Often in the press or online portals, you can find news that someone (usually a lone scientist) has discovered a cure for some extremely important disease. Such news stories appear and will continue to appear, and yet the new drugs they were talking about were as non-existent as they were. How does this happen? What is it that makes molecules, often so promising in laboratory studies, not end up as new drugs? The answer is ADME. But what is ADME?

What does ADME stand for?

ADME is an acronym used in pharmacology. It stands for Absorption, Distribution, Metabolism, and Excretion. In short, these are the processes that take place in our body in the context of foreign substances, including drugs. It is how drugs are absorbed, transported around our body, metabolized, and excreted that affects whether a drug is effective (reaches its destination) and safe (does not cause side effects).

Why is ADME important?

Imagine that you design a new therapeutic molecule. You test its activity in vitro and get amazing, even spectacular, results. So there is nothing left to do but test the molecule on animals. And there is a surprise. It turns out that in an animal model, the results are not very satisfactory. Why? Well. You have to ensure that the molecule still reaches its target and that it has a therapeutic effect. To do this, the properties of ADME should be studied in parallel with activity, and the results incorporated into the models used to design molecules.

What are ADME studies?

ADME can be studied at the in vitro and in vivo levels (determining the pharmacokinetic profile). During the discovery and even early development phases, in vitro testing plays a huge role. With appropriately designed analytical methods, it is possible to study intestinal drug absorption, hepatic metabolism, mode of elimination, and the form in which the molecule is eliminated. Among the most popular tests included in ADME are: LogD/LogP, Caco-2 absorption test, MDCK absorption test, PAMPA, metabolic stability, and metabolite identification.

Sometimes a T for toxicity is added to the ADME package. This results in an expanded suite of tests called ADMET or ADMETox.

So let’s divide ADME tests according to the property being tested.


  • Kinetic solubility
  • Thermodynamic solubility
  • LogP/ LogD
  • Caco-2 permeability assay
  • MDCK permeability assay

The first three of these tests, at first glance, have nothing to do with absorption. Nothing could be further from the truth. Let’s start with solubility. Only a dissolved compound is able to cross the intestinal-blood barrier. So it directly affects the possibility of absorption and correlates closely with bioavailability. I mentioned two solubility tests: thermodynamic solubility and kinetic solubility. How do they differ from each other? Let’s start with thermodynamic solubility. The result of this test tells about the solubility of a substance in thermodynamic equilibrium. The undissolved substance is added to a buffer of a certain pH until supersaturated, and then stirred for 24 hours for the system to reach thermodynamic equilibrium. The concentration of the solute in solution is then tested. Thermodynamic solubility is usually determined at three pH levels corresponding to the pH of the digestive system and plasma. It is this property that is crucial for the absorption of the substance.

The situation is slightly different with kinetic solubility. This test is performed to determine the solubility of a substance under conditions used in various in vitro tests. Usually, the starting point is a stock solution of the substance (for example, in DMSO). Solubility is tested by diluting the stock solution with the buffer under test to a specified concentration, e.g. 500 μM. After stirring for a specified time, e.g., 1 hour, the content of the substance in the tested solution is determined. Performing this test allows research teams to ensure that the test substance is present in the dissolved form under the conditions in which laboratory tests are performed.

The third non-obvious parameter determined by ADME is the partition coefficient between the water fraction and octanol, or so-called LogP. Octanol does not naturally occur in the body; however, this value closely correlates with the molecule’s ability to be absorbed into the body through passive absorption and also with the volume of distribution. The volume of distribution, in turn, tells whether the compound penetrates from the plasma into the tissues and is able to reach its therapeutic target.

Traditionally, the partition coefficient was determined by preparing a water/octanol system, adding the test compound to it, shaking the system vigorously, and then assessing the concentration of the compound in the individual fractions. Nowadays, it can also be determined using special columns for HPLC.

The PAMPA (Parallel Artificial Membrane Permeability Assay) test is often performed. This is a test that investigates passive transport across lipid membranes. For this test, special multi-well plates are used, with a bottom made of a special membrane. A lipid solution, e.g., soya lecithin, is applied to this membrane, which thus mimics a lipidic biological membrane. Solutions of the test substances are then added to the wells, and the plate is placed in a second (collection) plate, which contains buffer without the addition of the test substance. After a defined incubation time, the concentration in both plates is assessed, and the transport rate is determined.

The most developed laboratory tests in which uptake is investigated are cell assays using the Caco-2 and MDCK lines.

The Caco-2 line is a polyclonal cell line isolated from human colon cancer. Cultured under appropriate conditions, they adopt the morphology and functionality of small intestinal epithelial cells. They can therefore be used to test the transport of particles from the intestine into the blood. The superiority of this assay over the previously mentioned ones is that the cells are equipped with transporters and enzymes and therefore have complete gut-blood barrier functionality. Thus, it is possible to determine the rate of absorption, which consists of passive transport, active transport, return transport, and also metabolism (first-pass effect).

While the PAMPA test uses quite high concentrations of substances and thus simple UV-VIS spectroscopy can be used to determine concentrations, this is not possible with the Caco-2 test. High substance concentrations could be toxic to the cells and could interfere with the test result. The most commonly used concentration is 10 μM. In addition, the matrix is more complex. Cell metabolites, proteins, and sometimes the addition of BSA are used for the test. Therefore, the concentration in the samples is determined by LC-MS/MS analysis, usually using triple quadrupole or q-trap instruments.

