efflux transporter (p.glycoproteins)

1. P-glycoprotein transporters
Maryam kazemi
Ph.D student of pharmaceutics at SUMS

2. P-glycoproteins
belonging to the super family of adenosine triphosphate
(ATP)-binding cassette (ABC) transporters.
ABC efflux transporters in cancer cells such as :
• P-glycoprotein (P-gp/MDR1/ABCB1)
• multidrug resistance-associated protein 2 (MRP2/ABCC2)
• breast cancer resistance protein (BCRP/ABCG2)
limits the prolonged and effective use of chemotherapeutic
drugs .
• P-gp is the most importent efflux transporter in the body.

3. Structure of transporters
• P-Gp is an integral membrane protein, about 170–
180 kDa encoded by the MDR1 gene in humans
and contains 12 putative transmembrane domains
and two ATP binding sites.

6. Necessity of p-glycoproteins
• responsible for pumping substances out of cells
• Lypophilic substances are the substrates of efflux system .
• P-gp is an apical membrane transporter that is abundantly
expressed on the intestine mucosal membrane, kidney
proximal tubule epithelia, liver,placenta, and luminal blood–
brain barrier, where it functions to protect against
xenobiotics and cellular toxicants.

7. Substrate specificity of P-glycoprotein
and nature of the drug-binding site
• Most preferred substrates are amphipathic
and relatively hydrophobic.
• Many substrates, but not all, contain planar
aromatic rings and positively-charged tertiary
N atoms.

8. Substrates of p-glycoproteins
• 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

10. Mechanism of p-glycoprotein
• The efflux action of the protein follows a carrier
mediated primary active transport mechanism.
• In this process, the protein pump export needs direct
ATP requirement and the energy released from the
ATP hydrolysis gives the driving force for extrusion
process.
• The efflux takes place unidirectionally (out of the
cells into the extracellular space) and transfers only
one molecule at a time. Thus, P-gp is a uniporter
carrier protein.

12. inhibitors
• P-gp inhibitors were explored for overcoming
multidrug resistance and poor bioavailability
problems of the therapeutic P-gp substrates .
• competitive and non-competitive (non-transported)
inhibitors apart from the P-gp efflux kinetics .
• competitive inhibitors compete with the substrate
drugs for extrusion and occupy all the available
protein transport sites leaving no space for the P-gp
and substrate interaction.

13. • Non-competitive inhibitors neither bind to protein’s
transport site nor are translocated by the protein
efflux and hence are as well called as non-transported
inhibitors. They non-competitively inhibit the protein
efflux by binding to an allosteric modulatory site .

16. First generation inhibitors
• This class of inhibitors embodies those
pharmacological agents. Verapamil, an
antihypertensive calcium channel blocker,
trifluoperazine, cyclosporine, an
immunosuppressant, other antihypertensives
such as quinidine and reserpine, yohimbine,
antiestrogenic tamoxifen and toremifene, and
antineoplastic vincristine, all fall under this
category

17. • Since most of these compounds were P-gp
substrates themselves, they interacted with the
protein, competed with the other substrates and
acted as competitive inhibitors.
• Their P-gp inhibitory concentrations reached
high toxic levels due to which many of these
inhibitors failed in clinical trials

18. Second generation inhibitors
• Dexverapamil, the R-isomer of verapamil without
any cardiac activity, PSC 833 (valspodar), a
cyclosporine A analogue lacking
immunosuppressive character, MS-209 and
several other first generation drug derivatives or
analogues fall under this category.
• These resultant modulators still remained P-gp
substrates themselves and showed low protein
affinity

19. They don’t have pharmacologic effect
Better inhibitors than first generation
They are more specific
Low potency and high dose

20. Third generation
• Very high specifity
• High potency
• Lorafarnib,Laniquidar,Elacidar,zosuquidar,
tarquidar

21. Efflux studies invitro
• Caco-2-cells for in vitro permeability studies :
• Caco-2-cell lines:
• The Caco-2 cell line is a human colon adeno-
carcinoma cell line that differentiates in culture
and resembles the epithelial lining of the human
small intestine. It has been widely used as an in
vitro intestinal epithelial model for drug transport
and permeability screening of discovery
compounds.

23. Efflux Ratio (RE)
• Transport ratio=transport from apical to
basolateral/transport from basolateral to apical
• Assessing transport in both directions (apical
to basolateral (A-B) and basolateral to apical
(B-A)) across the cell monolayer enables an
efflux ratio to be determined which provides
an indicator as to whether a compound
undergoes active efflux.

24. • If the efflux ratio is more than 2:
Perhaps the drug has a carrier for absorption
• If the efflux ratio equal to 1 :
Perhaps the drug has simple diffusion for
transformation.
• If the efflux ratio less than 0.5:
Perhaps the drug is the substrate of efflux pump.

25. Thanks for your
attention