membrane transporters

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Information about membrane transporters
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Published on June 19, 2018

Author: 1508divya

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Pharmacokinetics : Membrane transport SLC , ABC , Vectorial transport , Pgp : Pharmacokinetics : Membrane transport SLC , ABC , Vectorial transport , Pgp Divya S ingh JR II Department of Pharmacology & Therapeutics Pharmacokinetics : Pharmacokinetics Pharmacon ( greek ) = drug Kineticos ( greek ) = putting in motion Pharmacokinetics is the movement of drug in , through and out of the body All pharmacokinetic processes involve the transport of the drug along the biological membranes Reference: Essentials of Medical Pharmacology –KDT, 13 th Edition Transporters : Transporters Membrane transport proteins are present in all organisms. These proteins Control the influx of essential nutrients and ions and the efflux of cellular waste , environmental toxins, drugs, and other xenobiotics Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Aquaporins : Aquaporins Bidirectional membrane channel Not ion channels – water is transported , uncharged molecule Driving force is osmotic gradient 13 AQP genes in human Physiological role of transporters : Physiological role of transporters Regulates the distribution and bioavailability of drugs Removal of toxic metabolites and xenobiotics from cells into urine , bile and interstitial lumen Transport of the drugs out the brain across the blood brain barrier Types of membrane transporters : Types of membrane transporters 2000 genes in humans code for transporter or transporter related protein 2 major superfamilies – ABC & SLC Most ABC proteins – primary active transports SLC family includes the facilitated transporters or ion coupled secondary active transporters Slide8: ABC 49 genes – classified into 7 families Unidirectional Widely recognized are P-glycoprotein & CFTR SLC 48 families with 315 transporters Bidirectional SERT & DAT are important examples Membrane transporters in therapeutic drug responses : Membrane transporters in therapeutic drug responses Pharmacokinetics Pharmacodynamics : transporters as drug targets Drug resistance Membrane transporters in therapeutic drug responses : Membrane transporters in therapeutic drug responses Pharmacokinetics Pharmacodynamics : transporters as drug targets Drug resistance Figure 1: Membrane Transporters : Figure 1: Membrane Transporters Role of the Membrane Transporter T in the pharmacokinetic pathway Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Biological membrane : Biological membrane Bilayer of phospholipid and cholesterol molecules High electrical resistance and relative impermeability to the membrane Extrinsic and intrinsic proteins are present in the lipid bilayer These proteins have enzymatic , carrier , receptor or signal transduction properties . Reference: Essentials of Medical Pharmacology –KDT, 13 th Edition Figure 2:Transport through the biological membrane : Figure 2:Transport through the biological membrane Organisation of the phospholipid bilayer and the membrane protein Reference : Ganong’s Review of Medical Physiology , 25 th edition Drug transportation across membrane : Drug transportation across membrane Passive diffusion Filtration Facilitated diffusion Active transport Primary active transport Secondary active transport Passive diffusion : Passive diffusion Down the elctrochemical /concentration gradient Lipid soluble drugs Three steps Partition from the aqueous phase to lipid phase Diffusion across lipid bilayer Repartition into the aqueous phase Filtration : Filtration Passage of drugs through pores in membranes or paracellular spaces Accelerated under hydrostatic or osmotic pressure gradient Lipid insoluble , size smaller than size of the pores can cross Filtration …: Filtration … MW > 100 or 200 daltons can not penetrate Pore size of cells 4 Ȧ capillaries 40 Ȧ Facilitated diffusion : Facilitated diffusion Down the electrochemical gradient Transporter-mediated membrane transport Does not require energy Steady state is achieved once the elctrochemical gradient is equal on both the sides of the membrane Figure 3 : facilitated diffusion: Figure 3 : facilitated diffusion The carrier (SLC) binds and moves the poorly diffusible substrate along its concentration gradient (high to low) and does not require energy Active transport : Active transport Against the electrochemical gradient Type of membrane transport Require energy Two types Primary active transport Secondary active transport Primary active transport : Primary active transport ATP hydrolysis is coupled directly with the substrate e.g., Na + K + ATPase Transporters belong to the superfamily of ATP Binding Cassettee (ABC) transporters The intracellular loops of ABC have ATPase activity Figure 4 : Primary active transport : Figure 4 : Primary active transport Primary active transport: the carrier (ABC) derives energy directly by hydrolysing ATP and moves the substrate against its concentration gradient (low to high) Secondary active transport : Secondary active transport Uses energy through an