Biological barriers, and the influence of protein binding on the passage of drugs across them

Abstract

Drug-protein binding plays a key role in determining the pharmacokinetics of a drug. The distribution and protein binding ability of a drug changes over a lifetime, and are important considerations during pregnancy and lactation. Although proteins are a significant fraction in plasma composition, they also exist beyond the bloodstream and bind with drugs in the skin, tissues or organs. Protein binding influences the bioavailability and distribution of active compounds, and is a limiting factor in the passage of drugs across biological membranes and barriers: drugs are often unable to cross membranes mainly due to the high molecular mass of the drug-protein complex, thus resulting in the accumulation of the active compounds and a significant reduction of their pharmacological activity. This review describes the consequences of drug-protein binding on drug transport across physiological barriers, whose role is to allow the passage of essential substances—such as nutrients or oxygen, but not of xenobiotics. The placental barrier regulates passage of xenobiotics into a fetus and protects the unborn organism. The blood–brain barrier is the most important barrier in the entire organism and the skin separates the human body from the environment.

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Molecular Biology Reports (2020) 47:3221–3231
https://doi.org/10.1007/s11033-020-05361-2
MINI REVIEW ARTICLE
Biological barriers, andtheinuence ofprotein binding
onthepassage ofdrugs acrossthem
KarolinaWanat1
Received: 28 November 2019 / Accepted: 27 February 2020 / Published online: 5 March 2020
© The Author(s) 2020
Abstract
Drug-protein binding plays a key role in determining the pharmacokinetics of a drug. The distribution and protein binding
ability of a drug changes over a lifetime, and are important considerations during pregnancy and lactation. Although proteins
are a significant fraction in plasma composition, they also exist beyond the bloodstream and bind with drugs in the skin, tis-
sues or organs. Protein binding influences the bioavailability and distribution of active compounds, and is a limiting factor
in the passage of drugs across biological membranes and barriers: drugs are often unable to cross membranes mainly due
to the high molecular mass of the drug-protein complex, thus resulting in the accumulation of the active compounds and a
significant reduction of their pharmacological activity. This review describes the consequences of drug-protein binding on
drug transport across physiological barriers, whose role is to allow the passage of essential substances—such as nutrients
or oxygen, but not of xenobiotics. The placental barrier regulates passage of xenobiotics into a fetus and protects the unborn
organism. The blood–brain barrier is the most important barrier in the entire organism and the skin separates the human
body from the environment.
Keywords Breast milk, Drug-protein binding, Skin barrier,
Protein binding, The blood–brain barrier, The placental
barrier.
Drug‑protein binding
Following absorption from the gastrointestinal system
or direct infusion into bloodstream, a drug can bind with
plasma proteins. The main proteins responsible for the bind-
ing in plasma are human serum albumin (HSA) and alpha-
1-acid glycoprotein (AAG) [1–3]. Their concentrations and
functions are listed in Table1 [4–6]. While the protein-drug
complex is relatively stable, the connection between mol-
ecules is reversible: molecules can join and separate, and the
equilibrium state is reached a few hours after the administra-
tion of a medicine [3].
The structure and properties of the drug determine the
extent of both: plasma protein binding (PPB) and protein
binding (PB) in the sense of the general process, because
these concepts should be distinguished. Lipophilicity
(described as logP) and acid–base properties have a signifi-
cant correlation with binding [7]. Hydrophobic and acidic
drugs (e.g. warfarin, ketoprofen, ibuprofen, diazepam) bind
preferably to HSA, while AAG connects with the basic
ones (e.g. bupivacaine, clindamycine) [6–9] which should
be taken into account while setting the therapy. Binding
can also increase the solubility of compounds, especially
hydrophobic ones, which would otherwise not be distributed
in the aqueous environment of plasma [10]. A connection
with the plasma proteins protects compounds from oxida-
tion, lowers their toxicity and increases their half-life; drugs
highly bound to the plasma proteins often reveal low first
pass-metabolism [10–12]. Volume of distribution depends
from PB as well and is decreased for drugs highly bound in
plasma or increased for those which bind in tissues [13–15].
