Drugs transporters

Prepared by
Alaa Ibrahim
Assistant lecturer of clinical pharmacology
Under supervision of
Pro.Dr. Sohair El Menshawy
Prof. of clinical Pharmacology

11/18/2012 Transporters 1

 1- Introduction
 2- Types of Transporters
 3- Structure of Transporters
 4- Mechanism of Action Of Transporters
 5- Regulation of Transporter Expression
 6- Physiological & Pharmacological role of
Transporters
 7- Novel Approaches To Bypass Drug
Transporters

 The basic mechanisms involved in solute
transport across biological membranes include
passive diffusion, facilitated diffusion, and
active transport
 Active transport can be further subdivided into
primary and secondary active transport.



Mechanism of
membrane
permeation

SECONDARY ACTIVE PRIMARY ACTIVE
TRANSPORTER TRANSPORTER


 Secondary active transport ( co-transport):
uses energy to transport molecules across a
membrane. In contrast to primary active transport,
there is no direct coupling of ATP; instead,
the electrochemical potential difference created by
pumping ions out of the cell is used.

The two main forms of this are
1- antiport: Na+ Ca++ exchanger
2- symport: glucose symporter which co-
transports one glucose molecule into the cell for
every two Na+

Types of 2ry Active Transporters

II- Symbort
( co-transport)
III- Antiport
( exchange)

Types of Membrane Transporters
8

 2000 genes in the human genome (7% of the total number
of genes) code for transporters or transporter-related
proteins.
 In considering the transport of drugs, pharmacologists
generally focus on transporters from two major
superfamilies, ABC (ATP binding cassette) and SLC (solute
carrier) transporters
 Most ABC proteins are primary active transporters,
which rely on ATP hydrolysis to actively pump substrates
across membranes
 The SLC superfamily includes genes that encode
facilitated transporters and ion-coupled secondary
active transporters
Transporters 11/18/2012

ABC (ATP binding
cassette)
 49 known genes for ABC  48 SLC families with 315
proteins that can be transporters have been
identified in the human
grouped into 7 subclasses
genome
or families (ABCA to  Many serve as drug targets
ABCG) or in drug absorption and
 the best recognized in the disposition
ABC superfamily are P-  Widely recognized SLC
glycoprotein (P-gp, transporters include the
encoded by ABCB1, also serotonin (5-HT) and
termed MDR1) and the dopamine transporters
cystic fibrosis (SERT, encoded by
transmembrane regulator SLC6A4; DAT, encoded by
(CFTR). SLC6A3).

Transporters 11/18/2012 9

Structure of ABC Transporters


 The common feature of all ABC transporters is that they
consist of two distinct domains, the transmembrane
domain (TMD) and the nucleotide-binding domain
(NBD).

 The TMD, also known as membrane-spanning domain
(MSD) or integral membrane (IM) domain, consists
of alpha helices, embedded in the membrane bilayer.

 It recognizes a variety of substrates and undergoes
conformational changes to transport the substrate across
the membrane.

 The sequence and architecture of TMDs is variable,
reflecting the chemical diversity of substrates that can be
translocated.

 The NBD or ATP-binding cassette (ABC) domain, on the
other hand, is located in the cytoplasm and has a highly
conserved sequence.

 The NBD is the site for ATP binding.

 In most exporters, the N-terminal transmembrane
domain and the C-terminal ABC domains are fused as
a single polypeptide chain, arranged as TMD-NBD-
TMD-NBD.

 Importers have an inverted organization, that is, NBD-
TMD-NBD-TMD, where the ABC domain is N-terminal
whereas the TMD is C-terminal


 Some ABC transporters have additional
regulatory class of proteins.
 In particular, importers have a high-
affinity binding protein (BP) that specifically
associates with the substrate in the periplasm
for delivery to the appropriate ABC transporter
 Exporters do not have the binding protein but
have an intracellular domain (ICD) that joins the
membrane-spanning helices and the ABC
domain.
 The ICD is believed to be responsible for
communication between the TMD and NBD


 The structural architecture of ABC transporters
consists minimally of two TMDs and two ABCs.
 Most exporters, such as in the multidrug
exporter are made up of homodimer consisting
of two half transporters
 A full transporter is often required to gain
functionality


 ABC transporters are active transporters, they
require energy in the form of (ATP) to translocate
substrates across cell membranes. These
proteins harness the energy of ATP binding
and/or hydrolysis to drive conformational
changes in the transmembrane domain
(TMD) and consequently transports molecules.

