Expert Opinion on Drug Metabolism & Toxicology

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Crosstalk of physiological pH and chemical pKa

under the umbrella of physiologically based
pharmacokinetic modeling of drug absorption,
distribution, metabolism, excretion, and toxicity

Lu Gaohua, Xiusheng Miao & Liu Dou

To cite this article: Lu Gaohua, Xiusheng Miao & Liu Dou (2021): Crosstalk of physiological pH
and chemical pKa under the umbrella of physiologically based pharmacokinetic modeling of drug
absorption, distribution, metabolism, excretion, and toxicity, Expert Opinion on Drug Metabolism &
Toxicology, DOI: 10.1080/17425255.2021.1951223

To link to this article: https://doi.org/10.1080/17425255.2021.1951223

© 2021 The Author(s). Published by Informa Published online: 31 Jul 2021.

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EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY
https://doi.org/10.1080/17425255.2021.1951223

REVIEW

Crosstalk of physiological pH and chemical pKa under the umbrella of

physiologically based pharmacokinetic modeling of drug absorption, distribution,
metabolism, excretion, and toxicity
a b
Lu Gaohua , Xiusheng Miao and Liu Douc
a
Research & Early Development, Princeton, New Jersey, USA; bDrug Metabolism and Pharmacokinetics, GlaxoSmithKline, Collegeville, Pennsylvania,
USA; cSchool of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, Guangdong, China

ABSTRACT ARTICLE HISTORY

Introduction: Physiological pH and chemical pKa are two sides of the same coin in defining the Received 6 May 2021
ionization of a drug in the human body. The Henderson-Hasselbalch equation and pH-partition Accepted 30 June 2021
hypothesis form the theoretical base to define the impact of pH-pKa crosstalk on drug ionization and
KEYWORDS
thence its absorption, distribution, metabolism, excretion, and toxicity (ADMET).
Absorption, distribution,
Areas covered: Human physiological pH is not constant, but a diverse, dynamic state regulated by metabolism, excretion and
various biological mechanisms, while the chemical pKa is generally a constant defining the acidic toxicity (ADMET);
dissociation of the drug at various environmental pH. Works on pH-pKa crosstalk are scattered in the Henderson-Hasselbalch
literature, despite its significant contributions to drug pharmacokinetics, pharmacodynamics, safety, equation; model-informed
and toxicity. In particular, its impacts on drug ADMET have not been effectively linked to the physio­ drug development (MIDD);
logically based pharmacokinetic (PBPK) modeling and simulation, a powerful tool increasingly used in modeling and simulation;
model-informed drug development (MIDD). physiologically based
Expert opinion: Lacking a full consideration of the interactions of physiological pH and chemical pKa in pharmacokinetic (PBPK)
a PBPK model limits scientists’ capability in mechanistically describing the drug ADMET. This mini- model; pH-partition
hypothesis
review compiled literature knowledge on pH-pKa crosstalk and its impacts on drug ADMET, from the
viewpoint of PBPK modeling, to pave the way to a systematic incorporation of pH-pKa crosstalk into
PBPK modeling and simulation.

1. Introduction species to different extents in a medium with varying pH.

pKa is the pH of the medium where a monoprotic drug is
In the context of pharmacology, physiological pH and the
50% ionized.
acid-base dissociation constant (pKa) are two sides of the
Physiologically based pharmacokinetic (PBPK) modeling
same coin when considering the effects of ionization or pro­
and simulation of drug ADMET has been increasingly applied
tonation on drug absorption, distribution, metabolism, excre­
by biopharmaceutical companies and regulatory agencies in
tion/elimination, and toxicity (ADMET). Physiological pH and
drug discovery and development. As will be discussed, it will
chemical pKa are two necessary and sufficient components to
not be possible for a PBPK model to accurately predict the
define the drug ionization in the biological fluid and/or tissue.
pharmacokinetics (PK), pharmacodynamics (PD), safety, and
In general, the pH of a fluid (and a tissue or an organ as
toxicity if it is missing key interactions between the physiolo­
well) is defined by the concentration of hydrogen ions (pro­
gical pH and the chemical pKa therefore, lacking the funda­
ton, H+) within the fluid (and/or tissue/organ) only, with a pH
mental description of drug ionization in a specific fluid/tissue/
of 7 considered as neutral. However, a neutral pH does not
organ.
always exist in the human body which consists of various
The impacts of ionization on a drug’s ADMET properties
acidic or alkalic fluids, tissues, and organs. As a typical exam­
have been a subject of interest to the scientific community
ple, the plasma is weakly basic, with its pH carefully regulated
from the dawn of modern pharmacology. A good example is
around 7.4 (7.35–7.45). Indeed, human physiological pH is not
cocaine, the first local anesthetic which was discovered by
a constant, but a diverse and dynamic state regulated by
Albert Niemann in 1860 [2]. An early study by Bignon in
various biological mechanisms, mainly by the respiratory and
1892 showed that alkalization increased the activity of cocaine
renal systems [1].
solutions [3]. Both free base and ionized forms of cocaine are
In contrast, pKa is a key physicochemical parameter defin­
necessary for its activity as local anesthetic. In fact, cocaine
ing the tendency of a drug (molecule or ion) to keep a proton
enters nerve fiber as neutral (free) base and its cationic form
(H+) at its ionization center(s). By definition, the pKa is
blocks nerve conduction by interacting at the inner surface of
a unique parameter which is specific to chemical structure.
the sodium channel – an excellent example of how
An ionizable drug can dissociate into separate, charged

CONTACT Lu Gaohua Gaohua.Lu@bms.com Quantitative Systems Pharmacology & Physiologically Based Pharmacokinetics, Clinical Pharmacology &
Pharmacometrics, Research & Early Development, Bristol Myers Squibb, Princeton, New Jersey, USA
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
2 L. GAOHUA ET AL.
Article highlights
● pH-pKa crosstalk determines the drug ionization and further impacts
on drug ADMET.
● However, the state-of-the-art PBPK models have not incorporated the
pH-pKa crosstalk systemically and consistently in modelling ADMET.
● In this review, we have thoroughly compiled our knowledge from
literature on hitherto elucidated impacts of ionization on drug
ADMET.
● The review with detailed information can serve as a guidance for
considering pH-pKa crosstalk in PBPK modelling and simulation.
modulation of a drug’s ionization state can affect the PK and
PD relationship or pharmacology of the drug.
In the middle of 20th century, organic chemists and biolo­
gists found that the chemical and biological reactivities
depend upon the degree of ionization of both the drug and
the receptor [4]. Shore and coworkers in the United States
Food and Drug Administration (FDA) demonstrated the
dependency of gastric secretion of weakly acidic or basic
organic electrolytes upon the ionization constant of the com­
pound, now known as the pH-partition hypothesis [5–8].
Undoubtedly, the pH-partition hypothesis has been the theo­
retical base for understanding the passive permeation pro­
cesses of drug absorption, exsorption, disposition, and
excretion. Most, if not all, PBPK models (including the state-
of-art commercial software platforms GastroPlusTM, Simcyp®)
utilize this fundamental pH-partition hypothesis.
The impacts of drug pKa and physiological pH on drug
solubility and permeability have been extensively discussed
[9]. Charifson and Walters recently reviewed potential advan­
tages and disadvantages of both acidic and basic drugs, pro­
vided new analyses based on available public data, and
discussed the effects of ionization state on drug metabolism
and PK (DMPK) properties including solubility, permeability,
oral bioavailability, metabolism and clearance, tissue distribu­
tion, volume of distribution, and protein binding [10].
Although there are many publications on the influence of
pH-pKa crosstalk on drug ADMET, to our knowledge,
Figure 1. pH and pKa impacts on drug ADMET.
challenges remain for PBPK modelers when integrating many
different information sources into PBPK modeling and simula­
tion. The purpose of the current mini-review is to summarize
the impacts of pH-pKa crosstalk on drug ADMET at the highest
level, from the viewpoint of PBPK modeling and simulation.
Figure 1 shows pH and pKa impacts on drug ADMET.
2. Physiological pH in the body
In general, the human physiological pH for most tissues is
around 7 ~ 7.4 with some exceptions (Figure 2). For instance,
the stomach pH is 1 ~ 3, small intestine pH is 4 ~ 7, and blood
pH is 7.3 ~ 7.45. Therefore, the same drug may exist in
different ionic states in different regions of the body.
Elsewhere in the body, changes in pH tend to be much smaller
and show less deviation from the pH of blood and human life
requires a tightly controlled pH level in the serum of about 7.4
(a slightly alkaline range of 7.3 ~ 7.45) to survive.
Knowledge of tissue and body fluid physiological pH is
important, especially when developing PBPK model for drug
transport and delivery through different tissues, such as eye,
inner ear, brain, skin, lung, and tumor tissues. Typical pH
values of various human body fluids are listed in Table 1
[11–14]. Although new information is continuously emerging,
the most recent examples of reported values are compiled
here together with their original sources.
2.1. Tear
Tear pH was measured in 44 normal subjects by immersing the
lip of a micro-combination glass pH probe in the tear fluid in the
inferior cul-de-sac [13]. The normal pH range was 6.5 to 7.6, with
a mean value of 7. pH on the ocular surface was 7.11. Older
women had a more alkaline pH than other subjects. A pH shift
from acid to alkaline was observed throughout the day [15].
2.2. Ear
Various fluids in the ear have varying pH [16]. The cochlear
perilymph pH is 7.3, the same as the cerebrospinal fluid pH in
 EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 3

Figure 2. Physiological pH values in the human body.