In addition drugs with higher affinity to a binding site on a
plasma protein can replace one with lower affinity and such
competition can lead to an uncontrolled rise in the concen-
tration of the free, unbound fraction of a drug [16]. This
can have serious consequences for narrow therapeutic index
(NTID) drugs, where the difference between therapeutic and
toxic doses is minimal (e.g. cardenolides, carbamazepine,
phenytoin or warfarin [17, 18]) and any changes in the con-
centration of unbound, active form may be poorly tolerated
* Karolina Wanat
karolina.wanat@umed.lodz.pl
1 Department ofAnalytical Chemistry, Medical University
ofLodz, Muszyńskiego 1, 90-151, Lodz, Poland
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3222 Molecular Biology Reports (2020) 47:3221–3231
1 3
by the organism. A sudden increase in the unbound fraction
of the drug may provide a toxic effect [19]. This can lead to
clinical consequences such as high risk of bleeding (warfa-
rin) [19] or cardiac arrest (cardenolides) [20].
Level of protein binding depends on the properties of a
drug but also on the surrounding environment for example,
the temperature or pH. The latter can change the ionization
state of the chemical compound [10, 21, 22]. The degree of
plasma protein binding is governed by two variables, these
being the unbound fraction of the drug in plasma (fu,p) and
the percentage of plasma protein binding (PPB%), as given
below in Eqs.1 and 2 [3]:
An important consideration, often omitted in the litera-
ture, is that of drug-protein binding occurring outside the
bloodstream. Compounds can bind with macromolecules
in skin, breast milk, tissues and organs including the pla-
centa [13, 23, 24], where they become ‘stuck’ and are thus
prevented from reaching site of pharmacological action
[23, 25]. These drugs may later pass into the plasma but
in an uncontrolled way, which disturbs the dosage and the
intended result of pharmacotherapy.
Transfer acrossbiological membranes
andbarriers
The cell membrane is a semipermeable phospholipid bilayer,
which separates cell organelles and cytoplasm from the envi-
ronment. The ability of a molecule to cross the membrane
depends on various factors including molecular weight, lipo-
philicity, ionisation state, the concentration on both sides
of the barrier and protein binding [26, 27]. Low-molecular,
lipid, unionised and unbound to plasma proteins molecules are
reckoned as good penetrators through membranes, although
(1)
f
u,p =
unbound drug concentration in plasma
total dose
(2)
PPB%
=
bound drug concentration in plasma
total dose
×
100%
extreme lipophilicity can cause accumulation in lipid environ-
ment [28, 29]. The mechanism of passive transport includes:
simple diffusion (the undisturbed movement of small, lipo-
philic and unionized molecules across membrane) and facili-
tated diffusion, where specialized membrane proteins transport
particles across barriers [30]. Active transport acts against the
concentration gradient and as such requires energy, which is
typically obtained by the hydrolysis of adenosine triphosphate
(ATP). One such family of membrane proteins which actively
transport drugs and other molecules across membranes is that
of the ATP-binding cassette transporters (ABC transporters).
They also contribute significantly to the passage of drugs
through the blood–brain barrier or placenta [31]. Crossing bio-
logical barriers is a far more difficult matter. Their structure is
more complex and there are additional mechanisms involved
which prevent the passage of xenobiotics. Transfer across each
barrier is explained in detail in the appropriate sections of this
review. The most important, and the most difficult to pass, is
the blood–brain barrier (BBB), which separates crucial organs
from the environment.
Binding with HSA and AAG macromolecules affects the
pharmacokinetic properties of pharmacologically-active
compounds by decreasing their bioavailability and slow-
ing their passage across biological membranes and barriers
[32–34]; proteins themselves hardly penetrate through the
cell membranes [35–37]. On the contrary new approaches
in target therapy also reveal that drug binding to the protein
carrier improves the effectiveness of several pharmacothera-
pies [38], e.g. a simple but effective mechanism was used
in anti-tumour pharmacotherapy. Drug-protein conjugates
penetrate into tumour circulation easily, through fenestrated
capillaries, and stay trapped inside [39]. Albumin is also
used as a protein carrier in commonly used drugs such as
levemir, methotrexate, doxorubicin or paclitaxel [40, 41].
The blood–brain barrier
The blood–brain barrier (BBB) protects the central nerv-
ous system (CNS), which controls the whole body.
