UPTEC-K12018 Examensarbete 30 hp September 2012 An investigation of protective formulations containing enzyme inhibitors Model experiments of trypsin Erika Billinger Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 AN INVESTIGATION OF PROTECTIVE FORMULATIONS CONTAINING ENZYME INHIBITORS. MODEL EXPERIMENTS ON TRYPSIN. ERIKA BILLINGER MASTER THESIS SPRING 2012 SVENSKA CELLULOSA AKTIEBOLAGET UPPSALA UNIVERSITY 2 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 ABSTRACT This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS- activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized. 3 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 POPULÄRVETENSKAPLIG SAMMANFATTNING Det är väl känt att avföring och urin är en viktig faktor som ger upphov till irriterad hud (dermatitis) för barn som använder blöjor. Tillsammans med avföringen följer enzymer från mag-tarmkanalen med och det är dessa enzymer som ”äter” på huden och ger upphov till dermatitis. Då urin avges i blöjan stiger pH vilket leder till en mer trivsam miljö för enzymerna som i sin tur leder till en ökad aktivitet och resulterar i en större area irriterad hud. Ytterligare anledningar till dermatitis är även den fuktiga miljön som skapas av stängd miljö och även friktionen som uppkommer då rörelser sker vid användning av blöjan. En skyddande formulering, i form av exempelvis en salva eller kräm, kan separera de skadliga ämnena från huden. Man kan även tänka sig använda en aktiv barriär innehållande aktiva substanser, exempelvis inhibitorer, som kan vara skyddande mot huden och samtidigt aktiv mot de skadliga ämnena från avföring och urin. Inhibitorerna i en salva eller kräm hämmar då enzymernas aktivitet och huden skyddas och blöjeksem undviks. Genom att undvika hudkontakt med de skadliga ämnena kan huden skyddas, åtminstone under en viss tidsperiod. Då avföringen innehåller ett matspjälkningsenzym trypsin har olika inhibitorer mot enzymet undersökts. För att även minimera risken för både toxiska och allergena effekter har två av dessa inhibitorer immobiliserats på NHS-aktiverad Sepharose. 4 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 TABLE OF CONTENTS An investigation of protective formulations containing enzyme inhibitors. Model experiments on trypsin. Abstract ............................................................................................................................................................. 3 Populärvetenskaplig sammanfattning ................................................................................................... 4 Introduction ..................................................................................................................................................... 6 What is an enzyme ............................................................................................................................................................... 7 Enzymes in feces .................................................................................................................................................................... 7 Trypsin ....................................................................................................................................................................................... 8 Substrate for trypsin ................................................................................................................................................. 9 Inhibitors ....................................................................................................................................................... 10 Aprotinin ................................................................................................................................................................................ 10 Soybean trypsin inhibitor ............................................................................................................................................... 11 α-1-antitrypsin .................................................................................................................................................................... 12 Leupeptin ............................................................................................................................................................................... 12 Antipain .................................................................................................................................................................................. 14 Immobilization ............................................................................................................................................. 15 Schiff bases ............................................................................................................................................................................ 15 NHS-activated Sepharose ............................................................................................................................................... 17 Materials & methods .................................................................................................................................. 18 Aprotinin ................................................................................................................................................................................ 18 Soybean trypsin inhibitor ............................................................................................................................................... 18 α-1-antitrypsin .................................................................................................................................................................... 18 Leupeptin ............................................................................................................................................................................... 18 Immobilization of α-1-antitrypsin ............................................................................................................................. 19 Coupling between NHS-activated Sepharose 4 Fast Flow and α-1-antitrypsin. .......................... 19 Test of immobilization .......................................................................................................................................... 19 Immobilization of leupeptin .......................................................................................................................................... 20 Coupling between NHS-activated Sepharose 4 Fast Flow and Leupeptin ...................................... 20 Test of immobilization of leupeptin ................................................................................................................ 20 Results ............................................................................................................................................................. 20 With each inhibitor ........................................................................................................................................................... 21 Experiments of immobilized inhibitor ...................................................................................................................... 25 α-1-antitrypsin ......................................................................................................................................................... 25 Leupeptin .................................................................................................................................................................... 26 Discussion and conclusions ..................................................................................................................... 29 References ..................................................................................................................................................... 30 Acknowledgements .................................................................................................................................... 32 Appendix ........................................................................................................................................................ 33 5 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 INTRODUCTION It is well known that feces and urine are two factors that affect the skin in a negative way and together they can cause skin irritation, dermatitis. The biggest difference between an adult skin and a baby’s skin lies