Absorption Basics - PDF

Absorption – basics By Rida Jamil  Absorption: is the drug absorbed from What does ADME the site of administration Distribution: where the drug travels in the body stand for? Metabolism: Where the drug is metab...

Absorption – basics By Rida Jamil  Absorption: is the drug absorbed from What does ADME the site of administration Distribution: where the drug travels in the body stand for? Metabolism: Where the drug is metabolised in the body (mainly the liver) Excretion: How the drug is passed out from the body (e.g. urine, faeces, bile and many other routes) How do drugs molecules move around the body? Bulk flow  The drug is transported through the bloodstream, lymphatics, cerebrospinal fluid or during passage through the GI tract. The chemical nature (hydrophobicity/ hydrophilic) makes no difference in this process Diffusion  The drug transporting over short distances or long distances. Drug that need to cross hydrophobic diffusion barriers are influenced by lipid solubility. Aqueous diffusion is crucial for drug transport as it delivers drug molecules to and from the non-aqueous barriers only happens with large MW drugs The rate of diffusion of a substance depends on the molecular size (diffusion coefficient  1/SQR of MW) Small molecules (200-1000kDa) can easily pass through various compartments, making it beneficial to study them in compartmental analysis Large molecules (16-150kDa) diffuse slowly, and examples are monoclonal antibodies or small interfering RNA drugs that can be a limitation in biopharmaceutical drugs How do drug molecules cross the cell barriers? Single layer membranes (e.g. epithelial barriers like gastrointestinal mucosa or renal tubule) consists of layer of cells connected to each other, so molecules need to transport through 2 membranes (inner and outer) to pass. Vascular endothelium (cell layer that separates intra and extra vascular compartments) varies from one tissue from the other. Gaps between endothelial cells are packaged with loose matrix of proteins that acts as filters, retaining large molecules and allowing smaller ones through MW of 80-100kDa permeate slowly and water passes through rapidly BBB and placenta have tight junctions between cells and endothelium encased in an impermeable layer of pericytes (peri endothelial cells)  prevents harmful molecules from penetrating brain or foetus and have major consequences for drug distribution & activity Other organs (e.g. liver & spleen), endothelium is discontinuous allowing free passage between cells. Liver has hepatocytes that take on several endothelial functions. Fenestrated endothelium occurs in the endocrine glands (transfer hormones or other molecules to the bloodstream/ pores in endothelium) Formation of endothelial-gland derived vascular endothelial growth factor (EG-VEGF) Endothelial cells line postcapillary venules that have specialised functions relating to leukocytes migrating and inflammation What are the 3 ways by which small molecules cross the cell membrane? 1) Diffusing directly through the lipid 2) Combination with solute carrier (SLC) or other membrane transporter 3) Diffusing through aqueous pores formed by special membrane glycoproteins (AQPs) that transverse the lipids 4) Small quantities of macromolecules may cross the barrier by pinocytosis (‘cell drinking’) What molecules are diffused directly through lipids? Diffusion through AQPs are important in the transfer of gases (e.g. CO2), but pores are small in diameter (0.4nm) to allow most drug molecules (which exceed more than 1nm in diameter) Drug distribution is not abnormal in patients with genetic diseases affecting AQPs Pinocytosis  involves invagination of part of the cell membrane and trapping within the cell of a tiny vesicle containing extracellular constituents Vesicle contents can be released within the cell or extruded from it’s other side > mechanism important for transport of some macromolecules, but not for small molecules  Nonpolar molecules (e-’s are uniformly distributed) dissolve freely in membrane lipids, and consequently diffuse readily across the membranes, in contrast to polar molecules in which e-’s are distributed non-uniformly Weak acids/ bases exist in either ionised (highly polar) or non- ionised state depending on pH The number of molecules crossing the membrane per unit of time is determined by permeability coefficient, P, along with the Diffusing concentration difference across the membrane (diffusion coefficient) through Permeant molecules must be present