Blood vessels, which are part of the BBB, are lined with
Table 1 Physicochemical properties of HSA and AAG
Plasma protein Protein family Concentration in plasma Function
Human serum albumin,
65kDa, 585 amino acids
Albumins 3.5–5g/dL Transport of compounds across the blood-
stream (mainly hydrophobic and acidic ones),
maintenance of the blood oncotic pressure,
antioxidant, anticoagulant and immunomodu-
lating properties
Alpha-1-acid glycoprotein,
44kDa, 183 amino acids
Globulins, acute phase
proteins
Depends from physiological condi-
tion
Transport of compounds across the bloodstream
(mainly ones with basic properties), AAG is
produced during the inflammatory state
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3223Molecular Biology Reports (2020) 47:3221–3231
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tightly-connected endothelial cells. These unique connec-
tions between the endothelial cells are called tight junctions
(TJs) and adherence junctions (AJs). The BBB is also com-
posed of a basement membrane, glial cells, pericytes and
surrounding neurons [42]. The close cell connections, viz.
TJs and AJs prevent the passage of molecules through the
intercellular space: transport can only take place through the
intracellular route, i.e. within the cells [43]. Further defence
is provided by unique metabolic activity of the barrier, with
enzymes such as γ-glutamyl transpeptidase (γ-GTP) or alka-
line phosphatase (AP) enabling chemical decomposition of
compounds which can cross the BBB from the bloodstream
[42, 44]. The CNS is also protected by the diversity of its
routes of xenobiotic transport mechanisms [42, 43, 45]. Of
the drug efflux transporters, i.e. those of ABC transport-
ers family P-glycoprotein (P-gp), breast cancer resistance
protein (BCRP) and multidrug resistance protein (MRP)
demonstrate the highest activity in the BBB [46]. These
transporters are responsible for drug distribution into the
CNS and they can remove compounds which cross the bar-
rier. Such efflux transporters have various substrates, includ-
ing anti-cancer drugs, such as doxorubicin or methotrexate,
antiepileptics, such as phenytoin and carbamazepine, and
antidepressants, such as venlafaxine and paroxetine. While
some drugs are not intended to act on the CNS, many others
have to penetrate the brain to reach the main site of their
activity and achieve successful therapy [46]. Drug transport
across the blood–brain barrier has been widely described by
Pardridge etal., with a series of articles providing a clear
review of various aspects of barrier structure, the transport
of drugs across it and the development of drugs for use in
the CNS [47–52]. New approaches to delivering CNS drugs
are also mentioned in other recent articles [53, 54].
The blood–brain barrier protects the CNS from harm-
ful substances but its main role is to provide nutrients and
oxygen, essential for the brain structures [42]. Oxygen mol-
ecules and drugs with low molecular weight and lipophilic
properties can easily cross the BBB by simple diffusion
[55]. Nutrition such as glucose, crucial for proper CNS
function, or amino acids are carried by specific transport-
ers (e.g. GLUT1 glucose transporter); macromolecules with
high molecular mass, such as insulin, are transported in the
process of endocytosis [43, 56]. Drugs can pass through
the BBB by transmembrane diffusion, especially those that
are lightweight or with high lipophilicity, or are carried by
transporters, as in the case of glucose [55]. Two parameters
(Eqs.3 and 4) describe the amount of a drug that is passed
into the CNS: log BB and log PS [57, 58]. Log BB repre-
sents the ratio between drug concentration in the CNS and
plasma, while log PS indicates the permeability of certain
surface; while the former is easier to obtain and more intui-
tive to understand, the latter is currently receiving more
research attention [57, 58]:
A number of studies have examined protein binding with
drugs and their ability to cross the BBB [55, 59–61]. Albu-
min, like other proteins, does not readily pass through the
barrier, and its drug-macromolecule complex, cannot cross.
