Basic Principles of Pharmacology - kinetics Block 2.3 (2018-19)

1. Basic Principles of Pharmacology - kineticsBlock 2.3 (2018-19) Prof Asghar Mehdi MBBS, DTCD, MCPS, Ph.D Department Of Pharmacology Sulaiman Alrajhi Colleges

2. Objectives • Drug transport. • Membrane types / barriers. • Permeability / movement across membranes – options. Factors affecting movements across membranes.** Ionization & pH – Ion trapping.** Protein binding & Others

3. Pharmacokinetic principlesAqueous diffusion – Drug transport. • The body is a series of interconnected compartments within each drug concentration is homogenous. • Dependent upon its route of administration and target area, every drug has to be absorbed, by diffusion, through a variety of tissue. • Movement between compartments involves penetration of non aqueous diffusion barriers that determines for how long an administered drug will remain in body.

4. Diffusion • Molecule by molecule – short distances. • Its ability to cross hydrophobic diffusion barriers – influenced by lipid solubility. • Bulk Flow • Via blood stream, lymphatics OR CSF. • CVS is the main source. • Unaffected with chemical nature of drug. Physical Barriers to Absorption

5. Membranes • Walls of capillaries: pores between the cells are larger than most drug molecules, allowing them to pass freely, without lipid solubility being a factor. • Cell membranes: this barrier is permeable to many drug molecules Depends on - lipid solubility & size of drug molecule.Small pores, 8 angstroms, permit small molecules such as alcohol and water to pass through.

6. Cell membranes:Barrier between aqueous compartments In the body • Single layer of cells separate intracellular from extra cellular compartments. • An epithelial barrier- GI mucosa / renal tubule – cells tightly interconnected – a molecule needs to traverse two cell membranes – inner & outer to pass from one side to other. • Vascular endothelium differs with anatomical disposition and permeability. Gaps between endothelial cells are packed with loose matrix of protein which acts as filters retaining large molecules. Department of Pharmacology

7. Cell membranes:Barrier between aqueous compartments In the body • Special areas such as PLACENTA AND BRAIN where junctions are tight & encased in impermeable layer of pericytes that prevents harmful things to traverse. • LIVER & SPLEEN endothelium is discontinuous allowing free passage b/w cells. • Fenestrated epithelium occurs in ENDOCRINE GLAND facilitating transfer of hormones to blood stream. • Endothelial lining of POST CAPILLARY VENULE allows migration of leucocytes without detectable leak of water or small ions.

8. Pharmacokinetic Principles • Drug should be able to reach its intended site of action after administration • Active drug molecule is sufficiently lipid soluble. • Inactive precursor prodrug chemical that is readily absorbed and distributed must be converted to active drug by biologic processes ( liver, GIT, any transport machinsm) • Drug delivery:1. direct application (IV, eye drop, inhlation)2. administration in one compartment like gut – absorption – distribution - permeation – elimination.

9. Mechanisms of permeation Fick’s Law “amount of a substance crossing a given area is proportional to the spatial gradient of concentration and the diffusion constant (D), that is related to molecular size and shape”. From: The Dictionary of Cell & Molecular Biology (Fifth Edition), 2013 • Aqueous diffusion: movement of molecules through watery intra- & extracellular spaces - governed by Fick's law. • Lipid diffusion: movement of molecules through membranes & other lipids - governed by Fick's law. • Transport by special carrier: Has limited capacity - transport ions, neurotransmitters, metabolites, and xenobiotics. • Solute carrier (SLC) transporters: passive & down the electrochemical gradient. • Organic anion transporters (OATs) • Organic cation transporters (OCTs) • ATP-binding cassette (ABC) transporters:

11. Simple diffusion Driving force is concentration gradient & energy independent. Drug transfer directly proportional to conc gradient across membrane. Down hill transport occur. down the gradient equilibrium exchange of solute

12. Aqueous diffusion • Aqueous diffusion occurs within larger aqueous compartments of body (interstitial space) and across Epithelial membrane tight junctions, Endothelial lining of blood vessels - through aqueous pores — permit passage of molecules as large as MW 20,000–30,000. • Drug molecules that are bound to large plasma proteins (albumin) do not permeate most vascular aqueous pores. If the drug is charged, its flux is also influenced by electrical fields (membrane potential)

13. Lipid diffusion • Lipid barriers that separate compartments of body are important limiting factor for drug permeation. • As lipid barriers separate aqueous compartments, Lipid : Aqueous Partition Coefficient of a drug determines how readily molecule moves between aqueous and lipid media. • In the case of weak acids and weak bases (which gain or lose electrical charge-bearing protons, depending on the pH), the ability to move from aqueous to lipid or vice versa varies with the pH of medium, because charged molecules attract water molecules. • The ratio of lipid-soluble form to water-soluble form for a weak acid or weak base is expressed by the Henderson-Hasselbalch equation

