fundamentals of membrane transporters and their role in in ......fundamentals of membrane...

PCEUT527 JD Unadkat, Ph.D. 1

Fundamentals of Membrane Transporters and their Role in

In Vivo PK/PD of Drugs Jash Unadkat, Ph.D.

Department of Pharmaceutics University of Washington

Seattle, WA 98195 http://depts.washington.edu/pceut/faculty_research/faculty_members/unadkat_jashvant.html

Fundamentals of Membrane Transporters


Transport-related ~5-10%

Protein Folding and degradation

13% Signal transduction

11%

Unclassified 8%

Ribosomal proteins 6%

Cytoskeletal 5%

Intermediary metabolism

28%

DNA /RNA metabolism

18%

1. Transporters - SLC series

2. ABC transporters

3. Pumps 4. Channels 5. Aquaporins

Importance of transporters



Why Transporters? Terminate pharmacological activity e.g. serotonin,

adenosine Need to supply cells with polar or charged nutrients

(e.g. amino acids, glucose uptake into cells) or to efflux polar molecules for physiological function eg. bile acids excretion into the gut.

To maintain intra- and extracellular milieu of the cell e.g. Na+-K+-ATPase pump, Na+-H+- exchanger

Protection mechanism, efflux of toxic and waste compounds e.g. MDR1 (P-glycoprotein)



Uptake: • solute is translocated by diffusion OR • receptor-mediated or non-receptor mediated

endocytic process (e.g. LDL and transferrin receptors)

Transport: • solute is translocated via a membrane protein, which

requires a conformational change during the process of translocation.

• Solute binding site is accessible to only one side of the membrane at any one time.

• Can be either facilitated (passive) or active

PCEUT527 JD Unadkat, Ph.D.

Lipid Bilayer

Simple Diffusion

Carrier-mediated Transport

Facilitative Transport Active Transport

Electrochemical gradient

Active vs Facilitative vs diffusion



Classification of transporters Facilitative and Active transporters Facilitative transporters: move solutes of a single class (uniporters) down a concentration (or electrical) gradient, not energy dependent but protein mediated (e.g. Na+-independent equilibrative nucleoside transporters). Saturable. Active (concentrative) transporters: can move solutes against a concentration gradient, energy dependent, protein mediated and saturable (e.g. P-gp).



Classification of transporters Active (concentrative) transporters: Primary transporters: generate energy themselves (e.g. ATP binding cassette or ABC of P-glycoprotein). Secondary transporters: Those that utilize energy (voltage and ion gradients) generated by a primary active transporter (e.g. Na+/K+-ATPase).

•Symporters: Secondary transporters which translocate two or more different solutes in the same direction (e.g. Na+-nucleoside transporters) •Antiporters: couple the transport of solutes in opposite direction (e.g. H+/organic cation exchanger in the kidney)



hCNT1 & hENT1 co-expressed in MDCK Cells Lai, Bakken and Unadkat J. Biol. Chem. 2002

hCNT1-CFP

hENT1-YFP

Vector

hCNT1-CFP

hENT1-YFP

Overlap

YFP vector



NUCLEOSIDE TRANSPORTERS EXPRESSION IN TANDEM

N B M P R - s e n s i t i v e ( hENT1)

P h l o r i d z i n s e n s i t i v e

N u c l e o s i d e N u c l e o s i d e

L u m i n a l ( B B M )

S e r o s a l ( B L M )

N a +

N a +

K + K +

N B M P R - i n s e n s i t i v e (hENT2)

N u c l e o s i d e

A T P

A D P + P i

N u c l e o s i d e

N a + N a + - K + A T P a s e

F i g . 1 . P r o p o s e d s c h e m e f o r n u c l e o s i d e t r a n s p o r t i n t h e i n t e s t i n a l e p i t h e l i a l c e l l .

