Transport Mechanisms 2013

8/10/2019 Transport Mechanisms 2013

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MEMBRANE TRANSPORT MECHANISMS

FOR DRUGS

A. INTRODUCTION: PharmacokineticsI. DEFINITION

II. THE FATE OF DRUGS IN THE BODY 3 phases: absorption, distribution, elimination

B. CLASSIFICATION OF MEMBRANE TRANSPORT MECHANISMS

C. DIFFUSION across the lipid matrix of the membrane

I. DETERMINANTS OF DIFFUSION area, concentration gradient, lipid solubility

II. DETERMINANTS OF LIPID SOLUBILITY1. For non-ionizable compounds presence of hydroxyl, alkyl, and halogen substituents2. For ionizable compounds the degree of ionization; the non-ionized molecule is membrane-diffusible

a. Determinants of the degree of ionization: pKa, pHb. Calculation of the ratio of NI/I molecules by the rearranged Henderson-Hasselbalch equation

D. CARRIER-MEDIATED TRANSPORT

I. CHARACTERISTICS capacity, selectivity, competition of substrates, driving force

II. CLASSIFICATION based on the driving force

1. FACILITATED DIFFUSION (ENT, GLUT transporters) driven by concentration gradient2. ACTIVE TRANSPORT driven by ATP directly or indirectly

a. PRIMARY ACTIVE TRANSPORTERS (Mrp, Mdr (= Pgp), BCRP transporters) exportersPgp-substrates are excluded from the CNSPgp-substrate drugs do not have CNS effects examples

b. SECONDARY ACTIVE TRANSPORTERSVariation 1. Na

+-dependent transport (NAT, NTCP, NIS, SGLT, SVCT2 transporters)

Variation 2. Membrane potential-dependent transport (OCT transporters)

c. TERTIARY ACTIVE TRANSPORTERS

Variation 1. Na

+

- and KG-dependent transport (OAT: OAKG exchangers)Variation 2. Na+- and GSH-dependent transport (OATP: OAGSH exchangers)Variation 3. Na

+- and H

+-dependent transport (PEPT, PCFT, DMT: XH

+cotransporters)

Variation 4. Na+- and H+-dependent transport (MATE: OCH+exchangers)

E. AN EXAMPLE FOR INTERACTION OF DRUGSWITH AN ENDOGENOUS METABOLITE AT TRANSPORTERSMechanism of hyperuricaemia induced by furosemide and some other acidic drugs


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A. INTRODUCTION: Pharmacokinetics

Definition

Two aspects of pharmacology:

1. PHARMACODYNAMICS: study of the effect of the drugon the body

2. PHARMACOKINETICS: study of the effect of the body on the drug

The body ABSORBS, DISTRIBUTES, and ELIMINATES drugs.

Pharmacokinetics isthe study of the

FATE, orDISPOSITION, or

MOVEMENT

of drugs in the body.

THE FATE OF DRUGS IN THE BODY 3 phases:

1. ABSORPTION

2. DISTRIBUTION

3. ELIMINATION

a. Chemically: BIOTRANSFORMATION

b. Physically: EXCRETION

Test question:Chloramphenicol is conjugated with glucuronic acid and theconjugate is excreted into urine. Which organ is important in theelimination of chloramphenicol?

A. LiverB. KidneysC. Both

Drugs are transported across membranesin the course of their absorption, distribution and excretion.


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B. CLASSIFICATION OF MEMBRANE TRANSPORTMECHANISMS

MEMBRANE TRANSPORT MECHANISMS

DIFFUSION SPECIALIZED TRANSPORT

via aqueouschannels (AQP)

across thelipid matrix

carrier-mediatedtransport

vesiculartransport

For smallhydrophilicunchargedmolecules

e.g., glycerol, urea,arsenite: As(OH)3

unimportantfor most drugs

IMPORTANTfor most drugs

for absorptionand

distribution

IMPORTANT

for several drugsforGI absorption andcellular uptake,

and

for many drugs andmost drug metabolites

(acidic conjugates)for excretion via biliary

and renal tubularsecretion

For proteins(rec-med. endocytosise.g., LDL, transferrin)

unimportantfor most drugs

Exceptions:- IF-Vit B12:rec-med. EC

- Folate:rec-med. EC*-AGs: adsorptive EC- Deferoxamine:

fluid-phase EC

*Folate receptoris utilized for delivery of folate-bound antitumor drugs (such asvinblastine), called folate conjugates, into tumor cells overexpressing the folate

receptor(drug targeting). Note:Folate is also taken up by carrier-mediated transportvia the proton-coupled folate transporter(PCFT; see later) across the luminalmembrane of enterocytes, thus PCFT also contributes to intestinal absorption of folate.

