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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5571_Библиотеки_им_академика_М_И_Перельмана
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targets. One exception is the capillary bed of the central nervous system (CNS), which
possesses a system of circumferential tight junctions that limit paracellular entry of
compounds. In this case, the ability of drugs to readily diffuse across the phospholipid
cell membrane is important for entry into the CNS as well as to other intracellular
target sites. At the same time, both absorptive (e.g., OATP in the liver) and exsorptive
(e.g., MDR1 in the CNS) transporters within the cell membrane can influence the
efficiency with which drugs can cross various cell barriers.
4.3.2 Volume of Distribution
The apparent volume of distribution is simply a proportionality factor between the
total amount of drug present in the body and drug concentration in a reference fluid
(typically plasma) at a given time. It is a theoretical value that reflects in part tissue
affinity. The volume of distribution at steady state (V
ss
) describes the extent of drug
distribution from the plasma pool into tissues in the body as
V
ss
¼ Vpþ KpV
t
where Vpis the volume of plasma, Vtis the volume of the extravascular space plus the
erythrocyte volume into which drug distributes, and K
p
is the steady-state tissue-to-
plasma drug concentration ratio.
For a drug that is confined to the blood, the apparent volume of distribution would
be about 0.08 L/kg (i.e., total blood volume). Similarly, a drug that distributes only
into total body water would have an apparent volume of distribution of about 0.6 L/kg.
Beyond these values, the volume of distribution only has mathematical significance.
For example, a volume of 4 L/kg means that about 2% of the drug is in circulation. The
rest of the drug may be moderately distributed to many tissues or concentrated in only
a few. Ions such as bromide do not readily pass through cell membranes but distribute
rapidly throughout extracellular water volume, having a volume of distribution of
about 0.4 L/kg [76]. On the other hand, neutral lipid-soluble drugs, such as antipyrine [77], readily diffuse through cell membranes and easily distribute throughout
extra- and intracellular water volume and can have a volume of distribution of about
0.6 L/kg (i.e., total body water). Compounds such as basic drugs, like amlodipine,
have a higher affinity for tissue than for plasma proteins, and can easily have volumes
of distribution greater than 10 L/kg [78]. This observation can, in part, be attributed to
the ion pairing that occurs between the positive charge of basic compounds and the net
negative charge of cell membrane constituents such as phospholipids, sialic acid
residues on glycolipids, and the carbohydrate-rich glycocalyx.
Table 4.1 summarizes some generalizations regarding acidic, neutral, and basic
compounds. A general comparison between acidic, neutral, and basic compounds and
volume of distribution is shown in Figure 4.7, with reference to lipophilicity [79].
Since half-life (t
1/2
) is determined by both the volume of distribution (Vb) and
clearance (CL), t
1/2
¼ ln2(Vb/CL), manipulation of the volume of distribution (with a
concomitant understanding of changes in clearance) can be used to change the drug
half-life and, potentially, the duration of effect.
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If a drug gradually diffuses from the plasma to various tissue compartments, the
volume of distribution actually increases with time. This is exemplified when a
plasma concentration–time profile exhibits a biexponential (two-compartment
model) decline following IV administration. Three different volumes of distribution
apply in this case: the early apparent volume of distribution of the central compartment (V
c
), the apparent volume of distribution at steady state (Vss) or distributional
equilibrium between plasma and tissue compartments, and the later maximal apparent
TABLE 4.1 General Volume of Distribution Trends for Acidic, Neutral, and Basic
Drugs
Compound
Type
Volume of
Distribution (L/kg) Binding Behavior Distribution Characteristics
Acidic <0.4 Plasma > tissue Drug mainly confined
to plasma with limited
tissue distribution
Neutral 0.4–1.0 Plasma tissue Drug uniformly distributes
between plasma and tissue
Basic >1.0 Tissue > plasma Drug may be more concentrated
in particular tissues
Figure 4.7 Illustration of differences in volume of distribution between acidic, neutral, and
basic compounds, as well as the relationship of volume of distribution to lipophilicity
(log D) [79].
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volume of distribution at pseudodistribution equilibrium (Vb). In the case of a
monoexponential concentration–time profile, the body behaves like a single compartment, and the three volume of distribution terms, V
c
, Vss, and Vbbecome the same
and are independent of time.
