P- GLYCO PROTEINDr Sirisha .K

Dept of Pharmacology

Slide index• Introduction• Membrane transporters• Structure• Physiological sites • Cellular localisation• Mechanism of action• Physiological relevance

• Pharmacokinetic importance

• P gp substrates• P gp inhibition• Pgp inhibitors• Pgp stimulators• Clinical implications• polymorphism

Introduction

• P-glycoprotein (P-gp) belongs to superfamily of ATP-binding

cassette (ABC) transporters.

• These proteins bind ATP and use the energy to drive the

transport of various molecules across all the cell membranes.

• The P-gp efflux transporter can functionally protect the body

against toxic xenobiotics and drugs by excreting these

compounds into bile, urine, and the intestinal lumen and by

preventing their accumulation in brain, gonads, and placenta.

Membrane transporters

They are membrane proteins that control influx of

essential nutrients and ions, and the efflux of cellular

waste, environmental toxins, drugs and other

xenobiotics.

They exhibit selectivity, saturability and competitive

inhibition by co-transported substances.

Membrane Transport Proteins

Selective Channels

transporters

Facilitated Diffusion

Primary Active Transport

Secondary Active Transport

UniportersATP-powered pumps

Symporters Antiporters

Need to study p glycoprotein

Although MDR is a multifactorial process, the

main obstacle is the expression of multidrug-

efflux pumps that lowers the intracellular drug

levels.P-glycoprotein (P-gp) is the longest identified

efflux pump involved in this.

structure

• P-gp is a 170 kDa membrane-bound protein, an energy-

dependent efflux transporter driven by ATP hydrolysis.

• It is composed of 2 homologous and symmetrical halves

(cassettes), each of which contains six TM domains that

are separated by an intracellular flexible linker

polypeptide loop, with an ATP-binding motif.

• There are two ATP-binding domains of P-gp, located in

the cytosol side.

Structure continued

• ATP binding site:• Walker A-ATP binding• Walker B- Magnesium ion• Signature C-?ATP hydrolysis

• Trans membrane domain:• TM1, TM4, TM6, TM10, TM11,TM12• TM1: pocket deciding drug size• TM6, TM12: drug binding sites

Physiological sites

Cellular localisation

• In liver, P-gp is found exclusively on the biliary

canalicular front of hepatocytes and on the apical

surface of epithelial cells in small biliary ductules.

• In pancreas, P-gp is found on the apical surface of

the epithelial cells of small ductules but not larger

pancreatic ducts.

• In kidney, P gp is found concentrated on the apical

surface of epithelial cells of the proximal tubules.

• Both colon and jejunum show high levels of P-gp on the

apical surfaces of superficial columnar epithelial cells.

• Adrenal gland shows high levels of P-gp, diffusely

distributed on the surface of cells in both the cortex and

medulla.

• Its expression is also detected in specialized epithelial cells

with secretory or excretory functions, trophoblasts in the

placenta, and on endothelial cells of capillary blood

vessels at blood–tissue barrier site

Mechanism of p gp

• Drug / substrate recognition:

Amino acids in TM1 are involved in the formation of a

binding pocket that plays a role in determining the suitable

substrate/drug size for P-gp, whereas Gly residues in TM2

and TM3 are important in determining substrate specificity.

ATP-binding and subsequent hydrolysis:

• Around 2molecules of ATP are hydrolyzed for every molecule

of the drug transported outside the cell.

• one in the transport of substrate and the other in effecting

conformational changes to reset the pump for the next

catalytic cycle.

• Alternate catalytic cycle of ATP hydrolysis and ADP release is

the rate-limiting step in the catalytic cycle and the substrates

exert their effect by modulating ADP release.

Efflux of substrate/drug through central pore:

• P gp intercepts lipophilic drugs as they move

through the lipid membrane and flips the drugs

from inner leaflets to the outer leaflet and to

extra cellular medium.

