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=
concentration.dissolved compound/
nonionized in water
Chapter • From In Vitro to In Vivo: Optimization of ADME and Toxicology Properties
19
The interaction between acompound and the binding
site of atherapeutically relevant biological macromolecule is the critical prerequisite for its suitability as adrug.
Another, no less important, requirement is the ability of
the substance to make its way from the site of application
through an often rather tortuous path to the target tissue
and nally into the binding pocket of the macromolecular target. To do this, the compound must penetrate
aqueous phases and lipid membranes. Depending on its
water and lipid solubility, it will end up in different compartments of the biological system. It is also modied by
metabolizing enzymes. After conjugation or degradation,
it is nally excreted through the kidney, bile, and/or intestine (Sects.9.1 and27.6).
In contrast to the biological activity of adrug, which
is called pharmacodynamics, the sum of all processes
affecting absorption, distribution, metabolism, and ex-
cretion, the so-called ADME parameters, is called phar-
macokinetics. Roughly speaking, pharmacodynamics
can be thought of as “the effect of the substance on the
organism” and pharmacokinetics as “the effect of the organism on the substance.” In recent years, this clear separation of denitions has begun to fade. The term pharmacodynamics has also been expanded more and more
to processes of pharmacokinetics. This is mainly due to
the increasing knowledge that transporters or enzyme
systems are responsible for properties such as absorption, distribution, or metabolism. Agrowing number of
structures of the involved enzymes and transporters has
been determined, allowing specic structure–activity relationships to be established for such systems (Sects.27.6
and30.10).
The pharmacokinetics of a given biological system
and the time dependence of the absorption, distribution,
and excretion processes are described by mathematical
models. The pharmacokinetics of every pharmaceutical
is scrupulously investigated and adosing scheme is determined before entry into clinical trials, especially during
clinical phasesI andII, which evaluate tolerability and
efcacy in humans. The isolation and structural elucidation of metabolites formed in humans helps to identify
the animal model that most closely resembles humans
in terms of metabolic properties. These species are then
used for toxicology studies, which are chosen to investigate possible teratogenic effects, and long-term studies
to investigate possible carcinogenic effects. In parallel,
individual metabolites of a pharmaceutical are investigated for their toxic side effects.
In the context of the rational design of new active
substances, asubstantial problem arises from the pharmacokinetic parameters and the toxicity: these investigations are only carried out for very few compounds because of the enormous experimental effort and the high
costs, and only for those compounds that are intended
for clinical development. This approach comes with
aserious danger: inadequate pharmacokinetic proper-
ties are only recognized in very late development stages,
and only then after considerable sums have already been
invested in the development of anew pharmaceutical.
In the mid-1990s, astudy showed that many unsuccessful development campaigns failed due to unsatisfactory
pharmacokinetics and intolerable toxicity. For these reasons, the search for in vitro models to predict ADME tox-
icity has intensied over the last 30years. Rather than
studying the pharmacokinetics of asingle compounds in
detail, the dependence of various pharmacokinetic parameters on the properties of many different compounds
is nowadays investigated. This provides a better understanding of the relationship between chemical structure
and pharmacokinetics. At the same time, it leads to the
establishment of general rules and numerous computer
models that are now used at an early stage in the design
of new drugs.
19.1 Rate Constants of Compound
Transport
The distribution of asubstance into phases of different
lipophilicities is measured as the partition coefcientP
(Sect.18.3). This denition is valid for systems at equilibrium. The distribution between the water and octanol
phases is considered as amodel system. The ratio of the
concentration of the nonionized form of an investigated
compound in the two phases is evaluated. In addition,
the pH value is adjusted during the measurement so that
the investigated compound overwhelmingly occurs in its
nonionized form. As ageneral rule, logP, the logarithm
of this value is used.
.octanol/water/
concentration.dissolved compound/
g
Biological systems are open systems that are kinetically
controlled. They can be temporarily found in adynamic
equilibrium. This condition can be compared to achromatographic process in which asubstance is in constant
exchange between the solid support and the mobile
phase. Locally, equilibria occur that are disrupted by the
continuous progression of the mobile phase. In contrast
to the relatively simple conditions in chromatography,
there are a plethora of different phases in biological
systems. Adrug is distributed throughout all of these
phases. Furthermore, metabolic processes are running in
parallel that lead to different metabolites.
To analyze these dynamic equilibria, the kinetic equi-
librium constants of the substance transport from the
aqueous phases into the lipid phases and in the reverse
direction must be known. It is astonishing that such
fundamental experimental investigations on organic
octanol
1k2
2
=−ˇk1+ c
1
=log P −log.ˇP + 1/ +constant
2
=−log.ˇP + 1/ +constant
. • Rate Constants of Compound Transport
substances were rst carried out by Bernard Lippold
in the mid-1970s, and later also by Han van de Waterbeemd. Lippold used athree-phase system: water/n-octanol/water (. Fig.19.1). After adding the substance to
one of the two aqueous phases, the time dependence of
the substance concentration in the different phases was
measured. From this, the equilibrium constant k1 for the
transport from water to the octanol phase and the rate
constant k2 in the opposite direction can be calculated.
In addition to the partition coefcientP, which is
described in Eq.19.1, avery simple correlation has been
shown for the dependence of k1 and k2 (Eq.19.2); β andc
are constants that depend on the system and not on the
structures of the substances.
