Fundamentals of Membrane Transporters and their Role in

In Vivo PK/PD of Drugs-I

Bhagwat Prasad, Ph.D.

Department of Pharmaceutics

University of Washington, Seattle, WA

bhagwat@uw.edu

How to Assess Role of Transporters in Drug Disposition?

-RIF +RIF

He et al., Mol. Pharm., 2014

18F-deoxyglucose, at 1 h

Imaging studies are challenging to perform. Need for alternative approaches.

Hepatic uptake and biliary excretion

of 11C-rosuvastatin in the rat-He et al., Mol. Pharm., 2014

Transport-related

~5-10%

Protein Folding

and degradation

13%Signal transduction

11%

Unclassified

8%

Ribosomal proteins6%

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?

1. Pharmacological activity e.g. serotonin, adenosine

2. 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 e.g., bile acids excretion into the gut.

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

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

Classification of transporters

1. 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.

2. Active (concentrative) transporters: can move solutes against a concentration gradient, energy dependent, protein mediated and saturable (e.g. P-gp).

Lipid

Bilayer

SimpleDiffusion

Carrier-mediated Transport

Facilitative Transport Active Transport

Electrochemicalgradient

Active vs Facilitative vs diffusion

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)

• 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)• Organic anion transporting polypeptides (OATPs)• Organic cation transporters (OCTs)• Nucleoside transporters • Oligopeptide transporters• Bile acid transporter

Selected drug transporters

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

I.V. or S.C.

Oral DoseBrain

Blood

ProximalTubule

RenalArtery

Blood

MaternalBlood

Syncytiotrophoblast

Urinary Excretion

Liver

GutWall

Gut Lumen

Fecal Excretion

Bile Duct

Portal Vein

P-gp expression

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

List of some substrates or inhibitors of P-gp

Cyclosporine, cortisol, dexamethasone, progesteroneOthers

Verapamil, amiodarone, quinidine, digoxinAntiarrythmics/Cardiac

Rifampin, quinolones, clarithromycin, erythromycin, azolesAnti-infectives

Nelfinavir, indinavir, ritonavir, saquinavir, amprenavir Antiviral

Vinblastine, topotecan, paclitaxel, doxorubicinAnticancer

• 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 IleumP-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. 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-glycoprotein and CYP3A co-localization

Portal Vein To liver

P-gp

Duodenum IleumP-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

Waterschoot & Schinkel Pharmacol. Rev. 63: 2011

Transporter-Metabolism Interplay

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

2.8x 11.5x 72xCompared with wt

2.0x 4.9x 17.1xCompared with wt

Transporter-Metabolism InterplayDocetaxel PK

Transporter-Metabolism Interplay

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

13457 8739 -3421 5527 -4515

• 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

17740 18553 412 11021 -2911

• 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

SubstratesBrain 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 knockoutmice 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 CsA11C-Verapamil

Post CsA11C-Verapamil

2.25

0.10

SUV

Val

ue

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 asthe - 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* mM - 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 femalesSasongko 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

Bra

in: blo

od r

atio o

f

tota

l [3 H

]-ra

dio

activity

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)

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

05.0

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

]-ra

dio

activity/d

ose (

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 ratio

AUC 0 to 20 Minutes ratio

AUC 0 to 40 Minutes ratio

Chan

ge (

%)

*

*

*

*

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 absorbing/elimination 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

• Additional slides

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.

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