Na+

hCNT1 hCNT2

Patil and Unadkat Am J Physiol 1997;272:G1314-20 Chandrasean et al. Biochem. Pharmacology 53: 1909-18, 1997



• Export transporters (ATP-Binding Cassette Transporters) • P-glycoprotein (P-gp) • Multidrug Resistance Associated Proteins (Mrp1-6) • Breast cancer resistant protein (BCRP)

Uptake transporters

• Organic anion transporters (OATs and OATPs) • Organic cation transporters (OCTs) • Nucleoside transporters • Oligopeptide transporters • Bile acid transporter

See next set of slides for additional transporters

Selected drug transporters


PCEUT527 JD Unadkat, Ph.D. 11 Giacomini et al. Nat Rev Drug Discov. 2010 Mar;9(3):215-36.



Giacomini et al. Nat Rev Drug Discov. 2010 Mar;9(3):215-36.



P-glycoprotein P-gp, encoded by MDR1, functions to

efflux xenobiotics from the cell; 170kDa P-gp is a member of the ATP binding

cassette superfamily of transport proteins ATP binding and hydrolysis is essential for

P-gp to function Effluxes lipophilic cationic drugs



Ambudkar et al., Annu.Rev.Pharmacol.Toxicol., Vol 39., 361-398., 1999.

~170kDa

http://pharmtox.annualreviews.org/content/vol39/issue1/images/large/pa39_0361_1.jpeg



Aside from anticancer agents, P-gp mediates the transport of

a large number of lipophilic cationic drugs, neutral drugs with bulky ring structures (steroids and cyclic peptides), and a few acidic drugs. Many drugs are capable of inhibiting P-gp.

From a physiologic standpoint, P-gp appears to guard against entry and promote elimination of xenobiotics.

Because of the widespread tissue expression of P-gp, modulation of P-gp function through drug interaction could result in concurrent changes in drug absorption, distribution and excretion.

P-glycoprotein ATP

ADP + Pi



I.V. or S.C.

Oral Dose

Brain

Blood

Proximal Tubule

Renal Artery

Blood

Maternal Blood

Syncytiotrophoblast

Urinary Excretion

Liver

Gut Wall

Gut Lumen

Fecal Excretion

Bile Duct

Portal Vein

P-gp expression

Endres et al., Eur J. Pharm. Sci., In Press, 2005



List of some substrates or inhibitors of P-gp

Cyclosporine, cortisol, dexamethasone, progesterone

Others

Verapamil, amiodarone, quinidine, digoxin Antiarrythmics/Cardiac

Rifampin, quinolones, clarithromycin, erythromycin, azoles

Anti-infectives

Nelfinavir, indinavir, ritonavir, saquinavir, amprenavir

Antiviral

Vinblastine, topotecan, paclitaxel, doxorubicin

Anticancer



Importance of P-gp in drug disposition Data presented will focus on in-vivo human

studies Drug interactions will be used to illustrate the

in vivo consequences of P-gp expression Examples will be of drugs where role of P-gp

in the disposition of these drugs is well established through studies in knock-out mice and P-gp expressing cell lines



P-gp-Based Drug Interactions Intestinal drug secretion (exsorption) mediated by

P-gp. • Inhibition of P-gp secretion • Induction of P-gp leading to increased intestinal

secretion or decrease in absorption – Effect of P-gp on absorption/excretion of

drugs in the intestine is often confounded/amplified by CYP3A4/5 metabolism and recycling, especially for highly extracted drugs


P-glycoprotein and CYP3A co-localization

Portal Vein To liver

P-gp

Duodenum Ileum P-gp

CYP3A

CYP3A

Drug

Metabolite

CYP3A

Metabolite

P-gp

CYP3A

Metabolite

P-gp

CYP3A

Metabolite

P-gp

P-gp: in vivo consequences on drug disposition

•P-gp and CYP3A enzymes have overlapping substrate selectivity •P-gp expression colon>ileum>jejunum>stomach (Fricker et al., Br. J. Clin. Pharmacol. 118:1841-7, 1996), while CYP3A expression and activity follows a reverse order

•↓ CYP3A metabolism ↑FG ↓P-gp transport ↑ OR ↔ FG – the latter when drug is not a substrate of intestinal enzymes and if the drug is completely absorbed



P-gp-Based Drug Interactions: Inhibition of Intestinal drug secretion e.g. talinolol-

verapamil, talinolol-digoxin, cyclosporine-grapefruit juice interaction, digoxin-clarithromycin Randomized, placebo-controlled, double-blind, cross-

over design Single 0.75mg of digoxin with or without 250 mg of

clarithromycin twice daily for 3 days (n=12 men) Three subjects received IV digoxin 0.1mg/kg with oral

placebo or clarithomycin Urine and plasma conc. of dig. Measured by RIA 1.7-fold increase in dig. AUC after oral dig. 1.2-fold increase in dig. AUC after IV dig.