4.3.2.1 In Vivo Tools to Assess Drug Distribution Whole animals can be used to
assess the volumes of distribution following IV administration (see Chapter 5),
although this value does not provide information about drug distribution to specific
organs or tissues. Specific tissue distribution may be assessed by collecting various
tissues and assessing total drug concentration with time following bolus or infusion
(steady state) administration.
A spatial tissue distribution of drug candidates can also be obtained using the
technique quantitative whole body autoradiography (Q WBA), which quantifies the
total radioactivity of tissues in thin sliced sections of the animal after the administration of a radiolabeled drug candidate. This technique does not distinguish between
intact drug and radiolabeled metabolites. Newer techniques such as matrix-assisted
laser desorption/ionization (MALDI) mass spectrometric imaging can simultaneously measure compound and metabolites distributed in whole-body tissue sections,
using nonradiolabeled compounds [80].
The comparison of the tissue distribution data with and without the use of chemical
transport inhibitors [81] or knock-out animals may also provide valuable information
about the mechanism of distribution.
4.3.3 Free Drug Concentration
Within blood, drug can bind to various components including red blood cells and
plasma proteins. As a consequence of binding, the concentration of total drug in whole
blood and plasma, and unbound drug in blood and plasma, can vary significantly.
Examples of plasma proteins in human blood are listed in Table 4.2. The two most
important proteins are albumin and a
1
-acid glycoprotein, which have a preference for
acidic and basic drugs, respectively, although basic drugs can bind to albumin as well.
TABLE 4.2 Representative Plasma Proteins in Human
Plasma Protein Molecular
Weight (kDa)
Concentration
(mM) Preference in Binding
Albumin 67 500–700 Acidic drugs
a
1
-acid glycoprotein 42 9–23 Basic drugs
Lipoproteins 200–2400 Variable Lipophilic and basic
drugs, triglycerides,
phospholipids, and
cholesterol
Cortisol-binding
globulin (transcortin)
53 0.6–1.4 Steroids
Source: From Ref. 83.
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Approximately 40% of the total albumin in the body is in the plasma, with the
remaining located mainly in interstitial fluid (Fig ure 4.6). Binding of drug to plasma
proteins is typically reversible, occurs with rapid equilibrium, and is saturable.
Lipophilicity is one, but not the only general descriptor influencing plasma protein
binding [82].
Characterization of drug protein binding is important in the understanding of
the overall pharmacokinetic and pharmacodynamic properties of the drug.
According to occupancy theory of drug response, receptor activation is assumed to
be directly proportional to the number of receptors occupied by a given drug ligand. It
is generally assumed that only free drug can exert pharmacological activity and that
unbound drug is free to move across cell membranes. Because the intended site of
activity may not be directly sampled, the pharmacodynamic relationship is typically
investigated when drug concentrations at the site of activity are in equilibrium with
those compartments that are accessible such as plasma or blood. Two important
aspects are important when correlating plasma concentrations with those at the
active site:
1. Drug has the appropriate physicochemical prope rties that allow it to diffuse
freely across tissue compartments and cell membranes and it is not subject to
transporter and/or metabo lic activity.
2. Unbound drug has direct access to the receptor.
In addition to the importance of free drug and pharmacolo gic activity, it is also
generally assumed that only unbound drug is subject to processes of hepatic and renal
clearance. The relationship between the free fraction of drug in blood (f
b
u
) and hepatic
clearance (CL
h
) is illustrated by considering the well-stirred hepatic clearance
model [84]:
CL
h
¼½Qhf
b
u
ðCL
int
=f
mic
u
=½Qhþ f
b
u
ðCL
int
=f
mic
u
Þ
where Q
h
is the hepatic blood flow (20 mL/min/kg in human), CL
int
is the intrinsic
hepatic clearance of the drug, and f
mic
u
is the fraction of drug not associated with
microsomes. The free drug concentration in red blood cells is related to free plasma
concentration as fol lows:
f
b
u
Cb¼ f
p
u
C
p
where Cband Cpare the total concentration in blood and plasma, respectively, and f
p
u
is
the free fraction of drug in plasma. It is generally assumed that free drug concentration
in red blood cells is the same as that in plasma, but drug can preferentially partition
into red blood cells and impact the interpretation of organ clearance [85]. The wellstirred hepatic clearance model would suggest that greater binding in blood or plasma
would reduce free drug concentrations within hepatocytes. Whether a change in blood
or plasma binding of a drug impacts overall hepatic clearance also depends on its
intrinsic hepatic clearance and microsomal association.