Table 1. pH values of human body fluids. extracellular pH is about 7.4. Therefore, the pH gradient is
pH reversed in the tumor tissue.
Fluid Mean Range
Blood, arterial 7.424 7.386–7.462
Blood: red cells 7.209 7.175–7.243 2.4. Skin
Blood: plasma, arterial 7.39 7.35–7.43
Blood: plasma, venous 7.398 7.378–7.418 The skin pH is also well known to be acidic with values as low
Bile, hepatic 7.5 6.2–8.5 as 5 at the skin surface and still below 7 in the horny layer;
Bile, gall bladder 6.0 5.6–8.0 although in atopic dermatitis and contact dermatitis the mean
Breast milk 7.01 6.4–7.6
Cerebrospinal fluid 7.349 7.327–7.371 values reported are a little higher (5.3 to 5.5 instead of 5–5.1),
Faces, adult 7.15 5.85–8.45 they are still quite acidic [18].
Faces, infant 4.9 4.6–5.2
Gastric juice, male 1.92 ± 1.28
Gastric juice, female 2.59 ± 2.08 2.5. Nervous system
Gastric juice, child 3.27 0.9–7.7
Gastric juice, newborn 2.52 1.2–7.4 Differences have been reported between general cellular and
Pancreatic juice 7.5–8.8
Saliva 6.4 5.8–7.1 interstitial pH and cerebrospinal fluid (CSF). The pHs for the
Semen 7.2–8.0 neural interstitial fluid, intracellular fluid, and cerebrospinal
Sweat 4.0–6.8 fluid are 7.44, 7.05, and 7.31, respectively [19,20].
Synovial fluid 7.434 7.31–7.64
Tear 6.5–7.6
Urine 5.75 4.8–7.5
Vaginal secretions 3.5–4.0 2.6. Lungs
The pH in the lung mass is 6.69 while the arterial blood pH is
between 7.38 and 7.43. The composition of the epithelial
the brain, while the cochlear endolymph pH is 7.5. In contrast, the lining fluid is mainly water (96%), salts, phospholipids, pro­
endolymph in the endolymphatic sac varies between 6.7 and 7.1. teins, and mucins with a pH about 6.6 in healthy indivi­
duals [21].
2.3. Tumor
Tumor tissue often has an acidic extracellular pH [17]. For
2.7. Milk
instance, in human breast cancer, the intracellular pH is 7.15 A dynamic change in human milk pH has been observed [22].
and 7.37 while the extracellular pH is 6.99 to 6.8. Note in The mean pH decreased from 7.45 for colostrum to 7.04 dur­
normal tissue, the intracellular pH is about 7 and the ing the second week of lactation. Thereafter, the pH of milk
4 L. GAOHUA ET AL.
remained between 7 and 7.1 until 3 months postpartum and
then increased gradually to 7.4 by 10 months.
2.8. Species differences
There exist species differences in physiological pH. It was
observed, for example, that gastric pH in the beagle dog was
significantly higher than that in human (1.8 vs. 1.1) and the
fasting intestinal pH in dog was also consistently higher than
that in human (7.3 vs. 6) [23]. McConnell and coworkers mea­
sured the mouse and rat gastrointestinal pH and reported the
stomach pH was 3.0 (fed) and 4.0 (fasted) for mouse, 3.2 (fed)
and 3.9 (fasted) for rat [24]. In contrast to dogs, the mean
intestinal pH in rodents was lower than that in man (pH< 5.2
in the mouse; pH< 6.6 in the rat). Thence, the difference of
ionization needs to be considered when translating the PBPK
models from preclinical species to human.
3. Distribution of pKa of marketed drugs
Lombardo et al. compiled a dataset consisting of 1,352 com­
pounds including mostly marketed drugs with a wide range of
fundamental physicochemical characteristics [25]. Among
those compounds with calculated pKa values, there were 457
neutral, 313 anionic, 472 cationic, and 97 zwitterionic com­
pounds. Clearly, most of the compounds are ionizable.
For a set of 661 drugs containing a single ionizable group
from the ChEMBL-18 database, there were 237 acids and 424
bases spanning a pKa range from 2 to 10 [10]. It was observed
that the acidic compounds completely ionized at pH greater
than 7, and basic compounds exhibit the same trend of dis­
sociation for pH less than 7. Interestingly, the authors noticed
the number of neutral compounds reported in the Journal of
Medicinal Chemistry decreased while the numbers of both
acids and bases were consistently increasing in the past two
decades.
The overall distribution of acid and base pKa values from
another set of 582 drugs was also reviewed [26]. Those com­
pounds were grouped into CNS and non-CNS drugs to inves­
tigate where differences existed. The distribution of pKa values
for single acids differed between CNS and non-CNS substances
with only one CNS compound having an acid pKa below 6.1.
The basic CNS substances also showed a marked cut-off with
no compounds having a pKa above 10.5. In a contemporary
set of 907 drugs, it was found that 64% of these compounds
contained an ionizable group [27]. Within this group of ioniz­
able compounds, 34% contained a single basic group while
only 20% contained a single acidic functional group. Oral
drugs showed that 78.6% contained an ionizable group,
while only 11.9% were neutral [27,28].
According to Manallack, the pKa distributions of drugs
seem to be determined by two main factors. The first is related
to the nature and frequency of occurrence of the functional
groups that are commonly observed in pharmaceuticals and
the typical range of pKa values they span. The other factor
concerns the biological targets these compounds are
designed to hit. This makes sense as the environmental pH
will define the drug ionization, which affects the drug PK and
PD (either the free or the ionized, or both, to drive the PD
effects).
4. pH effects on physicochemical properties
4.1. Crosstalk between pKa and pH
The Henderson-Hasselbalch equation below is the backbone
of pKa-pH crosstalk.
� �
½proton acceptor�
pH ¼ pKa þ log
½proton donor�
Based on the Henderson-Hasselbalch equation, the fraction
unionized or ionized is only defined by the pH of the drug-
dissolving medium and the pKa of the drug. In other words,
the degree of ionization of a drug in its solution depends
upon only two factors, namely the pH of the solution and
the pKa of the drug [4]. The pKa is a constant for any ionizable
drugs and thus, if the pH of the solution is controlled, as in the
case of the human physiological system, the degree of ioniza­
tion depends only on the nature of the ionizable drug being
investigated.
When 50% of an acidic or basic drug is ionized, its environ­
mental pH equals to its pKa. For the drugs with pKa’s between
6 and 8, they are always in equilibrium with at least 10% of its
ionic form at the physiologically important pH of 7. After
passing from a pH value which is one unit on either side of
the pKa to a pH value one unit on the other side, a given drug
is converted 10-fold from a substantially ionized to
a substantially non-ionized condition. For instance, for
a basic drug with pKa of 7, changing the environmental pH
from 6 to 8 results in its unionized from 9% to 91%. Hence,
even quite small changes in the pH value of a solution can
produce a very large alteration in the degree of ionization of
a drug, thus potentially resulting in a significantly modified
biological effect.
As aforementioned, most drugs are weak acids or weak
bases (with pKa around the physiological pH) and they exist
in physiological fluids, tissues, and organs at an equilibrium
between unionized and ionized forms. In general, only neutral
(free, unbound unionized) drug molecules can passively pene­
trate the physiological membrane, and at equilibrium, the
concentrations of the unbound unionized species are equal
on both sides. Increased accumulation of drug on the side of
a membrane where pH favors greater ionization of drug has
led to the pH-partition hypothesis. The majority of evidence
supporting the pH-partition hypothesis was from studies of
gastrointestinal absorption, renal excretion, and gastric secre­
tion of drugs.
4.2. Lipophilicity
Lipophilicity is a molecular parameter describing the partition
equilibrium of a solute between water and an immiscible
organic solvent such as octanol. Lipophilicity is usually mea­
sured by the partition coefficient (P) when the compound is in

