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).

1. 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?
ADME – A Key To An Effective And
Safe Drug – Selvita

2. 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.

3. Kinetic solubility
Thermodynamic solubility
LogP/ LogD
Caco-2 permeability assay
PAMPA
MDCK permeability assay
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.
Absorption

4. 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.

5. 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.

6. 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.

7. Metabolic stability
Identification of metabolites
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
Metabolism
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.

8. 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.

9. 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.

10. 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.

11. Distribution
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).

12. 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 development 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.
611 Gateway Blvd,
Suite 120
South San Francisco,
CA 94080, US
tel: +1 650 660 7899
sales@selvita.com
Website visite - www.selvita.com
Email -