 Both ABC importers and exporters have a
common mechanism in transporting substrates
because of the similarities in their structures.

 In this model, the substrate binding site alternates
between outward- and inward-facing
conformations. The relative binding affinities of
the two conformations for the substrate largely
determines the net direction of transport.
 For importers, since translocation is directed
from the periplasm to the cytoplasm, then the
outward-facing conformation will have higher
binding affinity for substrate. In contrast, the
substrate binding affinity in exporters will be
greater in the inward-facing conformation.


 This model presents two principal conformations
of the NBDs: formation of a closed dimer upon
binding two ATP molecules and dissociation to
an open dimer facilitated by ATP hydrolysis and
release of inorganic phosphate (Pi)
and adenosine diphosphate (ADP). Switching
between the open and closed dimer
conformations induces conformational changes
in the TMD resulting in substrate translocation


 Regulating the distribution and bioavailability of
drugs
 The removal of toxic metabolites and xenobiotics
from cells into urine, bile, and the intestinal lumen
 The transport of compounds out of the brain across
the blood–brain barrier
 Protection of hematopoietic stem cells from toxins


Regulation of Transporter
Expression
 Transcription of transporter mRNAs changes in
response to drug treatment and
pathophysiological conditions, resulting in
induction or down regulation.

 Recent studies have described important roles of
type II nuclear receptors, which form
heterodimers with the 9-cis-retinoic acid receptor
(RXR), in regulating drug-metabolizing enzymes
and transporters

 Such receptors include pregnane X receptor (
PXR), constitutive androstane receptor (CAR)
,farnesoid X receptor (FXR)& PPARα
(peroxisome proliferator-activated receptor α)
and retinoic acid receptor (RAR)

 These are ligand-activated nuclear receptors that,
as heterodimers with RXR, bind specific elements in
the enhancer regions of target genes.

 There is an overlap of substrates between
CYP3A4 and P-glycoprotein, and PXR mediates
coinduction of CYP3A4 and P-glycoprotein,
supporting their synergetic cooperation in

 Role of Transporters in Drug
Absorption


 Various transporters are expressed in the
brush-border membranes of intestinal epithelial
cells involved in the efficient absorption of
nutrients or endogenous compounds.

 The influx transporters expressed in the gut
improve drug absorption
Example: PEPT1, ASBT, OATP-B, OATP-D &
OATP-E

 PEPT1 mediates the transport of peptide-like
drugs such as β-lactam antibiotics, ACEIs
inhibitors and the dipeptide-like anticancer
drug bestatin

 However efflux transporters, such as P-gp, MRP2,
or BCRP, are expressed on the brush-border
membrane of enterocytes and excrete their substrates
into the lumen, resulting in limitation of net
absorption
 Activesecretion of absorbed drug is now is now
Active secretion of absorbed drug becoming
becomingas a significantas a significant factor
recognized recognized factor in oral drug
bioavailability
in oral drug bioavailability
 P-gp affects the absorption of many drugs because of
its broad substrate specificity
 The intestinal P-gp content correlates with the AUC
after oral administration of digoxin, a P-gp
substrate

Schematic
of role of
P-gp
intestinal
disposition
of
substrate.


 A report involving a patient undergoing a small
bowel transplant demonstrated that plasma conc. of
oral tacrolimus, a substrate of both P-gp and
CYP3A4, correlated well with the mRNA
expression of intestinal MDR1, but not CYP3A4

 These results suggest that intestinal P-gp, rather than
CYP3A4, is a good probe to predict intraindividual
variations in tacrolimus pharmacokinetics.