P-gp-Based Drug Interactions: Inhibition of Intestinal drug secretion e.g. digoxin-

clarithromycin (Rengelshausen et al., Br. J. Clin. Pharmacol. 56:32-38, 2003)



P-gp-Based Drug Interactions: Inhibition of Intestinal drug secretion? e.g. aliskein-

itraconazole

Mean ± SD plasma concentrations of aliskiren in 11 healthy volunteers after a single 150-mg oral dose of aliskiren on day 3 of a 5-day treatment with 100 mg itraconazole (first dose 200 mg) or placebo twice daily. Inset depicts the same data on a semi-logarithmic scale. Tapaninen et al. J. Clin Pharm, 2011;51:359

Alikserin is excreted predominately unchanged in the bile. F is low, about 2-3%. T½ is not affected.



Induction of intestinal secretion of digoxin by rifampin (Greiner et al., J. Clin. Inv. 104: 147-53, 1999). 8 volunteers - biopsies of small-bowel mucosa (second portion of the

duodenum) were obtained for immunohistochemistry or Western blot analysis.

Day 2, volunteers were randomized to a single oral dose of 1 mg digoxin (n = 4) or an intravenous infusion of 1 mg digoxin (n = 4) over 30 minutes.

On day 8, the volunteers took 600 mg rifampin once daily orally until day 23.

On day 17, all 8 volunteers underwent a second biopsy. Day 18, they received the oral or intravenous dose of digoxin in the same

manner as on day 2.

After 12 weeks, the identical protocol was repeated, except for a switch of digoxin administration (volunteers who received the oral dose first were now treated intravenously, and vice versa).



P-gp-Based Drug Interactions Induction of

intestinal secretion of digoxin by rifampin

Oral digoxin, 1 mg

IV digoxin, 1 mg



P-gp-Based Drug Interactions Induction of intestinal secretion of digoxin by St. John’s Wort extract

32

P-glycoprotein and CYP3A co-localization

Portal Vein To liver

P-gp

Duodenum Ileum P-gp

CYP3A

CYP3A

Drug

Metabolite

CYP3A

Metabolite

P-gp

CYP3A

Metabolite

P-gp

CYP3A

Metabolite

P-gp

Transporter-Metabolism Interplay

•P-gp and CYP3A enzymes have overlapping substrate selectivity •P-gp expression ileum>jejunum>stomach (Drozdzik et al., Mol. Pharm. 2014), while CYP3A expression and activity follows a reverse order

33

Waterschoot & Schinkel Pharmacol. Rev. 63: 2011


Paclitaxel

wt Pgp -/- Cyp3a -/- Cyp3a/Pgp -/-

Fh (fraction escaping liver) 0.22 0.61 0.84 0.96

Fg*Fa (fraction escaping gut) 0.47 0.25 0.30 0.47

34

2.8x 11.5x 72x Compared with wt

2.0x 4.9x 17.1x Compared with wt

Transporter-Metabolism Interplay Docetaxel PK

35


Dufeck & Thakker DMD 41:42, 2013

• P-gp in the upper intestine prevents metabolism of LOP where CYP3a activity is highest • In the lower intestine, CYP3a activity is lower, therefore LOP Fg is ↑ • As LOP dose ↑ Cyp3a (and P-gp) becomes saturated and Fg is ↑

P-gp +/+ P-gp -/-

Portal bioavailability (Fg) of oral loperamide in mice was measured by cannulating the hepatic portal vein



P-gp-Based Drug Interactions Inhibition of biliary and renal clearance of

digoxin by quinidine and quinine (Hedman Clin Pharmcol Ther 49:256-62, 1991; 47:20-6, 1990) Patients were studied, first on digoxin alone and then on

digoxin-quinine or digoxin-quinidine. Biliary (triple lumen catheter) and renal clearance of digoxin were measured at steady state.

