Lipid solubility and the brain uptake of compounds • Higher the Kow, the greater the brain uptake • Specific carriers can facilitate brain uptake (glucose, lucine, dopa) • Binding to plasma proteins can reduce uptake (phenytoin) Octanol / Water Partitioning Coefficient logP and ClogP http://www.biobyte.com/ • The logP is an important physicochemical parameter for oral absorption • Measure for the lipophilicity of a compound. • ClogP is the calculated log octanol/water partition coefficient, – It relates to solubility and influences the ability of a compound to permeate cell membranes – Too hydrophilic compounds (negative logP) cannot permeate membranes – Too lipophilic compounds (high logP) are insoluble, poorly permeate through membranes (get stuck in the lipophilic bilayer). 1 Predicting Favorable ADME in Drug Development - Lipinski’s Rule of Five A rule of thumb to evaluate: • If a chemical compound with a Lipinski's rule: An orally active drug has no more than one pharmacological /biological activity violation of the following : has properties that may make it a 1. No more than 5 hydrogen bond donors (N or O with >1 H likely orally active drug. atom) • Rule formulated by Chris Lipinski in 2. Not more than 10 hydrogen bond acceptors (N or O atoms) 1997, based on the observation that most medication drugs are relatively 3. A molecular mass < 500 daltons small and lipophilic molecules 4. An octanol-water partition coefficient (log P) < 5 * As with all ‘rules’, many exceptions! Predicts that poor absorption and permeability of potential drug candidates will occur if: 1) Number of H-bond donors > 5 2) Number of H-bond acceptors > 10 3) Molecular Weight > 500 4) ClogP > 5 Lipinski’s rule does not predict if a compound is pharmacologically active. • Rule is important to keep in mind during drug discovery when a lead structure is optimized step-wise to increase the activity and selectivity. • Candidate drugs that conform to the RO5 tend to have lower attrition rates during clinical trials Drug Metabolism / Biotransformation Learning objectives • Distinguish the differences between Phase I and Phase II reactions • Recognize the enzymes involved in these processes and that various factors (age, genetic predisposition/polymorphisms, co-exposure to other drugs, diet, illness) may affect these processes • Be familiar with at least one example of one of the following types of Phase I biotransformation reactions: Oxidation, Reduction, Hydrolysis. The same goes for the following types of Phase II biotransformation reactions: Conjugation with glucuronide, glutathione, sulfate. • Recognize that one of the possible consequences of biotransformation is the conversion of prodrugs to active drug, or active drug to reactive intermediates, and that depending upon their chemical nature, these intermediates can modify molecular targets through different mechanisms 2 Drug Metabolizing Enzyme System Phase I and Phase II Biotransformation • Phase I metabolism – ‘Functionalization’ • Addition or exposing of a reactive functional group such as -OH, -SH, -NH2 or –COOH – In general, Phase I metabolism prepares the xenobiotic for subsequent Phase II reactions • Phase II Reactions – Enzymatic reactions that conjugate large water- soluble, charged (polar) biomolecules to xenobiotics The Truck-Hitch-Trailer Analogy to Xenobiotic Biotransformation Phase 2 enzymes Phase 1 enzymes conjugate (transfer) Foreign Chemical add or expose a endogenous molecules* (xenobiotic) functional group to the functional group •lipophilic HITCH TRAILER •not charged Phase 1 enzymes add or •not lipophilic •not water soluble expose a functional group •usually not reactive •still lipophilic •water soluble products •poorly excretable •possibly reactive •Excretable •poorly water soluble •catalyzed by transferases •poorly excretable •catalyzed by P450s * sugars, amino acids, sulfates, acetyl groups 3 Biotransformation Enzyme-Containing Cells in Various Organs Organ Cell(s) . Liver Parenchymal cells (hepatocytes) Kidney Proximal tubular cells (S3 segment) Lung Clara cells, Type II alveolar cells Intestine Mucosa lining cells Skin Epithelial cells Testes Seminiferous tubules, Sertoli cells Mixed Function Oxidase System (MFO) (Phase I Metabolism) MFOs Contain: • Cyt. P450, • 2 flavoproteins (P450 reductases), • Cytochrome b5 4 CYP families in humans Humans have 57 genes and more than 59 pseudogenes divided among: • 18 families of cytochrome P450 genes • 43 subfamilies Function Members Names Family CYP1 drug and steroid (especially estrogen) metabolism 3 subfamilies, 3 genes, 1 pseudogene CYP1A1, CYP1A2, CYP1B1 CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2 drug and steroid metabolism 13 subfamilies, 16 genes, 16 pseudogenes CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 CYP3 drug and steroid (including testosterone) metabolism 1 subfamily, 4 genes, 2 pseudogenes CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4 arachidonic acid or fatty acid metabolism 6 subfamilies, 11 genes, 10 pseudogenes CYP4F22, CYP4V2, CYP4X1, CYP4Z1 CYP5 thromboxane A2 synthase 1 subfamily, 1 gene CYP5A1 CYP7 bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus 2 subfamilies, 2 genes CYP7A1, CYP7B1 CYP8 varied 2 subfamilies, 2 genes CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid biosynthesis) CYP11 steroid biosynthesis 2 subfamilies, 3 genes CYP11A1, CYP11B1, CYP11B2 CYP17 steroid biosynthesis, 17-alpha hydroxylase 1 subfamily, 1 gene CYP17A1 CYP19 steroid biosynthesis: aromatase synthesizes estrogen 1 subfamily, 1 gene CYP19A1 CYP20 unknown function 1 subfamily, 1 gene CYP20A1 CYP21 steroid biosynthesis 2 subfamilies, 2 genes, 1 pseudogene CYP21A2 CYP24 vitamin D degradation 1 subfamily, 1 gene CYP24A1 CYP26 retinoic acid hydroxylase 3 subfamilies, 3 genes CYP26A1, CYP26B1, CYP26C1 CYP27A1 (bile acid biosynthesis), CYP27B1 (vitamin D3 1-alpha hydroxylase, activates CYP27 varied 3 subfamilies, 3 genes vitamin D3), CYP27C1 (unknown function) CYP39 7-alpha hydroxylation of 24-hydroxycholesterol 1 subfamily, 1 gene CYP39A1 CYP46 cholesterol 24-hydroxylase 1 subfamily, 1 gene CYP46A1 CYP51 cholesterol biosynthesis 1 subfamily, 1 gene, 3 pseudogenes CYP51A1 (lanosterol 14-alpha demethylase) Cytochrome P450 Characteristics • Broad substrate specificity - – Most drug metabolizing CYPs are promiscuous re: substrate, – but some CYPs are highly specific to substrate, e.g., Cyp 19 (aromatase w/ testosterone substrate). – CYP enzyme levels induced by exposure to xenobiotics • Broad substrate specficity = Low catalytic efficiency (Kcat). – Drugs in general have t1/2 of 3 – 30 hrs, while endogenous substrates are much shorter (sec or min) – reflects lower Kcat of drug CYPs. • Broad substrate specificity of many CYPs accounts for much of reason for drug – drug interactions. – Multiple drugs may compete for binding site, altering metabolism of one or more of the drugs, thereby increasing t1/2 and plasma levels (Dixogin example). 5 Cytochrome P450’s • Limted number of CYPs (15) in families 1 – 3 are mostly responsible for drug metabolism. • In humans, 12 CYPs in particular important: CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5. • The most active in drug metabolism are the 2C, 2D, and 3A subfamiles, with CYP3A4 involved in metabolism of ~50% of clinically used drugs. Cytochrome P450 Substrate Binding • P450s possess active HEME group: • 2 types of substrate binding to P450 – Type 1 Binding: The substrate binds to a protein moiety near the active site. This brings the substrate within the region of the catalytic activity of the Heme-Fe. Fe in Low Spin – Type II Binding: The substrate binds directly to the Heme-Fe. Fe in High Spin Can determine whether Type I or Type II binding spectrophotometrically -Type I binding of Cyt. P450 to the substrate gives a characteristic AMAX of around 390nm. -Type II binding of Cyt. P450 to the substrate gives a characteristic AMAX of around 420nm. 6 Systems Involved in Phase II Metabolism Four primary enzymes: 1. Glucuronosyltransferase—glucuronic acid 2. Sulfotransferase—sulfate 3. Glutathione-S-transferase—glutathione (GSH) 4. Acetyltransferase—acetyl • enzymes are, for the most part, in the cytosol of cells • In all cases (except GSH) – requires conjugate activated to an Electrophilic Activated Donor Phase II Enzymes: Examples Glucuronidation and Sulfation of a Hydroxyl Group 7 UDP Conjugation - Phenytoin • Phenytoin – Common antiepileptic • Marketed as Phenytek, Dilantin • Insoluble in water • Stabilizes inactive state of voltage-gated Na channels • Induces CYP3A4, 2C19 • Interactions: • Coumarin and Warfarin increase serum phenytoin levels and prolong the serum half-life of phenytoin by inhibiting its metabolism • Pharmacokinetics – can be saturable, leading to highly variable plasma levels across minor dose changes Bioactivation and UDP Conjugation: Irinotecan • Irinotecan – cancer drug (mostly colon cancer). • Topoisomerase 1 inhibitor – leads to inhibition of DNA replication • Administered as ProDrug • Carboxyesterase activates to active drug (SN-38) 8 Gilbert’s Syndrome Drug – Polymorphism interaction • Generally benign condition present in ~10% of pop’n. • Diagnosed clinically because circulating bilirubin levels are 60 – 70% elevated. • Most common genetic polymorph associated with Gilbert’s is mutation in UGT1A1 gene promoter, leading to reduced expression of UGT1A1. • If drug undergoes Phase II with UGT1A1, it will compete with bilirubin metabolism conjugation,