(19.1)
(19.2)
The dependence of the rate constants k1 and k2 on the
partition coefcientP results from the combination of
both equations (Eqs.19.3 and19.4).
(19.3)
(19.4)
The experimental kvalues for 20different sulfonamides
and 15 further substances that were experimentally
determined by Han van de Waterbeemd are shown in
. Fig.19.2. Among the latter are neutral, acidic, ba-
sic, and even quaternary charged compounds with very
different molecular weights. The characteristic curve
shows that the rate constant k1 for the transfer from the
. Fig. 19.1 Three-compartment system for the determination of the
rate constants k1 and k2. At the beginning of the experiment the substance is dissolved in aqueous phaseA. Next the substance concentration is measured in phasesA, B, andC after different times until an
equilibrium is established between the individual phases
. Fig. 19.2 Experimentally determined rate constants k1 and k2 for
the transport of 20sulfonamides and 15further chemically different
substances with molecular weights between 100 and 500 Da. The
curves and correlation coefcientsr correspond to the tting of the
data with Eqs.19.3 and19.4
aqueous to the organic phase depends on the partition
coefcientP for relatively polar substances. It is thermodynamically controlled, which means that it increases
with increasing lipophilicity. However, there is apoint
at which the diffusion of the substance is limited by k1
at the maximally achievable value. More lipophilic substances cannot simply penetrate the organic phase faster.
The same applies in the opposite direction, where the
diffusion from the organic phase into the aqueous phase
is described by k2. In both cases, the chemical structure
plays arole in determining the value of the partition coefcientP. Since the rate constants are limited by diffusion,
there must be an apparent dependence on the molecular
size in this area. According to Fick’s law of diffusion,
the diffusion should be proportional to the radius of the
particle, parallel to the cube root of the volume, as arst
approximation. Due to the relatively low variability of
the molecular size of organic drugs and their conformational exibility, this effect is likely to be lost in the
noise of experimental error. In addition, it should not
be forgotten that the octanol/water system discussed is
very simple and only slightly approximates the complex
structural relationships of real membrane systems. For
this reason, more relevant models, such as the so-called
PAM PA or Caco-2models, are increasingly being used to
obtain experimental distribution data (Sect.19.6). Here,
more complex relationships are indicated. Obviously,
how acompound is distributed and structurally oriented
in the vicinity of membrane structures is important. At
the same time, these properties inuence how the penetration, and therefore the distribution, is to be described.
19
1
+log k2+constant
Chapter • From In Vitro to In Vivo: Optimization of ADME and Toxicology Properties
. Fig. 19.3 The rate constantk for the transport of drugs depends
nonlinearly on lipophilicity. This is valid for simple in vitro models as
well as for biological systems. The bottom curve describes the log k
values of the transport of barbiturates in an in vitro absorption model
from an aqueous phase, through an organic membrane into another
aqueous phase. Both curves in the center (gray dots) describe the de-
pendence of the absorption rate constantsk on the lipophilicity for
the absorption of homologous carbamates from the stomach (gastric
absorption) or the gut (intestinal absorption) of rats. The top curve
was determined for the entry of different drugs into the placenta from
the circulation. In all cases an increase in logk dependent upon logP
is seen, until amore-or-less-pronounced maximum for substances with
moderate lipophilicity. For very nonpolar substances, this curve falls,
and in rare cases aplateau is reached. The curves for gastric and intestinal absorption and for the penetration into the placenta run atter
than the curve for the invitro transport of barbiturates (below), because here no lipid barrier is present
19.2 Absorption of Organic Molecules:
Model and Experimental Data
The rate constant, k, for the penetration through the lipid
membrane from the aqueous phase is described by another equation, Eq.19.5. Here, the rate constants k1 and
k2 also describe the entry into the organic phase and the
transport in the opposite direction, respectively.
(19.5)
In the rst approximation, this equation should also
describe transportation processes in multicompartment
systems. Model calculations on arbitrary, complex systems show that this is indeed the case. They conrm that
there is bilinear dependence of the transport in different phases on the total lipophilicity of asubstance. For
multiple groups of drugs, for example, barbiturates, this
was demonstrated experimentally in simple in vitro model
systems (. Fig.19.3, bottom). The logk values increase
linearly upon penetration through an organic membrane,
which correlates with the increase of k1 with constant k2.
After passing through amaximum, they decrease with
aconstant k1 value and decreasing k2 value. This dependence was quantitatively summarized by Hugo Kubinyi
in the so-called bilinear model (Eq.19.6); a, b, β, andc
are constants, by which the nonlinear regression analysis
is ascertained.
(19.6)
Entirely analogous dependencies are observed with the
absorption of compounds, that is, out of the stomach or
intestines (. Fig.19.3, center). Active substances that
should be orally available should not be either very polar or very nonpolar. Substances with intermediate lipophilicity can cross the blood–placenta barrier more
easily than very polar or very nonpolar compounds
(. Fig.19.3, top). Anonlinear dependence on the lipophilicity for substance penetration through the blood–
brain barrier is particularly pronounced (. Fig.19.4).
The optimum for this barrier is in the range of log
P = 1.5–2.5. For central nervous system (CNS)-active
substances, an optimal lipophilicity around log P = 2
should be aimed for in order to facilitate penetration
across the blood–brain barrier.