14. carriers • Special carrier molecules exist for many substances that are important for cell function and too large or too insoluble in lipid to diffuse passively through membranes - peptides, aminoacids, and glucose. • These carriers bring about movement by Active transport or Facilitated diffusionand, unlike passive diffusion, are selective & saturable. • Because many drugs resemble naturally occurring peptides amino acids, or sugars, they can use these carriers to cross membranes. Carrier proteins used in active transport include: • Uniporters – move one molecule at a time • Symporters – move two molecules in same direction • Antiporters – move two molecules in opposite directions

15. Facilitated Diffusion • Down the concentration gradient • Through carrier proteins • Rate depends upon the amount of carriers available to which drug can bind Example ~ fluorouracil

16. ACTIVE TRANSPORT Cell uses energy Actively moves molecules to where they are needed Movement from an area of low concentration to an area of high conc (low  high) Carrier proteins, ion channels are required L-dopa example aquAporin’s • This is important for gases such as CO2 but pores are too small 0.04 nm to allow most drug molecules which are mostly more then 1.0 nm.

17. PINOCYTOSIS • Endocytosis Binding of molecule to receptors → in-folding of membrane → internalization → release into the cell. • Macromolecules enter on their own. • Polar substances like Vitamin B12 & iron combine with special proteins intrinsic factor & transferrin, respectively. • Exocytosis Expulsion of membrane-encapsulated things. • Done by most neurotransmitters.

18. Carrier mediated transport.Solute transporters (SLC) • It involves binding of a carrier molecule which is transmembrane protein. • It mediates passive movement of solutes down electro chemical gradient. • OCT – organic cation transporters – OCT1 in liverOCT2 in kidneys • OAT – organic anion transporters. • Transmembrane protein binds to one or more molecules or ions, changes conformation and releases them on the other side of membrane. • Unipolar – facilitated diffusion – without energy source – facilitate transmembrane equilibrium in direction of electrochemical gradient. • Carrier mediated transport involves binding step – hence saturation phenomenon Department of Pharmacology

19. Solute carrier transporters (SLC) • OCT1transports metformin, into hepatocytes. Single nucleotide polymorphisms (SNPs) that impair the function of OCT1 influence effectiveness of metformin. • OCT2concentrates drugs such as cisplatin (anticancer drug) in proximal renal tubules, resulting in its selective nephrotoxicity. • Other drugs in the group like carboplatin, oxaliplatin are not transported by oct2 and are less nephrotoxic. • Competition with cimetidine for OCT2offers possible protection against cisplatin nephrotoxicity. Department of Pharmacology

20. Carrier mediated transportATP-binding cassette (ABC) transporters • These are active pumps fueled by ATP - against the electrochemical gradient. • P-glycoprotein belongs to ABC transporter. • Present in renal tubular brush border membranes, bile canaliculi, GIT. • Often collated with SLC, such as drug that has been concentrated by OAT in renal tubular cells may be pumped out by P-gp in luminal membrane. • These are responsible for multidrug resistance in cancer cells. • Polymorphic variation in genes coding SLC’s and P-gp contributes to individual genetic variation in responsiveness to different drugs.

21. Pharmacokinetic principles Tissue is composed of cells which are encompassed withinmembranes, consisting of 3 layers, 2 layers of water-soluble complex lipid molecules (phospholipid) & layer of liquid lipid, sandwiched within these layers. Within layers are large proteins, such as receptors, trans versing all 3 layers. • The permeability of a cell membrane, for a specific drug, depends on a ratio of its water to lipid solubility. • Within the body, drugs may exist as a mixture of two interchangeable forms, either water (ionized-charged) or lipid (non-ionized) soluble. • The concentration of two forms depends on Pka,(pH at which 50% of the drug is ionized) and the pHof fluid in which it is dissolved. Department of Pharmacology

22. Diffusion through Lipid Non ionic molecules dissolve freely in membrane lipids and cross readily. Number of molecules crossing the membrane per unit area in unit time is determined by Permeability Coefficient p Conc diff across membrane Diffusion coefficient varies slightly between drugs, hence a close correlation between lipid solubility and permeability of cell membrane. Since molwt of drug is small – (200-1000 Da) & its square root is even negligible, hence diffusion coefficient is mainly dependent on molecular size large molecules diffuse more slowly then small ones

23. Permeation The movement of drug molecules into & within the biologic environment. It follows the fick's law. • What is fick’s law? Rate = (C1-C2) x area / thickness x permeability coefficient“drug molecules diffuse from a region of higher conc to lower conc, until equilibrium is attained - Sink condition Why do we have it? To predict rate of movement of molecules across a barrier. So, permeability is affected by: (1) Solubility (2) Concentration gradient (3) Surface area & vascularity

