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123
6.1 ADMET
6.1.1 Absorption
Obtaining a candidate that exerts a therapeutic effect at the site of action and is eliminated within
a specified time period is the primary objective of drug discovery [1]. The majority of drugs are
formulated for oral administration due to their convenience for patients to self-administer. Oral
administration of a drug necessitates traversing numerous cellular membranes in order to access
the site of action. The mechanism that controls how drugs cross biological membranes is essentially
the same. For instance, drug permeability across the blood–brain barrier (BBB), the gastrointestinal
tract barrier, and the human colon carcinoma cell line Caco-2 monolayers all utilize this
principle [2]. Two prevalent categories of mechanisms underlie the transmembrane transport of
drugs. The absorption of small-molecule drugs (with a molecular weight of around 150–1500 Da)
is most commonly achieved through passive transcellular diffusion, which refers to the process of
diffusion through the lipid bilayers. The process is propelled by concentration gradients, starting in
compartments with high concentrations and moving down to compartments with low
concentrations. Drugs with a high affinity for lipids tend to accumulate in the membrane, resulting
in a greater difference in concentration between the membrane and the cytosolic fluid. The process
of diffusion from the membrane to the cytosolic solution happens quickly because of the large
gradient that exists between the two. When the steady state is established, the concentration of
drugs that are not attached to the membrane should be the same on both sides of the membrane
[3, 4]. The physiology of the organ, including surface area, transit time, and pH gradient, as well as
the molecular characteristics of a drug, such as passive permeability, pK
a
, and other physicochemical
parameters, all have an impact on how well it absorbs. In the intestine, compounds with a molecular
weight of less than 200 can be taken in through the paracellular pathway, which is made up of the
spaces between cells [5]. No energy is required for the trans-membrane transportation of nonpolar
and uncharged drugs, which is why passive transport is the most common route for these types of
drugs. Predicting medication absorption and permeability primarily focuses on this channel since
passive diffusion is more significant for both the intestinal epithelium and the BBB [6, 7]. “Active
6
ADMET and Physicochemical Assessments in Drug Design
Ulviye Acar Çevik
1
, Ayşen Işik
2
, and Abdüllatif Karakaya
3
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey
2
Department of Biochemistry, Faculty of Science, Selçuk University, Konya, Turkey
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Zonguldak Bulent Ecevit University, Zonguldak, Turkey
6 ADMET and Physicochemical Assessments in Drug Design124
transport” is the second mechanism by which drugs are absorbed. Active transport is a key process
in drug efflux and the absorption of hydrophilic substances into cells. It is energy dependent (ATP
dependent) and can operate against a concentration gradient. For highly polar or charged drugs,
carrier proteins are essential for facilitating active transport. At its core, active transport is defined
by saturation, movement against an electrochemical gradient, and very high selectivity [8].
Permeability and solubility can be used to describe drug absorption, and the biopharmaceutics
classification system (BCS) has identified them as key parameters [9, 10]. Drug classification is
based on three dimensionless parameters: the absorption number, the dissolution number, and the
dose number. It also uses the basic drug properties of solubility and permeability. Highly soluble
drugs are those with a dose-to-solubility ratio of less than 250 mL in media with a pH range of
1.0–6.8 at 37°C. On the other hand, drug candidates that demonstrate an extent of absorption of at
least 85% in humans are classified as “highly permeable” [11]. Four distinct BCS classes have been
defined: BCS class 1 substances have high solubility and permeability, and gastric emptying or
drug dissolution limits drug absorption. BCS class 2 drugs have low solubility and high permeabil-
ity, limiting absorption. Permeability limits drug absorption in BCS class 3 compounds with high
solubility and low permeability. Low solubility and permeability compounds in BCS class 4 make
oral administration of drugs difficult. The BCS is useful for guiding the development of formula-
tions once a drug has been categorized. As an illustration, if the solubility of a component is trou-
blesome, it may be necessary to make further efforts in the development of formulations consisting
of, for instance, enabling formulations (Figure 6.1).