19.3 The Role of Hydrogen Bonds
The simple concept about the dependence of absorption on the octanol/water partition coefcients outlined
above has been questioned in recent years. While octanol
is arelevant model for lipid membranes in many respects
(Sect.4.2), it can only incompletely model the inuence
of hydrogen bonding. After equilibration in the octanol/
amounts of water, corresponding to a molar ratio of
octanol:water = 4:1. Substances with polar, solvated
groups, therefore, do not have to completely release their
water solvation shell upon entering the octanol phase.
Entry into abiological membrane is obviously different.
Apart from the dependence on lipophilicity, even poorer
membrane penetration is observed for substances that
can form an increasing number of hydrogen bonds. Similarly, aligand must release its water shell before it can be
accommodated in the binding site of aprotein.
The water/cyclohexane system is more suitable for describing such processes. Due to the nonpolar nature of
this hydrocarbon, the drug molecule cannot take its water shell with it when transitioning from water to cyclohexane. Many years ago, P.Seiler derived an increment IH
(Eq.19.7) from the differences in partition coefcients in
cyclohexane/water (loss of water shell) and octanol/water
(no loss of water shell) for different functional groups.
These IH values characterize the tendency of the groups
to form hydrogen bonds.
+
X
log
+ 0:16
2
O $ A−+H3O
+
2
O
. • Distribution Equilibria of Acids and Bases
. Fig. 19.4 The neurotoxicity (C=molar dose that induces aspecic
toxic effect) of homologous primary alcohols in the rat is ameasure of
their ability to cross the blood–brain barrier. Polar substances remain
predominantly in the circulation. In contrast, substances with moderate lipophilicity easily reach the central nervous system. Accordingly,
neither methanol (MeOH) nor ethanol (EtOH) exhibit pronounced
neurotoxicity. The high general toxicity of methanol (blindness) is not
due to its own action, but rather to the highly toxic metabolites formaldehyde and formic acid (acidosis). Short-chain alcohols such as amyl
alcohol (AmOH) are much more neurotoxic. The highly lipophilic decanol (DecOH) shows low toxicity
P
cyclohexane
IH=1:00
P
octanol
(19.7)
Seiler’s concept remained largely ignored. In 1988, Robin
Ganellin and coworkers described the CNS bioavailability of various substances, that is, their ability to cross
the blood–brain barrier, as a linear function of a∆log
Pvalue. This ∆log Pvalue is the difference between the
logP values in the cyclohexane/water and octanol/water
systems. The bioavailability of peptides also runs in arst
approximation parallel to the ∆log Pvalue or the number
of groups potentially involved in hydrogen bonding. In
fact, methylation of all NH groups of apeptide scaffold
can provide compounds with good bioavailability. The
requirements for good membrane penetration are similar
to those for high afnity at the binding site (Chap. 4).
Here again, the need to release relatively tightly bound
water molecules can have adetrimental effect on binding
afnity. It should not be forgotten that more complex
molecules can easily undergo conformational transitions
to ageometry that buries part of the hydrogen bonds
and, thus, their polarity intramolecularly (cf. cyclic peptidomimetics such as cyclosporine, Sect.10.1, . Fig.10.2).
Several other partitioning systems, such as heptane/
ethylene glycol, have been proposed as alternatives to
the octanol/water or cyclohexane/water systems for simulating penetration through alipid membrane. However,
even these systems cannot correctly reect the architecture of membranes with an inner lipophilic zone and
apolar, negatively charged outer rim. Another option is
the determination of the membrane/water partition coef-
cient, which is rather laborious experimentally. For this,
articial membranes or liposomes are used as models.
19.4 Distribution Equilibria of Acids
and Bases
Many drugs are acids (HA) or bases(B). They exist in
two forms through dissociation (Eq.19.8) or protonation
(Eq.19.9); one is usually anonpolar neutral form and
the other is apolar ionic form. The values of the partition coefcients of the ionic species are generally three
to ve orders of magnitude smaller than those of the
corresponding neutral molecule.
(19.8)
+H3O+$ BH++H
The partition equilibrium of an acid and its anion in
atwo-phase system depends on the pKa and pH of the
aqueous phase and the partition coefcients Pu and Pi
of the substance (. Fig.19.5). All components in each
phase must be in equilibrium with one another for the
total system to be in equilibrium. The dependence of the
partition coefcientP on pH, the pH partition prole, is
usually sigmoidal (i.e., S-shaped). Plateaus are observed
for the uncharged neutral form and for pH values where
so little of the neutral form is present that only the transfer of the charged species into the organic phase determines the measured partition coefcient (. Fig.19.6).
The charged species enters the organic phase as an ion
pair together with acounterion. The counterion is either the corresponding ion of the salt or the excess of
ions in the aqueous buffer. The partition coefcient of
the ion pair depends on the lipophilicity of the counter-
ion. The tetrabutylammonium salt of salicylic acid has
only aslightly lower partition coefcient than the neutral
form of salicylic acid. In contrast, the sodium salt of
salicylic acid has absolutely no tendency to partition into
the organic phase. Amino acids and other mixed acidic
and basic compounds yield pH partition proles with
amaximum between the pKa values of the two ionizable
groups (. Fig.19.6), this means when the zwitterionic
form is present.