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Protein binding is also important in the renal clearance (CLr) of drug
CL
r
¼ f
b
u
½GFR þðQrCL
s
int
Þ=ðQrþ f
b
u
CL
s
int
Þ½1 Fr
where GFR is the glomerular filtration rate, Q
r
is the renal blood flow, CL
s
int
is the
intrinsic clearance for active renal tubular secretion, and F
r
is the fraction of drug
reabsorbed from urine back into blood. Renal clearance generally trends lower with
increasing lipophilicity [86].
4.3.3.1 Tools to Assess Free (Unbound) Drug Conc entrations Microdialysis can
be used to assess free drug concentrations in various organs or blood circulation in
vivo [87]. There are also a number of in vitro methods available to assess unbound
drug concentrations in plasma, blood, or tissue homogenate including human
albumin-immobilized chromatographic columns, ultrafiltration, and equilibrium
dialysis [88–90]. Among them, equilibrium dialysis is considered the preferred
standard method and has more recently become amenable to higher throughput [91].
The technique is based on the establishment of an equilibrium between two chambers
separated by a semipermeable membrane; one chamber containing plasma, blood, or
tissue homogenate and the other only buffer. Drug is added to the buffer chamber and
its equilibrium allowed to be established between the two chambers (steady state
should be validated). Both temperature and pH during the experiment are critical in
order to accurately assess binding.
4.3.4 CNS Penetration
The blood–brain barrier (BBB) is the most important barrier for drug entry into the
central nervous system [92–96].
The brain capillaries form a continuous layer of endothelial cells joined together by
circumferential tight junctions, which highly restrict s paracellular diffusion of
molecules. Drug penetration across the BBB is essential for centrally active compounds; however, the BBB can also be exploited to minimize potential central side
effects [97].
Good brain penetration requires that a compound has good passive permea bility.
Lipophilic physicochemical properties have been shown to be particularly important
for CNS active drugs [98] (Table 4.3). Good brain penetration also requires avoidance
of P-glycoprotein (MDR1) susceptibility [94, 99, 100]. While other transporters have
been identified at the BBB [101], P-glycoprotein has been currently shown to have the
greatest clinical relevance [100]. P-glycoprotein is located at the luminal capillary
surface of the BBB and possesses a highly promiscuous substrate specificity. Greater
molecular weight, log P, polar surface area, and H-bonding increase the probability of
P-glycoprotein interaction [102, 103].
In addition to permeability across the BBB, brain distribution and, in particular,
unbound brain concentration are important for overall drug delivery to the brain [104–
108]. It is generally assumed that free brain concentration will be the primary driver of
physiological response. Consistent with this argument, central dopamine D
2
receptor
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occupancy has been shown to significantly correlate to free brain concentrations for a
number of antipsychotics, whereas total brain to plasma ratios were less useful [109].
In fact, simply considering total brain to plasma ratios has not generally been a useful
correlation to pharmacological action [106, 110]. Large amounts of drug in the brain
may be reflective of extensivebinding to brain tissue rather than free drug available for
interaction with its target receptor.
The relationship between free plasma, free brain, and total cerebrospinal fluid
(CSF) concentrations can be described by a three compartment model [106]
(Figure 4.8). It is important to understand that this equilibrium is only relevant for
freely diffusing drugs that are not subject to active transport (e.g., P-glycoprotein) or
metabolism between compartments [109, 111]. Under these circumstances, drug
concentration at the central target site can be predicted simply by measuring free
plasma or CSF concentration at steady state [112, 113].