Based on research into the World Drug Index, Lipinski came up with the concept of inadequate
absorption and penetration. This concept is known as the “rule of five.” This criterion specifies that
if a molecule surpasses two or more of the following restrictions, the medication will have poor
oral absorption or permeability. Molecular weight must be more than 500, the computed octanol–
water partition coefficient (log P) must be more than 5, the number of hydrogen bond (HB) donors
(OH and NH groups) must be more than 5, and the number of HB acceptors (N and O atoms) must
be more than 10 [12].
Last but not least, the characteristics of the drug, elements related to the formulation, and
physiological conditions all have an impact on the very complicated process of drug absorption
following oral administration. Before a drug reaches other body organs, oral bioavailability or drug
exposure in the systemic circulation depends on its capacity to be absorbed across the membranes
of the gastrointestinal tract and to be metabolized via the intestines and the liver. Immensely
important, then, is the development of drugs capable of penetrating the intestinal epithelium
throughout the course of drug discovery. As a result, given that the topic of drug absorption will be
covered in more depth, it is essential to underline the fact that solubility and membrane permeability
are genuinely critical factors in the process of absorption.
CLASS I
Highly soluble Poorly soluble
Highly
permeable
Highly
permeable
Highly
soluble
Poorly
permeable
Poorly
soluble
Poorly
permeable
CLASS II CLASS III CLASS IV
Figure 6.1 Biopharmaceutical classification system (BCS) of drugs.
6.1 ADMET 125
6.1.1.1 Solubility and Dissolution
The term “solubility” describes a compound’s capacity to dissolve in a certain solvent, such as
water, a buffer with a particular pH, a fasted-state intestinal fluid simulation, or a fed-state
intestinal fluid simulation [13]. The assessment of a drug’s oral absorption has been shown to
depend significantly on solubility despite the fact that this parameter is not always regarded as an
ADMET feature. Since solubility determines bioavailability and drugs must be in solution to pass
through the GI tract’s membrane, it is clear that this property is an important determinant of
absorption rates [14]. Aqueous solubility, which controls medication absorption, transfer, and
excretion from the body, is an essential characteristic that is essentially engaged in all phases of
drug development [15–17]. The effectiveness of drugs is mostly determined by their ability to
dissolve in water; hence, drugs that have a low dissolution rate or a low solubility will be destroyed
before they enter the blood circulation, and as a result, they will not provide the necessary
pharmacological effects[18, 19]. It is possible to evaluate a drug’s solubility in water by determining
its capacity to partition from lipid environments to aqueous settings. This ability is dependent on
the ionization of the drug that is being experimented with. The majority of drugs consist of weakly
basic or acidic compounds that are incapable of complete ionization in aqueous solutions and thus
ionize only partially. The solvent’s pH is typically considered to determine the above partition
behavior, and pK
a
is a common parameter used to characterize a compound’s dissolution process.
This is because drug ionization is very sensitive to the pH of the solvent. As a rule, ionized
medicines have significantly higher water solubility than their nonionized equivalents [14].
Recently, there has been an increase in the number of poorly soluble drugs, and concerns such as
poor absorbability, food effects, and the absence of pharmacokinetic linearity have also
emerged [20]. Numerous basic and acidic drugs frequently exhibit solubility profiles that depend
on pH levels. Consequently, their solubilities in the stomach and small intestine are distinct. When
the pH of the stomach is between 1 and 2, for instance, moderately basic substances may have
extremely good solubility. However, when the pH of the intestine is reached, the solubility may be
significantly lower than it is in the stomach [21]. Furthermore, the consumption of food can
influence solubility through its effects on stomach acidity and the elevation of bile acid levels.
Given the wide range of physicochemical and physiological parameters that influence drug
solubility, several strategies can be employed during the early stages of drug design to enhance the
oral drug’s solubility. One of these processes entails ionization into various salts: basic drugs are
ionized into salts using hydrochloride, citrate, tartrate, or other acid complexes, whereas acidic
drugs are ionized into sodium, calcium, and potassium salts. Better solubility usually follows salt
production. Sodium salts are the most prevalent form of basic drug salts, with 43% of the top 200
best-selling medications being in this form, and HCl salts are the most common form of acidic
drug salts, with 11% of the top 200 best-selling drugs in this form [22].
