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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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properties of both the drug and the route of administration [1]. The characteristics of a
drug that make it an ideal candidate for one route of administration do not necessarily
allow it to be effectively delivered via an alternative route.
4.2.1.1 Oral Administration As oral administration is the most convenient, safest,
and least expensive way to deliver a drug, it is the route most often used. Following
drug dissolution, the drug initially must pass intact through the oral cavity, esophagus,
stomach, and finally intestinal epithelium into the portal vein system, and subsequently through the liver before it is transported via the bloodstream to its systemic
target site (Figure 4.1). Because of the large surface area created in the small intestine
by the presence of villi and microvilli, the majority of drug absorption occurs in this
region [1]. In addition to the absorption surface area, several factors such as pH
stability, intestinal blood flow, the physical state of the drug (solution/solid dosage
form), solubility and dissolution of drug, and the luminal drug concentration also
influence oral absorption.
While the drug travels from the gastrointestinal tract to the systemic circulation,
the intestine and the liver can metabolize many drugs, decreasing the amount of drug
reaching the bloodstream (referred to as first-pass or presystemic clearance) [2, 3]. In
this regard, both the drug properties and the physiology of the gastrointestinal tract
determine overall drug oral absorption. Good oral bioavailability of a drug essentially
implies that the drug is able to reach the systemic circulation if administered by
mouth. The absorption of drugs via the oral rout e is a subject of intense and continuous
investigation in drug discovery and will be an important component of further
discussion here.
4.2.1.2 Parenteral Administration The parenteral route includes a number of
administration routes other than through the gastrointestinal tract [4] such as
intravenous, subcutaneous, intraperitoneal, and intramuscular, which have little in
common except that they require a hypodermic needle for delivery. These routes
bypass a number of physiologic barriers present with the oral route and can be
particularly useful for the delivery of biotherapeutic drugs, but may also provide rapid
systemic access when studying the biological effects of small molecules. The
intravenous and subcutaneous routes of administration are discussed here.
Intravenous Injection The intravenous (IV) route is a well-controlled and optim al
way to deliver a precise dose quickly into the body, not possible by other administration routes. IV administration requires delivery of a solution containing the drug
directly into the vein as a single dose through a needle puncture. The drug can also be
continuously infused by gravity or by an infusion pump through a catheter inserted
into a vein for greater control of drug concentrations. IV delivery can be used for more
irritating solutions, which might otherwise cause pain and damage to tissues if given
by the subcutaneous or intramuscular routes. There are both advantages and disadvantages to the use of IVadministration. An advantage is that drug injected directly
into the bloodstream can take effect more quickly than by any other route of
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administration. A disadvantage is that a high concentration of drug may be attained
rapidly, which may result in an unfavorable reaction or undesired side effects related
to the maximal plasma concentration.
Subcutaneous Injection A solution-containing drug can be administered by a needle
inserted into tissues just beneath the skin surface, where the drug diffuses into small
blood vessels and capillaries and then is carried away by the bloodstream or reaches
the bloodstream through the lymphatic vessels. The subcutaneous injection (SC)
route has been used for many biotherapeutics, such as protein drugs or vaccines,
because such drugs are not stable in digestive fluid and SC administration avoids
degradation of drug in the gastrointestinal tract. Biotherapeutics like insulin are large
in size and usually reach the bloodstream slowly through the lymphatic vessels, thus
prolonging absorption. Through SC injection, a drug can be prepared for controlled
release delivery in order to prolong drug absorption from the injection site for hours,
days, or longer. Certain drugs such as progestin for birth control may be given
subcutaneously by inserting plastic capsules under the skin for very long duration of
activity [5].
4.2.1.3 Other Routes of Administration Alternatively, drugs can be delivered
through various other routes of administration including pulmonary, nasal, sublingual, ocular, rectal, vaginal, and transdermal [1, 6]. These routes may be used when a
drug is needed, for example, to produce rapid absorption (e.g., sublingual for
nitroglycerin delivery) or for a local therapeutic effect (e.g., antiasthmatic drugs).