 BCRP is a member of the ABC transporter family
has only one ATP-binding cassette and six
transmembrane domains, suggesting that BCRP is a
half-transporter, which may function as a homo- or
heterodimer.
 BCRP plays a role in the secretion topotecan

 When both topotecan, a substrate of BCRP, and
GF120918, an inhibitor of both BCRP and P-gp,
were administered orally, the bioavailability of
topotecan was increased in P-gp-deficient mice
(over 6-fold) compared with mice given vehicle
alone


 BCRP is expressed not only in the intestine, but also
in the bile canalicular membrane and placenta .Thus,
treatment with GF120918 reduced the plasma
clearance and hepatobiliary excretion of topotecan .
 Furthermore, in pregnant GF120918-treated, P-gp-
deficient mice, the fetal penetration of topotecan was
2-fold higher than that in pregnant mice given
vehicle alone.
 These results indicate that BCRP plays an important
role in protecting the fetus from topotecan.


11/18/2012 Transporters
34

 Hepatic uptake of organic anions (e.g., drugs, LTs and
bilirubin), cations, and bile salts is mediated by SLC-type
transporters in the basolateral (sinusoidal) membrane of

11/18/2012
hepatocytes:
1- OATPs (SLCO) and OATs (SLC22) for anions

Transporters
2- OCTs and NTCP (SLC10A1) for cations & bile salts
 This uptake either by facilitated or secondary active
mechanisms

 ABC transporters such as MRP2, MDR1, BCRP,
BSEP, and MDR2 in the bile canalicular membrane of
hepatocytes mediate the efflux (excretion) of drugs and
their metabolites, bile salts, and phospholipids against a
35
steep concentration gradient from liver to bile.
 This primary active transport is driven by ATP hydrolysis

11/18/2012 Transporters
36
VECTORIAL TRANSPORT

 Vectorial transport of drugs from the circulating

11/18/2012
blood to the bile using an uptake transporter
(OATP family) and an efflux transporter

Transporters
(MRP2) is important for determining drug
exposure in the circulating blood and liver.
 Different examples illustrate the importance of
vectorial transport in determining drug exposure
in blood & liver:

37

1- HMG-CoA Reductase
Inhibitors:

11/18/2012
 Statins are cholesterol-lowering agents
that reversibly inhibit HMG-CoA reductase

Transporters
enzyme
inhibit cholesterol biosynthesis mainly in
the liver ( the main target), while exposure
of extrahepatic cells in smooth muscle to
these drugs may cause adverse effects
 Pravastatin, fluvastatin, cerivastatin,
atorvastatin, rosuvastatin are given in a
biologically active open-acid form 38

(relatively hydrophilic and have low
membrane permeability)

 However, most of the statins in the acid
form are substrates of uptake transporters,
so they are taken up efficiently by the liver

11/18/2012
and undergo enterohepatic circulation
 So, hepatic uptake transporters such as

Transporters
OATP1B1 and efflux transporters such as
MRP2 act cooperatively to produce
vectorial transcellular transport of
bisubstrates in the liver
 The efficient first pass hepatic uptake of
statins by OATP1B1 after their oral
administration helps to exert the
pharmacological effect and also minimizes
the escape of drug molecules into the 39
circulating blood limiting systemic adverse
effects

2- Temocapril is an ACE inhibitor
 Itsactive metabolite, temocaprilat, is
excreted both in the bile and in the urine

11/18/2012
whereas other ACE inhibitors are excreted
mainly via the kidney.

Transporters
 The special feature of temocapril among ACE
inhibitors is that the plasma concentration
of temocaprilat remains relatively unchanged
even in patients with renal failure.

 Temocaprilat is a bisubstrate of the OATP
family and MRP2, whereas other ACE
inhibitors are not good substrates of MRP2 40

 Taking these findings into consideration, the
affinity for MRP2 may dominate in
determining the biliary excretion of any

11/18/2012
series of ACE inhibitors.

Transporters
 Drugs that are excreted into both the bile
and urine to the same degree thus are
expected to exhibit minimum interindividual
differences in their pharmacokinetics.

41

3-Irinotecan (CPT-11):
 isa potent anticancer drug, but late-onset

11/18/2012
gastrointestinal toxic effects, such as severe
diarrhea, make it difficult to use CPT-11

Transporters
safely.

 Afterintravenous administration, CPT-11 is
converted to SN-38, an active metabolite, by
carboxy esterase. SN-38 is subsequently
conjugated with glucuronic acid in the liver.
SN-38 and SN-38 glucuronide are then
excreted into the bile by MRP2.
42

 Some studies have shown that the inhibition
of MRP2-mediated biliary excretion of SN-38
and its glucuronide by coadministration of

11/18/2012
probenecid reduces the drug induced
diarrhea, at least in rats.

