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Drug transport across cell membrane.

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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.

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Drug transport across cell membrane.

  1. 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. 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. 3. Transport Movement of drug across biological membranes or barriers is called drug transport.
  4. 4. Biological Membranes
  5. 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. 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. 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. 8. Passive Diffusion
  9. 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. 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. 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. 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. 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. 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. 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. 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. 17. Facilitated diffusion
  18. 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. 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. 20. • Up hill transport • Against electrochemical gradient • Energy dependent - Generated by membrane ATPase. • Follows Saturation Kinetics
  21. 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. 22. Primary active transport :
  23. 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. 24. Secondary active transport :
  25. 25. • One ion /solute (x) supplies driving force for transport of other solute (y) • Symporters : Na+glucose symporters • Antiporters : Na+/H+ exchanger
  26. 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. 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. 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. 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
  30. 30. • 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.
  31. 31. Best recognized transporter : • P glycoprotein (encoded by ABCB1 also called MDR1. • Cystic fibrosis transmembrane regulators (CFTR) (encoded by ABCC7).
  32. 32. • 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
  33. 33. 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
  34. 34. 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.
  35. 35.  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)
  36. 36. It is involved in both influx and efflux of substrate. Polymorphism with –SLC30A8 causes type 1 DM Polymorphism with –SLC22A4 & SLC22A5 causes IBD
  37. 37. • 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.
  38. 38. • 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
  39. 39. Membrane transporters in pharmacological pathways
  40. 40. Intestinal transporter
  41. 41. • Influx transporters: improve absorption eg. --PEPT-1 ( peptide like transporters), --OATP1 (organic anionic transporting polypeptide) # PEPT1 mediates transport of drugs : B-lactam, ACE Inhibitors
  42. 42. • Efflux transporters: limit absorption of drugs. • E.g --P glycoprotein --BCRP9 ( breast cancer resistance protein) --MRP2 (ABCC1)
  43. 43. E.g. --P-gp → substrate - Loperamide inhibitor - Quinidine --BCRP → substrate - Methotraxate inhibitor - Omeprazole Clinical applied aspect
  44. 44. Hepatic Transporters
  45. 45. 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
  46. 46.  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.
  47. 47. 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
  48. 48. Irinotecan Irinotecan ↓ Active metabolite SN 38 ↓ excreted in bile by MRP2 ↓ Diarrhea ↓ + Probenecid → inhibit MRP2 ↓ ↓ diarrhoea - Anticancer drug -GI effect→diarrhoea
  49. 49. 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.
  50. 50. • 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.
  51. 51.  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
  52. 52. (Rate Limiting) Organic Cation Transport
  53. 53.  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
  54. 54. Organic Anion Transport
  55. 55. 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.
  56. 56. • 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.
  57. 57. Transporters in Brain
  58. 58. • 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)
  59. 59. 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
  60. 60. 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
  61. 61. 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
  62. 62. • SLC6A1 /GAT1 → Tiagabine (Anti-epileptic)) • SLC6A2/NET → Desipramine (TCA) • SLC6A3/DAT → Cocaine & Amphetamine • SLC6A4/SERT → Fluoxetine & Paroxetine
  63. 63.  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
  64. 64. 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
  65. 65.  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
  66. 66. Thank you! 

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