Biliary clearance of digoxin was decreased by 45 and 34% by quinidine and and quinine respectively

Renal clearance of digoxin was decreased by 29% by quinidine but was unaffected by quinine



Inhibition of biliary clearance of digoxin by quinidine and quinine

Steady-state biliary clearance (ml/min) of digoxin in the absence and presence of quinidine or quinine

Control +Quinine Change (%)

+Quinidine Change (%)

Mean ±SD

134±57 87±39 -34±21 55±27 -45±15



Inhibition of renal clearance of digoxin by quinidine or quinine

Steady-state renal clearance (ml/min) of digoxin in the absence and presence of quinine or quinidine

Control +Quinine Change (%)

+Quinidine Change (%)

Mean ±SD

177±40 185±53 4±12 110±21 -29±11



P-gp-Based Drug Interactions: Blood-brain barrier Many classes of drugs excluded from CNS due to

expression of P-gp or other transporters at the blood-brain barrier e.g. anti-HIV protease inhibitors, narcotics

Efficacy and/or toxicity of drugs may be enhanced by overcoming this transporter barrier (e.g. anti-HIV protease inhibitors)

Difficult to conduct studies in humans



Substrates Brain to Blood Ratio mdr1(-/-)/mdr1(+/+)

Loperamide 13.5

Quinidine 14

Nelfinavir 40

Verapamil 9.5

Cetirizine (Zyrtec®) 2.3 ~ 8.7

Desloratadine (Clarinex®) 14



Questions In humans, is P-gp transporter at the blood-brain

barrier as important as that in knockout mice in excluding drugs from the CNS?

If so, can this activity be quantified non-invasively?

P-glycoprotein at the blood-brain and the placental barriers



2.25

0.10

SUV

Val

ue

MRI Pre CsA 11C-Verapamil

Post CsA 11C-Verapamil

2.25

0.10 SU

V V

alue

We measured the distribution of 11C-verapamil, a P-gp substrate, into human brain in the absence and presence of the P-gp inhibitor, CsA as the - used the noninvasive, quantitative technique, Positron Emission Tomography (PET)



Blood CsA

Ratio of AUCbrain:AUCblood

(20 min)

Ratio of AUCbrain:AUCblood

(45 min) N=12* µM - CsA + CsA % increase - CsA + CsA % increase

Mean 2.8 0.42 0.78 87 0.55 1.02 88 St. Dev. 0.4 0.04 0.10 19 0.10 0.18 20

Max. 3.2 0.55 0.98 148 0.68 1.31 128 Min. 2.1 0.30 0.56 62 0.38 0.75 65

•6 males, 6 females – no sig. difference between males and females Sasongko et al., Clin Pharmacol. Ther. 2005



P-gp at the blood-brain barrier Summary

P-gp activity at the human blood-brain barrier can be measured non-invasively using PET

Inhibition of this activity increases the distribution of the P-gp substrate verapamil modestly ~ 90%. This increase in verapamil distribution is not due to change in metabolism or protein binding of verapamil

Since CsA is the most potent systemic P-gp inhibitor available on the market, these data suggest that inadvertent drug interactions at the human BBB are likely to be modest



Does the rodent model grossly overestimate the importance of P-gp in humans?

Human PET +2.5 mg/kg/h CsA

20 Minutes post 11C-

Verapamil

45 Minutes post 11C-

Verapamil

Brain : Blood (single point) 100% ↑ 79% ↑

Brain : Blood (AUC) 87% ↑ 88% ↑

Rodent PET study At 1 hour post 11C-verapamil

Mdr1a(-/-) mice Sprague Dawley Rat+50 mg/Kg

CsA

950% ↑ 1,060% ↑



0

0.5

1

1.5

2

0 0 2.8 2.9

Human Rat

Brain

: blo

od ra

tio o

f

total

[3 H

]-rad

ioac

tivity

Blood CsA concentration (µM)

75%79%Increase vs. control

Comparison of human and rat data

Hsiao et al J Pharmacol Exp Ther. 2006



P-gp at the blood-brain barrier Conclusions

P-gp activity at the human blood-brain barrier can be measured non-invasively using PET

This technique could be used to investigate the effect of genetic, physiological (e.g. hormonal status) and other modulators (e.g. inducers, inhibitors) of P-gp activity at the blood-brain barrier

Discrepancy between rodent and human data is due to CsA concentrations. Rodent model is predictive of human data.