Transporters
 Itis expected that this agent will be used
clinically to prevent toxicity. Approaches
using intentional drug-drug interactions
(positive drug interactions) like this case
may become more important in the future

43

4- Troglitazone:
A thiazolidinedione insulin-sensitizing agent

11/18/2012
for the treatment of NIDDM
 Was withdrawn from the market because of

Transporters
liver toxicity . the mechanism underlying this
troglitazone-associated hepatotoxicity is at
present unclear, but it has been suggested
that a cholestatic mechanism is involved
 Troglitazone and, to a much greater extent
troglitazone sulfate, the main troglitazone
metabolite eliminated into bile, competitively
inhibit ATP-dependent taurocholate transport
44
via BSEP

 This inhibition of the hepatobiliary export of bile
salts by troglitazone and troglitazone sulfate may
lead to a drug-induced intrahepatic cholestasis

11/18/2012
possibly contributing to their hepatotoxicity

 Cholestasis induced by some drugs is mediated,

Transporters
at least in part, by inhibition of BSEP, resulting in
intracellular accumulation of cytotoxic bile salts. For
examples: cyclosporine, rifampicin,
glibenclamide & the cholestatic estrogen
metabolite

 One should consider the possibility that drugs
which inhibit BSEP may cause cholestasis
 The evaluation of BSEP inhibition will play an
important role in the identification of compounds 45
that could be a potential cause of cholestasis.

 During the past decade, molecular studies
have identified and characterized the renal
transporters that play a role in drug
elimination, toxicity and response.

 we now can describe the overall secretory
pathways for organic cations and their
molecular and functional characteristics

 Our understanding of organic anion transport
has progressed in a similar fashion.

 In some cases, transporters that are considered
organic anion or organic cation transporters
have dual specificity for anions and cations


 The OCT family of proteins is involved in
the uptake of organic cations into the
liver or kidney from blood. OCT1 and
OCT2 are expressed in epithelial cells of
the kidney, liver, and intestine, and appear
to be localized to the basolateral
membranes of the cells

 These transporters mediate the uptake of
a variety of organic cations, such as
dopamine, choline, 1-methyl-4-
phenylpyridinium (MPP+), N1-
methylnicotinamide, TEA, and cimetidine
ranitidine, metformin, procainamide, and

 Organic
cations cross the basolateral
membrane by three distinct transporters in
the SLC family 22 (SCL22): OCT1
(SLC22A1), OCT2 (SLC22A2), and OCT3
(SLC22A3).

 Organiccations are transported across this
membrane down their electrochemical
gradient (–70 mV).

 Transportof organic cations from cell to
tubular lumen across the apical membrane
occurs via an electroneutral proton–
organic cation exchange mechanism

 Transportersassigned to the apical membrane
are in the SLC22 family and termed novel
organic cation transporters (OCTNs). In
humans, these include OCTN1 (SLC22A4) and
OCTN2 (SLC22A5).

 These bifunctional transporters are involved not
only in organic cation secretion but also in
carnitine reabsorption.

 OCT2 play a housekeeping role in neurons,
taking up only excess concentrations of
neurotransmitters.

 OCT2also involved in recycling of
neurotransmitters by taking up breakdown
51
11/18/2012
Transporters

 The primary function of organic anion
transporters is the removal of xenobiotics
(include weak acidic drugs e.g. pravastatin,
captopril, pencillins & toxins)

 Two primary transporters on basolateral
membrane mediate the flux of organic anions
from intestinal fluid to tubular cell : OAT1(
SLC22A6) & OAT3 ( SLC22A8)
 organic anions are transported across the
basolateral membrane against an
electrochemical gradient in exchange with
intracellular α-ketoglutarate, which moves
down its concentration gradient


 The mechanism responsible for the apical
membrane transport of organic anions
from tubule cell cytosol to tubular lumen
remains controversial
 Some studies suggest that OAT4 may
serve as the luminal membrane
transporter for organic anions
 Other transporters that may play a role in
transport across the apical membrane
include MRP2 and MRP4,multidrug-
resistance transporters in the ATP binding
cassette family C (ABCC).