By knowing the log Pvalue of the neutral form and
the pKa value, the partition coefcient of asubstance at
neutral pH can be calculated. These concepts allow the
estimation of the absorption and distribution properties
of new compounds. Of course, these considerations are
only valid for drugs for which there is no transporter to
facilitate membrane penetration (Sects.22.7 and30.10).
Because of their importance, pKa values are now routinely measured by potentiometric titration in pharmaceutical research. However, it is often overlooked that the
(19.9)
19
Chapter • From In Vitro to In Vivo: Optimization of ADME and Toxicology Properties
denition of the pKa of acids and bases is only valid for
aqueous solutions. The addition of an organic solvent,
which changes the dielectric constant, shifts this value
(Sect.4.4). This is even more true for the binding site of
aprotein or the interior of amembrane. In some cases,
experimental values have been determined by NMR
spectroscopy and isothermal titration calorimetry.
19.5
Absorption Profiles of Acids and Bases
For example, the absorption of an active substance from
the intestine into the blood should depend on the pH of
the surrounding medium and the pKa of the substance,
just like the distribution between an aqueous buffer system
and an organic phase. Absorption should, therefore, follow very similar proles as the distribution. In the 1950s,
Brodie, Hogben, and Schanker formulated the pH parti-
tion theory to describe this effect. It states that the dependence of the absorption prole on the pH value, the pH–
absorption prole, is identical to the pH–partition prole
(Sect.19.4). This theory was conrmed by, among other
things, the investigation of the rate constant of absorption
of afew acids and phenols from the colon of the rat at
pH6.8. The neutral forms of the strong acids 5-nitrosalicylic acid (pKa = 2.3), salicylic acid (pKa = 3.0), m-nitrobenzoic acid (pKa = 3.4), and benzoic acid (pKa = 4.2)
display comparable lipophilicity with logP values between1.8 and 2.3. Under experimental conditions near
neutral pH, they are largely dissociated. Less than 0.1%
of the compounds in the neutral form. Therefore, they
are distinctly more slowly absorbed than the comparably
lipophilic, weakly acidic phenols p-hydroxypropiophe-
none (pKa = 7.8) and m-nitrophenol (pKa = 8.2), which
are more than 90% in their neutral form at pH6.8.
Neutral forms can diffuse through membranes;
charged forms are highly soluble in water. An equilibrium between the two forms is quickly established in an
aqueous medium and also at the phase boundaries. If the
pKa values of the substances are not more than 2–3units
. Fig. 19.5 Two-phase system with partition and dissociation equi-
libria for an acid HA (Eq.19.8). Ka is the dissociation constant, Pu and
Pi are the partition coefcients of the undissociated and ionic forms,
that is, neutral and charged species, respectively. Because there is usually adifference of several orders of magnitude between the Pu and
Pi values, in many cases the Pi value can be neglected. This leads to
considerable simplication of the corresponding mathematical models
. Fig. 19.6 The pH dependence of the distribution equilibrium of
acids and bases, the so-called pH distribution prole, follows simple
rules. Typically when an acid (red) or abase (blue) is present, sigmoidal, that is, S-shaped, curves are observed. For adibasic acid, for example, oxalic acid, the decrease in the partition coefcient continues with
increasing pH values (violet). In the presence of lipophilic counterions,
for example, the tetrabutylammonium salt of salicylic acid, the ion
pair displays avery high partition coefcient (magenta). Amino acids
with neutral side chains carry one basic amino group and an acidic
carboxyl group (green). Accordingly, they have amaximum partition
coefcient at the neutral point. Here, the majority of the substance is
indeed present as azwitterion, but a larger fraction is in the neutral
form than at lower or higher pH values
from the neutral value of pH7, the neutral form will be
present in the aqueous phase in aquite sufcient concentration of about 0.1–1%. It penetrates the membrane.
In the aqueous phase, it is immediately regenerated by
the dissociation equilibrium. In abiological system, the
distribution of such substances is rapid and effective
(. Fig.19.7), and it is even better the closer the pKa is to
neutral pH7. This also explains why so many drugs are
organic acids or bases. Because of the strongly deviating
pH values in the stomach and intestine, there is apoint
in the gastrointestinal tract where aneutral substance, an
acid or abase, can be well absorbed. If the pKa values
are too far away from the physiological pH values, for
example, amidines or guanidines with extremely high pKa
values, absorption can become problematic. This is also
true for zwitterionic compounds, such as amino acids,
and compounds with multiple acidic or basic groups in
the molecule. Because of the large volume available for
distribution, diffusion is predominantly from the gastrointestinal tract into the blood or tissues, and only to
anegligible extent in the opposite direction (. Fig.19.7).
The absorption of strongly acidic compounds outside
the range in which the compound exists as aneutral molecule, runs in rst approximation parallel to the difference pH − pKa, and for bases the difference is pKa − pH.
