Most drugs undergo some level of transformation once they enter the body. This metabolism process allows a drug’s active pharmaceutical ingredient (API) to achieve its desired effect and eliminate it from the body. But metabolism can also be tricky. Metabolizing a drug creates metabolites, which can have pharmacological and toxicological properties different from the drug.
Metabolites can stimulate or inhibit enzymes, cultivate a chemical reaction or defend against other organisms in vivo. Regulatory agencies such as the US Food and Drug Administration (FDA) require that if a metabolite exceeds 10% of total exposures to drug-related components in humans, they must be identified and characterized through metabolite identification (MetID) studies.
When are MetID studies used?
MetID studies in vitro are relatively simple, quick to conduct and can be used to predict metabolism in the body, potentially alleviating the need for experiments on test species. In vivo tests are more complicated.
MetID studies conducted inside the body use biological matrices like bile, urine and feces to provide data on a drug’s metabolic and clearance pathways. Liver microsomes and hepatocytes can also be analyzed to establish a correlation between in vivo and in vitro metabolism. Scientists can then use this correlation to understand and quantify the relationship between external/internal exposure and toxicological risk. The results can facilitate drug discovery, product development and chemical risk assessment.
Metabolism studies are crucial to understanding how a drug’s biotransformation impacts safety and efficacy and, as such, are used at all stages of drug development from discovery to clinical trials.
MetID Studies and PROTACs
Proteolysis targeting chimera (PROTAC® is a registered trademark of Arvinas. In this blog post, PROTAC specifically refers to the abbreviation of PROteolysis TArgeting Chimera as therapeutic modalities) drugs are a novel class of drugs that work by inducing the targeted degradation of disease-causing proteins. They have shown promise in the treatment of cancer, neurodegenerative diseases, and viral infections, among others, and are currently being investigated in preclinical and clinical studies. However, they usually have a high molecular weight and sometimes poor solubility and permeability in comparison with conventional small-molecule drugs. Because of this, they present several bioanalytical challenges.
PROTACs consist of two ligands (one binding to the target protein and another binding to an E3 ubiquitin ligase) and a linker. Their unique structures present challenges in finding the relevant metabolites they contain. One solution is to use a 14C-labeled MetID study to achieve the most comprehensive results during in vivo MetID studies. To achieve this, scientists must design 14C-labeled PROTAC molecules with appropriate labeling positions to reveal possible metabolites. This approach can maximize the data gathered from radio profiling and MetID studies.
Why MetID Studies for PROTACs are so Important
Industry research shows that the metabolic stability of PROTACs is more influenced by their shape and rigidity, which determine how the molecule interacts with the metabolizing enzymes. Research has also shown that one of the only ways to assess the potential metabolic liability of a PROTAC is through MetID studies.
One strategy to avoid the issues of low permeability and solubility associated with PROTACs is to incorporate heteroatoms into the molecule. This strategy can unveil metabolic soft spots, i.e., the locations in a molecule that are particularly susceptible to metabolic modification. These soft spots contribute to high pharmacokinetic clearance and, as such, must be identified and accounted for.
Predicting Efficacy and Toxicity
A PROTAC molecule’s linker impacts its metabolic stability, and linker cleavage metabolites can be formed. These metabolites can be close analogs of the protein of interest or E3 ligase ligands and potentially compete with the PROTAC to block its mode of action. A significant number of these metabolites can confound the PK/PD relationship.
Another risk for PROTACs is off-target degradation and small protein of interest or E3 binding metabolites. MetID studies in plasma and tissues such as liver, kidney, muscles and tumors, establish the relative abundance of the parent PROTAC molecule and each metabolite in each matrix, helping to understand and evaluate the in vivo efficacy and potential toxicity.
A Final Word
PROTACs are an exciting branch of medicine, and their strong pharmacological characteristics and targeting capabilities make for high-potential therapeutic options. But these types of drugs present unique challenges for those seeking to study in vivo and in vitro metabolism. MetID studies are vital to determine the relative abundance of metabolites and make PROTACs safer and more effective.
Because of the complexity of these studies, drug developers and sponsors who lack experience or expertise in this area should put their trust in a qualified lab testing partner to help out.
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