tritiated compounds are also widely utilized. Due to the shorter path length of the 3H isotope, the phosphor screens used for imaging cannot have a protective coating applied. Therefore, they are single use and this can add significantly to the cost of the study. When using tritium, there is also a potential for tritium exchange, where tritiated water is formed and subsequently removed during the freeze-drying process. Given the shorter half-life of tritium, radioactivity decay needs consideration as part of the quantification process. Radioactivity doses are typically targeted at 3.7–7.4 MBq/kg (100–200 µCi/kg) body weight for 14C or 37–74 MBq/kg (1000–2000 µCi/kg) for 3H. Other isotopes such as 125I, 35S and 32P can also be used. Published literature details autoradiography studies using other more specialized isotopes.
The pharmacokinetic properties of the drug under investigation in the rodent species are used as the basis of time-point selection. Time points selected should allow for the calculation of tissue half-lives and radioactivity exposure for each tissue, and should illustrate that the compound of interest has been absorbed, distributed, and eliminated, as appropriate. It is generally agreed that between five and seven sampling times, with one pigmented animal at each sampling time, are sufficient to provide comprehensive data on the distribution pattern and allow the elimination half-life of radioactivity to be calculated for most tissues. Tissue dissection studies typically require three animals at each sampling time and therefore QWBA uses far fewer animals in comparison to previous methods. Time-point selection should include a sampling time close to Tmax, with the remainder of samples distributed over a seven-day period based upon plasma or blood pharmacokinetic data, to try to capture the absorption (where applicable), distribution and elimination phases.
The purpose of a dosimetry calculation is to assess the cancer risk from the radioactivity associated with giving a dose of radioactivity to a human volunteer during the phase I AME clinical-trial process. Typically, this is administered as a single dose to obtain information regarding the absorption, kinetics, distribution, metabolism, and excretion of the radioactively labelled drug. The amount of radioactivity administered to human volunteers, who are not receiving the drug for therapeutic or diagnostic purposes, is governed by guidelines issued by the World Health Organization (WHO) and the International Commission on Radiological Protection (ICRP) in Europe, and the FDA in the USA. Data from the rat QWBA study are extrapolated to predict the concentration and duration of radiation exposure for various tissues within the human body (equivalent doses to tissues). The extrapolated data are used to estimate the maximum safe dose of radioactivity that can be administered to human volunteers. Finalized data are submitted to the Advisory Radioactive Substance Administration Committee (ARSAC) in the UK or the Radioactive Drug Research Committee (RDRC) in the USA for permission to administer the radiolabeled dose. Where dosimetry estimates prevent the administration of sufficient radioactivity by traditional methods, alternative methods such as accelerated mass spectrometry (AMS) should be employed.
In Europe, ICRP 60 guidelines define a list of critical and non-critical tissues associated with higher risk of cancer. Weighting factors are added to those tissues where a high incidence of cancer is observed. Dosimetry to the internal esophageal-gastrointestinal tract and urinary bladder surfaces, through transient passage of dose, is also taken into consideration using this methodology. In the US, ICRP 30 guidelines are used, in which the number of critical tissues is shorter and the five tissues with highest exposures to drug-related radioactivity are included within the calculation.
Melanin is found in the eye, inner ear, skin, hair, hair follicles, brain and lymph nodes of humans and many animal species. Melanin is synthesized in melanosomes (cytoplasmic organelles) within melanocytes. Although albino animals have melanocytes, they lack tyrosinase, an intermediate required in the formation of melanin. Melanin acts as a free-radical scavenger and inhibits UV-induced lipid peroxidation. The frontier orbital electrons in basic compounds are delocalized throughout the melanin polymer and, therefore, bind strongly to melanin within pigmented tissues. Under these conditions, elimination of compound-related radioactivity is typically linked to the turnover of melanin. Although no published value exists for the physiological turnover of melanin, when applicable, dosimetry calculations use a conservative estimate of 100 days. Given there is a link to UV-induced lipid peroxidation, binding of drugs to ocular melanin is not predictive of ocular toxicity, but knowledge that the test substance under investigation has an affinity for melanin should trigger the initiation of a phototoxicity study to meet regulatory requirements where the UV absorption of the drug falls within a pre-defined spectrum (typically 290–700 nm).
To support regulatory submission, the dose is often selected to correlate with the low dose used in toxicity testing and the effective clinical dose. This serves a dual purpose in that it can give support and provide confidence to the toxicology data and can generate human radiolabeled dose estimates. The dose vehicle should reflect that used in toxicity testing and in clinical studies where possible. The radiochemical purity of the test substance is critical, greater than 98% being the target. As the technique follows radioactivity only, if any impurity were to bind exclusively to a single tissue this could be misinterpreted as the distribution of parent compound or radiolabeled metabolites. The maximum specific activity of the radiolabeled material can influence dose selection, as a minimum of 37 MBq/kg is required for tritium labels and 3.7 MBq/kg for radiocarbon. Where specific radioactivity exceeds the recommended radioactive dose, dilution with unlabeled material is utilized. When isotopic dilution is required, it is important that both materials are co-dissolved rather than mixed to avoid differential absorption and ensure a homogenous distribution within the body.
QWBA is an important tool for determining the distribution of a radioactive test agent and therefore for providing insights into the absorption, metabolism, distribution and excretion of a drug. Data from QWBA animal studies helps guide the subsequent administration of radiolabeled test compounds to humans. We have extensive expertise in QWBA across our global sites and have established a collaborative approach across the scientists working on preclinical and clinical projects. This ensures the seamless transition of key metabolism information from animal studies into human studies, increasing efficiency, simplifying communication, enhancing safety and improving study outcomes.
Studies are performed in accordance with applicable animal welfare regulations in an AAALAC-accredited facility. Learn more about our commitment to animal welfare.