The fact that 75% of candidates for drug development are categorized as BCS classes 2 or 4 and
are not sufficiently soluble [23] provides further evidence that drug solubility is a limiting factor in
drug absorption, as stated previously. In order to minimize the risk of progressing drug candidates
throughout the development stage, it is critical to evaluate their solubility and any potential prob-
lems as soon as feasible. Limited solubility may impair absorption and, thus, drug likelihood.
During the early stages of the discovery process, kinetic and thermodynamic solubility are two
methods that can be utilized to quantify solubility [14]. The kinetic solubility approach is the
preferred method since it uses a reasonably high-throughput technology and is measured when a
tiny volume of DMSO is added to the aqueous buffer [24]. Thermodynamic solubility is
determined by introducing an aqueous buffer directly to the solid crystalline material and allowing
sufficient time for equilibrium to be established between the dissolved and solid components.
6 ADMET and Physicochemical Assessments in Drug Design126
In developing new formulations, this is essential since it represents the compound’s actual
solubility [25]. Kinetic solubility measures can be used to check for insolubility that happens in
standard in vitro potency and ADME tests. On the other hand, measurements of kinetic solubility
are not very relevant to the problems that arise in vivo. It is possible to achieve thermodynamic
solubility by adding the aqueous buffer directly to the solid crystalline material and then waiting
for a considerable amount of time for the dissolved and solid materials to reach a state of
equilibrium. The measurement process is time consuming and requires more material, even
though thermodynamic solubility is more important.
Poorly soluble drugs may have their absorption limited not only by their solubility but also by
their dissolution rate [26, 27]. A molecule’s solubility can be evaluated by measuring its dissolution
rate. A chemical with a rapid rate of dissolution will enter the solution quickly, leading to an
absorption phase that happens swiftly. This increases the likelihood that the molecule will be
absorbed within the GI transit time, and its solubility will remain constant. The dissolving rate of
the substance depends on its physical and salt form as well as its particle size. Decreasing the size
of the particles enhances the amount of solid material that comes into contact with the solvent,
hence accelerating the rate at which it dissolves [3]. One other approach to modifying the solubility
characteristics is to employ the principle of physical form [14]. In drug discovery, amorphous
solids are used most often. These are solids whose molecules are not organized in a specific way,
unlike crystals, which have molecules that are very well ordered. In comparison to their amorphous
counterparts, the compound’s crystalline forms are less soluble. Various crystalline structures,
such as polymorphs, salts, and solvates, can display varying levels of solubility. Water solubility can
be enhanced via the cocrystallization of drugs with solvents like ethanol or water, a process known
as hydrates [22].
To provide a brief summary, low oral bioavailability and a poor oral absorption rate are often the
outcomes of a low dissolution rate and poor water solubility. There is a correlation between the
lipophilicity of the substance and its low solubility in water. Accordingly, knowing a drug’s lipophi-
licity is essential for figuring out its permeability and oral absorption.
6.1.1.2 Lipophilicity
It has been reported that lipophilicity has a significant influence on a variety of pharmacokinetic
properties, including the absorption, distribution, and permeability of a pharmaceutical, as well as
the routes of drug clearance [28–30]. As a result, lipophilicity plays an important role in the
evaluation of the therapeutic suitability of a pharmaceutical. Between the point of application and
the location of the target protein, an active agent must pass through a number of lipid membrane
barriers and aqueous compartments in order to reach its destination. A sufficient amount of
lipophilicity is required in order to accomplish the desired distribution. This makes perfect sense
given that lipid membranes divide the many watery phases that make up biological systems. It is
consequently necessary for lipophilicity to be a factor in the transportation and distribution of tiny
molecules in such systems. These requirements mostly stem from the fact that biological targets
are lipid-based. For instance, neurotransmitter route, anatomical, and intracellular targets require
lipophilic agonists [31, 32].
When it comes to absorption, lipophilic chemicals and molecules with a molecular weight of
more than 500–600 Da are often well absorbed; yet, they are subject to biliary clearance at a
relatively rapid rate. This typically occurs immediately following absorption from the intestines
during the initial hepatic passage [33]. Only compounds that have moderate lipophilicity have a
fair probability of “migrating” into the aqueous phase as well as the lipid phase in order to arrive
at enough concentrations in the tissue that can be targeted. The reason is that highly lipophilic
6.1 ADMET 127
compounds are both more rapidly digested and potentially harmful from a toxicological standpoint.
