Lectura 2 - 05-08-22

Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, 2011, 33-51 33
CHAPTER 2
Physicochemical Characterization of NCEs in Early Stage Drug Discovery
György T. Balogh
Discovery Chemistry, Gedeon Richter plc., E-mail: gy.balogh@richter.hu
Abstract: In the last two decades, the role of physicochemical parameters has come to the forefront of early stage
drug discovery. The primary driver behind this revolution is the fact that the pharmacokinetic properties of hit
and lead compounds have become worse and worse. In this chapter, the theoretical background and measurement
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techniques for the more important physicochemical parameters are introduced from the perspective of the
ADME/T processes. Finally, during the last decade a number of significant ADME/T-physicochemical-based
structure relationships have been shown to be relevant.
INTRODUCTION
The prediction of physicochemical parameters can describe the PK characteristics of new drug candidates in early
stages of drug discovery and can decrease the high costs of delivery of new oral drug molecules to the marketplace
and can increase the chance of finding the optimal bioavailability and pharmacokinetic (PK) profile. Therefore,
physicochemical parameters attempt to predict the PK characteristics of new drug candidates in the early stages of
drug discovery. In addition, physicochemical parameters can be applied to determine drug formulation and storage
conditions. Today, inadequate PK profiles are one of the key reasons for the withdrawal of new chemical entities
(NCE) [1]. Owing to current commercial expectations, drugs are intended for oral therapy, while the discovery of
new leads by high-throughput techniques tends to result in (i) more lipophilic and therefore potentially less soluble
compounds, and (ii) compounds with higher numbers of hydrogen-bond donors and acceptors and larger molecular
volume [2]. A similar tendency can be expected during lead optimization if solubility/permeability, in parallel with
binding affinity/selectivity, is not adequately optimized. A wide variety of tools are available for predicting oral
absorption, ranging from in silico predictions across artificial membrane models (PAMPA) [3] to sophisticated in
vitro cell-culture models such as Caco-2, MDCK [4], and co-cultures of astrocytes with endothelial cells to mimic
the blood-brain barrier (BBB) [5].
Ionization
Drug release and Solubility
dissolution
Ionization , log P/D,

A bsorption H-bonds, Size,
Stability
Size, log P, Efflux transport
M etabolism and Solubility ,
Systemic Tissues
E xcretion circulation
D istribution
Ionization , log P/D,
Efflux transport Solubility , H-bonds,
Size, Stability
Copyright 2011. Bentham Science Publishers.
Central Nervous
Effect
System (CNS)
Ionization , log P
Figure 1: ADME process and related physicochemical parameters.

K. Tihanyi and M. Vastag (Eds)
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34 Solubility, Delivery and ADME Problems of Drugs and Drug Candidates György T. Balogh
The optimal physicochemical profile aims at selecting the right compounds while lowering the risk of eliminating
successful candidates. The main factors contributing to passive transport, such as solubility, lipophilicity and
permeability, will be discussed, together with measurement potentials and limitations. Relationships between
pharmacokinetic and pharmacodynamic stages and related physicochemical parameters are shown in (Fig. 1).
This section concentrates on measurable physicochemical parameters such as ionization (protonation/dissociation)

constants, lipophilicity and permeability and their application in lead selection and optimization. Due to the
increasing number of poorly soluble compounds in drug research, which influences the interpretation of results of in
vivo/in vitro preclinical models and ADME processes for drug candidates, solubility as a physicochemical parameter
is examined separately in chapter 3.
IONIZATION OF DRUGS IN AQUEOUS SOLUTION (PKA)
The vast majority of drugs (95%) contain ionisable groups. Most of them are basic (~75%, e.g. chlorpromazine), and
a smaller number are acidic or amphoteric (20%, e.g. warfarin and norfloxacine). Compounds are classified as
monoprotic (e.g. atenolol), diprotic (e.g. quetiapine) or poliprotic (e.g. doxycycline) compounds, based on the
number of functional groups gaining or loosing protons. The ionization of these molecules is highly dependent on
pH. The exceptions are the non-electrolytes, such as steroids, and the quaternary ammonium compounds, which are
completely ionized at all pH values, acting as strong electrolytes. The characteristic thermodynamic parameter
related to the pH, the charge state of a molecule, is the ionization or protonation/dissociation constant, pKa.
Determination of pKa is beneficial, as it determines the ADME processes as well as the pharmacodynamic characteristics
(receptor affinity) of medicinal substances. Moreover, it is useful for maximising chemical synthesis yields [6].
The ionization reaction for acids, bases, and ampholytes (ordinary and zwitterionic) may be represented by generic
forms:
Acids:
 A    H  
HA = H + A Ka =   
+ - 
 HA
Bases:
 B    H  
BH+ = B + H+ Ka =
 BH  
 
Ordinary ampholytes:
HABH+ = HAB = A-B Kabasic > Kaacidic

Zwitterionic ampholytes:
HABH+ = A-BH+ = A-B Kabasic < Kaacidic

The negative log of the quotients in the above equations yields Henderson-Hasselbalch equations, where p represent
the operator –log:
Acids:
 HA
pKa = pH + log
 A 
 
