Drug Absorption: Insights into Cell Membranes and Epithelial Barriers PDF

Drug Absorption: Insights into Cell Membranes and Epithelial Barriers Prof. Mohammad Issa Saleh 1 Pharmaceutical Perspectives on Cell Membranes 2 Introduction All living things are made of cells A typical cell consi...

Drug Absorption: Insights into Cell Membranes and Epithelial Barriers Prof. Mohammad Issa Saleh 1 Pharmaceutical Perspectives on Cell Membranes 2 Introduction All living things are made of cells A typical cell consists largely of cytoplasm, an aqueous liquid in which a wide range of biochemical processes occur The cytoplasm is held as an intact unit by a cell membrane, which surrounds it and prevents it from mixing with its surroundings 3 Introduction Depending on the cell type and function, a number of other structures may be present, particularly a nucleus, in which the cell genetic information is stored in the form of DNA The outer membrane of the cell must allow penetration of some substances and not others, i.e. it must be selectively permeable 4 Introduction Organs and tissues consist of cells surrounded by epithelia, functioning as the organ's 'outer membrane' similar to a cell's membrane These epithelia not only enclose the organ but also facilitate transport, barrier, and secretory processes, varying by organ Many epithelia protect organs from hostile environments, like skin or stomach lining, and often have rapid cell turnover and strong barrier properties. 5 Introduction To reach its site of action, a drug must move from an external site (e.g., skin surface or small intestine) to an internal site (e.g., bloodstream or cell cytoplasm) This involves passing through various tissues and epithelia, either by penetrating cell membranes or finding pathways between cells Overcoming these barriers is crucial in drug delivery and requires detailed knowledge of cell membrane and epithelial tissue structures and behavior. 6 The plasma membrane The plasma membrane retains cell contents and acts as a selective barrier, controlling substance entry and exit It is a highly organized structure with proteins that serve as structural elements, transport nutrients, and monitor the cell environment The bilayer, a carefully designed liquid crystal, is regulated by the cell to maintain specific fluidity and an optimal environment for internal processes. 7 The plasma membrane The phospholipid bilayer Dynamic behaviour of membranes Modulation of membrane fluidity by sterols Models of cell membranes Membrane proteins Membrane asymmetry 8 The phospholipid bilayer The detailed chemistry of the cell membrane was long unclear due to its many components from various organs We now know the bilayer's main scaffolding consists of surfactant molecules, mainly phospholipids Most membranes also contain proteins and sterols, but surfactant lipids alone can form the lipid bilayer. 9 The phospholipid bilayer Phospholipids are glycerol compounds with two fatty acid tails and a phosphate group linked to a small hydrophilic molecule These molecules, with tails of 12-24 carbon atoms and a hydrophilic head, include choline, ethanolamine, serine, and inositol 10 The phospholipid bilayer In water, surfactants like phospholipids form bilayer sheets with hydrophobic tails inward and polar headgroups outward, creating the most stable configuration These bilayers typically form spherical structures called liposomes when shaken in water. 11 The phospholipid bilayer 12 The phospholipid bilayer Common headgroup molecules are choline, ethanolamine, serine and inositol, and the resulting phospholipids are termed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol respectively 13 Dynamic behavior of membranes Although membranes are often depicted as regular structures, the reality is that the bilayer is much more disordered and dynamic. The most important dynamic processes are: Lateral diffusion, in which the lipid molecules can move in the plane of the bilayer Transverse diffusion or flip-flop, where a lipid molecule switches from one side of the membrane to the other 14 Dynamic behavior of membranes Lateral diffusion Transverse diffusion (Flip-Flop) 15 Dynamic behavior of membranes Since transverse diffusion involves moving the headgroup through the oily core of the bilayer, this is an extremely slow process, and in natural systems is generally catalyzed when required by specialized membrane proteins The most important factor in determining the dynamic behavior of the membrane is the transition temperature of the bilayer At low temperatures the lipid tails are held in a relatively ordered array in the bilayer core As the temperature is raised, little movement takes place until the transition temperature is reached 16 Dynamic behavior of membranes At transition temperature the lipid spacing increases slightly and the tails become much more disordered in arrangement The transition is often thought of as a gel-liquid melting of the bilayer, and in the fluid state the lipid molecules are relatively mobile to lateral diffusion; they diffuse at a speed of several microns a second and can move around a typical cell membrane on a timescale of seconds The transition temperature depends mainly on the structure of the fatty acid chains attached to the glycerol backbone, with unsaturated chains causing low transition temperatures (generally below 0°C) and saturated chains having higher transition temperatures. 