its unionized form. Partition coefficients are obtained as the
logarithm (logP) of the ratio of concentrations at equilibrium
between the organic solvent and the aqueous water [29].
The most commonly used measure of lipophilicity is the
n-octanol/water partition coefficient (logPo:w), which when
written in this way refers to the neutral form of the solute.
Frequently, the apparent distribution coefficient (logD) at
a specific pH (such as 7.4) is used for ionizable drugs. Unlike
logPo:w, which is valid only for neutral species, logDpH refers
to a pH-dependent mixture of all neutral and electrical species
present at that pH. Therefore, logPo:w is always larger than
logDpH as the ionized species tends to stay in water rather
than in the organic solvent. As a rule of thumb, if logDpH is
measured at different pHs (such as 2, 4, 7, and 10), the highest
logDpH is generally used as the surrogate of logPo:w.
From logPo:w and logDpH profile, the drug class (acidic,
basic, or neutral) and its pKa can be estimated using the
ionization fraction defined by the Henderson-Hasselbalch
equation.
4.3. Solubility
In general, the salt form of an organic compound is more
soluble in the aqueous medium than its unionized form,
thus switching from free base to salt form is one of the
effective approaches to the enhancement of the dissolution
rate of drugs with low solubility.
Indeed, an analysis of the aqueous solubility data based on
37,100 compounds extracted from PubChem indicated that
the ionizable compounds (acids, bases, and zwitterions) have
better solubility than neutrals and that the basic compounds
may even possess a slight (yet statistically significant) advan­
tage in solubility over acidic molecules [10]. The latter is
possibly due to an overall greater extent of ionization for
most bases at physiological pH. Solubility affects almost all
aspects of drug behavior and characterization from in vitro
assay reproducibility to in vivo oral bioavailability and
formulatability.
As ionization depends on the pH and the pKa, the aqueous
solubility of an ionizable drug is pH dependent. For an acidic
compound, the solubility increases when the pH in the solu­
tion is increased as more drug molecules are ionized in alka­
line conditions. In contrast, solubility of a basic compound is
increased when the pH of the solution is decreased. For
ampholyte/zwitterion compounds, the drug molecules are
normally non-charged at the physiological pH, resulting in
a low solubility. An increase in solubility of ampholyte and
zwitterion compounds can be observed when the solution pH
is significantly shifting high or low. Hence, pH-solubility pro­
files can be utilized to identify the type of ionization (acidic or
basic) and the intrinsic solubility of free base.
Local pH has a significant impact on drug PK through pH-
dependent solubility. For instance, phenytoin sodium is formu­
lated as an injection at pH 12 adjusted with sodium hydroxide
[30]. On injection of phenytoin sodium, the interstitial fluid
quickly reduces the pH of the injectable from 12 to normal
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 5
levels around 7, and phenytoin precipitates due to its poor
water solubility. A clinical implication is that a fall in plasma
levels may occur when patients are switched from oral to intra­
muscular administration. The drop is caused by slower absorp­
tion from the injection site, as compared to oral administration.
As a result, it may then take several days for the dose to be fully
absorbed after intramuscular injection [31].
Most of published data have clearly shown that weakly
basic drugs, which have low solubility at high pH, could
have impaired absorption in patients with high gastric pH,
thus leading to reduced and variable bioavailability. As this
reduction in exposure can lead to significant loss of efficacy, it
is imperative to understand the behavior of the compound as
a function of stomach pH to inform of any risk of bioavail­
ability loss in clinical studies [32].
One example is gefitinib as the first-line of non-small cell
lung cancer (NSCLC) treatment. It was shown that the clinical
outcomes (progression-free survival and overall survival) are
significantly affected when gastric acid-suppressing agents
(such as Histamine-1 receptor antagonists or proton pump
inhibitors) are co-administered [33,34]. Gefitinib is a weak
base with a pH-dependent solubility, which leads to its higher
solubility when the pH is less than 5. Thus, co-administration
of gastric acid suppressants would reduce gefitinib solubility.
Recently, FDA published a draft guidance on the evaluation
of gastric pH dependent drug interactions with acid-reducing
agents (ARA), mainly considering the pH-dependent solubility
of weakly basic drugs [35].
5. pH effects on drug absorption
5.1. Absorption process
Oral drug delivery is still the preferred and most convenient
route for systemic administration, due to ease of patient com­
pliance, cost-effectiveness, and flexibility in designing dosage
forms. However, oral drug absorption is a complex process
that is influenced by many factors, such as the properties of
the drug, its dosage form, and the physiological state of the GI
tract of patients. Figure 3 shows the drug absorption process.
After oral administration, the solid drug form (such as tablet or
capsule) disintegrates into granules and then further deaggre­
gates as fine particles. The drug leaves the dosage formulation
(tablet, capsule, granule, or fine particle) and dissolves into the
aqueous digestive fluids. All ionizable drug molecules will be
ionized, as determined by its chemical pKa and the surrounding
physiological pH following the aforementioned Henderson-
Hasselbalch equation. Both the ionized and unionized molecules
can bind to the fluid components, which include bile micelles and
food contents, a phenomena known as solubilization. All the
bound and unbound, ionized and unionized drug molecules can
diffuse and reach the GI mucosal membrane which is covered by
a thin unstirred water layer with a relatively constant pH
(Figures 3, 4).
Based on the classic pH-partition hypothesis, only the free
(unbound unionized) molecules can passively penetrate across
6 L. GAOHUA ET AL.
Figure 3. Drug absorption process.
the apical membrane of enterocyte. However, the unbound
ionized drug molecules can also penetrate across the cell
membrane of enterocyte, which has −40 mV membrane
potential, based on the Nernst-Planck equation [36].
Depending on pKa and pH, drug molecules may be fully
ionized within the unstirred water layer on the apical surface
of enterocytes. As the permeation is defined by the product of
the concentration gradient and the permeability-surface area,
if the permeability of ionized species is reasonably good,
although lower compared to the unionized counterpart,
there will still be reasonable absorption of the fully ionized
drug molecules when there is a high gradient of unbound
ionized concentration across the enterocyte membrane.
Simultaneously, the absorption process itself is influenced
by the nature and surface area of the GI mucosal membrane,
which varies from the stomach to the rectum, and by the
physicochemical properties of the luminal content, when
drug in solution or in solid form moves along the GI tract
with the luminal content at a variable speed. It is generally
assumed that drug absorption in the stomach is minimal due
to its small surface area; however, there is significant absorp­
tion from the stomach for acidic drugs as their free (unionized)
concentrations are high in the acidic stomach [5,6].
The following sections will discuss how physiological pH
and chemical pKa interact with each other to define the
absorption process.
5.2. pH in GI
5.2.1. Gut axial pH
Luminal pH changes dramatically along the whole GI. Evans
et al. measured the GI pH of 66 healthy subjects using a pH
sensitive radio-telemetry capsule passing freely through the GI
tract [37]. Signals were recorded with a portable solid-state
receiver and recording system, enabling unconstrained mea­
surements with normal ambulatory activities for up to
48 hours during normal GI transit. Capsule position in the
gut was monitored by surface location using a directional
detector. The stomach pH values of all subjects were highly
acidic (range 1 ~ 2.5). The mean pH in the proximal small
intestine was 6.6 for the first hour of intestinal recording, and
the mean pH in the terminal ileum was 7.5. There was a sharp
fall in pH with a mean of 6.4 as the capsule passed into the
cecum in all subjects. The pH then rose progressively from the
right to the left colon with a final mean value of 7.
A separate study had a similar observation that the intra­
luminal pH was rapidly changed from highly acid in the sto­
mach to about pH 6 in the duodenum [38]. The pH gradually
increased in the small intestine from pH 6 to about pH 7.4 in
the terminal ileum. The pH dropped to 5.7 in the cecum and
then gradually increased to pH 6.7 in the rectum.
Avdeef compiled the GI pH from various resources for
fasted and fed states, where the terminal ileum has the high­
est pH of 8 in both fasted and fed states and the rectal pH
ranges from 5 to 8 (Figure 4) [39].
5.2.2. Gut radial pH
While the luminal pH could be significantly affected by the
luminal component, the mucus layer between the luminal
fluid and the microvilli cells has a relatively constant pH,
ranging 5.2 ~ 6.2 (Figure 4) [39]. The thickness of the unstirred
water layer is about 30 ~ 100 μm in vivo and 300 ~ 700 μm in
isolated tissue, significantly larger than the sizes of drug mole­
cules, micelles, and some fine particles. Previously, it was
assumed that the particles did not exist in the mucus layer,
as shown in Figure 3. However, nano- and/or micro-size drug
particles could enter the mucus layer, resulting in a significant
decrease of the distance of drug molecule diffusion across the
unstirred water layer before getting absorbed, known as ‘par­
ticle drifting effect’ [40].
Since the mucus layer has a local pH independent on the
luminal pH, ionization within the mucus will be different from
that in the luminal fluid. Passive permeations of unbound
unionized and/or unbound ionized drug molecules across

Figure 4. GI physiological pH.
the apical membrane are determined by the mucus environ­
ment (binding and ionization), which is relatively stable, rather
than the luminal environment which is changing dynamically.
5.2.3. Varying GI pH
5.2.3.1. GI pH and gender. Gender difference in the small
intestinal pH was observed [41]. Gastric fluids are generally
more acidic in males than females (pH 1.92 in males vs. pH
2.59 in females), and the basal and maximal flow of gastric
fluid and acid secretion are both higher in men [42]. In
a sample of 365 healthy subjects, the average basal stomach
pH was 2.16 in men and 2.79 in women [43]. Such gender
difference in stomach pH is mostly due to the gender differ­
ence in stomach juice secretion. Healthy women secret sig­
nificantly less basal and pentagastrin-stimulated acid than
men, with a median 24 hour integrated acidity of 485 mmol/
h vs. 842 mmol/h [31].
5.2.3.2. GI pH and age. Russell et al. measured gastric and
duodenal pH in 79 healthy, elderly men and women (averaged
75 years old) under both fasted and fed conditions [44]. It was
observed in nine subjects that their fasted stomach pH was
greater than 5 and that in five of these subjects the stomach
pH remained greater than 5 postprandially. The gastric pH
values of approximately 50% of elderly subjects decreased
more slowly than in young subjects after administration of
a large meal. Therefore, the drugs and/or formulations that
need an acidic gastric environment for dissolution and/or
release may be poorly assimilated in elder subjects.
In 12 healthy children aged 8 ~ 14 years, the mean fasting
stomach pH was 1.5 and the duodenal pH was 6.4 [45]. The pH
gradually rose down the small intestine, reaching a peak value
of 7.4 in the distal ileum, then dropped to 5.9 in the cecum but
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 7
increased to 6.5 in the rectum. In 11 healthy adults, the median
pH was 7 in duodenum and dropped to pH 6.3 in the proximal
part, but rose to 7.3 in the distal part of the small intestine [46].
In the premature infants, the pH level of gastric contents
under fasting condition was 3.35. It increased to 7 after feed­
ing with Pre-Gallia formula or human milk and then decreased
from 7 to a minimum pH value of 3.35 [47]. These pH values
suggest either a high buffering capacity of the meal or that
the gastric acid secretion levels are not very high in premature
infants and contribute very little to the overall volume of the
gastric contents during the digestion period.
5.2.3.3. GI pH and food. Food intake stimulates GI secretions
including hormones and bile salts, which lower gastric pH, delay
stomach emptying and increase GI transit time. While feeding
can introduce a dynamic change to stomach pH within minutes
to hours, diet itself can change GI pH in the long term. In fact,
subjects on a vegan or vegetarian diet showed significantly
lower stool pH than those control subjects consuming ordinary
omnivorous diet (6.3 for vegan, 6.6 for vegetarian vs. 6.8 for
control) [48]. However, limited data on stomach pH has been
reported for vegan or vegetarian diets.
5.2.3.4. GI pH and drug. Many weakly basic drugs exhibit
pH-dependent solubility and are therefore subject to drug–
drug interaction mediated by stomach pH that can be mod­
ified by the co-administration of gastric acid reducing agents
(ARAs), such as Histamine H1-receptor antagonists and proton
pump inhibitors (PPIs) [49]. These interactions can sometimes
be clinically relevant – exposures of weakly basic drugs can be
reduced by an order of magnitude resulting in loss of efficacy.
For instance, the exposure of erlotinib decreased 50% when it
was co-administered with omeprazole [50]. Erlotinib labeling
8 L. GAOHUA ET AL.
recommends patients avoiding concomitant use of erlotinib
and PPIs if possible. FDA recently released its draft guidance
about drug–drug interaction mediated by stomach-pH chan­
ging due to ARAs [35].
Compared to the Histamin H2-receptor antagonists, PPIs
represent the gold-standard therapy in the gastric acid-
related disorders [51]. Omeprazole (a weakly basic compound,
pKa ~4) is a gastric anti-secretory agent. At low stomach pH,
greater than 99% omeprazole is ionized. In a study with eight
subjects treated with 40 mg QD for 5 days, the gastric pH rose
from 1.9 to 5 with morning dosage or 4.5 with evening
dosage, corresponding to a greater than 99% reduction in
hydrogen ion activity [52]. It was observed that the relative
bioavailability of omeprazole increased 2 ~ 3-fold from day 1
to day 5 of treatment, suggesting that increased absorption of
this acid-labile drug occurs with increasing inhibition of acid
secretion. PPIs, acting as perpetrators by gastric acid suppres­
sion, can inhibit drug oral absorption, for instance, the expo­
sures of atazanavir dropped 95% when it was co-administered
with lansoprazole [53].
Due to the serious clinical consequences of pH-dependent
drug–drug interaction, prediction methods would be crucial to
assess the drug–drug interaction risk associated with
a particular victim drug. Recent publications have presented
various methods applied across a range of drugs to simulate
gastric pH-dependent drug–drug interaction [54] as well as
food effects [55,56]. Oral absorption modeling is becoming
more impactful to respond to regulatory questions.
5.3. pH-triggered disintegration/deaggregation
Disintegration is a physical process related to the mechanical
breakdown of a tablet into fine particles/granules before dis­
solving (Figure 3). Disintegration and its following disaggrega­
tion help to increase the surface area of particles in contact
with the medium, therefore increasing the dissolution rate
from the solid form [57].
In an in vitro study about the influence of the medium pH
on the disintegration time of commercially available phos­
phate binder formulations, it was observed that the pH sig­
nificantly affected in vitro disintegration in the majority of
phosphate binders tested [58]. In this study, seven calcium
carbonate tablet formulations, two calcium acetate tablet for­
mulations, three aluminum hydroxide tablet formulations, and
three aluminum hydroxide capsule formulations were tested
at three pH conditions (distilled water, pH 5.1; simulated gas­
tric fluid, pH 1.5; and simulated intestinal fluid, pH 7.5) using
the USP standard disintegration apparatus at the time. It was
shown that 9 out of the 15 products tested were sensitive to
changes in pH, showing significant differences in disintegra­
tion time. The shortest disintegration time of 1 min was
observed in all three of the aluminum hydroxide tablets at
all pH tested, whilst the longest disintegration time of 99 min
was observed in one of the 3 aluminum hydroxide capsules in
the distilled water at pH 5.1. This study reveals how the
disintegration time can vary depending on the immersion
medium pH.
The pH change along the GI tract from pH of 1.5 at the
stomach to pH 7 at the colon has been used in colonic drug
delivery, using pH-responsive polymer coatings. For instance,
Eudragit S (a polymethacrylic acid methyl methacrylate ester
copolymer with a dissolution threshold of pH 7) is introduced
to effect release the drug substance from the formulation as
the intestinal pH increases distally to a maximum at the ileo­
cecal junction [59].
5.4. pH and drug instability
Drug stability investigation is one of the most important compo­
nents of drug quality evaluation. Hydrolysis is one common
degradation reaction and source of instability. Water, either as
a liquid solvent or from moisture in the air, contacts the pharma­
ceutical dosage forms to some degree, thence the potential for
this degradation pathway exists for most drugs and excipients. In
a study of the hydrolytic behavior of pravastatin at different pHs
and temperatures, it was observed that degradation of pravas­
tatin was dramatically influenced by both pH and temperature
[60]. At a high temperature of 80°C, the degradation constant
decreased between pH 3 ~ 9 although it increased between pH 9
and 12. Therefore, it is reasonable to conclude that, within the
physiological pH range, the stability of pravastatin increases
concomitantly with the increasing of pH. It should be noted
however that the result may not be generalizable to other drugs.
5.5. pH and intestinal microflora
The intestinal microflora is a complex ecosystem consisting of
over 400 bacterial species within the lower bowel. It was shown
that under in vitro conditions, pH was the strongest driver of
microbial community structure and function and microbial and
metabolic interactions among pH-sensitive fermentative species
[61]. The balance between bicarbonate alkalinity and formation
of fatty acids by fermentation determined the pH, which con­
trolled microbial community structure.
5.6. Modeling of dissolution rate
In most oral absorption models, the intrinsic dissolution rate of
a drug is described by the classical Noyes-Whitney equation or
Nernst-Brunner equation [62]. However, these equations were
developed for a planar surface where the concentration gra­
dient in the diffusion layer is linear at steady state. To allow for
the fact that the concentration gradient around a spherical
particle is not linear under pseudo-steady-state conditions,
Wang and Flanagan proposed a general solution of the diffu­
sion layer model (DLM) for dissolution from mono-dispersed
spherical particles under sink and non-sink conditions, and the
model was validated experimentally [63,64].
When applying the Wang and Flanagan DLM to describe
the drug dissolution in GI luminal fluid, the ionization of
a drug within the luminal fluid plays two significant roles.
First, the driving force for drug dissolution in the DLM is the
concentration gradient between the aqueous solubility at the
surface of particles and the bulk concentration in the luminal