within the membrane if rapid permeation should occur lipid Thus, 2 physiochemical factors namely, solubility and diffusivity expressed as the diffusion coefficient Solubility  Expressed as partition coefficient for substance distributed between the membrane phase and aqueous environment Diffusivity  Measure of the mobility within the lipid and expressed as a diffusion coefficient Diffusion coefficient varies between different drugs and important determinant is membrane permeability for conventional LMW drugs Lipid solubility can determine rate of absorption from the gut, penetration into different tissues and extent of renal elimination  Ionisation and membrane permeability affect only the rate at which drugs permeate membranes but also steady state distribution between aqueous compartments Lipid soluble drugs diffuse into cells where they are metabolised (e.g. esterase's); can make charged metabolite and effectively gets trapped into the cell Example: intracellular mediators like Ca2+ using fluorescent indicators (e.g. Ion Trapping fura-2), is loaded into cells as an uncharged ester and trapped intracellularly in its ionised form Used in drug development at intracellular sites of action within monocytes and macrophages using esterase-sensitive chemical motifs conjugated to drug (e.g. histone deacetylase inhibitor), forms pro- drug delivered to cells of monocyte macrophage and selectively express human carboxylase-1 (charged active drug trapped in monocyte cytoplasm (site of action)) H and Ionisation Weak acids or bases and exist in ionised and unionised forms The ratio of these 2 forms varies with pH The image shows the Henderson-Hasselbalch equation BH+ or A- has low lipid solubility and unable to permeate membranes except where specific transport mechanisms exist Lipid solubility of uncharged species, B or AH depends on chemical nature of drug and many uncharged species of drugs are lipid soluble and permit rapid membrane permeation Exceptions to the rule above is aminoglycoside antibiotics, where the uncharged molecule cannot cross membrane’s freely This is usually because of hydrogen-boding groups (e.g. hydroxyl group in sugar moieties – aminoglycosides) that render the uncharged molecule hydrophilic Sugar moieties: It is one of the most typical characteristics of biological molecules, particularly natural products (NPs) and latter small molecules have higher functions in signalling, intercellular and inter-organism communication or defence.  pH partition and ion trapping If pH difference between body compartments can alter steady-state distribution of drugs that are weak acids or bases and influence their ionisation Assumed that un-ionised species can cross the compartment freely Assumed that the ionised species cannot cross the compartment Concentration gradients produced by ion trapping can be very large if there is a large pH between compartments Such large gradients cannot be achieved as 1) Assumed totally impermeability of charged species unrealistic, small permeability will attenuate considerably as conc. difference can be reached and 2) Body compartments rarely approach equilibrium Neither gastric contents for renal tubular fluid stands still, and resulting in bulk flow of drug molecules reduces the conc. gradients well below the theoretical equilibrium conditions Attenuate: Decrease in absolute concentration of a drug by serial dilution; the more the substance is attenuated (diluted), the greater it’s effect It explains qualitative effects of pH changes in different body compartments on pharmacokinetics of weak acids and bases, in relation to renal excretion and penetration of the blood brain barrier pH partition not main determinant for absorption in the GI tract bcuz of enormous S.A of villi and microvilli in the ileum compared with smaller absorptive S.A of the stomach overriding its importance. Absorption of acidic drugs promoted by gastric acid emptying drugs (e.g. metoclopramide) and slowed by slowed gastric emptying (e.g. propantheline), even though acidic pH of stomach contents favours absorption of weak acids.  