Another example for drug targeting:Cholesterol-bound siRNA is used for gene silencing. It becomes incorporated intolipoprotein particles and then is taken up by cells through LDL receptors.


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C. DIFFUSION across the lipid matrix of the membrane

I. DETERMINANTS OF DIFFUSION:1. the area across which diffusion takes place2. the concentration gradient, i.e. the driving force3. the lipid solubility of the compound

II. DETERMINANTS OF THE LIPID SOLUBILITY:

1. For non-ionizable compounds (e.g., steroids, digitalis compounds):

a. the number of OH-groups e.g.: digoxin (3 -OH) vs. ouabain (6 -OH)b. the number of alkyl groups e.g.: testosterone vs. methyltestosteronec. the number of halogen atoms e.g.: hydro- vs.fludrocortisone (dyn + kin diff.)

2. For ionizable compounds (organic acids and bases)

In addition to 1. a.b.c.,the major determinantof lipid solubility is: THE DEGREE OF IONIZATION

O

COOH

C CH3

O

CH2

CH CH3

NH2

acetylsalicylic acid amphetamine

The NON-IONIZED SPECIES (R-COOH and R-NH2):is LIPOPHILIC and DIFFUSIBLEacross the lipid membrane.

The IONIZED SPECIES(R-COO-and R-NH3+)

is HYDROPHILIC and NON-DIFFUSIBLE across the lipid membrane.

The degree of ionization(or non-ionization)

depends on: - the pH of the solution- the pKaof the compound

calculatedby the Henderson-Hasselbalch equation:

For ACIDS: For BASES:

pHpK]ionized[

]nonionized[

log a apKpH]ionized[

]nonionized[

log

[NI]/[I] = 10pKa pH [NI]/[I] = 10

pH

pKa

Ionization of acids: R-COOH R-COO

-

+ H

+

Ionization of bases: R-NH2 + H+ R-NH3

+


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D. CARRIER-MEDIATED TRANSPORT

I. Characteristics:

1. Capacity: Tm, Km2. Selectivity for substrates

3. Competition between substrates4. Driving force

II. Classification based on the driving force:

1. FACILITATED DIFFUSION driven by concentration gradient

Equilibrative nucleoside transporter (ENT)

Glucose transporters (GLUT1 - GLUT5)

2. ACTIVE TRANSPORT driven by cellular energy (ATP)

a. Primary active transport driven by ATP directly

Multi-drug resistance transport proteins:- Mrp family: e.g. mrp2, mrp3

- Mdr family: e.g. mdr1 (P-glycoprotein)- BCRP family

b. Secondary active transport driven by ATP-dependent ion-gradient

Na+-dependent transporters:- Amino acid transporters (NAT)- Bile acid transporter (NTCP)- Concentrative nucleoside transporter (CNT)- Sodium-dependent glucose transporter (SGLT)

- Sodium-dependent vitamin C transporter (SVCT)- Na-iodide symporter (NIS)

Inside-negative membrane potential-dependent transporters:- Organic cation transporters (OCT1, OCT2, OCT3, OCTN1, OCTN2)

c. Tertiary active transport driven by ATP-dependent ion- and solute-gradient

Organic anion transporters (OAT): OAKG exchanger

Organic anion-transporting polypeptides (OATP): OAGSH exchanger

Peptide transporters (PEPT): PeptideH+-cotransportersDivalent metal transporter (DMT): DM-ionH+-cotransporters

Multidrug and toxin extrusion transporter (MATE): OC+H+exchanger


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1.FACILITATED DIFFUSION

driven by concentration gradientDirection: depends on the concentration gradient; equilibrative transport

EXAMPLES:

Equilibrative nucleoside transporter (ENT): uptake from blood of

Nucleosides: purine and pyrimidine nucleosides (e.g., uridine, cytidine)

Nucleoside analogues, e.g.:- Antiviral drugs: zidovudine, stavudine, lamivudine, zalcitabine,

abacavir, acyclovir, gancyclovir

- Anticancer drugs: 5-fluorodeoxyuridine (floxuridine)cytosine arabinoside (cytarabine)

difluorodeoxycytidine (gemcitabine)Note: There are sodium-dependent secondary active nucleoside transporters (calledconcentrative nucleoside transporters, CNT) as well (see later).