Each of these routes can require vastly different physicochemical drug properties for
optimal delivery.
4.2.2 Factors Determining Oral Bioavailability
Bioavailability is a term used to indicate the fractional extent of a dose of drug
reaching the systemic circulation. Oral bioavailability is the result of both absorption
and clearance processes. The important factors determining oral bioavailability
include drug solubility and dissolution, chemical and enzymatic stability in the
intestinal lumen, interacting luminal contents (food), gastrointestinal transit time,
enterocyte permeability, and intestinal and hepatic metabolism (Figure 4.1).
The bioavailability (F) of a compound is defined by the equation
F ¼ f
a
ð1 EgÞð1 EhÞ
where f
a
is the fraction absorbed representing all processes from dissolution of the
solid dosage form to the intestinal transport of the drug; and E
g
and Ehare the first pass
metabolic extraction/removal from the gut and the liver, respectively.
While the permeability, solubility, and metabolic stability of the compound may
be important individually, the interrelationship of these properties will determine
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the overall in vivo bioavailability of a drug. A drug with very low solubility may
be problematic for a high dose compound but acceptable for one with a low dose. The
maximum absorbable dose (MAD) model has shown some value in relating aqueous
solubility and intestinal permeability to absorption potential [7, 8]:
MAD ¼ SK
a
SIV SIT
where S is maximal solubility, K
a
is a rate constant of absorption, SIV is the small
intestinal volume, and SIT is transit time of the small intestine.
Overall PO bioavailability can be evaluated in vivo by comparing the dosenormalized area under the concentration (AUC) time curve following PO and IV
administration. A first assessment of PO bioavailability comes from animal
models. It is important to appreciate the potential differences between preclinical
animal models and human in order to under stand approaches to best predict human
performance.
As drug solubility and dissolution have been thoroughly discussed in Chapter 3,
this subject will not be reviewed in detail here. The following sections will primarily
discuss the aspects of the intestinal permeability of drugs. As metabolism, in general,
is covered in Section 4.4, the following discussion will only include some highlights
of intestinal metabolism.
4.2.2.1 Mechanism of Drug Absorption A drug can cross the intestinal mucosa
via a number of different pathways. The mechanisms involved in the passage of drug
across the enterocyte into the portal system include: (1) passive diffusion (paracellular
and transcellular); (2) active transport including facilitated uptake as well as active
efflux, and (3) receptor-mediated endocytosis [9, 10]. The absorption mechanisms
are not independent from each other, and will take place in parallel for susceptible
molecules. Figure 4.3 illustrates the mechanisms of intestinal absorption.
For typical drug design, the important mechanisms of absorption to consider are
passive permeability and carrier-mediated transport.
Figure 4.3 Mechanisms of absorption in the gastrointestinal tract.
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4.2.2.2 Passive Permeability Permeability is the velocity of drug traveling across
a biological membrane. It is a kinetic parameter related to lipophilicity, which in turn is
related to the partition and distribution coefficients. Physicochemical profiling of drug
molecules is important in assessing the potential for good oral absorption [11, 12]. At
the same time, physicochemical properties are not independent of each other, and
effective use is in the collective assessment of these parameters. Lipinski’s guidelines
have been useful in this regard, stating that good oral absorption is more probable for
molecules that have MW < 500, clog P < 5, hydrogen-bond donors <5, and hydrogenbond acceptors <10 [13]. Polar surface area (PSA) is another parameter that can
correlate with the permeability of drug candidates [14], with low PSA values
corresponding to high permeability.