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Physicochemical Characterization of NCEs in Early Stage Solubility, Delivery and ADME Problems of Drugs and Drug Candidates 35
Bases:
 BH  
pKa = pH + log  
 B
Generic Henderson-Hasselbalch equation:
pKa = pH + log
 proton donor 
 proton acceptor 
The Henderson-Hasselbalch equation indicates that the basic substance is completely ionized at pH values up to 2
units below and completely unionized at pH values greater than 2 units above its pKa. By contrast, the acidic drug
substance is completely unionized at pH values up to 2 units below and completely ionized at pH values greater than
2 units above its pKa. When the concentration of the free acid, HA (or conjugate acid, BH+), is equal to the
conjugate base, A- (or free base, B), the pH has the special designation pKa (Fig. 2).
1 1
0.9 [AH] [A- ] 0.9 [BH+] [B]

0.8 0.8
Dissociation ratio ( )
Dissociation ratio ( )
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
pK a pK a
0 0
1 1.5 2 2.5 3 3.5 4 4.5 5 7 7.5 8 8.5 9 9.5 10 10.5 11
pH pH
Figure 2: Proton dissociation – pH function of acids and bases

Figure 2: Proton dissociation – pH function of acids and bases.
In the case of zwitterionic ampholytes such as amino acids, acid/base functions are ionized as described above. In the pH
range between the pKa values of acid/base functions, a zwitterionic molecule is in a neutral ionization state (Fig. 3).
AHBH+ ↔ A-BH+ ↔ A-B

1
0.9
0.8
0.7
Dissociation ratio ()
0.6
0.5
0.4
0.3
0.2
0.1
pKa 1 pKa 2
0
1 2 3 4 5 6 7 8 9 10 11
pH
Figure 3: Proton dissociaton – pH function of zwitterions

Figure 3: Proton dissociaton – pH function of zwitterions.
This particular pH is known as the isoelectric point (pHi). pHi can be calculated from:
pKa acidic  pKabasic

pHi =
2
pKa Measurements
Potentiometric titration is the gold standard method for pKa measurements in the pharmaceutical industry [7], but
requires a relatively large amount (10-20 mg) of highly pure material with relatively high aqueous solubility.
Screening methods for pKa are typically based on differences in UV absorption or chromatographic retention at
various pHs [8]. For example, the spectrographic analysis (SGA) method creates a pH gradient in a flowing aqueous
fluid over a period of several minutes. Because the UV absorption of a chromophore is affected by an ionisable
centre that is within a few bonds of the chromophore, the UV absorption varies with pH and is recorded using a UV
detector. Analysis of UV absorption versus pH indicates the pKa [9]. A different approach is taken by recording the
capillary electrophoretic (CE) mobility of a compound in different buffers. Mobility increases with the degree of
ionization; a plot of mobility versus pH therefore indicates the pKa [10].
LIPOPHILICITY OF DRUGS
Meyer [11] and Overton [12] correlated the biological effect of drugs in relation to lipid solubility. Pauling
subsequently described the relationship between lipophilicity and anaesthetic potency for heterogeneous compounds
[13]. The thermodynamic characterization of lipophilicity was not seen as practical. Until now, only empirical scales
of lipophilicity have been of importance in practice, some expressing the changes in free energy associated with
solute transfer between two phases, others being dimensionless indices relating the partitioning data of given solutes
to a general standard.
Lipophilicity, however, is far from being a purely empirical tool in structure-activity analysis. It also helps in better
understanding intermolecular forces and intramolecular interactions in solutes. Intermolecular forces include the
following interactions between the solute, aqueous and organic phases [14-16]:
 hydrophobic interactions,
 ion-ion and ion-dipole interactions,
 van der Waals interactions,
 hydrogen bonds,
 charge transfer interactions.
Hydrophobicity and lipophilicity can be defined as follows: Hydrophobicity refers to the association of non-polar
groups or molecules in water deriving from the tendency of water to exclude non-polar molecules. Lipophilicity
represents the affinity of a molecule or a moiety to the lipophilic environment, which is applied mainly by medicinal
chemists to describe transportation in biological systems [17].
Partition of Drugs into Octanol and Anisotropic Systems (log Po/w, log Pmemrane)
Lipophilicity can be characterized by the distribution behaviour of solutes in a bi-phasic system, i.e. liquid-liquid
(e.g. n-octanol/water partition) or solid-liquid (e.g. retention on RP-HPLC). Berthelot and Jubgfleisch [18] defined
the partition coefficient in 1872, and Meyer [11] and Overton [12] applied it to explain the potencies of biologically
active substances. Gaudette and Brodie [19] shed light on its possible application to modelling lipophilic
characteristics and the application of the latter to modelling PK processes. The relationship between the partition of
certain drugs in heptane/buffer and the rate of their absorption into the cerebrospinal fluid was demonstrated.
However, application of partition as a lipophilicity parameter did not become widespread until 1964, and only the
Hansch [20] n-octanol/water system was generally applied. In 1971, Leo, Hansch and Elkins [21] published the first
comprehensive review of the partition coefficient, including their own measurements on some 800 values in the
octanol/water system.
As mentioned above, n-octanol/water partition can be generally described by log P0, which refers to the neutral form
of molecules:
log P0 = log [X0]oct/[X0]aq, where X is a generic ionisable molecule.
The lipophilicity profile can be given by the distribution ratio (log D) for ionisable compounds, considering changes in
partition with respect to pH. The shape of the profiles can be readily derived from pH-metric theory, and is described
by the four-equation partition model (Fig. 4). The log D – pH profile of monoprotic acid is shown in (Fig. 5).
A-
4 AH0
3 n-octanol 2
water
A- AH0
1
Figure 4: Four-equation partition model
Figure 4: Four-equation partition model.
5
0
log P
4.5
4
3.5
3
log D
2.5
1.5
1 I-
log P
0.5 oct
pK a pK a
0
3 4 5 6 7 8 9 10 11
pH
Figure 5: log D – pH profile of monoprotic acids