17 Plasma membrane lipids phases Lipids in liquid crystalline lamellar systems exist in two phases: solid (gel) and liquid (fluid or liquid-disordered) In the gel phase, acyl chains are immobile and ordered, while in the liquid phase, acyl chains and lipid molecules are highly mobile Transitions between these phases are crucial in membrane biology. The transition temperature is critical, as it can be close to room temperature, making membranes sensitive to slight temperature changes or lipid composition modifications. 18 Plasma membrane lipids phases Van der Waals interactions (hydrophobic attraction) between lipid acyl chains in liquid crystalline lamellae primarily determine lipid phases and organization These interactions also induce the assembly of various lipid lamellar phases 19 Plasma membrane lipids phases The gel phase (solid-ordered, So phase, or Lβ phase) features condensed, ordered acyl chains but is not crystalline The main transition in lipid lamellae is between the S0 phase and the liquid-disordered phase (Ld or Lα phase) 20 Plasma membrane lipids phases Additionally, there are two other phases: the ripple phase, an intermediate with curved bilayers the liquid-ordered phase (Lo phase) 21 Factors Affecting Lipid Phase Transitions: Unsaturation The fatty acid chains' structure (length and saturation) influences phase formation and transition temperatures between fluid and rigid phases Saturation leads to straight acyl chains, allowing closer lipid proximity and more rigid phases. Unsaturated chains, with double- bond kinks, result in a more open organization and greater bilayer fluidity 22 Factors Affecting Lipid Phase Transitions: Chain Length Longer fatty acid chains have higher surface areas than smaller ones, resulting in stronger Van der Waals interactions between long lipid chains This leads to increasing Transition temperature with increasing chain length, 23 Factors Affecting Lipid Phase Transitions: Sterols Sterols, such as cholesterol, play a large role in modulating the fluidity of membranes Sterols are small, rigid molecules that are largely hydrophobic, with the exception of a single hydroxyl group Most cell membranes contain sterols: sitosterol in plants, cholesterol in animals, and ergosterol in fungi, among others. 24 Factors Affecting Lipid Phase Transitions: Sterols Cholesterol is a major lipid in animal cell membranes Unlike phospholipids, it cannot form bilayers on its own but inserts into phospholipid bilayers with its steroid core in the hydrophobic acyl chain region and hydroxyl groups near the membrane interface. 25 Factors Affecting Lipid Phase Transitions: Sterols Cholesterol significantly impacts bilayer fluidity; higher cholesterol levels reduce fluidity, while lower levels increase it. This effect is due to van der Waals interactions between cholesterol's hydrophobic steroid rings and phospholipid acyl chains, reducing their mobility and increasing bilayer order 26 Factors Affecting Lipid Phase Transitions: Sterols Higher cholesterol concentrations transform liquid- disordered bilayers into a more rigid, liquid-ordered phase. However, in gel-phase lipid systems, cholesterol can increase fluidity. 27 Effect of sterols on transition temperature Sterols alter the fluidity of the cell membrane by ‘broadening’ the melting transition so that the membrane melts over a much wider temperature range than that observed for the lipid alone In the absence of sterols the bilayer melts over a small temperature range, causing a sharp peak in the thermogram 28 Effect of sterols on transition temperature In the presence of cholesterol the melting transition is much broader, and the thermogram peak spans several degrees 29 Membrane proteins While lipids primarily determine membrane structure and dynamics, membrane proteins, comprising up to 50% of membrane mass, play crucial roles in most biological processes 30 Membrane proteins Membrane-associated proteins are divided into peripheral and integral proteins Peripheral proteins do not penetrate deeply into the lipid bilayer and have a loose association Integral proteins are strongly anchored within the bilayer's core, often spanning the membrane multiple times and exposing functional domains on one or both sides. 31 Membrane proteins One of the most important groups of integral membrane proteins from a pharmacological viewpoint is the transport proteins These are responsible for moving substances into and out of the cell; for example, ATPase proteins pump ions across the cell membranes to maintain the required Na+/K+ electrolyte imbalance, and secrete H+ from the gastric parietal cells 32 Membrane proteins Proteins also recognize and transport nutrients such as small carbohydrates and amino acids into the cell, each protein transporting a small group of structurally similar compounds A second important group of membrane proteins are the cell surface receptors Glycoproteins are a group of integral proteins carrying polysaccharide chains which are responsible for cell recognition and immunological behavior 33 Membrane asymmetry Although liposomes are normally made with a similar lipid composition on both the inside and outside, living cells are much more asymmetric since they perform a range of processes