P-glycoprotein transporters

Maryam kazemi Ph.D student of pharmaceutics at SUMS

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.

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.

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.

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.

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

• Other drugs: Chloroquine, dexamethasone, morphine, phenytoin

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.

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.

• 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 .

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

• 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

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

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

Third generation

• Very high specifity • High potency • Lorafarnib,Laniquidar,Elacidar,zosuquidar,tarquidar

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.

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.

• 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.

Thanks for your attention