• A major reorganization of the TM domains

occurs throughout the entire depth of the

membrane resulting in central pore formation of

2-3 nm diameter and 5-6 nm in depth on binding

of ATP

Physiological significance

• Transport mechanisms for the extrusion of toxic xenobiotics and their metabolites from cellular environment

Example: 1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT)plastic derived xenobiotics

Blood

Intestine

Urine

Mucus

Feces

Liver

Lung

Kidney

MDRtumor

Testis

Brain

CSF

Placenta?

P gp substrates

• Neutral or cationic compounds form the substrates• Anticancer drugs: Actinomycin, cyclosporine-A, cisplatin,• Cardiovascular drugs: Atorvastatin, lovastatin, digitoxin,

losartan, • Antiviral drugs: amprenavir, indinavir, saquinavir,

nelfinavir, and ritonavir • Antibacterial agents: erythromycin, rifampin,

sparfloxacin, levofloxacin• GIT drugs: Cimetidine,domperidone, loperamide and

ondansetron • Other drugs: Chloroquine, dexamethasone, morphine,

phenytoin

Mechanisms of P gp inhibition

• Competitive inhibition: Itroconazole, verapimil

• ATP hydrolysis

• ATP hydrolysis blockage& Competitive inhibition: cyclosporine A

• Allosteric inhibition: flupenthixol

P glycoprotein inhibitors

• 1st Generation: • Verapamil, • Cyclosporine, • Erythromycin, • Ketoconazole,• Tamoxifen

• 2nd Generation: • Biricodar (VX-710)

Pharmacokinetic importance• ABSORPTION:

Enterocytes of the GI tract.

• DISTRIBUTION:

• Trophoblasts in the placenta

• Endothelial cells of capillary blood vessels

at blood–tissue barrier sites like blood

brain barrier

• METABOLISM: Intestinal CYP3A4-mediated bio

transformation and active efflux of absorbed drug by P gp are major determinants of bioavailability of orally administered drugs. • EXCRETION:

Renal P-gp forms a transepithelial tubular drug transport pathway that is responsible for the net urinary excretion of various xenobiotics and drugs

Drug interactions

• Digoxin: Absorbtion is reduced when given along with

rifampicin(inducer)Excretion is retarded when given with verapimil

(inhibitor)• Protease inhibitors• Azithromycin can modify the hepatobiliary excretion of

doxorubicin-a.

Clinical implications

ANTICANCER DRUGS:

Intrinsic resistance: Tumors arising from

tissues where MDR1/Pgp are expressed like

pancreatic carcinoma

Acquired resistance: Increased expression of

MDR1/P-gp is seen in tumours where cisplatin,

doxorubicin are used

PREVENTION OF RESISTANCE:

• Agents that are insensitive to P-gp-mediated drug

efflux:

Lamellarin D (LAM-D), a marine alkaloid a

potent proapoptotic agent used in prostrate ca and

leukaemia is an example of drug which is insensitive for

p gp

• P gp inhibitor:

verapimil, flupenthixol

a) ANTI-EPILEPTIC DRUG (AED) THERAPY:It has been hypothesized that overexpression of P-gp and other efflux transporters in the cerebrovascular endothelium in the region of the epileptic focus may lead to drug resistance in epilepsy

b) CHLOROQUINE-RESISTANT PLASMODIUM FALCIPARUM STRAINS: recently a study presented evidence for a saturable and energy-dependent chloroquine efflux system to be present in chloroquine-resistant strains.

c) PROTEASE INHIBITORS:Overexpression of multidrug transporters significantly reduces the accumulation of protease inhibitors (PIs) at major sites of virus replication

P gp polymorphism• These variations could contribute to the

interpatient variabilities in plasma protease inhibitor concentrations and might be of clinical use.

• Intracellular concentration of HIV PIs and antiretroviral efficacy is affected by variable P-gp expression, as a result of the polymorphism of MDR1

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