Based on this assumption, it appears that drugs which bind
more readily to proteins are less able to pass into the CNS
(‘free drug theory’ [34, 62]). This may be true for most
drugs, but there are some exceptions to the rule. Several
drugs which cross the BBB without difficulty, such as ben-
zodiazepines, steroids and a few hormones, demonstrate
higher concentrations in the CNS than their unbound plasma
fraction would indicate [63–66]. Similar observations were
made by Videbæk etal. (1999) (Table2) [67]. De Lange and
Danhof [68] collected several papers which describe highly
bound drugs (oxicams [69], imipramine and desimirpamine
[70], isradipine, darodipine [71]) which also penetrate the
BBB in surprisingly high extent (Table2). There are several
explanations of this phenomenon. Pardridge etal. claimed
that the conformation of the protein changes while interact-
ing with capillary walls and a drug molecule is freed from
a complex [64, 65, 72], Tanaka and Mizojiri ended up with
similar conclusion [66]. Another idea was protein-mediated
transport in which binding with protein (especially AAG)
enhances the BBB penetration [62]. Several authors claimed
that more permeable structure of capillary endothelium in
some regions may be the reason of the increased extraction
of a complex into the CNS [67, 68]. There is no doubt that
protein binding has a significant role for penetrating BBB; it
can either decrease the passage or affect it in the other way
with mechanisms still to be discovered. Mentioned studies
reveal that invivo analyses seem to be more applicable in
that case. The unique environment in the CNS or interac-
tions between proteins and brain capillaries apparently have
a high impact on the matter, therefore there is a substantial
difference between invitro and invivo results.
The placental barrier
The placenta is a unique connection between mother and
fetus. It is formed during the sixth week of pregnancy and
exists until the time of birth. Its main function is to deliver
nutrients and oxygen to the foetus and to remove waste and
metabolites. Throughout the pregnancy, the placenta also
adopts other roles: from the tenth week, it also produces
(3)
log BB
=
drug concentration in CNS
drug concentration in plasma
(4)
log PS
=observed permeability across BBB
cm
∕
s

surface area of brain capillary endothelium

cm2

g

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3224 Molecular Biology Reports (2020) 47:3221–3231
1 3
hormones such as chorionic gonadotropin (CG), human pla-
cental lactogen (HPL), relaxin, progesterone, testosterone,
oestrogens etc. and it manifests metabolic activity [73, 74].
Inside the placenta the blood vessels from a mother and a
child are tangled together but the blood itself does not mix;
despite this, sufficient exchange of substances is maintained
between the organisms [73]. Due to its high permeability,
the placenta acts more as a filter than an actual barrier [75].
Bacterial cells are retained within the placenta as are macro-
molecules, such as insulin or heparin, and immunoglobulins,
except IgG. Most drugs pass through the placental barrier,
including barbiturates, antibiotics, sulphonamides and alco-
hol [75]. Small molecules cross the barrier by simple diffu-
sion, while drugs also cross by facilitated diffusion or active
transport [75] or by endocytosis [73, 76–78]. It is assumed
that the penetration of drugs through the placenta is limited
mainly by protein binding rather than lipophilicity [79]. A
significant role in the active transport of drugs is played by
the ATP-binding cassette transporters, with the main ones
being glycoprotein P and breast cancer resistance protein.
They can either transport drug molecules to the fetal side
or return them into maternal circulation; of these, the latter
function is assumed to be more important, and plays a sig-
nificant role in forming the placental barrier [77].
During pregnancy, it is difficult to avoid pharmacother-
apy, and drug usage has increased in recent years. Drugs
are administered in the treatment of chronic diseases such
as epilepsy, diabetes, hypertension or they are prescribed
temporally to treat infections such as the common cold.
Additionally pregnant women often take over-the-counter
drugs and dietary supplements, without medical advice [80,
81]. The amount of a drug which crosses the placental bar-
rier is dependent on various factors: its physicochemical
properties, pharmacokinetics, the concentration gradient on
both sides of the barrier, the differences in pH in between
maternal and foetal plasma and the levels of protein binding
in both organisms [80, 81]. Protein binding is considered the
important property in determining drug transport through
the placenta, influencing both the speed and the extent of
this process [16, 74].
The distribution of a drug between mother and child is
limited also by the concentration of main plasma proteins.
These change continually over the course of pregnancy:
while the concentration of foetal albumin (alpha-fetopro-
tein, AFP) is lower than the maternal HSA level during the
initial stages of pregnancy, it can be up to 20% higher than
maternal HSA at childbirth [16]. The amount of foetal alpha-
1-acid glycoprotein also increases with the development of
the foetus; however, it never exceeds adult levels, remain-
ing about 30–40% lower [16]. There are also differences in
affinities to protein binding sites, with AFP attracting fewer
molecules than HSA in adult plasma [82]. Protein binding
can also occur in both maternal and foetal tissues; drug mol-
ecules can also form a repository in the placenta, from which
it can be released in uncontrolled way into the maternal or
foetal plasma [16].