In order to be completely functional, Caco-2 cells need to be cultured for 21 days in special multi-well plates with a semi-permeable membrane. During this time, the cells form a monolayer and differentiate to obtain the morphology and functionality of the small intestinal cells. The platelets are placed in the collection plates. Transport is studied in both directions, i.e., intestine blood (adding the compound solution to the top of the cells) and blood-gut (adding the compound solution to the stripping plate). In this way, it is possible to determine the so-called Efflux ratio, which tells what is the rate of efflux transport, carried out by special membrane proteins.


  • Metabolic stability
  • Identification of metabolites

When talking about drugs, we must bear in mind that for our organisms they are foreign substances, the so-called Xenobiotics. Organisms have developed a number of mechanisms to protect them from xenobiotics, as toxic substances can be found among them. Among these mechanisms, metabolism and excretion can be enumerated. While excretion seems obvious (the sooner we get rid of a harmful substance, the better), metabolism is no longer so. However, our organisms have an interesting ability to modify foreign substances so that they are less toxic or more easily excreted. It does this through their metabolism, i.e., chemical modification catalyzed by enzymes. Drug metabolism occurs throughout the body, but the main organ that does this is the liver.

Metabolism of xenobiotics is divided into two phases.

Phase I – functionalization occurs in the smooth endoplasmic reticulum. It is catalyzed by a family of enzymes collectively known as cytochrome P450, which change certain functional groups of chemical molecules in a red-ox process. They carry out reactions such as deamidation, deamination, hydroxylation, dehydroxylation, etc. As a result, the resulting molecules are usually less toxic. Sometimes, however, so-called reactive metabolites can be formed, which can cause further damage to the body by causing, for example, oxidation of proteins or nucleic acids.

Phase II – conjugation. Occurs in the cytoplasm of cells. It involves the attachment of highly hydrophilic functional groups to molecules such as the glucuronic acid residue. This is to facilitate the filtering of these molecules in the kidney and their removal with the urine.

Metabolism can be studied at two levels: biochemical and cellular. At the cellular level, this can be done using isolated hepatocytes. However, this is rarely used due to the high cost of obtaining hepatocytes and also the rather high requirements of these cells. The most commonly used are the subcellular fraction of microsomes (containing the endoplasmic reticulum) and the S9 fraction (containing microsomes and cytoplasm). The test compound is incubated with the subcellular fraction with the addition of appropriate cofactors, and the loss of substance over time is examined by LC-MS/MS. By using this technique, it is also possible to identify metabolites.

The study of metabolism is extremely important at an early stage of research. The enzymes responsible for metabolism are characterized by high inter-species variability. It is possible that a molecule is metabolized extremely rapidly in rodents, e.g. by fractions derived from the liver of mice or rats, while being completely absent from metabolic pathways in humans. This can greatly complicate issues of selecting an appropriate animal model for in vivo studies or subsequent attempts to extrapolate results to the human body.

On the occasion of metabolism, it is worth mentioning the study of cytochrome P450 inhibition. This family of proteins metabolizes many drugs. The 3A4 isoform of this enzyme alone catalyzes the metabolism of more than 80% of all small-molecule drugs. Not surprisingly, there can be interactions between different drugs due to their mutual metabolism. If a molecule is metabolized, it simultaneously becomes an inhibitor of the enzyme that is responsible for its metabolism. When other drugs are taken, this can lead to an increase in their concentration and subsequent side effects. For some drugs, this is extremely important, e.g., in the case of neurological drugs, which often have a narrow safety window, and the blood-brain barrier makes it difficult to remove them from the brain.

Metabolism does not only occur in the liver. Various enzymes that can induce it are found, among others, in the plasma. These include, for example, esterases responsible for breaking down esters. Therefore, stability tests are often performed in plasma. For this purpose, the test substance is added to the plasma, and its plasma content is determined at successive time points. This is an important test also for the reliability of another test that is performed as part of ADME – binding to plasma proteins. For some molecules, stability testing in plasma is extremely important are so-called ‘prodrugs’, i.e. molecules that do not have therapeutic activity on their own, but acquire it after metabolization. Often, scientists who design drugs construct them in the form of esters so that they are more easily absorbed. Then, through the action of esterases present in the plasma, the pro-drug molecule is metabolized to its active form.


The absorption of a drug into the body does not yet mean that it will reach its target. Neurological drugs need to reach the brain to bind to their receptors there. Others have to go inside cells to be affected by their metabolism or to combine with intracellular receptors. And even if they reach the target organs, it must be remembered that only unbound molecules show a therapeutic effect. Many drugs, however, use porters when traveling through our bodies and bind to plasma proteins.

For this reason, an extremely important test that is performed as part of ADME is the study of binding to plasma proteins. Normally, the analyzed compound is added to the plasma, and dialysis is performed. Only the unbound fraction of the compound is able to pass into the dialysate. After a defined dialysis time, a determination of the concentration of the substance in both fractions is performed (in the fraction with plasma after prior denaturation of the proteins to release the test substance).

In vitro ADME studies are able to answer many questions. A very powerful tool is physiology-based pharmacokinetics modeling, known as PBPK (physiology-based pharmacokinetics modeling), which uses results from in vitro and in vivo studies to predict drug behavior in the body. However, not everything can be verified only at the in vitro level. Sometimes the properties of a molecule make it difficult to perform these tests. This can happen when the molecule is highly hydrophobic or unstable in plasma. Through in vivo studies, calculations in PBPK models can also be validated and improved. We will discuss how in vivo pharmacokinetics studies are performed, the types of these studies, and the information that can be obtained in the next article on ADME.