On the other side, drugs that have a very low lipophilicity are likewise undesirable since they will
not have the impact that is required. When ions or membrane-bound receptor-interacting
substances are enriched in the surrounding membrane, they can frequently reach their targets
more readily. In order to achieve this, the compounds must possess lipophilic properties or possess
a substantial lipophilic group that can be used to secure them within the membrane. At the same
time, appropriate drug formulations need to demonstrate a high degree of water solubility and an
acceptable degree of lipophilicity to evaluate the most effective oral absorption in addition to the
necessary deposition and activity. Lipophilicity and solubility have an inverse relationship,
meaning that increased lipophilicity results in decreased water solubility. This is characterized by
the partition coefficient that exists between the aqueous phase and the lipid phase. A measurement
is taken of the distribution of octanol and water in the model that is the most basic.
The partition coefficient (P):
P C C
no w octanol water/
/
The partition coefficients are calculated by taking the logarithm (log P) of the ratio of equilib-
rium concentrations of the aqueous water to the organic solvent [34]. Log P is included in Lipinski’s
“rule of 5” because, at high log P values (>5), solubility is frequently poor, impeding absorption,
and/or molecules partition into membranes, rendering them impervious to enterocytes [21]. This
is the reason why log P is included in the rule. To provide a more comprehensive understanding of
the connection between the log P value (lipophilicity) and the solubility/permeability [14]:
A drug’s log P value:
i) Between 0 and 3 is considered optimal for passive diffusion.
ii) <1 indicates that a compound’s solubility will be high due to its hydrophilic nature, but its
permeability will be low.
iii) A value greater than 3 suggests that a molecule has a high affinity for lipids, may have limited
solubility, and is likely to undergo metabolism and/or be excreted through the bile.
The distribution coefficient, log D, represents the proportion of the compound’s total concentra-
tions (including both ionized and unionized forms) in two solvents (namely, the aqueous phase at
a given pH and n-octanol). It is important to note that the log P will change depending on the con-
ditions under which it is measured as well as the partitioning solvent that is selected. On the other
hand, log D represents the log distribution coefficient at a specific pH degree. The equilibrium
between ionized and nonionized species in the two solvents is considered in the analysis [35].
Distribution coefficient
Unionized
Unionized
o
aq
, D Ionized Distribution coefficient
aq
,
og logL D
10
When taking into consideration the connection between lipophilicity and ADME, log D is
utilized more frequently than log P for ionizable compounds [13]. It is common practice to use
log D at a pH of 7.4 as a means of providing an indicator of the lipophilicity of a drug at the pH of
blood plasma.
Calculating log P is a straightforward and rather reliable process. However, determining log D pH
is more complex as it necessitates knowledge of the pK
a
. Drug solubility, lipophilicity, permeabil-
ity, and absorption are among the physicochemical and ADME characteristics that are
6 ADMET and Physicochemical Assessments in Drug Design128
significantly impacted by the ionization state of compounds, as indicated by the pK
a
value [36, 37].
The ionization constant (pK
a
) is traditionally represented by the Henderson–Hasselbalch equa-
tion, which quantifies the proportion of ionized and unionized chemicals at a specific pH.
pH K
Ionized compound
for we
a
p log
Unionized compound
aaak acid
pH K
Ionized compound
for we
a
p log
Unionized compound
aaak base
6.1.1.3 Permeability
Permeability is a significant physicochemical characteristic of compounds that has a direct impact
on the pace at which drugs are absorbed. One of the primary factors that determine the ADME/PK
of a drug and its metabolites, as well as their exposures to various tissues, is its permeability.
Permeability is the factor that influences the molecule’s capacity to pass through the biological
membrane. The drug’s interdependent physicochemical qualities, including its molecular size,
polarity, and lipophilicity, are a significant factor in determining its effectiveness of permeability.