Table 2 Influence of protein binding on drug penetration into the CNS
* Calculated from unbound fraction data available in the reference paper
** Not available
*** Bovine serum albumin, used as a replacement for HSA
Drug Pharmacological activity Plasma protein binding* CNS penetration Reference
Isoxicam Non-steroidal anti-inflammatory drugs 96,5% in human serum Increased (in the presence of HSA and
AAG)
[69]
Meloxicam 99,7% in human serum Increased (in the presence of AAG)
Imipramine Antidepressants − 52% to HSA − 67% to AAG Higher than predicted from the unbound
fraction (for both proteins)
[70]
Desimipramine − 61% to AAG Higher than predicted from the unbound
fraction (for both proteins)
Isradipine Calcium channel antagonists − 91% to HSA − 92% to AAG Higher than predicted from the unbound
fraction (for both proteins)
[71]
Darodipine − 86% to HSA − 96% to AAG Higher than predicted from the unbound
fraction (for both proteins)
Propranolol Beta blocker NA** Low, compatible with the prediction (in
the presence of BSA***) Higher than
predicted from the unbound fraction (in
the presence of aag)
[62]
Flumazenil GABA receptor antagonists − 39% to HSA Higher than predicted from the unbound
fraction (HSA)
[67]
Iomazenil − 58% to HSA Higher than predicted from the unbound
fraction (HSA)
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3225Molecular Biology Reports (2020) 47:3221–3231
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In vitro experiments with propofol using a human pla-
centa model by He etal. [79, 83] found propofol clearance to
correlate with the concentration of fetal albumin. It appears
that the potential to cross into the placenta is significantly
dependent on binding with alpha-fetoprotein: an increase
of alpha-fetoprotein concentration results in greater drug
penetration. It was also found that infiltration across the
placental barrier diminishes as the concentration of mater-
nal HSA rises. Elsewhere, [84] it was found that HSA has
a great influence on citalopram and fluoxetine placental
transport, with its presence in the perfusion solute increased
the degree of penetration; this effect was correlated with
the affinity of the drugs to HSA: the passage of fluoxetine
(PPB% = 94%) was significantly lower than that of citalo-
pram (PPB% = 50%).
The placenta is considered a very weak barrier against
xenobiotics and most of the administered drugs can easily
cross it. Plasma protein binding appears to affect this process
because it significantly limits placental transit, but alpha-
fetoprotein concentration increase which can enhance the
passage is also an important matter. The accumulation of
drugs in the placenta is still underestimated and it needs to
be studied in detail to get a clearer picture of the processes
that can affect fetal safety during pharmacotherapy.
Skin barrier
The skin is the largest human organ, and one which separates
the internal environment from the surroundings and protects
it from various pathogens. As a barrier, the skin also pre-
vents the penetration of many chemical compounds. This
poses a challenge for the design of dermatological prepara-
tions, which are quite common in modern pharmacotherapy,
mainly due to their easy and convenient application and lack
of side effects typical for the oral administration. Dermato-
logical application can also enhance the systemic activity
of a drug [85]. It has previously been assumed that most of
the administered drug particles are absorbed into the skin
circulation, thus allowing them to pass into the bloodstream,
and that the process was regulated by the skin structure and
condition, the structure of the drug and the type of pharma-
ceutical formulation [85, 86]. However, later studies suggest
that the most important factors determining skin penetration
are the structure and properties of the drug [85]. The per-
meability of the skin varies across its surface in response to
changes in its structure, for example, variation in the num-
bers of follicles or the thickness of the stratum corneum [87].
Externally administered drugs can bind with the proteins
within the skin layers, which can be desirable if only local
action is intended: the drug will accumulate at its site of
activity and will not cause any adverse systemic effects.
However, in the case of transdermal drugs such skin pro-
tein binding will disturb their flow into the circulation, slow
the passage through the skin and reduce the overall amount
of active molecules in the system. Previously, it was found
that highly protein binding drugs achieved lower concentra-
tion in plasma and the time of skin penetration was longer
[88]. A 2008 study [89] examining the different pharmaco-
dynamics of tacrolimus and pimecrolimus with regard to
their ability to penetrate the skin found that pimecrolimus is
more likely to bind non-specifically with various skin pro-
tein than tacrolimus, thus yielding a lower systemic con-
centration (Table3). Similarly, benzocaine has also been
found to accumulate in the skin through non-specific binding
(Table3) [90].