Figure 5: log D – pH profile of monoprotic acids.
These equilibria and the corresponding four equations can be seen below:
1. Ionization equilibrium in aqueous media (“aqueous pKa”):

 H     A 
pKa = log    
 HA
2. Partition of the neutral species between water and octanol:
 HA0 
0   oct
log P = log
 HA0 
  aq
3. Partition of the anionic species between water and octanol:
 A 
I-   oct
log P = log
 A 
  aq
4. Ionization equilibrium in aqueous media (“Scherrer pKa”):

 H     A 
oct     oct
pKa = log
 HAoct
Because the ratio of the concentration of ionized and neutral species changes with pH, the distribution ratio is also
pH-dependent. For monoprotic acid:
 A0    A 
  oct   oct
D=
 A0    A 
  aq   aq
By combining this equation with the four-equation model:
P 0  P I    H    K a
D=
1   H    K a
Although lipophilicity was traditionally defined for n-octanol/water systems, several other solvent pairs were also
used. Furthermore, differentiation of its log P value with classical log P measured by the n-octanol/water system
proved to be a good model system for tissue-specific partition of drugs (Table 1).
Table 1: Tissue specific log P models
log P Tissue specificity

BBB penetration model [22]
log Pn-octanol/water – log Pcyclohexane/water
H-bond, lipophilicity of partial alkyl chain
Cardio-selective penetration model[23]
log Pn-octanol/water – log PPGDP/water
Proton donation
Skin-selective penetration model [24]
log Pn-octanol/water – log Pheptane/water
H-bond
Leahy et al. [25] described the differences in the H-bonding characteristics of the lipid components of membranes
and n-octanol. Therefore, lipid membranes can contain largely amphiprotic (modelled by octanol), proton donor
(modelled by chloroform) and acceptor groups (as in phospholipids). Leahly upheld the use of propylene glycol
dipelargonate (PGDP) as model for phospholipid membranes (Fig. 6).
Other interactions can be formed between drugs and lipid membranes such as ion pairs. Yeagle et al. [26] showed in
a 31P NMR study that N-methyl hydrogens of phosphatidycholine were found in close proximity to phosphate
oxygens in neighbouring phospholipids, suggesting that the surface of the bilayer was a “shell” of interlocking
electrostatic associations. Boulanger et al. [27] proposed a three site model after studying the interaction of
tetracaine and procaine with multilamellar eggPC vesicles as a function of pH. The membrane-bound species
comprises two different locations: a weakly bound charged surface site and a strongly bound uncharged deeper site.
Herbette and co-workers [28,29] investigated the chemical structures of drugs bound to liposomes. The
dihydropyridine ring of amlodipine was found to be buried in the vicinity of the carbonyl groups of the acyl chains.
The side chain primer amine (positively charged at pH 7.4 in lipid membranes) functional group of amlodipine
pointed toward the aqueous phase, so the positive charge was located close to the negative oxygen atoms of the
phosphate. In summary, Avdeef et al. [30] suggested a model of phospholipid membrane-water tetrad equilibria of
ionisable drugs (e.g. amlodipine - PC (Fig. 7)).
H3C
CH3
H3C
Figure 6: Structure of propylene glycol dipelargonate (PDFP).
Figure 7: Phospholipid membrane – water tetrad equilibria.
In this study, the n-octanol-water and liposomal-water partitions of ionisable drugs were compared, highlighting the
difference between liposomal partitions of ionized species compared to n-octanol partitions (Table 2). The partition
increment of ionized compounds in liposomes can be explained by the ion-pair formation mentioned above.
Table 2: n-octanol – water and liposomal–water partition of ionisable drugs
Compound Log Poct0 log PoctI(+/-) log Pmem0 log PmemSIP

Chlorpromazine 5.40[31] 1.67[31] 5.40[32] 4.45[32]
Diclofenac 4.51[31] 0.68[31] 4.34[33] 2.66[33]
[33] [33] [33]
Ibuprofen 4.13 -0.15 3.87 1.94[33]
Metoprolol 1.95[34] -1.10[34] 2.00[33] 1.25[33]
Propranolol 3.48[34] 0.78[34] 3.45[33] 2.61[33]
[33] [33] [33]
Warfarin 3.54 0.04 3.46 1.38[33]
Lipophilicity Measurements
This section discusses the state-of-the-art of automated lipophilicity measurement methods. Screening techniques
apply a 96-well plate design using reversed phase HPLC and capillary electrophoresis (CE) to measure partition.
Traditional approaches apply HPLC for quantification: for example, HPLC at 5 min per well requires 8 hours per
96-well plate for one injection of the water phase, assuming no repeat injection [8]. Zavlavski et al. [35] have
described a deep-well-based method, where the n-octanol and aqueous phases are individually sampled and analyzed
using flow injection analysis (FIA) into a UV detector. The most commonly used C18-based column in HPLC
simulates the partitioning of compounds between the lipophilic medium (the octadecylsilane stationary phase) and
the aqueous mobile phase. Rapid gradient reversed phase HPLC are generally applied for lipophilicity
measurements because of the poor water solubility of lead-like compounds [36,37]. According to the above-
mentioned HPLC methods, log P and log D parameters can be described by the chromatographic retention capacity:
t R  t0 Cstationary Vstationary Vstationary