Passive diffusionis not a saturable process and so cannot be inhibited by other drugs
unless the physical properties of the drug or membrane are affected in some way. The
process of crossing the gastrointestinal tract follows the solubility–diffusion model,
which is defined by the following equation derived from Fick’s law:
Flux rate ¼½D A K DC=X
where D represents the diffusion constant, which is inversely proportional to the
molecular weight of the drug; A represents the area of the membrane available for
permeability; K represents the membrane partition coefficient, which is represented by
lipid solubilityand is the most important determinant of the speed of transfer across the
membrane; DC is the concentration gradient across the membrane; and X is the
thickness of membrane. The permeability coefficient, a useful parameter for comparing drugs, is defined as [D K]/X.
The luminal surface of the gastrointestinal tract is lined with a monolayer of
epithelial cells tethered together through apical tight junctions. There are two passive
permeability pathways available for drug molecules: transcellular and paracellular
diffusion. Transcellular diffusion involves the movement of drug through the epithelial cells by passing directly through the cell membrane. In contrast, paracellular
transport is defined as drug movement into bloodstream between epithelial cells by
simple diffusion [15, 16]. The small paracellular pore size limits the permeation of
hydrophilic compounds [17], where increasing molecular size decrease s the rate of
paracellular penetration. Paracellular permeability is both size and charge dependent [18]. Generally, paracellular permeability is relevant for smaller drugs with
molecular weights of less than about 300 Da. Paracellular transport is preferred for
drugs with a positive charge and log D < 0.
The transcellul ar passive mechanism is the preferred pathway for drugs that are in
the 500 Da range or less, are mode rately lipophilic (lo g D > 0), and uncharged. As the
majority of drugs are relatively lipophilic in nature, the transcellular route is thought
to be the major mechanism of permeability for most marketed drugs [19, 20].
4.2.2.3 Tools to Assess Passive Permeability A number of tools are available to
assess the passive permeability of molecules and are discussed below.
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In Vivo Models for Assessing Permeability The extent of the drug absorption from
the gastrointestinal tract can be evaluated by comparing the PO dose plasma AUC
with the AUC of the compound in samples collected from a hepatic portal vein
cannulated animal [21, 22].
In Situ and Ex Vivo Models for Assessing Passive Permeability Absorption processes can be assessed in situ and ex vivo through perfusion of a drug solution through
an isolated intestinal segment [23–25]. Two ends of an intestinal segment are
cannulated, setting up a closed circulation loop through which a buffered drug solution
is circulated. Drug absorption is assumed from a measurement of drug disappearance
from the circulating solution, which may not always be the case.
The permeation of a drug across intestinal mucosa can also be studied ex vivo in an
using chamber, which consists of two sampling chambers with a section of intestinal
mucosa mounted between the chambers [21, 26]. The Using chamber provides a
physiological system to measure the transport of ions, nutrients, and drugs across
various epitheli al tissues [27].
Another ex vivo technique used for studying drug absorption is the inverted gut
sac [28]. It is prepared by removing the small intestine from the animal, inverting it over
a glass rod and dividing it into segments. Sacs (mucosal side out) are then submerged in
culture medium containing the drug, and accumulation within the inner compartment is
measured. Under optimal conditions, sacs remain viable for up to 120 min [21].
Cell Cultur e Systems for Assessing Passive Permeability A major method of
permeability assessment is the use of various cell culture systems such as Caco-2,
MDCK, HT29, and LLCK-1 cell lines. In this method, the immortalized cells are
grown on a semipermeable filter, generating a cell monolayer with tight junctions.
Theoretically, any cell line that could form a cell monolayer with tight junctions may
be applied in the permeability assessment.
Among the cell lines, Caco-2 has received the greatest attention and characterization. These cells are of human colon adenocarcinoma origin and develop microvillus that mimic the morphology of intestinal epithelial cells as well as the expression
of some drug transporters [29]. However, the long time needed for cell differentiation
(18–21 days in culture) hinders its high-throughput use in the pharmaceutical
industry. A 5 day Caco-2 system has been developed for permeability assessment,
however, its transporter expression and functionality are not optimal [30].