As previously stated, lipophilicity is commonly assessed using the partition coefficient. In general,
a molecule with a high partition coefficient has a greater ability to move between an aqueous fluid
and a biological membrane, which results in higher permeability. Conversely, a high partition coef-
ficient value leads to the drug being trapped in the membrane, preventing it from further distribut-
ing across the membrane and blood. Concerning molecular size, molecules with high molecular
weight exhibit low permeability. The permeability of a molecule depends on the equilibrium of the
intermolecular forces between the drug and both water and the membrane. There are a number of
research that have discussed the hydrogen bonding parameter as a means of describing drug per-
meability. The majority of the findings have demonstrated that HB acceptor descriptors are con-
nected with less value when it comes to predicting the permeability of the human intestinal
epithelium [38, 39]. Furthermore, there exists an inverse link between the polarity of a molecule
and its permeability. Polar molecules that have several HB acceptors and donors interact better
with water [40].
Multiple investigations have demonstrated that drug absorption varies across different regions.
The pH gradient in the digestive system, ranging from acidic (pH 2–3) to basic (pH 8–9), has been
identified as a significant determining factor. Most of the process of absorbing nutrients from the
intestines takes place in the small intestine, specifically in the duodenum, jejunum, and ileum. The
pH in this part of the intestine typically ranges from 4 to 7[41, 42]. In order to provide an explanation
for this occurrence, the pH-partition hypothesis [43] proposes that the lipophilic membrane can
only allow uncharged molecules to pass through it. The pH-partition hypothesis states that only
free drugs, which are unionized and unbound, are able to passively pass across the cell membrane.
It is widely acknowledged that the unbound unionized molecule exhibits a greater permeability,
typically between three and four log units, in comparison to its ionized counterpart [44]. Therefore,
in order to pass through the majority of membranes, the drug must possess a relatively low polarity.
At a pH level found in living organisms, ionized molecules have a tendency to engage with lipid
membranes that carry a negative charge. This interaction often leads to a low level of permeability,
with the molecules arranged in the following order: neutrals, bases, zwitterions, and finally,
acids [45].
6.1 ADMET 129
Passive permeability impacts all aspects of drug candidate ADME properties, including as
gastrointestinal absorption, first-pass extraction, tissue distribution, cell penetration, clearance
mediated by enzymes and transporters, renal reabsorption, enterohepatic circulation, and drug–
drug interactions. The advantage of passive permeability is that it is not reliant on the concentration
of the substrate; as a result, it is nonsaturable, which results in linear absorption. Compounds with
high passive permeability can easily pass through cell membranes. This property has several
advantages, including effective absorption when taken orally, the ability to be used in modified-
release dosage forms, increased exposure to the brain, reduced clearance by the kidneys, and a
lower risk of drug–drug interactions mediated by transporters [39]. For most tissues, cell types, and
animal species, scaling factors allow for the translation of passive permeability. Scaling factors are
required to account for variations in lipid compositions [46]. Some examples of in vitro cell-based
or artificial membrane assays that can be used for permeability measurements are Caco-2,
LLC-PK1, MDCK, and PAMPA. Caco-2 is a cell line derived from human epithelial colon
adenocarcinoma that exhibits characteristics of both enterocytes and colonocytes [47]. Furthermore,
the findings produced in these cells show a strong association with the permeability and
bioavailability of the human jejunum in vivo [48, 49]. MDCK cells, derived from the distal renal
tissue of canines, emerged thereafter as a more expeditious and economically advantageous
substitute for Caco-2 cells [50]. PAMPA, or parallel artificial membrane permeability assay, is a
widely used and cost-effective screening tool that does not require the use of cells. It is commonly
used alongside cell-based assays [51]. Although PAMPA is unable to simulate active transport, this
limitation is counterbalanced by the fact that over 90% of medicines are absorbed through passive
diffusion [52, 53]. The permeability of PAMPA at both pH 7.4 and 5.5 shows a strong correlation
with the permeability of Caco-2 in small datasets [54]. Because of its adaptability to high-
throughput as well as its flexibility with experimental circumstances (various lipid compositions/
range of pH conditions), PAMPA is an effective screening approach that can be utilized in the early
stages of drug discovery [55]. In summary, membrane permeability is essential for the absorption
of medications via the biomembrane of the gastrointestinal system and is also vital for their
distribution to all other bodily regions. Hence, it is imperative to establish principles that facilitate
the attainment of favorable passive permeability, as well as other essential qualities, in order to
effectively facilitate drug design.