There are exceptions to this approximation. Highly lipophilic compounds require amore detailed description of
the pH–absorption prole. The neutral forms of these
=
nonionized forms
/
. • Absorption Proles of Acids and Bases
. Fig. 19.7 aA moderately polar neutral substanceN
is absorbed very well from the stomach as well as from
the intestines. It is quickly distributed in the circulation
so that back-transport does not play anotable role. bAn
organic acid HA (pKa = 4) is absorbed well from the
stomach, as long as it is not too polar, because it exists
there overwhelmingly in the neutral form. The absorption
is facilitated by the fact that the free acid is in considerably lower concentration in the blood than in the stomach. The formation of an anion shifts the concentration
gradient in this direction. The absorption is slower from
the gut because there the equilibrium lies overwhelmingly
on the side of the ionized form. cA weak base (pKa = 5)
is relatively poorly absorbed from the stomach because
it is predominantly present in its polar, protonated form.
It is well absorbed in the intestine, where it exists in its
neutral form. dA strong base with apKa = 9 cannot
be absorbed via the stomach. The equilibrium indeed
lies heavily on the side of the protonated form in the
intestines, but the nonpolar form is available in adequate
quantities. Therefore, the substance can be absorbed.
When asubstance reaches apKa value of more than11,
the concentration of the neutral, bioavailable form is too
low for good absorption to take place
substances enter the lipid phase as soon as they come
near the membranes. The neutral molecule is being con-
stantly removed from the dissociation equilibrium, which
is established in the aqueous phase. However, it is very
quickly replenished by this equilibrium. In equilibrium,
there is acontinuous transport of substance from the
aqueous phase into the membrane. The small amounts of
the uncharged neutral form are the door through which
the entire process takes place. The rate of transition into
the lipid layer does not depend on the (often very low)
concentration of the neutral form, but rather on
The total concentration of the compound,
-
The rate constants of the dissociation equilibrium,
-
and
The diffusion constant of the compound.
-
Accordingly, ashift in the pH–absorption prole is ob-
served in biological systems for lipophilic acids and bases
relative to the pH–partition prole, which is referred to
as pH shift. This always occurs in the direction towards
the neutral point, which means to higher pH values for
acids and to lower pH values for bases. The greater the
lipophilicity of an acid or base, the greater the observed
shift in the absorption prole. The log Pvalue and the
pKa values cannot be considered separately when assessing how well asubstance is absorbed. Their combination is crucial. For the design of new drugs, this means
that asubstance with an unfavorable partition behavior,
which means with apKa value that is too high or too
low, can be favorably modied in the desired direction
by increasing its lipophilicity. To describe the pH dependency of the distribution equilibrium, adistribution
coefcientD was introduced as asupplement to the par-
tition coefcientP. For this, the ratio of the sum of all
concentrations of ionized and nonionized forms of an
investigated compound in the two phases are considered.
The pH value is adjusted for measurement in abuffer
solution so that the addition of the investigated compound does not shift the pH. Usually logD, logarithm
of the distribution coefcient, is used here (Eq.19.10).
.Octanol=Buffer/
Sum.Conc..SovatedSubstance/
g
Sum.Conc:.Solvated Substance/
(19.10)
Octanol; ionized=
nonionized forms
Buffer; ionized=
/
Chapter • From In Vitro to In Vivo: Optimization of ADME and Toxicology Properties
19
19.6 What Is the Optimal Lipophilicity
of aDrug?
Lipophilicity plays an important role in the assessment
of adrug’s therapeutic potential. This applies to absorp-
tion, distribution, metabolism, and excretion. With the
exception of substances that are absorbed via atrans-
porter, absorption is usually better when compounds are
more lipophilic. This advantage is limited by the solu-
bility in aqueous phases, which decreases signicantly
with increasing lipophilicity. The following comparison
may help to illustrate the low water solubility of some
hydrophobic drugs. The neuroleptic chlorpromazine
(19.1, . Fig.19.8) with log P = 5.4 has awater solubil-
ity of 2.55 mg/L at 24 °C in its neutral form. Marble with
14 mg/L and sand with 10 mg/L are much more solu-
ble in water. Due to its basic nature (pKa = 9.3), chlorpromazine can be converted to its hydrochloride, which
is much more water soluble at about 10 g/L. However, it
does not approach the solubility of common salt, which
is 350 g/L.
These factors depend on the intermolecular interactions in the crystalline solid and can vary greatly between
different polymorphic crystal modications in which the
drug molecule has been crystallized. Therefore, correlations for predicting bioavailability consider the melting
point as another simple parameter to estimate the stability of apolymorphic form, in addition to lipophilicity and solubility. Furthermore, computational methods
are used to investigate whether additional polymorphic
forms may exist with more suitable properties. In addition to solubility, dissolution kinetics is important for
galenic formulations, namely the nal drug product. It
determines the amount of substance that is dissolved
. Fig. 19.8 Chlorpromazine 19.1, haloperidol 19.2, and sulpiride
19.3 are neuroleptics with typical side effects that are associated with
dopamine antagonists. Clozapine 19.4 is different from these substances in its binding prole on the dopamine receptors (. Table19.2)
as well as in its side effects
during the gastrointestinal passage. This amount can be
inuenced by several factors, such as, the following:
Increasing the surface area by grinding the crystals
-
into miniscule particles (micronization),
Growing a modied crystal with better solubility
-
properties,
Crystallization under special conditions to afford
-
amore uniform (usually smaller) size, or crystals with
lattice defects,
Changing the salt form,
-
Adding solubility-mediating additives, and
-
Embedding the drug as amorphic solid solutions of
-
easily dissolvable polymers.