Recently, the use of MDCK cells has increased in popularity due to ease of
handling and shorter differentiation times (4–5 days). MDCK cells are of canine
origin and kidney phenotype. Since these cells lack human P-glycoprotein expression,
they are useful as a good background model for over-expression of human transporters
such as P-glycoprotein [31]. With any of the cell culture systems, it is important to
understand potential sources for variation [32]. Transport and metabolic properties of
cultured cells can vary due to culture conditions, seeding density, passage number,
confluency, filter support, monolayer age, and stage of differentiation. Therefore,
permeability assays typically include various positive and negative controls to allow
for facilitated comparison between studies and laboratories.
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The distinction between paracellular and transcellular permeability is commonly
madewiththe use of invitro epithelialmonolayercell lines. Any cellline,suchas Caco-2
and MDCK, which form tight junctions can be used for this purpose. The paracellular
pathway is investigatedby opening it with the use of EDTA to chelate calcium, which is
importantfortight junctional integrity [33–35]. The tightjunctionscan also be disrupted
by cytochalasin D [36]. The disruption of tight junctions increases the space for
paracellular compounds to penetrate while leaving the integrity of transcellular
permeability intact. During testing, mannitol (paracellular probe) and propranolol
(transcellular probe) are commonly used as a positive and negative controls,
respectively [37].
Noncell Systems for Assessing Passive Permeability Two common cell membrane
mimetic systems include immobilized artificial membrane (IAM) HPLC columns [38,
39] and the parallel artificial membrane permeability assay (PAMPA) [40, 41]. IAM
columns contain covalently bound phosphatidylcholine analogs that serve as a surface
for drug membrane interactions and are highly amenable to high-throughput screening [42]. The PAMPA system uses a hexadecane or other lipid-filled membrane as a
lipophilic barrier and has been shown to correlate to cell culture and human jejunum
permeability [43, 44]. Both of these systems are used as a measure of passive
permeability only and not for assessment of potential active transport (see below).
Highly lipophilic compounds can be difficult to assess with these systems because of
issues with high retention to lipophilic matrix.
In Silico Tools for Assessing Passive Permeability Computational methods for the
prediction of drug permeability are increasing [45]. Numerous commercial software programs are available for the prediction of intestinal absorption. In silico
prediction models are especially useful tools for virtual screening, when a medicinal
chemist investigates the permeability effects of various substitutions before actually
synthesizing compounds. However, the prediction capability of software varies and
the user should avoid over-interpreting the results. The computational models are
most useful when the in silico predictions can be validated against in vitro permeability data from a testing set of compounds (see Chapter 2 for a comprehensive
discussion).
4.2.2.4 Transporter-Mediated Permeability A number of membrane transporters
within the intestinal tract can influence drug disposition [46]. They are responsible for
two major permeability mechanisms: active uptake and efflux. Figure 4.4 illustrates
the distribution of drug transporters in epithelial cells of the intest ine.
Epithelial cells in the intestine contain several uptake and ATP-dependent efflux
transporters in the luminal membrane including the organic transporting polypeptide
family (OATP/SLCO), oligopeptide transporter (PEPT/SLC15A), monocarboxylic
acid transporter (MCT/SLC16A), apical sodium-bile acid co-transporter (ASBT/
ALC10A2), P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (MRP2/
ABCC2), and breast cancer resistance protein (BCRP/ABCG2). On the basolateral
membrane, the transpor t systems include the organic cation transporter 1 (OCT1;
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SLC22A1), heteromeric organic solute transporter (OSTa-OSTb), and multidrug
resistance protein 3 (MRP3; ABCC3). Some drugs utilize these transporters for
systemic availability. For example, the PEPT system is a proton coupled transporter
that plays a critical role in the oral absorption of b-lactam antibiotics, anticancer
drugs, and dopamine receptor antagonists [46].