6.1.2 Distribution
Another important process that ends with the target organ being exposed to the drug is drug
distribution, which occurs after the drug is absorbed from the gastrointestinal luminal fluid into
the systemic circulation. This involves the drug moving from the blood into different compartments
within tissues and organs, such as the interstitial space and the intracellular space [56]. Due to the
fact that it has the potential to influence the quantity of drug that is ultimately delivered to the
active sites, the process of drug distribution is of great significance [57]. Fat, muscle, and brain
tissue are among the many areas that a drug can reach after it has been absorbed. The distribution
of drugs is typically very quick, and the majority of small lipophilic compounds are able to quickly
move across lipid membranes through a process that is passive. Passive diffusion does not allow
large or highly polar molecules to flow across lipid membranes. Instead, these compounds need
particular transporters to enter the tissue [58]. A drug’s concentration in tissue may be significantly
greater than its plasma concentration if it enters a tissue through an active transporter mechanism.
The drug molecules can be transported to several organs for action, including the skin, tumors,
brain, and other tissues that contain receptors. Additionally, some drugs can be disseminated to
6 ADMET and Physicochemical Assessments in Drug Design130
the liver and kidneys, which are responsible for disposal. A number of factors, including the drug’s
interaction with blood components like plasma proteins and the rate of blood flow, as well as its
molecular size and lipophilicity, impact the distribution process, which mostly takes place in the
systemic circulation, or blood. Several in vitro assays, including as lipophilicity, solubility, plasma
stability, and plasma protein binding, have been established by researchers in order to evaluate the
distribution process [59]. The enormous volumes of distribution that occur as a consequence of
lipophilic positively charged compounds partitioning into biological membranes are a consequence
of their positive interactions with the phospholipid bilayer. Neutral compounds do not exhibit any
electrostatic interaction with the surface of membranes. As a result, the rise in lipophilicity will be
the primary factor that determines their capacity to partition into membranes. Even though
negatively ionized substances bind strongly to serum albumin – the most abundant plasma
protein – they have a poor affinity for membranes and, as a result, a low volume of distribution[60].
When it comes to neutral and basic compounds, lipophilicity is once again the most important
factor in albumin binding [61]. There is a correlation between a drug’s distribution and its binding
strength to tissue proteins, with higher log D values indicating stronger binding [62]. Excessive
lipophilicity not only enhances the dispersion volume but also the oral absorption rate. Lipophilic
compounds, on the other hand, are more susceptible to metabolism, which results in a higher
clearance [63]. This, in turn, reduces the drug’s half-life and decreases its bioavailability.
Another crucial metric for determining the distribution process is the assessment of aqueous
solubility at physiological pH. Insoluble compounds can have a negative impact on the ADME
analysis, as a portion of the molecule will form a precipitate, potentially rendering the action sites
unavailable [59].
Plasma protein binding is a critical component that significantly impacts drug distribution and,
by extension, the pharmacokinetic and pharmacodynamic behavior of drugs in general. There are
7% proteins in plasma, with human serum albumin being the most significant protein for drug
binding, followed by α1-acid glycoprotein and lipoproteins [64]. The amount of drugs that are freely
available in the plasma and can be distributed to the active sites is determined by analyzing the
plasma protein binding data. As a result of the majority of the drug fraction binding to the plasma
proteins during the process of drug absorption into the bloodstream, the amount of drug that is
accessible to reach the target is decreased. This, in turn, determines the efficacy of the drug, as well
as its metabolism and other pharmacokinetic parameters. Conversely, drugs that have a lower
affinity for plasma proteins have a greater ability to be distributed to organs and tissues. On the
other hand, drugs that strongly bind to plasma proteins are limited to the blood vessels and have a
relatively small volume of distribution [59]. The temporary localization, reduced or eliminated
pharmacological effects or hazardous actions, and difficulty of the attached molecule to penetrate
biological membranes or diffuse into tissues make plasma protein binding of foreign compounds,
and drugs in particular, an essential process. Among the plasma proteins that have the ability to
attach to foreign compounds, albumin exhibits a broad capacity to bind several molecules.