existing electrochemical gradient Involves the solute carrier (SLC) transpoters Secondary active transport …: Secondary active transport … Further divided into symport and antiport Symport / cotransport describes movement of driving ion and transported solute in the same direction. E.g., Na + Glucose transporter SGLT 1 Antiport / exchange transport occurs when the driving ion and the transported solute move in opposite directions. e.g., Na + /Ca +2 exchanger ,NCX Figure : symport and antiport : Figure : symport and antiport Symport : the carrier moves the substrate ‘A’ against its concentration gradient by utilizing energy from downhill movement of another substrate ‘B’ in the same direction D. Antiport : the carrier moves the substrate ‘A’ against its concentration gradient and is energized by the downhill movement of another substrate ‘B’ in the opposite direction Pinocytosis : Pinocytosis Transport across the cell in particulate form by formation of vesicles Applicable to protein and other big molecules Vitamin B 12 absorption from the gut after binding to intrinsic factor Figure 5 : Classification Of Membrane Transports : Figure 5 : Classification Of Membrane Transports Red circles depict substrate , size of the circle depict concentration of drug , arrows depict the direction of flux of drug ,black squares depict the ion that supply the driving source for the transport , blue ovals depict the transport proteins. Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Basic mechanism of membrane transport : Basic mechanism of membrane transport Transporters Vs Channels: Transporters Vs Channels Channels open state & closed state Turnover rate constants : 10 6 to 10 8 sec -1 Transporters confirmational change in the shape of the protein- transport of the protein across the membrane Turnover rate constants : 10 1 to 10 3 sec -1 Characterised by saturability and inhibition of the substrate Kinetics of transport : Kinetics of transport The flux of a substrate (rate of transport) across a biological membrane via a transporter-mediated process is characterized by saturability The relationship between the flux v and substrate concentration C in a transporter-mediated process is given by the Michaelis-Menten equation : V = Vamx = maximum transport rate ,Km = michaelis constant   Slide31: The Km and Vmax values can be determined by the Eadie – Hofstee plot Made by plotting V vs V/C for determining Vmax and Km The slope is –Km and the y intercept is Vmax Figure 6 : Eadie Hofstee plot of transport data : Figure 6 : Eadie Hofstee plot of transport data Blue line depicts the transport of substrate in the presence of competitive inhibitors Red line depicts the transport of transport in the presence of non competitive inhibitors Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Transport structure : Transport structure Based on the secondary structure of the proteins based on molecular analysis ABC and SLC transporters are multimembrane spanning proteins ABC transporters: ABC transporters ABC transporters have NBDs (nucleotide binding domains) which act as the motor units Figure 7: Model of ABC transporter function : Figure 7: Model of ABC transporter function Exchange of ATP with ADP and after extrusion of the substrate molecule exchange of ADP with ATP completes the cycle Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition SLC Transporter : SLC Transporter This group contains channels , facilitators , secondary active t ransporters . SLCs use an alternating access , the gated pore mechanism In the gated pore mechanism the transporter exposes a single solute binding site interchangeably at either side of the membrane barrier Gated pore mechanism : Gated pore mechanism Figure 8 : Alternating access to SLC transporter : Figure 8 : Alternating access to SLC transporter R otation of two broken helices facilitates alternating access of substrates to the intracellular and extracellular sides of the plasma membrane Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Rocker Switch Mechanism : Rocker Switch Mechanism Variation of the gated pore mechanism Here two distinctive protomers are joined in the cytoplasmic side by a connecting loop, supporting a rocker switch mechanism Figure 9 : Alternations in the gated pore mechanism : Figure 9 : Alternations in the gated pore mechanism R ocker switch represents example models of a facilitated glucose transporter, GLUT2. Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Vectorial transport: Vectorial transport Transport of an ion or molecule across an epithelium occurs in a certain direction The ABC transporters mediate only unidirectional efflux, SLC transporters mediate either drug uptake or drug efflux. Vectorial transport implies a   nonuniform distribution of transport proteins on the plasma membranes of two faces of the epithelium Examples of vectorial transport : Examples of vectorial transport Asymmetrical transport across a monolayer of polarized cells, such as the epithelial and endothelial cells of brain capillaries Efflux of drugs via brain endothelial cells and brain choroid plexus epithelial cells Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Examples of vectorial