Because of its importance, high-throughput solubility
measurement techniques have been established in recent
years. As mentioned above, large-scale computer simulations are nowadays used to predict the polymorphic
crystal forms of new drug molecules in order to assess
whether there might be adifferent crystal form that could
positively inuence dissolution behavior, solubilization
kinetics and, thus, bioavailability.
Cell cultures are also increasingly used as in vitro
models to study drug absorption. Athin layer of cells
from human colon carcinomas (so-called Caco-2, HT29
or MFCH cell lines) is grown in atwo-chamber system.
Drug transport can be monitored from either the apical
or basolateral side. Since these cells also express transporters, the involvement of specic transport mechanisms can also be studied. These models are less suitable
for studying the possible consequences of drug metabolism because the metabolizing enzymes (Sect.27.6) are
expressed at reduced levels in these cells.
In vitro models have also been developed to study
blood–brain barrier penetration. However, these models are relatively labor-intensive and the results can
often only be compared within aseries of structurally
related compounds. Articial membrane assay systems
(PAM PA, from parallel articial membrane permeability assay) can be constructed to allow high-throughput
screening. In addition, the penetration behavior in liposomes can be evaluated by surface plasmon resonance.
When experimentally determining the absorption of
various substances, results obtained with saturated solutions of the substances should not be compared with results obtained with solutions of constant concentration.
In the rst case, the absorption rate of highly lipophilic
substances decreases linearly due to the decrease in solubility with lipophilicity. In the second case, the absorption
rate often remains at amore or less constant value, even for
highly lipophilic substances. Acomparison of such different experimental conditions is likely to lead to erroneous
conclusions. Further confusion arises when the terms ab-
sorption and bioavailability are used incorrectly (Sect.9.1).
Absorption of acompound may be excellent, but bioavailability may be poor. Lipophilic compounds and substances
. • Computer Models and Rules to Predict ADME Parameters
with amolecular weight greater than 500–600 Da are often well absorbed but suffer from very rapid biliary elimination. This usually occurs during the rst liver passage
(rst-pass effect, Sect.9.1) immediately after absorption
from the intestine. To achieve good bioavailability, the lipophilicity must not be too high. The route of excretion also
depends on lipophilicity. In general, extremely lipophilic
substances are metabolized more rapidly, but they are also
of greater toxicological concern. Hydrophilic substances
and polar metabolites, even after conjugation with polar
groups, are excreted via the kidneys. Lipophilic substances
are usually excreted by the liver and then by the intestine.
Such substances often undergo oxidative metabolism, with
the potential for the formation of toxic metabolites.
Substances that interact with membrane-bound receptors or ion channels can often reach their targets more
easily if they are enriched in the surrounding membrane.
To achieve this, the substances should be lipophilic or
carry alarge lipophilic group with which they can be
anchored in the membrane (Sect.4.2, . Fig.4.2).
19.7 Computer Models and Rules
to Predict ADME Parameters
In addition to setting up appropriate test systems to
systematically record parameters that determine pharmacokinetic properties, much effort has been devoted to
establishing rules and computer models to predict favorable ADME properties. First and foremost is the Rule of
Five, developed by Chris Lipinski at Pzer. It states that
an active substance should not violate more than two of
the ve criteria listed in . Table19.1. These simple rules
are derived from experience and are often used to preselect compounds for screening. Tudor Oprea (Albuquerque, New Mexico, USA) has further rened these rules
and extended them to include the occurrence of certain
structural building blocks, such as the maximum number
of rings of acertain size. Programs such as CLOGP, or
ACD/pKa and Pallas/pKa have been developed to estimate lipophilicity and pKa values. Meanwhile, the simple
Ro5 has come under increasing criticism as more and
more examples of successful drugs that do not obey the
rule have become known. These so-called “beyond Ro5”
drugs typically bind to at and groove-shaped binding
sites. Chemically, they often belong to the class of macrocycles. Solvation enthalpies are used to attempt to predict
solubility. Predictions of permeability, absorption, and
bioavailability are based on empirical correlation models.
Experimental observations are related to the chemical
structure of the investigated molecules. The methods
used are derived from the QSAR models presented in
Chap.18. The properties to be predicted are described by
models based on intuitively chosen or more or less obvious descriptors. Usually, molecular parameters are used,
which are often derived from molecular surface contribu-
. Table 19.1 Criteria for the Rule of Five
Molecular weight ≤ 500 Da
Octanol–water partition coefcient log P ≤ 5
Number of H-bond donor groups not more than5
Number of H-bond acceptor
groups
tions and may be determinant for the target properties.
In addition to routine regression analysis, more recent
mathematical models such as neural networks, nearest
neighbor classiers, decision trees, or machine learning
techniques such as support vector machines are applied.
In addition to the easy-to-evaluate Rule of Five, the
following criteria should be considered for rational design: substances that act in the periphery, such as cardiovascular drugs, should be relatively polar. Of course,
a certain degree of minimal lipophilicity is necessary
for their absorption. Because of the risk of central side
effects or the formation of toxic metabolites, this lipophilicity should not be exceeded too much. As ageneral
rule, it is better to be alittle less potent than to have all
the other problems! Agood therapeutic window is much
more valuable than apicomolar afnity to aprotein.