Nevertheless, it is important to note that other plasma proteins, such as globulins, also play a role in
this process. Numerous factors, including variations in species, shifts in pH, and hormonal impacts,
have the potential to modify the binding of foreign molecules. The interaction between drugs or
chemicals and plasma proteins is occasionally crucial, as a chemical that forms a strong bond with
a plasma protein will exhibit an extended duration of effect. Suramin, a potent therapeutic drug for
trypanosomiasis, exhibits a significant affinity for plasma proteins. The drug provides protection for
a period of at least three months with just a single dose [65]. Permeability barriers allow concentration
gradients to be formed and sustained, which in turn supports biological specialization and
complexity. Because of this, diffusion across membranes via extracellular gaps is more likely to
6.1 ADMET 131
occur in most tissues than in those with continuous membrane barriers due to the structure of cells.
It is at the level of the capillary endothelium that the effective tight junction takes place in
permeability barriers, such as those found in the brain, the placenta, and among the thymus. Blood–
organ barriers are the name given to these barriers. In comparison to other organs, the brain has
unique characteristics that affect a drug’s penetration. A crucial characteristic of the brain is its total
isolation from the bloodstream by the BBB [66]. It is necessary for the drug to get through the BBB
in order for it to enter the brain through simple diffusion. Hence, the capacity of drugs to permeate
the BBB is crucial in the process of drug development. For drugs that are active in the central
nervous system (CNS), a high level of penetration is required, whereas a low level of penetration
may be desirable in order to reduce the adverse effects on the CNS that are associated with
pharmaceuticals that have a peripheral site of action [67]. Since passive diffusion allows some drugs
to pass through the BBB, physicochemical features, including charge, lipophilicity, and molecular
weight, are crucial for substances discovered in the CNS [68]. The distribution of a drug can be
determined either by analyzing its systemic exposure or by directly measuring its presence in
tissues. Typically, the majority of data is acquired by the former approach. It is possible to determine
the extent of drug distribution throughout the body by using a theoretical volume known as the
apparent volume of distribution at the steady state (V
dss
) of a molecule. Despite the fact that V
dss
does not have any physiological significance, it is a major pharmacokinetic parameter that is directly
related to the elimination half-life (T
1/2
) and protein binding. Furthermore, it is not dependent on
the total clearance (CL) of a drug. A liter per kilogram of bodyweight (L/kg) is a common unit of
measurement for V
dss
. V
dss
> 1 L/kg (>100% of body weight) suggests that the drug is concentrated
in specific tissue compartments. As a result, V
dss
is a valuable parameter that may be used when
determining the relative amount of drug that is present in the tissues or outside of the central
compartment [69].
T =
V
T
V
1 2
dss
1 2
dss
0 693
CL
/
/
.
Half life
Volume of distributionsss
ClearanceCL
6.1.3 Metabolism
Drugs are compounds that are not native to the body and are converted by a wide range of enzymes
into other products that are referred to as metabolites. Metabolites typically exhibit more polarity
and higher solubility in water, and are more easily eliminated through urine. Metabolism, also
known as biotransformation, refers to the process by which a drug undergoes a chemical transfor-
mation in order to become a metabolite. After being absorbed in the intestines, the drugs next
travel through the hepatic portal system to the liver, which is where the majority of the drug’s
metabolism takes place. Mostly, the liver is responsible for the metabolism of a wide variety of
xenobiotics, which includes the metabolism of drugs and hazardous chemicals [70]. There is a cor-
relation between the physicochemical properties of medicines and their metabolic clearance, for
example, a high log D 7.4 for lipophilicity and a high metabolic clearance [71]. Many metabolizing
enzymes make xenobiotics more soluble in water, which helps the body get rid of them.