Important consequences of pH partition mechanism Free base trapping of some antimalarial drugs (e.g. chloroquine) in acidic environment in the food vacuole of malaria parasite contributes to disruption of haemoglobin digestive pathway that underlies their toxic effect on the parasite Urinary acidification accelerates excretion of weak bases and reduces excretion for weak acids Urinary alkalisation accelerates excretion of weak acids and reduces excretion for weak bases Increasing plasma pH (e.g. administration of sodium bicarbonate) causes weakly acidic drugs extracted from the central nervous system (CNS) into the plasma Reducing plasma pH (e.g. acetazolamide) causes weakly acidic drugs to be concentrated into the CNS, increasing their neurotoxicity Alkalisation of urine is used to treat overdose in aspirin, bicarbonate and acetazolamide, these each increase pH and salicylate elimination, bicarbonate reduces Acetazolamide increases distribution of salicylate into the CNS Carrier mediated transport Cell membranes possess specialised mediated transport mechanisms that regulate entry and exit of important molecules (e.g. sugars, amino acids, neurotransmitters and metal ions) Divided into SLC transporters and ATP binding cassette (ABC) transporters Some transporters facilitate passive movement of solutes down the concentration gradient, and some are active pumps fuelled by ATP Some of these transporters transport xenobiotic drugs These transporters are mainly located in the blood brain barrier, GI tract, renal tubule, biliary tract and placenta  ABC transporters They are ATP-Binding Cassette transporters are membrane proteins that couple substrate transport to ATP hydrolysis, and they are either importer or exporters of substrates (NOT BOTH) Exporters: MRP1 & P-gp Importers: found mainly in prokaryotic species (e.g. maltose uptake transporter or methionine uptake transporter) Most cells expressing these transporters are polarised and expressed either on the apical or basolateral side (NOT BOTH) They protect body against xenobiotics and have some normal physiological role (e.g. bile transport in the liver or regulation of insulin) ABC transporters interfere with the circulation of drugs and pump compounds back into the bloodstream BCRP, MDR3 & MRP1 display multi-drug resistance  P-glycoprotein (ABCB1) They are the second most important class of transporters responsible for multidrug resistance in cancer cells, many express on ATP-dependent pump with broad specificity called MRP1 Expressed in animals, fungi, bacteria and may have evolved as a defence mechanism against toxins Present in renal tubular brush border membranes, bile canaliculi, astrocyte foot processes in brain micro vessels & GI tract Polymorphisms of genes that encode this transporter and SLCs contribute to individual genetic variation and responsiveness to different drugs causes competition between drug, leading to drug- drug interactions Collie dogs lack MDR1 (encodes P-gp transports toxins from CSF across the BBB), leads to ivermectin (anthelminthic drug) causes neurotoxicity in many border collies ABCB1 gene on chromosome 7 in humans encodes for this transporter Main roles are to remove xenobiotics from enterocytes, transport xenobiotics from bile across canicular membrane, prevents xenobiotic transport in the brain and xenobiotics transported into lumen of kidney on brush-border membrane Cancer cells can express Pgp at high levels causing drug resistance  BSEP (ABCB11) Described as ‘sister of P- glycoprotein’ Transports bile salts and got renamed to Bile Salt Export Pump Exclusively expressed in hepatocytes (mainly at canicular membrane) Main role is to transport bile salts across the canicular membrane of hepatocyte Patients with rare genetic disease called PFIC2 (progressive familial intrahepatic cholestasis) have inherited defect in BSEP  MDR3 (ABCB4) It is a specific translocase (floppase) for phosphatidylcholine Translocates phosphatidylcholine (PC) from the inner to the outer leaflet of the canicular membrane for extraction into the lumen by bile salts Forms micelles with bile salts to protect hepatocyte biliary membrane Genetic mutations of ABCB4 leads to distinct but related hepatobiliary diseases: progressive familial intrahepatic cholestasis type 3 (PFIC3), intrahepatic cholestasis of pregnancy (ICP) and gallstones. It also transports Anthracyclines, vinca alkaloids (derived from periwinkle plant) and taxanes (chemotherapy agent derived from plant taxus) Anthracyclines: A class of drugs used in cancer MRP1 Multidrug Resistance Associated protein (MRP), MW 190KDa and are ATP- dependent high molecular weight membrane proteins 12 different MRPs are now known Variety of functions ranging from protection of xenobiotics to challenging ions Facilitate extrusion of numerous glutathione, glucuronate and sulphate conjugates Expressed in numerous tissues in the body, including brain, testis, lungs and very low levels