A brief presentation about the transport of drugs across the cell membrane including the many mechanisms and various transporters and a brief overview of the ABC and SLC superfamily of transporters.

1. Drug Transport across Cell
Membrane
Dr. Salman H. Rizvi
JR-1
Dept. of Pharmacology
Dr. V. M. Govt. Medical College
Solapur
(08/08/2018)

2. • Introduction
• Mechanism of transport
• Role of transporters
• ABC Superfamily
• SLC Superfamily
• Vectorial Transport
• Intestinal transporters
• Hepatic transporters
• Renal transporters
• Transporters in brain
• Blood brain barrier
• Transporters in Regulatory Sciences

3. Transport
Movement of drug across
biological membranes or barriers
is called drug transport.

4. Biological Membranes

5.  Bilayer (100 A° thick).
 Made up of phospholipid & cholesterol.
 Polar groups are (phospholipid heads) oriented
at the two surfaces and with nonpolar
(hydrocarbon tails) embedded in the matrix to
form a continuous sheet.
 This makes it electrically resistant and relatively
impermeable.
 Their specific lipid and protein composition
differs according to cell/organelle type.

6.  Some intrinsic proteins extend through full
thickness of membrane, surround fine aqueous
pores while others are adsorbed on the surface
have enzymatic or signal transduction
properties.
 Paracellular spaces or channels also exist.
 Biological membranes are highly dynamic
structures.

7. 1. Passive Diffusion
2. Carrier mediated Transport
i) Facilitated Diffusion
ii) Active Transport
a) Primary
b) Secondary  Symport (Cotransport)
 Antiport (Counter-transport)
3. Endocytosis
i) Phagocytosis
ii) Pinocytosis
4. Filtration

8. Passive Diffusion

9. • Transfer process from higher concentration
gradient to lower concentration gradient.
• Its called passive because it doesn’t require
energy.
• Non-ionised drugs can diffuse passively
through the biological barriers at a rate
proportional to their lipid:water partition
coefficient gradient.

10. • For weak electrolytes (partially ionised) drugs,
diffusion would depend on-
1. Degree of ionisation
2. pH of the surrounding environment
3. Lipid:water partition coefficient of their
undissolved form.

11. Influence of pH on weak electrolytes.
Weakly ACIDIC drugs
-- Form Salt with bases
-- Phenytoin, phenobarbitone,
salicylate, penicillin-V
-- Ionize more at alkaline pH
-- Absorbed readily from acidic
environment - Stomach
Weakly BASIC drugs
--Form Salt with acids
-- Atropine, morphine,
amphetamine,
-- Ionize more at acidic pH
-- Absorbed readily from alkaline
environment - Intestine

12. Arelationshipthatexistsbetweendegreeofionizationofa
weakelectrolyteandthepHofsurroundingmediumisgiven
byHenderson-Hasselbalchequation.
Implications of this equation :
1. Stronger acids/ weak bases have a lower pKa value.
2. Weakly acidic drugs – stomach
Weakly basic drugs – intestine
Net absorption from intestine usually exceeds
that from stomach (short transit time and
limited surface area ).

13. 3. At pH = pKa, the drug is 50% ionized and 50%
unionized.
4. For each unit change in pH, there is 10 fold
change in ratio of log
[ionized form/ unionized form] of a drug.
5. Strongly acidic/ basic drugs and quaternary
ammonium compounds remain predominantly
ionized and are poorly absorbed.
6. Basic drugs attain higher concentration
intracellularly. (pH 7.0)

14. 7. Ion Trapping – When a weak electrolyte crosses a
biological membrane to encounter a pH in which it
turns into ionic form and cant diffuse out, e.g when an
unionized form of acidic drugs which crosses gastric
mucosa (pH=2), reverts to the ionized form within
mucosal cells and then only slowly passes to the ECF.
Aspirin (pKa=3.5)-reasonforgastricmucosaldamage.
#Difference in pH across membranes can result in
differential drug distribution.
8. Acidicdrugsionizemoreinalkalineurineandviceversa.
Onceionized,theycannotdiffusebackintotubules,get
excretedfaster.Alkalinization/Acidificationof urine.