DRUG ACTION IN THE BRAIN


56

• Transporters involved in the neuronal reuptake

of the neurotransmitters and the regulation of
their levels in the synaptic cleft belong to two
major superfamilies, SLC1 and SLC6

• Transporters in both families play roles in reuptake
of γ-aminobutyric acid (GABA), glutamate, and
the monoamine neurotransmitters NA, 5-HT, and
dopamine. These transporters may serve as
pharmacologic targets for neuropsychiatric
drugs.

57

• SLC6 family members in the brain involved in the

reuptake of neurotransmitters into presynaptic
neurons include the NA transporters (NET, SLC6A2),
the dopamine transporter (DAT, SLC6A3), the
serotonin transporter (SERT, SLC6A4), and several
GABA reuptake transporters (GAT1, GAT2, and
GAT3)
• The SLC6A family regulate the concentrations and
dwell times of neurotransmitters in the synaptic
cleft
• The extent of transmitter uptake also influences
subsequent vesicular storage of transmitters.

• The transporters can function in the reverse
direction.

58

SLC6A1 (GAT1), SLC6A11 (GAT3), andTransporters 11/18/2012

SLC6A13 (GAT2).
• GAT1 is the most important GABA transporter in
the brain, expressed in GABAergic neurons and
found largely on presynaptic neurons

• GAT3 is found only in the brain, largely in glial
cells. GAT2 is found in peripheral tissues,
including the kidney and liver, and within the
CNS in the choroid plexus and meninges

59


• GAT1, GAT2, and GAT3 are approximately 50%
identical in amino acid sequence
• The presence of GAT2 in the choroid plexus and
its absence in presynaptic neurons suggest that this
transporter may play a primary role in maintaining
the homeostasis of GABA in the CSF.

• GAT1 and GAT3 are drug targets.
• GAT1 is the target of the antiepileptic drug
tiagabine, which acts to increase GABA levels in the
synaptic cleft of GABAergic neurons by inhibiting
the reuptake of GABA.
• GAT3 is the target for the nipecotic acid
derivatives that are anticonvulsants.

60


• SLC6A2 (NET):
• is found in central and peripheral nervous tissues
as well as in adrenal chromaffin tissue
• A major role of NET is to limit the synaptic dwell
time of NA and to terminate its actions, salvaging
NA for subsequent repackaging.
• NET participates in the regulation of many
neurological functions, including memory and
mood.
• NET serves as a drug target; the antidepressant
desipramine is considered a selective inhibitor of
NET.
• Other drugs that interact with NET include other

61


• SLC6A3 (DAT)
DAT is located primarily in the brain in
dopaminergic neurons present mainly on
presynaptic neurons at the neurosynapatic
junction & also present along the neurons, away
from the synaptic cleft.

The primary function of DAT is the reuptake
dopamine, terminating its actions

DAT is involved in functions like mood, behavior,
reward, and cognition

Drugs that interact with DAT include cocaine,
amphetamines, and the neurotoxin MPTP

62


• SLC6A4 (SERT)
SERT is located in peripheral tissues and in the
brain along extrasynaptic axonal membranes
SERT clearly plays a role in the reuptake and
clearance of serotonin in the brain
Substrates of SERT :
1- 5-HT
2- Tryptamine derivatives
3- 3,4-methylene-dioxymethamphetamine
(MDMA; ecstasy) neurotoxin
4- Fenfluramine

63

• The serotonin transporter has been one of the

most widely studied proteins in the human
genome
WHY ?

1- It is the specific target of the antidepressants in
the selective serotonin reuptake inhibitor class
SSRI
(e.g., fluoxetine and paroxetine)
2- One of several targets of tricyclic
antidepressants TCA (e.g., amitriptyline)
3- The important role of 5-HT in neurological
function and behavior, genetic variants of
SERT have been associated with many
behavioral and neurological disorders

64


• Membrane transporters play a critical role
in the development of resistance to
1- anticancer drugs,
2- antimicrobial agents
3- anticonvulsants.
• N.B) P-glycoprotein is overexpressed in
tumor cells after exposure to cytotoxic
anticancer agents
• P-glycoprotein pumps out the anticancer
drugs

65


• Other transporters, have been implicated in
resistance to anticancer drugs include:
1- Breast cancer resistance protein (BCRP)
2- The organic anion transporters
3- several nucleoside transporters