More potent P-gp inhibitors will be needed to overcome human BBB and tumor P-gp barriers (e.g. LY335979)



P-gp at the placental barrier Is it important in protecting the fetus from

toxic drugs and xenobiotics? Does it exclude drugs that are intended to

treat the fetus e.g. prophylaxis with anti-HIV protease inhibitors?

Does the activity change with gestational age?



P-gp at the placental barrier

Fig. 1. Concentrations of L-652,280 (avermectin analog) in CF-1 mouse fetuses with different P-glycoprotein genotypes. Lankas et al., Reprod Toxicol 1998, 12(4):457-63



Pregenotyped mice were mated and females received vehicle or 1.5 mg/kg/d L-652,280 on Gestation Days 6 through 15. On Gestation Day 18, animals were euthanized, and the fetuses were examined for cleft palate. Lankas et al., Reprod Toxicol 1998, 12(4):457-63

P-gp at the placental barrier



P-gp-Based Drug Interactions: Placental barrier

Inhibition of efflux of anti-HIV protease inhibitors across the placenta by P-gp inhibitors (Schinkel et al., J. Clin. Inv. 96:1698-705)


PCEUT527 JD Unadkat, Ph.D. 52 Molsa et al., Clin Pharmacol Ther. 2005 78:123-31

Transplacental clearance index of (A) indinavir determined in the maternal-to-fetal and fetal-to-maternal directions for each placenta. Lines join data from the same placenta. Sudhakaran et al, Antimicrob Ag Chemo2005, 49:1023–1028

P-gp activity in the perfused Human Placenta

PET – before CsA

PET – during CsA

PET – pixel-by-pixel subtraction of A from B

MRI

-

The Effect of CsA on Tissue Distribution of [11C]-Radioactivity

Fetal liver

Maternal brain

Maternal brain

Maternal liver

Fetal liver

Maternal brain

Maternal liver

Uterine wall

Heart

Fetal liver

Maternal liver

A

B

C

D

5.0

0 5.0

0 1.45

0.5

The Effect of CsA on Tissue

Distribution of [11C]-Radioactivity

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

0.01

0.10

1.00

10.00

0 10 20 30 40

Time (min)

[11C

]-rad

ioac

tivity

/dos

e (L

-1)

Maternal brainPlasma Fetal liver

KidneysMaternal liver

Heart Lungs

Vertebrae

Spleen

0.01

0.10

1.00

10.00

0 10 20 30 40

Uterine wall

Gallbladder

0.01

0.10

1.00

10.00

0 10 20 30 40

Placenta

The Effect of CsA on Tissue Distribution of [11C]-Radioactivity

-100

0

100

200

300

400

AUC 0 to 9 Minutes ratioAUC 0 to 20 Minutes ratioAUC 0 to 40 Minutes ratio

Cha

nge

(%)

*

*

*

*

Brain Fetalliver Maternal

liver Maternalgallbladder

Kidneys Heart Lungs Vertebrae Spleen Uterinewall

Placenta

*

AUC0-9

AUC0-20

AUC0-40

Conclusions

• Changes in placental P-gp activity with gestational age: • Will be a barrier to target drugs to the fetus • Will affect the efficacy and/or toxicity of drugs to the fetus

• Drug interactions at the BPB are likely to be modest

• Imaging is powerful tool to identify MDR tumors or to

determine the contribution of factors (genetics, physiological, pathological or drugs - inducers, inhibitors) on P-gp activity at the human BBB and BPB

P-gp drug interactions at the Blood-Placental Barrier



P-gp based drug interactions (both induction and inhibition) are likely : to be frequent due to the promiscuity of the transporter

AND the strategic location of P-gp in eliminating/absorbing organs

to be particularly pronounced in the intestine due to the high concentration of drugs experienced by the enterocytes (unless P-gp is already saturated!).

to increase (or decrease) the availability of drugs to privileged sites such as the CNS and the placental-fetal unit resulting in either enhanced (or decreased) drug toxicity or efficacy or both

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