Capacity factor: k '     P (or D ) 
t0 Cmobile Vmobile Vmobile
The linear relation between capacity factor k’ and the partitioning coefficient can be written as:
log P (or log D )  a  b  log k ' ,
where b reflects the degree of similarity of the chromatographic partition system to the n-octanol-water system [38].
Several other columns have been used to estimate lipophilicity with a similar theoretical background. The polymer-
based (octadecyl-polyvinyl alcohol, OCD) column is thought to have advantages compared to the C1-based column.
This column is capable of suppressing the ionization of acids and bases between pH 2.0 and 12.0, allowing for the
measurement of log P for neutral molecules. In the polymer-based LC, ODP mimics n-octanol while the mobile
water:methanol mobile phase mimics the aqueous phase, leading to a more realistic approximation than that
provided by acetonitrile. Immobilized Artificial Membrane (IAM) columns are prepared by covalently binding
various phospholipid monolayers to silica particles [39,40,41]. In contrast to non-modified octadecylsilane columns,
the amphiphilic environment of IAM can take ion pairs with ionized compounds, as mentioned in section 2.a. CE
has also been applied for lipophilicity screening. In this application, microemulsion electrokinetic chromatography
(MEEKC) is used, which is an electrodriven separation technique. The partition is realized between water and an
oil-in-water microemulsion. The microemulsion consists of surfactant-coated nanometre-sized droplets of oil
suspended in an aqueous buffer. This experiment can be automated using 96 parallel capillary channels [42,43].
PERMEABILITY OF DRUGS (PASSIVE DIFFUSION)
The partitioning and solubility of drugs into lipid phases are crucial factors in pharmacokinetics. The bioavailability
of drugs is mainly determined by their ability to move across the lipid bilayer of intestinal epithelial and other
endothelial cell linings. These processes are described by permeability as an essential parameter. In general,
permeability is defined as the velocity of a compound’s passage through a lipid membrane barrier. Drug permeation
across the cell membranes can be realized by (Fig. 8): passive diffusion, paracellular transport, endocytosis, active
transport (influx: transport into the cells or efflux: out of the cells).
Apical side
A B C D E
Basolateral side
Figure 8: Drug permeation mechanisms across the cell membranes: (A) passive diffusion
(transcellular transport), (B) paracellular transport, (C) endocytosis, (D) active influx, (E)
active efflux
Figure 8: Drug permeation mechanisms across the cell membranes: (A) passive diffusion (transcellular transport), (B)
paracellular transport, (C) endocytosis, (D) active influx, (E) active efflux.
Paracellular permeation mainly depends on the size (the critical upper limit is 200 Da) and the charge of the drugs.
Passive diffusion across cell membranes and transcellular permeation through cells are the most important
mechanisms of drugs for crossing lipid membranes. Consequently, these drug transport processes are among the key
factors for bioavailability. Therefore, significant interest in the development of high-throughput in vitro models for
predicting drug absorption across different human tissue barriers (mainly gastrointestinal epithelia, the blood-brain
barrier, etc.) exists. Several in vitro permeability-measurement methods have been developed in the last two
decades, including biological cell layers (Caco-2, MDCK cells) and artificial membranes (PAMPA models, parallel
artificial membrane permeability assays). This section concentrates primarily on artificial membrane models. The
use of artificial membranes to investigate passive permeation processes has a long history, dating back more than 30
years [44-50].
Initially, Mueller and co-workers prepared thin membranes using different phospholipid-organic solvent systems
[51,52]. Efforts were made to overcome the limitations of the fragile membranes with the use of membrane
supports, such as polycarbonate filters [53]. Thomson et al. studied the use of polycarbonate filters, and applied the
so-called BLM single bilayer membranes (“Black Lipid Membranes”) [54]. Ghosh used cellulose nitrate
microporous filters as a scaffold material to set down octanol into the pores, then, under controlled pressure
conditions, displaced some of the oil in the pores with water. This created a membrane with parallel oil and water
pathways, intended to serve as a possible model for part of the outermost skin layer, the stratum corneum [55].
Kansy et al. [3] introduced a new permeability assay called the “parallel artificial membrane permeability assay”
(PAMPA). The following “permeability measurements” section will focus on PAMPA and its assay evolution.
Permeability Measurements
PAMPA is a non-cell culture-based technique, which uses two aqueous buffer solution wells separated by an
artificial membrane. The artificial membrane is composed of a lipid inorganic diluent supported by a porous
commercial filter plate matrix (Fig. 9) – Millipore’s PAMPA system.
The test compound is first diluted in a buffer and placed in a well on the donor plate. The compound moves from the
donor well by passive diffusion, through the artificial membrane and into the acceptor well. The rate of permeation
is determined by the compound’s effective permeability [56,57]:
2.303  V V    V  VD   CD  t   
Pe    A D   log 1   A      ,
A   t   SS   VA  VD    1  R   VD   CD  0   
Figure 9: Millipore’s PAMPA system.
where A is the filter area (cm2), t is time (s), SS is the steady-state time (s), CD(0) is the total concentration of the
compound (mol/cm3) in the donor well at time = t, VD is the volume in the donor well, VA is the volume of the
acceptor well and R is the retention factor:
CD  t  VA  C A  t 
R 1  .
CD  0  VD CD  0 
Effective permeability is a kinetic measurement comprised of two steps: first, movement through the unstirred water
layer (Pu); then movement across the membrane itself (Pm) (Fig. 10).
1  1 1  1
   .

