Pharmacological Strategies for facilitating the excretion of Homogentisic acid in Alkaptonuria DominicWilliams
Background MRC Centre for Drug Safety Science Helpmeasure HGA in patients & rodents Led to a BBSRC Integrative Mammalian Biology funded PhD Amy Lawrence (ct 2010) utline the relationship between adverse drug reactions and drug metabolism utline of proposed studies in In vitro homogentisate 1,2-dioxygenase K mice translation to AKU patients
Centre Strategy for Investigating ADRs Investigation of the chemical Investigation of the patient
Integrated Mechanistic Drug Safety: Patient Drug Class SAR SMR STR Animal Chemistry Clinical Problem Research Question Man Bioanalysis Mechanism utcomes Biomarkers In vitro Clinical Samples
Integrated Mechanistic Drug Safety: Chemical SAR SMR STR In vitro Drug / Compound Chemical Studies Bioanalysis Man Chemical Validation Biological Validation Animal Clinical Validation & Application
Integrated Mechanistic Drug Safety Drug Class SAR SMR STR Animal Chemistry Clinical Problem Research Question Man Bioanalysis Mechanism utcomes Biomarkers In vitro Clinical Samples Dynamic & Iterative SAR SMR STR In vitro Drug / Compound Chemical Studies Bioanalysis Man Chemical Validation Biological Validation Animal Clinical Validation & Application
Mechanistic Classification of Adverse Drug Reactions TYPE A (augmented) predictable exaggeration of pharmacological effect dose dependent TYPE B (idiosyncratic) unpredictable apparently dose-independent less common more severe TYPE C (chemical) predictable from chemical structure eg. Paracetamol Park et al., 1998
Drug Metabolism Physiology:- To facilitate the (rapid) excretion of potentially toxic foreign compounds and prevent accumulation during chronic exposure Lipophilic drug Drug metabolism Phase I CHEMICAL REACTIVITY xidation Reduction Hydrolysis Water soluble metabolite Phase II CNJUGATIN WITH PLAR GRUP Glucuronidation Sulphation Glutathione conjugation etc. Excretion Urine Bile Cytochrome P450 Transferases
Drug Disposition Physiological, Pharmacological & Toxicological DRUG Cellular accumulation Toxicity Phase I/II/III bioactivation Stable metabolites Chemically reactive metabolites bioinactivation Excretion
Consequences of drug bioactivation DRUG Cellular accumulation Toxicity Phase I/II/III bioactivation Chemical Stress Carcinogenicity Stable metabolites Excretion Chemically reactive metabolites bioinactivation Modification of: nucleic acid enzyme transporter signalling protein receptor random autologous protein Necrosis Apoptosis Hypersensitivity
Drug Metabolism: Pharmacology DRUG Phase I/II Drug Cellular accumulation RESPNSE Concentration in Plasma Stable metabolites Metabolism Disposition Absorption Excretion Excretion Drug plasma level Pharmacological exposure
Drug Metabolism: Toxicology DRUG Phase I/II Drug Cellular accumulation RESPNSE Concentrations in affected organs Stable metabolites Metabolism Disposition Absorption Excretion Excretion Drug & metabolites Pharmacological & Toxicological exposure
Drug Metabolism: Chemical toolbox Use drug/chemicals to perturb biological processes elucidates biological mechanisms informs toxicology / drug safety assists in biomarker discovery & drug development Apply knowledge of drug metabolism to homogentisic acid Does it undergo further chemical changes Can we facilitate further excretion knowledgeof rate / route Determine metabolism & fractional clearance of HGA homogentisic acid (HGA)
Metabolic comparison of HGA& Aspirin Physiocochemical properties Predominant route of excretion Metabolism Aspirin Weak acid pka 3.5 ~80% Biliary Renal in verdose Hydrolysis Phase 1: hydroxylation Phase 2: glycine conjugation glucuronidation Homogentisic acid Weak acid pka 4.4 Renal Biliary?? C H C H H C CH 3 H Aspirin Homogentisic Acid
Metabolism of Aspirin C H C Glucuronid e Aspirin C CH 3 H Acyl Glucuronide hydrolysis C H NH CH 2 CH C H Glucuronid e Phenolic Glucuronide H Salicylic Acid hydroxylation H Salicyluric acid: conjugation with glycine saturatable H C H Amino acid conjugation H Gentisic Acid Amino acid conjugation
Fractional clearance of Aspirin Therapeutic dose Low overdose High overdose 600mg (n=45) plasma SA 240-630mg/L; Urine ph 6 (n=24) plasma SA 715-870mg/L; [i.v, 1.26% sodium bicarbonate] Urine ph 7 (n=13) Ph. Glucuronide % recovery Therapeutic dose 11% Low overdose 23% High overdose 15% C H Glucuronid e Phenolic Glucuronide Salicylic acid % recovery Therapeutic dose 9% Low overdose 32% High overdose 65% H C H Salicylic Acid hydroxylation C H Gentisic Acid H H SU acid % recovery Therapeutic dose 75% Low overdose 47% High overdose 22% H NH CH 2 CH Salicyluricacid (saturatable) Gentisic acid % recovery Therapeutic dose 5% Low overdose 10% High overdose 7% Patel et al., 1990
Testable hypothesis applied to HGA Determine metabolism & fractional clearance of HGA in HGD -/- mice & man Urine from 2 patients showed 2 spots on paper chromatography with different R F 1 spot is water soluble / ether insoluble Hydrolysis determined this to be glycine Constitutes up to 25% of the HGA in the urine Can glycine supplementation increase HGA excretion?