which are obviously directional The phospholipid composition of the inside and outside layers of the membrane is different in most cells; for example in the erythrocyte membrane phosphatidylcholine occurs predominantly on the outside of the cell and phosphatidylethanolamine predominantly on the inside Glycolipids are normally oriented so that the polysaccharide segment is outside the cell, since it is responsible for immunogenicity and tissue adhesion. Integral proteins always have a specific orientation in the membrane that depends on their function 34 Epithelial Tissue 35 Epithelial Tissue Epithelial tissues are large sheets of cells covering body surfaces and lining organs exposed to the outside world, as well as forming much of the glandular tissue This includes the skin, airways, digestive tract, and urinary and reproductive systems Hollow organs and body cavities not exposed to the exterior, like blood vessels and serous membranes, are lined by endothelium, a type of epithelium 36 Epithelial Tissue All epithelia are highly cellular with little extracellular material and form specialized cell junctions. They exhibit polarity with structural and functional differences between the apical (exposed) and basal (underlying) surfaces The basal lamina, composed of glycoproteins and collagen, attaches to the reticular lamina from connective tissue, forming the basement membrane. 37 Generalized Functions of Epithelial Tissue Epithelial tissues protect the body from physical, chemical, and biological damage Acting as gatekeepers, they control permeability and allow selective transfer of materials All substances entering the body must cross an epithelium Some epithelia have features for selective transport of molecules and ions 38 Generalized Functions of Epithelial Tissue Many epithelial cells secrete mucus and specific chemicals For example, the small intestine releases digestive enzymes, and respiratory tract cells secrete mucus to trap microorganisms and particles Glandular epithelium contains many secretory cells 39 Classification of Epithelial Tissues Epithelial tissues are classified by cell shape and number of layers Cell shapes are squamous (flattened), cuboidal (boxy), or columnar (tall) Simple epithelium has one cell layer where all cells rest on the basal lamina, while stratified epithelium has multiple layers with only the basal layer on the lamina Pseudostratified epithelium has a single layer of irregularly shaped cells that appear layered Transitional epithelium is a specialized stratified type with variable cell shapes 40 Simple squamous epithelium Simple squamous epithelium consists of thin, scale-like cells( ‫مثل‬ ‫ )الحراشف‬with flat, elliptical nuclei The endothelium lines lymphatic and cardiovascular vessels with a single layer of squamous cells, facilitating rapid chemical passage This epithelium is found in lung alveoli, kidney tubules, and capillary linings 41 Simple cuboidal epithelium In simple cuboidal epithelium, the nucleus of the box-like cells appears round and is generally located near the center of the cell These epithelia are active in the secretion and absorptions of molecules Simple cuboidal epithelia are observed in the lining of the kidney tubules and in the ducts of glands 42 Simple columnar epithelium, Like the cuboidal epithelia, this epithelium is active in the absorption and secretion of molecules Simple columnar epithelium forms the lining of some sections of the digestive system (stomach and small intestine) and parts of the female reproductive tract 43 Simple columnar epithelium, Ciliated columnar epithelium is composed of simple columnar epithelial cells with cilia on their apical surfaces These epithelial cells are found in the lining of the fallopian tubes and parts of the respiratory system, where the beating of the cilia helps remove particulate matter 44 Pseudostratified columnar epithelium Pseudostratified columnar epithelium is a type of epithelium that appears to be stratified but instead consists of a single layer of irregularly shaped and differently sized columnar cells Pseudostratified columnar epithelium is found in the respiratory tract, where some of these cells have cilia 45 Stratified squamous epithelium A stratified epithelium consists of several stacked layers of cells. This epithelium protects against physical and chemical wear and tear The top layer may be covered with dead cells filled with keratin Mammalian skin is an example of this dry, keratinized, stratified squamous epithelium The lining of the mouth cavity is an example of an unkeratinized, stratified squamous epithelium. 