Pe  Pu , Donor Pu , Acceptor  Pm

Donor well Membrane Acceptor well

concentration
Pu,Donor Pm Pu,Acceptor
BulkDonor UWLDonor UWLDonor BulkAcceptor
Figure 10: Drug transport through artificial membrane system. (UWL, unstirred water layer)
In vivo, overall permeability is controlled by the membrane, as the unstirred water layer (UWL) is very small [58].
In PAMPA experiments, the unstirred water layer is several times thicker than it is in the human intestine.
Corrections of PAMPA data must therefore be made, as otherwise measurements would merely represent the
properties of the unstirred layer and not the membrane itself [59].
When all molecules are in neutral form, Pe is at maximum. Correction for the Pu gives Pm, the permeability through
the membrane. The Pm for an ionisable compound is related to the pH of the bulk solution. Intrinsic permeability
(P0) is equal to the maximum Pm, when the only species present is the neutral species.
Pm  fu  P0 ,
where the fraction of uncharged species (fu) is defined for monoprotic acids as:
 HA 1
fu  
 HAtot 10 pH  pKa 
1 
According to the two equations above, effective permeability for a weak acid is:
1

1

10  pH  pKa 
1 .
Pe Pu P0
PAMPA is performed in a 96-well plate format, and can be rapidly quantified using a UV plate reader (Fig. 9).
Because there are no transport proteins present, only passive diffusion is tested and there is no metabolism [60]. The
PAMPA system has undergone considerable improvements in the last ten years. Improvements in the system can be
classified according to three main changeable parameters: the artificial membrane, the pH of the buffer in donor and
acceptor wells, and incubation conditions. As shown in Table 3, changes in all parameters have moved towards
shortening the duration of the assay, enabling the PAMPA system to mirror the in vivo conditions of the biological
membranes more and more closely.
Table 3: Evolution of the PAMPA system
Membrane Buffer System Incubation parameters

