Pharmacological Strategies for facilitating the excretion of Homogentisic acid in Alkaptonuria. DominicWilliams

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1 Pharmacological Strategies for facilitating the excretion of Homogentisic acid in Alkaptonuria DominicWilliams

2 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

3 Centre Strategy for Investigating ADRs Investigation of the chemical Investigation of the patient

4 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

5 Integrated Mechanistic Drug Safety: Chemical SAR SMR STR In vitro Drug / Compound Chemical Studies Bioanalysis Man Chemical Validation Biological Validation Animal Clinical Validation & Application

6 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

7 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

8 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

9 Drug Disposition Physiological, Pharmacological & Toxicological DRUG Cellular accumulation Toxicity Phase I/II/III bioactivation Stable metabolites Chemically reactive metabolites bioinactivation Excretion

10 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

11 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

12 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

13 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)

14 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

15 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

16 Fractional clearance of Aspirin Therapeutic dose Low overdose High overdose 600mg (n=45) plasma SA mg/L; Urine ph 6 (n=24) plasma SA mg/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

17 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?

18 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?

19 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

20 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

21 Drug Metabolism: Paracetamol oxidation Detoxication GLUCURNIDE SULPHATE Bioactivation verdose GSH CVALENT BINDING TXICITY

22 Mechanism of Nrf2-regulated Gene Induction NHCCH 3 NCCH 3 H 900 GSH depletion Adduct formation Protein oxidation Nrf2 Nuclear Nrf2 (% control) TXICITY Nrf2 Keap1 0 Proteasomal proteolysis Paracetamol (mg/kg) Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009

23 Hepatic nuclear translocation of Nrf2 in paracetamol-treated mice Nuclear Nrf2 (% control) TXICITY Paracetamol(mg/kg) meh mrna/18s H-1 mrna/18s GCLC mrna/18s controls Visualize genes 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

24 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% NAPQI (µm) Copple et al. Hepatology 2008

25 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% NAPQI (μm) Copple et al. Hepatology 2008

26 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% *** * * Time (h) Copple et al. Hepatology 2008

27 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

28 Homogentisic acid oxidation HGA serum concentrations of µM 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., Martin & Batkoff, Free RadicBiolMed. 1987;3(4):

29 Potential Pathways in HGA Excretion. H H CH 2 CH P450/MP/Auto Quinone reductase NQ 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?

30 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 m/z, amu m/z, amu Mass Spec 167 Intensity (cps) Absorbance units 1,400 1,250 1,125 1, e6 1.00e6 8.00e5 6.00e5 4.00e5 2.00e HPLC XIC of +Q1 Time (min) BQA: 167 amu ~ 90% yield > 97% purity Time, min

31 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

32 Acknowledgements University of Liverpool: Amy Lawrence Jim Gallagher & AKU group Jonathan Jarvis Andrew Preston BBSRC: CDSS: Kevin Park James Maggs Xiaoli Meng