fluid. The aqueous solubility at the surface of particles
depends on the pKa of the drug and the micro-
environmental pH at the surface of particles, following the
aforementioned Henderson-Hasselbalch equations. Secondly,
the effective diffusion coefficient used in the DLM to define
the dissolution rate is also affected by the ionization, because
the binding of ionized drugs to the bile micelle is supposed to
be lower than their unionized counterpart. Therefore, if the
drug is highly ionized, the diffusion coefficient may be lower.
The total impact of ionization on dissolution rate is case-by-
case, as the solubility and the diffusion coefficient show dif­
ferent directions when pH is changing.
5.7. Passive permeability
Based on the pH-partition hypothesis, only free (unbound
unionized) drug can passively penetrate across the cell mem­
brane. It is generally accepted that the unbound unionized
molecule has higher permeability (normally 3 ~ 4 log unit)
than its ionized counterpart [65]. Hence, to get across most
membranes, the drug must be relatively nonpolar.
Although the penetration of unbound unionized species
would be higher than the unbound ionized species, the
unbound ionized drug molecules do penetrate across the
cell membrane of enterocytes, which have −40 mV mem­
brane potential, following the Nernst-Planck equation.
Ionized molecules at physiological pH tend to interact with
negatively charged lipid membranes, generally resulting in
a low permeability in an order of neutrals, bases, zwitter­
ions, and then acids [10]. Increasing lipophilicity may
increase permeability, especially for ionizable drugs, i.e.
acids, bases, and zwitterions.
The penetration of unbound unionized drug molecules
across cell membranes, specifically the apical membrane of
the enterocytes in the GI tract, is also dependent on the
gradient of unbound unionized concentration across the
membrane. Due to the constant pH gradient between the
unstirred water layer and the intracellular fluid of the enter­
ocytes (~6.5 vs. ~7), the weakly basic drugs are more ionizable
in the unstirred water layer than in the intracellular fluid. The
difference results in about 0.5 log unit (~3-fold) high ionized
fraction of drug in the unstirred water layer, even though the
unbound unionized drug concentration in the unstirred water
layer is in equilibrium with that in the enterocyte. Therefore,
the unstirred water layer with a constant pH of 6.5 could be
a natural barrier of the enterocytes.
Notably, the drug penetration across the apical membrane
of enterocyte is also dependent on the drug binding in the
unstirred water layer and in the enterocyte. If the drug is
significantly bound in the enterocyte such as by lysosomal
trapping, it is expected that the unbound unionized fraction
will be lower in the enterocyte than that in the unstirred water
layer with less binding proteins and therefore resulting in an
accumulation of drug within the enterocyte. Resultantly, low
Cmax and large Tmax will be observed in the plasma concentra­
tion-time profile, though the drug has been well penetrated
from the gut lumen into the enterocyte.
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 9
6. pH effects on drug distribution
6.1. Distribution process
After being absorbed from the GI luminal fluid into the sys­
temic circulation, the process of drug movement from blood
into various tissue and organ compartments like the interstitial
space and the intracellular space is called drug distribution,
another important process resulting in exposure of the target
organ to the drug. Various factors influence the dynamics of
drug distribution, including cardiac output, regional blood
flow, alterations in capillary permeability, plasma protein bind­
ing, and affinity of tissue protein binding. Active transporters
and metabolic enzymes may contribute to the drug distribu­
tion and disposition within the tissue/organ as well.
Drug distribution within plasma, tissue, and organ is pre­
dominantly determined by the chemical pKa and the physio­
logical pH if the drug is not a substrate of the transporter or
enzyme. As mentioned previously, the pH within the human
body is heterogeneous, with the plasma pH slightly varying
around 7.4, while local pH at various tissues can vary signifi­
cantly (Figure 2). Within a solid tissue such as the liver, the
extracellular pH is almost the same as that of the perfusing
plasma; however, the intracellular pH is lower, around 7, and
the lymphatic pH is between 7 and 7.4. Due to the ionization
difference across the cell membrane and based on the pH-
partition hypothesis, it is expected that cations accumulate
intracellularly and anions extracellularly. At steady state, how­
ever, the free (unbound unionized) concentration should be in
equilibrium across any semi-permeable membranes, regard­
less of the type of tissue and organ, cell or its organelle.
Notably, different tissues and fluids within the human body
have different pH, and these are mostly time-dependent as
well (as discussed in Section 2). These pH differences are
important especially for tissue-targeted drug delivery. It is
critical to consider the ionization and pH gradient when devel­
oping PBPK models for tissue-targeted drug delivery.
6.2. Ionization and tissue distribution
Only minor differences were observed between the binding
of acidic, basic, neutral, and zwitterionic substances to var­
ious tissues, although basic drugs tend to be stored in
tissues with a pH that is lower than their pKa values (e.g.
lung) [10]. For tissues with a lower pH, a greater fraction of
ionized basic species can electrostatically interact with the
negatively charged cell constituents (i.e. membrane phos­
pholipids and/or acidic cellular compartments in which
accumulation may occur). Binding of bases was stronger to
hepatocytes compared to neutral or acidic molecules for
a given lipophilicity, although tissue distribution of basic
drugs is highly dependent on lipophilicity. Only minor dif­
ferences in binding to brain tissue were found between
acidic, basic, neutral, and zwitterionic substances, which
were more influenced by lipophilicity. This is in contrast to
overall CNS penetration for which basic substances showed
greater brain exposures than neutrals followed by zwitter­
ions and then acids [26].
10 L. GAOHUA ET AL.
6.3. Tissue/plasma partition coefficient (Kp) prediction
The pH of intracellular fluid is typically slightly lower than that
of the extracellular fluid (approximately 7 vs. 7.4) (Figure 5).
This drives a slight pH partitioning in accordance with the pKa
of the drug and the Henderson-Hasselbalch relationship.
Likewise, mammalian cells maintain an electric potential
(approximately −40 mV) which can drive electrochemical par­
titioning in accordance with the degree of ionization and
relative intrinsic permeability of the neutral and ionized
species.
Various approaches have been applied to define the tissue/
plasma partition coefficient (Kp) and the steady-state distribu­
tion volume (Vss), two key parameters used in PBPK modeling
and simulation. The mechanistic model of Kp prediction was
first developed by Poulin and Theil based on the equilibrium
of unbound concentration between plasma and tissue, with­
out consideration of ionization difference between plasma
and tissue [66]. The model was later expanded by Rodgers
and Rowland to incorporate the ionization difference in the
intracellular vs. extracellular space, following the pH-partition
hypothesis [67–69]. Depending on the intracellular pH, which
is about half log unit lower than plasma pH, positively charged
cations accumulate intracellularly. Furthermore, the intracellu­
lar acidic phospholipid is negatively charged; therefore, an
electrochemical interaction with the positively charged cations
introduces more accumulation of basic compounds within
a cell. In the method of Rodgers and Rowland, the ratio of
unbound unionized concentration in the intracellular vs. that
in the extracellular space, known as Kpuu, is 1 at the steady-
state, in accordance with pH-partition hypothesis.
Additional mechanisms can be incorporated in the predic­
tion of Kp and Vss. One example is the impact of electrostatic
potential of cell membranes (Figure 5). A negatively charged
cell membrane will benefit the passive permeation of posi­
tively charged cations into the intracellular space [70]. In this
case, Kpuu is larger than 1 for basic compounds and less than 1
for acidic compounds. In other words, the cell membrane
potential and the passive permeability of ionized compounds
would result in a high Vss predicted for basic compounds but
Figure 5. Physiological pH in tissue compartments.
a low Vss for acidic compounds, compared to the classic
Rodgers and Rowland’s method.
Acids generally have lower Vss values, also due to higher
plasma protein binding. Basic molecules often have higher Vss
values (due to lower protein binding) favoring increased half-
life. These compounds can show significant inter-organ varia­
tion [10].
6.4. Ion trapping
Another example to add an extra complex mechanism to Kp
prediction is to consider the ionization difference in the intra­
cellular organelles and the intracellular plasma due to their
different pH (Figure 5). A typical phenomenon is known as
lysosomal trapping.
Lysosomal trapping of ions has been considered as one of
the major mechanisms for high Vss of basic compounds [65].
Both GastroPlusTM and Simcyp® have incorporated the lysoso­
mal trapping into their mechanistic models to predict Kp and
Vss for ionizable compounds.
As evidenced by the hepatic accumulation of chloroquine,
the acidic interior of lysosomes is able to trap the positively
charged cations [71]. However, it is important to consider that
the accumulation of basic compounds into lysosomes would
raise the intra-lysosomal pH; therefore, the capability of lyso­
somal accumulation of these basic compounds will be
reduced when those cations accumulate within lysosomes.
Although greater pH and electrochemical partitioning can
occur in subcellular compartments where larger gradients
exist (lysosome pH 5, mitochondrial membrane potential of
approximately −170 mV), the impact of these effects to overall
Vss can often be negated by the dominant relative effect of
the plasma unbound fraction (fup) and the relatively small
aqueous volumes of these intracellular organelle spaces [72].
6.5. pH and fup
General trends for plasma protein binding are well estab­
lished, in order from greatest to least: acids, neutrals, zwitter­
ions, and then bases [10]. Albumin and alpha-1-acid