Glucose transporters(GLUT1 - GLUT5):

mediate uptake of glucose from bloodinto cells orexport of glucose from cells into blood (depending on the conc gradient)

(RBC:GLUT1, hepatocyte:GLUT2, skeletal muscle:GLUT4)

GLUT also takes up dehydroascorbate into cells, which is then reducedto ascorbate intracellarly

Note: There are sodium-dependent secondary active glucose transporters (SGLT) as well (seelater).

XX


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2.a.PRIMARY ACTIVE TRANSPORT (export)

driven by ATP directly

EXAMPLES: 3 families of multi-drug resistance transport proteins:

Mrp family, e.g.:

Mrp1 and Mrp3: e.g.in hepatocytes (in the sinusoidal membrane) transports organic acid-typedrugs/metabolites into the blood

(e.g., chloramphenicol-glucuronide, valproic acid-glucuronide).

Mrp2: e.g.in hepatocytes (in the bile canalicular membrane)

transports organic acid-type drugs (e.g. cefoperazone, methotrexate)andglucuronides (e.g., bilirubin-digluc., phenolphthaleine-gluc., ezetimibe-gluc.)into the bile.

Mdr family, e.g.:mdr1 (Pgp): transports large basic molecules (see next page)and some neutral

molecules (e.g. digoxin)- in enterocytes,luminal membrane exports drugs into the gut

- in renaltubule cells, luminal membrane exports drugs into the tubules- in braincapillary endothelial cells, luminal membrane

pumps drugs back into the blood = BBB (see table on next p.)

BCRP (Breast Cancer Resistance Protein)familytransports mainly large basic molecules (e.g., mitoxantrone, topotecan, prazosin,glybenclamide), but also dehydroepiandrosterone sulfate(used as a model substrate)

- in enterocytes,luminal membrane exports drugs into the gut

- in renaltubular cells, luminal membrane exports drugs into the tubules- in braincapillary endothelial cells, luminal membrane

pumps drugs back into the blood = BBB

ADP

ATP

X1


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2.b. SECONDARY ACTIVE TRANSPORT (import)Variation 1. Na+-dependent transport

driven by the NaK-ATPase-generated invardly directedNa+-gradient

EXAMPLES:

Na

+

-coupled amino acid transporter (NAT):e.g. L-DOPA uptake into the brain

Bile acid transporter (NTCP) (Na+-coupled):e.g. taurocholic acid and -amanitin uptake into liver (also by OATP1B3)

Concentrative nucleoside transporter (CNT) (Na+-coupled):e.g.purine and pyrimidine nucleosides uptake into cells (salvage)

Sodium-dependent glucose transporter (SGLT): absorption of glucose from the intestinal lumen

reabsorption of glucose from the renal tubular lumen

absorption of glucose from the CSF into the choroid plexus

Sodium-dependent vitamin C transporter (SVCT): uptake of ascorbic acid into cells

Na-iodide symporter (NIS): takes up I-into the thyroid (and other monovalent anions: ClO4

-, SCN

-)

ATP2 K

+

3 Na+

1

ADP

1

XX--

2Na

+


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2.b. SECONDARY ACTIVE TRANSPORT (import)Variation 2. Membrane potential-dependent transport

driven by the NaK-ATPase-generatedinside-negative membrane potential

EXAMPLE:

Organic cation transporters: OCT1, OCT2, OCT3, OCTN1, OCTN2mediate uptake of small m.w. organic cationsfrom the blood into cells, e.g.:

into liver cells(OCT1)

uptake of metformin(antidiabetic drug)inhibits gluconeogenesisinto renal proximal tubular cells(OCT2)

tubular secretion of metformin urinary excretion

tubular secretion of amiloride delivery to coll. duct (site of action)

uptake of cisplatin tubular injury,an unwanted effect of cisplatin

Substrates:cimetidine, ranitidine, procainamide, carbachol, pindolol, metformin,

amiloride, triamterene, cisplatin

-

-

-

-

-

- - - - -

ATP

2 K+

3 Na+

ADP

1

XX++ 2

-

-

-

- - - - -

- - - - -

-

-

-

--

-

-

OCT


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2.c. TERTIARY ACTIVE TRANSPORT (import)Variation 1. Na+- and

KG-dependent transport

driven by ATP-dependent Na+and KG-gradients

Blood side Tubule lumen

EXAMPLE:

Organic anion transporter (OAT)

in the BLM of proximal renal tubules (see above)

works as an organic anion-ketoglutarate exchanger

mediates the uptake of

-acidic dugs: e.g. penicillin, loop diuretics (furosemide), thiazide diuretics- many acidic conjugates of drugs:

e.g. - chloramphenicol-glucuronide,- valproic acid-glucuronide,- salicyl-glycine (also called salicyluric acid, the main metabolite of aspirin)

Such drugs aretaken upinto proximal convoluted tubular cells by OAT1,followed by exportat the luminal membrane (by Mrp4 and OAT4)= renal tubular secretion of acidic drugs or drug conjugates.