transport… : Examples of vectorial transport… Efflux and influx of drug molecules in intestine in both apical and basolateral membrane Absorption of nutrients and bile acids in the intestine Hepatobiliary and excretion of the drugs from the blood to the lumen and in the intestinal absorption of the drug Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Figure 10 : Transendothelial and Transepithelial flux : Figure 10 : Transendothelial and Transepithelial flux Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Transporters involved in pharmacokinetics : Transporters involved in pharmacokinetics Transporters in the liver and kidney have important roles in removal of drugs from the blood and hence in metabolism and excretion. Hepatic transporters : Hepatic transporters Bisubstrate vectorial transport Uptake by OATP1B1 Efflux by MRP2 Gemfirozil inhibit uptake of active hydroxy form of statin by OATP1B1 , increasing its plasma concentration leading to toxicity Bosentan action prolonged by using inhibitors of OATP1B1 and OATP1B3 Figure 11 : Transporters in liver : Figure 11 : Transporters in liver Transporters function in the uptake and efflux of drugs across the sinusoidal membrane and efflux of drugs into the bile across the canalicular membrane . Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Organic cation transport : Organic cation transport Substrates : Endogenous- choline , N- methylnicotinamide Xenobiotics – cimetidine , ranitidine , metformin , varnecilline Genetic variations in OCT 1 and OCT2 are associated with altered elimination and response of antidiabetic drug metformin Figure 12: Organic Cation Transporters in kidney : Figure 12: Organic Cation Transporters in kidney Organic cation secretory transporters in the proximal tubule Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Organic anion transport : Organic anion transport Substrate – pravastatin , captopril , PAH , pencillins OAT1 exhibits saturable transport of organic anions such as PAH Figure 13: Organic Anion Transporters in kidney : Figure 13: Organic Anion Transporters in kidney Reference: Goodman & Gillman’s The Pharmacological Basis of Therapeutics , 13 th Edition Figure 14 : urate excretion : Figure 14 : urate excretion P-glycoprotein : P-glycoprotein Belongs to the ABC efflux transporters Responsible for decreased drug accumulation Mediates the development of resistance to anticancer drugs Also functions as transporter in blood brain barrier Figure 14 : physiological sites of P-glycoproteins : Figure 14 : physiological sites of P-glycoproteins Slide56: Table 1: Substrate for Pgp Analgesic Morphine Antibiotic Tetracycline , rifampacin Anticancer Etoposide , vincristine , danurubicin Antiemetics Ondansetron Antidepressants Vanelefexine Antifungals Itraconazole HIV protease Inhibitors saquinavir , Indinavir Antidiorrheal Loperamide Antiepileptics Phenytoin, carbemazipine Cardiac glycoside Digoxin Slide57: TABLE 2: Inhibitors of Pgp , 1 st generation Antiarrhytmics Verapamil , amiodarone Antibiotic Clarithromycin , Erythromycin Anticancer Actinomycin D , Vinblastine Calcium channel blocker Verapamil , Nefidipine Proton pump inhibitors Pantoprazole , Lansoprazole Antidepressant Sertraline Steroid Tamoxifen Inhibitors of Pgp , 2nd Generation : Inhibitors of Pgp , 2 nd Generation 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 . P- gp substrates with low protein affinity Inhibitors of Pgp , 2nd Generation …: Inhibitors of Pgp , 2 nd Generation … They don’t have pharmacologic effect Better inhibitors than first generation They are more specific Low potency and high dose Inhibitors of Pgp , 3rd Generation : Inhibitors of Pgp , 3 rd Generation Very high specifity High potency Lorafarnib Laniquidar Elacidar zosuquidar , tarquidar Pgp and drug resistance : Pgp and drug resistance Play a critical role in development of resistance to Anticancer Antmicrobial Antiepileptics Genetic variations in membrane transporters : Genetic variations in membrane transporters 2 common SNPs in OATP1B1 (SLCO1B1) are associated with elevated plasma level of pravastatin Muscle spasm with the use of simvastatin Genetic variations in SLC22A – variation in clearance of antidaibetic medication Genetic variations in membrane transporters …: Genetic variations in membrane transporters … Genetic variations in ABCG2 – response to XOIs allopurinol and rosuvastatin Polymorphisms in MRP2, MRP 4 – deposition of antiviral drug tenofovir Transporters in regulatory science: Transporters in regulatory science Transporters are major determinants of variation in therapeutic response and adverse drug response The U.S. FDA has issued a draft clinical pharmacology guidance on performing drug-drug interaction studies during clinical drug development Reference : FDA , 2012 Transporters in regulatory science …: Transporters in regulatory science … The guidance presents information on how to use in vitro data for transporter studies to make decisions about whether to conduct a clinical drug-drug interaction study Transporters OATP1B1 , OATP1B3, Pgp , BCRP, OCT2, MATE1, OAT1, and OAT3 are included in the FDA guidance Slide66: Thank you

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