TABLE 4.3 Calculated Physicochemical Properties of CNS Drugs [98]
Property Mean Range
MW 319 151–655
cLog P 3.43 0.16–6.59
cLog D 2.08 1.34–6.57
Polar surface area (A
˚
´
) 40.5 4.63–108
H-bond donors 0.67 0–3
H-bond acceptors 1.85 0–7
Positive charges 1.19 0–3
Negative charges 0 0
BCSFBBBB
C
u
p
C
p
C
csf
C
u
b
C
b
Target
Brain
Blood
CSF
Figure 4.8 Relationship between drug concentration in plasma, brain, and CSF compartments
for a freely diffusing molecule. Key: C
p
, Cb, and C
csf
are total plasma, brain, and CSF
concentrations, respectively; C
u
p
and C
u
b
are unbound plasma and brain concentrations,
respectively; BBB, blood–brain barrier; BCSFB, blood–CSF barrier.
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4.3.4.1 Tools to Assess CNS Delivery of Drugs In terms of experimental tools in
drug discovery, at a minimum, the three most import ant aspects of assessing
compounds for CNS delivery are as follows:
.
passive transcellular permeability,
.
human P-glycoprotein substrate susceptibility.
.
free brain concentrations .
The first two parameters will characterize the rate of BBB penetration, while the
last parameter relates to the extent of brain distribution.
Tools to Assess Passive Transcellular Permeability into CNS There are a number of
in vitro transwell cell permeability models available to assess passive permeability,
where Caco-2 and Madin–Darby canine kidney (MDCK) cells are the most
common for industrial use. The major criterion for any in vitro BBB permeability
model is that it possesses a restrictive paracellular pathway [114]. Much work has
been done to characterize in vitro brain endothelial cell lines for this purpose, but these
tend to be limited by high paracellular permeability or lack high-throughput capacity [100, 115]. Noncell-based in vitro models such as PAMPA methodology have
shown some value in assessing passive permeability of potential drug candidates as
well [116].
Tools to Assess P-Glycoprotein Substrate Susceptibility Assessment of P-glycoprotein substrate specificity is typically performed in the cell types discussed above,
where the stably transfected human MDR1-MDCK cell line has received significant
attention [117, 118]. The P-glycoprotein knockout mouse is also a valuable tool and
has shown widespread use in assessing the in vivo function of the efflux transporter in
drug disposition [119, 120]. There have been reports of species differences in the brain
uptake of P-glycoprotein substrates [121] and some breeds of dog, such as Collie,
actually lack functional P-glycoprotein [122]. In general, compounds found to be
substrates in rodent models are also likely to be substrates in higher species [100, 118,
122]. It is important to appreciate that the majority of marketed CNS active drugs have
weak to no human P-glycoprotein interaction [100].
Tools to Assess Free Brain Concentrations of Drugs Techniques used to assess free
brain concentrations include in vivo microdialysis and in vitro binding to brain tissue
homogenate or slices [106, 123]. While microdialysis is a direct measure of free
in vivo brain concentrations, there are challenges of nonspecific drug binding and
the methodology is quite labor intensive and low throughput. Greater use is being
made of indirectly estimating free brain concentrations by combining in vivo
assessments of total brain to plasma concentration ratios and estimates of in vitro
brain binding [104, 105, 107, 108]. Total brain to plasma ratios are successfully
assessed by either measurement of total brain and plasma AUC values following a
single PO dose or with terminal brain, plasma, and CSF concentrations following a
steady-state IV infusion. Care should be taken initially with single point plasma and
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brain measurements following single PO doses without an understanding of potential
differences in peripheral and central absorption and clearance rates. When using in
vitro brain homogenate binding techniques, consideration should be given to the
ability of a compound to move freely between intracellular and extracellular
compartments, where brain slices retain greater cellular integrity [123].
4.4 ELIMINATION
Elimination is the process by which a drug is irreversibly removed from the systemic
circulation by metabolism and/or excretion. Metabolism, also referred to as biotransformation, is the chemical alteration of a drug or substance in the body by the
action of enzymes. Metabolites occasionally can be converted back to parent
compound; thus metabolism is an elimination process only if the metabolite is
excreted or lost from the body irreversibly. Excretion is the irreversible removal of
unchanged drug and/or metabolites from the body by all possible routes [124].
Quantitatively, renal excretion and hepatic biliary secretion are the two most
important excretion mechanisms, although drugs and metabolites can also be excreted
from lung, sweat, tears, and other bodily fluids.