glycoprotein are major drug-binding proteins in serum.
Albumin is an alkaline protein, so acidic and neutral drugs
primarily bind to it while basic drugs are predominantly
bound to alpha1-acid glycoprotein, and this binding is often
high and is very pH sensitive. Since acids typically have higher
plasma protein binding and thus lower distribution volume,
they may require high metabolic stability in order to obtain
acceptable half-lives. Increasing lipophilicity may also increase
protein binding regardless of ionization class.
In general, the unbound concentration in the whole blood
or in the plasma is the most important determinant of PK/PD
of a drug. Many PK analyses require unbound blood or plasma
concentrations, including prediction of clearance, volume of
distribution, drug–drug interactions, tissue-specific uptake,
and so on [73].
Plasma protein binding, i.e. the free fraction fup, is pH-
dependent. Based on literature data, Hinderling and
Hartmann have analyzed the relationship between unbound
fraction and pH [74]. The basic drugs showed a consistent
behavior with the unbound fraction decreasing with increas­
ing pH, and a large majority of the acids displayed pH-
dependent binding as well – the binding increased for some
and decreased for others with increasing pH. The authors also
observed a large difference in the pH sensitivity of the plasma
protein binding among individual compounds. The unbound
fraction in plasma for some bases and acids increased up to
136% and 95%, respectively, at pH values seen in severe
acidemia or alkemia. These changes in unbound fraction are
unlikely to have significant effect on unbound plasma drug
concentration. However, they could have clinical relevance
with respect to the interpretation of changes in total plasma
drug concentration. Since only unbound drugs have pharma­
cological effects and can be hepatic metabolized or renal
excreted, these changes can be clinically relevant with drugs
having narrow therapeutic windows.
In practice, measuring the total concentration of drug in
plasma is more convenient than measuring its unbound con­
centration. To arrive at unbound plasma concentration from
the measured total plasma concentration, a separate in vitro
determination of the plasma protein binding of a drug is
usually carried out in serum or in plasma in the laboratory.
The plasma PK profiles and parameters are mathematically
adjusted using the in vitro measured unbound fraction. In
vitro measurement of plasma protein binding or the drug
unbound fraction is known to be affected by protein, drug,
free fatty acid concentrations, lipoprotein partitioning, tem­
perature, pH, and the presence or absence of other drugs or
displacing agents within plasma samples. Experimental errors
in the determination of unbound fraction caused by lack of
adequate pH control in in vitro assays will have unpredictable
impacts on the PK calculations.
Konchansky and colleagues examined the effect of variable
pH on measured values of fup during equilibrium dialysis
experiments using a diverse set of 55 drugs [73]. The ratio of
the unbound fraction at pH 7.4 (10% CO2) vs. pH 8.7 (air) was
more than two-fold for 40% of the 55 compounds tested. Only
one of the 55 compounds tested had a ratio less than 0.9. It
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 11
was observed that changes in plasma protein binding due to
pH increases, which often occurred during the equilibrium
dialysis experiment, were not species-specific but drug-
specific, though cationic compounds had a higher likelihood
of displaying pH-dependent binding. The study emphasized
the importance of effectively controlling pH in plasma protein
binding studies in vitro and in vivo. Similarly, it is essential in
the PBPK modeling and simulation to consider a calibration of
the unbound fraction fup measured in the in vitro system of
a pH different from the physiological pH.
6.6. pH and blood/plasma partition
Cytosol of the red blood cells (RBCs) has a pH range between
7.1 and 7.3, slightly lower than the plasma pH 7.4. Such a small
difference in pH is accountable for the passage of slightly
positively charged electrolytic drug into the RBCs from plasma
as acidic conditions favor the ionization of basic drugs [75].
Like other cells, ionization within RBCs prevents the transloca­
tion of ionized species from the cell, based on the pH-partition
hypothesis and low membrane permeability of ionized spe­
cies. It was observed in an in vitro study that for both quinine
and cryptolepine, a decrease in pH caused an increase in RBCs
partitioning, due to various mechanisms related to permeabil­
ity, solubility, lipophilicity, and stability. For acidosis, the dose
for both quinine and cryptolepine should be decreased to
prevent accumulation in the RBCs [74].
6.7. pH and transporters
The expression and activity of transporter P-glycoprotein
(P-gp) plays a role in the multidrug resistance of tumors [76].
Growing solid tumors often show pronounced hypoxia or
extracellular acidosis. The impact of an acidic environment
on the expression and activity of P-gp and on the cytotoxicity
of chemotherapeutic agents has been studied, using prostate
carcinoma cells which were exposed to an acidic extracellular
environment (pH 6.6) for up to 24 h. It was observed that P-gp
activity more than doubled after 3 ~ 6 h of incubation in acidic
medium, whereas cellular P-gp expression remained constant,
indicating that increased transport rate was the result of func­
tional modulation. In parallel, the cytotoxic efficacy of daunor­
ubicin showed pronounced reduction at low pH, an effect that
was reversible upon co-incubation with a P-gp inhibitor.
Indeed, a reduction of intracellular Ca2+ concentration by
35% under acidic conditions induced a higher transport rate
of P-gp, an effect comparable to that found on inhibition of
protein kinase C. These data indicate that P-gp activity is
increased by acidic pH presumably as a result of lowered
intracellular Ca2+ levels and inhibition of protein kinase
C. These findings may explain the reduced cytotoxicity of
chemotherapeutic agents in hypoxic/acidic tumors.
Organic anion-transporting polypeptide (OATP) 1A2 is
expressed on the apical sides of intestinal and renal epithelial
cells and is considered to be involved in the intestinal absorp­
tion and renal reabsorption of drugs [77]. The pH-dependency
of transport kinetics of human OATP1A2 was observed in
12 L. GAOHUA ET AL.

HEK293 cells. Indeed, OATP1A2 exhibited bimodal saturation a wide variety of nucleophilic compounds containing
kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 a nitrogen, sulfur, phosphorous, or selenium atom [85,86].
(5.62 mM), the Km value of the high-affinity site was ~8-fold Basic amines tend to be FMO substrates although there is
higher at pH 7.4 (43.2 mM). Therefore, transport properties of some overlap with CYPs (e.g. CYP2D6), whereas non-basic
OATP1A2 under lower pH conditions, such as those found in compounds tend to be oxidized by CYPs, with the degree of
the microenvironments of the small intestinal mucosa and N-substitution also playing a distinct role. Tertiary amines are
distal tubules, may differ from those seen under neutral pH generally FMO substrates in contrast to primary amines that
conditions. are generally better CYP substrates; secondary amines are less
clear-cut [10]. In the case of N-oxidation, when a molecule
possesses more than one basic nitrogen atom, the preferred
7. pH effects on drug metabolism
site is generally the most basic center.
7.1. Ionization and enzyme substrate
A major group of drug-metabolizing enzymes is the microso­ 7.2. pH and enzyme activity
mal cytochrome P450 (CYP450) family. CYP3A4, the most
Various environmental factors can affect the rate of enzyme-
abundant P450 enzyme in the liver, is responsible for meta­
catalyzed reactions through reversible or irreversible changes
bolizing approximately 40 ~ 50% of the drugs used in clinical
in the protein structure. Most enzymes have a characteristic
practice. Among the FDA-approved small molecule drugs
optimum pH at which the velocity of the catalyzed reaction is
(2005 − 2016), Hu et al. found that neutral and basic com­
maximal, and above and below which the velocity
pounds with molecular weight (MW) ≥ 360 g/mol tend to be
declines [87].
primarily metabolized by CYP3A4, whereas acidic compounds
As the pH changes, the ionization of groups both at the
with MW < 360 g/mol are most likely to be primarily metabo­
enzyme’s active site and on the substrate can alter, influencing
lized by other CYP enzymes [78].
the rate of binding of the drug molecule to the active site of
CYP2D6, which is expressed not only in the liver but also in
the enzyme. These effects are often reversible. Noteworthy,
the brain, is responsible for the metabolism of the second
the optimum pH of an enzyme might not be identical to that
highest number of drugs metabolized by P450 enzymes after
of its normal intracellular surroundings. In other words, the
CYP3A4. CYP2D6 tends to oxidize substrates that contain
local pH can exert a controlling influence on enzyme activity.
a protonated basic nitrogen at physiological pH and planar
The pH optimum of cytochrome P-450 reactions usually is
aromatic ring [79].
reported to be at 7.4 [88]. However, Jensen and Dalgaard
CYP2C9, which is expressed in a number of tissues through­
studied the metabolism of M1-muscarinic agonist Lu 25–109
out the body including GI tract, prefers neutral and acidic
in human liver microsomes [89]. The authors observed several
substrates, and the majority of substrates are weakly acidic
fold increases of the metabolites mediated by CYP1A2 at pH
compounds, although CYP2C9 also catalyzes the
8.5 compared to pH 7.4. This phenomenon was also reported
N-demethylation of a number of basic drugs (e.g. amitripty­
for cyclosporin A and CYP3A4, for benzphetamine and CYP2B/
line, fluoxetine, and zopiclone) [80]. CYP2C9 and CYP2C19 are
3A4 in human liver microsomes [90], and for testosterone and
structurally similar, but CYP2C19 does not have the same
CYP3A4 in rat liver microsomes [91].
affinity for acids as that found for CYP2C9 [10].
A general characteristic of FMO forms is that maximal
Monoamine oxidases (MAOs) are flavin-containing enzymes
enzyme activity is typically observed for a pH range of
that are responsible for catalyzing the deamination of bio­
8.5 ~ 11 [92–97]. This is substantially higher than the pH
genic and xenobiotic amines. MAO exists in two isoforms in
used for the determination of activity for other microsomal
most tissues, namely MAO-A and MAO-B. The substrates of
enzymes such as P450 between 7.2 and 7.6 [88]. Human FMO5
MAOs range from weakly basic to highly basic (primary
showed a broader range and greater functional activity from
amines, also some secondary and tertiary amines). MAO-A
pH 6 to 11, and its pH dependence profile for functional
preferentially metabolizes bulkier amines, while MAO-B prefers
activity differed significantly from that of other FMO enzymes
to metabolize smaller amines.
[98]. Taniguchi-Takizawa et al. used benzydamine
Flavin-containing monooxygenases (FMOs) are present
N-oxygenation as an index for FMO activity and found that
widely in both hepatic and extrahepatic tissues including the
high benzydamine N-oxygenation activities of recombinant
liver, kidney, lungs, skin, brain, and other tissues, with the
hFMO1 and hFMO3 and human kidney microsomes were
highest concentration occurring in liver, kidney, and lungs
observed at pH 8.4 compared to pH 7.4 [99]. Similarly, the
[81,82]. Multiple FMO forms have been identified in most
pH optimum of hFMO1 and hFMO3 for the oxidation of thia­
mammalian species, including humans, with the gene family
cetazone was found to be pH ~9 [100]. Krueger et al. reported
consisting of five functionally active members (FMO1-5) that
hFMO2.1 having a maximal activity at pH 9.5, and authors also
exhibit at least 80% amino acid identity for orthologous forms
confirmed its presence in lung tissue from a heterozygous
(i.e. human FMO1 and rat FMO1) and 51 to 58% identity for
individual by Western analysis [95].
homologous forms (i.e. human FMO1 and human FMO3)
Many psychoactive drugs (e.g. imipramine) are substrates
[83,84]. FMOs catalyze the NADPH-dependent oxidation of
of the brain FMO, so the FMO-mediated metabolism of these