Tissue-specific membranes
Human serum albumine in
(BBLM) Brush-Border Lipid Membrane
Acceptor well
BBB, MDCK
Stirred donor well
Cosolvent in Donor and
Single lipid membranes
Acceptor well
Phosphatidylcholine, Phosphatidylserine, Incubation at physiological
Phospatidyletanolamine, Cardiolipin, temperature (37 oC)
Gradient-pH system
etc.
Donor well: pH 2 – 8
Incubation at room temperature
Acceptor well: pH 7.4
Solvent membranes
n-Octanol, cyclohexane, hexadecane,
Iso-pH system
dodecane
Membrane Layer
More simple permeability models have demonstrated that some solvents alone can provide adequate results for
simple passive diffusion testing. Octanol permeability is an important factor to be explored, as it is the principal
basis for the lipophilicity scale in pharmaceutical research [61]. Generally used dodecane-, hexadecane- or
cyclohexane-coated filter membrane systems were studied to determine the role of hydrogen-bonding and
electrostatic effects in different lipid membrane permeability assays [62, 63].
As most pure phospholipids are solids, they can not disperse into filters. A non-polar solvent (mainly dodecane) is
therefore usually used to dissolve the phospholipids before filter coating. In several studies, single lipid systems
have been applied, such as PC dissolved in dodecane as the model membrane barrier [3, 64]. This choice was
inspired by the prevalence of PC in mammalian membranes. In addition, single lipid systems can be used to study
the formation of ion-ion interactions between phospholipids with different ionization states (PC(0), PS (-1), CL(-2))
and ionized drugs. Others use tissue-specific artificial lipid mixtures or lipid compositions isolated from different
tissues for studying tissue-specific drug permeability. Sugano and co-workers have attempted to replicate in vivo
brush-border membrane conditions using a highly biomimetic phospholipid mixture (BBLM-PAMPA). Avdeef and
co-workers have described a cost-effective lecithin-based lipid combination referred to as the gastrointestinal tract
lipid composition (Double-Sink, DS-PAMPA) [65]. Di and co-workers have studied a blood-brain barrier-specific
permeability model (BBB-PAMPA) using a polar lipid fraction of the rat brain [66].
Buffer Systems
Another variable that often changes in PAMPA systems is the pH of buffers. In considering pH on the two sides of
the PAMPA, there are two possibilities. If the pH values of buffers are similar on both sides (donor and acceptor) of
the sandwich, it is called an iso-pH system; if pH values differ on the donor and acceptor sides, it is called a
gradient-pH system. Iso-pH (pH 7.4donor-7.4acceptor) is concordant with most tissue membrane transport processes,
such as the blood-brain barrier, renal and hepatic transport processes, but not with the gastrointestinal tract (GIT),
where the pH on the apical side of the GIT epithelia varies from 5.5 to 8.0, while the pH on the basolateral side is
7.4. Therefore, in designing the ideal screening strategy, it appears to be important to use gradient-pH techniques. If
the in vivo GIT conditions need to be mimicked and the investigated molecule is ionized at physiologically
important pH values, at least three effective permeability measurements should be attempted: pH 5.5donor-7.4acceptor,
pH 6.5donor-7.4acceptor, pH 7.4donor-7.4acceptor [67]. The pH effects of ionisable drugs on permeability are illustrated in
(Fig. 11) (in-house data), for a series of weak bases, acids and amphoters.
The effect of cosolvents and surfactants on drug permeability has been also studied. In general, cosolvents are
expected to decrease the membrane-water partition coefficients. In addition, the decreased dielectric constant of the
cosolvent-water solutions should give rise to a higher proportion of the ionisable molecule in the uncharged state
[30]. These two effects are contradictory. In general, increasing levels of cosolvents were observed to lead to
decreasing permeabilities. However, cosolvents made the weak acid more permeable with increasing cosolvent
levels, an effect consistent with the increasing pKa and with the decreasing dielectric constant of the cosolvent
mixtures. Another and important role of cosolvents in permeability assays is that they may increase the aqueous
solubility of molecules with limited solubility. Ruell et al. described a cosolvent procedure based on the use of 20 %
(v/v) acetonitrile in a universal pH buffer. For the first time, they could measure the intrinsic (1.3 cm/s) permeability
of the molecules, which have an intrinsic aqueous solubility of less than 10 ng/mL [68].
The effects of serum protein on drug absorption can be also investigated by PAMPA systems. If serum albumin is
added to the acceptor wells (basolateral side), then Pe(D→A) is faster then Pe(A→D), as the concentration of free drugs in
the unstirred water layer (UWL) is decreased by albumin-drug interactions. This effect increases the concentration
gradient between the acceptor-donor sides, causing augmented permeability of the drug in the direction of the
acceptor side [69].
Incubation Conditions
Stirring the donor side solution results in increased Pe(D→A), as for serum albumin. Stirring the acceptor side mimics
the blood stream of the capillary vessels at the basolateral side of the intestinal epithelia, which decreases the drug
concentration from the UWL of the acceptor side [70]. Moreover, increasing the incubation temperature from room
temperature to a physiologically adequate temperature of 37 oC has two beneficial effects on the permeability
processes. On the one hand, the increased temperature reduces membrane integrity, which results in easier
permeability between lipid molecules. On the other hand, enhanced convection reduces the thickness of the UWL,
and also increases the mixing of solutions on both sides and the solubility of the compound. These effects
significantly increase the permeability process, thereby halving incubation time.
Cl
Haloperidol Indomethacin
HO O Cl
N
N pKa = 8.65
100.0
90.0 O
O 90.0
80.0 O
80.0
OH
70.0
70.0 pKa = 4.42
60.0
F 60.0
Pe (cm/s*10 )
6
Pe (cm/s*10 )
6
50.0
50.0
40.0
40.0
30.0 30.0
20.0 20.0
10.0 10.0
0.0 0.0
5.4 6.5 7.4 5.4 6.5 7.4
pH pH
8.0
Difloxacin
7.0
6.0
O O
F
5.0 HO
Pe (cm/s*10 )
6
pKa1 = 5.85
4.0 N N
N
3.0
pKa2 = 7.35
2.0
F
1.0
0.0
5.4 6.5 7.4
pH
Figure 11: Permeability – pH profile of ionizable drugs