15. • Transmembrane proteins embedded in the cell
membrane serve as carriers/transporters.
• Transporters form an intermediate complex
with the substrate.
• Much slower than flux through channels.
• Specific for the substrate/ type of substrate.

16. • Follows saturation kinetics.
• Competitively inhibited by analogues
which utilize the same transporter.
• What is the difference between Channels
and Transporters?
•Types depending on energy use:
--Facilitated diffusion
--Active transport

17. Facilitated diffusion

18.  Down hill transport
 Carrier moves drug along its concentration
gradient
 No energy required, follows Saturation Kinetics
 Drugs →Anti cancer drugs, antiviral drugs,
amino acids in brain, certain vitamins:
riboflavin, thiamine, vit B12

19. • Plot of rate of absorption against drug concentration.
-- linear relationship at a lower range
-- rate of ascent of the curve decreases and eventually
the curve becomes flat.
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8
Drug Concentration
Rate
Of
Absorption

20. • Up hill transport
• Against electrochemical gradient
• Energy dependent - Generated by membrane
ATPase.
• Follows Saturation Kinetics

21. • Drugs : 5-fluorouracil, Choline, Digitalis.
• Blocked by inhibiting cell metabolism or by reducing
ATP levels.
Sodium cyanide or Sodium fluoride or
2,4-dinitrophenol.
• Depending on the source of driving force, it can be
Primary or Secondary

22. Primary active transport :

23. • Direct energy is required
• Carried by ATP Binding Cassette group of
transporters
• Unidirectional flow from cytoplasm to ECF
or organelles.
• E.g anticancer agents, digoxin, anti-retroviral
(protease inhibitors).

24. Secondary active transport :

25. • One ion /solute (x) supplies driving force for
transport of other solute (y)
• Symporters : Na+glucose symporters
• Antiporters : Na+/H+ exchanger

26. Endocytosis:-
• Cellular uptake of exogenous complexes inside
membrane-derived vesicles.
• At the expense of cellular energy.
1)Phagocytosis:-
• Rare process for engulfing relatively larger
molecules.
• E.g. bacteria/ foreign bodies by macrophages
botulinum toxin, allergens

27. 2)Pinocytosis:-
o Fluid uptake – engulfs a fluid/ drug in solution.
o Stages
• Trapping of macromolecular
solutes into invaginations.
• Fusion of membrane to form
vesicle.
• Pinching off of the vesicle and
passing of solute/drug inside
the cell.
o E.g. Insulin, Immunoglobulin in
neonates’ gut, lipid droplets.

28. Filtration:-
o Physical process where passage of drugs occurs through
pores or paracellular spaces.
o Depends on
• 1. Molecular weight / size
• 2. Pressure gradient
• 3. Protein binding – free form
o E.g Glucose, urea, Alcohol

29.  Membrane proteins.
 Control Influx of essential nutrients & ions.
 Efflux of cellular waste, environmental toxins,
drugs and other xenobiotic.
 Regulates bioavailability & distribution of
drugs
 Transport of compounds out of brain across
blood brain barrier

31. • Involved in primary active transporters
• Energy - from ATP hydrolysis
• 7 families (ABCA TO ABCG) consisting of 49
known members.
• Unidirectional flow from cytoplasm to ECF
or organelles.

32. Best recognized transporter :
• P glycoprotein (encoded by ABCB1 also
called MDR1.
• Cystic fibrosis transmembrane
regulators (CFTR) (encoded by ABCC7).

33. • Transporter Name : P-gp MDR1 (ABCB1)
• Tissue Distribution : Liver, Kidney, Intestine,
BBB, BTB.
• Physiological Function : Natural
detoxification system against xenobiotic.
• Inhibitors: Quinidine, Verapamil,
Spironolactone, Clarithromycin & Ritonavir

34. oSubstrates -
• Anticancer drugs: Etoposide, Doxorubicin,
Vincristine
• Ca2+ channel blockers: Diltiazem, Verapamil
• HIV protease inhibitors: Indinavir, Ritonavir
• Antibiotics/Antifungals: Erythromycin,
Ketoconazole
• Hormones: Testosterone, Progesterone
• Immunosuppressant: Cyclosporine,
Tacrolimus
• Others: Digoxin, Quinidine, Fexofenadine,
Loperamide

35. Applied
Tariquidor and Laniquidar are under trial, they
block the efflux of drugs by inhibiting P-gp.
Ivacaftor, a first-in-class drug, was recently
approved for treatment of Cystic Fibrosis, it acts
on CFTR (ABCC7).
It is termed as a potentiator, increases the
probability that the mutated Cl- channel remains
in the opened state.