24. Solubility CHARGED (by ionization or polarity) → aqueous solubility UNCHARGED → lipid solubility Weak acids & bases can be ionized or non-ionized according to their pKa (pH at which 50% are ionized & 50% are non-ionized) and surrounding Ph. Does Henderson-Hasselbalch equation come to mind? Log(protonated/un-protonated) = pKa – pH

25. What does means for weak acids & bases if pH is belowpKa→ Protonated > unprotonated if pH is above pKa→Unprotonated < protonated ACIDS Protonated form of weak acid & un-protonated form of weak base is neutral BASES

26. Ionization & clearance Both the ionized & non-ionized are filtered as long as they're free & unbound. Difference is: • The non-ionized is actively secreted, & actively or passively reabsorbed. • The ionized is trapped in filtrate - actively secreted & NOT reabsorbed • Excretion: ionized > nonionized • Acidification of urine (by NH4Cl or vitamin C) → ionization of weak bases → excretion • Alkalinization of urine (NaHCO3)→ ionization of weak acids → excretion • Alkalinization of blood → ionization of weak acids → leaving the CNS to plasma

27. Effect of pH and ionization. • Most drugs are weak bases or weak acids and exists in both unionized and ionized forms – ratio depending on the Ph • As most drugs are weak acids or bases, only uncharged species can diffuse across lipid membranes- hence Ph partition. • An acidic drug being concentrated in compartment with high Ph & vice versa. • Concentration gradient thus produced is ion trapping.

28. ION TRAPPING A weak acid (e.g. aspirin , p K a 3.5) and a weak base (e.g. pethidine , p Ka8.6) would be distributed at equilibrium between three body compartments, namely plasma (pH 7.4), alkaline urine (pH 8) and gastric juice (pH 3). Within each compartment, the ratio of ionised to un-ionised drug is governed by the p K a of the drug and the pH of that compartment. It is assumed that the un-ionised species can cross the membrane, and therefore reaches an equal concentration in each compartment. The ionised species is assumed not to cross at all. The result is that, at equilibrium, the total (ionised + un-ionised) concentration of the drug will be different in each compartment, with an acidic drug being concentrated in the compartment with high pH (‘ion trapping’), and vice versa. It moves towers left in acid environment and to right in basic environment. Department of Pharmacology

29. Ion Trapping: • Kidney: Nearly all drugs filtered at glomerulus. Most drugs in a lipid-soluble form are reabsorbed by passive diffusion. • To increase excretion: change the urinary pH to favor the charged form of the drug: • Weak acids: excreted faster in alkaline pH Weak bases: excreted faster in acidic pH Other sites: stomach contents small intestine breast milk aqueous humor (eye) vaginal secretions prostatic secretions

30. Binding of drugs to Plasma Proteins • Albumin being the most important plasma protein which binds with drugs. This binding is dependent on • Concentration of free drug. • Affinity for binding sites. • Concentration of proteins. • A & B are two drugs which compete for binding to albumin, increasing the drug A will render more of free drug B • In plasma many drugs exists in bound form upto 99%. • The fraction of free drug (pharmacologically active) can be less than 1%. • However small difference in plasma binding may have significant effect on free drug concentration and drug effect. • Such difference in binding of plasma proteins varies significantly between humans and animals. • Extensive protein binding slows drug elimination.

31. Partition of body into fat & other tissues. • Fat presents a large nonpolar compartment. However only few drugs are affected because the fat-water partition coefficient is relatively low. • Morphine can cross the blood brain barrier because it is lipid soluble but the fat-water partition coefficient is 0.4 - LOW, does not accumulate in body fats. • Thiopental has fat-water coefficient of 10 (high), hence accumulates substantially in body fat and therefore its use is limited for induction of anesthesia. • Blood supply to fats is low – less than 2% of cardiac output. Hence drugs are delivered to body fat slowly.

32. Lipid-Water Partition Coefficient • The ratio of concentration of the drug in two immiscible phases: nonpolar liquid or organic solvent (representing the membrane);aqueous buffer, pH 7.4 (representing the plasma) • Higher lipid / water partition coefficient Greaterthe rate of transfer across membrane Polarity of a drug, by increasing ionization will the lipid/ water partition coefficient Polarity of a drug, decreasing ionization will the lipid/ water partition coefficient

33. Factors Affecting Absorption pH Lipid solubility Weak acids are absorbed in acidic medium where it remains in unionized form weak bases are absorbed in basic medium where it remains in unionized form Lipid-soluble drugs are absorbed more rapidly Water-soluble drugs are absorbed rarely

34. Factors Affecting Absorption Molecular size Ionization smaller the molecular size, faster will be the absorption Example: • Corticosteroids • Aspirin • unionized species absorbed readily • ionized species absorbed slowly

35. References • Basic & clinical pharmacology by Katzung: 13th edition. • Rang & Dale Pharmacology: 8th edition. • Pharmacology by Gollan: 3rd edition. • Goodman and Gilman’s the Pharmacological Basis of Therapeutics 9th ed., McGraw-Hill.