These results suggest that protein binding in the skin
should be carefully studied in case of dermatological for-
mulations, especially for highly protein binding drugs. Albu-
min is present in the skin [91] so the correlation between
binding to HSA in plasma and skin could be a useful tool
in pharmaceutical design. Nowadays, only the process of
skin sensitisation is widely examined, which is supposed to
be the result of non-covalent, reversible binding of various
compound with skin proteins, including albumin [92, 93].
Drug penetration intobreast milk
During lactation, similarly to pregnancy, it can be difficult
to avoid the use of any medicines. Many women abandon
breastfeeding when they take drugs, but often unnecessarily.
The penetration of most xenobiotics into milk is quite low
and only a fraction is typically ingested by an infant [94,
95]. The amount of a drug in breast milk is estimated using
the M/P ratio for a particular drug (Eq.5): this represents
the ratio between concentration of the drug in milk and in
maternal plasma:
Table 3 Influence of protein binding on skin penetration
Drug Pharmacological activity Protein binding in skin Skin permeability Reference
Pimecrolimus Calcineurin inhibitors High, non-specific binding to human skin proteins Lower penetration than in
the case of tacrolimus
[89]
Tacrolimus Low, non-specific binding to human skin proteins –
Benzocaine Local anaesthetic Accumulation of significant amount of benzocaine in skin Low penetration [90]
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3226 Molecular Biology Reports (2020) 47:3221–3231
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This parameter should be calculated for each drug indi-
vidually and can be obtained from clinical studies, observa-
tions of single, medical cases or derived mathematically,
using chemometric methods [96–101]. Drugs with M/P
value lower than 1 are considered as safe for breast-feeding
child.
Breast milk is regarded as the best nourishment for a new-
born infant, and for first six months of life, it can be its only
food. Milk is produced in the mammary glands by special-
ised cells called lactocytes [102]. It is composed of a mixture
of water and carbohydrates, proteins, lipids, vitamins and
various other nutrients [103], with the composition changing
over the course of lactation [104, 105]. During the first stage
of lactation, breast milk, colostrum, is composed mainly of
structural proteins and proteins which support the immune
system e.g. lactoferrin or immunoglobulins [106, 107]. This
is later replaced by transitional milk, which has higher levels
of carbohydrates and lipids and it is more nutritious than
colostrum. Mature milk is produced around the third week
after birth, and it consists of around 7% carbohydrates, 4%
lipids and less than 1% proteins [102, 106, 108]. This is
later replaced by transitional milk, which has higher levels
of carbohydrates and lipids and it is more nutritious than
colostrum. Mature milk is produced around the third week
after birth, and it consists of around 7% carbohydrates, 4%
lipids and less than 1% proteins [109].
Drugs mostly penetrate into breast milk by simple dif-
fusion along a concentration gradient. This process is also
limited by various factors connected with the compound
structure: molecular weight, lipophilicity, protein binding
or pKa [94, 110]. The pKa of a drug plays an important role
on its accumulation in milk: the mean pH of breast milk
ranges from 7.1–7.2 while that of plasma is around 7.4 [102,
105]. Weak bases become ionized in breast milk, trapping
them inside the mammary gland and preventing their return
to maternal plasma [111]. In addition, drugs with high lipo-
philicity can also accumulate in the lipid phase of breast
milk, and while protein binding can prevent the passage of
molecules into milk, drugs also bind with the breast milk
proteins themselves [112]. The composition of the protein
phase consists of alpha-S1, alpha-S2, beta- and kappa-
caseins, alpha-lactoalbumin, beta-lactoglobulin, plasma
albumin and lactoferrin, as well as immunoglobulins A, M,
G and lysozyme and alpha-1-acid glycoprotein [104, 113].
However, drug binding is typically weaker in breast milk
than in plasma [111].
Drug transfer into breast milk is still a difficult subject
for invivo study. Although clinical studies have been per-
formed, they are usually based on very small groups of sub-
jects or describe individual cases. Short-term use of drugs,
(5)
M/P = drug concentration in milk
drug concentration in plasma
during infection for example, seems to be less problematic
than in the case of long-term pharmacotherapy. Women
suffering from chronic conditions such as multiple sclero-
sis, epilepsy or psychiatric disorders, or those undergoing
anticancer therapy, often want to maintain breast-feeding.