Substances that act on membrane-bound proteins and
substances that act in the central nervous system should
have amoderate to high log Pvalue of > 1. To avoid the
development of toxic metabolites, the inclusion of the
following is recommendable:
Easily conjugated groups, for example hydroxyl,
-
amino, or carboxyl groups,
Preconceived metabolic cleavage points such as ester
-
or amide bonds, and
Oxidizable groups that lead to nontoxic and easily
-
excretable metabolites, for example, methyl groups.
Of course, this strategy should not be exaggerated, otherwise the substances will be excreted too quickly. The
biological half-life is then reduced to avalue that makes
therapeutic administration in humans impossible.
The structural consideration of properties that lead
to optimal bioavailability, adequate biological half-life,
and nontoxic metabolites is aproblem in the search for
new drugs. Structure-based drug design initially focuses
on the t of aligand to its binding site. Often, aspects
related to pharmacokinetics and metabolism are not
adequately considered at this stage. Disappointments at
the end of asuccessful optimization in the preclinical
phase, or at the latest in the clinic, punish such aonesided approach. As the spatial structures of transport-
ers, channels, and metabolic enzymes become increasingly
available, structure-based design concepts can be used to
test the cross-reactivity of proposed or developed ligands
on these target structures.
not more than
2 • 5 = 10
Chapter • From In Vitro to In Vivo: Optimization of ADME and Toxicology Properties
19
Binding to the potassium-ion-transporting hERG
ion channels leads to their blockage. This can lead to
life-threatening cardiac arrhythmias (Sect. 30.3). For
this reason, QSAR models have been developed to
screen molecules for potential hERG channel binding.
Methods have been developed to dock ligands directly
into the spatial structure of the channel. Another system
that has recently been structurally characterized is the
membrane-bound glycoprotein GP170. It is atransporter
capable of expelling drugs from the cell (Sect.30.10). It is
desirable to avoid interactions with this protein as much
as possible. Another large family of enzymes worthy of
attention are the cytochrome P450 metabolic enzymes
(Sect.27.6). Here one tries to estimate how drugs interact
with these proteins and how they are metabolized. This
opens up awide eld for structure-based design.
19.8 From In Vitro to In Vivo Activity
Compounds are rst tested in simple in vitro models, such
as enzyme inhibition or receptor binding, in cell culture
and later in organs and animal models. In general, the
simplest model where the results are predictive of the
expected effect in animals or humans is chosen. To do
this, it is necessary to derive quantitative relationships
between the different test models, known as activity–ac-
tivity relationships. These describe the relationship between biological activity, e.g., between in vitro and in vivo
data. In the best case, they even allow extrapolation to
therapeutic effects in humans from the determination of
afnity in abinding or inhibition assay.
Conrmation of acorrelation between asimple test
model and atherapeutic effect is often more important
than the derivation of astructure–activity relationship.
Once the relevant quantitative relationship has been established, inexpensive and rapid assays can be used instead of time-consuming and costly animal experiments.
This signicantly reduces the number of animal experi-
ments. But that is not the only benet. The use of automated molecular testing systems allows reliable characterization and standardization of compound proles.
19.9 Compartmentalization: Natural
Ligands Are Often Unspecific
Prior to biological testing of acompound, the following
questions must be answered: What is the therapeutic goal
to be achieved and how will it be achieved? Therapeutic
concepts are derived from the pathophysiology of the
disease mechanism. Regulatory intervention with drugs
should restore the original physiological state as far as
possible. To mimic the natural ligands of enzymes and
receptors, the drug must have sufcient specicity and be
able to clearly access the target site.
Nature operates on two orthogonal principles with respect to endogenous substances: specicity of action and
usually highly pronounced spatial compartmentalization.
Hormones act predominantly systemically, which means
they are released at one site in the body and transported
by the bloodstream to another, completely different site.
There they exert their effect. Other substances, such as
neurotransmitters, act strictly locally. In the context of
the picture of lock and key (Sect.4.1), Nature prefers to
have amaster key that can act on different locks. It acts
only at the site where it is produced and is removed once it
has served its purpose. Neurotransmitters are synthesized
in nerve cells, stored, and released when the cell is stimulated at the synaptic cleft (Sect.22.5). There they bind
to specic receptors and stimulate the neighboring nerve
cell. The effect is quickly dissipated after reuptake into
the cell or after degradation, for example, by monoamine
oxidases (amines), esterases (acetylcholine), or peptidases.
Nature’s efciency is demonstrated most impressively
by the diversity with which small molecules such as
adrenaline and noradrenaline (Sect.1.4) can be used as
hormones and neurotransmitters. There is aplethora of
different receptors and receptor subtypes for these substances, allowing the same molecule to have completely
different effects. The amino acid sequence of aparticular
receptor, and thus its binding site, can be altered relatively easily at the gene level. The evolution of complex
biosynthetic pathways for nonpeptidic ligands, often involving multiple enzyme-catalyzed steps, is much more
complicated. Accordingly, almost all neurotransmitters
and many hormones are derived in asimple way from the
central intermediates of the metabolism of, for example,
amino acids. On the other hand, the steroid hormones
(Sect.28.3) demonstrate that Nature can achieve very different effects with aset of chemically similar structures
and evolutionarily and structurally related receptors,
such as the estrogens, gestagens, androgens, glucocorticoid steroids, and mineralocorticoid steroids.