Figure 11: Permeability – pH profile of ionizable drugs.
PHYSICOCHEMICAL PARAMETERS IN ADME PROCESSES
The function of physicochemical parameters for pharmacodynamy and pharmacokinetics has been investigated over
the last 50 years. However, its application in pharmaceutical research has come to the forefront only in the last
decade. Prior to the 1990s, preclinical investigations of drug candidates were conducted using in vivo animal
models. As a result, pharmacokinetic properties were adversely selected. Molecule banks composed of millions of
compounds founded as a result of the rapid development of combinatorial chemistry in the 1990s accelerated the
development of high-throughput screening (HTS) techniques. However, these screening models provide starting
points (hits) with generally poor pharmacokinetic properties. This phenomenon can be largely explained by the
difference between the models used for HTS – i.e. receptor-based assays during an earlier period, and cell-based
assays more recently – and in vivo processes. This recognition is spurring the leading pharmaceutical companies to
seek out rules that can help to rapidly pre-filter compounds and accelerate hit-to-lead optimization. In 1997, Lipinski
and co-workers described, for the first time, the physicochemical and chemical properties of drugs absorbed by
passive transport [2]. After the turn of the millennium, an emerging new approach – hit-lead-candidate strategy – has
necessitated the reconsideration of the Lipinski rules, a revision process that was initiated by Lipinski himself in
2004 [71]. Using the original rules as starting points, several new regularities have now been determined. For
example, the Veber rules [72] were determined based on bioavailability measurements for known drug compounds
performed on rats. The BBB permeability relation was defined by Pardidge [73], and the rule of three, which deals
with the lead-like compounds, by Oprea [74] and co-workers. It is important to note that to better understand and
predict ADME properties and to optimize drug candidates, physicochemical parameters cannot be separated;
instead, their overall effects should be investigated. Absorption of candidates by passive transport can be also
predicted by intrinsic lipophilicity (log P0). The optimal interval of log P0 for oral bioavailability is between 0 and 3.
Compounds with log P0 values below this interval are usually weakly permeable, while those above it have low
aqueous solubility. This leads to a more accurate assessment of lipophilicity, as the absorption relation can be set by
taking the proton dissociation properties into account. In general, for compound series having stronger acidic and
basic moieties, lipophilicity is lower and aqueous solubility is increased [75,76]. It is therefore determined by the
ionisation state present (according to pKa) at the physiological pH interval – i.e. pH 7.4 or log D7.4 should be
considered – during absorption.
In case of log D7.4 < 1, compounds are expected to have moderate solubility but inadequate absorption and brain
penetration. If the MW is less than 200 Da, paracellular transportation is more probable. At the same time, these
compounds show high kidney clearance. For 1 < log D7.4 < 3, the compound is ideally absorbed by passive transport.
In case of 3 < log D7.4 < 5, the compound is likely to have better permeability but weaker intestinal absorption
because of its weaker water solubility. For log D7.4 < 5, compounds are expected to show good membrane
permeability but weaker intestinal absorption, due to the poor solubility. For log D7.4 > 5, absorption is poor due to
low solubility; however, in this lipophilicity range metabolic (or hepatic) clearance is accelerated [77,78].
In absorption mechanisms, another two-parameter system is the Biopharmaceutics Classification System (BSC), which
analyses the role of oral absorption of the compound due to the relationship of solubility and permeability (Fig. 12).
Class II Class I
Lipophilic Drugs Amphiphilic Drugs

High
Permeability Solubility Ideal for oral absorption
enhancement strategy
Class IV Class III
Risk-philic Hydrophilic Drugs

Low
Permeability Poor in vivo – in vitro Prodrug strategy
correlations
Low High
Solubility Solubility
Figure 12: Biopharmaceutical Classification System (BSC)

Figure 12: Biopharmaceutical Classification System (BSC).
The proton-dissociation has an important role among the physicochemical properties of those compounds absorbed
in the brain. In general quasi-ideally lipophilic compounds are negatively charged at pH 7.4 (acidic) and show a
distorted BBB penetration profile.
A possible explanation is that the ratio of lipids with negatively charged moieties are generally higher in the BBB;
therefore, in contrast to other lipid membranes, the negative charges repel the negatively charged compounds more
extensively. At the same time, at pH 7.4 for positively charged basic compounds, this negative charge of the BBB is
attracted so that brain permeability is improved [79,80]. The relation between active transportation and the
physicochemical properties of lead-like compounds is discussed in the case of P-gp efflux transport. The P-gp efflux
of drug compounds is slightly dependent on their ionization state.
Regarding lipophilicity, accelerated efflux can be observed in the range of log P 3-5. Within this interval, basic
compounds tend to be transported by efflux in a linear proportion to their lipophilicity values [81].
The relation for drug partitioning is closely related to ionization state and lipophilicity. In general, basic compounds
(positively charged at pH 7.4) have increased partitioning volume, while acidic compounds (negatively charged at
pH 7.4) have decreased partitioning volume. This phenomenon can be explained by the tendency of negatively
charged compounds to bind to human serum albumin [77,82]. The relation between the partitioning and lipophilicity
properties of compounds can only be shown for basic and neutral molecules. For these compounds, the higher the
lipophilicity, the greater the partitioning volume [81,83].
It is not possible to describe metabolic pathways using only physicochemical properties. However, some related
clearance processes – firstly hepatic clearance, and secondly the inhibition of certain cytochrome P450 isoenzymes –
and the physicochemical properties of drug compounds have been shown to be connected. Decreased hepatic
clearance properties have been shown for acidic compounds, and slightly increased clearance has been observed for
basic compounds [84]. The relationship between lipophilicity and clearance has been well characterised for mainly
acidic compounds. For acidic compounds, increased lipophilicity led to decreased in vivo clearance [81]. For 2C9
CP450 enzymes, inhibition rose for neutral and acidic compounds. Moreover, in investigating lipophilicity
parameters, rising lipophilicity resulting in higher 2C9 isoenzyme binding was observed [85]. For CP450 3A4
isoenzymes, increased enzyme inhibition was found for neutral and basic compounds, while it decreased for acidic
compounds. For 3A4 isoenzymes, neutral and basic compounds resulted in the type of enzyme inhibition that is
increased by lipophilicity [86]. The physicochemical description of toxicological processes usually deals with the
hERG potassium channel causing QT-elongation, and phospholipidosis, which is related to membrane binding. In
studying the ionisation properties/abilities of compounds, hERG inhibition increased for compounds with basic
centres, and this risk is higher with the increasing number of basic centres [87-89]. In addition to increasing
lipophilicity, a higher probability of hERG enzyme inhibition was observed [88,90].
Phospholipidosis (PLD) is a lipid storage disorder characterized by the accumulation of phospholipids within cells,
and has been found to be induced by several drugs [91-93]. This excess accumulation of phospholipids can occur in
many organs, e.g. the liver [94], CNS [95], adrenal system [96] etc. Drug-induced phospholipidosis has been
observed as a distinct formation of lamellar inclusion bodies, which is thought to lead to cell dysfunction [97].
However, there is no evidence that lamellar inclusion bodies are linked to toxicity, or that they are a consequence of
drug actions. In general, drugs that induce PLD are referred to as cationic amphiphilic drugs (CADs), which
generally contain a hydrophobic part consisting of an aromatic ring, and a hydrophilic group with one or more
nitrogen groups bearing a net positive charge at physiological pH [93,98]. The positive charge, in combination with
the amphiphilic character, allows CADs to partition by electrostatic and hydrophobic interaction into cellular
membranes, in particular those containing anionic lipids (e.g. phosphatidylserine, cardiolipine). To this end, the
mechanistic basis of PLD induction has been suggested to be a complex formation with acidic phospholipids,
resulting in the inhibition of lysosomal phospholipid degradation by acidic phospholipase. Various physicochemical
approaches to predicting the PLD of drugs have been reported. Recently, Ploemen et al. developed a simple
approach, which employs log P and the dissociation constant of the most basic functional group (pKa – MB) [99]:
((pKa-basic)2 + (ClogP)2) ≥ 90 → predicted positive
provided pKa ≥ 8 and ClogP ≥1,
and Pelletier et al. modified the Ploemen model [100]:
((pKa-basic)2 + (ClogP)2) ≥ 50 → predicted positive
provided pKa ≥ 6 and ClogP ≥2.
As discussed above, physicochemical parameters play important roles for every ADME/Tox process. Neither of the
processes can be described by only one parameter, but require a combination of parameters. Various pharmacological
research centres have attempted to identify and interpret the correlation between physicochemical parameters, chemical
structure and ADME properties via complex models and combined graphical interpretations. Recently published
models are: the Golden Triangle for clearance and oral absorption, by Johnson et al. [101]; the in silico ADMET
Traffic Lights system, by Lobell et al. [102]; Interpretable AMDET Rules of Thumb systems by Gleeson et al.
[81,103]; radar plot visualisation of bioavailability by Faller and Urban [104]; and a number of comprehensive analyses
of publications by Waring [105], Keserű and Makara [106] and Leeson and Spingthorpe [107] for drug candidates, and
of publications by Wunberg et al. [108] and Manly et al. [109] for Hit-to-Lead processes.
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Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, 2011, 33-51 33