3 Na+

ATP2 K

+

1

ADP

1

XX--

3KG=

KG=2

Na+

OAT1


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2.c. TERTIARY ACTIVE TRANSPORT (import)Variation 3. Na+- and H+-dependent transport

driven by ATP-dependent Na-ion- and H+-gradients

Intestinal lumen Blood side

EXAMPLES:

Peptide transporter (PEPT)(H+-coupled, in the BBM of enterocytes):

uptake of ACE inhibitors and -lactam antibiotics intestinal absorption

N

S

COOH

O

N

H

N

S

C

CCH

2

COOH

C

O

HNH2N COOHNH

NH2

C

O

HOOC

ceftibuten CEDAX

lisinoprilLISOPRESS

PRINIVIL

NOTE: These drugs are dicarboxylic acids. In the gut, they are deprotonated and have 2 negativecharges. Thus, they could not be absorbed by diffusion. Their absorption is made possible by PEPT.

Proton-coupled folate transporter (PCFT)(in the BBM of enterocytes):

uptake of folate intestinal absorption

uptake of heme intestinal absorption heme-bound ironDivalent metal transporter (DMT)(H+-coupled, in BBM of enterocytes):

uptake of Fe++ (and Cd++ !!!) intestinal absorption of non-heme Fe++

2K+

3Na+

1

XX--

3

H+

H+

2Na

+

ATP

ADP

PEPT


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2.c. TERTIARY ACTIVE TRANSPORT (export)Variation 4. Na+- and H+-dependent transport

driven by ATP-dependent Na+and H+gradients gradients

Blood side Tubule lumen

EXAMPLE:

Multidrug and toxin extrusion transporter (MATE)

Working as protonorganic cation exchanger,MATE exports organic cationsfrom the excretory cells into excreta:

from the liver cellsinto the bile canaliculi

from the renal tubular cellsinto the tubular lumen tubular secretion of metformin and cimetidine (which are taken up

into the tubular cell by OCT2, an inside-negative potential-driven secondaryactive transporter)

Substrates:cimetidine, ranitidine, procainamide, carbachol, pindolol, metformin,amiloride, triamterene (all are organic cations)

Inhibitor:pyrimethamine (a DHFR inhibitor; used for treatment of malaria and toxoplasmosis)

- - - - -

ATP

2 K+

3 Na+

ADP

1

XX++ 2

-

-

-

- - - - -

H+

-

-

-

-

-

-

OCT2

22 K

+ Na+

3

H+

H+

MATE1


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E. AN EXAMPLE FOR INTERACTION OF DRUGSWITH AN ENDOGENOUS METABOLITE AT TRANSPORTERS

Mechanism of hyperuricaemia induced by furosemideand some other acidic drugs

Apical surfaceBasolateral surface

Furosemide

Urate

Furosemide*

Urate

-KG2-

OAT1

GLUT9

OAT4

* Other hyperuricaemic drugs: Loop diuretics

Thiazides

Large dose of aspirin

Pyrazinamide

pyrazinic acid

This figure demonstrates that tubular secretion of furosemide (and some other acidic drugs see listed) is coupled to the reabsorption of urate.

Furosemide is taken up from the blood into the renal proximal tubular cell by the tertiary-

active transporter OAT1, an organic acid-ketoglutarate exchanger, located in thebasolateral membrane of these cells. Then, furosemide is transported across the apical(luminal) membrane of the tubular cells by OAT4 in exchange for urate (and by Mrp4 - notshown). Subsequently urate is exported from the cell into the blood via GLUT9 (a glucosetransporter) across the basolateral membrane by facilitated diffusion.

Not indicated in this figure that furosemide, thiazides and salicyl-glycine (the major metaboliteof aspirin) are exported from the proximal tubule cell across the luminal membrane into thetubule not only by OAT4, but also by MRP4. MRP4 is a primary active transporter that usesATP directly to drive the transport process. When relatively small doses of aspirin areadministered, salicyl-glycine is secreted largely via OAT1-MRP4. At high aspirin doses,however, OAT4 is increasingly involved in the export of salicyl-glycine from the proximaltubular cells into the tubular fluid, which is coupled to urate reabsorption. This explains whylarge dose of aspirin causes hyperuricaemia.

Transport Mechanisms 2013

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