46 Transitional epithelium Transitional epithelium, found only in the urinary system (ureters and bladder), changes shape as the bladder fills When empty, the epithelium is convoluted with cuboidal apical cells As the bladder fills, it stretches, and the apical cells transition from cuboidal to squamous This epithelium appears thicker when the bladder is empty and more stretched when full. 47 drug transport across the gastrointestinal epithelium 48 Mechanisms of transport across the gastrointestinal membrane There are two main mechanisms Transport across membrane of drug transport across the gastrointestinal epithelium: Transcellular Paracellular transcellular (across the cells) and paracellular (between the cells). The transcellular pathway Passive is further divided into simple diffusion passive diffusion, carrier- mediated or membrane Membrane transporter processes and transporters transcytosis. Transcytosis 49 Mechanisms of transport across the gastrointestinal membrane 50 Passive diffusion Passive diffusion is the primary transport route for small lipophilic molecules Drug molecules move from a high concentration in the lumen to a lower concentration in the blood The transport rate depends on the drug's physicochemical properties, membrane nature, and concentration gradient 51 Passive diffusion The drug first partitions between gastrointestinal tract fluids and the lipoidal membrane of the epithelial lining It then diffuses across the epithelial cells to the blood in the capillaries Once in the blood, the drug is rapidly distributed, maintaining a lower concentration at the absorption site 52 Passive diffusion Passive diffusion is the process by which molecules spontaneously diffuse from a region of higher concentration to a region of lower concentration This process is passive because no external energy is expended. The rate of transfer is called flux 53 Passive diffusion Passive diffusion is the major absorption process for most drugs The driving force for passive diffusion is higher drug concentrations on the mucosal side compared to the blood Passive diffusion is described using Fick's law of diffusion 54 Fick's law where dQ/dt = rate of diffusion D = diffusion coefficient K = lipid water partition coefficient of drug in the biologic membrane that controls drug permeation A = surface area of membrane h = membrane thickness CGI= the concentrations of drug in the gastrointestinal tract Cp = the concentrations of drug in the plasma. 55 Passive diffusion: Fick’s Law These equations indicate that the rate of gastrointestinal absorption of a drug by passive diffusion depends on the surface area (A) of the membrane that is available for drug absorption. Thus the small intestine, primarily the duodenum, is the major site of drug absorption due to the very large surface area resulting from the presence of villi and microvilli 56 Factors influencing passive diffusion Thickness of the hypothetical model membrane, h, is a constant for any particular absorption site Drugs usually diffuse very rapidly through capillary plasma membranes in the vascular compartments, in contrast to diffusion through plasma membranes of capillaries in the brain 57 Factors influencing passive diffusion In the brain, the capillaries are densely lined with glial cells, so a drug diffuses slowly into the brain as if a thick lipid membrane existed. The term blood–brain barrier is used to describe the poor diffusion of water-soluble molecules across capillary plasma membranes into the brain. However, in certain disease states such as meningitis these membranes may be disrupted or become more permeable to drug diffusion. 58 Factors influencing passive diffusion The degree of lipid solubility of the drug influences the rate of drug absorption. The partition coefficient, K, represents the lipid–water partitioning of a drug across the hypothetical membrane in the mucosa. Drugs that are more lipid soluble have a larger value of K 59  The diffusion coefficient, and hence rate of absorption, is influenced by the molecular Passive diffusion weight of the drug. Small molecules diffuse rapidly and so will cross the membrane more quickly than large, slowly-diffusing molecules 60 Passive diffusion: Sink conditions Once in the blood, the drug is quickly carried away from the absorption site by gastrointestinal circulation It becomes diluted in the systemic circulation, body tissues, other fluids, and undergoes metabolism and excretion Plasma protein binding further lowers the concentration of free drug in the blood 61 Passive diffusion: Sink conditions The blood acts as a 'sink' for the absorbed drug, maintaining a low concentration at the absorption site and a high when CGI>>CP the term concentration gradient across the gastrointestinal membrane during absorption. the previous equation become: 62 Informal HW A: drugs with similar MW (250) and different partition coeffecient B: drugs with similar MW (400) and different partition coeffecient 63 Informal HW Explain the pattern observed in the previous figure (the initial increase in absorption rate as partition coefficient increases followed by a decline in the absorption rate at high partition coefficient values) Explain the difference between series A and B 64 Membrane transporters Most drugs are absorbed transcellularly by passive diffusion However, some compounds and many nutrients use membrane transporters for absorption A carrier or membrane transporter binds a drug and transports it across the membrane (Figure) 65 Membrane transporters This carrier-mediated absorption is like a shuttle process across the epithelial membrane The drug forms a complex with the transporter on the apical cell membrane of the polarized enterocyte (small intestinal cell) This complex crosses the membrane, releases the drug on the other side, and the carrier returns to its original position to transport another drug molecule 66 Membrane transporters Membrane transport is saturable and structurally selective for the drug and shows competition kinetics for drugs of similar structure 67 Membrane transport Membrane transport includes: Active transport Facilitated transport Active transport requires energy to move materials against a concentration gradient, from lower to higher concentration, using ATP hydrolysis or the transmembrane sodium gradient 68 Membrane transport Facilitated transport allows solutes like glucose and amino acids to cross membranes down their electrochemical gradients without energy expenditure, at a faster rate than expected based on molecular size and polarity Unlike active transport, facilitated transport cannot move substances against a concentration gradient. 