drugs might contribute to local pharmacodynamic modulation
within the human brain. Bhagwat et al. reported the optimum
pH for N-oxidation of imipramine was found to be 8.5 in
human brain microsomes, so it is conceivable that FMOs are
significantly involved in the local metabolism and modulation
of pharmacological and/or toxic effects of psychoactive
drugs [101].
For the FMOs in animals, the purified FMOs from mouse
kidney and liver showed pH optima of 9.2 [102]. The activity of
dog FMO1 showed a 4.5-fold increase in S-oxidase activity as
the incubation pH increased from 7.6 to 9.0 [103]. Krueger
et al. reported that monkey FMO demonstrated a maximal
activity at pH 9.5 [95].
Glucuronosyl transferases are a family of enzymes that
catalyze the transfer of glucuronic acid from UDP-glucuronic
acid to acceptor molecules containing hydroxyl, phenol, car­
boxylic acid, thiol, or amine groups. UDP-
glucuronosyltransferases (UGTs) are expressed almost ubiqui­
tously throughout the body, with high expression in the liver
and the intestines [104].
Chang et al. explored the impact of incubation pH on the
glucuronidation of raloxifene, mycophenolic acid, and ezeti­
mibe, which are basic, acidic, and neutral compounds, respec­
tively [105]. The glucuronidation was examined in human liver
microsomal incubations by monitoring the production of glu­
curonide metabolites at a pH range of 5.4 ~ 9.4. Compared to
physiological pH, unbound intrinsic clearance was 11- and 12-
fold higher at pH 9.4 for raloxifene 4′-glucuronide and ralox­
ifene 6-glucuronide, respectively, whereas a 10-fold increase
was observed at pH 5.4 for mycophenolic acid glucuronide. In
contrast, ezetimibe glucuronidation did not vary as the pH
deviated from 7.4. One explanation for the phenomena may
be the ionization of key amino acids making up the catalytic
site is sensitive to shifting incubation pH; thus, it affects sub­
strate affinity for UGTs. Secondly, this may be due to mem­
brane permeability changes, as adjusting the incubation pH
can neutralize the charge on the acidic and basic compounds.
Neutralizing compounds would make them more readily able
to move across biological membranes. Shifting the incubation
pH may also enhance membrane permeability by changing
the substrate binding to membrane channels. Glucuronidation
in microsomes often under predicts in vivo glucuronidation,
the higher rate of glucuronidation may give better in vivo
correlation. The results suggest that not only the assay condi­
tions of microsomal glucuronidation should be considered
while conducting PBPK modeling on the drugs cleared by
glucuronidation, but also the impact of pH on extrahepatic
metabolism should be investigated.
Sulfate conjugation as catalyzed by the cytosolic sulfotrans­
ferases (SULTs) is known to be involved in the biotransforma­
tion of a variety of drugs. There are thirteen SULTs that are
classified into four distinct gene families, designated as SULT1,
SULT2, SULT4, and SULT6 [106]. Yamamoto et al. studied pH
dependence of the major acetaminophen SULTs, SULT1A1,
SULT1A3, and SULT1C4 [107]. The pH-dependence of the sul­
fation of acetaminophen differed considerably among the
three human SULTs. SULT1A1 showed a high level of activity
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 13
with pH ranging 6.5 ~ 8, whereas SULT1A3 displayed a distinct
optimum at pH 9.5. SULT1C4 showed a pH optimum ranging
7 ~ 8.5. The differential pH-dependence profiles of these three
SULTs may reflect their distinct substrate binding and/or cat­
alytic residues involved in mediating the sulfation of
acetaminophen.
7.3. Hepatic intracellular pH
Depending on the cell and tissue type, pH of intracellular fluid
can vary in the range of 6.8–7.2, with a relatively consistent
intracellular fluid pH of 7 in the hepatocytes in humans [108].
Physiologically, pH of the intracellular fluid can be affected by
many factors, such as metabolic inhibition, temperature, trans­
port of weakly basic/acidic compounds into the cell [109].
Intracellular pH can also be directly modulated by extra­
cellular pH. It was observed that intracellular pH of cultured
hepatocytes in bicarbonate:CO2 medium was relatively con­
stant at 6.85–7.05 over the external pH range of 7–8 [110].
Below an external pH of 7, intracellular pH fell below 6.8.
Varying the pressure of CO2 between 15 and 40 mmHg did
not alter the extracellular versus intracellular pH curve.
An extracellular pH of 7.4 and an intracellular pH of 7 result
in a half-log unit gradient of pH between plasma and the
intracellular environment, which theoretically would lead to
a pKa-dependent change in the ratio between intracellular
and extracellular concentrations. The liver is the major route
of drug elimination. Given changing ionization based on the
chemical properties (such as compound class and its pKa’s) of
the drug and the differences in the physiological environment
(such as pH and proteins) of intracellular and extracellular fluid
across the membrane of hepatocytes, it has been proposed
that hepatic models for clearance should be modified to
account for changes in hepatic intracellular concentra­
tion [111].
7.4. Well-stirred liver model
In the approach of in vitro-in vivo extrapolation (IVIVE) which
empowers the translational PBPK modeling and simulation,
the in vivo hepatic clearance is derived from the in vitro intrin­
sic clearance. In vitro clearance is determined from various
assays such as hepatocytes, microsomes, S9 or recombinants,
with various liver tissue models such as the well-stirred, par­
allel-tube, dispersion, or multiple-compartmental liver models
[112,113]. Although the well-stirred liver model is the most
used model, often the IVIVE with the well-stirred liver model
under-predicts the in vivo human hepatic clearance. Among
various approaches to improve the IVIVE clearance prediction,
correction of ionization has shown the most success, which
was based on the observation that pH difference exists in the
extracellular and intracellular fluid (7.4 and 7, respectively)
[111]. An ionization factor FI, defined as the ratio of the union­
ized fractions in plasma and that in intracellular fluid, has been
introduced to the traditional well-stirred liver model. It was
demonstrated that accounting for this ionization factor FI may
yield the IVIVE-predicted hepatic clearance up to 6.3-fold
14 L. GAOHUA ET AL.
greater than that obtained using the traditional equation for
the strong diprotic basic compounds, and up to 6.3-fold smal­
ler for the strong diprotic acidic compounds. For triprotic acids
and bases, the difference could be as much as 15-fold. It has
been demonstrated that the model performance of predicting
both clearance and drug–drug interactions can be significantly
improved after considering the ionization correction in the
PBPK model [108,114].
8. pH effects on drug excretion
8.1. Urine pH modification and renal excretion
On average, urine pH is close to 6.3. The extremes of urine pH
can be 4.5 and 7.5 under forced acidification and alkaliniza­
tion, respectively. These extremes contrast with the narrow
range of plasma pH of 7.3–7.5, thus a large pH gradient may
exist between plasma and urine. Urine pH can be altered by
diet, drugs, and the clinical state of a patient. In addition, urine
pH varies through the day.
Renal clearance itself is a complicated PK parameter con­
sisting of filtration, secretion, and reabsorption. Both acids and
bases were subject to significantly greater renal clearance than
neutral or zwitterionic molecules [10]. The effect of urine pH
on renal excretion and systemic disposition has been observed
for many drugs [115]. For ionizable drugs, mostly being weak
acids and weak bases, urine pH is one of the key parameters
affecting reabsorption and therefore drug PK, PD, and toxi­
city [116].
Changing the urine pH will directly affect the drug ioniza­
tion in the renal tubules; therefore, the passive secretion and
reabsorption between tubular urine and tubular cells resul­
tantly influence the renal clearance and systemic exposure of
drugs. Urine pH effect on drug disposition has not been well
characterized in humans.
Memantine, a weak base with a pKa of 10.27, is predomi­
nantly excreted unchanged via the kidneys. It has been
demonstrated that memantine plasma concentrations depend
on urine pH [117]. Alkaline urine pH results in a reduced renal
excretion and renal clearance compared to acidic urine pH.
The reduced renal clearance at alkaline urine pH can be
explained by pH-dependent tubular reabsorption under
these alkaline conditions because the ratio of unionized mem­
antine in alkaline urine (e.g. pH 8) is considerably higher
(0.005) than in acidic urine (e.g. pH 5), where the ratio of
unionized drug is very low (0.000005). Under acidic conditions,
tubular reabsorption seems to be unlikely, and tubular secre­
tion must be considered. As a result, the renal clearance of
memantine at acidic pH will exceed the expected glomerular
filtration rate.
8.2. pH in kidney and tubule
Though less relevant to PBPK modeling and simulation,
urine pH is one of the major factors that can modulate
kidney stone formation. Manissorn and coworkers used arti­
ficial urine to systematically evaluated effects of pH (ranging
4 ~ 8) on kidney stone formation, including calcium oxalate
(CaOx) crystallization, crystal-cell adhesion, crystal internali­
zation into renal tubular cells, and binding of apical mem­
brane proteins to the crystals [118]. Microscopic
examination revealed that CaOx monohydrate (COM) was
crystallized with greatest size, number, and total mass at
pH 4 and least crystallized at pH 8, whereas CaOx dihydrate
(COD or weddellite) was crystallized with the vice versa
order. The greatest degree of crystal-cell adhesion was
observed at the most acidic pH and least at the most
basic pH. Crystal internalization into renal tubular cells was
maximal at the neutral pH 7. No significant difference was
observed in binding capacity of the crystals to apical mem­
brane proteins at different pH. The authors concluded that
the acidic urine pH may promote CaOx kidney stone forma­
tion, whereas the basic urine pH (i.e. by alkalinization) may
help to prevent CaOx kidney stone disease.
While only the unbound drug in the renal perfusing
blood can be filtrated through the glomerulus, the
unbound fraction in blood or plasma is pH-dependent, as
aforementioned. Therefore, the renal clearance depends on
the systemic plasma pH. The more the unbound drug, the
more the renal filtration. Compared to the systemic plasma
pH, however, it is the renal tubular pH that directly and
significantly affects the drug secretion and reabsorption
(Figure 6). To explore the interplay of various mechanisms
involved in renal excretion, mechanistic PBPK models have
been developed for kidney in the last decade [119,120].
8.3. Modeling of renal excretion
To the best of our knowledge, the mechanistic kidney model
(Mech-KiM) implemented in the Simcyp® simulator was the
first PBPK model to integrate drug characteristics to renal
physiology in predicting the contributions of glomerular filtra­
tion, active and passive secretion, active and passive reabsorp­
tion, and metabolism to renal elimination and excretion [119].
The Mech-KiM model has been used by industry and regula­
tors in the PBPK modeling of various market drugs, such as
oseltamivir carboxylate, cidofovir, cefuroxime, and peme­
trexed [121,122].
Huang and Isoherranen also developed a 35-
compartmental kidney model to simulate the renal clearance,
including filtration and pH-dependent passive reabsorption,
from in vitro permeability data [120]. The model was verified
using 46 compounds, including neutrals, acids, bases, and
zwitterions. The feasibility of incorporating active secretion
and pH-dependent bidirectional passive diffusion into the
model was demonstrated using para-aminohippuric acid,
cimetidine, memantine, and salicylic acid. The developed
model enabled the simulation of renal clearance from in vitro
permeability data, with predicted renal clearance within two­
fold of observed for 87% of the drugs assessed.