36.  Facilitate transporters & ion coupled secondary
active transporters.
52 SLC families with 395 transporters present in
human genome.
Many serve as drug targets or in drug
disposition.
Mostly are Facilitative transporters or
sometimes Secondary Active transporters.
 E.g. Serotonin transporter (SERT→SLC6A4)
Dopamine transporter (DAT →SLC6A3)

37. It is involved in both influx and efflux of
substrate.
Polymorphism with –SLC30A8 causes type 1 DM
Polymorphism with –SLC22A4 & SLC22A5 causes IBD

38. • Asymmetrical transport across monolayer of polarized
cells.
• Important in transfer of solute across the epithelial &
endothelial barriers.
• ABC transporters → unidirectional efflux.
• SLC transporters → either drug uptake or efflux.

39. • Intestine → absorption of nutrients & bile acids.
• Liver → hepatobiliary transport.
• Kidney → tubular secretion.
• Brain → barrier functions.
capillaries
• Different examples illustrate the importance of
vectorial transport in determining the drug
exposure in circulating blood & liver.
1. HMG COA reductase inhibitors
2. ACE inhibitors
3. Irinotecan

40. Membrane transporters in
pharmacological pathways

41. Intestinal transporter

42. • Influx transporters: improve absorption
eg. --PEPT-1 ( peptide like transporters),
--OATP1 (organic anionic transporting
polypeptide)
# PEPT1 mediates transport of drugs :
B-lactam, ACE Inhibitors

43. • Efflux transporters: limit absorption of
drugs.
• E.g --P glycoprotein
--BCRP9 ( breast cancer resistance
protein)
--MRP2 (ABCC1)

44. E.g. --P-gp → substrate - Loperamide
inhibitor - Quinidine
--BCRP → substrate - Methotraxate
inhibitor - Omeprazole
Clinical applied aspect

45. Hepatic Transporters

46. SLC transporters:
Basolateral membrane of hepatocyte.
Uptake of organic anions (drugs, billirubin),
cations & bile salts.
OATPs → anions.
OCTs (organic cation transport protein) &
NTCP → cation & bile salt

47.  Present in bile canalicular membrane of
hepatocyte.
 MRP2, MDR1, BCRP, BSEP & MDR2.
 Mediate efflux of drugs & their metabolites, bile
salts & phospholipids→ Against concentration
gradient from liver to bile.

48. HMG COA reductase inhibitors
• Inhibit cholesterol biosynthesis.
• Statins :- Parvastatin
Fluvastatin
Atorvastatin
• OATP1B1 →uptake
• MRP2 → efflux
• Gemfibrosil inhibits OATP1B1
and increases its concentration
in systemic circulation leading
to toxicity
• Genitic polymorphism also
affectstheactivityof thistransporter.
Gemfibrosil

49. Irinotecan Irinotecan
↓
Active metabolite SN 38
↓
excreted in bile by MRP2
↓
Diarrhea
↓
+ Probenecid → inhibit
MRP2
↓
↓ diarrhoea
- Anticancer drug
-GI effect→diarrhoea

50. Bosentan
 Bosentan is taken up in the liver by OATP1B1
and OATP1B3 and subsequently metabolized
by CYP2C9 and CYP3A4.
 Transporter-mediated hepatic uptake can be a
determinant of elimination of bosentan.
 Inhibition of its hepatic uptake by
cyclosporine, rifampicin, and sildenafil can
affect its pharmacokinetics.

51. • Renal transporters play an important role in drug
elimination ,toxicity and response.
• For the transepithelial flux of a compound, the
compound must traverse two membranes
sequentially, the basolateral membrane facing
the blood side and the apical membrane facing
the tubular lumen.
• Of the two steps involved in secretory transport,
transport across the luminal membrane appears
to be rate-limiting.