A review by Constantinescu etal. [114] examined the usage
of various immunosuppressive drugs, including azathio-
prine, belatacept, corticosteroids, cyclosporine A, everoli-
mus, sirolimus and tacrolimus, during lactation. A study of
methylprednisolone levels in the breast milk of two lactating
women, one of them after renal transplantation and the other
with multiple sclerosis [115, 116] found that methylpredni-
solone passes poorly into milk, which could be related with
its high PPB%, estimated to be around 79% (Table4) [116].
A 2013 study of antiepileptic drugs by Davanzo etal.
[117] reviewed a body of pharmacokinetic and clinical data,
including relevant infant dose (RID), and toxicity guidelines
taken from LactMed [118] and Hale [119]. Older-generation
drugs such as carbamazepine, phenobarbital, phenytoin and
valproic acid were found to be relatively safe, even pheno-
barbital, which weakly binds with plasma proteins in mater-
nal plasma (20–45% [120]). The overall conclusion was that
neither pharmacokinetic or literature toxicity parameters are
good predictors of the drug penetration into breast milk. The
penetration of cisplatin across the placenta [121] and into
breast milk (Table4) [121, 122] was also studied. The drug
was found to demonstrate poor penetration into milk as its
platinum ion binds strongly with plasma proteins [120, 122].
However, cisplatin is contraindicated during lactation, prob-
ably due to the fact that that it accumulates during repeated
dosage.
Postpartum depression or anxiety also requires a long-
term treatment. SSRIs (selective serotonin reuptake inhibi-
tors) are believed to be the safest drugs for lactating women
because their high PPB% values, among other factors, pre-
vent them from crossing readily into milk (Table4) [123].
One exception is paroxetine, as it has been linked with an
increased risk of heart dysfunction [124]. A detailed reviews
about CNS drugs usage during lactation by Eberhard-Gran
etal. and by Weissman etal. [125, 126]. They provide data
regarding drug secretion into breast milk and recommenda-
tions for use. The latter study also points out the negative
correlation between PB and M/P values [125]. Further infor-
mation about the use of antidepressants is also given in a
review by Lanza di Scalea and Wisner [127].
In most of the cases described, where the excretion of the
drug into milk is very low, one of the main reasons men-
tioned is high plasma protein binding. This may indicate that
milk penetration may be the most PPB dependent of all the
barriers described in this review. Another issue to consider
is milk protein binding, which further reduces the amount of
medicine an infant ingests. Experts claim that breastfeeding
should not be interrupted during pharmacotherapy unless it
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3227Molecular Biology Reports (2020) 47:3221–3231
1 3
is necessary, which seems to be a reasonable solution. How-
ever, there should be sufficient evidence that the medicine is
safe for breastfed infants or wouldn’t interrupt the lactation.
Summary
Drug-protein binding has a significant influence on the phar-
macokinetic properties of most compounds. It can limit the
bioavailability of active compounds by controlling their
passage through biological membranes; however, binding
to plasma proteins allows hydrophobic drugs to be trans-
ported in the aqueous environment of the human organism.
The drug-protein complex is less likely to cross the placental
barrier or to enter breast milk, which decreases the nega-
tive effect of medicines on breastfeeding infants; however,
some drugs can accumulate in placental tissues or in milk
by binding with proteins in these regions, and upon their
later release, enter the foetus or infant in uncontrolled way.
Passage through the blood–brain barrier is more complicated
by mechanisms which protect the central nervous system,
such as active efflux and the use of strong protein binding
mechanisms. Additional unknown mechanisms that lead to
the penetration of several protein-bounded drugs make this
matter even more complex. Skin penetration is an impor-
tant issue for transdermal drugs because they have a strong
impact on their bioavailability and protein-binding interacts
with this process.
Protein binding is relatively simple to study invitro, but
its effect on crossing biological barriers in a living organ-
ism could be difficult to grasp with these methods. This is
probably due to the complexity of the entire barrier-crossing
process and additional side-effects that simply cannot be
obtained in the laboratory.
Acknowledgements This work was supported by an internal grant of
the Medical University of Lodz no. 502–34-106.
Compliance with ethical standards
Conflict of interest The author declares no conflict of interest, finan-
cial or otherwise.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
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