Testable hypothesis applied to HGA Treat in a similar manner to aspirin (any weak acid) overdose patients Alkalinisation of the urine with sodium bicarbonate Enhances urinary excretion of weak acids Traps weak acids in ionized state (ion trapping) Prevents reabsorptionby renal tubules Relationship between the renal clearance of salicylate and urinary ph (Smith et al., 1946) Can urinary alkalinisation increase the elimination of HGA?
The Hepatocyte: Defence Against Chemical Stress 1 st line defence 2 nd line defence 3 rd line defence DRUG METABLISM ANTIXIDANT RESPNSE APPTSIS Stress Chemical / metabolite Reactive oxygen species Basal expression of genes co-ordinating cell defence: Phase II enzymes, antioxidant proteins Induction of genes co-ordinating cell defence: Phase II enzymes, antioxidant proteins Suicide of the cell: apoptosis NECRSIS GSH Transcription factor: Nrf2 Increasing levels of chemical stress
Mechanism of Nrf2-regulated Gene Induction Chemical Chemical (metabolite) GSH depletion repletion Adduct formation Protein oxidation Keap1 Nrf2 Nrf2 Proteasomal proteolysis Nrf2 ARE Nrf2 Target genes Cell defence proteins Glutamate Cysteine Ligase Glutathione transferases NAD(P)H quinone oxidoreductase Haem oxygenase Glucuronyl transferase Catalase Restore cellular redox status Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009
Drug Metabolism: Paracetamol oxidation Detoxication GLUCURNIDE SULPHATE Bioactivation verdose GSH CVALENT BINDING TXICITY
Mechanism of Nrf2-regulated Gene Induction NHCCH 3 NCCH 3 H 900 GSH depletion Adduct formation Protein oxidation Nrf2 Nuclear Nrf2 (% control) 800 700 600 500 400 300 200 100 TXICITY Nrf2 Keap1 0 Proteasomal proteolysis 0 200 400 600 800 1000 Paracetamol (mg/kg) Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009
Hepatic nuclear translocation of Nrf2 in paracetamol-treated mice Nuclear Nrf2 (% control) 900 800 700 600 500 400 300 200 100 0 TXICITY 0 200 400 600 800 1000 Paracetamol(mg/kg) meh mrna/18s H-1 mrna/18s GCLC mrna/18s 2.5 2 1.5 1 0.5 0 2 1.5 1 0.5 0 1.5 controls Visualize 0 12500 genes 1 0.5 Individual Nrf2-dependent genes APAP: 530mg/kg 1hour controls controls Nrf2- * regulated Genes apap apap ** apap ** meh H-1 GCLC 5 salines 5 APAP 5 salines 5 APAP 5 salines 5 APAP 5 salines 5 APAP 28S 18S Cell defence Goldring et al, Hepatology, 2004 Williams et al., Chem. Res. Toxicol, 2004
In vitro Nrf2 activation by NAPQI H N CH33 N CH 3 Nrf2 β-actin H paracetamol NAPQI N-acetyl p-benzoquinoneimine(napqi) Hepatotoxic APAP metabolite Mouse hepatoma cells - Hepa 1c1c7 Nrf2 translocation 1 hour Relative Nuclear Nrf2 200% 150% 100% 50% 0% 0 5 10 50 100 250 NAPQI (µm) Copple et al. Hepatology 2008
In vitro Nrf2 activation by NAPQI H N CH33 N CH 3 100% *** H paracetamol NAPQI N-acetyl p-benzoquinoneimine(napqi) Hepatotoxic APAP metabolite Mouse hepatoma cells - Hepa 1c1c7 Relative GSH 75% 50% 25% *** *** *** *** Nrf2 translocation GSH depletion 1 hour 1 hour 0% 0 25 50 75 100 NAPQI (μm) Copple et al. Hepatology 2008