Ionization , log P/D,

Figure 1: ADME process and related physicochemical parameters.

This section concentrates on measurable physicochemical parameters such as ionization (protonation/dissociation)

IONIZATION OF DRUGS IN AQUEOUS SOLUTION (PKA)

HABH+ = HAB = A-B Kabasic > Kaacidic

HABH+ = A-BH+ = A-B Kabasic < Kaacidic

0.9 [AH] [A- ] 0.9 [BH+] [B]

Figure 2: Proton dissociation – pH function of acids and bases

AHBH+ ↔ A-BH+ ↔ A-B

Figure 3: Proton dissociaton – pH function of zwitterions

pKa acidic  pKabasic

log P0 = log [X0]oct/[X0]aq, where X is a generic ionisable molecule.

Figure 4: Four-equation partition model

Figure 4: Four-equation partition model.

Figure 5: log D – pH profile of monoprotic acids

1. Ionization equilibrium in aqueous media (“aqueous pKa”):

3. Partition of the anionic species between water and octanol:

4. Ionization equilibrium in aqueous media (“Scherrer pKa”):

By combining this equation with the four-equation model:

Table 1: Tissue specific log P models

log P Tissue specificity

Figure 6: Structure of propylene glycol dipelargonate (PDFP).

Figure 7: Phospholipid membrane – water tetrad equilibria.

Table 2: n-octanol – water and liposomal–water partition of ionisable drugs

Compound Log Poct0 log PoctI(+/-) log Pmem0 log PmemSIP

t R  t0 Cstationary Vstationary Vstationary

log P (or log D )  a  b  log k ' ,

PERMEABILITY OF DRUGS (PASSIVE DIFFUSION)

Figure 9: Millipore’s PAMPA system.

Donor well Membrane Acceptor well

BulkDonor UWLDonor UWLDonor BulkAcceptor

Table 3: Evolution of the PAMPA system

Membrane Buffer System Incubation parameters

Figure 11: Permeability – pH profile of ionizable drugs

PHYSICOCHEMICAL PARAMETERS IN ADME PROCESSES

Lipophilic Drugs Amphiphilic Drugs

Class IV Class III

Risk-philic Hydrophilic Drugs

Figure 12: Biopharmaceutical Classification System (BSC)

((pKa-basic)2 + (ClogP)2) ≥ 90 → predicted positive

provided pKa ≥ 8 and ClogP ≥1,

and Pelletier et al. modified the Ploemen model [100]:

((pKa-basic)2 + (ClogP)2) ≥ 50 → predicted positive

provided pKa ≥ 6 and ClogP ≥2.

[83] Ghafourian T, Barzegar-Jalali M, Hakimiha N, Cronin MTD. Quantitative structure-pharmacokinetic relationship

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