4.4.1 Elimination Versus Clearance
The terms elimination and clearance are often used interchangeably, particularly with
respect to the mechanism of clearance, including in this chapter. However, elimination refers to the physiological process of removing drug from the systemic circulation, while clearance is a pharmacokinetic parameter defined as the quantitative
measure of the rate at which a substance is removed from the blood, and is usually
derived from blood or plasma concentration data.
4.4.2 Metabolism Versus Excretion
In a pharmaceutical discovery setting, in vivo pharmacokinetic studies in preclinical animal species are used to elucidate the elimination pathways in animals
and predict the principal elimination pathways of drug candidates in humans. As a first
step, compound is dosed intravenously and the parent compound concentrations
are determined in plasma (or blood) and urine. The contribution of renal excretion
can be determined from the urine concentrations, and the remainder of the elimination
is likely to be hepatic, especially for lipophilic compounds. Hepatic elimination
occurs via metabolism or biliary secretion, or a combination of the two mechanisms.
The contribution of biliary secretion to elimination can be assessed by dosing the
compound intravenously in a bile-duct cannulated animal, such as a rat, and
determining the amount of parent compound secreted into bile. When a parent
compound is initially metabolized, the elimination mechanism is metabolism,
even though the metabolites may be subsequently excreted in urine and feces
(via bile).
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4.4.3 Drug-Free Fraction and Clearance
An important assumption in clearance is that only the free compound is available to be
cleared from systemic circulation whether by biotransformation or excretion. The
compound must be unbound to plasma proteins or other cellular or tissue components
in order to be eliminated because protein-bound compounds cannot be readily
metabolized or transported across membranes by hepatic or renal transporters (see
earlier discussion in Section 4.3.3).
4.4.4 Lipophilicity and Clearance
Lipophilicity is the major determinant of clearance for neutral drugs and drug-like
compounds. In general, lipophilic drugs are most often eliminated by hepatic
metabolism, whereas hydrophilic drugs tend to be eliminated renally. A relationship
between log D
7.4
and clearance has been established (Figure 4.9). For drugs with log
D
7.4
below 0, excretion is predominantly renal, and renal excretion decrease s as log
D
7.4
becomes more lipophilic, while metabolic clearance increases as log D
7.4
becomes more lipophilic [79].
4.4.5 Transporters and Clearance
In addition to lipophilicity, another key determinant of clearance is affinity for
transporter proteins in the liver and kidney, especially for charged molecules and
compounds containing both lipophilic and hydrophilic functionalities.
4.4.5.1 Hepatic Uptake Transporters In the liver, drugs enter hepatocytes by
either passive diffusion through the cellular membrane or by hepatic uptake transport
Figure 4.9 Relationship between lipophilicity and unbound renal (squares) and metabolic
clearance (triangles) for a range of neutral drugs in man [79].
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proteins, or by a combination of these two mechanisms. Hepatic uptake transporters
are especially important for anionic or cationic compounds of molecular weight
>400. For ionizable compounds of molecular weight >50 0, hepatic uptake may even
serve as the rate-limiting step in clearance [125]. Important hepatic uptake transporters are shown in Figure 4.10.
4.4.5.2 Biliary Transporters and Secretion While lipophilic, low-molecular
weight compounds can easily diffuse out of hepatocytes through the cellular
membrane, hepatic efflux transporters are required for secretion of ionizable and
higher molecular weight compounds into the bile. Important biliary transporters are
shown in Figure 4.10.
4.4.5.3 Renal Transporters Renal transport mechanisms are important for the
excretion of drugs that are positively or negatively charged at physiological pH and
are discussed below in Section 4.4.7.1.
4.4.6 Metabolism
Metabolism by biotransformation enzymes is the primary mechanism of elimination
for many drugs and other xenobiotic substances, especially for lipophilic compounds.
Drug metabolism and biotransformation are often used interchangeably and refer to
the structural modification of a chemical by enzymes in the body. The drug or
xenobiotic compound can be metabolized by direct modification, referred to as a
Phase I reaction, and by the addition of a conjugate functionality, which is termed a
Phase II reaction.
Figure 4.10 Hepatic uptake and biliary secretion transporters.
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