Figure 6. Physiological pH in renal tubule.
9. pH and toxicity/safety
9.1. pH and pharmacodynamics
In cellular assays, the zwitterions tend to be more active than
basic and neutral compounds while the acids generally tend
to be less active, though there are still a significant number of
acids with potent cellular activity [10].
9.2. pH and receptor binding
It has been demonstrated in Chinese hamster ovary (CHO) cells
that the affinity and binding kinetics of Histamine H1 receptor
antagonists (levocetirizine, fexofenadine, and desloratadine) are
differently affected by an acidic environment (pH 5.8) that can be
encountered during inflammation status [18]. Simulations based
upon clinical data indicated that, albeit apparently modest in
amplitude, these changes in binding properties could have sig­
nificant effects on the PD of antihistamines in the clinical situa­
tion. Indeed, under acidic conditions and at their therapeutically
active doses, the levocetirizine binding profile would be
enhanced, that of fexofenadine would remain largely unchanged
and that of desloratadine would be impaired.
9.3. pH and toxicity
pH of exposure mediums can influence the toxicity and
bioaccumulation of ionizable compounds [123]. An ionizable
compound is generally more toxic and more bioaccumula­
tive when in the neutral state. The change in lipophilicity
when a neutral compound becomes ionized, the electrical
attraction, and the ion trap have been identified as 3 major
processes to explain the behavior of ionizable compounds
with changing pH. Literature data indicated that toxicity and
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 15
bioaccumulation were higher for acids at lower pH values,
whereas the opposite was true for bases. The effect of pH
was most pronounced when pH-pKa was in the range of
−1 ~ 3 for acids, and −3 ~ 1 for bases. It was observed that
the factor by which toxicity and bioaccumulation changed
with pH was correlated with the lipophilicity of the com­
pound. For both acids and bases, the correlation was posi­
tive, albeit significant only for acids. Limited information was
available for amphoteric and zwitterionic compounds.
9.4. Ionization and safety
9.4.1. Selectivity
Basic compounds are generally known to bind to a variety of
receptors and transporters. Highly basic positively charged
compounds are generally more prone to promiscuity than
neutral, zwitterionic, or acidic compounds. Increased lipophili­
city also generally contributes to overall promiscuity [10].
9.4.2. Cellular distribution
Represented by lysosomal trapping, bases often become
sequestered in acidic organelles of many different cell types
and may thereby contribute to various toxicities.
Lipophilic basic molecules, especially cationic amphiphiles,
can cause phospholipidosis by distributing to membranes and
lysosomes and can also be influenced by overall compound
lipophilicity.
Both strongly acidic and basic drugs have been associated
with mitochondrial dysfunction. Strong lipophilic acids tend to
have the greatest propensity to cause uncoupling of oxidative
phosphorylation, while basic drugs can accumulate into the
acidic mitochondrial cytosolic space in tissues such as liver
and pancreas.
16 L. GAOHUA ET AL.

10. Further considerations 10.2. pH and pKa manipulation

10.1. Impacts of uncertain pKa or pH 10.2.1. Change pH for better PK
The pHs of the injectables are always adjusted for better
10.1.1. Uncertainty in pH and pKa measurements
performance. Phenytoin sodium is a typical example. Dilantin
As one of the major physicochemical parameters of drugs,
(phenytoin sodium injection, USP) is a ready-mixed solution of
various methods have been developed to measure pKa [124].
phenytoin sodium in a vehicle containing 40% propylene
The physiological pH can also be measured using various
glycol and 10% alcohol in water for injection, adjusted to pH
methods, non-invasively and continuously. For example,
12 with sodium hydroxide [30]. A fall in plasma levels may
a telemetric SmartPill® capsule system can be used to gener­
occur when switching from oral to intramuscular administra­
ate the intra-gastric pH profile over time, while the capsule is
tion. Such a drop in exposure is possibly due to slower absorp­
transited from stomach to colon [125].
tion from the precipitates at the injection site, due to the poor
pH is the negative of the base 10 logarithm of the hydro­
water solubility of phenytoin.
gen ion concentration in the medium and pKa is the pH of the
medium where the drug is 50% ionized. Since both pH and
10.2.2. Change pKa for better PK
pKa are logarithmic measurement, one-unit difference in the
Fluorination, namely introducing fluorine into a drug mole­
values of pH or pKa corresponds to 10-fold difference in
cule, can alter electron distribution, which can impact on
ionization. Slight uncertainty in the pH and pKa measurements
the pKa, dipole moment and even the chemical reactivity
may have significant impacts on the calculation of ionization;
and stability of neighboring functional groups [129].
therefore, the aqueous solubility, passive permeation, and
Fluorine can reduce the basicity of compounds, which
further drug ADMET. This is true especially in the case where
can improve bioavailability due to better membrane per­
the physiological pH and the chemical pKa are comparable. If
meation of the compound. Recently, Velcicky et al.
the difference between pH and pKa is big enough, the impact
reported their approach based on specifically placed fluor­
of uncertain pH and pKa may not be such significant, as the
ine within a pyrrole-based MK2 (MAP-activated protein
ionization is reaching its limitations (either fully ionized or fully
kinase 2) inhibitor scaffold for manipulation of its physico­
unionized).
chemical and ADME properties [130]. While preserving tar­
get potency, the novel fluoro-derivatives showed greatly
10.1.2. Temperature matters
improved permeability as well as enhanced solubility and
While the human body has a representative temperature dif­
reduced in vivo clearance, leading to significantly increased
ferent from the environmental room temperature, when
oral exposure.
applying pKa measured in in vitro systems under the room
temperature 20 ~ 25°C to in silico PBPK model of the human
body with a representative temperature 37°C, one needs to 10.3. Extension of Henderson-Hasselbalch equation
consider the difference in temperature, as pKa itself is tem­
10.3.1. Common ion effect and salt effect
perature dependent [124,126]. In general, pKa of an acidic
Henderson-Hasselbalch equation is the basis to define the
compound or a basic compound will increase or decrease
drug ionization within a medium. Changing the medium pH
and then the acidic or basic drug is more ionizable at
shifts the equilibrium of the fraction of drug that is ionized
a specific pH and more soluble when temperature is increased.
and unionized. For instance, more basic drug molecules are
For instance, the pKa of fentanyl, a weak base, decreased from
ionized when the environmental pH is decreased by adding
8.6 to 7.89 as the temperature was increased from 15°C to
hydrochloric acid (HCl), resulting in high total concentration
47.5°C [126]. Such data indicated the necessity to calibrate the
in the medium, corresponding to enhanced solubility due to
solubility measured at room temperature 20 ~ 25°C to that at
acidification of the medium. However, due to the common
37°C before applying to PBPK model.
ion effect, a decrease of solubility for a basic salt is frequently
observed when adding extremely high HCl to adjust the
10.1.3. Circadian rhythm of physiological pH
medium pH in the pH-solubility experiments. The same is
The physiological pH in plasma and several fluid/tissue/organs
true for an acidic salt when using sodium hydroxide to adjust
is tightly regulated within narrow ranges. GI is a typical case
the medium pH. Common ions (Cl− and Na+) added to the
where pH is changing tremendously, depending on location,
medium in general shift the acid-base equilibrium to the
feeding, age, gender, and drug itself and so on. Plasma pH has
reactant’s side causing precipitation, a mechanism used to
a circadian rhythm with a peak in the afternoon (e.g. 3 PM)
purify the compound in drug discovery and development.
and a trough in the middle-night (e.g. 3 AM) [127]. Urinary pH
The common ion effect generally decreases the solubility of
has a diurnal pattern as well [128]. To the best of our knowl­
the salt form (but not the free base, which does not have
edge, however, this detailed pH variation remains to be incor­
common ion).
porated into PBPK model, even though it might be one of the
In contrast, adding a new, uncommon ion to the medium
critical physiological parameters that contribute to the intra-
can facilitate strong ionic interactions with the products. This
individual (or inter-occasion) variabilities.