52.  Cations secreted in proximal tubules
 Cations →endogenous compounds e.g. choline &
dopamine.
 Primary function for secretion →eliminate body
xenobiotic, positively charged drugs & their
metabolites.
 E.g. Cimetidine
Ranitidine
Metformin
Procainamide

53. (Rate Limiting)
Organic Cation Transport

54.  Primary function → removal of body xenobiotics
including weakly acidic drug e.g. parvastatin,
captopril, & penicillins.
 Two primary transporters on basolateral membrane
→ OAT1 & OAT3 →Flux organic anions from
intestinal fluid to tubular cells.
 OAT4 luminal membrane transporter

55. Organic Anion Transport

56. Pharmacological Relevance.
oDrugs showing their action via:
• Thiazide diuretics act at
SLC12A3 transporter,
on Na+Cl- symporter.
• Loop diuretics act at
SLC12A1 transporter, on
Na+K+2Cl- symporter.
• K+ sparing diuretics act at
SCNN1 transporter,
on ENaC.

57. • Recent studies have suggested that genetic
variations of OCT1 and OCT2 are associated
with alteration in renal elimination and
response to Metformin.
• Polymorphisms in ABCG2 have been
associated with reduced response to
Allopurinol and Oxypurinol.

58. Transporters in Brain

59. • Involved in neuronal reuptake of neurotransmitters
→ SLC1 & SLC6 transporters
• SLC6 responsible for reuptake of :-
1. Norepinephrine transporters (NET/SLC6A2)
2. Dopamine transporters (DAT/SLC6A3)
3. Serotonin transporters (SERT/SLC6A4)
4. GABA transporters (GAT)

60. Act as pharmacological targets for
neuropsychiatric drug.
SLC6 regulate concentration of
neurotransmitter in synaptic cells
o Function in reversible direction
o Depend on Na+ gradient to transport
their substrate

61. GAT
GAT1 → GABA transporter present on
presynaptic neuron
GAT3 → on glial cells
GAT2 → Absent on presynaptic neuron
present on choroid plexus
primary role to maintain homeostasis
of GABA in CSF
Drug target

62. SLC6A2/ NET
 On CNS, PNS &
adrenal chromaffin
tissue
 Limit synaptic
dwell time of
noradrenaline &
limits its action
 Regulation of
memory & mood
SLC6A3/DAT
 Mainly on
presynaptic
neurons
 Reuptake of
dopamine &
terminate
dopamine action
 Regulation of
behavior, mood,
reward, cognition
SLC6A4/ SERT
 On CNS, PNS &
axonal membrane
 Reuptake &
clearance of
serotonin in brain

63. • SLC6A1 /GAT1 → Tiagabine (Anti-epileptic))
• SLC6A2/NET → Desipramine (TCA)
• SLC6A3/DAT → Cocaine & Amphetamine
• SLC6A4/SERT → Fluoxetine & Paroxetine

64.  Drugs acting on CNS have to cross BBB.
 Functionally, the BBB is
o (1) partly physical,
o (2) partly a consequence of selective permeability
o (3) partly a consequence of the enzymatic destruction of
certain permeates by enzymes in the barrier.
 In this efflux transporters play role.
 P-glycoprotein extrudes its substrate drugs on luminal
membrane of brain capillary endothelial cells into
blood.
 Limiting brain penetration

65. The U.S. FDA has issued a draft clinical pharmacology
guidance on performing drug-drug interaction studies
during clinical drug development (FDA, 2012). 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.
Although only a handful of transporters (OATP1B1,
OATP1B3, P-gp, BCRP, OCT2, MATE1, OAT1, and
OAT3) are included in the FDA guidance, more might
be included in the list which cause drug-drug
interactions

66.  Goodman L. S, Goodman & Gilman’s
Pharmacological Basis of Therapeutics, 13th
Edition, New York; New Delhi, TataMcGraw-Hill
Education, 2017.
 Katzung B. G, Basic and Clinical Pharmacology,
14th Edition, New York; New Delhi, TataMcGraw-
Hill Education, 2017.
 Tripathi K.D, Essentials of Medical Pharmacology,
7th Edition, New Delhi, Jaypee Brothers
Publications, 2013.
 Sharma H. L, Principles of Pharmacology, 3rd
Edition, New Delhi, Paras Medical Publisher, 2018

67. Thank you!
