Drugs transporters

Prepared by

Alaa Ibrahim Assistant lecturer of clinical pharmacology

Under supervision of

Pro.Dr. Sohair El MenshawyProf. of clinical Pharmacology

11/18/2012 1Transporters

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 TRANSPORTER

PRIMARY ACTIVE 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

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

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Transporters

ABC (ATP binding cassette)

49 known genes for ABC proteins that can be grouped into 7 subclasses or families (ABCA to ABCG)

the best recognized in the ABC superfamily are P-glycoprotein (P-gp, encoded by ABCB1, also termed MDR1) and the cystic fibrosis transmembrane regulator (CFTR).

48 SLC families with 315transporters have been identified in the human genome

Many serve as drug targets or in drug absorption and disposition

Widely recognized SLCtransporters include the serotonin (5-HT) and dopamine transporters (SERT, encoded by SLC6A4; DAT, encoded by SLC6A3).


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

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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 xenobioticsfrom 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

efficient detoxification


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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, ACEIsinhibitors 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

Active secretion of absorbed drug is now becoming

recognized as a significant factor 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

Active secretion of absorbed drug is now

becoming recognized as a significant factor

in oral drug bioavailability


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.


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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

hepatocytes:

1- OATPs (SLCO) and OATs (SLC22) for anions

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

steep concentration gradient from liver to bile.

This primary active transport is driven by ATP hydrolysis

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VECTORIAL TRANSPORT11

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Vectorial transport of drugs from the circulating

blood to the bile using an uptake transporter

(OATP family) and an efflux transporter

(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:

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1- HMG-CoA Reductase

Inhibitors:

Statins are cholesterol-lowering agents

that reversibly inhibit HMG-CoA reductase

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

(relatively hydrophilic and have low

membrane permeability)

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However, most of the statins in the acid form are substrates of uptake transporters, so they are taken up efficiently by the liverand undergo enterohepatic circulation

So, hepatic uptake transporters such as 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 minimizesthe escape of drug molecules into the circulating blood limiting systemic adverse effects

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2- Temocapril is an ACE inhibitor Its active metabolite, temocaprilat, is

excreted both in the bile and in the urine whereas other ACE inhibitors are excreted mainly via the kidney.

The special feature of temocapril among ACE inhibitors is that the plasma concentrationof temocaprilat remains relatively unchangedeven 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

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Taking these findings into consideration, the

affinity for MRP2 may dominate in

determining the biliary excretion of any

series of ACE inhibitors.

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.

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3-Irinotecan (CPT-11): is a potent anticancer drug, but late-onset

gastrointestinal toxic effects, such as severe

diarrhea, make it difficult to use CPT-11

safely.

After intravenous 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.

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Some studies have shown that the inhibition

of MRP2-mediated biliary excretion of SN-38

and its glucuronide by coadministration of

probenecid reduces the drug induced

diarrhea, at least in rats.

It is 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

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4- Troglitazone:A thiazolidinedione insulin-sensitizing agent

for the treatment of NIDDM

Was withdrawn from the market because of

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

via BSEP

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This inhibition of the hepatobiliary export of bile salts by troglitazone and troglitazone sulfate may lead to a drug-induced intrahepatic cholestasispossibly contributing to their hepatotoxicity

Cholestasis induced by some drugs is mediated, at least in part, by inhibition of BSEP, resulting in intracellular accumulation of cytotoxic bile salts. For examples: cyclosporine, rifampicin,glibenclamide & the cholestatic estrogenmetabolite

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 that could be a potential cause of cholestasis.

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During the past decade, molecular studies have identified and characterized the renal transporters that play a role in drugelimination, 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

N-acetylprocainamide11/18/2012 49Transporters

http://pharmrev.aspetjournals.org/content/55/3/425.long

Organic cations cross the basolateral

membrane by three distinct transporters in

the SLC family 22 (SCL22): OCT1

(SLC22A1), OCT2 (SLC22A2), and OCT3

(SLC22A3).

Organic cations are transported across this

membrane down their electrochemical

gradient (–70 mV).

Transport of organic cations from cell to

tubular lumen across the apical membrane

occurs via an electroneutral proton–

organic cation exchange mechanism11/18/2012 50Transporters

Transporters assigned 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.

OCT2 also involved in recycling of neurotransmitters by taking up breakdownproducts, which in turn enter monoamine


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 basolateralmembrane 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


• 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.

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• 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.

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Transporters

SLC6A1 (GAT1), SLC6A11 (GAT3), and 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 glialcells. GAT2 is found in peripheral tissues, including the kidney and liver, and within the CNS in the choroid plexus and meninges

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• 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.

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• 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

tricyclic antidepressants and cocaine

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• SLC6A3 (DAT)DAT is located primarily in the brain in

dopaminergic neurons present mainly on presynaptic neurons at the neurosynapaticjunction & also present along the neurons, awayfrom the synaptic cleft.

The primary function of DAT is the reuptakedopamine, 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

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• 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

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• 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 tricyclicantidepressants 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

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• 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 cytotoxicanticancer agents

• P-glycoprotein pumps out the anticancer drugs

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• 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

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• Tumors arising from tissues where MDR1\P-gp

is highly expressed show intrinsic resistance to

different chemotherapeutic agents , although

acquired resistance is often correlated with an

increased expression of MDR1\P-gp

Inhibition of P-gp represents a promising approach for

treatment of multidrug-resistant tumors

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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.


Reversal agents 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 thiomersform 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


Transporters are 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.

Various approaches 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

Health & Medicine

Transcript of Drugs transporters