69 Membrane transporters The small intestine contains over 400 membrane transporters, but only a few are involved in absorption These transporters are located on the apical (brush border) or basolateral membrane of enterocytes. 70 Membrane transporters: uptake and efflux The small intestine membrane transporter are classified as uptake or efflux transporters Intestinal efflux They belong to two main super- families: solute carrier (SLC) for uptake and ATP-binding cassette Intestinal (ABC) for efflux uptake Efflux is about expelling substances from the cell. Uptake is about absorbing substances into the cell 71 SLC transporters Members of the SLC family of transporters facilitate the transport of various substrates, including amino acids, peptides, nucleosides, sugars, bile acids, neurotransmitters, and vitamins These transporters are typically concentrated in specific segments of the gastrointestinal tract, where they preferentially absorb their respective substances For instance, bile acid transporters are found only in the ileum Each transporter has a specific substrate it can carry, though some have broader specificities Drugs resembling natural substances can be transported by the same mechanisms. 72 SLC transporters The SLC superfamily includes key transporters for drug absorption and disposition, such as proton-dependent oligopeptide transporters (PEPT1 and PEPT2), organic anion transporters (OAT), organic cation transporters (OCT), nucleoside transporters, plasma membrane monoamine transporter (PMAT), and monocarboxylate transporters (MCT) 73 SLC transporters Many peptide-like drugs, such as penicillins, cephalosporins, ACE inhibitors, and renin inhibitors, rely on peptide transporters for absorption Nucleoside analogues for antiviral and anticancer drugs depend on nucleoside transporters L-dopa and a-methyldopa are transported by amino acid carriers, with L-dopa having a faster permeation rate due to its higher affinity for the carrier 74 ABC transporters The most studied transporters in the intestine are the ABC family of efflux transporters: P-glycoprotein (P-gp), multidrug-resistance- associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) These are abundant at the apical membrane of enterocytes, limiting the effective intestinal absorption and bioavailability of many drugs, such as statins, antibiotics, HIV protease inhibitors, immunosuppressants, and anticancer drugs ABC transporters use ATP to pump substrates against a concentration gradient 75 Transcytosis Transcytosis is a transcellular transport mechanism where a cell engulfs extracellular material into a vesicle (endocytosis), moves the vesicle across the cell, and ejects the material through the opposite membrane (exocytosis) Endocytosis includes four main processes: clathrin-mediated endocytosis, macropinocytosis, caveolin-mediated endocytosis, and phagocytosis. 76 Transcytosis Transcytosis is generally not significant for the oral absorption of drugs in solution, though it can absorb macromolecules like proteins and particles Nanoparticles are absorbed more effectively than microparticles There is ongoing debate about using this method for peptide and protein drugs 77 Transcytosis Transcytosis allows some viruses and bacteria to enter the lymphatic system via absorption by enterocytes and specialized cells (M cells) in the gut- associated lymphoid tissue (GALT) 78 Paracellular pathway The paracellular pathway transports materials through aqueous pores between cells rather than across them Cells are connected by tight junctions, which occupy about 0.01% of the epithelial surface area The tightness of these junctions varies, with absorptive epithelia like the small intestine being leakier 79 Paracellular pathway The importance of the paracellular pathway decreases along the gastrointestinal tract as the number and size of pores decrease This pathway is crucial for transporting ions like calcium, sugars (e.g., mannitol), amino acids, peptides, and small hydrophilic charged drugs (log P < 0) that don't easily penetrate cell membranes 80 Paracellular pathway The typical molecular mass cut- off for paracellular transport is 250 Da, though some larger drugs can also be absorbed this way Examples of drugs using this route include cimetidine, loperamide, and atenolol. 81 Which Absorption Path Dominates Drug Absorption? Different mechanisms of oral drug absorption can occur simultaneously in the small intestine Typically, the fastest route dominates absorption Passive diffusion is the primary mechanism for many lipophilic compounds, while carrier-mediated processes govern transporter substrates Some small hydrophilic compounds (molecular weight less than 300) are absorbed through the paracellular junction. 82 83