In vitro Nrf2 activation by NAPQI H N CH33 N CH 3 H paracetamol NAPQI N-acetyl p-benzoquinoneimine(napqi) Hepatotoxic APAP metabolite Mouse hepatoma cells - Hepa 1c1c7 Nrf2 translocation 1 hour GSH depletion 1 hour GSH resynthesis >8h % Control GSH 160% 120% 80% 40% 0% *** * * 0 4 8 12 16 20 Time (h) Copple et al. Hepatology 2008
In vivo Induction of Nrf2 by Model Hepatotoxins HN CH 3 N CH 3 GSH Depletion Covalent Binding Nrf2 Induction Required Yes H Br Br Br Required Yes CCl 4 CCl 3 May occur Yes H 2 N 2 S Cl H 2 N 2 S Cl May occur Yes N N C 2 H H C 2 H H Randle et al., 2008
Homogentisic acid oxidation HGA serum concentrations of 50-400µM depending on dietary state of individual 1 HGA can be auto-oxidised giving superoxide, hydrogen peroxide & peroxide radical 2 Auto-oxidation rate is oxygen dependent xidised product is benzoquinone acetic acid:- C H Reaction inhibited by reducing agents + catalase HGA/Fe 3+ can degrade (depolymerise) hyaluronicacid in conc n. & time dependent manner 1 Seegmilleret al., 1961 2 Martin & Batkoff, Free RadicBiolMed. 1987;3(4):241-50.
Potential Pathways in HGA Excretion. H H CH 2 CH P450/MP/Auto Quinone reductase NQ1 2. - CH GST GS H H CH Phase II metabolism NADP+ NAD+ NADPH NADH GGT Dipeptidase NAT H H Glucuronide H CH Amino acid conjugate Acyl Glucuronide H CH H Maintain amino H acid conjugation glucuronidation H of HGA. Cys H CH H Glucuronide CH reduction of BQA H detoxicationof BQA (GSH). NAcCys H H CH chronosis?
Does HGA or BQA elicit chemical stress? In vitro: Expose cells to HGA and BQA Chemical synthesis of BQA (Wolffe et al., 1989) H C H 80mg HGA 2ml Tetrahydrofuran 220mg sliver oxide C H Intensity, cps Intensity, cps 2.6e7 2.2e7 1.8e7 1.4e7 1.0e7 6.0e6 2.0e6 3.4e6 3.0e6 2.6e6 2.2e6 1.8e6 1.4e6 1.0e6 6.0e5 2.0e5 HGA (M+1) BQA (M+1) 123 H 100 110 120 130 140 150 160 170 180 123 m/z, amu 100 110 120 130 140 150 160 170 180 m/z, amu 151 149 169 Mass Spec 167 Intensity (cps) Absorbance units 1,400 1,250 1,125 1,000 875 750 625 500 375 250 125 1.17e6 1.00e6 8.00e5 6.00e5 4.00e5 2.00e5 0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 0.00 0 HPLC XIC of +Q1 Time (min) BQA: 167 amu ~ 90% yield > 97% purity 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min
Does HGA or BQA elicit chemical stress? -next steps In vitro: Expose cells to HGA and BQA Chemical synthesis of BQA (Wolffe et al., 1989) Currently assessing BQA stability (cf NAPQI; available to collaborators) In vivo: in HGA 1,2,dioxygenase -/- mice assess HGA and BQA concentrations in tissues assess rate of degradation of HGA vsformation of BQA do K mice have more NQ1 enzyme vswt mice analysis of HGA and metabolites in human urine
Acknowledgements University of Liverpool: Amy Lawrence Jim Gallagher & AKU group Jonathan Jarvis Andrew Preston BBSRC: CDSS: Kevin Park James Maggs Xiaoli Meng