InnovatIve strategIes for stabIlIzatIon formulatIons

1 InnovatIve strategIes for stabIlIzatIon of therapeutIc peptIdes In aqueous formulatIonsChristina AvantiThe research presented in this thesis was performed within the framework of project D6-202 of the Dutch Top Institute : Milica Stankovic Leviny ZachreinaPrinting of this thesis was supported by generous contributions from: University of Groningen Faculty of Mathematics and Natural Sciences of the University of GroningenISBN: 978-94-6182-122-5 Copyright 2012 C. AvantiAll rights reserved.

2 No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any form or by any means, mechanically, by photocopying, recording or otherwise, without the written permission of the design: Bao Tung Pham and Christina AvantiLayout & printing: Off Page, AmsterdamRIJKSUNIVERSITEIT GRONINGENI nnovatIve strategIes for stabIlIzatIon of therapeutIc peptIdes In aqueous formulatIonsProefschriftter verkrijging van het doctoraat in deWiskunde en Natuurwetenschappenaan de Rijksuniversiteit Groningenop gezag van deRector Magnificus, dr.

3 E. Sterken,in het openbaar te verdedigen opmaandag 2 juli 2012om uurdoorChristina Avantigeboren op 3 april 1968 Kota Baru, Indonesi Promotor: Prof. dr. FrijlinkCopromotor: Dr. HinrichsBeoordelingscommissie: Prof. dr. Groothuis Prof. dr. Driessen Prof. dr. W. JiskootcontentsPART ONEINTRODUCTIONC hapter 1 General Introduction 9 Chapter 2 Current strategIes for stabIlIzatIon of Therapeutic Peptides in Aqueous formulatIons 13 PART TWOTHE USE OF DIVALENT METAL IONS AND CITRATE BUFFERS TO STABILIZE OXYTOCIN IN AQUEOUS SOLUTIONC hapter 3A New Strategy to Stabilize Oxytocin in Aqueous Solutions : I.

4 The Effects of Divalent Metal Ions and Citrate Buffer 35 Chapter 4A New Strategy To Stabilize Oxytocin in Aqueous Solutions: II. Suppression of Cysteine-Mediated Intermolecular Reactions by a Combination of Divalent Metal Ions and Citrate 51 PART THREETHE USE OF DIVALENT METAL IONS AND ASPARTATE BUFFER TO STABILIZE OXYTOCIN IN AQUEOUS SOLUTIONC hapter 5 Insight into the Stability of the Zinc-Aspartate-Oxytocin Complex 79 Chapter 6 Aspartate buffer and divalent metal ions affect the oxytocin conformation in aqueous solution and protect it from degradation 95 PART FOURTHE USE OF EXTREMOLYTES TO STABILIZE PROTEIN IN AQUEOUS

5 SOLUTIONC hapter 7 Extremolytes: Are There Universal Stabilizers for Proteins in Aqueous Solution? 121 Summary, Concluding Remarks, and Global Perspective 137 Samenvatting, Conclusies, Aanbevelingen, en Mondiaal Perspectief 145 Acknowledgments 153for all the women in for a better chance to live the life with the new born ..Every year 166 000 women die of bleeding after child birth, and more than 50% of these deaths occur in sub-Saharan Africa (Clyburn et. al., 2007)1general IntroductIon1general IntroductIonAlthough successful developments in the field of peptide synthesis increased the availability of peptide drugs [1], a significant part of the world s population is still facing serious problems associated with insufficient access to several essential drugs which are peptide in nature.

6 The major cause for this problem is the lack of stable formulatIons that withstand transport, storage and distribution, particularly in rural and remote areas of developing (tropical) countries that lack a cold chain. An important active pharmaceutical peptide is oxytocin, a nonapeptide which is the drug of first choice to treat bleeding after childbirth or post-partum hemorrhage (PPH) [2]. Although the availability of this drug has greatly declined maternal mortality rates in the developed world, PPH remains a leading cause of maternal mortality elsewhere [3].

7 Current commercial formulatIons of oxytocin are insufficiently heat-stable to withstand tropical conditions [4,5]. Cleavage of the disulfide bridge was found to be the major degradation pathway [6,7].The aim of the present thesis was to develop heat-stable formulatIons for polypeptide drugs, in particular oxytocin and investigate the mechanism of stabIlIzatIon based on degradation products formed during thermal stress before and after thesis is divided in four parts.

8 In part 1 (Chapter 2) a general introduction is given on the stability of peptide drugs in aqueous solution and current stabIlIzatIon technologies. Part 2 (Chapter 3 and 4) describes the use of a combination of divalent metal ions and citrate buffer to improve stability of oxytocin in aqueous solution, whereas part 3 (Chapter 5 and 6) is about the effect of zinc ions to improve stability of oxytocin in aspartate buffer. Finally, part 4 (Chapter 7) describes an attempt to find the universal stabilizer for polypeptides by investigating various extremolytes using lysozyme and insulin as model Chapter 2, we review the most common degradation pathways of peptides, such as hydrolysis, deamidation, isomerization, racemization, oxidation, disulfide exchange, dimerization, and further aggregation that cause loss of potency of pharmaceutical peptides.

9 In this chapter we also review different strategIes to overcome these instabilities, such as pH optimization, the use of buffers, antioxidant and other additives. In Chapter 3, we describe an investigation on the effect of monovalent (Na+ and K+) and divalent (Ca2+, Mg2+, and Zn2+) metal ions in combination with citrate and acetate buffers at pH of on the stability of oxytocin in aqueous solution. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by reversed-phase high performance liquid chromatography (RP-HPLC) and size exclusion chromatography (HP-SEC) after 4 weeks of storage at either 4 C or 55 C.

10 We also measured the interaction between oxytocin and Ca2+, Mg2+, or Zn2+ in citrate buffer in comparison with acetate buffer by using isothermal titration calorimetry (ITC). In Chapter 4, we identified various degradation products of oxytocin in citrate-buffered solution after thermal stress at a temperature of 70 C for 5 days and the differences in degradation pattern in the presence and absence of divalent metal ions. Degradation products of oxytocin in the citrate buffer formulation with and without divalent metal ions were analyzed using liquid chromatography mass spectrometry/mass spectrometry (LC MS/MS).