A significant family of enzymes that are responsible for the metabolism of drugs is known as the
microsomal cytochrome P450 (CYP450) family [72]. The enzyme CYP3A4, which is the most
prevalent P450 enzyme in the liver, is accountable for the metabolism of around 40–50% of the drugs
that are utilized in clinical practice. Because they reveal useful information regarding the possibility
6 ADMET and Physicochemical Assessments in Drug Design132
of drug exposure, the fate of metabolites in preclinical species to humans, and clinical interactions
caused by inhibition and induction, CYP inhibition assays have become an important factor in the
exploration and development of new medications [73]. The following reactions can be catalyzed by
CYPs: oxidation of carbon and oxygen, carboxylation of nitrogen and oxygen, aromatic hydroxylation,
oxidation of nitrogen and sulfur, deamination, and dehalogenation. Humans have around 100
isoforms of CYPs. The primary site of action is predominantly in the liver. The human body contains
12 different CYPs, all of which are recognized to play a significant role in the metabolism of
xenobiotics. CYP2C, CYP2D, and CYP3A subfamilies are the most active with regard to the
metabolism of drugs. CYP3A4 is the enzyme that is most frequently expressed and is involved in the
metabolism of nearly 50% of all drugs for treatment [74]. Following CYP3A4 in terms of the number
of medicines that are metabolized by P450 enzymes, CYP2D6 is responsible for the metabolism of
the second biggest number of drugs. The brain and liver are two places where this enzyme is
expressed. CYP2D6 has a tendency to oxidize substrates that have a protonated basic nitrogen at a pH
that is typical for the body and a flat aromatic ring [75]. Monoamine oxidases (MAOs) are enzymes
that contain flavin and are found in the mitochondria. They play a crucial role in catalyzing the
process of deamination for both biogenic and xenobiotic amines. Most tissues have two isoforms of
MAO, called MAO-A and MAO-B [76, 77]. Flavin-containing monooxygenases, also known as
FMOs, are found in a wide variety of tissues, including those found in the liver, kidney, lungs, skin,
brain, and other tissues. However, the liver, kidney, and lungs in particular have the highest
concentrations of these enzymes [78]. There are now five distinct isoforms of FMOs that have been
identified and described. FMO1 and FMO3 have a significant involvement in the adult human liver.
The primary substrates for FMO1 are imipramine and orphenadrine. In addition to inhibiting FMO-
dependent pathways, methimazole also inhibits CYP2B6, CYP2C9, and CYP3A4 [79]. Many
nucleophilic molecules with nitrogen, sulfur, phosphorous, or selenium atoms are oxidized by FMOs
using NADPH [80]. While nonbasic molecules are more likely to be oxidized by CYPs, the degree of
N-substitution also plays a significant effect. Basic amines are often FMO substrates, but there is
some overlap with CYPs (e.g., CYP2D6). In contrast to primary amines, which are typically better
CYP substrates, tertiary amines are typically FMO substrates. Secondary amines, on the other hand,
are less clear-cut [45]. For N-oxidation, if a molecule has many nitrogen atoms, the more basic
nitrogen center is typically the chosen location.
The primary purpose of metabolic transformation is to enhance the drug’s hydrophilicity.
Metabolism is commonly categorized into two distinct stages: Initially, in a phase known as Phase I,
the molecule undergoes functionalization, resulting in the incorporation or release of polar groups
such as alcohols, phenols, and amines by processes such as dealkylation, hydroxylation, heteroatom
oxidation, and oxidative dehalogenation. In addition, both esters and amides undergo hydrolysis.
Phase I enzymes modify drugs’ functional groups. Because of this, phase I metabolism is frequently
referred to as the functionalization phase of the drug metabolism process. If the parent molecule
has sufficient solubility and/or transporter selectivity, both unmodified parent compound and
Phase I metabolites are eliminated through bile and urine. Phase II reactions mostly encompass
conjugation reactions, including glucuronide, sulfate, and glutathione conjugations. Phase II
reactions typically yield very hydrophilic molecules. For instance, the presence of the strongly
polar glucuronide moiety typically results in the swift elimination of a drug from the body.
Nevertheless, not all phase II reactions produce a metabolite that is more polar. The
biotransformation reactions are facilitated by enzymes that are found in several organs and tissues,
with the liver being the most crucial one overall [81–84].
To provide a brief summary, it has been recently estimated that drug metabolism is one of the
primary characteristics that has been demonstrated to be taken into serious consideration during the