The utility of these uptake transporters as a platform delivery vehicle provides new
knowledge for strategies to enhance intestinal absorption of drugs [47]. In contrast to
the uptake transporters, ATP-dependent efflux pumps may serve as a barrier to oral
absorption [48–50]. Transporter-mediated drug absorption is saturable and affected
by competitive inhibitors [51]. Therefore, drug–drug interactions (DDI) may be more
of a concern for efflux transporters [52, 53]. Drug transporter research is still a
relatively young area of investigation, but is rapidly progressing in terms of transporter function, substrate specificity, spectrum, and kinetics, and overall implications
for drug discovery and development. Based on the current understanding of transporter molecular biology and the role of transporters in pharmacokinetics, guidelines
for drug transporter interaction studies have been proposed [54].
4.2.2.5 Tools to Assess Transport-Mediated Permeability A number of tools are
available to assess the transport-mediated permeability of molecules and are discussed below.
In Vivo Systems for Assessing Transport-Mediated Permeability Confirmingin vitro
findings in in vivo dynamic living system can provide greater confidence and
understanding of the interaction with the transporters. Genetic knockout or naturally
occurring transporter-deficient animal models and chemical knockout models are
popular assays used in drug discovery and development to characterize transporter
effects. Numerous transporter gene knockout mice have been characterized in recent
years and are commercially available [55]. For example, the Mdr-1 gene knock out
model has been used in assessing the impact of P-glycoprotein on ADME in drug
discovery and development [22, 56].
Extrapolation of preclinical transport findings to the clinic remains challenging
because of species differences in transporter expression, substrate affinity, physiological function, the interplay between transporters and enzymes, and occasionally
unpredicted differential regulation of transporters between genetically modified and
wild-type animals [57]. Recently,humanized transporter animal models and stem cell
Figure 4.4 The distribution of drug transporters in epithelial cell of the intestine.
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derived cell lines have been introduced as promising tools that may prove to be useful
in the near future to study discovery compounds and transporter interactions [58].
In Vitro Systems for Assessing Transpo rt-Mediated Permeability Cell-based assay
systems include gene transfected cell lines and polarized cell lines. Caco-2 cells are
known to express many of the drug transporters existing in epithelial cell of the
gastrointestinal tract [59]. Polarized transport in bidirectional transport studies is
useful as a first indication of transporter involvement. Chemical knockdown experiments using the co-administration of a transporter-specific inhibitor with test compound identify the involvement of specific transporters. Transporter cDNA stably or
transiently expressed in various cell lines can also be used to characterize drug
transporter interactions. Recently, several laboratories have developed polarized
cells that express both an uptake transporter in their basolateral membrane and an
efflux transporter in their apical membrane to mimic in vivo vectoral transport
[60–62]. However, whether these doubly transfected cells represent the true in vivo
situation is still under discussion [63].
Cell membrane preparations from transporter gene over-expressed insect cells are
commercially available for characterizing drug interactions with transporters.
For ATP-dependent efflux pump assessments, substrate-dependent ATP hydrolysis
methods have been developed. The simplicity of these assays is an advantage in
high-throughput applications [64]. However, as the assay only monitors ATPase
activity and not substrate transport, a high incidence of false positives might
occur. Recently, inverted cell membrane vesicle preparation have been employed
primarily to study efflux transporter-mediated uptake activity [65]. The advantage of
this type of assay is to enable detailed substrate and inhibitor kinetic measurements for
SAR analyses. In addition, the assays are readily automated for high-throughput
screening.
In Silico Tools for Assessing Transport-Mediated Permeability Compared to the
general prediction tools for intestin al absorption, commercial models for predicting
specific transporter interactions are still limited. With the increasing availability of
higher quality data sets, the application of computational modeling algorithms to
assess transporter substrate/inhibitor interactions has advanced [66–70].
4.2.2.6 Solubility and Permeability The Biopharmaceutics Classification System
(BCS) (Figure 4.5) has commonly been used in pharmaceutics to classify discovery
compounds in terms of their permeability and solubility and is adapted by the FDA to
rationalize the critical components related to oral absorption (http://www.fda.gov/
AboutFDA/CentersOffices/CDER/ucm128219.htm).