Often, the spatial distribution of biosynthesis or the
release of areceptor ligand or the distribution from membrane-bound receptors or enzymes plays adecisive role in
the specicity of an effect. Different effects of the same
ligand can be achieved by locally restricted release or by
the presence of different receptors. The differentiation is
not only between certain organs or areas, but also between individual cells and cell compartments. For example, the concentration of dopamine in different regions of
the rat brain has been determined. While in some regions,
such as the caudate nucleus (lat.: Nucleus caudatus), an
important synaptic site for the motor and olfactory systems, concentrations of up to 100 ng dopamine per mg
protein are reached, while most other areas of the brain
only contain between 0.2 and 10 ng/mg. Even in the Sub-
stantia nigra of the midbrain, dopamine levels are only
5–6 ng/mg. Degeneration of dopaminergic neurons in this
area leads to Parkinson’s disease in humans. It is known
. • Specicity and Selectivity of Drug Interactions
from labeling experiments that the distribution and population density of receptor subtypes in distinct areas of
the brain and other tissues can be very different.
19.10 Specificity and Selectivity
of Drug Interactions
How specic should adrug act? There is no absolute
answer to this question. Since drugs are almost always
administered orally or intravenously, they act systemically, which means on the whole organism. The lack of
restriction to aspecic organ or compartment must be
compensated for by agreater specicity. In any case, the
drug must be as specic as necessary to achieve asuccessful therapy with tolerable side effects.
In the case of enzyme inhibitors, substances that are
specic enough to inhibit only one particular enzyme are
preferred. Nonspecic inhibitors that simultaneously in
hibit several serine or metalloproteases would have adevastating effect on an organism. For example, athrombin
inhibitor designed to reduce an increased risk of thrombosis should not also act as an inhibitor of the closely
related plasmin, which causes brinolysis and leads to the
dissolution of blood clots that have already formed. The
situation with kinase inhibitors (Sect.26.3) is somewhat
different. Because of the similarity among kinases, one
member of the family can easily do the work of another
related kinase that has been blocked. In doing so, it will
reduce the therapeutic effect to zero. Here, abroad-spectrum kinase inhibitor that can simultaneously block an
entire family of proteins may be desirable. Abroad-spectrum action that inhibits multiple isoenzymes of aparasite
equally well may also be advantageous for antibacterial or
antiparasitic compounds (e.g., plasmapepsins, Sect.24.7).
Receptor agonists and antagonists should also
be highly selective. β-Agonists used to treat asthma
(Sect.29.3) must be β2-specic so that they do not induce
an unwanted increase in heart rate or blood pressure.
Often, asingle active agent cannot achieve the desired
therapeutic response. The simultaneous use of several
drugs is often indicated for the treatment of arterial hypertension (Sect.22.10). More complex, multifactorial
disease processes must be treated by targeting multiple
mechanisms. Due to the low dosage of the different components, the nonspecic side effects of the individual
components fade into the background.
Specicity is critical to the efcacy of drugs acting in
the CNS. Advances in genetic engineering have given us
an explosion of knowledge about receptors, but also adilemma. We know the exact receptor prole of established
compounds. We know what specicity must be achieved
to mimic aparticular type of effect. However, in many
cases we do not know what that prole of acompound
should look like to achieve abetter therapeutic effect. An
example should illustrate this point. Neuroleptics and
many antidepressants (Sect.1.6) act on neuroreceptors.
The classical neuroleptics chlorpromazine 19.1 and halo-
peridol 19.2 (see Sect. 19.9), used in the treatment of
schizophrenia, are relatively unspecic dopamine receptor antagonists (. Table19.2). The mixed neuroleptic/
antidepressant sulpiride 19.3 acts simultaneously on D2
and D3 receptors. All of these drugs have side effects on
the musculoskeletal system, as seen in Parkinson’s disease
(see Sect.9.4), which is caused by alack of dopamine.
Because of their mode of action, it was assumed that
the side effects of neuroleptics were inevitable consequences of the antagonism of dopamine receptors. Then
an atypical neuroleptic, clozapine 19.4, was introduced
(. Fig.19.8). It does not have the side effects described
above. Today we know that clozapine, unlike the other
neuroleptics, is much more potent at the D4 receptor than
at the D2 and D3 receptors (. Table19.2). However, at
the concentrations at which clozapine acts on the D4 receptor and which can be detected in the cerebrospinal
uid of treated patients, clozapine also binds to certain
serotonin and muscarinic receptors, in some cases with
higher afnity. It is, therefore, possible that the antago-
. Table 19.2 The natural neurotransmitter dopamine binds with higher afnity to dopamine receptors of the D1-type. The classic
neuroleptics chlorpromazine 19.1, haloperidol 19.2, and (S)-sulpiride 19.3 are different from clozapine 19.4 (. Fig.19.8) in one point:
they have no comparable selectivity for the D4 receptor
Substance D
Dopamine 0.9 < 0.9 7 4 30
Chlorpromazine 19.1 30 130 3 4 35
Haloperidol 19.2 80 100 1.2 7 2.3
(S)-Sulpiride 19.3 45,000 77,000 25 13 1000
Clozapine 19.4 170 30 230 170 21
Binding to the dopamine receptors, Ki in nM
-Type D2-Type
1
D
1
D
5
D
2
D
3
D
4