• N.B) The overexpression of multidrug-resistance
protein 4 (MRP4) is associated with resistance
to antiviral nucleoside analogs

66


• Tumors arising from tissues where MDR1P-gp
is highly expressed show intrinsic resistance to
different chemotherapeutic agents , although
acquired resistance is often correlated with an
increased expression of MDR1P-gp

Inhibition of P-gp represents a promising approach for
treatment of multidrug-resistant tumors

I- Reversal agents:
They are also called as chemosensitizers as
they inhibit P-gp efflux of drugs and increase
the absorption of drugs intracellularly so these
agents may be co-administered with
therapeutic agent as competitive inhibitors.


 Reversalagents for P-gp as per the
generation:

A) First-generation agents
These agents had their own pharmacological action.
These agents were used in high dose as they were not
selective to inhibit P-gp so they resulted into high
toxicity which impedes their use to inhibit P-gp:
1- Cyclosporine: (hepatic, renal, myeloid and
neurotoxicity)
2- Verapamil: (cardiotoxicity)


B) Second-generation agents :
These agents were selective and less toxic than the first
generation agents. Many chemotherapeutic agents are
substrate of P-gp and CYP 3A4. Same way, second
generation agents were also substrate of the CYP 3A4.
So these may lead to unpredictable absorption and
metabolism and these ultimately resulted into modified
bioavaibility:
1-Valspodar (R-enantiomer of Verapamil )
2-Biricodar


C) Third-generation agents
 These agents were not the substrates of CYP 3A4
so used to overcome drawback of second generation
agents and these agents
selectively and potentially inhibit P-gp.
1-Tariquidar XR9576
2- Zosuquidar LY335979
3- Laniquidar R101933


II- Natural and Synthetic Polymers:
A- Natural polymers:
They are obtained from natural source. For example:
1-Anionic gums:
Xanthan gum P-gp inhibitor at 0.05%
Gellan gum :P-gp inhibitors at 0.05%
Dextran Xanthan gum :P-gp inhibitor at 0.05%
2- From Green tea: Polyphenols
3- From Grapefruit Juice: Various Polysaccharides
like D-glucose and/or D-glucuronic acid


B- Synthetic polymers
 Synthetic polymers can be synthesized by monomer
polymerization or by natural polymers modifications
or by combination of natural substances with
synthetic substances. e.g.: Detergents based on
Polyethylene glycol, Dendrimers and Thiomers.

 The mechanism to inhibit P-gp by these polymeric
surfactants is believed to be mediated by modifying
the function of cell membrane lipid.


 Thiomers:
These polymers have been newly implemented in
the pharmaceutical area.
Polymers having thiol group has shown to have
superior penetration improving properties.
Recently, many researchers have suggested that
thiomers is having P-gp pump inhibitory
activity due to thiol group because thiomers
form disulfide bond between cysteine group of P-
gp and free thiol group of thiomers.
For example: α-Chitosan–thiobutylamidine
(chito– TBA)


 III- Nanocarrier drug delivery system:
Liposomes:
Liposomes are vesicles made up of bilayer or
multilayers that contain phospholipids and
cholesterol enveloping hydrophilic aqueous region.
Both lipophilic and hydrophilic drugs can be
encapsulated within this nanocarrier and is available
for absorption at the intestinal membrane surface.
Neutral phospholipids are selectively effluxed out by
P-gp so there would be competition for P-gp when
neutral phospholipids administered with P-gp
substrates.


CONCLUSION


 Transportersare membrane proteins that are present
in all organisms. These proteins control the influx of
essential nutrients and ions and the efflux of cellular
waste, environmental toxins, and other xenobiotics.

 The functions of membrane transporters may be
facilitated (equilibrative, not requiring energy) or
active (requiring energy).

 Drug-transporting
proteins operate in
pharmacokinetic and pharmacodynamic pathways,
including pathways involved in both therapeutic and
adverse effects


 Membrane transporters play a critical role in the
development of resistance to anticancer drugs,
antiviral agents, and anticonvulsants.

 Variousapproaches have been developed to
overcome the effect of membrane transporters
include reversal agents, polymers & liposomes
which could enhance the effect of substrate drugs of
membrane transporters


THANK YOU

Drugs transporters

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