‘salt effect’ therefore shifts from the reactants to the ionized
products, therefore increasing solubility of the drug. Both the
common ion effect and salt effect are examples of using Le
Chatelier’s principle to modulate solubility.
10.3.2. Critical micellization concentration and pH
Oral absorption is a pathway of primary interest in many PBPK
applications. Bile acids are amphipathic molecules formed
from cholesterol metabolism that play an important role in
oral absorption. Bile salt solubilization is one of the major
mechanisms contributing to solubility enhancement of lipo­
philic weakly basic compounds in the proximal GI, but not in
the distal GI. It is well known that the unconjugated bile acids
are insoluble at neutral pH while their conjugated salts are
highly soluble. From the Henderson-Hasselbalch equation, the
solubility of both the unconjugated and conjugated forms
rises exponentially with increasing pH. As the concentration
of the conjugated anions reaches the critical micellization
concentration (CMC), micelle formation occurs and solubility
becomes practically unlimited [131]. Various bile acids and bile
salts have different CMCs, which depend on the pKa of bile
acids and bile salts as well as the environmental pH. In gen­
eral, CMC increases with the increasing pH until it reaches the
critical micellization pH, above which the CMC is pH-
independent. Lipophilic basic drugs tend to precipitate when
the dissolved species move from the strong acidic stomach to
the weakly acidic jejunum and ileum. However, the bile salts
are secreted from the bile duct into the duodenum, transit
through the small intestine and are re-absorbed at the end of
ileum. Bile salts allow the lipophilic drugs to be dissolved
within micelles, preventing precipitation. It is therefore impor­
tant to define the correct CMC for each bile salt at each GI
segment, enabling a proper description of solubilization, dis­
solution and precipitation in the PBPK modeling and
simulation.
10.4. Extension of pH-partition hypothesis
10.4.1. Transporters
The current review on pH-pKa crosstalk, its impacts on drug
ADMET and its incorporation in the PBPK modeling and simu­
lation work is mostly based on the traditional pH-partition
hypothesis established in the 1950s for the passive permea­
tion process [5–8]. However, Kell recently proposed ‘no trans­
porter means no transport’ based on recent insights from
experiments using targeted CRISPR-Cas9 deletion of solute
carries genes [132]. In the perspective article from Kell, drugs
pass through the cell membrane by hitchhiking on membrane
transporters may be substantially underappreciated, implying
‘passive diffusion’ through membrane bilayers is negligible.
Such a novel hypothesis may fundamentally change the cur­
rent approach for PBPK modeling and simulation and the
paradigm of drug discovery and development. Given the dra­
matic implications and outstanding quantitative questions the
hypothesis raises, it remains challenging to entirely accept and
adopt at this moment.
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 17
10.4.2. New mechanisms
There are several additional developments beyond the tradi­
tional pH-partition hypothesis in the PBPK modeling area. One
concerns the permeation of bound drugs across cell mem­
brane. Tsao and colleagues have constructed a ‘facilitated-
dissociation’ model in which the interaction of the ligand-
albumin complex with the cell surface enhanced the dissocia­
tion of that complex to provide unbound ligand for uptake
into the hepatocytes [133]. The ‘facilitated-dissociation’ model
enabled an accurate description of the enhanced update
clearance in the in vitro hepatocyte experiment with various
amounts of albumin added [134,135]. Poulin and coworkers
have summarized several recent literatures supporting the
protein-facilitated uptake mechanism(s) and further discussed
the interplay among the effects of pH gradient, protein-
facilitated uptake, metabolism, active transport, and permea­
tion limitation in cells [136,137].
The second development of the pH-partition hypothesis is
the passive permeation of unbound ionized species across the
cell membrane. As aforementioned, the cell membrane is nor­
mally negatively charged; therefore, the positive charged
cations can penetrate the cell membrane because of the elec­
tronic potential, following the Nernst-Planck equation. Various
PBPK models have considered the passive permeation of ionized
species across cell membrane [36,70,138,139]. To the best of our
knowledge, however, such a penetration of unbound ionized
drug molecules across the apical membrane of enterocytes
remains to be considered in mechanistic oral absorption model.
10.5. Feedback from PD to PK in PBPK modeling and
simulation
As mentioned, pH in the GI tract has a significant impact on
the oral absorption for all ionizable drugs administrated orally.
However, some drugs like PPIs (such as omeprazole) are mod­
ulating the GI pH, while their PKs are depending on GI pH
itself. For this reason, a feedback loop from PD to PK is
required in the PBPK/PD modeling and simulation to model
PK and PD simultaneously.
Another example where a PD-PK feedback loop is impor­
tant for quantitative accuracy is for those bile acid transporter
inhibitors (iBATs, such as elobixibat), where the solubility of
the iBATs in the GI tract is enhanced by bile salts while the bile
acid re-absorption is inhibited by iBATs.
Some urinary pH modifiers (such as sodium bicarbonate) can
change the urinary pH, therefore affecting the renal section and/
or re-absorption, which may impact on the PK of drug itself.
10.6. Biologics
The neonatal Fc receptor (FcRn) is characterized by its high
affinity to bind albumin and immunoglobulin G (IgG) at low
pH. It plays an important role in regulating the half-lives of
both plasma albumin and IgG through a pH-dependent recy­
cling mechanism [140]. The endosome is slightly more acid­
ified than the blood (pH 6 vs. 7.4). The FcRn-bound albumin
18 L. GAOHUA ET AL.
and IgG in the acidified endosome are protected from degra­
dation and can be recycled when these albumin and IgG meet
the physiological pH again on the membrane surface of
endothelial cells. The half-lives of antibodies can often be
long, around 21 days for IgG, due in substantial part to the pH-
dependent FcRn recycling mechanism [141].
11. Conclusion
Although it is not an exhaustive review, we have collated and
compiled the majority of information in the literature about
the interaction of the physiological pH and the chemical pKa
and its impacts on drug ionization and ADMET from the view­
point of PBPK modeling and simulation. Our analysis of pH-
pKa crosstalk has been focused mainly on absorption pro­
cesses, where drug solubility, dissolution, and passive penetra­
tion are directly impacted by ionization. Beyond absorption,
the pH-pKa interaction also has a significant impact on drug
disposition, where the tissue/plasma partition coefficient (Kp)
and the ion trapping within organelles are normally predicted
using various mechanistic models based on pH-partition
hypothesis. Additional impacts of pH-pKa crosstalk on meta­
bolism, excretion, and toxicity are being increasingly recog­
nized. The pH-partition hypothesis has been the theoretical
basis for PBPK modeling and simulation when describing
these ADMET processes mathematically and mechanistically.
A better understanding of the interaction of physiological pH
and chemical pKa therefore enables a full consideration of the
ionization of drugs within the physiological systems, regard­
less of whether modeling healthy or disease states, when
developing a PBPK model to support drug discovery and
development.
12. Expert opinion
Majority of our experiences on pH-pKa crosstalk and pH-
partition hypothesis in PBPK modeling and simulation are
related to the passive processes of oral absorption and tissue
distribution, both of which are determined by the free con­
centration gradients across cell membrane. Ideally, all the
mechanistic oral absorption and distribution PBPK models
should have been based on the same assumption of pH-
partition hypothesis for the passive membrane permeation.
However, to the best of our knowledge, many PBPK models
have not fully incorporated the pH-pKa crosstalk and pH-
partition hypothesis systemically and consistently. For exam­
ple, the ‘industry standard’ GastroPlusTM and Simcyp® PBPK
platforms predict the tissue/plasma partition coefficient (Kp),
a key parameter defining tissue concentration kinetics and
steady-state distribution, using mechanistic models that are
based on pH-partition hypothesis. However, GastroPlusTM
ACAT model (V.9.7) is using the gradient of the total luminal
concentration and the unbound enterocyte concentration to
define the passive oral absorption. In contrast, Simcyp® ADAM
model (V.18.2) uses the total dissolved amount to define the
passive oral absorption [142].
The OrBiTo project, a multi-million EU-funded international
public–private partnership established in late 2012, has high­
lighted multiple issues in these models that ultimately led to
unsatisfactory accuracy in the prediction of drug oral bioavail­
ability [143]. However, it should have been recognized that none
of the oral absorption models compared in OrBiTo project has
properly implemented the basic pH-partition hypothesis for the
passive process of oral absorption/exsorption. One of our con­
cerns is, without systemic and consistent incorporation of pH-
pKa crosstalk and pH-partition hypothesis in the existing oral
absorption and PBPK models, that any conclusion and sugges­
tion from the comparison of model performances may be mis­
leading. We suggest the ‘industry standard’ oral absorption
models should be revisited to review their fundamental model­
ing assumptions, as missing pH-partition hypothesis in the mod­
els should be aware to the industrial end-users while these
models are increasingly used by the pharmaceutical companies
and regulatory agencies in drug discovery and development.
Our understandings on pH-pKa crosstalk and ionization in
defining drug ADMET are accumulated in the past centuries
and will be continued in the future with efforts from different
disciplines. As evidenced in this review, the impacts of ioniza­
tion on oral absorption (through modifying solubility, dissolu­
tion, disintegration, and permeation and so on) and tissue
distribution (through modifying free fractions across the cell
membrane) have been well explored, whilst the impacts of
ionization on drug metabolism, transporter-mediated elimina­
tion and tissue accumulation and toxicity remain further study.
Undoubtedly, PBPK model is providing us a theoretical tool/
platform to integrate our understandings systemically, consis­
tently and mechanistically. Among all the impacts the pH-pKa
crosstalk has on drug ADMET, we need to identify the key
mechanisms where pH-pKa crosstalk plays the most important
roles and to implement them into PBPK models first. In our
opinion, independent and scattered works on ionization
impacts on distribution volume, IVIVE of drug metabolism
and drug–drug interaction prediction will benefit from
a consortium by bringing all scientific thoughts together in
a systemic and consistent way.
Looking forward, we expect more emphasis on the funda­
mental understanding of pH-pKa crosstalk and pH-partition
hypothesis in PBPK modeling and simulation, where new
aspects and functionalities are continuously being incorpo­
rated in the next 5–10 years. More confidence will be achieved
about our PBPK model and its prediction when we know our
model basic assumptions are correct.
Acknowledgments
The views and opinions expressed here are those of the authors and do
not necessarily reflect the official policy, practice, or position of the
organizations that the authors work. The authors acknowledge
 EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 19

proofreading by and scientific discussions with Dr. Brian Schmidt of BMS, 13. Abelson MB, Udell IJ, Weston JH. Normal human tear pH by direct
and Drs. Claire Beaumont, Simon Teague, and Kunal Taskar of GSK. measurement. Arch Ophthalmol. 1981 Feb;99(2):301.
14. Valentin J. Basic anatomical and physiological data for use in radi­
ological protection: reference values. A report of age- and
Declaration of interest gender-related differences in the anatomical and physiological
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