Effect of fluorofamide as urease inhibitor on helicobacter sp in vivo and in vitro

HO CHI MINH CITY – UNIVERSITY OF SCIENCE FACULTY OF CHEMISTRY MEDICINAL CHEMISTRY SEMINAR DRUGS DISCOVERY AND DEVELOPMENT TOPIC 1: STOMACH ULCER EVALUATING METHOD EFFECT OF FLUOROFAMIDE AS UREASE INHIBITOR ON HELICOBACTER SP IN VIVO AND IN VITRO Location • The most dilated part of Mouth Esophagus the digestive system • Between the esophagus and small intestine • Volume is about 1200- Stomach Small intestine Colon 1500 mL Anus The four main sections of the stomach: • Cardia (tâm vị) • Fundus (thượng vị) • Body or corpus (thân vị) • Pyloric part (mơn vị) Reservoir Breaking food into smaller fragments which are mixed with the gastric juice Acid secretion Hydrochloric acid (pH 1-2) in the gastric juice break food down even more, forming a semiliquid substance called chyme Enzyme secretion Releasing enzymes, hormones (gastrin, secretin, and gastric inhibitory peptide) Role in gastrointestinal Also involved in a small degree of absorption (water, caffeine, ingested ethanol) Plays a role in controlling secretion Functi on What is a stomach ulcer? • Are painful sores in the stomach lining • Occur when the thick layer of mucus (protects your stomach from digestive juices) is reduced → Digestive acids to eat away at the tissues that line the stomach → AN ULCER Symptoms of stomach ulcers A burning sensation or pain in the middle of your abdomen between your chest and belly button → More intense if stomach empty → A few minutes to several hours The causes of peptic ulcer • Helicobacter pylori (H pylori) • Long-term use of nonsteroidal anti- inflammatory drugs (NSAIDs) (aspirin, ibuprofen (Advil, Motrin IB,… ), naproxen sodium (Aleve)) • 1% by Zollinger-Ellison syndrome and others What is Helicobacter pylori? • Gram-negative bacilli with a spiral pattern of 2-4 µm long with a diameter of 0.5-1 µm and 2-6 cilia at one end were identified under a microscope • Live mainly in the lining of the stomach lining Mechanism of H pylori infection 10 F O H N FLUOROFAMIDE P NH2 NH2 • N-[diaminophosphinyl]-4-fluorobenzamide O • Synthesized by Dr A Bayless, Norwich-Eaton Pharmaceuticals (U S Patent No 4,182,881) • Fairly nontoxic (LD50 = 2.125 mg/kg) • Potent urease inhibitor (ED50 ≈ 100 nM) 11 BACTERIA STRAINS & GROWTH CONDITIONS Variable atmosphere incubator (Don Whitley Scienti®c Ltd) Helicobacter sp (type strain WO 881) 37oC Blood agar 12 A EFFECT OF pH AND UREASE ON H PYLORI SURVIVAL Saline control Wash Urea (50 mM) Saline Urea + Fluorofamide Phosphate - buffer (pH 7.4) Incubate 30mins 37oC (both 50 mM) F H N O O H2N Enzyme Urease NH3 + O P NH2 NH2 Incubate 30mins 37oC Dilute 1:10 with saline Adjust pH with HCl CO2 NH2 H O pH monitoring UREA NH3 decreases Plate and grow days 13 A EFFECT OF pH AND UREASE • Urease protects H pylori from exposure to acid by alterating the pH of the medium, through the production of ammonia At pH = 5.0: pH ≤ 4.0: → ✓no loss of virus viability ✓unchanged pH no loss of virus viability ✓no loss of virus viability ✓unchanged pH unchanged pH ✓no loss of virus viability ✓unchanged pH severe loss of virus viability ✓no loss of virus viability ✓unchanged pH change in pH Buffering capacity of the medium at pH < 4.0 H pylori urease is irreversibly inactivated 14 IN VITRO B EFFECT OF UREASE INHIBITION ON UREASE ACTICITY IN INTACT H PYLORI Saline control Incubate H pylori Fluorofamide 50/150/300/500nM 30mins 37oC Diluted 1:10 Incubate 30mins Phosphate buffer (pH 5.5) containing mM urea pH monitoring Samples removed Weather-Burn Amonniac analysis + 10% TCA Centrifuge 15 IN VITRO B EFFECT OF UREASE INHIBITION ON UREASE ACTICITY IN INTACT H PYLORI • Figure 3A: Fluorofamide at extremely low dose (ED50 = ~ 100nM), effectively removed the ability of the bacteria to raise the pH • Figure 3B: The release of ammonia from urea was measured, which confirmed that the change in pH was due entirely to urea hydrolysis 16 IN VIVO C.1 DOSING Ferrets male + female 760 - 1418 g naturally infected with H mustelae (1) Saline control (2) Fuorofamide mg/kg (3) Fuorofamide 50 mg/kg 24 hrs Killed by anesthetic overdose Stomachs removed 17 IN VIVO C.1 DOSING Fundus 4% formaldehyde Wax embed 24 hrs Stain Antrum (Warthin-Starry method) – none seen – minimal – mild – moderate – marked – very marked 18 IN VIVO C.1 DOSING 19 IN VIVO C.2 THE EFFECT OF FLUOROFAMIDE ON UREASE [14C] UREASE BREATHE TEST Saline control After 24 hrs Expired 14CO2 Fluorofamide 50 mg/kg O Enzyme Urease 14 C H 2N 14 Urea 14C (5 µCi/1mL saline) NH4+ + H 14CO3- Blood NH2 H O C-Urea Lung 14 CO2 in breath 2:1 (v/v) 2-ethoxyethanol + ethanolamine 20 ... secretin, and gastric inhibitory peptide) Role in gastrointestinal Also involved in a small degree of absorption (water, caffeine, ingested ethanol) Plays a role in controlling secretion Functi on What... hrs Stain Antrum (Warthin-Starry method) – none seen – minimal – mild – moderate – marked – very marked 18 IN VIVO C.1 DOSING 19 IN VIVO C.2 THE EFFECT OF FLUOROFAMIDE ON UREASE [14C] UREASE BREATHE... loss of virus viability ✓unchanged pH change in pH Buffering capacity of the medium at pH < 4.0 H pylori urease is irreversibly inactivated 14 IN VITRO B EFFECT OF UREASE INHIBITION ON UREASE