in the liver Encoded by ABCC1 on chromosome 16, contributes to MDR Prefers amphiphilic organic anions  MRP2 Highly expressed on the bile canaliculus of the hepatocyte, also in the apical membranes of kidney & intestine Eliminates drug glucuronides in the bile like Diclofenac, Morphine and Fexofenadine Eliminates bilirubin from the body (liver bile) In Dublin Johnson syndrome there is no MRP2 due to mutations, allowing high levels of bilirubin glucuronide in their plasma (seen in ABCC2 gene) Usually, a benign condition but may see jaundice in pregnancy or with some drugs Jaundice: medical condition causing yellowing of the skin or whites of the eyes, arising from excess of the pigment bilirubin causing destruction of bile duct by liver disease or excessive breakdown of RBCs ABCC2 gene provides instructions for producing a protein called MRP2 BCRP (ABCG2) Similar location to P-gp and high levels are in lactating breasts Secretion of xenobiotic into milk which has implications for breast-fed infants Restrictions on use of certain drugs by nursing mothers BCRP evidence suggests imatinib is a substrate for BCRP and CML stem cells appear to have higher levels of ABCG2 than more mature CML cells (these are less sensitive to imatinib and remain after mature cells are eliminated) GWAS displayed genetic polymorphism in an amino acid change in BCRP as a risk factor for gout (accumulation of uric acid as crystals in joints occurs resulting in pain and inflammation) Further work displayed uric acid is a substrate for BCRP and unstable form of this protein results in poor ability to excrete this compound Uric acid generated by purine metabolism and accumulation in the body and causes effects in joints (high levels in synovial fluid) and kidney (uric acid kidney stones) Makes an unstable protein by the second mutation H155A SLC transporters Solute carriers and have 52 different gene families exist These all code for membrane proteins but these proteins have a variety of functions including roles in normal physiology There are 2 types of proteins: Facilitative transporters and Secondary active transporters Facilitative transporters: Allow substrates to flow downhill with their electrochemical gradients Secondary active transporters: Substrates that can flow uphill against their electrochemical gradient by coupling of transport to that of a co-substrate THESE ARE NOT ATP DEPENDANT Usually have 12 transmembrane domains and often present on membrane of polarised cell with specific location (apical or basolateral  NOT BOTH) OATP family Organic anion-transporting polypeptide These are encoded by SLOC1 to SLOC6 genes Families 1 and 2 are the most important in relation to drug disposition (SLOC1 and SLOC2 also called OATP1 and OATP2) These proteins are important in drug transport across sinusoidal membrane into the liver OATP1B1, OATP1B3 & OATP2B1 most important in the liver Kidney also has OATPs present but are more limited in the role of renal excretion Drug substrates normally are anionic with a high molecular weight and generally are plasma protein bound Examples of OATP1B1 substrates are statins, rifampicin and benzylpenicillin OAT family Complete separate family from OATPs Members of the SLC22A subfamily Key role in renal excretion but expressed elsewhere (e.g. liver & small intestine) OAT1, 2 & 3 found on the basolateral membrane of proximal tubule cells, facing blood vessels OAT4 on apical membrane of proximal tubule, facing urine (can sometimes transport in both directions) OAT1 to 3 coupled to dicarboxylic acid (e.g. alpha-ketoglutarate) transport (this is sodium dependent)  anionic drug transport in renal proximal tubule cells There are many substrates for this transporter making it still unclear, they are likely to be sulphated or glucuronide conjugates and don’t undergo metabolism (e.g. penicillin's) may also be substrates Examples: OAT1 = Tetracycline, OAT2 = Diclofenac, OAT3 = Rosuvastatin & Benzylpenicillin & OAT4 = Diclofenac glucuronide OCTs Organic cation transporters Members of the SLC22A so some sequence homology to OATs OCT1 and 2 are the most important cationic transporters in human drug disposition OCT1 = Important transporter on the sinusoidal face of the liver though found elsewhere in the body also substrates include metformin and cisplatin OCT2 = High levels on the basolateral membrane of the kidney and substrates include metformin, cisplatin and cimetidine PEPT transporters Peptide transporters PEPT1 & PEPT2 and are members of the POT protein family  these