The classification system allows pharmaceutical companies to bypass clinical
bioequivalence studies if their drug product meets the specifications detailed in the
guidance [11, 71]. The BCS is a simplification of Fick’s first law. The diffusion
equation reduces simply to a product of permeability and solubility, which can be
characterized in vitro based on certain assumptions. The scientific rationale in
the BCS is that if the highest dose of a drug candidate is readily soluble in the fluid
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volume of the gastrointestinal tract (250 mL in human) and the drug is more than 90%
absorbed, then the in vitro drug product dissolution profiles should allow assessment
of equivalence of different drug formulations. In principle, the BCS classification
system can be applied to clinical development as well as to postapproval changes in
drug manufacturing. Therefore, early investments in optimizing the permeability and
solubility of a drug candidate in discovery research can provide significant savings in
future development costs.
4.2.2.7 First Pass Drug Metabolism Oral bioavailability can be affected by
chemical reactions in the gastrointestinal tract, which includes the potential formation
of a drug complex, hydrolysis by gastric acid or digestive enzymes in the intestinal
lumen, and intestinal and liver metabolism (Phase I and II metabolism). The
enterocytes express most of the metabolic enzymes that exist in intestine, including
UDP-glucuronyltransferases, sulfotransferases, esterases, and cytochromes P450s
(3A4 major). Therefore, drugs that interact with liver metabolic enzymes may also be
chemically removed during passage through the gut wall [72] (see also Section 4.4).
The distribution of metabolizing enzymes and transporters is uneven along the
intestinal tract. The metabolic activity of Phase I and II metabolism is higher in the
duodenum and jejunum than in the ileum and colon in both rat and human [23].
Conversely, P-glycoprotein mRNA levels increase longitudinally along the intestine,
with the lowest levels in the stomach and highest in the colon [22]. There may also be
an interplay between substrates for both P-glycoprotein and CYP3A4 substrates
where efflux cycling can increase the time that a drug interacts with CYP3A4 within
the enterocyte [73].
Tools to Assess First Pass Metabolism From in vivo pharmacokinetic studies, it is
difficult to clearly identify the first pass extraction contribution from the intestine and
liver. However,transgenic mouse models with selective organ expression of CYP3A4
offer the opportunity to investigate the first-pass metabolism of the anticancer agent
docetaxel by the gut wall, and not the liver [74]. The study helped explain the major
cause of low oral bioavailability in humans and interpatient differences in efficacy and
safety following oral therapy with CYP3A4 substrates.
High solubility Low solubility
High permeability
Class I Class II
Low permeability Class III Class V
Figure 4.5 Biopharmaceutics classification system.
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The extent of first pass drug metabolism by the liver can be evaluated by comparing
the plasma AUC of the compound dosed PO with the AUC of the compound
administered into the portal vein of hepatic portal vein cannulated animals, bypassing
the process of oral absorption (see Section 4.4).
4.3 DISTRIBUTION
The fate of a drug following absorption begins with an understanding of its
distribution. The factors that determine its ultimate delivery to the intended target
site must be appreciated and are discussed in the following sections.
4.3.1 Drug Distribution
The distribution of drug refers to the reversible movement of the compound from one
compartment to another within the body (Figure 4.6). Following drug absorption into
blood, the rate and extent of distribution between the blood and various tissues will
depend both on the physicochemical prope rties of the drug and the biological
properties of tissue compartments. Useful information as to the rate and extent of
drug distribution can be derived from an assessment of drug concentrations in blood or
plasma.
Movement of drug from the intravascular to the extravascular space is typically
rapid as the mean aqueous pore diameter in capillary membranes is of the order of
50–100 A˚(Figure 4.6). Therefore, most drugs have ready access to cell surface
Figure 4.6 Schematic representation of drug distribution between the intravascular and
extravascular spaces [75]. Drug available for distribution is influenced by both drug absorption
and clearance. The free drug hypothesis suggests that only unbound drug moves between
compartments and is at relatively equal concentrations at steady state.
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