transporters are encoded by SLC15A subfamily PEPT1 expressed in intestine and kidney PEPT2 expressed in kidney only Both show similar substrate specificities with penicillins, ACE inhibitors & valcyclovir Proton-dependent transporters  in the kidney, PEPT1/2 contribute to drug reabsorption from the urine within the proximal tubule MATE transporters Multidrug and toxin extrusion (MATE) family were first identified in bacteria where, they have membrane transport activity towards cationic compounds 2 MATE genes are now identified as members of the SLC47 family MATE1  expressed in the kidney and liver MATE2  expressed only in the kidney Export pumps which contribute to biliary and renal excretion of cations Transport is proton dependent Transporter proteins roles in the liver and kidney Xenobiotics often need to be transported into the hepatocyte across the sinusoidal membrane for both metabolism and to reach targets (e.g. HMG-CoA reductase) Compounds usually following metabolism need to be transported out either across the canicular membrane (to bile) or sinusoidal membrane (back to the blood for renal excretion) Basolateral membrane  Compounds are either excreted by tubular secretion or reabsorbed by returning to circulation (bloodstream) Brush border membrane  Compounds cross this membrane to enter the lumen for final renal excretion and reabsorption may occur Both roles in the liver & kidney is when diclofenac metabolised by CYP2C9 & UGT2B7, UGT2B7 produces acyl glucuronide (DF-AG - a proportion of DF is metabolized and/or conjugated). Furthermore, OAT2 and 4 excretes DF-AG, these transporters transport the parent compound and get biliary excretion of DF-AG  Drug-drug interactions involving transporters Inhibition of transport is caused in OAT and ACBC1 (P-gp) OAT  penicillin and probenecid and valuable as penicillin is cleared more slowly P-gp  Digoxin excretion affected by a range of drugs (e.g. erythromycin, statins) and is problematic at high levels causing toxicity P-gp, MRP2 and OATP1B transporters induce PXR, this has potential for drug-drug interactions when rifampicin is prescribed due to induction, results in rapid clearance of other drugs (e.g. cyclosporin, digoxin) and St John’s Wort has very similar effects  Pharmacogenetic s of transporters OATP1B1  genetic polymorphism with higher plasma levels of some statins due to impaired ability to enter hepatocyte. Higher plasma level may lead to toxic levels of statins in muscle cells, leading to potentially fatal rhabdomyolysis where muscle protein dissolves OCT2 linked to nephrotoxicity with anti-cancer drug cisplatin and may also involve MATE2 and drug accumulates in tubule cells causing Fanconi syndrome.  Binding of drugs to plasma proteins When drugs are bound to plasma proteins, the free drug fraction is less than 1% and the bound fraction of the drug is 99% Small differences in protein binding has large effects on free drug concentration and effects This is common between human plasma and plasma taken from another species used in FIH studies, used in pre-clinical testing Albumin is the most important protein in the plasma and mainly binds acidic drugs (e.g. warfarin, sulphonamides and NSAIDs) and a smaller number of basic drugs (e.g. TCAs and Chlorpromazine) Amount of drug bound to protein depends on 1) Concentration of free drug, 2) Affinity for binding sites and 3) Concentration of protein Furthermore, depends if you are a Class 1 or 2 drug  this contributes to drug-drug interactions and drug distribution Partition of drugs into fat and other tissues Fat acts as a reservoir for non-polar drugs mainly general anaesthetics, which are lipid soluble Many drugs have a low fat: water partition coefficient, indicating minimal fat sequestration (e.g. Morphine crosses BBB and has a low partition coefficient 0.4, making fat soluble negligible, however thiopental has a partition coefficient of 10, leading to accumulation in body fat, restricting it’s use short-term induction of anaesthesia) Body fat’s low blood supply slows down drug delivery, delaying equilibrium between fat and water, making the partitioning significant for few highly lipid soluble drugs used acutely (e.g. general anaesthetics) High administration of BZDs can lead to significant accumulation of fat, additionally some drugs can be accumulated progressively in fat if ingested intermittently Other tissues also accumulate drugs like, chloroquine binds to melanin in retina, causing ocular toxicity, Tetracyclines accumulate in bones and teeth